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INTRODUCTION
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Knowledge of this manual is required for the operation of the instrument. Would you therefore please
make yourself familiar with the contents of this manual and pay special attention to hints concerning the
safe operation of the instrument.
The specifications are subject to change; the manual is not covered by an update service.
©
Unless expressly authorized, forwarding and duplication of this document, and the utilization and
communication of its contents are not permitted. Violations will entail an obligation to pay
compensation.
All rights reserved in the event of granting of patents or registration of a utility model.
Issued by
Carl Zeiss MicroImaging GmbH
07740 Jena, Germany
Phone:
+49 (0) 3641 64-3400
Fax:
+49 (0) 3641 64-3144
E-mail:
[email protected]
www.zeiss.de/lsm
II
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
INTRODUCTION
Carl Zeiss
How to make best use of the LSM 5 LIVE operating instructions
A few symbols in these operating instructions will help you to recognize the nature and purpose of
information immediately:
The WARNING symbol warns against hazards for the user that might arise when operating the
laser.
This WARNING symbol warns against hazards from dangerously high voltages.
The CAUTION symbol warns against faults and hazards that might arise during operation and
which might cause damage to the unit.
The NOTE symbol will help you to optimally solve your work problem. It represents a practical
tip which will help you to find out which settings and methods are capable of improving or
accelerating a procedure.
The HOT SURFACE symbol warns against hazards for the user that might arise when touching
the lamp housing during operation.
The MAINS PLUG symbol remembers service personal to pull the mains plug before opening the
device housing.
Depending on the problem, these operating instructions will supply you with various possibilities:
• If you want to know where to find certain general areas of information, refer to the following outline
of sections to get a general overview.
• You will find a detailed table of contents at the start of every chapter. There you will see at a glance
what topics are dealt with in detail.
Always remember:
03/06
The time you invest in getting acquainted with the product will pay
for itself many times over in your application task.
B 45-0019 e
III
INTRODUCTION
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Contents
1
Notes on Device Safety
This section contains general notes on device safety, safe operation, and possible hazards
caused by failure to observe the instructions.
2
LSM 5 LIVE - Setup Requirements
The Setup Requirements section outlines the installation and supply requirements of the
LSM 5 LIVE Microscope Systems, together with the relevant specifications.
3
Introduction to Laser Scanning Microscopy
Here you will find an introduction to Laser Scanning Microscopy, with an explanation of the
principles of confocal imaging. The section also outlines the ways to present LSM image series
in three dimensions, and introduces you to the performance features of your LSM 5 LIVE.
4
Operation
In the Operation section you will find the most important steps and procedures of the
LSM menu structure. The step-by-step description how to get an image will be shown by
typical application examples including the WINDOWS XP graphic user environment.
5
Tools
This section contains a description of the use of the tools for setting the microscope.
6
3D for LSM
This section contains a description of the LSM 3D software package (basic program and addons). At the same time, all functions and settings are presented in a systematic form and in the
order in which they can be reached from the basic menu via sub-menus and dialog boxes.
7
Annex
The annex contains the Application-specific Configurations, special notes and information for
using the LSM microscope and the brochure The confocal Laser Scanning Microscopy.
8
Certification
9
Brief Operating Manual
This section contains the brief instructions of the LSM 5 LIVE Microscope Systems.
10
IV
Laser safety warning labels
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
CHAPTER 1
NOTES ON DEVICE SAFETY
Contents
Carl Zeiss
NOTES ON DEVICE SAFETY
CONTENTS
Page
1
NOTES ON DEVICE SAFETY .............................................................................................1-2
1.1
General..............................................................................................................................1-2
1.2
Warning and Information Labels ........................................................................................1-3
1.3
Regulations ......................................................................................................................1-10
1.4
Protection against diffuse laser excitation emitted around the objective and sample .........1-12
1.5
Protection against white light emitted around the objective and sample by an X-cite
fluorescence illumination..................................................................................................1-13
1.6
Notes on Setting up the Microscope System .....................................................................1-13
1.7
Power Requirements ........................................................................................................1-15
1.8
Notes on Handling the Laser Components........................................................................1-16
1.9
Notes on Handling HBO and X-Cite Light Sources ............................................................1-18
1.10
Physical Dimensions .........................................................................................................1-18
1.11
Environmental Requirements............................................................................................1-18
1.12
Notes on Handling the Computer and Data Media ...........................................................1-19
1.13
Notes on Care, Maintenance and Service .........................................................................1-20
1.14
1.14.1
1.14.2
User Interface...................................................................................................................1-21
Mounting and Dismounting Lamps, TPMT and Switching Mirror ......................................1-21
Mounting and Dismounting the Scan Heads.....................................................................1-23
03/06
B 45-0019 e
1-1
NOTES ON DEVICE SAFETY
General
Carl Zeiss
1
NOTES ON DEVICE SAFETY
1.1
General
LSM 5 LIVE
LSM 5 LIVE DuoScan
The LSM 5 LIVE laser scanning microscope, including its original accessories and compatible accessories
from other manufacturers, may only be used for the purpose of microscopic techniques.
Laser Scanning Microscopes (LSM) are intended for high resolution imaging of biological or material
samples, whereby in contrast to wide field microscopy the specimen is illuminated raster-fashion with a
focused laser beam and the optical arrangement prevents light from out-of-focus regions of the
specimen contributing to image formation.
Installation and commissioning of the LSM 5 LIVE system must be performed by authorized
Carl Zeiss service staff. The system should not be used prior to instruction by a Carl Zeiss
representative.
The manufacturer will not assume liability for any malfunction or damage caused by any thing
other than the intended use of the LSM 5 LIVE or individual modules or parts of it, nor by any
repair or other service operation performed or attempted by persons other than duly authorized
service staff. Any such action will invalidate any claim under warranty, including parts not
directly affected by such action. This also includes the modification of the system computer
with new cards, etc. by the user. The use of a camera at the base port of Axiovert 200 M
Combi stands is not allowed for reasons of laser safety. Any manipulation will result in the loss
of warranty of laser safety.
Please read also the notes on device safety and manuals of the microscope, the HBO, the HAL and
additional optional devices if ordered as the UV Laser, the piezo focusing device and heating inserts.
As the system is largely operated via menus on a computer, you should be acquainted with the principles
of the operating system and its WINDOWS, WINDOWS 2000 or Windows XP graphical user interface. The
respective manuals are supplied together with the programs.
The LSM 5 LIVE is a device that belongs to laser hazard class 3B. The system is equipped with
safety interlocks that comply with laser hazard class 3B and 4. WHO recommendations
concerning health and industrial protection when handling laser devices must be observed. The
operator of the unit must also observe all and any relevant statutory accident prevention
regulations. The user is referred to the safety data sheet provided together with the manual.
1-2
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
1.2
NOTES ON DEVICE SAFETY
Warning and Information Labels
Carl Zeiss
Warning and Information Labels
The warning and information labels attached on the LSM 5 LIVE must be observed. Check
whether all of the labels shown below are provided on your instrument, and contact Carl Zeiss
Germany or one of the service agencies if you should discover that any of the labels should be
missing. You will receive a free replacement.
Description of labels
Caution: Faults and hazards that might arise during operation which might cause damage to
the unit or injury to the user.
Attention: Laser irradiation hazards possible when operating the system.
Attention: High voltage.
Pull the mains plug before opening the device housing.
Caution: Hot surface.
Caution: UV radiation.
Caution: Fingers can be caught.
The arrow points to the opening where laser light comes out during operation of the system.
Other labels on the system include one of the above depicted symbols and a detailed
description of the handling instructions. See also the following drawings of the system parts.
03/06
B 45-0019 e
1-3
Carl Zeiss
NOTES ON DEVICE SAFETY
Warning and Information Labels
LSM 5 LIVE
LSM 5 LIVE DuoScan
1:1
E
TIV
JEC
RO
OB
ZE
OR
CT
FLE
RE
S
CU
FO
Fig. 1-1
1-4
Warning and information labels on the Axiovert 200 M microscope with the LSM 5 LIVE
scanning module
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
Fig. 1-2
03/06
NOTES ON DEVICE SAFETY
Warning and Information Labels
Carl Zeiss
Warning and information labels on the Axio Imager.Z1 microscope with scanning module
B 45-0019 e
1-5
Carl Zeiss
NOTES ON DEVICE SAFETY
Warning and Information Labels
LSM 5 LIVE
LSM 5 LIVE DuoScan
04
01
07
02
06
03
05
08
0
10
90
Fig. 1-3
1-6
Warning and information labels on the Axioskop 2 FS MOT microscope with scanning module
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
Fig. 1-4
03/06
NOTES ON DEVICE SAFETY
Warning and Information Labels
Carl Zeiss
Warning and information labels on LSM DuoScan (systems LSM 5 LIVE DuoScan only)
B 45-0019 e
1-7
Carl Zeiss
Fig. 1-4
1-8
NOTES ON DEVICE SAFETY
Warning and Information Labels
LSM 5 LIVE
LSM 5 LIVE DuoScan
Warning and information labels on laser components
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
NOTES ON DEVICE SAFETY
Warning and Information Labels
CO
HE
RE
Carl Zeiss
nT
En
TE
RP
RIS
E
AK
PE
CH
AR
SE
E4
En
TE
RP
RIS
E
E5
CO
HE
RE
nT
Fig. 1-5
03/06
um
xim
ma ter
wa ure
ss
pre p.s.I.
60
T
OU
ter
wa
7
J3
E4
J6
Warning and information labels on UV laser components (LSM DuoScan only)
B 45-0019 e
1-9
NOTES ON DEVICE SAFETY
Regulations
Carl Zeiss
1.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Regulations
Extensive knowledge of the hardware/the system is indispensable for safe operation of the LSM 5 LIVE.
Read these operating instructions and all device publications belonging to the system conscientiously before operating the LSM 5 LIVE! You can obtain additional information on the hardware configuration delivered and on optional system extensions from the manufacturer or via
the service hotline.
⇒ The LSM 5 LIVE has been designed, built and tested in conformity with the following regulations and
guidelines:
DIN EN 61010-1 (IEC 601010-1) "Safety requirements for electrical equipment for measurement,
control and laboratory use"
DIN EN 60825-1 (IEC publication 60825-1) "Safety of laser equipment", taking relevant CSA and
UL specifications into account
DIN EN 61326: “Electrical equipment for control technology and laboratory use – EMC-requirements”
Low voltage directive: 73/23/EWG
EMC directive: 89/336/EWG
⇒ The company works according to a certified Environment Management System according to
ISO 14001.
The Product was developed, tested and produced in accordance with the valid regulations and guidelines
for environmental law of the European Union.
The product and its accessories have been classified as instrument category 9 (laboratory equipment or
comparable standard). The product and its accessories agree with the EU-regulations 2002/95/EG (RoHS)
and 2002/96/EG (WEEE), if applicable for the product.
Carl Zeiss has installed a process for taking back and recycling the instruments within the member states
of the European Union, which takes care of the appropriate utilization according to the said EU
guidelines.
For details on the disposal and recycling please refer to your relevant Carl Zeiss sales or service
organization.
The product must not be disposed in the household waste or through the municipal disposal
organizations. In case of resale the seller is obliged to inform the buyer, that the product has to be
disposed according to the said regulations.
1-10
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
1.4
NOTES ON DEVICE SAFETY
Protection against diffuse laser excitation …
Carl Zeiss
Protection against diffuse laser excitation emitted around the objective and sample
Though the radiation levels around the objective and sample is very low (comparable to a common laser
pointer used for e.g. talks), avoid to approach this area closer than 10 cm with your eye.
Also limit the time of skin exposure of your hands to the time needed to place the sample and to mechanically set up the acquisition.
Switch off the scanning while doing such manipulation around the objective and sample!
In case of scanning, don’t stare into the beam!
To further reduce the radiation level of fluorescent emission and excitation light around the microscope,
Carl Zeiss delivers antiglare screens for all microscopes.
Axiovert 200M: 000000-1109-741
Axio Imager.Z1/M1: 452163-0000-000
Axioskop 2 FS mot: 452163-0000-000
Fig. 1-6
Antiglare screens for Axiovert 200M (left) and Axio Imager and Axioskop 2 FS (right)
Use these screens for improved working ergonomy!
03/06
B 45-0019 e
1-11
Carl Zeiss
1.5
NOTES ON DEVICE SAFETY
Protection against white light …
LSM 5 LIVE
LSM 5 LIVE DuoScan
Protection against white light emitted around the objective and sample by an X-cite
fluorescence illumination
Follow the guidelines in the X-Cite manual provided by EXFO Inc..
Don’t use a neutral beamsplitter (e.g. “NT80/20” position in LSM DuoScan or LSM 5 DUO systems) in the
P&C modules while looking through the eyepieces. Strong white light might project into your eyes.
In case such strong light is projected into your eye, the natural lid closure effect will protect you from any
damage. Nonetheless such exposure is unpleasant and should absolutely be avoided.
1.6
Notes on Setting up the Microscope System
Installation and commissioning of the LSM 5 LIVE system must be performed by authorized
Carl Zeiss service staff. The system should not be used prior to instruction by a Carl Zeiss representative.
The LSM 5 LIVE laser scanning microscope is delivered in several crates:
The LSM 5 LIVE must be set up so as to ensure that the minimum clearance between the wall
and the rear of the system is no less than 0.5 m. This clearance is needed for adjustment and
maintenance operations.
Do not set up the unit in the proximity of heat sources such as radiators or direct sunlight. To avoid heat
build-ups, the ventilation slots on the microscope system must not be covered up.
The system must not be set up in areas with potential danger by explosives.
The unit must be connected to a properly installed socket outlet with earthing contact by means of the
mains cables supplied. Continuity of PE connection must not be affected by the use of extension leads.
The system contains components with dangerous voltage. The system must not be opened by
anybody else than authorized Carl Zeiss Service staff. Before opening the main plug has to be
disconnected.
Before connecting the mains cables, please check whether your mains voltage corresponds to
the voltage specified on the rating plate of the laser module.
For reasons of laser safety, all ports must either be equipped with the corresponding device
(scan head, camera, HBO lamp etc.) or covered with the counterpart of the laser safety kit
provided.
1-12
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
NOTES ON DEVICE SAFETY
Protection against white light …
Carl Zeiss
Maintenance, repair, modification, addition, removal or exchange of components, or other
interference with the equipment beyond the operations described in this manual may only be
carried out by the manufacturer Carl Zeiss or by persons expressly authorized by Carl Zeiss to do
so.
This applies especially to the microscope system, the laser scanning module, lasers, the PC
system, the power supply units, cable connections, safety interlock devices and other system
components.
Please note that the LSM 5 LIVE are high-precision opto-electronic instruments. Inexpert
handling may easily impair their function or even damage them.
The openings for ventilation must not be covered.
There are hot surfaces on the HBO and HAL lamp.
When sliding the compact Laser module in and out of the System electronic rack take care not
to catch your fingers.
The HBO 50 and 100 W, XBO 75 W and X-cite 120 lamps used on the light microscopes
incident light path emit UV light which is harmful to the human eye and skin when observed
without appropriate filters. Never remove the lamp and look direct into the emitted light.
After installation or conversion of the LSM system, authorized specialized staff must carefully check that it
is in a proper condition and, particularly, that covers protecting against laser radiation are provided.
Tube openings or other unused mounts should always be protected against dust and moisture with the
corresponding device components or with termination covers/blind plugs.
By establishing a corresponding workplace environment, please ensure that the formation of electrostatic
charges of electronic components is avoided.
To avoid vibrations during operation, the LSM 5 LIVE should only be operated in conjunction with the
system table (vibration damping).
03/06
B 45-0019 e
1-13
NOTES ON DEVICE SAFETY
Power Requirements
Carl Zeiss
1.7
LSM 5 LIVE
LSM 5 LIVE DuoScan
Power Requirements
The LSM 5 LIVE comes with a mains power supply cord and plug, either CEE red (3/N/PE
400/230 V/16 A), or NEMA L 14-30P (2/N/Ground 120/240 V/30 A), and with the matching
mains socket outlet.
A ground wire (AWG10 green/yellow) is supplied because it is necessary to ground the system.
The connecting part on both ends of the cable is a cable eye with 8 mm inner diameter.
A suitable grounding point must be installed in the room.
For systems (220 ... 240 V AC) equipped with X-Cite 120 the mains socket outlet must be
equipped with a fuse having minimum tripping characteristic C according to IEC/EN 60898.
Line voltage
220 … 240 V AC (±10 %)
100 … 125 V AC (±10 %)
Line frequency
50...60 Hz
50...60 Hz
− Max. current
3 phases at 16 A
2 phases at 25 A
− Power
Phase 1 = 1.9 kVA max.
Phase 1 = 3.2 kVA max.
Phase 2 = 1.5 kVA max.
Phase 2 = 2.8 kVA max.
LSM incl. VIS laser
Phase 3 = 2.6 kVA max.
− Power Consumption
5000 VA max.
5000 VA max.
208...240 V AC
208...240 V AC
(±10 %) 50 / 60 Hz
(±10 %) 50 / 60 Hz
1 phase at 63 A
1 phase at:
Argon UV laser
−
−
Line Voltage
Max. current
208 V: 34 Amps
Note: For Line Voltage 220 V the connector and power plug are rated for
63 Amps, However wiring and fuse
should be rated for 32 Amps.
240 V: 29 Amps
7000 VA max.
7000 VA max.
Class of protection
I
I
Type of protection
IP 20
IP 20
Overvoltage category
II
II
Pollution degree
2
2
−
Power consumption
230 V: 31 Amps
1-14
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
1.8
NOTES ON DEVICE SAFETY
Notes on Handling the Laser Components
Carl Zeiss
Notes on Handling the Laser Components
The LSM 5 LIVE are laser hazard class 3B instruments. This moderate and high-risk class
embrace medium-power and high power lasers. You must take care not to expose yourself to
the radiation of such lasers. In particular, never look into the laser beam! Only personnel which
has been instructed on laser safety is allowed to operate the system.
The following laser types are currently intended for use in the LSM 5 LIVE. The use of any other lasers as
the ones listed below is not authorized.
Laser
Class
Power
1 Diode laser 405 nm
3B
50 mW
2 Diode laser 440 nm
3B
16 mW
3 Diode laser 488 nm
3B
100 mW
4 DPSS laser 532 nm
3B
75 mW
5 Diode laser 635 nm
3B
35 mW
6 Ar laser 351/364 nm (UV, for DuoScan only)
4*
80 mW
* Laser type class 4, if mounted on laser module with fiber output class 3B.
Please note that for the maintenance of the UV Laser it is recommended to run the laser at
maximum power once a day if the laser is not used frequently or only at low power levels. This
enables the Autofill process which keeps up the correct tube gas pressure. This operation
prolongs the life time of the tube and prevents complete tube failure if the laser is not used
for a prolonged period of time. For details please refer to the Operator’s Manual of the UV
laser.
Please contact Carl Zeiss if you intend to use a different laser other than the ones above.
If used properly, the LSM 5 LIVE will not pose any laser radiation risks for operating staff. Nevertheless,
you should observe the following warnings:
03/06
B 45-0019 e
1-15
Carl Zeiss
NOTES ON DEVICE SAFETY
Notes on Handling the Laser Components
LSM 5 LIVE
LSM 5 LIVE DuoScan
• If necessary - insofar as specified by law - inform the laser protection officer before commissioning the laser.
• The laser modules are equipped with a key-interlock.
• Always store keys for laser key switches and, if applicable, keys for further laser power
supply units, where they are inaccessible to persons not authorized to operate the laser.
• A red LED on the front of the scan head lights up when one or all of the lasers are switched
on.
• Do not place any reflecting objects into the beam path.
• Never open any covers or panels.
• Never look into the laser beam, not even to simply view the specimen, whether with the aid
of optical instruments or without. Otherwise you risk going blind!
• Do not leave any empty objective positions of the nosepiece uncovered.
Suitable protective measures must be taken if gases, dust or vapors hazardous to health,
secondary radiation or explosive objects should arise on the specimen as a result of laser
radiation.
1-16
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
1.9
NOTES ON DEVICE SAFETY
Notes on Handling HBO and X-Cite Light Sources
Carl Zeiss
Notes on Handling HBO and X-Cite Light Sources
For LIVE systems equipped with a rearport mounted scan head be aware that the mirror cube in
the reflector turret leads to a strong back reflection of HBO light into the specimen plane and
the ocular lens. When observing the specimen through the ocular lens the use of the mirror
cube position should be avoided. The light flash is not harmful but unpleasant. The reflex of
closing the eyelid is sufficiently protective. To avoid this situation an additional filter LP 397
(from filter cube 01, # 48 80 01) can be mounted into the mirror cube which prevents the back
reflection of the HBO UV light in the ocular plane.
1.10
Physical Dimensions
Length (cm)
Passively damped anti-vibration table
Width (cm)
Height (cm)
Weight (kg)
130
100
75
137
Scanning Module LSM 5 LIVE
55
17
30
19,5
Scanning Module LSM DuoScan
39
15
13
7
Microscope
50
35
50
20
Laser Module LIVE
66
52
22
58
Laser Module, UV (for DuoScan only)
140
20
20
60
System Electronic Rack
110
70
58
90
Plug-in unit external laser (UV)
66
52
22
9
Power supply for Ar (UV)
50
50
30
30
Cooling unit for Ar (UV)
80
45
50
30
1.11
Environmental Requirements
1. Operation, specified performance
T = 22 °C ± 3 °C without interruption (24 h a day
independently whether system is operated or
switched-off)
2. Operation, reduced performance
T = 10 °C to 35 °C, any conditions different from 1.
and 5.
3. Storage, less than 16 h
T = -40 °C to 55 °C
4. Storage, less than 6 h
T = -55 °C to 70 °C
5. Temperature gradient
± 0.5 °C/h
6. Warm up time
1 h, for high-precision and/or long-term measurements ≥ 3 h
7. Relative humidity
< 65 % at 30 °C
8. Operation altitude
max. 2000 m
9. Loss of heat
4 kW
03/06
B 45-0019 e
1-17
Carl Zeiss
1.12
NOTES ON DEVICE SAFETY
Notes on Handling the Computer and Data Media
LSM 5 LIVE
LSM 5 LIVE DuoScan
Notes on Handling the Computer and Data Media
The computer used as standard in your LSM system is an IBM-compatible high-end Pentium computer
with WINDOWS XP operating system.
Do make sure, though, that you receive your LSM system with the operating system installed,
with initialization and start files set up and with the LSM program also installed.
When working with the hard disk, it is important to know that the more data it contains, the
slower its operation will become. Therefore, data that you do not need permanently should be
stored on other external devices.
When handling diskettes, avoid data losses by protecting them against extreme temperatures,
moisture and magnetic fields. The data on a diskette is stored in the form of magnetic signals.
To some extent, monitors, telephones or even lamps generate magnetic fields that might
destroy this data. Also, never open the metal cover on diskette cases. A diskette´s surface can
also be destroyed by touching it.
When handling CDs, CD ROMs or DVDs, do not touch the data side of the disc (the side of the
disc with no label or printing).
Do not apply paper labels or write on any part of the disc, data side or label side. If dust or
fingerprints get on the disc, wipe it with a soft cloth from the center to the edge, but do not
use benzine, paint thinner, record cleaner, or static repellent. This can damage the disc.
Do not place the disc in any place where it is exposed to direct sunlight or high temperatures.
Backup your data on a regular basis.
Do not install any other software without talking to your Carl Zeiss representative.
Never turn your computer off before you have terminated the LSM program and run down the
WINDOWS XP operating system. Otherwise, the program and/or data files may get lost.
1-18
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03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
1.13
NOTES ON DEVICE SAFETY
Notes on Care, Maintenance and Service
Carl Zeiss
Notes on Care, Maintenance and Service
The manufacturer of the unit cannot be held liable for damage resulting from operating errors, negligence or unauthorized tampering with the device system, particularly as the result of removal or
replacement of parts of the unit or as the result of the use of unsuitable accessories from other manufacturers.
Any such action will also render all warranty claims null and void and laser safety might no longer be
warranted.
You are well advised to arrange a service agreement with your nearest Carl Zeiss representative to
guarantee perfect functioning of the microscope system in the long term.
Modifications and conversion work on the components of the system must only be carried out by the
manufacturer, by the service agency or by persons authorized and trained for this purpose by the
manufacturer.
Damaged units or parts may only be repaired or maintained by the responsible service agency.
During maintenance or repair carried out by the service personnel the customer is requested to stand
aside and wear a pair of laser safety goggles if needed.
Before opening the housing of the halogen lamp switch off all laser units.
Care operations that may be carried out by operating staff are limited to cleaning painted and glass
surfaces.
• Before cleaning the instrument make sure the main power supply is disconnected.
• Cleaning painted surfaces
To do this, use a clean cloth that has been moistened in a mixture of water and some detergent; do
not use any solvent, however. Dry with a lint-free cloth.
• Cleaning glass surfaces
Glass surfaces that have become soiled or which are marked with fingerprints may be rubbed with a
clean optical cleaning cloth.
If soiling is persistent, dip the optical cleaning cloth into a mixture of distilled water and a small
quantity of detergent.
To complete cleaning, lightly breathe on the glass surface and rub it dry with a clean cloth. Lint or dust
is best removed with a clean brush.
• Make sure that no cleaning liquid penetrates into the system.
• Dust filters in the ventilation entries of the system electronic rack have to be replaced every 6 month.
For replacement please contact your local service representative.
03/06
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1-19
NOTES ON DEVICE SAFETY
User Interface
Carl Zeiss
1.14
LSM 5 LIVE
LSM 5 LIVE DuoScan
User Interface
All user interface ports are equipped with a safety interlock system which warrants laser safety.
These interlock devices must not be manipulated. Other interfaces which are not described here
are service interfaces and are only to be operated by authorized Carl Zeiss service personnel.
The following devices can be mounted and dismounted by the user:
1.14.1
−
Halogen and HBO lamp
−
Scan head
Mounting and Dismounting Lamps, TPMT and Switching Mirror
The ports of the lamps, the switching mirror and the transmission PMT are equipped with hardware
interlock devices. At most ports (all ports of the Axioskop 2 FS MOT and the Axio Imager.Z1 as well as the
HBO port of the Axiovert 200 M) the following interlock devices are present and have to be operated in
the following way:
Interlock with sensor ring and contact ring:
Fig. 1-7
Sensor ring mounted to the interface
ports on the microscope side
Fig. 1-8
Contact ring mounted to the lamp,
TPMT or switching mirror
The interlock is working when the sensors of the sensor ring (Fig. 1-7/1) are depressed by the pins on the
contact ring (Fig. 1-8/1). Whenever this is not the case, for example if the distance between the two
devices is too large, the laser will be blocked and the system cannot be used.
In case the system is not operating following the removal or attachment of any device on a port
with safety interlock check again the connection of the Contact ring to the Sensor ring.
1-20
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LSM 5 LIVE
LSM 5 LIVE DuoScan
NOTES ON DEVICE SAFETY
User Interface
Carl Zeiss
For dismounting the lamps, TPMT or switching
mirror slightly unscrew the contact ring first
(Fig. 1-8/2 or Fig. 1-9/2) so it can be moved away
from the sensor ring (Fig. 1-9/1). Then unscrew the
lamp, TPMT or switching mirror (main screw
Fig. 1-9/3) which is in one of the recesses of the
sensor ring (Fig. 1-7/2). Hold the device to be
dismounted with one hand while unscrewing to
keep it from dropping. The now empty port has to
be closed with the blind cap to restore the
functionality of the system. Use the main screw of
the port to fix the cap. Make sure the pins of the
cap depress the sensors of the sensor ring.
Do not remove the sensor ring from
the microscope. This might result in
failure of laser safety and a non
operating system.
Fig. 1-9
HBO lamp mounted to
Axiovert 200 M with sensor
ring and contact ring for laser
safety
Fig. 1-10
Blind cap for closing any port
equipped with an interlock device
For mounting any lamp, TPMT or the switching
mirror back onto the microscope reverse the steps
for dismounting the device. Be careful not to bend
the pins on the contact ring when screwing the
device onto the microscope port.
For the Axiovert 200 M transmission port and the
two ports available on the motorized switching
mirror no sensor ring is present. Instead the
sensors are directly installed at the Axiovert 200 M
transmission port or the two ports of the motorized switching mirror.
03/06
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1-21
Carl Zeiss
NOTES ON DEVICE SAFETY
User Interface
1.14.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Mounting and Dismounting the
Scan Heads
The scan heads are connected to the microscope
via an integrated safety interlock. They can be
moved between two microscopes. Make sure the
system is shut off completely before starting the
following procedure:
Be aware that the LSM 5 LIVE scan
head weights up to 19,5 kg.
Moving
the
scan
heads
between
Axiovert 200 M, Axioskop 2FS MOT and Axio
Imager.Z1:
Fig. 1-11
Fig. 1-12
Electronic connection port of the
LSM 5 LIVE scan head
• Loosen the three screws on LSM 5 LIVE or LSM
DuoScan scan head (Fig. 1-12/1 or Fig. 1-13/1).
Port connection between LSM 5 LIVE
and Axiovert 200 M
Fig. 1-13
Fastening screws of the scan
head at the front of the tube
on Axioskop 2 FS MOT and
Axio Imager.Z1
• Slowly pull the scan head away from the microscope port or the tube. For mounting the scan head
onto a microscope, make sure the pins and the electronic connections of the safety interface match
closely. Fasten the three screws on the LSM 5 LIVE or LSM DuoScan scan head (Fig. 1-12/1 or Fig.
1-13/1).
1-22
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LSM 5 LIVE
LSM 5 LIVE DuoScan
NOTES ON DEVICE SAFETY
User Interface
Carl Zeiss
• Use the three fastening screws (Fig. 1-14/1 and 2) to attach or detach the LSM DuoScan to or from
the microscope stand.
Fig. 1-14
Fastening screws and connections on LSM DuoScan
To ensure functioning of the system and laser safety the following connections have to be changed:
1.
The connection of the microscope to the safety interface of the system is located either on the
additional Safety-Box (Axioskop 2 FS MOT, Fig. 1-15/1) or on the rear side of the microscope
(Axiovert 200 M or Axio Imager.Z1; Fig. 1-16/1 and Fig. 1-17/1). This connection has to be
unplugged from the microscope which is not in use and plugged into the microscope to be used
following the exchange of the scan head.
Fig. 1-15
03/06
Safety-Box of Axioskop 2 FS MOT
with main connection to safety
interface (1)
B 45-0019 e
Fig. 1-16
Connection of Axio Imager.Z1
to safety interface (1) and
electronics (2)
1-23
Carl Zeiss
NOTES ON DEVICE SAFETY
User Interface
LSM 5 LIVE
LSM 5 LIVE DuoScan
1
Fig. 1-17
Fig. 1-19
1-24
Connection of Axiovert 200 M to
safety interface (1)
Fig. 1-18
Four CAN connections (1) are
available on the rear side of the
electronics module. The microscope
in use has to be connected to one
of them
2.
The plug for the main connection of the
microscope to the electronics is situated on
the rear side of the electronics rack. It is
either of the four CAN connections shown
in Fig. 1-18. Only ONE microscope can be
connected to the electronics at a time. The
connection has to be plugged in and
unplugged at the electronics rack. The
corresponding
connections
on
the
microscopes are shown in Fig. 1-16/2,
Fig. 1-19/1 and Fig. 1-20/1. Remove the
connection of the microscope no longer in
use at the electronics rack and plug in the
connection of the microscope which should
be used instead.
B 45-0019 e
03/06
CAN connections on the
Axiovert 200 M (1). One of the
plugs is occupied with the
connection to the electronics
LSM 5 LIVE
LSM 5 LIVE DuoScan
3.
NOTES ON DEVICE SAFETY
User Interface
After the connections of the non used
microscope are disconnected and the ones
of the used microscope are connected, the
system can be switched on again. Before
initializing the system with the LSM
Software make sure to use the right
database according to the microscope in
use. It can be chosen via the icon Stand
Select.
Fig. 1-20
03/06
Carl Zeiss
B 45-0019 e
The Axioskop 2 FS MOT is connected
to the electronics via the Interface
Control. The CAN connection is
fixed (1) to the control box
1-25
LSM 5 LIVE
LSM 5 LIVE DuoScan
CHAPTER 2
LSM 5 LIVE - SETUP REQUIREMENTS
Carl Zeiss
Contents
LSM 5 LIVE/LSM 5 LIVE DuoScan SETUP REQUIREMENTS
CONTENTS
Page
2
LSM 5 LIVE - SETUP REQUIREMENTS ..............................................................................2-2
2.1
General Remarks................................................................................................................2-2
2.2
System Components and Recommended Setup..................................................................2-2
2.3
Room Requirements...........................................................................................................2-2
2.4
Minimum Space Requirements of the System .....................................................................2-3
2.5
Dimensions (Overview) .......................................................................................................2-3
2.6
Schematic Outline of a System ...........................................................................................2-4
2.7
2.7.1
Space Requirements...........................................................................................................2-4
LSM DuoScan with Ar UV Laser ..........................................................................................2-5
2.8
Power Requirements ..........................................................................................................2-6
2.9
Physical Dimensions ...........................................................................................................2-9
2.10
Dimension of Shipment Crates ...........................................................................................2-9
2.11
Environmental Requirements............................................................................................2-10
2.12
Vibrations ........................................................................................................................2-10
2.13
Microscopes.....................................................................................................................2-11
2.14
Scanning Module LSM 5 LIVE ...........................................................................................2-12
2.15
Laser Module LIVE............................................................................................................2-12
2.16
Laser Module UV for LSM DuoScan UV ............................................................................2-12
2.17
System Overview LSM 5 LIVE ............................................................................................2-13
2.18
System Overview LSM 5 LIVE DuoScan .............................................................................2-15
03/06
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2-1
LSM 5 LIVE - SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
2
LSM 5 LIVE - SETUP REQUIREMENTS
2.1
General Remarks
LSM 5 LIVE
LSM 5 LIVE DuoScan
The LSM 5 LIVE is a user friendly, high performance laser scanning system with unique technological
performance. It is made of high precision components representing latest technological developments
and a design that was guided by ergonomics, best possible performance and every day usability. The
system is highly modular and flexible, but also easy to service and open for upgrades.
2.2
System Components and Recommended Setup
The core of the system is a vibration isolating, air damped microscope table with a breadboard for
fixation of the system and possible accessories. We strongly recommend the use of our Science Desk
tables, since these have the most compact size that allows the setup of the various system configurations
and future upgrades. A wide range of special accessories for physiological, molecular or cellbiological, or
technical experiments (e.g. posts, shelves, grounding, faraday cages, cabinets etc.) is available worldwide
by our partner Melles Griot. The science desk is a professional, open system platform for your
experiments. A wide and a narrow version of the science desk table is available, matching the side- or
rear port configurations of the microscope stands.
The microscope table is complemented by the laser and electronic system rack, which can be positioned
at the side or at the back of the microscope table. This rack contains all electronic and laser components
the LSM 5 LIVE needs, and is again designed with best performance, usability and future upgrades in
mind. For setup and service purposes, free access is needed to the long side of the rack. Hence approx.
50 cm space should be provided to the next wall.
Use of the LSM 5 LIVE is mainly done by operating the software. Consequently, we provide an ergonomical computer workplace that fits the microscope table and should best be positioned on the right
side of it. We recommend the use of the 45° triangle extension to get the best ergonomical setup of the
complete system, but also a 0° or a 90° orientation to the microscope table is possible. The best overall
system design depends on the shape of your room (see below).
2.3
Room Requirements
The LSM 5 LIVE needs approx. an area of 7 m². The room for the LSM 5 LIVE should therefore offer a
minimum size of 10 m². Due to the modular design, both square shaped rooms (approx. 3,2 m x 3 m) or
rectangular shaped rooms (approx. 2.6 m x 3.9 m) match the setup requirements of the LSM 5 LIVE. Due
to the modern diode technology of the lasers, the system will produce much less heat and noise than
older confocals. Nonetheless an lab typical air condition or ventilation should be provided. A constant
temperature of 23 °C would be ideal for constant and best system performance. Details should be
discussed with your local Zeiss LSM specialist.
2-2
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LSM 5 LIVE
LSM 5 LIVE DuoScan
2.4
LSM 5 LIVE - SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
Minimum Space Requirements of the System
For a list of the components dimensions see below; in general, a space of 1.8 m x 3.6 m for systems with
a 0° or 45° oriented computer workplace, or 2.8 m x 2.6 m for systems with a 90° oriented computer
workplace is required. As mentioned, free access to the long side of the electronic and laser system rack
is required for service and setup of the system. If possible, we also recommend to have a free space of
30 cm to the next wall all around the system.
2.5
Dimensions (Overview)
Component
Overall dimensions
Remarks
Science desk microscope table (wide)
125 cm x 100 cm
Side port systems
Science desk microscope table (narrow)
100 cm x 125 cm
Side- and Rear port systems
Electronic and laser system rack
75 cm x 115 cm
Long side must be
accessible
Computer workplace (0° or 90°)
120 cm x 80 cm
Computer workplace diagonal (45°)
130 cm x 160 cm
Recommended for
ergonomics
Complete system in ideal configuration, wide 180 cm x 360 cm
table, 45° computer workplace
Complete system in ideal configuration, 180 cm x 340 cm
narrow table, 45° computer workplace
Most often used setup
Complete system square setup, 90° computer 280 cm x 260 cm
workplace orientation
03/06
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2-3
LSM 5 LIVE - SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
2.6
Schematic Outline of a System
Fig. 2-1
2.7
Schematic outline of a system
Space Requirements
Fig. 2-2
2-4
LSM 5 LIVE
LSM 5 LIVE DuoScan
LSM 5 LIVE with microscope table and PC table (dim. in mm)
The System rack contains all electronics and laser unit.
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
2.7.1
LSM 5 LIVE - SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
LSM DuoScan with Ar UV Laser
We recommend placing the cooling unit of the Ar laser (UV) in a separate room to prevent heat
accumulation and vibration. Length of the water hose: 400 cm
Fig. 2-3
Space requirements for LSM DuoScan using an AR UV Laser (measurements in mm)
Fig. 2-4
Lab container for UV laser setup
The lab container cart 000000-0465-515 is recommended to provide space for the setup of the external
UV-laser, the UV laser power supply and the plug in unit for external laser.
03/06
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2-5
LSM 5 LIVE - SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
2.8
LSM 5 LIVE
LSM 5 LIVE DuoScan
Power Requirements
The LSM 5 LIVE comes with a mains power supply cord and plug, either CEE red (3/N/PE
400/230V/16A), or NEMA L 14-30P (2/N/Ground 120/240V/30A), and with the matching mains
socket outlet.
A ground wire (AWG10 green/yellow) is supplied because it is necessary to ground the system.
The connecting part on both ends of the cable is a cable eye with 8 mm inner diameter.
A suitable grounding point must be installed in the room.
Line voltage
220 … 240 V AC (±10 %)
100 … 125 V AC (±10 %)
Line frequency
50...60 Hz
50...60 Hz
− Max. current
3 phases at 16 A
2 phases at 25 A
− Power
Phase 1 = 1.9 kVA max.
Phase 1 = 3.2 kVA max.
Phase 2 = 1.5 kVA max.
Phase 2 = 2.8 kVA max.
LSM incl. VIS laser
Phase 3 = 2.6 kVA max.
− Power Consumption
5000 VA max.
5000 VA max.
208...240 V AC
208...240 V AC
(±10 %) 50 / 60 Hz
(±10 %) 50 / 60 Hz
1 phase at 63 A
1 phase at:
Argon UV laser
(LSM DuoScan only)
− Line Voltage
− Max. current
208 V: 34 Amps
Note: For Line Voltage 220 V the connector and power plug are rated for
63 Amps, However wiring and fuse
should be rated for 32 Amps.
− Power Consumption
230 V: 31 Amps
240 V: 29 Amps
7000 VA max.
7000 VA max.
Class of protection
I
I
Type of protection
IP 20
IP 20
Overvoltage category
II
II
Pollution degree
2
2
2-6
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LSM 5 LIVE
LSM 5 LIVE DuoScan
Fig. 2-5
03/06
LSM 5 LIVE - SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
Multipoint connectors
B 45-0019 e
2-7
LSM 5 LIVE - SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
1
2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Key-interlock Laser ON/OFF
Door interlock interface
Fig. 2-6
Key-interlock Laser ON/OFF and interface for connection of door interlock
The door interlock interface is covered with a green plug to bypass a door interlock.
• To use the interface remove the top of the green plug and the bypass wire.
• Then connect the wires of the door interlock at the same position.
Two door interlocks can be connected.
2-8
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LSM 5 LIVE - SETUP REQUIREMENTS
Physical Dimensions / Dimension of Shipment Crates
LSM 5 LIVE
2.9
Carl Zeiss
Physical Dimensions
Length (cm)
Passively damped anti-vibration table
Width (cm)
Height (cm)
Weight (kg)
130
100
75
137
Scanning Module LSM 5 LIVE
55
17
30
19.5
Scanning Module LSM DuoScan
39
15
13
7
Microscope
50
35
50
20
Laser Module LIVE
66
52
22
58
140
20
20
60
66
52
22
9
110
70
58
90
Power supply for Ar (UV)*
50
50
30
30
Cooling unit for Ar (UV)*
80
45
50
30
Water hose for Ar (UV)*
700
Fiber optic cable, UV*
300
Fiber optic cable, VIS(ible)
300
Cables
350
Laser Module UV *(DuoScan only)
Plug-in unit external laser (UV)*
System Electronic Rack
2.10
Dimension of Shipment Crates
Crate containing
Length (cm)
Width (cm)
Height (cm)
Weight (kg)
Passively damped antivibration table
145
115
115
150
System Electronic Rack and
Laser module
135
90
100
300
LSM, Microscope, Computer
135
90
100
150
Additional Hardware
Components
135
90
61
100
UV laser unit
125
55
50
100
UV cooling unit
120
60
90
50
03/06
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2-9
LSM 5 LIVE - SETUP REQUIREMENTS
Microscopes
Carl Zeiss
2.11
LSM 5 LIVE
LSM 5 LIVE DuoScan
Environmental Requirements
1. Operation, specified performance
T = 22 °C ± 3 °C without interruption (24 h a day
independently whether system is operated or
switched-off)
2. Operation, reduced performance
T = 10 °C to 35 °C, any conditions different from 1.
and 5.
3. Storage, less than 16 h
T = -40 °C to 55 °C
4. Storage, less than 6 h
T = -55 °C to 70 °C
5. Temperature gradient
± 0.5 °C/h
6. Warm up time
1 h, for high-precision and/or long-term measurements ≥ 2 h
7. Relative humidity
< 65 % at 30 °C
8. Operation altitude
max. 2000 m
9. Loss of heat
4 kW
2.12
Vibrations
Vibrations under operation conditions
(with system table)
Shipping shock (LSM 5 box)
5 µm pp at 5 Hz
3g
10 µm pp at 10 Hz
10 µm pp at 20 Hz
2-10
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03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
2.13
LSM 5 LIVE - SETUP REQUIREMENTS
Microscopes
Carl Zeiss
Microscopes
Inverted Axiovert 200 M SP
Upright Axioskop 2 FS MOT
Upright Axio Imager.Z1
All ICS objectives from Carl Zeiss and their accessories can be
accommodated.
Z motor
DC servomotor, opto-electronically coded
Least Z interval:
Piezo Objective focus
50 nm (Axiovert 200 M SP)
100 nm (Axioskop 2 FS MOT)
10 nm (Axio Imager.Z1)
Piezo-driven single objective drive
Max. travel 250 µm; resolution 5-10 nm
In the unlikely case of extreme fluctuations of the external power net or
electromagnetical radiation, the piezo crystal will vary and disturbance in
the image is visible. Note that this is not a defect and the piezo drive will
not be damaged.
03/06
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2-11
LSM 5 LIVE - SETUP REQUIREMENTS
Scanning Module / Laser Module LIVE
Carl Zeiss
2.14
LSM 5 LIVE
Scanning Module LSM 5 LIVE
Scanners
2 individually driven galvanometric scanners
Scanning speed
Up to 120 frames/sec (512 × 512 pixels)
Field resolution
Max. 512 × 2048 pixels (optional adjustable for each axis)
Field of view
10 × 10 mm² with a 1.25× objective
Zoom
0.5× ... 2×, continuous control
Channels
Up to 2 channels simultaneously:
Optional transmitted light detection mode
Dynamic range
12-bit DAC for each detection channel
Apertures
1 or 2 individual variable aperture
Computer controlled automatic alignment
2.15
Laser Module LIVE
Single-mode polarization preserving fiber
Laser beam attenuation (AOTF) for all lasers
Diode laser (405 nm, 50 mW)
Diode laser (440 nm, 16 mW)
OPSS laser (488 nm, 100 mW)
DPSS laser (532 nm, 75 mW)
Diode laser (635 nm, 35 mW)
In the unlikely case of extreme fluctuations of the external power net, the laser diode will switch off or
the laser power might vary. Note that this is not a defect and the laser will not be damaged.
2.16
Laser Module UV for LSM DuoScan UV
Single-mode polarization preserving fiber
Laser beam attenuation (UV-AOTF)
Ar UV Laser (351/364 nm, 80 mW)
2-12
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03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
2.17
03/06
LSM 5 LIVE - SETUP REQUIREMENTS
System Overview LSM 5 LIVE - Material
Carl Zeiss
System Overview LSM 5 LIVE
B 45-0019 e
2-13
Carl Zeiss
LSM 5 LIVE - SETUP REQUIREMENTS
System Overview LSM 5 LIVE - Material
LSM 5 LIVE
LSM 5 LIVE DuoScan
04
08
01
07
02
06
03
05
100
90
2-14
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03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
2.18
LSM 5 LIVE - SETUP REQUIREMENTS
System Overview LSM 5 LIVE - Material
Carl Zeiss
System Overview LSM 5 LIVE DuoScan
04
08
01
07
02
06
03
05
100
90
03/06
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2-15
LSM 5 LIVE
LSM 5 LIVE DuoScan
CHAPTER 3
INTRODUCTION TO LASER SCANNING MICROSCOPY
Contents
Carl Zeiss
INTRODUCTION TO
LASER SCANNING MICROSCOPY
CONTENTS
Page
3
INTRODUCTION TO LASER SCANNING MICROSCOPY ...................................................3-2
3.1
Principle of Laser Scanning Microscopy...............................................................................3-2
3.2
Three-Dimensional Presentations of LSM Image Stacks .......................................................3-3
3.3
Optical Diagram of the LSM 5 LIVE (Schematic) ..................................................................3-4
3.4
3.4.1
3.4.2
3.4.3
Performance Features of the LSM 5 LIVE ............................................................................3-5
Optical and Mechanical Aspects .........................................................................................3-5
Microscope Equipment of the LSM 5 LIVE System...............................................................3-6
Computer Hardware and Software.....................................................................................3-8
03/06
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3-1
Carl Zeiss
INTRODUCTION TO LASER SCANNING MICROSCOPY
Principle of Laser Scanning Microscopy
3
INTRODUCTION TO LASER SCANNING MICROSCOPY
3.1
Principle of Laser Scanning Microscopy
LSM 5 LIVE
LSM 5 LIVE DuoScan
To yield information on their inner structure by conventional transmitted-light microscopy, specimens
have to be very thin and translucent; otherwise image definition will be poor. In many cases it is a
problem to satisfy these requirements.
The essential considerations have led to trailblazing changes in conventional microscopy and supplied a
successful solution to the above problem.
• Unlike the practice of even illumination in conventional microscopy, the LSM technique projects the
light of a point light source (a laser) through a high-NA objective onto a certain object plane of
interest as a nearly diffraction-limited focus. However, if not for another "trick", the stray light
produced outside the object plane, or the fluorescence of fluorescent specimens, would disturb the infocus image of object point of interest, resulting in a blurred image of poor contrast. The problem is
therefore, how to capture only the light coming immediately from the object point in focus, while
obstructing the light coming from out-of-focus areas of the specimen.
• The light reflected, or the fluorescence light
produced, at the focus of the high-NA objective
is projected onto a variable aperture diaphragm
by the same objective and a tube lens. The
focus inside the specimen and the aperture are
situated at optically conjugate points (confocal
imaging). The decisive advantage of this
arrangement is the fact that essentially no other
light than that coming from the object plane of
interest can pass the narrow aperture and be
registered by a detector. Unwanted light
coming from other specimen areas is focused
outside the aperture, which passes only a small
fraction of it. The smaller the aperture, the less
stray light or fluorescence from out-of-focus
areas will get on the detector. The image point
thus generated is largely free from blur caused
by unwanted light.
• In order to obtain an image of the selected
object plane as a whole, it is necessary to scan
the object plane in a point-by-point, line-by-line
Fig 3-1
Principle of confocal imaging
raster by means of an XY light deflection
system. The detectors - as a rule,
photomultipliers - convert the optical information into electric signals. This allows the image of any
object plane to be generated and stored within less than a second. By a defined focusing (Z axis)
movement it is possible to look at any object plane of interest. By scanning a succession of object
planes in a specimen, a stack of slice images can be produced.
This way, the LSM technique in conjunction with ICS optics (Infinity Color-Corrected System) has brought
decisive improvements over conventional microscopy in terms of resolving power and confocal depth
contrast:
Object features in the order of 0.2 μm can be resolved, and height differences of less than
0.1 μm made visible, without the use of interference methods.
3-2
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03/06
LSM 5 LIVE
LSM 5 LIVE Duo Scan
3.2
INTRODUCTION TO LASER SCANNING MICROSCOPY
Three-Dimensional Presentation of LSM Image Stacks
Carl Zeiss
Three-Dimensional Presentations of LSM Image Stacks
One of the advantages of the LSM technique is that it can present structures in three dimensions. This
opens up many ways to process images. Outlined below are some of the possible methods to extract
spatial information from stacks of slice images.
• Gallery
The simplest presentation of 3D information is a gallery showing the individual slice images (sections)
of a stack arranged side by side, with each slice apart from the next by a defined, selectable interval
on the Z axis.
• Virtually infinite depth of focus
The entire set of data can be imaged as a single projection. The computer establishes an image
composed of all in-focus optical sections. The image produced by this so-called composite method has
a virtually infinite depth of focus, since the result is made up of information from in-focus planes only.
• Rotary animation
A sequence of projections is computed, with the specimen being apparently rotated by a certain angle
from image to image, for example by a full turn about an axis. If such a sequence is displayed on the
monitor screen in rapid succession, the visual effect is that of a rotating three-dimensional object.
• Stereo image pairs
The computer establishes a pair of images corresponding to those we see with the right and the left
eye, respectively. The two images forming the stereo pair can be shown on the monitor side by side.
They can be seen as a 3D image with suitable optical aids. Another possibility is to present both
images in registration, with one image in the red channel and the other in the green one (anaglyph).
Viewed through red and green color filters in a spectacle frame, which only pass the image intended
for the respective eye, the two images form a 3D image in the brain
• Color-coded height slices
Each level, i.e. each slice is assigned a different color. For direct evaluation, a color scale is shown,
indicating the actual height above the bottom slice.
• Orthogonal sections
This computation produces a triplet of mutually perpendicular sectional images.
• Oblique sections
A section through the stack is made along an oblique plane defined by the selection of five
coordinates, i.e. X, Y, Z, angle of rotation, and angle of tilt.
• Topography (option)
A computing program for surface topography presentations (as required in materials research) is
available.
• Physiology (option)
With a special software, kinetic processes can be tracked, which is especially of interest to physiology.
• Image VisArt (option)
Three-dimensional display of floating transparent structures (cells) or opaque structures (material)
• 3D Deconvolution (option)
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Carl Zeiss
3.3
INTRODUCTION TO LASER SCANNING MICROSCOPY
Optical Diagram of the LSM 5 LIVE
LSM 5 LIVE
LSM 5 LIVE DuoScan
Optical Diagram of the LSM 5 LIVE (Schematic)
AOTF
y
(d, x, y)
AOTF Acousto-Optical Tunable Filter
Fig. 3-2
Optical path, schematic (2-channel configuration)
The diagram above is a schematic representation of the LSM 5 LIVE system.
Laser light is focused onto the specimen through an objective in a diffraction-limited mode. Light emitted
at the focal plane and at planes below and above it is directed via an Y scanner onto the Achrogate
beam splitter, which separates the emissions from the excitation light. The fluorescences are separated
from each other by a Secondary Dichroic Beam Splitter and directed to individual photodetectors (CCD
Line1 and CCD Line2).
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LSM 5 LIVE
LSM 5 LIVE DuoScan
INTRODUCTION TO LASER SCANNING MICROSCOPY
Performance Features of the LSM 5 LIVE
3.4
Performance Features of the LSM 5 LIVE
3.4.1
Optical and Mechanical Aspects
Carl Zeiss
The highly integrated system design makes for the shortest possible optical paths, top-grade optical
precision and high stability. The compact scanning module can be fitted to an inverted (Axiovert 200 M
SP) or upright (Axioskop 2 FS MOT, Axio Imager.Z1, Axio Imager.M1) microscope in less than three
minutes. On the Axiovert 200 M, the scanning module may be mounted either to the base port directly
below the microscope or to the side port.
For the VIS (visible-light) Laser Module, the user can select from up to four lasers with wavelengths of
635, 532, 488, 440, and 405 nm. Coupling of the laser light is through polarization-preserving singlemode optical fibers. One beam collimator for the visible ranges provides optimum adaptation of the
respective laser wavelength to the objective used and, thus, optimum correction for Z aberrations.
The two simultaneous image acquisition channels, usable for fluorescence are ideal for the investigation
of multiple fluorescence specimens. In the two channels, the diameters of the aperture and their XY
positions can be optimized, and the desired emission filter placed into the beam path, by servo-motor
control. In the simultaneous registration of multiple fluorescences, identical optical sections can be
obtained in each confocal channel. This is of importance, e.g., with the FISH method (fluorescence in-situ
hybridization) used for genome analysis in cytogenetic studies.
It is possible to superimpose a multiple fluorescence image on a brightfield, differential interference or
phase image.
In addition to the emission filters for all standard and special applications, available in motor-controlled
filter wheels, the user can easily install his own emission filters in two of the channels.
The high-NA C-APOCHROMAT objectives specially developed for the LSM technique reach the physical
limit in resolving power, and can be used throughout the 350...700 nm spectral range with the same
high quality, producing brilliant images.
A mirror scanner system, controlled by Realtime Electronics, offers several advantages. The large
deflection angle of the scanning mirrors allows a wide area to be scanned. With a 1.25x objective, the
object area scanned is 10 x 10 mm².
The scanning field size can be freely selected between 4 x 1 and 512 x 2048 pixels.
It is possible to carry out fastest XY scans without having to rotate the specimen itself under laser
radiation load.
Selection of the specimen detail of interest for zooming is fast and convenient, and the zoomed image is
automatically centered. This saves the job of specimen centration with the microscope stage.
Using a bi-directional scanning facility will double the scanning rate to 120 frames/sec (at 512 x 512
pixels); if two different lasing wavelengths are used for the scanning, two fluorochrome dyes can be
viewed and documented in a quasi-simultaneous mode. This will absolutely prevent "bleeding".
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INTRODUCTION TO LASER SCANNING MICROSCOPY
Performance Features of the LSM 5 LIVE
Carl Zeiss
3.4.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Microscope Equipment of the LSM 5 LIVE System
The LSM 5 LIVE system is equipped either with the inverted Axiovert 200 M SP microscope or with the
upright Axio Imager.Z1, Axioskop 2 FS MOT or Axio Imager.M1 microscopes.
Referring to the delivered operating manual "Axiovert 200 M" only differences to this manual will be
explained.
(1) Stand
a) The motorized objective nosepiece 5x H DIC is firmly fixed to the stand, where no operating elements
can be found for the nosepiece. Operation will be done LSM 5 software controlled. The "Restriction of
revolver height to protect the objectives when changing the objectives motorized" is inactivated. The
nosepiece will be moved down automatically before each motorized objective change.
b) The reflector mount is motorized and provided with the Axiovert 200 M reflector turret. The reflector
turret has 5 positions: One transmitting light position, which is identical to the LSM position, and four
further positions for fluorescence filter sets (reflector modules). If you want to use more than five
conventional fluorescence filter sets, it is advisable to use a further reflector turret. When changing the
reflector turret position you must make sure that the turret will click into position, since otherwise the
image area will be cut.
c) The stand has a motorized focusing drive (fine coarse). Sensitivity of the focusing drive is adjusted to
the delivered objectives by the manufacturer. If you want to use other objectives, sensitivity and
parfocality can be adjusted via the Axioset program.
d) The stand features an integrated power supply for the internal motors and stand electronics. The
power supply can be switched on at the right side of the stand. External power supply units will be used
for the mercury vapor short arc lamp.
e) The analyzer slider for conventional DIC methods will be operated from the right side and is located
just below the nosepiece.
When the rod is pushed in, the analyzer is located in the beam path. In LSM-mode the analyzer must not
be located in the beam path, analyzer rod must be pulled out.
f) The stands dispose of five additional ports: two side ports, front ports and base ports.
The side port or the front is equipped with the LSM 5 special interface, one of the others with the TV
interface. The LSM 5 LIVE scanning module can be mounted to the special interface port. Different
camera systems can be adapted to the TV interface using the TV adapters 452982/83/92/94/95/97/980000-000.
(2) Specimen stages and fine focus drives
a) Mechanical stage
The stage must be mounted with the coaxial drive on the right side of the stand.
b) Scanning stage
c) Piezo objective focus drive
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LSM 5 LIVE
LSM 5 LIVE DuoScan
INTRODUCTION TO LASER SCANNING MICROSCOPY
Performance Features of the LSM 5 LIVE
Carl Zeiss
(3) Transmitted-light illumination
a) The illuminator support contains a security circuit, which activates a shutter preventing laser light from
reaching the stand when the support is moved to back. A complementary shutter built-in the stand
prevents laser light from reaching the eye pieces during scanning mode.
b) The illuminator support is equipped with a rotatable polarizer. The Axiovert 200 M description
contains the adjustment for DIC mode during conventional observation.
For scanning transmitted light DIC mode the polarizer in the transmitted light support works like an
analyzer and must be adjusted in such a manner, that direct laser light will be blocked.
The conventional analyzer slider in the stand is not allowed be located in the beam path because of the
laser light already is polarized.
c) On the illuminator support as an option there is mounted a LSM 5 software controlled switching mirror
fully motorized. Alternatively the light is directed to the LSM 5 T-light detector or enables conventional
transmitted-light observation.
d) The focusing screen for conventional transmitted-light is located in a support in front of the halogen
lamp housing.
e) Further information to halogen lamp and condensers you will find in the Axiovert 200 M operating
manual.
(4) Reflected light fluorescence
All Axiovert 200 M fluorescence accessories exceptional the reflector slider can be used.
Further information you will find in the Axiovert 200 M operation manual.
(5) Imaging optics
Optovar sliders are not usable.
The analyzer for conventional DIC mode will be operated from the right side and is located just below the
nosepiece.
Use of sliders with auxiliary objects (473704/14-0000-000) is not possible.
(6) Photo equipment
The stand hasn’t have an integrated SLR-port, but microscope cameras, as described in the
Axiovert 200 M operation manual, can be used.
(7) TV adaptation
The TV port aside and the tubes can be used as described in the Axiovert 200 M operation manual.
The TV interface side port or base port can be used with TV adapters 44 or LSM adapters.
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Carl Zeiss
3.4.3
INTRODUCTION TO LASER SCANNING MICROSCOPY
Performance Features of the LSM 5 LIVE
LSM 5 LIVE
LSM 5 LIVE DuoScan
Computer Hardware and Software
The LSM 5 LIVE is controlled through a standard high-end Pentium PC. Linking with the electronic control
system is via an ultrafast interface. The PC comes with the WINDOWS XP operating system.
The instrument is fully motorized, permitting fast change-over between methods as well as automatic
operation. Parameters once set or complex examination sequences once established can be saved and
reproduced; this way, complete application programs can be loaded and executed by pushbutton control.
The software of the LSM 5 LIVE offers perfect facilities for individual settings of functions and parameters.
Conversion of the light signals into a digital image is effected by means of four 12-bit A/D converters,
each of which can generate 4096 brightness levels.
The software provides a enormously wide range of image processing functions, including all standard
2D/3D (stereo, projection) functions same as sophisticated 3D reconstruction capabilities (surface and
alpha rendering), digital processing of voxels and 3D measurement functions (surface areas, volumes).
As all files and images are recorded in MS Access databases, elegant image database editing is just as
easy as transferring the records to other programs.
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LSM 5 LIVE
LSM 5 LIVE DuoScan
IMPORTANT NOTES FOR CHAPTER 4
Carl Zeiss
IMPORTANT NOTES FOR CHAPTER 4
Bleach series
When doing bleach series on LSM DuoScan systems, the time stamps and bleach
markers indicated in the images will depend on the start position of the scanners in
the image frame. Since these are not identical for the imaging and the
photomanipulation scanners, sometimes imaging seems to start too early after a
bleach event. Ignore the last two digits in the time stamps and bleach markers in
this case.
Macro
When using the modify images macro to convert time series data into Z stacks, an
arbitrary section step of 1 µm is applied to the stack. This will also lead to an
arbitrary time stamp when reconverting the Z stack into a time series. The order of
the images however is preserved under all conditions.
Fast Z-stack and When acquiring fast Z-stacks with line scan mode, the starting point of the stacks
line scan acquisi- and the vertical dimensions can differ depending on uni- or bidirectional scanning
mode. This is a mechanical effect which increases with rising line scanning speed.
tion
Avoid extremely fast speeds in the hundred(s) of frames per second range when
acquiring Z-stacks.
Piezo objective
focus units
03/06
The piezo objective focus units 1325-210, 1325-212 and 1325-213 with an
increased travel range of 250 µm are controlled by a 14 bit logic. Due to the
increased travel range, this implies a smallest step of 15 nm, in contrast to former
fine focus solutions with a smaller travel range but a smallest step size of <10 nm.
B 45-0019 e
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IMPORTANT NOTES FOR CHAPTER 4
Carl Zeiss
4-II
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LSM 5 LIVE
LSM 5 LIVE Duo Scan
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
CHAPTER 4
OPERATION
Purpose
Carl Zeiss
OPERATION
CONTENTS
Page
4
OPERATION......................................................................................................... 4-9
4.1
Purpose ........................................................................................................................... 4-9
4.1.1
4.1.2
4.1.3
4.1.4
4.1.5
Software ........................................................................................................................... 4-9
Windows and Window Elements ..................................................................................... 4-10
Convention for the Text in this Manual ........................................................................... 4-11
Backup............................................................................................................................ 4-11
Software Operation......................................................................................................... 4-11
4.2
Switching on the System............................................................................................. 4-12
4.2.1
4.2.2
Switching on the HBO 100.............................................................................................. 4-13
Starting the LSM 5 Program ............................................................................................ 4-13
4.3
Main Menu ................................................................................................................... 4-15
4.4
File Menu ...................................................................................................................... 4-17
4.4.1
4.4.2
4.4.3
4.4.3.1
4.4.3.2
4.4.3.3
4.4.3.4
4.4.3.5
4.4.3.6
4.4.3.7
4.4.3.8
4.4.3.9
4.4.3.10
4.4.4
4.4.5
4.4.6
4.4.7
4.4.7.1
4.4.7.2
4.4.8
Create New Image Database ........................................................................................... 4-18
Open Existing Image Database ........................................................................................ 4-19
Image Database Window ................................................................................................ 4-19
Form Display Mode ......................................................................................................... 4-21
Gallery Display Mode ...................................................................................................... 4-23
Table Display Mode......................................................................................................... 4-24
Load an Image into the Image Display Window ............................................................... 4-24
Load Image with Reduced Resolution (Subset) ................................................................. 4-25
Update Image Database Display (Refresh) ........................................................................ 4-25
Copy / Paste Image(s) to the CLipboard ........................................................................... 4-25
Display Images with Certain Features (Filter) .................................................................... 4-26
On Filter Function............................................................................................................ 4-28
Delete Function .......................................................................................................... 4-28
Save an Image to the Image Database ............................................................................. 4-29
Import of Images............................................................................................................. 4-32
Export of Images ............................................................................................................. 4-33
Multi Print....................................................................................................................... 4-34
Overlay Toolbar............................................................................................................... 4-34
Printing Images ............................................................................................................... 4-36
Exit ................................................................................................................................. 4-36
4.5
Acquire Menu ............................................................................................................... 4-37
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4-1
Carl Zeiss
4.5.1
4.5.1.1
4.5.1.2
4.5.1.3
4.5.1.4
4.5.2
4.5.2.1
4.5.2.2
4.5.2.3
4.5.2.4
4.5.2.5
4.5.3
4.5.3.1
4.5.3.2
4.5.3.3
4.5.3.4
4.5.3.5
4.5.3.6
4.5.3.7
4.5.3.8
4.5.3.9
4.5.4
4.5.4.1
4.5.4.2
4.5.4.3
4.5.4.4
4.5.5
4.5.5.1
4.5.5.2
4.5.6
4.5.6.1
4.5.6.2
4.5.6.3
4.5.6.4
4.5.6.5
4.5.6.6
4.5.6.7
4.5.6.8
4.5.7
4.5.8
4.5.8.1
4.5.8.2
4.5.8.3
4.5.8.4
4.5.8.5
4.5.9
4.5.9.1
4.5.9.2
4-2
OPERATION
Purpose
LSM 5 LIVE
LSM 5 LIVE DuoScan
Laser Control ...................................................................................................................4-38
Switching on the Enterprise UV Laser ...............................................................................4-38
Opening / Closing the Laser Control window ...................................................................4-38
Function Description ........................................................................................................4-39
Settings............................................................................................................................4-39
Microscope Control..........................................................................................................4-40
Open the Microscope Control Window ............................................................................4-40
Microscope Control Window for Axio Imager.Z1 ..............................................................4-41
Microscope Control Window for Axiovert 200 M .............................................................4-46
Working in LSM Mode .....................................................................................................4-50
Microscope Control for Axioskop 2 FS MOT.......................................................................4-50
Configuration Control......................................................................................................4-51
Open / Close the Configuration Control Window .............................................................4-52
Spectra Button .................................................................................................................4-52
Config Button ..................................................................................................................4-53
Settings for Single Track in the Channel Mode .................................................................4-55
Settings for Multi Track in the Channel Mode ..................................................................4-60
Transmitted light preset ...................................................................................................4-64
Ratio Settings Panel..........................................................................................................4-65
Camera Detection Panel...................................................................................................4-66
LSM 5 LIVE / DuoScan Panel .............................................................................................4-67
Scan Control ....................................................................................................................4-68
Open / Close the Scan Control Window ...........................................................................4-69
Frame ..............................................................................................................................4-71
Line..................................................................................................................................4-87
Camera Control ...............................................................................................................4-88
Edit ROI (Region Of Interest).............................................................................................4-90
Open / Close the Edit ROI Window...................................................................................4-90
Function Description ........................................................................................................4-90
Time Series.......................................................................................................................4-94
Open / Close the Time Series Control Window .................................................................4-94
Start Series Panel..............................................................................................................4-95
Stop Series Panel..............................................................................................................4-97
Cycle Delay / Time Interval Panels .....................................................................................4-98
Marker Panel..................................................................................................................4-100
Time Series of a Frame ...................................................................................................4-102
Time Series of a Frame over Z Stack (option)...................................................................4-104
Time Series with Mean ROI.............................................................................................4-106
Stripe bleach function (LSM 5 LIVE without LSM DuoScan) .............................................4-108
Edit Bleach (LSM 5 LIVE DuoScan) ..................................................................................4-110
Open / Close the Edit Bleach Window ............................................................................4-110
Settings Panel ................................................................................................................4-111
Bleach Parameter Panel ..................................................................................................4-112
Excitation of Bleach Track Panel .....................................................................................4-113
Start / End a Bleaching Procedure...................................................................................4-113
Stage .............................................................................................................................4-114
Open / Close the Stage and Focus Control Window .......................................................4-114
Focus Position Panel .......................................................................................................4-115
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LSM 5 LIVE
LSM 5 LIVE DuoScan
OPERATION
Purpose
Carl Zeiss
4.5.9.3
4.5.9.4
4.5.10
Stage Position Panel ...................................................................................................... 4-116
Tile Scan Panel .............................................................................................................. 4-118
VIS, TV and LSM Buttons............................................................................................... 4-120
4.6
Process Menu.............................................................................................................. 4-121
4.6.1
4.6.1.1
4.6.1.2
4.6.1.3
4.6.1.4
4.6.1.5
4.6.2
4.6.2.1
4.6.2.2
4.6.3
4.6.3.1
4.6.3.2
4.6.4
4.6.4.1
4.6.4.2
4.6.4.3
4.6.5
4.6.5.1
4.6.5.2
4.6.6
4.6.7
4.6.7.1
4.6.7.2
4.6.7.3
4.6.7.4
4.6.8
4.6.9
4.6.9.1
4.6.9.2
4.6.9.3
4.6.9.4
4.6.10
4.6.10.1
4.6.10.2
4.6.10.3
4.6.10.4
4.6.11
4.6.11.1
4.6.11.2
4.6.11.3
4.6.12
4.6.12.1
4.6.12.2
Add .............................................................................................................................. 4-121
Open / Close the Add Window...................................................................................... 4-121
Source Panel ................................................................................................................. 4-122
Destination Panel .......................................................................................................... 4-122
Add Panel ..................................................................................................................... 4-123
Preview Panel................................................................................................................ 4-123
Subtract ........................................................................................................................ 4-124
Open / Close the Subtract Window ............................................................................... 4-124
Performance of the Subtract Function ........................................................................... 4-124
Multiply ........................................................................................................................ 4-125
Open / Close the Multiply Window................................................................................ 4-125
Performance of the Multiply Function............................................................................ 4-125
Ratio ............................................................................................................................. 4-126
Open / Close the Ratio Window .................................................................................... 4-126
Performance of the Ratio Function ................................................................................ 4-126
Formula Panel ............................................................................................................... 4-127
Copy (Channel) ............................................................................................................. 4-127
Open / Close the Copy Window .................................................................................... 4-127
Performance of the Copy Function ................................................................................ 4-128
Duplication (Image) ....................................................................................................... 4-128
Filter ............................................................................................................................. 4-129
Open / Close the Filter Window..................................................................................... 4-129
Image Panel .................................................................................................................. 4-129
Filter List and Edit Panel................................................................................................. 4-129
Preview Panel................................................................................................................ 4-132
Contrast........................................................................................................................ 4-132
Interpolate .................................................................................................................... 4-133
Open / Close the Interpolate Brightness and Contrast Window...................................... 4-133
Image Panel .................................................................................................................. 4-133
Interpolation Panel ........................................................................................................ 4-134
Preview Panel................................................................................................................ 4-135
Channel Shift ................................................................................................................ 4-135
Open / Close the Channel Shift Window................................................................... 4-135
Image Panel.............................................................................................................. 4-136
Shift Panel ................................................................................................................ 4-136
Preview Panel ........................................................................................................... 4-137
Unmix ........................................................................................................................... 4-137
Open / Close the Unmix Window.............................................................................. 4-138
Source Panel............................................................................................................. 4-138
Definition of Channels Panel..................................................................................... 4-138
Ion Concentration ......................................................................................................... 4-141
Open / Close the Ion Concentration Window............................................................ 4-141
Function Description................................................................................................. 4-141
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Carl Zeiss
OPERATION
Purpose
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.6.12.3
4.6.12.4
4.6.12.5
4.6.12.6
4.6.12.7
4.6.12.8
4.6.13
4.6.14
4.6.15
4.6.16
4.6.17
4.6.17.1
4.6.17.2
4.6.17.3
Single Wavelength Dyes – Offline Calibration ............................................................4-142
Ratiometric Dyes .......................................................................................................4-143
Ratiometric Dyes - Online Ratio..................................................................................4-143
Ratiometric Dyes - Calibration....................................................................................4-144
Ratiometric Dyes - Equation Calibration (Grynkiewicz) ...............................................4-145
Options for Calibration Image Selection (Equation- or Titration Calibration) ...............4-145
Image Scaling.................................................................................................................4-145
Copy Window Contents.................................................................................................4-145
Copy Full Resolution.......................................................................................................4-146
Paste Bitmap ..................................................................................................................4-146
Kinetic ...........................................................................................................................4-146
Open / Close the Kinetic Analysis window .................................................................4-146
Function Description..................................................................................................4-147
Example: FRAP Performed in a Nucleus Expressing GFP Labeled Proteins ....................4-148
4.7
3D View Menu.............................................................................................................4-152
4.7.1
4.7.1.1
4.7.1.2
4.7.1.3
4.7.1.4
4.7.2
4.7.2.1
4.7.2.2
4.7.2.3
4.7.2.4
4.7.3
4.7.3.1
4.7.3.2
4.7.3.3
4.7.3.4
4.7.4
4.7.4.1
4.7.4.2
4.7.4.3
3D DepthCod (Color Coded Depth Map).......................................................................4-152
Open / Close the Depth Coding Window .......................................................................4-152
Source Panel ..................................................................................................................4-153
Depth Coding Panel .......................................................................................................4-153
Preview Panel.................................................................................................................4-155
Projection.......................................................................................................................4-155
Open / Close the Projection Window..............................................................................4-155
Source Panel ..................................................................................................................4-155
Projection Panel .............................................................................................................4-156
Preview Panel.................................................................................................................4-157
Stereo ............................................................................................................................4-159
Open / Close the Stereo Images Window .......................................................................4-159
Source Panel ..................................................................................................................4-159
Stereo Images Panel .......................................................................................................4-160
Preview Panel.................................................................................................................4-162
3D DeConVolution (DCV)...............................................................................................4-163
Open / Close the 3D Deconvolution Window .................................................................4-164
Source Panel ..................................................................................................................4-165
Deconvolution Panel ......................................................................................................4-165
4.8
Macro Menu ................................................................................................................4-167
4.8.1
4.8.2
4.8.2.1
4.8.2.2
4.8.2.3
4.8.2.4
4.8.3
4.8.4
4.8.4.1
4.8.4.2
4.8.4.3
4.8.5
Macro Language ............................................................................................................4-167
Macro Control ...............................................................................................................4-168
Open / Close the Macro Control Window.......................................................................4-168
Edit Macro Function .......................................................................................................4-168
Assign Macro to Button Function ...................................................................................4-171
Editing and Debugging of Macros ..................................................................................4-172
Overview of Available Macros ........................................................................................4-173
Sample Macros ..............................................................................................................4-175
Distance Macro ..............................................................................................................4-175
Profile Macro .................................................................................................................4-175
Multiple Time Series Macro ............................................................................................4-176
Kollimatic.......................................................................................................................4-177
4-4
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LSM 5 LIVE
LSM 5 LIVE DuoScan
OPERATION
Purpose
Carl Zeiss
4.8.6
4.8.7
4.8.8
4.8.9
4.8.9.1
4.8.9.2
4.8.9.3
Optimize Macro ............................................................................................................ 4-186
ReInit Macro ................................................................................................................. 4-186
Image Match (Scanfield Transformation) Macro............................................................. 4-187
Visual Macro Editor ....................................................................................................... 4-189
Open / Close the Visual Macro Editor Window .............................................................. 4-189
Function Description ..................................................................................................... 4-190
Editing a Macro............................................................................................................. 4-195
4.9
Options Menu............................................................................................................. 4-198
4.9.1
4.9.2
4.9.2.1
4.9.2.2
4.9.2.3
4.9.2.4
4.9.2.5
4.9.2.6
4.9.2.7
4.9.2.8
4.9.2.9
4.9.2.10
4.9.2.11
4.9.2.12
4.9.2.13
4.9.2.14
4.9.2.15
4.9.2.16
4.9.2.17
Dye DB Function ........................................................................................................... 4-199
Settings Function .......................................................................................................... 4-200
Open / Close the Settings for user : ... Window ............................................................. 4-200
Autosave....................................................................................................................... 4-201
Database General.......................................................................................................... 4-202
Database Table Viewer.................................................................................................. 4-203
Database Gallery Viewer ............................................................................................... 4-203
Import / Export.............................................................................................................. 4-203
Scan Information........................................................................................................... 4-204
Image Status Display ..................................................................................................... 4-204
Print Status Display........................................................................................................ 4-204
Recording / Reuse..................................................................................................... 4-205
Time Series ............................................................................................................... 4-205
Mean of ROIs ........................................................................................................... 4-205
Temporary Files ........................................................................................................ 4-206
Program Start ........................................................................................................... 4-207
Hardware ................................................................................................................. 4-207
Image Display ........................................................................................................... 4-207
Save ......................................................................................................................... 4-208
4.10
Maintain Menu........................................................................................................... 4-210
4.10.1
4.10.1.1
4.10.1.2
4.10.2
4.10.2.1
4.10.2.2
4.10.2.3
4.10.3
4.10.3.1
4.10.3.2
4.10.3.3
4.10.3.4
4.10.4
4.10.5
4.10.6
4.10.7
Scanner......................................................................................................................... 4-210
Electrical Calibration ................................................................................................. 4-210
Important Notes and Hints........................................................................................ 4-211
Objective....................................................................................................................... 4-212
Objective Change ..................................................................................................... 4-212
Focus Speed Change ................................................................................................ 4-214
Parfocality Correction ............................................................................................... 4-214
Pinhole Adjustment....................................................................................................... 4-215
Open / Close the Pinhole and Collimator Window .................................................... 4-216
Pinhole Panel............................................................................................................ 4-216
Collimator panel....................................................................................................... 4-217
Aperture and collimator Adjustment......................................................................... 4-218
Camera......................................................................................................................... 4-220
Reboot.......................................................................................................................... 4-220
HW/Admin.................................................................................................................... 4-220
Test Grid ....................................................................................................................... 4-220
4.11
Window Menu............................................................................................................ 4-221
4.11.1
Full Screen .................................................................................................................... 4-221
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Carl Zeiss
OPERATION
Purpose
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.11.2
4.11.3
Close All Image Display Windows...................................................................................4-221
Scan and System Information .........................................................................................4-222
4.12
Help Menu ...................................................................................................................4-223
4.12.1
4.12.2
4.12.3
Help...............................................................................................................................4-223
About ............................................................................................................................4-223
FRAP Guide....................................................................................................................4-223
4.13
Display and Analysis of Images .................................................................................4-224
4.13.1
Structure of the Image Display Window .........................................................................4-224
4.13.2
Display Modes................................................................................................................4-225
4.13.3
Select - Chan .................................................................................................................4-226
4.13.3.1
Assigning Another Color to a Channel.......................................................................4-227
4.13.3.2
Switching a Channel of a Multi Channel Image off or on...........................................4-227
4.13.3.3
Switching to Monochrome Image Display ..................................................................4-227
4.13.3.4
Defining a New Color ................................................................................................4-227
4.13.4
Select - Zoom.................................................................................................................4-228
4.13.5
Select - Slice...................................................................................................................4-229
4.13.6
Select - Overlay ..............................................................................................................4-230
4.13.6.1
Functional Description ...............................................................................................4-230
4.13.6.2
Buttons in the Overlay Toolbar...................................................................................4-231
4.13.7
Select - Contr.................................................................................................................4-234
4.13.7.1
Ramp ........................................................................................................................4-235
4.13.7.2
PolyLine.....................................................................................................................4-235
4.13.7.3
Spline........................................................................................................................4-236
4.13.7.4
Gamma .....................................................................................................................4-236
4.13.8
Select - Palette ...............................................................................................................4-237
4.13.8.1
Editing and Storing a Palette......................................................................................4-238
4.13.8.2
Delete a Palette .........................................................................................................4-239
4.13.8.3
Import a Palette.........................................................................................................4-239
4.13.9
Select - Anim .................................................................................................................4-239
4.13.10
Select - Reuse.................................................................................................................4-241
4.13.11
Select - Crop ..................................................................................................................4-242
4.13.12
Select - Copy..................................................................................................................4-243
4.13.13
Select - Save...................................................................................................................4-243
4.13.14
Select - Save As..............................................................................................................4-244
4.13.15
Display - xy.....................................................................................................................4-244
4.13.16
Display - Split xy .............................................................................................................4-245
4.13.17
Display - Ortho...............................................................................................................4-246
4.13.17.1 Ortho - Select Function..............................................................................................4-247
4.13.17.2 Ortho - Distance Function..........................................................................................4-248
4.13.17.3 Ortho - 2D DeConVolution Function..........................................................................4-249
4.13.18
Display - Cut ..................................................................................................................4-250
4.13.19
Display - Gallery .............................................................................................................4-251
4.13.20
Display - Histo ................................................................................................................4-253
4.13.20.1 Display - Histo - Overview ..........................................................................................4-253
4.13.20.2 Area Function............................................................................................................4-255
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LSM 5 LIVE DuoScan
OPERATION
Purpose
Carl Zeiss
4.13.20.3 Colocalisation Function............................................................................................. 4-257
4.13.21
Display - Profile ............................................................................................................. 4-261
4.13.21.1 Function ................................................................................................................... 4-261
4.13.21.2 Function Elements .................................................................................................... 4-263
4.13.21.3 Display - Mean ......................................................................................................... 4-265
4.13.22
Display - 2.5 D .............................................................................................................. 4-266
4.13.22.1 Direct Setting in the Image ....................................................................................... 4-266
4.13.22.2 Setting via Scrollbars................................................................................................. 4-267
4.13.23
Display - 3D (Image VisArt)............................................................................................ 4-268
4.13.23.1 Shadow Projection.................................................................................................... 4-269
4.13.23.2 Transparency Projection............................................................................................ 4-272
4.13.23.3 Surface Rendering .................................................................................................... 4-273
4.13.23.4 Appearance (Settings)............................................................................................... 4-274
4.13.23.5 Series ....................................................................................................................... 4-275
4.13.24
Display - Topography for LSM ....................................................................................... 4-278
4.13.24.1 Channel Selection..................................................................................................... 4-279
4.13.24.2 Generate Topography............................................................................................... 4-279
4.13.24.3 Topography Thresholds ............................................................................................ 4-280
4.13.24.4 Processing by Filtering .............................................................................................. 4-281
4.13.24.5 Display Modes .......................................................................................................... 4-284
4.13.24.6 Context Menu of the 3D Display Mode..................................................................... 4-287
4.13.24.7 Measurement Functions ........................................................................................... 4-294
4.13.24.8 3D Measurement Functions ...................................................................................... 4-304
4.13.24.9 Export Data .............................................................................................................. 4-308
4.13.24.10 Topo ReUse .............................................................................................................. 4-308
4.13.25
Display - Prev. ............................................................................................................... 4-309
4.13.25.1 Context Menu for Scan Information Text .................................................................. 4-310
4.13.25.2 Context Menu for Topography Images ..................................................................... 4-310
4.13.25.3 Arranging and Printing the Print Preview .................................................................. 4-310
4.13.26
Display - Info................................................................................................................. 4-311
4.13.27
Additional Display Mode in Time Series ......................................................................... 4-312
4.13.27.1 Display - Mean ......................................................................................................... 4-312
4.14
Image Optimization ................................................................................................... 4-315
4.14.1
Single Channel.............................................................................................................. 4-315
4.14.1.1
Requirements ........................................................................................................... 4-315
4.14.1.2
Confocal Aperture / Detector Gain / Ampl. Offset ..................................................... 4-319
4.14.1.3
Exposure Time, Scan Average and Pixel Depth .......................................................... 4-321
4.14.2
Multiple-channel ........................................................................................................... 4-322
4.14.2.1
Requirements ........................................................................................................... 4-322
4.14.2.2
Image Optimization .................................................................................................. 4-323
4.15
Shut-Down Procedure................................................................................................ 4-325
4.15.1
4.15.2
4.15.3
4.15.4
Exiting the LSM Program ............................................................................................... 4-325
Shut Down the WINDOWS Operating System................................................................ 4-326
Turning Power Off ........................................................................................................ 4-326
Turning off the HBO 100............................................................................................... 4-326
4.16
Software and Hardware Options.............................................................................. 4-327
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OPERATION
Purpose
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.16.1
4.16.2
Software ........................................................................................................................4-327
Hardware.......................................................................................................................4-329
4.17
Courses on "How to Operate the System in an Optimized Way" ...........................4-330
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OPERATION
Purpose
4
OPERATION
4.1
Purpose
Carl Zeiss
This chapter describes the operation of the LSM 5 LIVE Laser Scanning Microscope exemplified by typical
applications in conjunction with the LSM 5 software and its graphic user environment.
When starting up and operating the microscope system, mind the operating instruction manuals for the
Axio Imager.Z1, Axiovert 200 M and Axioskop 2 FS MOT microscopes:
− B 46-0046
Axio Imager, Operating Manual
− B 40-080
Axiovert 200 M, Operating Manual
− B 40-076
Axioskop 2 FS MOT, Operating Manual
4.1.1
Software
The LSM 5 software, Version 4.0:
− controls the microscope, the scanning and laser modules, tools (filters, stand, Axioset) and the
image acquisition process
− displays and analyzes the images
It is based on the network-capable graphic 32-bit Microsoft ® WINDOWS XP operating system.
Portions © Copyright 1981 - 2001, Microsoft Corporation. All rights reserved.
The installation of the software for the LSM 5 LIVE and the basic settings of the equipment
components are carried out by Carl Zeiss service staff. This job includes the creation of a
customized software configuration in line with the specific hardware components of the
customer's microscope system.
The LSM 5 software is menu-controlled and normally uses its own windows for the activation of the
various functions; within these windows, further submenus (panels) can be displayed and removed.
The screen layout used in this manual is based on the former Windows ® standard layout and
may vary from yours.
Images of the specimens to be examined, created by scanning, are displayed in separate Image Display
windows.
Theoretically, the number of simultaneously opened windows for software operation or image display is
unlimited, but should not be too excessive so that an overview is still possible.
Identical functions, e.g. Laser Control, can be performed in several software windows. Changes made
by the software are recorded immediately and are automatically transferred to all the other windows
concerned.
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OPERATION
Purpose
Carl Zeiss
4.1.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Windows and Window Elements
Window element
Description / Explanation
Window (e.g.: Laser Control window)
− Window displayed after activation of a
function button (e.g.: Laser button in the
toolbar of the Main menu).
Panel (e.g.: Lasers panel)
− Limited function range within a window
List box or selection box
− Selection of one of the displayed options at a
click of the mouse.
− Open the box by clicking on the arrow
button.
Input box
− Input of text or numeric values via the
keyboard.
Scrollbar with slider
− Setting of numbers in the relevant input box
by moving the slider or clicking on the arrow
buttons or clicking on the slider and moving
via the arrow keys of the keyboard. Press the
Shift or Ctrl key while clicking on the arrow
button to change the numeric values in
coarse or fine steps.
Check box
− Activates / deactivates setting options.
Button
− Selection / performance of a function via
mouse click.
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4.1.3
OPERATION
Purpose
Carl Zeiss
Convention for the Text in this Manual
All the originally used terms of the software interface, e.g.
− names of windows,
− panels,
− input boxes,
− list / selection boxes,
− check boxes,
− menu items,
− names of buttons and
− keyboard keys,
are displayed in bold letters to allow easier identification.
4.1.4
Backup
System backup
− A complete backup is contained on the enclosed backup CD-ROM.
User files backup
The following user-generated files need to be included in a backup procedure (keep directory structure):
−
−
−
−
−
−
−
Image database files: *.mdb (but not system_configuration_*.mdb)
LSM Image files: *.lsm
Exported images: *.* (*.Tiff, *.LSM-Tiff, *.BMP, …)
Palette files: AIM \ Palette \ *.lut
Filter files: AIM \ Filter \ *.krn
Pibhole (aperture setting files: AIM \ PH*.pos
Log files: AIM \ *.log
The following files generated during the system integration should also be included in a backup
procedure:
−
−
−
−
4.1.5
Parameter file for pinhole (aperture) setting: AIM \ *.set
Parameter file after pinhole (aperture) adjustment: AIM \ *.adj
Scanner files: AIM \ bin \ *.bin
Microscope stand database: AIM \ database \ system_configuration_*.mdb
Software Operation
The LSM 5 software can be operated using the mouse, the PC keyboard, or both.
The operation of the mouse and the keyboard is identical to that of the Microsoft ® WINDOWS
operating system and is therefore not dealt with in detail in this manual.
If required, see the Microsoft manual or online help for relevant information.
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OPERATION
Switching on the System
Carl Zeiss
4.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Switching on the System
The LSM 5 LIVE system is equipped with a main power switch located at the front of the System
Electronic Rack and two further switches labeled System/PC and Components. The main switch has to
be set to ON to be able to switch on and off the system using the System/PC and Components
switches. The main switch can be used to switch off the complete system with one switch only. The
electronics to run the computer and the microscope are switched on with the System/PC switch. The
lasers and the scan head are switched on with the Components switch. These switches are also
accessible via the remote power control switch (Fig. 4-1).
Note that the full performance and correct adjustment of all internal elements is achieved after a
warm-up time of 40 min.!
• When set to ON the REMOTE CONTROL switch labeled
System/PC provides power to the microscope and the
computer. This allows to use the microscope and the
computer without running the LSM Software.
The drives for floppy discs and CD/DVD of the
computer must not contain any data storage item.
• To switch on the system completely put the
Components switch also to ON. Now the complete
system is ready to be initialized with the LSM Software.
• After switching on the computer type in the user name
and password to log on to the computer.
Fig. 4-1
REMOTE CONTROL switch
• After entries, confirm by clicking the OK button or
Enter.
− The WINDOWS XP operating system desktop appears
on the screen, showing a number of icons.
Change Filters icon
Change Filters
The Change Filters tool is used to update the filter data in the software after a
change of filters in the reflector turret. See printed manual CHAPTER TOOLS.
Stand Select icon
Stand Select
The Stand Select tool permits a new or updated database to be assigned to the
LSM 5 software program. This function should preferably be performed by authorized
service personnel. See printed manual CHAPTER TOOLS.
LSM 5 LIVE icon
LSM 5 Live
4-12
Start the LSM 5 software program for the LSM 5 LIVE laser scanning microscope.
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LSM 5 LIVE DuoScan
4.2.1
OPERATION
Switching on the System
Carl Zeiss
Switching on the HBO 100
• If the HBO 100 is required, switch it on via the toggle switch of the HBO 100 power supply.
4.2.2
Starting the LSM 5 Program
The LSM 5 software program can be operated in
two different modes (with or without connected
instrument system).
− In the on-line mode, the entire program
package (image recording and analysis) is
available, while only a part of the software
functions (image analysis only of already
stored images)
Fig. 4-2
Starting the LSM 5 software
− In the off-line mode no hardware functions
are available. Of course, the off-line mode
can also be started when the instrument
system is connected. In that case, it is not
necessary that the lasers and the microscope
are switched on.
• Double-click on the LSM 5 LIVE icon on the desktop of WINDOWS to start the LSM 5 software
program (Fig. 4-2).
− The LSM 5 LIVE Switchboard window appears on the screen (Fig. 4-3).
Fig. 4-3
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LSM 5 LIVE Switchboard menu
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4-13
OPERATION
Switching on the System
Carl Zeiss
Online Mode
LSM 5 LIVE
LSM 5 LIVE DuoScan
Clicking on this button activates the complete LSM hardware (on-line
mode).
Please note that the Online Mode button must be activated
before clicking the Start button. Otherwise, the hardware can
not be controlled by the LSM 5 software.
Offline Mode
This item allows you to process and analyze previously acquired images
with the LSM 5 software. In this mode, control of the hardware (laser
module ...) is not possible (off-line mode).
Start
Use of this button starts the software system.
After the start, instrument initialization is
performed and can be monitored in the
Initialization window and interrupted with a click
on the Cancel button, if required.
Fig. 4-4
OFF LINE Initialization window
Depending on the selected option (Online Mode
Images or Offline Mode), initialization is
performed in the offline or online mode.
Some printers (for example KODAK Thermo Printer) will produce an error message "hard key
not found" in case the printer is not switched on.
Remedy: turn on the printer before starting the LSM 5 software.
Don’t switch off the KODAK printer during the scanning process.
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LSM 5 LIVE DuoScan
4.3
OPERATION
Main Menu
Carl Zeiss
Main Menu
The major functions can be selected in the Main menu either via the pull-down menus in the menu bar
or via the Main menu toolbar which can be displayed or removed as required.
Further subordinate toolbars are available below this toolbar, depending on which button has just been
pressed (File, Acquire, etc.).
In the standard setting of the LSM 5 software, the toolbars are automatically displayed after clicking
Start.
However, since the LSM 5 software is operated more conveniently with the help of the toolbars,
only this method of function activation will be described in the following.
• Click on the Start button in the LSM 5 LIVE Switchboard window.
− The LSM 5 LIVE Main menu appears on the screen.
The Acquire button is active automatically, and the submenus selectable in it are shown in the second
(bottom) toolbar.
Fig. 4-5
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LSM 5 LIVE Main menu
File button
Open, save, import and export of image data. Printing individual
or several images on one page. Ending (Exit) the Main menu.
Acquire button
Calling up and setting the necessary operating parameters.
During the preparation for and execution of laser scan image
acquisition, this menu item is used as the working dialog
between the computer and the microscope.
Process button
Used for processing of acquired images.
3D View button
Used for three-dimensional reconstruction.
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OPERATION
Main Menu
Carl Zeiss
Macro button
Options button
LSM 5 LIVE
LSM 5 LIVE DuoScan
Makes it possible for the user to store frequently used processes
(Macro recorder) and to run them automatically (Macro play). It is
possible to write new macros or to edit existing ones.
For custom-configuration of software and hardware options.
This menu item enables access to the coloring table.
In the Settings for User window you can specify essential
operating modes and informative help, organized by tabs, which
have an effect on the user interface.
Maintain button
4-16
Service mode for adjustment and setting of other parameters
(e.g. objectives).
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4.4
OPERATION
File Menu
Carl Zeiss
File Menu
The functions of the File menu permit images and the relevant information to be managed and handled
completely in a database system. You can also create your own databases. The databases allow images to
be stored, loaded and deleted. The additional functions Import and Export permit images from other
systems to be made available to the LSM 5 software, or the export of images to other software packages.
The Print function allows individual or several images to be arranged on a print page for printout. The
Main menu can be ended via the Exit function.
In the pull-down Main menu File two additional functions not displayed in the Main Menu Toolbar are
available.
Clicking Recent Database opens up a choice of the last 5 databases opened by the program. Clicking
the appropriate line will open up that database. A similar menu exists for Recent Image Files.
• In the Main menu toolbar, click on File.
− This opens another, subordinate toolbar in the Main menu.
Fig. 4-6
File menu
Fig. 4-7
Subordinate File menu toolbar
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OPERATION
File Menu
Carl Zeiss
4.4.1
LSM 5 LIVE
LSM 5 LIVE DuoScan
Create New Image Database
The New function permits the creation of a new
image database.
• Click on the New button in the
subordinate toolbar of the Main menu.
File
− This opens the Create New Database
window for the selection of drives,
directories and subdirectories.
Fig. 4-8
• Enter the name of the image database you
want to create in the File name text box, e. g.
Convallaria.
Create New Database window
• If you want to create the image database in a
certain folder (drive / directory), click on the
arrow button next to the Create in box.
− This opens a drop-down list box showing all
folders available for selection.
To move up one layer of folders, click on the
• If you want to create a new folder, click on the
button. Cancel allows you to stop the process.
button.
• Click on the Create button.
− This creates the new image database in the selected drive and directory.
− The database files of the LSM 5 software have the filename extension *.mdb.
The Convallaria.mdb window appears, presenting the opened image database with 0 of 0 image
entries (see Image Database window, page 4-19).
The new image database can be used to store an acquired or processed image (see Saving an Image,
page 4-29).
The start settings and the extent of the parameters displayed in the image database window can
be changed as required via the Settings function in the Options subordinate toolbar (see
Settings Function, page 4-200).
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LSM 5 LIVE DuoScan
4.4.2
OPERATION
File Menu
Carl Zeiss
Open Existing Image Database
The Open function allows one or several databases
to be opened. The images stored in the database(s)
are displayed in thumbnail form; they can be
selected and loaded into the Image Display
window (see page 4-224).
• Click on the Open button in the File
subordinate toolbar of the Main menu.
− This opens the Open Database window for
selection of image databases.
• If you want to load an image database from
another folder (drive / directory), click on the
arrow button to the right of the Look in box.
Fig. 4-9
Open Database window
− This opens a drop-down list box in which
you can select from all available folders.
The window displays the various image databases with the file extension *.mdb.
• Open the image database by a double click on the respective key icon (e.g. Convallaria.mdb), or click
on the name of the image database for selection and open it by clicking on the Open button.
− This opens the image database window, e.g. Convallaria.mdb - AIM, in which you can select
from a variety of options.
• Click on the
selection.
4.4.3
button in the title bar of the Database window to close this window and make no
Image Database Window
The Image Database window allows you to choose one of three different display modes:
− Form
− Gallery
− Table
To choose the required mode, activate the relevant button on the right-hand side of the Image
Database window. Loading of images into the Image Display window is possible in every display mode.
The buttons on the right have the following functions:
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Form button
Show image database in form display mode.
Gallery button
Show image database in gallery display mode.
Table button
Show image database in table display mode.
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OPERATION
File Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Load button
Load image and parameter from image database to Image
Display window.
Subset button
Load image and parameter with size reduction from image
database to Image Display window.
Reuse button
Reuse scan parameters of the selected image without loading the
image.
Refresh button
Refresh Image Database window.
Copy button
Copy selected images to clipboard.
Paste button
Paste images from clipboard into image database.
Filter button
Select or edit search filter from image in the image database.
On Filter button
Switch search filter on or off.
Delete button
Delete selected images from the image database.
The status line, which displays the following current parameters, is at the bottom of the Image
Database window:
Total: …
Display of storage volume of the entire image database
Selected: …
Display of storage volume of the selected image(s)
Current: …
Display of storage volume of the current image
Clipboard: …
Display of storage volume of the image(s) contained in the clipboard
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LSM 5 LIVE DuoScan
4.4.3.1
OPERATION
File Menu
Carl Zeiss
Form Display Mode
When a image database is opened, the Form display mode is used, if no other settings were made under
Settings in the Options subordinate toolbar.
Fig. 4-10
Image Database window (Form display mode)
In the Options menu in the function Settings it is possible to define
− the start mode of the image database (Form, Gallery, Table)
− the Recordset displayed (first, last, middle) and
− the parameters shown.
The number of the image currently displayed from a set of images is indicated in the Recordset text box.
• From the image database you can display images using the recording slider, or in one of the following
ways:
show the next image
show the previous image
show the last image of the image database
show the first image of the image database
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Carl Zeiss
OPERATION
File Menu
LSM 5 LIVE
LSM 5 LIVE DuoScan
The currently selected image is displayed as thumbnail in the Image Display window on the right. In the
case of Z Stacks or time series, the Slice slider appears on the screen beside the Image Display window.
• You can browse through the series by dragging the Slice slider using the mouse.
• Click on the Load button to open the selected image.
A double-click on the Image Display window of
the database will also load the selected image.
For a description of the toolbars Select and
Display see Display and Analysis of Images,
page 4-224.
The name of the image is displayed in the Name input box
of the Image Database window.
• If you want to change the name, click on the input box
and enter the new name directly via the keyboard.
• The Description and Notes input boxes allow you to
subsequently add descriptions or special notes on the
recorded image via the keyboard.
Fig. 4-11
Opened image displayed in the Image
Display window
The acquisition parameter settings of the image are
displayed in the Acquisition panel.
Changes to an original scan image are automatically recorded in the Processing panel under History. If,
for example, the image was added to the database via the clipboard, the entry Imported file will be
shown under History.
Under Image Size, the size of the image in pixels for XY(Z) and the number of used channels are
displayed.
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4.4.3.2
OPERATION
File Menu
Carl Zeiss
Gallery Display Mode
• Click on the Gallery button. All images of the image database, e.g. Convallaria.mdb, (image series)
are shown in a tiled arrangement of thumbnails on the screen.
Fig. 4-12
Database window (Gallery display mode)
In the Options menu in the function Settings it is possible to define
− the start mode of the image database (Form, Gallery, Table)
− the recordset displayed (first, last, middle) and
− the parameters shown.
• To select one of the images of the database for normal-size presentation, double-click on the desired
image. The same can be achieved by clicking on the desired image in the gallery and then clicking on
the Load button.
• To select several single images press & hold down the Ctrl-key and select each desired image by a click
of the mouse. If several images have been selected, they will all be opened and displayed one after the
other.
• To select a number of consecutive images press & hold down the Shift-key, click on the first and the
last image to be selected. All the images between these two will also be included in the selection. If
several images have been selected, they will all be opened and displayed one after the other.
Selected images are highlighted in blue. Furthermore, the current image selected last receives a patterned
frame.
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OPERATION
File Menu
Carl Zeiss
4.4.3.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Table Display Mode
• Click on the Table button.
− All images of the image database, e.g. Convallaria.mdb, (image series) are shown in Table display
mode on the screen.
Fig. 4-13
Database window (Table display mode)
In the Options menu in the function Settings it is possible to define
− the start mode of the image database (Form, Gallery, Table)
− the recordset displayed (first, last, middle) and
− the parameters shown.
• To select one of the images of the database for normal-size presentation, double-click on the desired
line. The same can be achieved by clicking on the desired image in the table and then clicking on the
Load button. If several images have been selected, they will all be opened and displayed one after the
other.
The selection of several images is performed in the same way as in the Gallery mode, i.e. by
pressing the Shift and Ctrl keys.
4.4.3.4
Load an Image into the Image Display Window
• Click on the Load button to load the selected images into the Image Display window or double click
on the appropriate image in the database window.
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4.4.3.5
OPERATION
File Menu
Carl Zeiss
Load Image with Reduced
Resolution (Subset)
The Subset function allows images to be loaded
with reduced resolution. For this purpose, the
image pixels in XY(Z) are reduced. It is also possible
to reduce the number of slices (in stacks and time
series).
• Click on the Subset button to open the Load
with reduction in size window.
• Enter one value each for n under Pixel (x),
Pixel (y), Pixel (z) and Stack (Time) in the
Load every nth panel.
• If required, turn on the
Fig. 4-14
Load with reduction in size window
Load 12 bit as 8 bit check box.
• Click on the Load button to load the selected images with reduction in size, time slices and stack
slices.
• Use Cancel to exit the window without any selection.
4.4.3.6
Update Image Database Display (Refresh)
• Click on the Refresh button to update the Image Database window.
4.4.3.7
Copy / Paste Image(s) to the CLipboard
• Select the image(s) to be copied. You can use the Shift and Ctrl keys for multiple selection.
• Click on the Copy button.
− The image(s) is(are) copied to the clipboard and can be inserted in either the same or another
database or in other software packages.
• Click on the Paste button to insert the image in the current database.
Identical to the WINDOWS function "Drag & Drop", one or several images can be copied or moved from
one database to another.
The Form mode allows only one image to be copied or moved. The Gallery and Table modes permit
several images to be copied or moved simultaneously by multiple selection (keeping the Shift key pressed
on clicking).
• Open the relevant databases and position both windows side to side.
• Select the required images (multiple selection by keeping the Shift key pressed) from one database.
• Click on a selected image and keep the mouse button pressed, move the mouse button to the
window of the other database (a small rectangular appears near the mouse button) and release the
mouse button again (Drag & Drop).
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OPERATION
File Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
The images are now being moved to the other image database, i.e. they are deleted from the first image
database and are then only available in the second image database.
• If the images shall only be copied, also press the Ctrl key during the Drag & Drop procedure (in
addition to the rectangular, a "+" sign will also appear near the mouse button).
The images will then be available in both image databases.
4.4.3.8
Display Images with Certain
Features (Filter)
The Filter function permits the display of the
database to be modified in such a way that only
images with certain features are displayed. This
requires the definition and following activation of a
relevant filter. Defined filters can be stored,
reloaded and also deleted.
Fig. 4-15
Filter window
Edit panel
The following features can be used as filter functions under Field:
Name
Words or row of letters from the name of the image
Description
Words or row of letters from the description of the image
Scan Mode
Scan Mode in which the image was created: Stack or Plane
Date / Time
Date / Time of image acquisition
# Planes (z)
Pixel size of the image in the Z-direction (e.g.: 10)
# Lines (y)
Pixel size of the image in the Y-direction (e.g.: 512)
Samples (x)
Pixel size of the image in the X-direction (e.g.: 512)
Z-Step
Distance of Z Slices in a Z Stack in µm
User
Name of the user as entered in the WINDOWS NT login
Time series
Selection of time series
• Open the Field list box and select the filter feature (e.g.: Scan Mode).
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OPERATION
File Menu
Carl Zeiss
The following operator signs can be activated under Operator:
=
equals
>
larger
<
smaller
>=
larger, equals
<=
smaller, equals
<>
smaller, larger
• Select the relevant operator sign (e.g.: =) from the Operator list box.
The relevant value or a combination of characters for the filter function (Field) is entered under Value via
the keyboard:
• Enter the relevant text or value (e.g.: Stack).
• Click on Add. The defined filter feature is displayed in the work box of the Edit panel and is therefore
activated (e.g.: Scan Mode = stack).
If a further filter feature is to be linked with the already defined one, proceed as follows:
• Activate the relevant entries under Field and Operator and enter a value or text (e.g.: Name =
Convallaria) under Value.
If groups of letters shall be searched, the * sign can be entered for undefined letters (e.g.: if you
search for the letter row Conv, enter Conv*).
• Activate the required link type And, Or or Not with a click of the mouse (e.g.: And).
• Click on the Add button. The created filter feature is added to the work box of the Edit panel (e.g.:
AND Name = Convallaria).
The Modify button enables you to edit an already defined filter feature:
• Activate the required feature on the work box.
• Make the necessary changes under Field, Operator and Value. Select the link type And, Or or Not.
• Click on Modify. The filter feature will be changed accordingly.
The Delete button enables you to delete a defined filter feature:
• Activate the required feature in the work box.
• Click on Delete. The filter feature will be deleted from the work box.
• Clicking on OK will activate the filter (the entire set of filter features) displayed in the work box and
close the Filter window. On Filter is activated right on in the Database window and the filter
function will be performed. Only those images which fulfill with the defined filter features will then be
displayed. The procedure is interrupted via Cancel.
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OPERATION
File Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
If required, the filter features displayed in the work
box can be stored.
• Click on the Add to List button. The Add to
List window will be opened.
• Enter a name for the filter and click on OK. The
filter will be included in the Filter List panel.
Fig. 4-16
Add Filter to List window
Filter List panel
All the stored filters are displayed in the Filter List
panel and can be activated any time at a click of
the mouse.
• Click on the name of the required filter. The
linked filter features will be displayed in the
work box.
Fig. 4-17
Filter List panel
Filters which are no longer needed can be deleted.
• Click on the filter to be deleted in the Filter List panel.
• Click on Remove from List. The filter will be removed.
4.4.3.9
On Filter Function
The On Filter function is a toggle switch to activate or deactivate selected filter settings.
4.4.3.10
Delete Function
• Select the images to be deleted from the image database.
• Click on the Delete button. Confirm the safety inquiry then displayed by pressing OK.
− The images and the acquisition parameters will be removed from the image database.
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4.4.4
OPERATION
File Menu
Carl Zeiss
Save an Image to the Image Database
The Save function allows to store an image together with the acquisition parameters (and processing
information) to be stored in an image database.
In the Options menu in the function Settings it is possible to define an Autosave function. When
Autosave is off, the Save dialogue is the Save As dialogue.
Proceed as follows to save an acquired or an edited / processed image:
• Click on the Save or Save As button in the File subordinate toolbar of the Main menu.
− The Save Image and Parameter As window appears on the screen.
Save
Stores a newly created or changed image. Newly created images must be given a name and
assigned to an existing or new database.
Save As
Stores a previously stored and called up image under a different name. If images are called up
and stored again, the original data and time display will be retained.
Clicking on either of these buttons opens the Save As window to create and open an image
database.
check box is activated, the images are stored in a compressed
When the Compress Files
form.
• If necessary, enter a description of the image or comments on it in the appropriate text boxes.
• The default display in the User text box is the name of the logged-on user. If you want, you can enter
a different user name for the current image.
• Click on the Open MDB button if you want to open an existing image database in which you want to
save the current image. Click on the New MDB button if you want to create a new database to save
the current image.
Fig. 4-18
Save Image and Parameter As window
• Enter the name of the image in the Name text box, e.g. Conv-7.
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OPERATION
File Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• Click on the New MDB button.
− This opens the Create New Database window in which you can create a new image database,
e. g. Projections.mdb (see Create New Image Database, page 4-18).
− After creating the new database Projections.mdb - AIM window appears.
Fig. 4-19
4-30
Save Image and Parameter As window and Create New Database
window
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Fig. 4-20
OPERATION
File Menu
Carl Zeiss
Database window
• Click on the OK button in the Save Image and Parameter As window.
− The Projections.mdb - AIM window now shows the saved image.
− The Recordset box indicates the current number of the image in the image series contained in this
database.
• In the Description text box you can enter, for example, the configuration of the image.
• In the Notes text box you can enter further information about the image content.
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OPERATION
File Menu
Carl Zeiss
4.4.5
LSM 5 LIVE
LSM 5 LIVE DuoScan
Import of Images
The Import function enables the import of externally created images into the Image Display window
and the image database of the LSM 5 software.
• Click on the Import button in the File subordinate toolbar of the Main menu.
− This opens the Import Images window.
Fig. 4-21
Import Images window
• Select the data medium and the directory where the relevant image is contained in the Look in
selection box.
• Select the image file by clicking on it.
− The selected image will be shown for checking in the Image Display window (on the left) together
with the relevant details (size, channels, storage volume).
• Select the image type (Single Image or Image Series) in the Image type selection box.
• Click on Open.
− The image is displayed in a new Image Display window.
All the usual image and movie formats (e.g. .tif, .jpg, .bmp, .pcx, .avi, .mov etc.) are supported.
When importing series, please make sure to select the first image for the representation of the
entire series and to select the Image Series option under Image type.
• Finally, save the image in the desired image database via the Save As function.
• In Processing History this file is marked as imported file.
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4.4.6
OPERATION
File Menu
Carl Zeiss
Export of Images
The Export function allows the export of acquired images and images loaded from the image database.
• Select the image to be exported.
• Click on the Export button in the File subordinate toolbar of the Main menu.
− This opens the Export Images and Data window.
• Under Save in, select the data medium and the directory to which the image is to be exported.
• Enter a name for the image under File name.
• Select the image format into which the image is to be exported under Image type (Single Image
with raw data, Contents of the Image Display window, Full resolution).
• Chose a compression level. For some file formats lossless compression or various other compression
levels are available. The degree of losses for the image quality are listed according to the type of
compression.
• Click on the Save button.
− The image is stored on the relevant data medium / directory.
All the usual image and movie formats (e.g. .tif, .jpg, .bmp, .pcx, .avi, .mov etc.) are supported.
When stacks or time series are exported, each frame is stored as an individual image.
Fig. 4-22
03/06
Export Images and Data window
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OPERATION
File Menu
Carl Zeiss
4.4.7
LSM 5 LIVE
LSM 5 LIVE DuoScan
Multi Print
This function permits you to arrange several
images on one print page and to print them out
together.
• Click on the Multi Print button in the File
subordinate toolbar of the Main menu.
− This opens the Print - AIM window.
The main area of the Print – AIM window is used
for the display of the print page in the selected
paper orientation and for the arrangement of the
images to be printed.
The Print toolbar with the following buttons is
displayed on the right-hand side of the window:
Fig. 4-23
4.4.7.1
Print - AIM window
Paste
Paste from clipboard to sheet.
Delete
Delete marked image.
Print
Start printing.
Setup
Printer setup.
Landsc.
Landscape paper orientation.
Portrait
Portrait paper orientation.
Zoom slider
Zoom function for page preview.
Overlay Toolbar
The following functions can be performed on activation of the buttons in the Overlay toolbar (left-hand
side):
Arrow (selection) button: Activation of the mouse button for selection, resizing or
movement of an overlay element in the Image Display window.
Resizing: Click on the handle and hold down the mouse button, drag the handle, release
the mouse button.
Movement: Click on the line and hold down the mouse button, move the entire element,
release the mouse button.
Line button: Creation of a straight line in the Image Display window.
Click and hold down the mouse button, draw a line in any required direction, release the
mouse button to end the procedure.
Rectangle button: Creation of a rectangle in the Image Display window.
Click and hold down the mouse button, draw a rectangle in any required direction,
release the mouse button to end the procedure.
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OPERATION
File Menu
Carl Zeiss
Closed polyline button: Creation of a closed polyline figure in the Image Display
window.
The first click sets the starting point, each additional click adds a further line, a click with
the right mouse button closes the figure and ends the procedure.
Open polyline button: Creation of an open polyline figure in the Image Display
window.
The first click sets the starting point, each additional click adds a further line, a click with
the right mouse button ends the procedure.
Ellipse button: Creation of an ellipse in the Image Display window.
The first click sets the center point, the displayed line permits the determination of the
first dimension, the second click sets the first dimension, the second dimension and
rotation direction can then be determined, the third click sets the second dimension and
direction and ends the procedure.
Closed free-shape curve button: Creation of a closed Bezier figure in the Image
Display window.
The first click sets the starting point, each additional click adds a further line, a click with
the right mouse button closes the figure and ends the procedure.
Open free-shape curve button: Creation of an open Bezier figure in the Image Display
window.
The first click sets the starting point, each additional click adds a further line, a click with
the right mouse button closes the figure and ends the procedure.
Circle button: Creation of a circle in the Image Display window.
Clicking and holding down the mouse button sets the center point, drag the diameter
and release the mouse button to end the procedure.
Line with arrow button: Creation of a line with arrow in the Image Display window.
Click and hold down the mouse button, drag the line in any required direction, release
the mouse button to end the procedure.
A (Text) button: Creation of a text box in the Image Display window.
After clicking on A, the Text window will be displayed, and text can be entered via the
keyboard. The Font ... button enables you to select the font style and size in the Font
window. The entered text will be displayed in the left upper corner of the Image Display
window after clicking on OK and can be moved to the required position using the
mouse.
The Text window can also be activated with a double-click on a created text box, and the
entered text can be edited subsequently.
Insert opens up a further window which allows you to annotate coordinates, time and
Z-position with either automatic or user definable units and precision. This annotation is
updated during image acquisition and can be exported with the image. The annotation
can be stamped into already existing images.
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OPERATION
File Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Recycle bin button: All the overlay elements and dimensions dragged to the scanned
image are deleted. If one overlay element was marked before, this element is now
deleted from the scanned image.
Line button:
This button allows you to determine the line thickness of the area outline.
Color button: After clicking the Color button, the Color selection box will be opened.
The colors displayed in the Color selection box can be assigned to the overlay elements
with a click of the mouse. The currently selected color is displayed in the Color button. A
selected color is automatically assigned to the currently selected overlay element and then
to all the elements created afterwards.
4.4.7.2
Printing Images
To print several images on one page, proceed as follows:
• Use the Overlay functions to additionally illustrate the graphics and images to be printed.
• Select the paper orientation by clicking on the Landsc. or Portrait button.
• Open the image to be printed or select it from the relevant image database.
• Click on the Copy button. The image is copied to the clipboard.
• In the Print - AIM window, click on the Paste button.
The copied image appears in the work area of the Print - AIM window. You can click on it with the
mouse and move it to any position on the print page or you can adapt the image size.
• Proceed in the same way with all other images you want to print.
• Finally, arrange all images on the print page as required.
• Click on the Print button to start the printout.
• Close the Print - AIM window by clicking on the
4.4.8
button.
Exit
• Make sure to save all required images in the image database or export them.
• Close all open windows of the LSM program by clicking on the closing icon
of each window.
in the top right corner
• Click on the Exit button in the File subordinate toolbar of the Main menu.
− The LSM 5 LIVE Main menu will be closed and the LSM 5 LIVE Switchboard menu appears on the
screen.
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4.5
OPERATION
Acquire Menu
Carl Zeiss
Acquire Menu
• In the Main menu toolbar, click on Acquire.
− This opens another, subordinate toolbar in the Main menu.
Fig. 4-24
Acquire menu
For preparing and acquiring a scanning image, it is recommended to call up and use the tools of the
subordinate toolbar in the following order:
• Conventional microscope setting.
• Laser setting.
• Configuring the optical system for the Scanning Mode.
• Setting of scan parameters.
• EditROI permits up to 99 regions within a frame to be defined and analyzed (option).
• TimeSeries permits user-specific time series to be selected for the scan procedure.
• Upon selecting Stage you can set the focus (Z coordinate) and the Z step size between successive
slices. If the optional, motorized X/Y-stage is connected, the X and Y-positions of the sample can also
be selected.
• The VIS, TV and LSM buttons switch the beam path and indicate which beam path has been set in
the binocular tube of the microscope (VIS for viewing, TV for camera observation, LSM for laser
operation with monitor observation).
For the scanning process, the LSM button in the toolbar subordinate to the Acquire item must
be activated.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.1
LSM 5 LIVE
LSM 5 LIVE DuoScan
Laser Control
The Laser Control panel shows the types,
excitation wavelengths and operating status of all
lasers available.
The subordinate laser settings panel shows the
relevant and currently set Maximum Power,
Wavelength, Status and Tube Current (only
Sapphire 488) of the current laser.
The buttons On, Off and Standby permit the
current laser to be set in the required status.
Fig. 4-25
The name of the selected laser (Toptica ..., etc.) is
displayed in the headline of this setting panel for
checking.
Laser Control window
4.5.1.1
Switching on the Enterprise UV
Laser
• If the UV laser is required, switch it on via the
toggle switch (4-26/1) of the power supply.
− It will be ready for operation after a few
seconds.
Fig. 4-26
Power supply of UV-laser
4.5.1.2 Opening / Closing the Laser Control window
• Click on the Laser button in the Acquire subordinate toolbar.
− This opens the Laser Control window, which shows all lasers connected to the system.
When the setting of the required lasers has been finished, the Laser Control window can be closed
again.
• Click on the Close button to close the Laser Control window.
− The Laser Control window will be closed.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.1.3 Function Description
Lasers panel (upper)
List of available lasers, including the display of relevant wavelengths and
switching status.
Selection of the laser to be switched on / off and setting of the laser
output is performed in the subordinate setting panel.
Laser settings panel (lower) Switch on / off the required laser or set Standby operation.
Display Maximum Power, Wavelength and Status of the relevant laser.
4.5.1.4 Settings
• Click on the desired laser on the (upper) Lasers panel.
− This highlights the selected laser.
On the lower panel of the Laser Control window, activate the laser as follows:
This applies to the 405 Diode laser (405 nm):
• Click on the On button directly (not on Standby)
This applies to the Toptica and the Sapphire lasers (635 / 488 nm):
• Click on the Standby button.
− Wait for the laser to heat up, until the Status ready - Standby message appears (approx. two
minutes).
• Click on the On button.
− Status ready - On appears.
This applies to Compass 315M laser (532 nm):
• After selecting the laser, click on the On button.
−
The required laser for image acquisition is now available.
After switching on the lasers in the laser control window and their status ready the system can
be used for imaging. However, for quantitative imaging it is recommended to let the system
warm up for 40 minutes.
This applies to the Coherent UV-laser 653 II (Enterprise) and the Ar-multiline laser
• Click on the Standby button.
− Wait for the laser to heat up, until the Status ready - Standby message appears (approx. two
minutes).
• Click on the On button.
− Status ready - On appears.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• Use the Output [%] slider to set the laser power which is ideal for the measurement job. Thus, the
laser needed for image acquisition is available.
Argon:
Set output between 25 and 100 % of the maximum tube current. Optimum operation
is at 8 A (lowest laser noise). However, the laser life is reduced if the laser is constantly
operated at 8 A. Therefore, 8 A should be used only if this is absolutely necessary.
Enterprise:
Set output between 50 and 100 % of the maximum tube current. Optimum operation
is at 20 A (Tube Current; lowest noise). However, the laser life is reduced if the laser is
constantly operated at 20 A. Therefore, 20 A should be used only if this is absolutely
necessary.
To switch on the Enterprise laser, proceed as follows:
• The internal water cooling LP 5 is running.
• Start the PC, wait until NT system is booted.
• Switch on the power supply of the Enterprise laser, power potentiometer turned to maximum.
• Start the LSM 5 software.
Please bear in mind that a cooling phase of at least 5 minutes is required between switching off
of the laser via the software and switching off of the entire system via the REMOTE CONTROL
main switch or the Power Supply switch of the Enterprise UV laser.
If the LSM 5 software is already running and you want to use the UV laser, do the following:
• Close the LSM 5 software.
• Switch on the power supply of the Enterprise, power potentiometer turned to maximum.
• Start the LSM 5 software again.
4.5.2
Microscope Control
The Microscope Control (Micro button) window permits motorized functions (objective and reflector
change, condensor, filter and diaphragm settings) and the illumination mode (transmitted or reflected
light) of the connected microscope to be controlled via the software.
Without any difference to software control, these microscope functions can also be operated directly on
the stand via the relevant controls. In that case, any changes are recorded by the software and displayed
in the relevant windows / panels.
4.5.2.1
Open the Microscope Control Window
• Click on the Micro button.
− This opens the Microscope Control window on the screen.
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OPERATION
Acquire Menu
Carl Zeiss
After conclusion of the conventional setting of the connected microscope, the Microscope Control
window can be closed again.
• Click on the Close button in the Microscope Control window.
− The Microscope Control window will be closed.
4.5.2.2
Microscope Control Window for Axio Imager.Z1
• Click on the Micro button in the main frame.
• The microscope window opens in the last saved configuration.
• By clicking on the More / Less button the microscope window is displayed with or without detailed
microscope beampath panel.
Reflected Light button
The shutter is switched on and off. When using the X-Cite 120
additional controls are available. See below for description.
Reflector button
Push and click reflector cube can be selected via graphical pop-up menu.
Objective button
Objective can be selected via graphical pop-up menu.
Condensor button
Numerical aperture of the condensor is set via input box or slider. Turret
position selected from graphical pop-up menu (only for motorized
condensors). By clicking on the Close button the value is stored and the
Condensor frame is closed.
Field Stop button
Opening of luminous-field diaphragm (transmitted and reflected light)
can be set via input box or slider. By clicking on the Close button the
value is stored and the Field Stop frame is closed.
Filter button
Transmission values for attenuation filter (transmitted and reflected light)
is set via input box or slider for the front or rear filter wheel in
accordance with the available filter steps. By clicking on the Close
button the value is stored and the Filter frame is closed.
Aperture Diaphragm
button
Diaphragm opening is set via input box or slider. By clicking on the Close
button the value is stored and the Aperture Diaphragm frame is
closed.
Transmitted Light button
Transmitted light is switched on / off via ON button in the Transmitted
Light frame, setting of light intensity can be varied via input box or
slider. 3200 K color temperature for photo documentation can be
switched on via 3200 K button in the Transmitted Light frame. By
clicking on the Close button the Transmitted Light frame is closed.
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4-41
OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Recording of microscope settings
The upper part of the Axio Imager Control
window shows the recording functionality of
microscope configurations.
Complete microscope
created and applied.
configurations
can
be
The Store button permits existing microscope
configurations to be stored under any name.
The Apply button permits existing
microscope configurations to be loaded.
stored
The Delete button permits existing microscope
configurations to be deleted.
The Assign button permits the assignment of a
microscope configuration to a button.
Load a microscope configuration
An existing microscope configuration can be
loaded as follows:
• Click on the arrow button.
Fig. 4-27
− This opens a list box of all stored microscope
configurations.
Axio Imager.Z1 Control window
• Browse through the microscope configurations
by clicking, or use the scroll bar at the side of
the list box.
• Click on the desired microscope configuration.
− The selected microscope configuration is shown in the first line of the Microscope Configurations
list box.
• Click on the Apply button.
• Click on the Close button to close the microscope window.
Only those microscope settings which are encoded and motorized can be reloaded.
Store a Microscope Configuration
A newly created or changed microscope configuration can be stored under a new name as follows:
• Enter the desired name in the first line of the microscope setting list box.
• Click on the Store button.
• The actual configuration with the chosen name is added to the microscope settings list.
• Click on the Close button to close the microscope window.
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LSM 5 LIVE DuoScan
OPERATION
Acquire Menu
Carl Zeiss
Delete a Microscope Configuration
A no longer required microscope configuration can be deleted as follows:
• Select the microscope configuration to be deleted from the microscope configuration list box.
• Click on the Delete button.
• Click on the Close button to close the microscope window.
Assignment of a microscope configuration to
a button
A microscope configuration can be assigned to a
button as follows:
• Click on the Assign button.
• This opens the Assign-Microscope-SettingsTo-Button window.
Fig. 4-28
• Click on the arrow in the Button list box and
select a button out of the list.
Assign-Microscope-Settings-To-Button
window
With increasing numbers the buttons are arranged from the upper to the lower row from lefthand side to right-hand side.
• Click on the arrow in the Settings list box and select a microscope configuration.
• Click on the Apply button. A new button with the name of the selected microscope configuration has
been created.
• Click on the Close button to close the Assign-Microscope-Settings-To-Button window.
• Click on the Close button to close the microscope window.
Conventional setting of the microscope Axio Imager
• Click on the VIS button in the Acquire subordinate toolbar.
• Place specimen on microscope stage.
− The cover slip must be facing up.
• Click on the Micro button to open the Microscope Control window.
• Via the Objective button, select the required objective as follows:
− Open the graphical pop-up menu by clicking on the Objective button.
− Click on the objective you want to select.
− The selected objective will automatically move into the beam path.
• Use the focusing drive (4-29/5) to focus the required object plane.
• Select specimen detail by moving the stage in X and Y using the XY stage fine motion control (4-29/6
and 7).
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OPERATION
Acquire Menu
Carl Zeiss
Fig. 4-29
LSM 5 LIVE
LSM 5 LIVE DuoScan
LSM 5 LIVE with Axio Imager.Z1
Microscope settings on Axio Imager for transmitted-light observation
• Set the reflector turret position to None and click on the On button for transmitted light.
• Control the brightness of the halogen lamp with the potentiometer (4-29/4) or the Intensity % slider
in the Transmitted Light panel.
• Set the required transmission value of the gray filters in the Filter frame.
• Set the condensor and the luminous-field diaphragm for KÖHLER illumination.
With Transmitted Light activated (On), the halogen lamp is automatically occluded in the laser scanning
mode.
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LSM 5 LIVE DuoScan
OPERATION
Acquire Menu
Carl Zeiss
Microscope settings on Axio Imager for reflected-light observation (Epi-fluorescence)
• Turn on the HBO 100 W power supply with switch (4-29/1).
• Click on the RL reflected light button. The shutter opens.
To avoid excessive bleaching of biological samples, expose the specimen to the minimum
possible irradiation, i.e. keep the irradiation time as short as possible. For this, close the field
stop to the necessary field of illumination and use additional filters if available.
• By clicking on the reflector turret button, select the reflector module (filter sets) to suit the type of
fluorescence excitation. Proceed as follows:
• Click on the reflector turret button.
• Click on the desired reflector module.
− The reflector turret moves the selected reflector module into the beam path.
The FITC filter set consists of an excitation filter for the 450 - 490 nm spectral range, an FT color
splitter for 510 nm and an LP long pass filter, which passes emission light wavelengths greater
than 510 nm (FSET 09 = FITC, FSET 15 = Rhodamine, FSET 01 = DAPI).
Other filter sets:
DAPI:
BP 365 FSET01
FT 395
LP 397
Rhodamin:
BP 546 FSET15
FT 580
LP 590
The filter sets described in this section are included in the standard configuration, but other sets
are available on request.
If the X-Cite 120 fiber coupled lamp for
reflected-light illumination is used a
further dialog is opened when clicking the
Reflected light button. The lamp internal
shutter and the attenuation function can
be controlled via the LSM Software. The
lamp has to be switched on first using the
main switch on the lamp housing.
Fig. 4-30
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X-Cite 120 control panel
4-45
OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Remote
Enables the attenuation control via LSM Software. This is also enabled
when clicking the ON button.
ON
Opens the shutter in the reflected light path and the internal shutter of
the lamp. Both shutters are closed automatically when switching to LSM
mode.
Shutter
Opens and closes the shutter in the reflected light path independent of
the status of the internal lamp shutter and the attenuation.
Intensity
Sets the light via input box or slider.
Lamp Hours
Indicates the total time the lamp has been in use.
The aperture setting on the condensor of the Axio Imager.Z1 is performed in fixed steps.
4.5.2.3
Microscope Control Window for Axiovert 200 M
Transmitted Light button
Transmitted light is switched on / off via ON button in the Transmitted
Light frame, setting of light intensity can be varied via input box or
slider. 3200 K color temperature for photo documentation can be
switched on via 3200 K button in the Transmitted Light frame. The
transmission light control potentiometer on the stand is disabled via the
Remote button. By clicking on the Close button the Transmitted Light
frame is closed.
Condensor button
Numerical aperture of the condensor is set via input box or slider. Turret
position selected from graphical pop-up menu (only for motorized
condensors). By clicking on the Close button the Condensor frame is
closed.
Objective button
Objective can be selected via graphical pop-up menu.
Reflector button
Push and click, reflector cube can be selected via graphical pop-up
menu.
Tube Lens button
Push and click, tube lens can be selected via graphical pop-up menu.
Reflected Light button
The shutter is switched on and off. When using the X-Cite 120
additional controls are available. See below for description.
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LSM 5 LIVE DuoScan
OPERATION
Acquire Menu
Carl Zeiss
Recording of microscope settings
The upper part of the Axiovert Control window
shows the recording functionality of microscope
configurations.
Complete microscope
created and applied.
configurations
can
be
The Store button permits existing microscope
configurations to be stored under any name.
The Apply button permits existing
microscope configurations to be loaded.
stored
The Delete button permits existing microscope
configurations to be deleted.
The Assign button permits the assignment of a
microscope configuration to a button.
Load a microscope configuration
An existing microscope configuration can be
loaded as follows:
• Click on the arrow button.
− This opens a list box of all stored microscope
configurations.
Fig. 4-31
Axiovert 200 Control window
• Browse through the microscope configurations
by clicking, or use the scroll bar at the side of
the list box.
• Click on the desired microscope configuration.
− The selected microscope configuration is shown in the first line of the Microscope Configurations
list box.
• Click on the Apply button.
• Click on the Close button to close the microscope window.
Only those microscope settings which are encoded and motorized can be reloaded.
Store a microscope configuration
A newly created or changed microscope configuration can be stored under a new name as follows:
• Enter the desired name in the first line of the microscope setting list box.
• Click on the Store button.
• The actual configuration with the chosen name is added to the microscope settings list.
• Click on the Close button to close the microscope window.
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Carl Zeiss
OPERATION
Acquire Menu
LSM 5 LIVE
LSM 5 LIVE DuoScan
Delete a microscope configuration
A no longer required microscope configuration can be deleted as follows:
• Select the microscope configuration to be deleted from the microscope configuration list box.
• Click on the Delete button.
• Click on the Close button to close the microscope window.
Assignment of a microscope configuration to a button
A microscope configuration can be assigned to a button as follows:
• Click on the Assign button.
• This opens the Assign-Microscope-Settings-To-Button window.
• Click on the arrow in the Button list box and select a button out of the list.
With increasing numbers the buttons are arranged from the upper to the lower row from lefthand side to right-hand side.
• Click on the arrow in the Settings list box and select a microscope configuration.
• Click on the Apply button. A new button with the name of the selected microscope configuration has
been created.
• Click on the Close button to close the Assign-Microscope-Settings-To-Button window.
• Click on the Close button to close the microscope window.
For the conventional setting of the Axiovert 200 M, proceed as follows:
• Click on the VIS button in the Acquire subordinate toolbar.
• Place specimen on microscope stage.
− The cover slip must be facing down.
• In the Objective list box, select the required objective.
• Use the focusing drive (4-32/4) to focus the required specimen plane.
• Select specimen detail by moving the stage in X and Y via the XY stage fine motion control (4-32/3
and 2).
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LSM 5 LIVE DuoScan
OPERATION
Acquire Menu
Carl Zeiss
1:1
ZE
OB
RO
JEC
TIV
RE
FO
Fig. 4-32
CU
E
FLE
CT
OR
S
LSM 5 LIVE with Axiovert 200 M BP
Microscope settings on Axiovert for transmitted-light observation
• Click on the Reflected Light button and set the shutter to the Closed position.
• Click on the Transmitted light button. Click on the On button in the Transmitted Light panel and
set the transmitted light intensity via the slider or click on 3200 K. Click on Close to close the
Transmitted Light panel.
• Click on the Condensor button and set the aperture via the slider in the Condensor panel. Set the
filter in the Filter selection box. Click on Close.
• Click on the Objective button and select the objective by clicking on it.
• Click on the Reflector button and select the None.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Microscope settings on Axiovert for reflected-light observation (Epi-fluorescence)
• Turn on the HBO 100 power supply switch (4-32/1).
• Click on the Reflected Light button and set the shutter in the Open position.
• Click on the Reflector button and select the desired filter set by clicking on it.
• The filter is automatically moved into the beam path to enable observation in epi-fluorescence.
• Click on the Tube Lens button and select the tube lens.
• Click on the Objective button and select the objective.
Fig. 4-33
If the X-Cite 120 fiber coupled lamp for
reflected-light illumination is used a
further dialog is opened when clicking
the Reflected light button. The lamp
internal shutter and the attenuation
function can be controlled via the LSM
Software. The lamp has to be switched
on first using the main switch on the
lamp housing.
X-Cite 120 control panel
Remote
Enables the attenuation control via LSM Software. This is also enabled
when clicking the ON button.
ON
Opens the shutter in the reflected light path and the internal shutter of
the lamp. Both shutters are closed automatically when switching to LSM
mode.
Shutter
Opens and closes the shutter in the reflected light path independent of
the status of the internal lamp shutter and the attenuation.
Intensity
Sets the light via input box or slider.
Lamp Hours
Indicates the total time the lamp has been in use.
4.5.2.4
Working in LSM Mode
Switchover to the scanning mode is then required.
• Click on the LSM button in the Acquire subordinate toolbar.
4.5.2.5 Microscope Control for Axioskop 2 FS MOT
For setting the Axioskop 2 FS MOT, proceed in the same way as with Axio Imager.Z1 and Axiovert 200 M.
Since the Axioskop 2 FS MOT is not motorized (except the external Z drive and the tube), all microscope
settings have to be made manually.
Especially, the change of objectives is made manually. The used objective must be set in the Scan
Control window.
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LSM 5 LIVE
LSM 5 LIVE DuoScan
4.5.3
OPERATION
Acquire Menu
Carl Zeiss
Configuration Control
The setting of the beam path for the scanning procedure, i.e. the definition of channels (PMT
photomultiplier) and tracks and the setting of the attenuators of the various laser lines is performed in
the Configuration Control window.
A track is:
- a set of parameters for the detection channels and for illumination (wavelength and intensity)
- scanned simultaneously and identified and handled by the system with one name
The Configuration Control window has a different appearance, depending on which selection button
has been activated (Single Track and Multi Track). Use the Single Track and Multi Track buttons to
toggle between the two image acquisition modes single tracking and multitracking.
Performed settings can be stored as Track Configurations for single tracking.
In case the number of available channels is not sufficient for the scanning procedure, further tracks can
be added and configured. The combination of these tracks can also be stored as Recording
Configurations for multitracking (option). A recording configuration may contain the maximum of 4
tracks. Regardless of the number of included tracks, the maximum of 8 channels (incl. transmission) can
be used in a recording configuration in multitracking.
If several tracks have been activated, they are processed one after the other during the scan procedure.
If the maximum number of channels to be used in a Single Track or a Multi Track has already been
achieved, it is no longer possible to add further channels or tracks.
If a second track or further tracks are used, the scan parameters can be changed as required. This avoids
"cross-talk" from one channel to another when different tracks are used.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.3.1
LSM 5 LIVE
LSM 5 LIVE DuoScan
Open / Close the Configuration
Control Window
• Click on the Config button in the Acquire
subordinate toolbar.
− The Configuration Control window is
opened with the display last selected.
The
Beam
Path
and
Channel
Assignment panel differs according to
the hardware configuration supplied.
• Click on the Close button to quit the
Configuration Control window.
Fig. 4-34
Configuration Control window,
Single Track activated
4.5.3.2
Spectra Button
The Spectra button opens the Detection Spectra
& Laser Lines[Help74] window. This window
displays the laser wavelengths activated for
excitation (as colored vertical lines) and the
activated channels (as colored horizontal bars).
The color of the bar corresponds to the one assigned to the relevant channel. Non-active channels receive a gray bar over the entire spectral
range.
The length and position of the bar corresponds to
the emitted spectral range which is overlaid with
the filter and beam splitters selected in the Configuration Control window or number of selected
channels of the META detector.
Fig. 4-35
4-52
Detection Spectra & Laser Lines window
• Click on the Spectra button to open the
Detection Spectra & Laser Lines[Help75]
window and to check the settings you made.
The window is closed via Close.
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LSM 5 LIVE DuoScan
OPERATION
Acquire Menu
Carl Zeiss
All amendments made in the Configuration Control or Laser Control window are updated on-line in
the Detection Spectra & Laser Lines window.
• A click on the Laser button enables you to open the Laser Control window, switch lasers on and off,
if required, and control the laser output.
4.5.3.3
Config Button
The Config button permits existing track
configurations to be loaded, stored under any
name, or deleted.
(1)
Fig. 4-36
Track panel
Fig. 4-37
Track Configurations window
Load a track configuration
A configuration stored in the system, whether
factory-supplied or user-created, can be accepted
or selected for active operation as follows:
• Click on the Config button, the Track
Configurations window appears on the
screen.
• On the Store / Apply Configuration panel, click on the arrow button
.
− This opens a list box of all stored track configurations.
• Browse through the configurations by clicking, or use the scroll bar at the side of the list box.
• Click on the desired configuration.
− The selected configuration is shown in the first line of the Configurations list box (e.g.:
FITC/Rhod).
• Click on the Apply button.
− This results in the stored instrument parameters being taken over for active use. The track
configuration selected before is overwritten.
The optical diagram of the configuration selected appears on the Beam Path and Channel
Assignment panel. The newly loaded track has been automatically activated for the scanning
procedure. The Track Configurations window is closed automatically.
In the Options menu in the function Settings it is possible to define the parameters to be used
when applying a track configuration.
(2)
Store a track configuration
A newly created or changed track configuration can be stored under a new name as follows:
• Click on the Config button, the Track Configurations window appears on the screen.
• Enter the desired name in the first line of the Configurations list box.
• Click on the Store button.
• Close the window by clicking on Close.
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Carl Zeiss
OPERATION
Acquire Menu
LSM 5 LIVE
LSM 5 LIVE DuoScan
During storage via the Store/Apply function, all the data of the Beam Path and Channel Assignment
and the Detector Gain, Ampl. Offset, Ampl. Gain and Data Depth (8 / 12 Bit) scan parameters of the
current track (single tracking) will be stored.
(3)
Delete a track configuration
A no longer required track configuration can be deleted as follows:
• Click on the Config button, the Track Configurations window appears on the screen.
• Select the configuration to be deleted from the Configurations list box.
• Click on the Delete button.
• Close the window by clicking on Close.
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LSM 5 LIVE DuoScan
4.5.3.4
OPERATION
Acquire Menu
Carl Zeiss
Settings for Single Track in the Channel Mode
The settings of the beam path for the scanning procedure with regard to the main beam splitter
(Achrogate), secondary dichroic beam splitter (NFT), emission filters (EM) to be used and the assignment
of channels, excitation wavelengths and laser intensities are performed in the Beam Path and Channel
Assignment panel.
The setting can be performed manually or by using existing track configurations.
• Click on the Single Track button, unless it has already been activated.
− The Configuration Control window for single tracking is displayed.
(1)
Beam Path and Channel Assignment panel
The Beam Path and Channel Assignment panel displays the selected track configuration which is used
for the scan procedure.
You can change the settings of this panel using the following function elements.
Activation / deactivation of the excitation wavelengths (check box) and setting of
excitation intensities (slider). Open the Laser Control window via the Laser button.
Selection of the main beam splitter (Achrogate) or secondary dichroic beam spliter (NFT)
position through selection from the relevant list box.
Selection of an emission filter through selection from the relevant list box.
Activation / deactivation of the selected channel for the scanning procedure by
assigning an existing color icon or defining a new one. Deactivation of the channel via
deactivation of the check box.
For the configuration of the beam path, please refer to the application-specific configurations depending
on the used dyes and markers and the existing instrument configuration (e.g.: module LSM 5 LIVE
1 channel biomedical configurations – VarioOne GB, 488, 532 (635) nm, 1 channel FL) listed in the
ANNEX of the printed manual.
The assignment of the numbered emission filters (1-2), NFT secondary dichroic beam splitters and
Achrogate main beam splitters in the Beam Path and Channel Assignment panel is shown in the
Configuration Control window (Fig. 4-38). The numbers of the emission filters correspond to those of
the channels lying behind (PMT photomultipliers).
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OPERATION
Acquire Menu
Carl Zeiss
Fig. 4-38
(2)
LSM 5 LIVE
LSM 5 LIVE DuoScan
Configuration Control window
Beam path - Achrogate main beam splitters and NFT secondary dichroic beam splitters
• On the Beam Path and Channel Assignment panel, click on the Achrogate main beam splitters
(see Fig. 4-38).
− This opens a graphical pop-up window of all beam splitters available.
• To select a beam splitter, click on the respective line of the list.
− The selected beam splitter moves into the beam path.
• Proceed accordingly to configure the NFT secondary dichroic beam splitters
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LSM 5 LIVE DuoScan
(3)
OPERATION
Acquire Menu
Carl Zeiss
Beam path - Emission filter
• On the Beam Path and Channel Assignment
panel, click on the
emission filter symbol.
− This opens a graphical pop-up window of all
available emission filters (e.g. BP for band
pass, or LP for long pass) with their
wavelengths.
• To select an emission filter, click on the
respective filter in the pop-up window.
− The emission filter selected moves into the
beam path in front of the PMT
photomultiplier.
• Depending on the application, it may be
necessary to insert additional mirrors, secondary
dichroic beam splitters or neutral glass filters
between the Achrogate main beam splitter and
the PMT photomultiplier. To select these
symbols.
components, click on the respective
For channels 1 and 2, it is possible to
change the filters directly on the LSM 5
LIVE scan module (see CHAPTER ANNEX:
Filter Change in the Detection Beam Path
of Channels 1 and 2 of the printed
manual).
(4)
Fig. 4-39
Configuration Control window
Fig. 4-40
Channel Color Selection window
Beam path - Activation / Deactivation of
Channels and Channel Color Assignment
• On the Beam Path and Channel Assignment
.
panel, click on the channel symbols, e.g.
− This opens the Channel Color Selection
window on the Beam Path and Channel
Assignment panel.
• Click on the desired color bar.
This changes the color of the channel symbol.
• To close the Channel Color Selection box,
click on the Close button.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Further colors for the corresponding channel can
be produced as follows:
• Clicking on the Define button will open a
further Channel Colors window.
All the available colors are shown as buttons in the
Current Set of Channel Colors panel.
• Via a reticule in the Define Color panel, any
desired color can be produced.
• Clicking on the Add button allows the color to
be used for further channel coloring.
• Choose the desired color with the reticule (the
reticule is in the left corner at the bottom of the
color range).
• Define the brightness by use of the scroll bar.
• Use the Add button to add the color to the
color range.
• To delete a defined color, click on the relevant
color button and then on the Remove button.
Fig. 4-41
Channel Colors window
Standard colors (black for OFF, red, green, blue and white) cannot be removed.
• Click on the Close button to close the Channel Colors window.
− Newly defined colors are accepted and displayed in the Channel Color Selection window. They
can then be used in the same way as standard colors.
The PMT1 photomultiplier is activated / deactivated by the check box.
• Proceed in the same way for the other PMT photomultiplier.
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(5)
OPERATION
Acquire Menu
Carl Zeiss
Beam path - Laser attenuation
• On the Beam Path and Channel Assignment
panel, click on the Excitation button.
− This opens a dialog box of all available lasers
with their wavelengths and their usable
attenuation.
• To select the desired laser line, activate the
check box for Line Active.
• Use the Transmission [%] slider to set the
utilizable laser intensity (recommendation: start
at 50 %).
− The transmittance of the attenuator changes
accordingly.
Fig. 4-42
Configuration Control window
• This allows you to adapt the laser intensity very
sensitively to the job. Activate the check box for
Line Active.
By clicking on the Excitation button you can check at any time which lasers are available for
active operation.
If you deactivate Line Active, the laser wavelengths for the lasers are deselected by means of
the attenuator, i.e. these lasers change into standby status.
Excitation filters, emission filters, Achrogate main beam splitters and NFT secondary dichroic
beam splitters can be switched online, channels (PMT photomultipliers) only off-line.
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OPERATION
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Carl Zeiss
4.5.3.5
LSM 5 LIVE
LSM 5 LIVE DuoScan
Settings for Multi Track in the
Channel Mode
The Multi Track function permit several tracks to
be defined as one configuration (Recording
Configuration) for the scan procedure, to be
stored under any name, reloaded or deleted.
The maximum of four tracks with up to 8 channels
can be defined simultaneously and then scanned
one after the other. Each track is a separate unit
and can be configured independently of the other
tracks with regard to channels, emission filters and
dichroic beam splitters.
• Click on the Multi Track button.
− The Configuration Control window for
multitracking appears, which means that the
List of Tracks panel is additionally displayed.
The tracks required for multitracking can either be
configured manually one after the other (identical
to single tracking) and then stored as recording
configuration, or already existing recording
configurations can be used and changed as
required.
It is also possible to load already stored track
configurations (single tracking) in a recording
configuration.
Fig. 4-43
(1)
Configuration Control window,
Multi Track activated
Beam Path and Channel Assignment panel
The Beam Path and Channel Assignment panel displays the track configuration of the track currently
selected in the List of Tracks panel (highlighted in blue or gray).
The settings for this panel are performed separately for each track, in the same way as for single tracking.
To do this, select the track to be configured from the List of Tracks panel (see the following description
of the List of Tracks panel).
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(2)
OPERATION
Acquire Menu
Carl Zeiss
List of Tracks panel
In the List of Tracks panel, the available tracks are
displayed with names, activated channels and laser
lines.
The sequence of tracks to be processed can be
changed for the scan procedure.
The Add Track, Store/Apply Single Track and
Remove buttons permit individual tracks to be
added, saved or deleted.
Fig. 4-44
List of Tracks panel
In addition, this panel is used to activate / deactivate the tracks for the scan procedure.
• To activate or deactivate one or several tracks for the scan procedure, activate / deactivate the check
box of the relevant tracks.
The configuration of the selected track is displayed in the Beam Path and Channel Assignment - ...
panel.
• To select a track for the display of the beam path configuration, click on its name.
− The selected track is highlighted in gray or blue.
When you switch from multitracking to single tracking, the track selected in the multitracking
mode (highlighted in blue or gray) is always transferred and automatically activated for the scan
procedure. All other tracks are deactivated, and they remain deactivated when you switch back
to the multitracking mode afterwards.
The following functions are available in the List of Tracks panel.
Add Track button
An additional track is added to the configuration list. The maximum of
four tracks can be added. One track each with basic configuration is
added, i.e.: one ChL 1 channel is activated, all laser lines are switched
off, emission filters and dichroic beam splitters are set in accordance with
the configuration last used.
Remove button
The single track previously marked in the List of Tracks panel in the
Name column is deleted.
Store/Apply button
Opens the Track Configurations window. A selected track defined in a
Recording Configuration can also be stored as a single track for single
tracking applications. Also, it's possible to load a single track in a
multitracking configuration.
A click on this arrow button will move the selected track (highlighted in
blue) one position upwards in the list box.
A click on this arrow button will move the selected track (highlighted in
blue) one position downwards in the list box.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
When adding new tracks, the following sequence should be followed:
• Add a track by clicking on the Add Track button.
• Determine the configuration of the track in the Beam Path and Channel Assignment panel or select
an existing one via the Store/Apply Single Track button of the List of Tracks panel.
• Store the name of a track configuration defined via the Store/Apply button of the List of Tracks
panel. The new track name will then be displayed in the List of Tracks panel.
If this way of storing is performed, the created track will also be available as a single track and can
therefore also be activated individually.
• Add the next track via the Add Track button and then configure and store it again.
The name of a track can also be changed directly in the List of Tracks panel. In that case, however, the
edited track is not available as a single track configuration, but only within the recording configuration.
To edit a track name within Recording Configurations, proceed as follows:
• To select the track, click on the relevant track name in the List of Tracks panel. Then click on the
name again to open the text editing field.
• Change the track name via the keyboard. Use Esc to undo the procedure.
• Click once in the area outside the text editing box to close this box.
The channels of the individual tracks with the relevant scan parameters can be displayed in the
Scan Control window after activation of the Channels button. The description of channel 1 in
Track 1, for example, is ChL1-T1.
(3)
Config button in Multi Track mode
The Config button in the Multi Track mode permits all tracks to be loaded, stored under any name, or
deleted.
(a)
Load a Channel Mode Configuration
An existing recording configuration can be loaded as follows:
• Click on the Config button, the Channel
Mode Configurations window appears on the
screen.
• On the Store / Apply Configuration panel,
click on the arrow button .
Fig. 4-45
Channel Mode Configurations window
− This opens a list box of all stored recording
configurations.
• Browse through the configurations by clicking,
or use the scroll bar at the side of the list box.
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LSM 5 LIVE DuoScan
OPERATION
Acquire Menu
Carl Zeiss
• Click on the desired configuration.
− The selected configuration is shown in the first line of the Configurations list box (e.g.: DAPI).
• Click on the Apply button.
− The program loads those parameters of the selected Channel Mode Configuration which have
been activated in the Options menu under Settings / Recording Configuration (see Settings
Function, page 4-200). The Channel Mode Configurations window is automatically closed.
The optical diagram of the configuration selected appears on the Beam Path and Channel
Assignment panel. The entire recording configuration has been activated for the scanning
procedure.
(b)
Store a Channel Mode Configuration
A newly created or changed recording configuration can be stored under a new name as follows:
• Click on the Config button, the Channel Mode Configurations window appears on the screen.
• Enter the desired name in the first line of the Configurations list box.
• Click on the Store button.
• Close the window by clicking on Close.
During storage via the Config button, all the data of Beam Path and Channel Assignment and the
Detector Gain, Ampl. Offset, Ampl. Gain and Data Depth (8 / 12 Bit) scan parameters of all the defined
tracks (multitracking) are stored. Furthermore, the used objective, the Frame Size, Zoom, Rotation &
Offset and Scan Direction parameters are stored.
(c)
Delete a Channel Mode configuration
A no longer required recording configuration can be deleted as follows:
• Click on the Config button, the Recording Configurations window appears on the screen.
• Select the configuration to be deleted from the Configurations list box.
• Click on the Delete button.
• Close the window by clicking on Close.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.3.6
LSM 5 LIVE
LSM 5 LIVE DuoScan
Transmitted light preset
To add a transmitted light image, the light level of
the Halogen lamp can be preselected in the
configuration control window. The illumination is
activated when the actual scan is started. To
separate the transmitted light signal from the
emitted fluorescence, choose e.g. the BP 700-750
in Ch1.
Fig. 4-46
4-64
Transmitted light preset
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LSM 5 LIVE DuoScan
4.5.3.7
OPERATION
Acquire Menu
Carl Zeiss
Ratio Settings Panel
The Ratio Settings panel permits you to activate two additional Ratio channels.
• Click on the Ratio button.
− The Ratio Settings panel is displayed at the bottom of the Configuration Control window. The
settings of the selected tracking mode (Single Track / Multi Track) remain unchanged.
• The Ratio Settings panel is only available in the Single Track and Multi Track mode.
The following function elements are provided in the Ratio Settings panel:
Activation of the Ratio channel (R1, R2) through assignment of an existing color
or definition of a new one. Activation / deactivation of the Ratio channel via the
check box.
Selection of the channels of which the ratio is to be formed from the relevant list
box. Source 2 in ratio settings includes the option to select "1st Image" for R1
and/or R2 (e.g. to calculate F/F0 for single wavelength dyes).
A suitable color can be assigned to each of the two Ratio Channels R1 and R2, in the same way as for
the photomultiplier channels.
The channels of which a ratio will be formed are selected via the Source 1 and Source 2 list boxes.
• Click on the
opened.
arrow button to select the required channel for Source 1 and 2 from the list box now
The ratio to be formed between the selected channels can be defined more precisely using one
of the four preset formulas in the Scan Control window after activation of the Channels
button and a click on the relevant ratio button (e.g.: R1) for online display of radiometric or
single wavelength dyes. The Set by min/max function (in Scan Control window - Channel mode)
allows the definition of the display scaling according to the expected minimal and maximal
values
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OPERATION
Acquire Menu
Carl Zeiss
4.5.3.8
LSM 5 LIVE
LSM 5 LIVE DuoScan
Camera Detection Panel
The use of this function permits the use of a Zeiss
AxioCam (HRm, HRc MRm) camera as an
alternative external detector.
• Click on the Config button in the Acquire
subordinate toolbar of the main menu.
− The Configuration Control window opens.
• Activate one of the Single Track or Multi
Track buttons and click on the Camera button.
− The Beam Path and Channel Assignment
panel for camera detection is opened.
Control buttons
Fig. 4-47
Configuration Control window;
camera detecting activated
TV
Menu for selecting a display
color for the camera image.
Reflector
Selects a beam splitter for the
excitation/emission.
Add Track
Adds a second track to the
acquisition in Multi Track
mode, e.g. a different
fluorescence filter cube or
transmitted light.
If TV and LSM tracks are mixed, the active detection port of the microscope has to be set
according to the first track.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.3.9 LSM 5 LIVE / DuoScan Panel
The use of this function permits the use of a
LSM 5 LIVE or a LSM DuoScan in combination with
a T-PMT as an alternative transmitted light
detector.
• Click on the Config button in the Acquire
subordinate toolbar of the main menu.
− The Configuration Control[Help92] window
opens.
• Activate one of the Single Track or Multi
Track buttons and click on the LSM 5 LIVE or
LSM DuoScan button.
− The Beam Path and Channel Assignment
panel for camera detection is opened.
If LSM 5 LIVE and LSM DuoScan tracks
are mixed, the active detection port of
the microscope has to be set according to
the first track.
Fig. 4-48
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Configuration
Control
window;
LSM 5 LIVE / Duo Scan activated
4-67
OPERATION
Acquire Menu
Carl Zeiss
4.5.4
LSM 5 LIVE
LSM 5 LIVE DuoScan
Scan Control
The scan parameters for image acquisition are set
in the Scan Control window.
The microscope must be in the LSM mode, i.e. the
LSM button on the tube of the microscope stand
must be pushed or the LSM button in the Acquire
subordinate toolbar must be activated to set the
LSM mode.
The scanning actions are started via the buttons on
the right-hand side of the Scan Control window,
and the scan parameters are set in the main part of
the window.
An acquired image is displayed in a separate
Image Display window. If an Image Display
window is not yet available, a new Image Display
window is automatically opened during the
acquisition.
The following scanning modes can be performed:
Line
Fig. 4-49
Scan Control window
− scanning of a line in the XY-plane
(Line, Line + Time Series)
− scanning of a line with different Z-values
(Line + Z Stack, Line + Z Stack + Time Series)
Frame
− scanning of an XY frame (Frame, Frame + Time Series)
− scanning of XY frames with different Z-values (Frame + Z Stack, Frame + Z Stack + Time Series)
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4.5.4.1
OPERATION
Acquire Menu
Carl Zeiss
Open / Close the Scan Control Window
• Click on the Scan button in the Acquire subordinate toolbar of the Main menu.
− This opens the Scan Control window, which shows all lasers connected to the system.
• Click on the Close button to quit the Scan Control window.
The following main function buttons are available in the Scan Control window:
Generally available buttons
When the button is activated, the following panels are available for the
Mode button
setting of the scanning parameters for the line and frame modes:
Objective Lens, Image Size & Line Step Factor, Speed, Pixel Depth,
Scan Direction & Scan Average and Zoom, Rotation & Offset.
Channels button
When the button is activated, the Channel Settings and
Excitation of Track ... panels are available for the setting of the
channels and the laser excitation.
Line button
Activates the Line scan mode.
Frame button
Activates the Frame scan mode.
Z Stack button
Activates the Z Stack scan mode, display of additional buttons on the
right-hand side of the Scan Control window.
Z Settings button
When the button is activated, the Z Settings panel is available for the Zscan parameter definition. The Z Stack scan mode must be active.
Close button
Closes the Scan Control window.
New button
Opens a new Image Display window.
Find button
Automatic optimization of image brightness and contrast. The settings
for the Find function can be varied as required using the Maintain
menu.
Fast XY button
Continuous scan with high speed. This function should be used to a
limited extent and only for a short period of time. Fast XY switches
temporarily to 512 x 512 frame size.
Single button
Single scan (named Start in the Z Stack mode).
Stop button
Stops the current scan procedure, no matter in which window the
button is pressed (also see the Time Series Control window).
Cont. button / Finish button
Continuous scan (not available in Spot Scan mode and Z Stack mode).
If you select the option Frame for Mode and the option Continuous for
Number in the Pixel Depth, Scan Direction & Scan Average panel,
the Finish button is displayed instead of the Cont. button. In this case,
continuous averaging is performed when you have started the scan. If
you click on the Finish button, the scan/averaging process is stopped
after the scan of the current image has been completed.
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Carl Zeiss
OPERATION
Acquire Menu
LSM 5 LIVE
LSM 5 LIVE DuoScan
Additional button in the Line mode
Automatically defines a line in the center of the Image Display window
Line Sel button
(Frame) for creation of the intensity profile; using the mouse, the line for
the intensity profile can then be positioned anywhere in the Image
Display window.
Additional buttons in the Z Stack mode
Triggers the scan of a stack.
Start button
XYscan button
Triggers a single XY-scan
XYcont button
Triggers continuous XY-scan.
Line Sel button
To prepare the Range function, a cutline is created in the scanned XYframe to determine the position at which the XZ-scan through the
specimen is to be produced. Using the mouse, the line for the XZ-scan
can be positioned anywhere in the scan frame. The cutline can be
defined either as a straight line or free shape curve.
Range button
Produces an XZ-scan through the specimen within the limits determined
in Num Slices and Interval; the cutline is determined via the Line Sel
function.
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4.5.4.2
OPERATION
Acquire Menu
Carl Zeiss
Frame
When the Frame button is activated, a frame of
variable size is scanned pixel by pixel and line by
line. The laser beam is moved over the specimen
line by line.
The scan parameters and the channels (single
detector) are set via the Mode and Channels
buttons, and the laser settings can be checked
again or changed.
(1)
Mode
When the Mode button is activated, the
Objective Lens, Image Size & Line Step Factor,
Pixel Depth, Scan Direction & Scan Average
and Zoom, Rotation & Offset panels are
displayed in the Scan Control window.
Objective Lens, Image Size & Line Step Factor
panel
• Open the Objective list box and select the
objective to be used via a click of the mouse
(identical to Microscope Control). When using
immersion oil objectives, make sure to perform
immersion as required.
• Select the Frame Size from the default sizes via
the buttons 128, 256, 512, 768, 1024 or enter
the required values via the keyboard.
Recommended setting to start with: 512 x 512
pixels.
Fig. 4-50
Scan Control window - Mode/Frame
Fig. 4-51
Objective Lens, Image Size & Line Step
Factor panel
− It is also possible to enter different values for
X and Y. The value for Y is freely selectable
between 1 and 1024 pixels (integers). The
value for X depends on the chosen image
format.
− The most common image formats can be set
directly with the Frame size buttons. Note
that more formats are available in the format
pull down list.
• Use the Scan Speed slider to adjust the
exposure time.
• Previous the slider is shown the number Frames
per Second. Start with 2 to 4 frames per
second.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
The time slider displays the following information:
− Frames per second (it is possible to type in the required FPS directly, or to move the slider
accordingly),
− Pixel time and
− Frame (scan) time.
Pixel Depth, Scan Direction & Scan Average
panel
• Select 8 Bit or 12 Bit Data Depth, i.e. 256 or
4096 gray values.
• Select the Unidirectional or Bi-directional
Scan Direction.
Fig. 4-52
Pixel Depth, Scan Direction &
Scan Average panel
−
Unidirectional: The laser scans in one
direction only, then moves back with beam
blanked and scans the next line.
−
Bi-directional: The laser also scans when
moving backwards, i.e. the Scan Time is
halved.
Unidirectional scan is always optimal regarding
image quality. For possible artefacts caused by
bidirectional scan (partially caused by fluorochromes behaviour), (jitter or flicker) the Scan Corr
Y correction slider is available. Move slider to either
side in continuous scan mode till images are stable.
The Auto button sets the right correction
automatically.
Fig. 4-53
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OPERATION
Acquire Menu
Carl Zeiss
Example
Fig. 4-54
Example for Scan Correction
Intensity fluctuations at the end of the frame (left image) are fully compensated by the Auto button (right
image).
Average
• Select the Line or Frame mode for averaging.
• Select the desired scan average method Mean or Sum in the Method selection box.
• Select the desired scan average from the available values 2, 4, 8 and 16 in the Number selection box
or Continues (only for Frame average mode).
The greater the number of averages selected for Mean average Method, the better the image
quality will be; the scanning time will be prolonged accordingly.
Averaging can be performed in different ways, depending on whether the Mean or Sum method has
been activated.
If you are using the Mean method, the image information is generated by adding up all scans pixel by
pixel and then calculating the mean value.
In the Sum method, the pixel values of all scans are only added up, without a mean value being
calculated.
To create the image information using the Line average mode, each line (depending on the setting) is
scanned 2, 4, 8 or 16 times during Scan Average, and then the average value per pixel is calculated. This
minimizes noise interference during the scanning procedure.
If the Frame average mode is used to create the image information, the complete frame is scanned 2, 4,
8 or 16 times, depending on the setting. The average value is recalculated after each frame scan.
The Frame average mode also permits continuous averaging.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
For this, select the Continuous option in the Number selection box.
If you have selected the Continuous option, the Finish button for ending continuous averaging is
displayed instead of the Cont. button. Use the Single button in this case to start continuous scanning.
When you click on the Finish button, the scan currently in progress will be completed before the process
is stopped.
Zoom, Rotation & Offset panel
In this panel, the scan range is set for zoom and
offset in relation to the field of view of the
microscope. The diagonals of the outer square on
the right-hand side correspond to the field of view
of the microscope.
Fig. 4-55
The inner square contained in it (rectangle in the
case of differently set frame size) represents the
scan range and immediately shows the changes
made to zoom and offset.
Zoom, Rotation & Offset panel
The blue line at the top of the scan range is helpful
for orientation when the scan range is rotated in
the direction of the field of view.
• Set the desired zoom factor via the slider (Zoom) or by clicking on the arrow buttons.
− The zoom factor can be set continuously in the range from 0.5 to the maximum of 2.0, and is
displayed in the relevant input box. The value 0.5 corresponds to factor 1, and value 2.0 to factor
4, related to the field of view of 18 mm in the intermediate plane. Clicking on button 1 enables
immediate resetting to the zoom factor 1.
− Recommended setting to start with: Zoom 1.
• Move the scan area by clicking on the 4 arrow buttons (Offset).
− The offset of the scan area from the center of the field of view is displayed online in µm for X and
Y.
− A click on the center button will recenter the scan area to the field of view.
− Clicking, holding and drawing the rectangle with the mouse permits the scan area to be moved
directly within the field of view.
− Recommended setting to start with: Offset X = 0, Y = 0
During the scan procedure, the functions Objective change, Speed, Scan Corr, Zoom,
Rotation and Offset can be influenced online.
By clicking on the Reset button the scan zoom is set to 1 and the XY offsets are set to the zero position.
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(2)
OPERATION
Acquire Menu
Carl Zeiss
Channels
If the Channels button is activated, the Channel
Settings and Excitation of Track ... panels are
displayed in the Scan Control window.
Channel Settings panel
In the Channel Settings panel, the channels (incl.
ratio channels) defined in the Configuration
Control window are listed track by track as
selectable buttons.
Depending on the selected Channels button (e.g.
ChL1-T1), the currently used settings of Pinhole
(confocal aperture), Detector Gain, Amplifier Offset
and Amplifier Gain are displayed.
• The slider near Pinhole (Confocal Aperture)
enables you to change the aperture size of the
relevant channel.
− The aperture size is indicated in µm, Optical
Slice and Airy Units. The Airy value
depends on the aperture of the objective,
excitations and the emission wavelength.
− A small aperture size will increase the depth
of focus, but reduce the light intensity
received by the PMT photomultiplier.
− When you vary the aperture size, an Optical
Slice value is displayed. For optimum depth
resolution, Airy values should be small, but in
fluorescence applications not below 1.0 to
keep the intensity loss within a reasonable
limit.
Fig. 4-56
Scan Control window – Channels
− A click on the 1 button sets the aperture size
to 1 Airy unit. A click on the Max button
sets the aperture size to the maximum.
• The sliders (and the relevant arrow buttons) near Detector Gain, Ampl. Offset and Exposure Time
enable you to set the photomultiplier of the selected channel during continuous scanning.
− Detector Gain: Setting of the sensitivity of the detector - setting of image contrast and brightness
(values available between 0 and 50)
− Amplifier Offset: Setting of the electronic offset - background of the image can be set (values
available between -2 and 0.1)
− Exposure Time: Setting of exposure time with display field for frame rate (Frames per Second)
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Gain and Find
The Find button always sets an ideal gain for a
clear background, best sensitivity and full grey
resolution. This is maximally a gain value of 25.
One can manually increase this gain by moving the
gain slider beyond 25 and use the digital post
amplification by doing this. Be aware that from
value of 37,5 on, background artefacts can be
visible, since those are amplified too.
When using 12 bit grey resolution, gaps can occur
in the histogram of the acquired image, in case the
Gain slider is moved beyond a gain level of 25.
Use the Amplifier Offset slider to remove
underexposed pixels in the image background (no
blue pixels visible with “range” palette).
Fig. 4-57
Gain and Find
The parameters Detector Gain, Ampl. Offset and Ampl. Gain are described in section
Confocal Aperture / Detector Gain / Ampl. Offset (see page 4-319) in the context of image
optimization.
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OPERATION
Acquire Menu
Carl Zeiss
The parameters of a ratio channel are set in a
separate dialog box.
• Click on the button of a ratio channel (e.g. R1).
The dialog box for the setting of the ratio
parameters is displayed.
Clicking on the required tabs enables you to
choose from four formulas (Type 1 to 4) for ratio
calculation. The relevant decimal values can be
entered in the input boxes via the keyboard. The
entered values remain unchanged even after
switchover to another formula and can be
reactivated any time.
The formula type activated last is always used for
ratio formation during the scan procedure. If the
input box does not contain any value at all or no
suitable value, the useful value last used will be
activated.
The ratio channels are displayed in the Image
Display window (see Fig. 4-61).
• Select the required formula and enter the
relevant values.
Letters can be entered into the formula fields
which will be valued as 1; it is also possible to
make no entry, which will also be valued as 1, but
will not be displayed.
Set by min/max (in Scan Control window Channels mode) allows the definition of the
display scaling according to the expected minimal
and maximal values.
Fig. 4-58
Channel Settings panel of a
Ratio Channel
Fig. 4-59
Excitation of Track ... panel
Excitation panel
• In the Excitation panel you can select other
lasers and vary laser intensities (in the same way
as in the Laser Control or Configuration
Control window).
All parameters under Channels can be varied
online.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Acquisition of a frame
Once you have set up your parameter as defined in the above section, you can acquire a frame image of
your specimen.
• Click on the Single button in the Scan Control
window. The system will automatically start the
acquisition of a frame. The individual channels
and the overlay image can be viewed by
changing to the Split xy mode. This button is
located on the right-hand side of the Image
Display window.
The following scan image shows the result with
two defined tracks and the overlay (see Fig. 4-61).
See the appropriate Channel Settings panel in
the Scan Control window (Fig. 4-60).
Fig. 4-60
Channel Settings panel for two defined
tracks plus Ratio channel
Fig. 4-61
4-78
Image Display window with two tracks plus ratio track (Split xy
mode)
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(3)
OPERATION
Acquire Menu
Carl Zeiss
Z Stack
This function permits a series of XY-images to be
produced in different focus positions (Z slices).
When the Z Stack button is pressed, the
Z Settings button is automatically activated and
the Z Settings panel is displayed in the Scan
Control window. However, it is possible at all
times to switch over to setting / changing the scan
parameters or the PMT photomultipliers and lasers
via the Channels and Mode buttons.
The additional XYscan, XYcont, Line Sel and
Range buttons are available on the right-hand
side of the Scan Control window, and the
labeling of the Single button changes to Start.
The Z Stack function is deactivated by clicking
again on the Z Stack button.
Z Settings panel - overview
The parameters of the Z Stack to be created are
defined and displayed online in the Z Settings
panel.
Fig. 4-62
Scan Control window – Z Settings
Stack Z Size:
The dimension of the Z Stack in µm. The stage (nosepiece) is moved in such a way that
the stack size, dependent on the refractive index, is achieved optically.
Focus Position:
The current Z position. If the refractive index (Refr. Corr.) changes, the value of the
focus position in relation to the "0" also changes (online).
Z Slice:
Opens the Optical Slice window (Fig. 4-63). The Optical Slice window contains three
buttons (Optimal Interval: ... µm, Optimal Pinhole Diameter (confocal aperture)
and Undo) to allow the setting of the optimal interval and the optimal aperture size of
fluorescence stacks. Both values influence each other and depend on the objective
used.
In the case of a fixed aperture size, half the value of the smallest aperture size used is
taken to determine the optimum interval. Accordingly, the aperture size to be used in
the case of a preset interval is determined by doubling the value of the selected
interval. Undo resets the just before altered values.
The Optical Slice window displays the following information:
Black: Stack Z Size (µm) = intervals x (number of slices - 1)
Optimal Interval = depending on the objective used and the aperture size setting
Red and other colors: Presentation of the actual data set by the operator helps to
optimize stack creation.
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OPERATION
Acquire Menu
Carl Zeiss
Fig. 4-63
LSM 5 LIVE
LSM 5 LIVE DuoScan
Optical Slice window
Tabs
Z Sectioning:
Tab for setting of Number of Slices, Interval and Current Slice via slider / arrow
button.
Mark First/Last: Tab for determination of the Z-value for the first and last XY-image of the stack,
combined with manual focusing or Stage control.
Hyperfine
Z Sectioning:
Tab for production of a Z Stack using the optional Piezo focus for objectives.
First:
Scanning / Display of the beginning (first XY-image) of the stack.
Mid:
Scanning / Display of the center (XY-image in the center) of the stack.
Last:
Scanning / Display of the end (last XY-image) of the stack.
Refr. Corr.:
Considers the different refractive index between the immersion medium of the
objective (n') and the embedding medium of the specimen (n), which can be set
between 0.5 and 3 via the slider / arrow buttons
Ratio =
n
n'
X:Y:Z=1:1:1
Clicking on this button will set the Z-interval in such a way that the voxel has identical
dimensions in the X-,Y- and Z-directions (cube).
Auto Z Corr.
This function permits the set values of the scan parameters Detector Gain, Ampl.
Offset and Ampl. Gain (as measure for the brightness level) to be varied between
two freely selectable slices of a stack to be recorded. During the scan procedure, the
interim values of these three parameters are automatically linearly interpolated
between the initial and end values (see page 4-84).
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OPERATION
Acquire Menu
Carl Zeiss
The parameters of a Z Stack can be defined using the Z Sectioning tab, the Mark First/Last tab or - if
the optional Piezo focus for objectives is connected - the Hyperfine Z Sectioning tab.
Z Sectioning tab
Num Slices:
Entry of the number of sections (single XY-images) to be recorded with the stack via
the slider / arrow buttons. The entry does not influence the interval.
Interval:
Entry of the step width (Z-distance between the single XY-images) via slider / arrow
buttons. The entry has no influence on Num Slices.
Current Slice:
Display of the current position of the slice within the stack. Change of position via
slider / arrow keys. Reset of the current slice position in the center of the stack by
clicking on the C button. Of course, the borders of the stack are also changed if the
current slice position is changed.
Keep Interval:
The interval remains constant when the stack limits or number of slices are changed.
Keep Slices:
The number of slices remains constant when the stack limits or interval are changed.
The Stack Z Size is slightly adjusted to hold the number of slices with no
interval changes or the interval size if the number of slices is varied.
Fig. 4-64
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Scan Control window - Z Sectioning tab activated
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• Click on the Range button.
− The XZ-scan will be performed and displayed in the Image Display window. At the same time, the
position of the current slice is shown with a green line and the positions of the first and last slice
with two red lines.
Fig. 4-65
Scan Control window and Image Display window
• Moving the green line (current slice) enables you to change the current focus position (moving the
stage or nosepiece in the process). The stack limits are also changed, while interval and Num Slice
remain unchanged.
• Shifting one of the red lines enables you to change the stack size; in that case, the interval size is
matched, and the Num Slice remains constant.
− Changing the values of Num Slice, Interval and Current Slice in the Z Sectioning tab will, of
course, also change the positions of the red and green lines in the Image Display window.
• A click on the Start button will start the recording of the Z Stack.
− The settings of the entire Scan Control window (Mode, Channels, Z settings) will be used when
the stack is produced.
In Frame mode the stack is acquired as a stack of images in xyz. In Line mode the stack is
displayed by a xz image. Continuous imaging of the xz stack is also possible.
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OPERATION
Acquire Menu
Carl Zeiss
Mark First/Last tab
The determination of the optimum stack size is
performed here via focusing during a continuous
scan.
• Click on the XYcont button.
−
A continuous XY-scan of the set focus
position will be performed.
−
If you have reduced the scan speed or have
set image averaging, you should use the fast
scanning mode to find the lowest and
highest points of focus. These settings are
made under Mode in the Scan Control
menu, or directly via the FAST XY button.
Fig. 4-66
Mark First/Last tab
• Use the manual focusing drive or the Stage and Focus Control window (see Stage, page 4-114) to
focus on the upper position of the specimen area where the Z Stack is to start.
• Click on the Mark First button to set the upper position of the Z Stack.
• Then focus on the lower specimen area where the recording of the Z Stack is to end.
• Click on the Mark Last button to set this lower position.
• Keep Slice enables the Num Slices slider which allows to vary the number of slices. The limits of the
Z Stack remain constant, the interval is matched accordingly.
• Keep Interval enables the Interval [µm] slider which allows to vary the interval between the slices.
The number of slices is matched accordingly, the limit of the Z Stack is adjusted.
• Click on the Start button to start the recording of the Z Stack.
In case the upper and lower limits of the stack have been switched round, automatic matching will be
performed by the software, since the stage of the Axio Imager.Z1 always moves from bottom to top and
the nosepiece of the Axiovert 200 M always moves from top to bottom.
Setting via Range is not possible via the Mark First/Last function, i.e. the lines cannot be
shifted.
The Fast Z Line functions is not available in frame mode.
When you change from Mark First/Last to Z Sectioning or vice versa, the values are updated
in the Z Sectioning tab.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Hyperfine Z Sectioning tab
Activation of this tab is only possible if the piezo
objective focusing device has been connected.
The piezo objective focusing device can be
controlled via software (see Stage, page 4-114).
Fig. 4-67
Hyperfine Z Sectioning tab
The accuracy of the piezo objective focusing device
regarding the step width in the Z-direction lies in
the range of 60 nm.
The piezo objective focusing device allows stacks
to be produced considerably quicker than via the
focus of the microscope stand.
The focus position remains unchanged.
• Clicking on the Leveling button moves the piezo objective focus to the zero position, while the motor
focus moves into the opposite direction at the same time, i.e. the position of the object in relation to
the objective remains unchanged. This function is used to set defined initial conditions.
• Use the slider or the arrow keys to set the number of slices for the Z Stack.
• Use the slider or the arrow keys to set the size of the interval.
Num Slices and Interval can be varied independently of each other within the piezo objective focus
work range of ±250 µm. When change is made to Z Sectioning, or vice versa, values are also taken
over, provided they are within the piezo objective focus work range.
If a larger range is set for the Z Stack under Z Sectioning or Mark First/Last, the Interval is matched
accordingly when changing to Hyperfine Z Sectioning, while Num Slice remains constant.
• Use XYcont, Line Sel and Range to determine the parameters of the Z Stack (identical to Z
Sectioning).
If the green line (Current Slice) is shifted after the creation of Range, the focus position will change (the
piezo objective focusing device remains in the center position). The red lines (stack limits) can only be
changed symmetrically to the Current-Slice position within the piezo objective focusing device work
range.
Since the piezo objective focusing device moves from bottom to top during the creation of the
Z Stack, top and bottom of the Axiovert 200 M have been switched round.
Bidirectional Z-Scan in time series (4D)
Similar effects can occur when using the fast piezo
Z-drive in Bidirectional mode with multiple stacks.
Like for X/Y-scanning, there is a uni- and a bidirectional mode here too.
Fig. 4-68
Corr Z Slider
• To compensate possible image deviations in the
Z-direction in bidirectional focus mode, use the
Corr Z slider by typing in the deviation
occurring in the adjacent image stacks of a time
series.
If only one time point (stack) is acquired,
the Corr Z slider has no effect.
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OPERATION
Acquire Menu
Carl Zeiss
Auto Z Corr.
The function Auto Z Correction allows a linear variation of Detector Gain, Ampl. Offset, and Ampl. Gain
values between the different slices of a stack.
• Click on the Auto Z button, the Auto Z
Brightness Correction panel opens.
The buttons Set A and Set B permit definition of
two distinct gain / offset settings at two different Z
positions A and B.
Pressing the Move A and Move B buttons permits
the defined Z-position to be directly approached.
The Enable test check box permits simulation of
the value changes for Detector Gain, Ampl.
Offset, Ampl. Gain and Attenuation in the Scan
Control window without the scanners being in
operation.
Fig. 4-69
Z Brightness level control panel
If a Z Stack is performed and the Auto Z Brightness Correction window is opened this
correction is automatically performed equal whether the Enable test box is enabled or disabled.
• Use the focusing drive to set the Z-position where the brightness level correction is to be started.
• In the Scan Control window, set the initial values for Detector Gain, Ampl. Offset and Ampl. Gain.
If required, start the continuous scan procedure for this purpose. Click on the Set A button.
• Use the focusing drive to set the Z-position where the brightness level correction is to be ended.
• Set the end value for Detector Gain, Ampl. Offset and Ampl. Gain in the Scan Control window.
Click on the Set B button.
• If required, check the change of the set values by activating Enable test.
After the start of the scan procedure, the brightness level values are linearly interpolated between the
defined references A and B.
Note that the total Z range where the interpolation takes place can exceed the Z reference A
and B.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Acquisition of a Z Stack
Once you have set up your image as defined in the above section, you can collect a series of confocal
images through the different focal planes of your specimen.
• Click on the Start button on the Scan Control window. The system will automatically start the
creation of a Z Stack. Be careful not to bump the air table or the microscope until Z sectioning is
completed. Each successive Z Slice can be viewed by changing to the Gallery Mode. This button is
located on the right-hand side of the image.
A black bar will be shown under the image and will move from left to right, showing that the LSM 5 LIVE
is in the process. The laser will automatically stop scanning when the Z Stack is completed.
The entire stack of images can be saved using the Save or Save As buttons on the right-hand side of the
image.
Fig. 4-70
4-86
Image Display window of a Z Stack
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4.5.4.3
OPERATION
Acquire Menu
Carl Zeiss
Line
In the Line mode, fluorescent or reflected light
along a freely definable line is displayed in the
form of an intensity profile.
All the possibilities of creating an image (Frame,
Z Stack) are also available in the Line mode.
The Line and Frame buttons are activated
alternately and exclude each other.
• Set all the parameters for the Scan procedure
(Mode and Channels or Z Settings) in the
same way as for the scanning of a frame or a
Z Stack.
• Then click on the Line Sel button.
Fig. 4-71
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Scan Control window - Mode/Line
4-87
OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.5.4.4
Camera Control
The use of this function permits the control of the
external CCD-camera settings.
(1) Open / Close the Scan control window
for camera control
• In the Configuration Control window, activate
the Camera button.
• Click on the Scan button in the Acquire
subordinate toolbar of the main menu.
• Click on the Close button.
Fig. 4-72
Scan Control window - Mode, settings
for camera control
(2)
Function description
Mode button
Displays the selected objective, frame size and pixel depth.
Frame Size
Selects between square formats or free defined frame sizes.
Format
Selects between a range of default camera resolutions. The 5x5 binning mode can be
used for focusing without delay of the image display.
Data Depth
Sets the pixel depth.
Zoom/Offset
Shifts a subregion in the frame.
Reset
Resets the frame/subregion to default value selected in Format.
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OPERATION
Acquire Menu
Fig. 4-73
Carl Zeiss
Scan Control window - Channels, settings for camera control
Channels button
Displays the activated channels and possible settings.
Exposure time
Sets the exposure time of the camera.
Find
Starts a prescan and sets the exposure time automatically.
In case of a camera multitracking, only one channel should be selected in
Configuration Control in order to speed up the find function.
Fast X/Y
Starts a fast online scan mode, e.g. for focusing. Also, the 5x5 binning mode can
be used (to be set in Mode / Format).
Single
Starts a single image acquisition (The Image Display window appears.).
Cont.
Starts acquisition of a series of images (The Image Display window appears.).
Crop
Defines a ROI for camera acquisition in the Image Display window. Note that this
is just a Crop function, while the whole sample is illuminated. Rotation of the ROI
is not possible.
Info button
Shows the acquisition parameters in the Image Display window.
Close
Close the Scan Control window.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.5
LSM 5 LIVE
LSM 5 LIVE DuoScan
Edit ROI (Region Of Interest)
A scan image allows certain areas (ROIs) to be defined. Definition and activation of the ROIs for the
analysis is performed in the Edit ROI window.
4.5.5.1
Open / Close the Edit ROI Window
• Click on the Edit ROI button in the Acquire subordinate toolbar of the Main menu. The Edit ROI
window appears on the screen and the ROIs defined last are visible in the Image Display window.
• Click on the Close button in the Edit ROI window. The Edit ROI window is closed and the ROIs
disappear from the Image Display window.
Fig. 4-74
Edit ROI window and Image Display window with ROIs
When Edit ROI is activated and the first ROI is drawn in the Image Display window, the Use ROI is
activated automatically.
4.5.5.2
Function Description
The following functions are available on the right-hand side of the Edit ROI window:
Close button
The Edit ROI window is closed.
Remove button
An entry marked in ROI Lists (stored ROI configuration) is deleted.
Add to Lists
button
The Add ROI List window is opened.
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(1)
OPERATION
Acquire Menu
Carl Zeiss
ROI Lists panel
In the ROI Lists panel, all the currently defined and
stored ROI configurations are shown.
• Click on the ROI configuration which you want
to use for the scan procedure.
− The selected ROI configuration is highlighted
in blue and displayed in the opened Image
Display window.
Fig. 4-75
ROI Lists panel
Fig. 4-76
Interactive ROI Definition panel
• To produce a new ROI configuration, an already
stored configuration can be activated, changed
and stored under a new name using the Add
to List button.
• To delete a stored ROI configuration from the
list, click on its name first (highlighted in blue)
and then on the Remove button.
(2)
Interactive ROI Definition panel
In the Interactive ROI Definition panel, the
parameters of the ROI configuration just selected
from the ROI Lists panel are displayed.
Furthermore, it contains all the functions required
for the creation of ROIs.
The X and Y values for Center Position and
Dimension can be edited.
• Activate the relevant text box with a mouse
click and enter the new value via the keyboard.
• If you click outside the edited text box, the new
value will be taken over and the ROI figure be
shifted to the new position.
The upper part of the panel gives an overview of
all the individual figures stored under the selected
name according to type, position within the Image
Display window (in pixels) and greatest dimension
in X and Y (in pixels). The origin of the position
indication lies in the left top corner of the Image
Display window.
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Carl Zeiss
OPERATION
Acquire Menu
LSM 5 LIVE
LSM 5 LIVE DuoScan
Check box (e.g.: 1 - 4): Clicking on this check box allows a ROI to be deactivated.
The tick disappears from the check box, as does the relevant marked area from the
scanning image. Clicking on the check box again will reactivate the ROI.
Arrow button: Activation of the mouse button to change the size or move the
ROIs in the Image Display window.
Rectangle button: Draw of a rectangle in the Image Display window; click and
keep mouse button pressed, drag the rectangle in any direction, let go off the
mouse button to end the procedure.
Bezier button: Draw of a bezier figure in the Image Display window; first click
sets the starting point, each additional click adds a line, double-click on the
starting point closes the figure and ends the procedure.
Ellipse button: Draw of an ellipse in the Image Display window; first click sets
the center point, displayed line permits determination of the extension, second
click sets the first dimension, then the second dimension and the rotation direction
can be determined, third click sets the second dimension and direction and ends
the procedure.
Circle button: Draw of a circle in the Image Display window; click and keep the
mouse button pressed to set the center point, drag the diameter, let go off mouse
button again to end the procedure.
Polyline button: Draw of a polyline figure in the Image Display window; first
click sets the starting point, each further click adds a line, double-click on the
starting point closes the figure and ends the procedure.
Recycle bin button: All the ROIs dragged to the scanning image are deleted. If an
area outline was marked before, this area is now deleted in the scanning image.
Auto / Color button: A defined color from the list of colors can be assigned to the
ROIs. In that case, the same color is assigned to all the individual figures. In the
Auto position, the outlines of the dragged ROIs are automatically colored
differently.
Line button: This button allows you to determine the line thickness of the area
outline. This is for display purposes only. The scanned line is not effected.
Fit Frame Size to bounding Rectangle of all ROIs check box: If this check box
is ticked, the scan procedure is displayed only within a rectangle which is defined
by the greatest extension in X and Y of all the individual figures together, i.e. the
pixel number and the data quantity of the Image Display window are reduced.
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OPERATION
Acquire Menu
Carl Zeiss
• In the toolbar of the Interactive ROI Definition panel, click on the symbol of the area you want to
use to mark the region of interest in the scanning image. Five different area symbols are available in
the form of buttons.
• Click on the marking area and keep the mouse button pressed to drag the area into the region of
interest in the scanning image. The marking area will be numbered automatically and entered in the
Interactive ROI Definition panel with its position and dimension parameters and the appropriate
number.
• The dragged marking area is marked by clicking on its outline; its size can be changed by clicking on
the marking points. Clicking on the area edge beside the marking points allows repositioning of the
area on the scanning image.
The digits of the ROIs can be shifted independently of the contours of the figure.
• If you have framed all the required ROIs in
accordance with steps 2 to 4, you can store
these ROIs under any required name via the
Add to Lists button.
• The Add ROI List window will appear. Enter
any required name to store the ROIs and click
on the OK button.
• This stored ROI configuration appears in the
ROI Lists panel of the Edit ROI window.
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Fig. 4-77
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Add ROI List window
4-93
OPERATION
Acquire Menu
Carl Zeiss
4.5.6
LSM 5 LIVE
LSM 5 LIVE DuoScan
Time Series
The Time Series Control window allows the
definition of parameters for time series.
The Time Series function offers the following
options for the creation of image series:
• Definition of break times between 0.1 ms and
10 hours.
• Determination of the number of steps from 1 to
10,000 for one scanning procedure.
• Setting of markers.
• Interruption of time control via pause function,
and resume of the time series function.
• Triggering of time series via:
− numeric input
− external trigger pulses
− time (of the PC)
4.5.6.1
Open / Close the Time Series
Control Window
• Click on the Time Series button in the Acquire
subordinate toolbar of the Main menu.
Fig. 4-78
Time Series Control window
The Time Series Control window appears on the
screen.
• Click on the Close button to close the Time
Series Control window.
The following functions are available on the right-hand side of the Time Series Control window:
Close button
Closes the Time Series Control window.
New button
Opens a new Image Display window.
Start T button
Starts the Time Series.
Stop button
Stops the entire Time Series. A current scan is interrupted.
Pause button
Interrupts the Time Series. Button labeling is changed to Resume. A current scan
is performed until the end. When the button is pressed again, the Time Series is
immediately continued with the next scan procedure.
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Mean ROI button
OPERATION
Acquire Menu
Carl Zeiss
Creates a Time Series with the intensity values of the Frame or the default ROIs. An
average value is formed of the intensity values of the Frame or the ROIs
determined. These average values are displayed in an extended Image Display
window as a function of the time which has passed.
The status line, in which the phases of the current Time Series or notes for the user are displayed, is in the
lower part of the Time Series Control window.
4.5.6.2
Start Series Panel
In this panel, the parameters for the start of the
time series are set.
Fig. 4-79
Start Series panel
The following functions are available:
Manual button
The time series is started manually with a click on the Start T or Start B button.
Trigger button
The time series is started via a trigger signal from Trigger Control.
Time button
The time series is started when the set time is reached. The internal computer time
applies.
Time input box
Input of the time for the start of the time series (Time button activated).
Trigger in list box
Selection of the trigger key (1-4) with which the start is to be triggered (Trigger
button activated).
Trigger out list box Selection of the trigger keys (1-4) for the out signal.
Pre-Scan
If this box is checked before starting the Time Series a continuous scan is started
, which then appears on the
but no images are acquired until the button Go
lower right hand side of the control window, is clicked.
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OPERATION
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Carl Zeiss
(1)
LSM 5 LIVE
LSM 5 LIVE DuoScan
Start via Trigger
For the start via trigger control (Trigger button
activated), first determine the trigger key which is
to trigger the start of the Time Series.
Fig. 4-80
Start Series panel
• Open the Trigger in list box with a click on the
arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger1).
It is also possible to trigger an out signal via trigger control.
• Open the Trigger out list box with a click on the arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger1).
In this example, the scan procedure is triggered on pressing key 1 of the trigger control, and an out signal
is given at the same time.
When starting a Time Series via Trigger, the Start T or Start B button must be pressed first.
Waiting for Trigger will then be displayed in the status line.
Then the relevant trigger key on the Trigger Control must be pressed to start the first scan
procedure of the Time Series.
(2)
Start via Time
For the start via the time set on the PC (Time
button activated), the start time must be entered
first in the Time input box.
Fig. 4-81
Start Series panel
• Click in the Time input box to open it.
• Enter a start time via the keyboard. Then click
outside the input box once to close it again.
When starting a Time Series via the time, the Start T or Start B button must also be pressed in
this case. Waiting for Start Time will be displayed in the status line.
The Time Series is started when the starting time has been reached.
The starting time for the Time Series can be changed online.
(3)
Pre-Scan
The Pre-Scan allows to image the sample continuously before actually starting the
image acquisition.
This function should only be used for starting the time series manually.
After clicking the Start or Start B button, the button Go appears on the
lower right hand side of the control window. By clicking this button the
actual image acquisition is started.
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4.5.6.3
OPERATION
Acquire Menu
Carl Zeiss
Stop Series Panel
In this panel, the parameters for the end of the
time series are set and the number of cycles is
determined.
Fig. 4-82
Stop Series panel
The following functions are available:
Manual button
The time series is finished manually with a click on the Stop button.
Trigger button
The time series is finished via a trigger signal.
Time button
The time series is finished when the set time has been reached. The internal
computer time applies as the set time.
Number input box / Determination of the number of images acquired or image stacks for the time
series.
arrow keys / slider
Time input box
Input of the time for the end of the time series (Time button activated).
Trigger in list box
Selection of the trigger keys (1-4) with which the end is to be triggered (Trigger
button activated).
Trigger out list box Selection of the trigger keys (1-4) for the out signal.
• Use the slider near Number to select the images or image stacks for the time series.
(1)
Stop via Trigger
To end the Time Series via Trigger Control (Trigger
button activated), first determine the trigger key
which is to end the Time Series.
• Open the Trigger in list box with a click on the
arrow button.
• Choose one of the trigger keys 1 to 4 (e.g.
Trigger2).
Fig. 4-83
Stop Series panel
It is also possible to trigger an out signal via Trigger Control.
• Open the Trigger out list box with a click on the arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger2).
In this example, the Time Series is ended on pressing key 2 of the Trigger Control, and an out signal is
given at the same time.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
If the entered number of cycles has been processed without a trigger impulse having been given
to end the procedure, the Time Series is finished.
If a trigger signal is given before the cycles have been processed, the Time Series will only be
interrupted. Waiting for Trigger will be displayed in the status line. The Time Series can now
be continued via a new trigger signal or ended via Stop.
(2)
Stop via Time
To end the Time Series via the time set on the PC
(Time button activated), the end time must first be
entered in the Time input box.
• Click on the Time input box to open it.
Fig. 4-84
Stop Series panel
• Enter the end time via the keyboard. Then click
outside the input box once to close the box.
The Time Series is interrupted when the end time has been reached.
If the entered Number of cycles has been processed, the Time Series is finished.
If the number of cycles has not yet been processed, the Time Series is only interrupted. Waiting
for Start Time is displayed in the status line. The Time Series can now be continued by entering
a new start time, or finished via Stop.
The end time for the Time Series can be changed online.
4.5.6.4
Cycle Delay / Time Interval Panels
Depending on the settings in the Time Series tab
(see Options menu, Settings), the time series
interval is defined either as a Cycle Delay or Time
Interval. Accordingly, either the Cycle Delay
panel or the Time Interval panel is displayed in
the Time Series Control window.
Cycle Delay is the interval between the end of
one scan process and the beginning of the next.
Fig. 4-85
Cycle Delay panel
Time Interval is the interval between the
beginning of one scan process and the beginning
of the next.
The Cycle Delay (or Time Interval) panel permits
the intervals to be activated and changed.
Fig. 4-86
4-98
Time Interval panel
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OPERATION
Acquire Menu
Carl Zeiss
Function description
Cycle delay or Time List of the stored sets of Cycle Delays or Time Intervals for time series.
Interval list box
Apply button
Application of the sets of delays for time series selected in the list box.
Store button
Storage of sets of delays for time series.
Delete button
Deletion of sets of delays for time series from the list box.
Time buttons
Activation of the time for the time series set for the relevant button.
Time input box /
Determination of the cycle time for the currently activated Time button.
arrow buttons / slider
Unit buttons
Selection of time units: min, sec or ms.
Trigger in list box
Selection of the trigger key (1-4) to be used to activate the Time button for the
delay time.
Trigger out list box
Selection of the trigger key (1-4) for the out signal.
Setting delay time or time interval
• The delay time or time interval to be used during the Time Series is set to a default value by activating
a Time button.
For this purpose, the relevant time must be assigned to the Time button first.
• Activate a Time button with a click of the mouse.
• Set the required delay time or time interval via the slider (arrow keys or input box) near Time. The set
time is displayed online on the button. Select the time unit by clicking on the relevant button near
Unit.
You can assign different times to all the six Time buttons and store this assignment either as a set of
delays or of time intervals.
• Enter a name in the Cycle Delay list box or Time Interval list box and click on Store to store the set
of delays.
Activating delay time or time interval
If required, a set of delays or time intervals can be activated again quickly.
• Open the list box with a click on the arrow button and select the required set with a click of the
mouse.
• Then click on the Apply button to activate the set. The stored delays are assigned to the Time
buttons.
Sets of delays or Sets of time intervals which are no longer required can be deleted.
• Open the list box and select the required set.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• Click on the Delete button. The set will be removed.
The Time buttons can also be activated via keys 1 to 4 of the Trigger Control.
• Click on the required Time button.
• Open the Trigger in list box with a click on the arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger3).
It is also possible to trigger an out signal via Trigger Control.
• Click on the required Time button.
• Open the Trigger out list box with a click on the arrow button.
• Choose one of the trigger keys 1 to 4 (e.g. Trigger3).
In this example, the relevant Time button is activated on pressing key 3 of the Trigger Control, and an
out signal is given at the same time.
The delays or time intervals can be changed online with a click on another Time button. The
new delay will be applied immediately.
A change of the delay during a Time Series is displayed in the Image Display window if the
Gallery button (Display toolbar) is activated.
4.5.6.5
Marker Panel
The setting of a marker permits information about
the moment in the current time series and any
required comment to be assigned to the current
scan. The time indication is set automatically, while
comments must be defined before.
The markers (red squares) are visible in the Image
Display window if the Gallery button (Display
toolbar) is activated.
Fig. 4-87
Marker panel
On storage of the image, all the markers, including
the time indication and the comments, are stored
along with the image contents.
Function description
Marker list box
List of the stored combinations of markers.
Apply button
Application of the marker combinations selected from the list box.
Store button
Storage of a combination of markers.
Delete button
Deletion of a combination of markers from the Marker list box.
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OPERATION
Acquire Menu
Set 1-7 button
Setting of a marker during the scan procedure.
Description
input box (1-7)
Entry of the comments for the marker.
Trigger in
list box (1-7)
Selection of the trigger key (1-4) with which the marker is to be set.
Trigger out
list box (1-7)
Selection of the trigger key (1-4) for the out signal.
Carl Zeiss
Setting marker for the current scan
• A marker for the current scan is set by clicking on one of the Set 1 to 7 marker buttons.
The assignment of any required comment for the marker must be performed as follows:
• Click in the Edit Text box of the required marker key (e.g.: Set 1) to open the editing box.
• Enter the comments via the keyboard. Then click outside the editing box to close this box again.
You can assign comments of any required length to all the seven Set buttons and store this assignment
as a combination of marker keys.
• Enter a name in the Marker list box and click on Store to store the combination.
If required, a combination of markers can be activated again quickly.
• Open the Marker list box with a click on the arrow button and select the required combination with a
click of the mouse.
• Then click on the Apply button to activate the combination. The relevant comments are displayed in
the Edit Text boxes of the Set buttons.
Combinations which are no longer required can be deleted.
• Open the Marker list box and select the required combination.
• Click on the Delete button. The combination will be removed.
The marker buttons can also be activated via keys 1 to 4 of the Trigger Control.
• Click on the required Set button.
• Open the Trigger in list box with a click on the arrow button.
• Select one of the trigger keys 1 to 4 (e.g. Trigger4).
It is also possible to trigger an out signal via Trigger Control.
• Click on the required Set button.
• Open the Trigger out list box with a click on the arrow button.
• Select one of the trigger keys 1 to 4 (e.g. Trigger4).
In this example, the relevant Set button is activated on pressing key 4 of the Trigger Control, a marker is
set in the Scan and an out signal given at the same time.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.6.6
LSM 5 LIVE
LSM 5 LIVE DuoScan
Time Series of a Frame
• Set the relevant parameters for time control in the Start Series, End Series and Cycle Delay panels.
• Start the Time Series with a click on the Start T or Start B button.
• If you use Trigger Control, confirm the relevant Trigger key to start the Time Series with the first scan
procedure.
• Use the Set 1 to Set 7 buttons to set markers during the scanning procedure which will allow you to
evaluate interesting scanning images later.
Time end will finish time series even if you have created a program which would exceed the
time end.
If a time series is interrupted before its programmed end, the programmed number of images
will be taken over in the database. However, only those images are stored which were created
before interruption of the time series. This is due to the fact that the original image parameters
are to be taken over via the Reuse function.
If a stop time for time series is entered via the Trigger button or the Time button, the recording
of the series will not be definitely finished. It is possible to either continue the series via new
settings of Trigger and Time or to definitely finish the time series via the Stop key.
The following example of a scanning image was taken using the Time Series function. Both the time
and the markers set during the scanning procedure are projected in the image series in different colors.
If the cursor is moved to a marker position in the scanning image, the relevant information on the image
detail is automatically provided in an additional window.
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Fig. 4-88
OPERATION
Acquire Menu
Carl Zeiss
Image Display window of a Time Series Scan
The image markers have different colors with the following meaning:
• red:
manually set marker with time indication and comments
• blue:
automatically set marker with change of delay
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OPERATION
Acquire Menu
Carl Zeiss
4.5.6.7
LSM 5 LIVE
LSM 5 LIVE DuoScan
Time Series of a Frame over Z Stack (option)
• First, set all parameters required for recording a Z Stack in the Scan Control window.
• Then set the parameters required for recording the time series in the Time Series Control window
(identical procedure as for the time series of a frame).
• Start the time series by clicking on Start T.
− Complete stacks are now recorded at the defined time intervals. The result is displayed in the form
of the combined Image Display window of the stack and time series (4D).
Fig. 4-89
Image Display window of a Z Stack and a Time Series Scan
The additional Z, Time and Z + Time buttons are available in the Gallery toolbar of the Image Display
window.
When you click on the Z button, the individual frames of the Z Stack are displayed for the selected Time
Slice. When you click on the Time button, the individual frames of the time series are displayed for the
selected Z Slice.
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OPERATION
Acquire Menu
Carl Zeiss
For Z Stacks over the time (4D) following offline functions will be enlarged:
− Slice (Z slider and Time slider)
− Gallery (Z, Time and Z + Time buttons)
− 3D (slider for single time index)
To select the Z or Time Slices, use the appropriate sliders which are displayed if the Slice button in the
Image Display window has been activated.
When you click on the Z + Time button, all individual frames will be displayed.
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4-105
OPERATION
Acquire Menu
Carl Zeiss
4.5.6.8
LSM 5 LIVE
LSM 5 LIVE DuoScan
Time Series with Mean ROI
• Set all the parameters in the same way as for Time Series of a frame.
• Then click on the Mean ROI button in the time series frame.
A mean intensity profile of the defined ROIs (To be defined using the Edit ROI, see page 4-90) is created
as a function of time.
Fig. 4-90
Image Display window of a Time Series with Mean ROI
The Image Display window of the Mean ROI function is structured differently than that of a frame.
On the left-hand side of the Image Display window, the intensity time profiles per ROI are displayed
graphically.
The Select and Display toolbars, which are also available in the standard Image Display window, are
positioned in the center.
The Scan Mean of ROIs toolbar with further function elements is additionally displayed on the righthand side. The major purpose of these function elements is to vary the display of the recorded Mean ROI.
By selecting the appropriate options (see Options menu, Settings – Scan Mean of ROIs) you can
activate the following additional functions:
− Display of the live image in the Image Display window of the Mean ROI function (used ROIs only)
− Scan of the complete image (if Live Image has been activated)
− Saving of the complete time series (if Live Image has been activated)
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OPERATION
Acquire Menu
Carl Zeiss
The following functions are available:
Display of the data of the ROIs used for the creation of the MeanROI
(identical to the Edit ROI window). If the check box of a ROI is
deactivated, the ROI's intensity values are no longer displayed in the
Intensity-Time diagram.
1 button: Intensity values for ROI and Channels are displayed in a
diagram. Chan button: Intensity values are displayed separately for each
channel used. ROI button: Intensity values are displayed separately for
each ROI used. Mono button: Switches between color and
monochromic display of intensity profiles.
Automatic button: Automatic scaling of the display of Intensity-Time
diagrams. Time Range button: Display of Intensity-Time diagrams is
scaled depending on the Time Range set in the input box shown on the
left. Number Cycles button: Display of Intensity-Time diagrams is
scaled depending on the Number Cycle set in the input box shown on
the left.
Show Image button: Shows the scan image in the Image Display
window to the side of the intensity diagram. This button is active only if
the Live Image option is activated. Copy Table button: The table of
intensity values is copied to the clipboard. Show Table button: The
table of intensity values is displayed at the bottom left of the Image
Display window. Save Table button: The table of intensity values can
be stored as a text file.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.7
LSM 5 LIVE
LSM 5 LIVE DuoScan
Stripe bleach function (LSM 5 LIVE without LSM DuoScan)
Bleaching of selectable rectangular image regions with the LSM 5 LIVE
Stripe Bleach is activated by the EditBleach button in the main menu. The Bleach Control window
opens and can be used in the same way as in any other Zeiss LSM 5, e.g. the LSM 510. The only
difference is that the bleach region has to be rectangular.
Fig. 4-91
EditBleach Button
Hence the Define Region button only allows to select the upper and lower border of a bleach region. It
is possible to select more than one bleach region.
Bleach Regions are drawn directly in the image window. The bleach wavelength is choosen in the
Excitation (laser) wavelength menu on the bottom of the bleach control dialogue. Check the required
line and set the power with the sliders. A single bleach event can be started interactively by pressing the
Bleach button.
Fig. 4-92
4-108
Stripe Bleach Function
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Fig. 4-93
OPERATION
Acquire Menu
Carl Zeiss
Image Window
Bleach series
To set up a time series with a bleach event, the bleach region is selected as described above. In the Time
Series window, a time series is set up.
Fig. 4-94
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Time Series Button
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
In the upper part of the Bleach Control window,
the time parameters can be set.
Check the required boxes and type in the number
of the images where the bleach event is required.
The number has to be lower than the overall
number of images choosen in the time series
dialogue. Start the bleach series with StartB (!) in
the Time Series window.
Fig. 4-95
Settings for Bleach Series
4.5.8
Edit Bleach (LSM 5 LIVE DuoScan)
The use of this function permits the setting of
bleaching parameters for spot, line or frame
bleaching.
4.5.8.1
Open / Close the Edit Bleach
Window
• Click on the Edit Bleach button in the Acquire
subordinate toolbar of the Main menu. The
Bleach Control[Help190] window appears on the
screen.
• Click on the Close button to close the Bleach
Control window.
The following functions are available on the righthand side of the Bleach Control window:
Fig. 4-96
4-110
Close button
The Bleach Control window
is closed.
Bleach button
Starts the bleaching
procedure.
Stop button
Ends the bleaching
procedure.
Bleach Control Window
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4.5.8.2
OPERATION
Acquire Menu
Carl Zeiss
Settings Panel
The Settings[Help191] panel allows you to determine
when, where and how the bleaching process shall
be done (only works in connection with time
series).
Furthermore, all the settings of the Bleach
Control window can be stored, reactivated or
deleted in this panel.
a
Fig. 4-97
Settings panel
Bleach after
number scans:
If this check box is ticked , the bleaching procedure is automatically performed
in combination with a time series. Under Scan Number, you must enter after
how many scanning procedures bleaching is to be performed.
Scan Number:
Number of Scans in a time series, after performance of which the bleaching
procedure shall be started.
Bleach repeat after
number scans:
If this check box is ticked , the bleaching procedure is automatically performed
in combination with a time series. Under Scan Number, you must enter after
how many scanning procedures bleaching is to be repeated.
Scan Number:
Number of Scans in a time series, after performance of which the bleaching
procedure shall be repeated.
Different Z
Position:
If this check box is ticked , you can set the current stage position as the one in
which the bleaching will be done by clicking the Mark Position Z button. This
function is only available using the Line or Frame scanning mode.
Different Scan
Speed
If this check box is ticked , the speed for scanning during the bleach process
can be defined independently of the scan speed during image acquisition. A
lower speed results in a longer pixel dwell time which increases the efficiency of
bleaching.
Different XY Spot
Bleach Position:
If this check box is ticked , you can set a different XY position for spot
bleaching. This function is only available using the Spot scanning mode. Click on
the Spot Select button in the Scan Control window. A new image is produced
and two crosshairs appear in the image. The red crosshair marks the spot that
will be imaged. The green crosshair marks the spot that will be bleached. Move
the center of the crosshairs to the desired positions and perform bleaching.
Store settings of the Bleach Control
Proceed as follows to store the entire settings of the Bleach Control window:
• Enter a name in the Settings list box and click on Store to store the settings.
If required, stored settings for the bleaching procedure can be reactivated quickly.
• Open the Settings list box with a click on the arrow button and select the required name with a click
of the mouse.
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OPERATION
Acquire Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• Then click on the Apply button to activate these settings. The Bleach Control window will be
updated accordingly.
Delete settings of the Bleach Control
Settings which are no longer required can be deleted.
• Open the Settings list box and select the required name.
• Click on the Delete button. This stored setting will be removed.
The bleaching procedure can also be activated via keys 1 to 4 of the Trigger Control.
• Open the Trigger in list box with a click on the arrow button.
• Select one of the trigger keys 1 to 4 (e.g. Trigger4).
It is also possible to trigger an out signal via trigger control.
4.5.8.3
Fig. 4-98
Bleach Parameter panel
Bleach Parameter Panel
The Bleach Parameter[Help193] panel allows you to
determine how often the bleaching process shall
be performed, and to select the area for bleaching
in the scan image via the Define Region button.
• Enter the number of iterations of the bleaching
procedure in the Iterations input box.
• Click on the Define Region button.
− The
Bleach
appears.
Regions[Help194]
window
The definition of bleach regions corresponds to the
Edit ROI function and is performed in the same
way (see Edit ROI, page 4-90).
ROIs already defined with Edit ROI are also
available in the Bleach Regions window. They can
be activated directly, modified - if required - and
stored under a new name.
ROIs newly defined in the Bleach
Regions window will then also be available in the
Edit ROI window.
Fig. 4-99
4-112
Bleach Regions window
• Define the required bleach regions in the scan
image or use an existing ROI.
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4.5.8.4
OPERATION
Acquire Menu
Carl Zeiss
Excitation of Bleach Track Panel
In the Excitation of Bleach Track panel you can
select the lasers and laser intensities for bleaching.
The setting of the lasers for the bleaching procedure corresponds to that for the scanning procedure and must be performed accordingly (see
Laser Control, Configuration Control and Scan
Control).
• Select the required laser wavelength and its
intensity under Excitation.
• If required, switch the relevant laser to On
(Laser button).
• If more than one ROI has been defined under
Bleach Parameters, a different laser intensity
and laser line can be defined for each region.
Toggle between the buttons indicating the
number of the ROI and check the laser line and
intensity for each ROI.
4.5.8.5
Fig. 4-100
Excitation of Bleach Track panel
Start / End a Bleaching Procedure
• The bleaching process will be started via the Bleach button. However, it is also possible to start the
bleaching process via the Bleach button in the Time Series Control[Help201] window or to combine it
with a time series.
When a trigger key is activated to start the bleaching procedure, the Waiting for Trigger message first
appears in the status line of the Bleach Control window. In that case, the bleaching procedure is started
after activation of the relevant trigger key.
• The bleaching process can be finished via Stop[Help202] in the Bleach Control window.
Stop does not only stop the bleaching process, but the entire scanning process.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.9
LSM 5 LIVE
LSM 5 LIVE DuoScan
Stage
The following software description applies to
systems which are equipped with a motorized
stage.
This window enables you to activate both the
motor focus and the scanning stage.
The Focus Position and Stage Position panels
include the function keys for the performance of
defined moves and the display of the current Z and
X, Y positions.
By use of an LSM 5 LIVE scanhead on an
Axiovert 200 M sideport system care should be
taken when moving the motorized XY scanning
stage to the maximum positions, so that fingers
are not bruised between scan head and stage.
4.5.9.1
Open / Close the Stage and Focus
Control Window
• Click on the Stage button in the Acquire
subordinate toolbar of the Main menu.
Fig. 4-101
Stage and Focus Control window
− The Stage and Focus Control window
appears on the screen.
• Click on the Close button in the Stage and
Focus Control window to close this window.
The following functions are available on the right-hand side of the Stage and Focus Control window:
Close button
The Stage and Focus Control window is closed.
Start button
Starts the tile scanning procedure.
Stop button
Ends the scanning procedure.
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4.5.9.2
OPERATION
Acquire Menu
Carl Zeiss
Focus Position Panel
Focus buttons (Z Moves)
Clicking on the ▲ button moves the specimen
stage / nosepiece upwards.
Clicking on the Z button sets the current Z-position
to zero.
Clicking on the ▼ button moves the specimen
stage / nosepiece downwards.
Fig. 4-102
Focus Position panel
Focus Step slider
0.1 µm is the smallest value which can be set, and 100 µm the highest.
• Clicking on the arrow keys changes the step size by 1 µm.
• Pressing the CTRL key and clicking changes the step size by 0.05 µm.
• Pressing the Shift key and clicking changes the step size by 10 µm.
Work button
Pressing the Work button moves the specimen stage / nosepiece back to the Work position. This is the
position last set before the Load button was pressed.
Load button
Clicking on the Load button lowers the specimen stage / nosepiece to make it easier for you to change
the specimen (or objective).
Focus Wheel check box
Clicking on this check box activates / deactivates the focus wheel of the microscope.
Use of the optional piezo objective focusing device
The HRZ Step slider is used to set the step width of the fine focusing device.
• Use the arrows ▲ and ▼ of HRZ to move the fine focusing device upwards or downwards in steps.
As soon as the focus position is changed (via handwheel or software), the piezo objective focus is
automatically leveled.
• A click on the L button moves the piezo objective focus in the center position of its travel range and
the focus position is reset accordingly. Therefore, the same Z-level remains visible (the current position
is not set to zero).
The motor focus of the stand is operated in the same way via the relevant buttons. Moving into the
Work or Load position is always performed via the motor focus and not via the piezo objective focus.
Please see CHAPTER ANNEX of the printed manual for further information on the piezo
objective focus.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.9.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Stage Position Panel
The Stage Position panel shows a symbolic
specimen carrier in the left upper.
The buttons for moving to a position and mark it
are below or on its right.
The Current Position display for X and Y is below.
Below that, you will find the Marks selection box
of marked positions and the possibility to activate
and delete them.
Moving the scanning stage
Fig. 4-103
Stage Position panel
The scanning stage can be moved using the
joystick, or software-controlled using the Stage
XY buttons, or manually.
Stage XY buttons
• Clicking on the arrow buttons moves the stage in X or Y direction.
• Clicking on the Center button moves the stage in the XY = 0 position.
XY Step slider
1 μm is the smallest value which can be set for XY movement, and 100 μm the highest.
Manual check box
This check box activates / deactivates the motor control of the stage and the joystick, if available.
If Manual is active, the scanning stage can be moved manually via the knurled screws. The Move To and
Center function buttons in Stage Position are without a function. The Current Position is updated.
You can zero the display via ZERO and mark manually set positions (Mark pos.).
The scanning stage cannot be moved via the software or the joystick.
If Manual is deactivated, the scanning stage can be moved via the software or the joystick. All the
functions of the Stage Position window are available.
Current Pos(ition) field
Current Pos displays the currently set stage position in relation to the zero position.
Marks selection box
Clicking on the arrow button displays the table of the session-related marked specimen areas. The table
includes the ordinal number, the X-position and the Y-position. Click on the appropriate mark to select it
for operation.
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OPERATION
Acquire Menu
Carl Zeiss
Move To button
Clicking on the Move To button moves the stage to the position selected before from the Marks
selection box.
Remove
The Remove command enables a selected position to be deleted from the table. The position then also
disappears from the specimen carrier display.
The selected position is deleted, the position with the next number in sequence moves up one
number.
Remove All
The Remove All command deletes all the entries marked in the current session.
Speed selection box
Clicking on the arrow key displays the table of the available speeds for stage movement. Click on the
appropriate speed to select it for operation.
Zero button
Zeros the Current Position display and thus sets the currently set stage position to 0 in relation to X and
Y. The already marked object areas thus receive new X and Y-coordinates.
Mark Pos. button
Mark Pos. allows the Current Position to be marked. This marked position is then stored in the Marks
selection box in sequence. The marked position is shown on the specimen carrier with a cross and its
ordinal number.
HRZ Zero button
Zeros the Current Position display and thus sets the currently set stage position to 0 in relation to X and
Y. The already marked object areas thus receive new X and Y-coordinates.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.9.4
Fig. 4-104
LSM 5 LIVE
LSM 5 LIVE DuoScan
Tile Scan Panel
This function permits a frame to be created as an
overview image of the specimen with a maximum
size of 4096 x 4096 pixels. According to settings,
such a frame is divided in XY-tiles of 1 x 1 to the
maximum of 15 x 15. A tile of special interest
(target) can then be selected for scanning.
Tile Scan window
The application of the Tile Scan function requires
an objective with a minimum magnification factor
of 2.5x.
Tiles Numbers X / Y input box
Input of the number of tiles for X or Y from which the frame is to be composed.
Tile Size X / Y display
Display of the size of a single tile in µm (corresponds to the value selected in the Scan Control window).
Frame Size X / Y display
Display of the frame size of the tile scan for X or Y. Specification in pixels and µm.
Move To button
If the Move To button is activated, a rectangle with a target allowing the selection of the region of
interest is positioned in the center of the scanned frame. Click and hold down the left mouse button to
drag the rectangle to the required specimen area. When you release the mouse button, the stage moves
to the selected position.
Mark button
If the Mark button is activated, marks previously set in the Tile Scan image are displayed, and further
marks can be added at spots of special interest by a mouse click in the Tile Scan image. By activating the
Move To button, the stage can be moved to the individual marks set in Tile Scan in the same way as it is
moved to the marks set in the Stage Position panel.
Creating an overview image
• Set the number of tiles for the frame in the Tiles Numbers X / Y input boxes of the Tile Scan
window.
− The resulting frame size is displayed on-line.
• Click on Start.
− The overview frame is scanned and displayed on the screen in a new Image Display window.
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Fig. 4-105
OPERATION
Acquire Menu
Carl Zeiss
Image Display window of a Tile Scan
• Activate the Move To button.
• In the tile scan image, move the target to the required spot of the frame (dragging with the mouse).
− The microscope stage then travels to the selected position.
Or:
• Activate the Mark button.
• Set a mark at the spot of interest by clicking with the mouse in the Tile scan image. A cross with the
consecutive number of the mark is displayed in the Tile Scan image. The new mark is also displayed in
the specimen carrier (Stage Position panel) and included in the Marks selection box.
• Select the mark in the Marks selection box and click on the Move To button in the Stage Position
panel. The stage moves to the selected position.
• Then click on the Single button in the Scan Control window to scan the selected area as a single
image.
− The single image is scanned and displayed in a new Image Display window.
Overlay functions cannot be activated in the Tile Scan Image Display window.
The created overview frame can then be stored like any other scan image. If a stored overview
frame is opened again, the rectangle with target will appear again. However, it can be deleted
using the Overlay function.
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OPERATION
Acquire Menu
Carl Zeiss
4.5.10
LSM 5 LIVE
LSM 5 LIVE DuoScan
VIS, TV and LSM Buttons
The VIS, TV and LSM buttons are included in the Acquire subordinate toolbar of the Main menu.
They switch the beam path and indicate which beam path has been set in the binocular tube of the
microscope:
− VIS:
observation via the eyepieces of the binocular tube, lasers are off
− TV:
camera observation (if connected) via camera adapter of the binocular tube
− LSM:
screen observation via laser excitation using the LSM 5 LIVE and software evaluation
If the beam path of the microscope is changed manually via buttons on the tube this is recorded
by the software and the relevant button is activated automatically.
Vice versa, the beam path can be "switched" via activation of the appropriate button in the
software.
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4.6
OPERATION
Process Menu
Carl Zeiss
Process Menu
• In the Main menu toolbar, click on Process.
− This opens another, subordinate toolbar in the Main menu.
Fig. 4-106
Process menu
The functions of the Process menu permit already stored scan images to be subsequently linked and
processed using mathematical functions and algorithms.
4.6.1
Add
The Add function links two channels each of one
or two images into a new channel through
addition. The channel created in this way can be
stored via the Save As function.
4.6.1.1
Open / Close the Add Window
• Click on the Add button in the Process
subordinate toolbar of the Main menu.
− This opens the Add window.
• Click on the Close button to quit the Add
window.
Fig. 4-107
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Add window
4-121
OPERATION
Process Menu
Carl Zeiss
4.6.1.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Source Panel
In the Source 1 panel, the first image source for
the addition process is determined. The current
image is displayed in the display box of the image
selection box.
Fig. 4-108
Source 1 panel
Proceed as follows to select an image via the
image selection box:
• Click on the arrow button. The image selection box is opened and all the currently loaded images are
displayed in a minimized form.
• Click on the required image. This image will then appear in the display box of the image selection box
and has been selected as Source 1.
Use the Click into window button to directly select the opened image:
• Click on the Click into window button first and then double-click on the relevant Image Display
window. The selected image will then be displayed in the display box of the image selection box and
has been activated as Source 1.
The channel which is to be used for the Add operation is selected via the Channel selection box:
• Click on the arrow button. The Channel selection box is opened and shows all the recorded channels
of the relevant image.
• Click on the required channel to activate it.
In the Source 2 panel, the second image source for the addition process is determined. The procedure is
identical to that for Source 1.
• Select the image for Source 2 and the relevant channel.
4.6.1.3
Destination Panel
In the Destination panel, it is determined in which
Image Display window the Add operation is
performed, and the data format which the newly
created image shall have.
Fig. 4-109
Destination panel
The Add operation can be performed in an already
opened window or in a new Image Display
window.
• Click on the arrow button of the image selection box to open this box.
• Click on the relevant image if the Add operation shall be performed in an existing Image Display
window.
or
• Click on New Image 8 bit or New Image 12 bit to use a new Image Display window.
You can also use the Click into window button for image selection.
Clicking on the New 8 bit or New 12 Bit button enables you to determine directly and quickly
whether the new image is to be created in the 8-bit or 12-bit format.
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OPERATION
Process Menu
Carl Zeiss
If an existing Image Display window is used to perform the Add function, you must determine whether
an existing channel shall be overwritten with the Add operation or whether a new channel shall be
added.
• In the Channel selection box, click on the channel which shall be overwritten, or click on New for a
new channel.
4.6.1.4
Add Panel
In the Add panel, the currently set formula for the
Add operation is displayed. The editable input
boxes permit the formula to be changed with any
numeric values.
Fig. 4-110
Add panel
• Click in the required input box and enter the relevant value.
• Click on the Apply button to perform the operation in the activated window or a new Image Display
window.
• The new image can then be stored via the Save As function.
4.6.1.5
Preview Panel
The Preview function enables you to preview the
result of the defined Add operation in a preview
window.
Fig. 4-111
Preview panel
Preview check box with a click
• Activate the
of the mouse. The Add - Preview Image
Display window is displayed with the operation
result.
• Deactivate the Preview check box to close the Add - Preview Image Display window.
After a change of the formula in the Add panel, click in the Add - Preview Image Display
window for an update.
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OPERATION
Process Menu
Carl Zeiss
4.6.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Subtract
The Subtract function links two channels each of
one or two images into a new channel by
subtraction. The channel created in this way can be
stored via the Save As function.
4.6.2.1
Open / Close the Subtract Window
• Click on the Subtract button in the Process
subordinate toolbar of the Main menu.
− This opens the Subtract window.
• Click on the Close button to quit the Subtract
window.
4.6.2.2
Fig. 4-112
4-124
Subtract window
Performance of the Subtract
Function
This function is performed in the same way as the
Add function (see Add, page 4-121). The only
difference is that the mathematical formula is
based on subtraction.
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4.6.3
OPERATION
Process Menu
Carl Zeiss
Multiply
The Multiply function permits two channels each
to be linked into a new channel by multiplication.
The channel created in this way can be stored via
the Save As function.
4.6.3.1
Open / Close the Multiply Window
• Click on the Multiply button in the Process
subordinate toolbar of the Main menu.
− This opens the Multiply window.
• Click on the Close button to quit the Multiply
window.
4.6.3.2
Performance of the Multiply
Function
This function is performed in the same way as the
Add function (see Add, page 4-121). The only
difference is that the mathematical formula is
based on multiplication.
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Multiply window
4-125
OPERATION
Process Menu
Carl Zeiss
4.6.4
LSM 5 LIVE
LSM 5 LIVE DuoScan
Ratio
The Ratio function permits to create a new image
or image series by offsetting two images or image
series against each other. The channel created in
this way can be stored via the Save As function.
4.6.4.1
Open / Close the Ratio Window
• Click on the Ratio button in the Process
subordinate toolbar of the Main menu.
− This opens the Ratio window.
• Click on the Close button to quit the Ratio
window.
4.6.4.2
Performance of the Ratio Function
This function is performed in the same way as the
Add function (see Add, page 4-121).
However, self edited formulas can be used for
image calculation.
Fig. 4-114
4-126
Image Calculation window
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4.6.4.3
OPERATION
Process Menu
Carl Zeiss
Formula Panel
The formulas can be typed in using the keyboard.
• Click the Operators button to open the
permissible operators list.
• Select the operator(s) from this list (Fig. 4-115)
and click the Insert button.
The operators will be inserted into the formula
at the cursor position. By highlighting the
selected operator a description of the function
of this operator is displayed in the lower part of
the operators list.
The calculation works pixel by pixel and starts with
the upper left pixel irregardless of the image size of
the two images or image series.
When choosing images of different data
depth the check box next to Intensity
range 1…0 should be marked. The
image intensity for all images will then be
normalized to values between 1 and 0.
4.6.5
Fig. 4-115
Operators window
Fig. 4-116
Copy window
Copy (Channel)
The Copy function permits one channel each of an
existing image to be copied and stored as a new
image.
The selection of Source, Channel and Destination is
made in the same way as in the Add function (see
Add, page 4-121).
4.6.5.1
Open / Close the Copy Window
• Click on the Copy button in the Process
subordinate toolbar of the Main menu.
− This opens the Copy window.
• Click on the Close button to quit the Copy
window.
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OPERATION
Process Menu
Carl Zeiss
4.6.5.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Performance of the Copy Function
• Select Source, Channel and Destination and then click on the Apply button.
− The image of the copied channel is then displayed in a new window or in the Image Display
window activated for it.
• The new image can be stored via the Save As function.
For Z Stacks or Time Series, the entire series of the selected channel is copied.
4.6.6
Duplication (Image)
This function permits images (including Z Stacks and Time Series) to be duplicated completely.
• If several images have been opened, select the image to be duplicated.
• Click on the Dup button in the Process subordinate toolbar of the Main menu.
− The selected image is duplicated and displayed in a new Image Display window.
• Use the Save As function to store the image under a new name.
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4.6.7
OPERATION
Process Menu
Carl Zeiss
Filter
The filter function permits the subsequent
processing of scanned images via the integrated
Lowpass, Sharpness and Median filters.
Furthermore, User-defined filters can be installed
by the user. User-defined filters can be stored,
reloaded and removed.
4.6.7.1
Open / Close the Filter Window
• Click on the Filter button in the Process
subordinate toolbar of the Main menu.
− This opens the Filter window.
• Click on the Close button to quit the Filter
window.
4.6.7.2
Image Panel
In the Image panel, the image or channel to be
processed is selected.
Fig. 4-117
Filter window
The currently selected image is displayed in the
image selection box.
Proceed as follows to select an image via the image selection box:
• Click on the arrow button. The image selection box is opened and all the currently loaded images are
displayed in a minimized form.
• Click on the required image, which will then appear in the display box of the image selection box and
will be available for filtering.
You can also use the Click into window button to select the image.
• Open the Channel selection box with a click on the arrow button and select the channel to be
processed.
4.6.7.3
Filter List and Edit Panel
In the Filter List panel, the filters and the matrix size (Kernel Size) are selected.
The matrix of the selected filter and the set filter parameters Factor, Divisor and Offset are displayed in
the Edit panel.
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OPERATION
Process Menu
Carl Zeiss
(1)
LSM 5 LIVE
LSM 5 LIVE DuoScan
Kernel size
The size of the filter matrix can be modified here.
The effect of a filter increases along with the
matrix size. However, this also increases the time
required for filtering.
• Select the required matrix size by clicking on
one of the selection buttons 3 x 3, 5 x 5 or
7 x 7.
(2)
Lowpass filter
With the lowpass filter, the gray value of each
center pixel is replaced with the average value of
the surrounding neighbor pixels. The viewed
neighbor pixels are defined by a square. The
modified pixel now is the center pixel of the filter
matrix.
Fig. 4-118
Filter List and Edit panel (Lowpass)
Image noise will be reduced by the application of
the lowpass filter. The cutoff of regions will blur.
Local maxima will be flattened. The dynamic range
will be reduced considerably.
This filter permits the matrix size to be modified
only in the 3 preset steps.
(3)
Sharpness filter
With the sharpness filter, the original image is
filtered with a lowpass filter first. The result of this
filtering is then subtracted from the original image.
This will improve image sharpness.
The matrix size can be modified in the 3 preset
steps.
Furthermore, divisor values ranging from 1 to 78
can be entered. The higher the divisor value, the
lower the image sharpness.
Fig. 4-119
4-130
Filter List and Edit panel (Sharpness)
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(4)
OPERATION
Process Menu
Carl Zeiss
Median filter
With the median filter, the gray value of each
center pixel is replaced with the median value of
the surrounding neighbor pixels. The viewed
neighbor pixels are defined by a square. The
modified pixel now is the center pixel of the filter
matrix.
The median value is defined as the middle value
(not average) of all the gray values sorted in
ascending order within a matrix.
Image noise will be reduced by the application of
the median filter. The cutoff of regions will slightly
blur. Local maxima will be flattened. The dynamic
range will be reduced considerably.
The settings of this filter can not be modified.
(5)
Fig. 4-120
Filter List and Edit panel (Median)
Fig. 4-121
Filter window (User-defined filter)
User-defined filter
The User-defined function permits you to create
your own filters. In addition to the Kernel Size,
the parameters Factor, Divisor and Offset can be
modified here.
The filter result can be subtracted from the original
image via the Subtract from Source check box.
Proceed as follows to store User-defined filters:
• Click on the Add To List button and enter a
name in the Add Filter To List window. The
name will be included in the Filter List.
Proceed as follows to activate stored, Userdefined filters:
• Click on the name of the filter in the Filter List.
The filter will then be activated immediately.
Proceed as follows to delete User-defined filters:
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OPERATION
Process Menu
Carl Zeiss
LSM 5 LIVE
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• Click on the name of the filter in the Filter List
and then on the Remove button. The filter will
be deleted.
• After selection of the required filter, click on the
Apply button to start the filter procedure.
Fig. 4-122
Add Filter to List panel
− Filtering will be performed and displayed in
the current Image Display window.
• In the case of images with several channels,
activate the xy button in the Display image
toolbar to display all the channels. Each channel
must be filtered separately.
• Use the Save As function to store the newly
created image.
4.6.7.4
Fig. 4-123
Preview panel
Preview Panel
The Preview function allows you to have the result
of the Filter operation displayed as a preview
image.
Preview check box with a click of the mouse. The Filter - Preview Image Display
• Activate the
window with the filter result will be displayed.
• Deactivate the Preview check box to close the Filter - Preview Image Display window.
After a change of the filter settings, click in the Filter - Preview Image Display window once
to update it.
4.6.8
Contrast
The Contrast function permits the subsequent modification of contrast and brightness of the stored
image.
• Open the image to be processed and click on the Contrast button.
− The function is performed with firmly set parameters and the result is displayed in a new Image
Display window. The procedure can be repeated as often as required.
• The newly created image can be stored using the Save As function.
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4.6.9
OPERATION
Process Menu
Carl Zeiss
Interpolate
This function permits the continuous contrast and
brightness change in a Z- stack or Z- stacks over
time through interpolation between the starting
and end values. This permits the subsequent
compensation of signal loss when imaging further
into deeper tissue regions where the excitation
efficiency decreases but also the detection
efficiency. Interpolation can be defined for the
entire image or only for individual channels.
The Modify Series macro can be used to
convert time series data for use of the
Interpolate function.
4.6.9.1
Open / Close the Interpolate
Brightness and Contrast Window
• Click on the Interpolate button in the Process
subordinate toolbar of the Main menu.
− This opens the Interpolate Brightness and
Contrast window.
Fig. 4-124
Interpolate Brightness and Contrast
window
Fig. 4-125
Image panel
• Click on the Close button to quit the window.
4.6.9.2
Image Panel
The image to be processed is selected in the
Image panel.
The currently selected image is shown in the
display box of the image selection box.
Proceed as follows to select a series via the image
selection box:
• Click on the arrow button. The image selection box will be opened and all the currently loaded images
will be displayed in a minimized form.
• Click on the required image, which will then appear in the display box of the image selection box and
has been selected for the interpolation procedure.
You can also use the Click into window button for image selection.
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OPERATION
Process Menu
Carl Zeiss
4.6.9.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Interpolation Panel
In the Interpolation panel, the parameters for the
interpolation procedure are set.
• Use the Start Image slider to select the slice at
which the interpolation procedure shall start.
Clicking on the First button permits the fast
selection of the first slice in the series.
• Use the Brightness and Contrast sliders to set
the image brightness and contrast for the first
slice (Start Image).
Fig. 4-126
Interpolation panel
• Use the End Image slider to select the slice at
which the interpolation procedure shall end.
Clicking on the Last button permits the fast
selection of the last slice in a series.
• Use the Brightness and Contrast sliders to set
the image brightness and contrast for the last
slice (End Image).
• Use the available Channel buttons (e.g.: Ch1) to select the channel for interpolation or click on the All
button if the entire image is to be interpolated.
• Having set the parameters, click on the Apply button. Interpolation will be performed in a new Image
Display window.
• The newly created image (series) can be stored using the Save As function.
If you activate the Overwrite Source Images check box, interpolation will be performed in the
current Image Display window.
If you activate the Ignore Images less than "Start Image" and greater than "End Image"
check box, only the slices lying between Start Image and End Image will be taken into
consideration for interpolation. Otherwise, brightness and contrast will also be changed for the
other slices.
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4.6.9.4
OPERATION
Process Menu
Carl Zeiss
Preview Panel
The Preview function enables you to see the result
of interpolation for one slice each in a preview
window.
Fig. 4-127
• Activate the
Preview panel
Preview check box with a click of the mouse.
− The Interpolate C&B - Preview Image Display window will be displayed. At the same time, the
Slice slider with the relevant input box and arrow keys and the two buttons Start and End are
displayed in the Preview panel.
• Use the slider or input box / arrow keys to set the slice which shall be displayed in the preview
window.
• Clicking on the Start or End button permits the fast activation of the Start Image or End Image for
previewing.
• Deactivate the Preview check box to close the Interpolate C&B - Preview Image Display window.
4.6.10
Channel Shift
The Channel Shift function is used to produce a
congruent image with relation to the pixels of the
various channels.
This pixel correction function
important in UV applications.
4.6.10.1
is
particularly
Open / Close the Channel Shift
Window
• Click on the Shift button in the Process
subordinate toolbar of the Main menu.
Fig. 4-128
− This opens the Channel Shift window.
Channel Shift window
• Click on the Close button to quit the window.
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OPERATION
Process Menu
Carl Zeiss
4.6.10.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Image Panel
• Click on the arrow button. The image selection
box will be opened and all the currently loaded
images are displayed in a minimized form.
Fig. 4-129
Image panel
• Click on the required image, which will then
appear in the display box of the image selection
box and has been selected for the Shift
function.
You can also use the Click into window
button for image selection.
4.6.10.3
Shift Panel
• Select the channels required for processing in
the Shift panel by clicking on the Ch1 or Ch3
will appear in the button
buttons. A tick
when the channels are activated.
Fig. 4-130
Shift panel
and
buttons to
• Use the scrollbar or the
select the pixel shift in the horizontal and
vertical direction.
• Click on the Apply button to activate the
setting.
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4.6.10.4
OPERATION
Process Menu
Carl Zeiss
Preview Panel
• If Preview is activated, a preview of the shift
is shown in a separate Image Display window.
Fig. 4-131
Preview panel
The following image shows the result of a pixel shift via the Shift function. This image change can be
stored in the image database via the Save or Save As buttons.
For applications requiring 3- or 4-channel scanning, proceed in the same way as described for the 1- or 2channel mode.
Fig. 4-132
4.6.11
Image Display window with channel shift
Unmix
The Unmix functionality permits to extract the emission of single fluorescence dyes (e.g. GFP only, YFP
only etc.) from the overall emission band of strongly overlapping multifluorescence signal intensities by a
pixelwise linear unmixing procedure.
Mathematically, experimental fluorescence spectra of monolabelled samples are taken as an external
reference. Up to 8 different reference signals can be varied in this least-square-fit based algorithm to
produce an 8 channel multifluorescence stack without any partial overlap between the channels.
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OPERATION
Process Menu
Carl Zeiss
4.6.11.1
LSM 5 LIVE
LSM 5 LIVE DuoScan
Open / Close the Unmix Window
• Click on the Unmix button in the Process subordinate toolbar of the Main menu.
− This opens the Linear Unmixing window.
• Click on the Close button to quit the Unmix window.
4.6.11.2
Source Panel
In the Source panel the image source for the linear unmixing process has to be defined.
This has to be a Stack, a Stack Z series or a Stack T series.
Proceed as follows to select an image via the image selection box:
• Click on the arrow button. The image selection box is opened and all the currently loaded images,
stacks, time series with a Lambda dimension are displayed in a minimized form.
• Click on the required image. This image will then appear in the display box of the image selection box
and has been selected.
4.6.11.3
Definition of Channels Panel
In the Channels panel the number of reference spectra (number of fluorescence channels) can be
selected from the channel selection boxes.
• Select the references fluorescence dye spectra which are present in the sample with the check boxes.
• If necessary change the colors of the relevant fluorescence channel.
• A new window with the resulting channels of the unmixing procedure opens immediately.
There are several settings that can be chosen for linear unmixing.
Autoscale balances the intensity of the unmixed images.
Generate channels with Residuals shows the difference between the offered spectrum and the
resulting image in intensity values. The higher the intensity in this additional channel the worse the fit of
the spectra to the dataset. This occurs for example when pixels are saturated and indicates to repeat the
image acquisition with different settings to avoid overexposure or in extreme cases to chose different
reference spectra.
Ask for Background ROI, when this is checked a window appears before the calculation starts which
asks for the spectrum of the background which is then subtracted from the images prior to unmixing.
Advanced Linear Unmixing restarts the unmixing process again if negative values are generated. The
negative values are set to zero and ignored for the next unmixing calculation.
Ignore Overexposed Pixels and Ignore Underexposed Pixels will exclude this pixels for calculation of
the unmixed images.
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OPERATION
Process Menu
Carl Zeiss
Multichannel Unmixing allows to apply the
unmixing algorithm to a multichannel image (up to
8 channels) without the use of reference spectra.
The calculation of residuals and the subtraction of
background based on a background spectrum are
not available when this procedure is chosen. It will
not work for heavily overlapping signals.
Try to avoid saturation of fluorescence
signals in the stack to be unmixed. This
will generate a high signal in the residuals
channel.
To get the highest quality unmixing
results, please define an extra background
channel, if possible.
Fig. 4-133
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4-139
OPERATION
Process Menu
Carl Zeiss
Fig. 4-134
4-140
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Image Display window after unmixing
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4.6.12
OPERATION
Process Menu
Carl Zeiss
Ion Concentration
The use of this function (option)
permits the calibration of ion
concentrations in physiological
experiments.
4.6.12.1
Open / Close the Ion Concentration
Window
• Click on the Ion Conc button in the Process
subordinate toolbar of the main menu.
− This opens the Ion Concentration window.
• Click on the Close button.
Fig. 4-135
4.6.12.2
Ion Concentration window
Function Description
Ion Conc button
Activates the Ion Concentration window.
Source window
Selects input of images to be processed.
Destination window
Select output and pixel depth of processed image.
Calibration window
Sets the six different calibration options, according to the dyes used (single
wavelength, ratiometric) and required method.
Show Curve button
Shows resulting calibration curve.
Image scaling window
Sets min. and max. concentration.
Preview window
Activates Preview function.
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OPERATION
Process Menu
Carl Zeiss
4.6.12.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Single Wavelength Dyes – Offline Calibration
• Subtract background/autofluorescence image from raw images to obtain.
• Perform equation- or titration calibration (compare F with a calibration curve → titration calibration or
put F values in calibration formula).
Fig. 4-136
4-142
Ion Concentration window
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4.6.12.4
OPERATION
Process Menu
Carl Zeiss
Ratiometric Dyes
• Fura-2, Indo-, SNARF, Cameleon, Ratiometric Pericam, Phluorin, ...
• Display fluorescence ratio R over time
• Display fluorescence ratio R corrected for background/autofluorescence over time
• Calculate absolute ion concentrations (pixel by pixel) via titration calibration (known ion concentrations
applied to the cells – in situ – or in solutions – in vitro or equation calibration where possible [Fura-2,
Indo-, SNARF]
• Calculation of R eliminates artifacts and uncertainties caused by
− inhomogenous dye distribution
− photobleaching
− may be applied with moving cells
4.6.12.5
Ratiometric Dyes - Online Ratio
R(t1) = F1(t1) / F2(t1), R(t2) = F1(t2) / F2(t2) ...
Fig. 4-137
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OPERATION
Process Menu
Carl Zeiss
4.6.12.6
LSM 5 LIVE
LSM 5 LIVE DuoScan
Ratiometric Dyes - Calibration
• Subtract background/autofluorescence images from raw images to obtain
Rkorr [=F1-F1Background)/(F2-F2Background)]
when calibration reference is not obtained with the experimental sample (in situ)
• Calculate ratio R
• Perform equation- or titration calibration (compare R with a calibration curve -> titration calibration or
put R values in calibration formula)
Fig. 4-138
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4.6.12.7
OPERATION
Process Menu
Carl Zeiss
Ratiometric Dyes - Equation
Calibration (Grynkiewicz)
Fura-2, Indo-1,..
KD (dissociation constant) taken from literature
Rmin: derived from ion-free state of the dye (e.g. 0
Ca2+)
Rmax: derived from ion-bound state of the dye
(e.g. saturated with Ca2+)
Fmin2 and Fmax2 are the minimum and maximum
fluorescence intensities at wavelength 2
Fig. 4-139
Ion Concentration button
Rmin, Rmax, Fmin2 and Fmax2 may be determined in the cells under investigation (in situ) or in solutions (in
vitro)
Calibration parameters may be saved and reloaded (*.cal)
4.6.12.8
Options for Calibration Image Selection (Equation- or Titration Calibration)
• Click into image window.
• Select source channel(s).
• Optional background subtraction
• Optional calculation of parameters from overlay region(s)
4.6.13
Image Scaling
The use of this function permits the scaling of a imported image in voxel dimensions. Setting the values
will provide the voxel dimensions in the X-,Y- and Z-directions ( Z-direction not for 2D images).
4.6.14
Copy Window Contents
The use of this function permits to duplicate the displayed image.
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OPERATION
Process Menu
Carl Zeiss
4.6.15
LSM 5 LIVE
LSM 5 LIVE DuoScan
Copy Full Resolution
The use of this function permits to display the current image with full resolution in a new Image Display
window.
4.6.16
Paste Bitmap
This function allows to paste bitmap images from the clipboard into a Image Display window.
Images can be incorporated from other programs such as MS Excel, MS Powerpoint or MS Word.
4.6.17
Kinetic
The use of this function (option) permits
− a correction of FRAP data for bleaching and background
− the fitting of the FRAP data to a mono exponential or double exponential model for intensity
recovery.
4.6.17.1
Open / Close the Kinetic Analysis window
• Click on the Kinetic button in the Process subordinate toolbar of the main menu.
− The Kinetic Analysis window opens.
• Click on the Close button.
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4.6.17.2
OPERATION
Process Menu
Carl Zeiss
Function Description
Click into window
Allows to select the image series of the FRAP experiment to be analyzed by
clicking into the appropriate image window.
Channel
pull down menu
Allows to select single channels or all channels for analysis.
Kinetic Model
pull down menu
Allows to select the mathematical model (mono or double exponential model) for
fitting the data.
Background Region Displays a button Select (Click) that allows to chose the region of interest which
checkbox
represents the mean background intensity that should be used to correct the
data. First Click Select (Click), than click into the ROI that has been drawn using
the drawing tools in the Mean ROI image display. If a region has been selected a
cross appears next to the button.
Reference Region
checkbox
Displays a button Select (Click) that allows to chose the region of interest which
represents the fluorescence intensity of a reference cell which has not been
bleached. The mean intensity within that region is used to correct the data at
each time point for any bleaching artifact that occurred during the imaging
process. First Click Select (Click), than click into the ROI that has been drawn
using the drawing tools in the Mean ROI image display. If a region has been
selected a cross appears next to the button.
Combine Regions
checkbox
Opens a further dialogue with the following options: Add Region, New Group,
Remove Group and Remove All. It provides the possibility to chose more than
one ROI for analysis and also to group them together according to the
experimental set up. First click Add Region, than click into the ROI that has been
drawn using the drawing tools in the Mean ROI image display. For each added
ROI a cross is displayed in the line. Click Add Region for each ROI to be added.
Clicking New Group puts the cursor in the next line and further ROIs or Group of
ROIs can be added for analysis.
Apply
Performs the calculations and the fitting of the data.
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OPERATION
Process Menu
Carl Zeiss
4.6.17.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Example: FRAP Performed in a Nucleus Expressing GFP Labeled Proteins
Display of the image series in the Mean ROI display mode: The drawing tools are used to define the ROI
to be analyzed (ROI 1), the background ROI (ROI 2), and the reference ROI (ROI 3). The reference ROI
must be a neighboring cell which has been imaged with the same laser intensity over time identical to the
cell, which has been bleached to induce FRAP. Make sure the whole cell or cell compartment of interest is
imaged and therefore illuminated.
Use the Slice submenu in the Select toolbar of the image window to display the image right
after the bleach event. This makes it easy to chose the ROI for analysis. The region should be
slightly smaller than the original region that has been bleached. The latter is listed in the Mean
of ROIs list in the image window.
In the ROI Mean Display three ROIs are defined. See Additional Display Mode in Time Series, page
4-312 for further details.
Fig. 4-140
Image window displaying the first bleached image of a images series
• Open the Kinetic dialogue and chose the image series for analysis.
• Mark the checkboxes for background and reference region.
• Press Select (Click) for the Background Region, than click into the ROI number 2 (Background region)
in the image. The value of the mean intensity of this region will be subtracted from the intensity values
within the ROI to be analyzed.
• Press Select (Click) for the Reference Region. The intensity values of the ROI to be analyzed will be
corrected for changes in intensity calculated for the reference region. The correction is done for each
time point taking the actual intensity difference in the reference region into account.
• If no other option is chosen the remaining ROI or ROIs are used for analysis. Each ROI is analyzed
separately.
• Chose the Kinetic Model in the pull down list, then press Apply.
− The result of the fit is displayed in the table. The result can be copied to the clipboard (Copy
Results) or saved as a text file (Save Results). The following values are calculated and shown:
− The final signal intensity in the analyzed ROIs following recovery I0 (of the fitted curve).
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OPERATION
Process Menu
Carl Zeiss
− The amplitude of the fitted curve (which equals the mobile fraction) I1 mobile fraction.
− The fitted parameter T1 (seconds).
− The rate constant for the exchange of molecules between the bleached region and the surrounding
area K (per second).
− The part of the immobile fraction of the protein I delta immobile fraction.
Show Table opens a further table beneath the results. It shows all intensity values being corrected for
background intensity and intensity loss of the reference region.
The values can be saved as a text file (Save Table) or copied into Excel via clipboard (Copy Table).
The data can be normalized optionally when marking the checkbox Normalize in the Kinetic Analysis
Diagram toolbar.
The calculation of the parameters is based on the same ROIs unless other ROIs or moved ROIs
are selected again. The Kinetic Display is always available once the analysis has been performed.
The analysis is not stored with the image.
Note that this modeling is a very basic approach to your experiment. It offers a first hint on the
possible presence of only one or, in case of a bad fit, more than one mobile fractions of the
labeled protein within the cell or cell compartment examined.
The half time of the recovery can be calculated using the following formula:
Fig. 4-141
t half =
ln 0.5
− T1
Image window displaying the analysis of FRAP data using a mono
exponential fit
If the analysis is done using the double exponential fit the fitted curve displays the mean of the fitted
values for the two different mobile fractions. The table shows the following additional parameters:
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OPERATION
Process Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
− The amplitude of the two curves, displayed as one (which equals each part of the mobile fractions)
I1 and I2.
− The fitted parameters T1 and T2 (seconds) for each mobile fraction.
− The rate constant for the exchange of molecules between the bleached region and the surrounding
area K1 and K2 (per second) for each mobile fraction.
Fig. 4-142
Image window displaying the analysis of FRAP data using a double
exponential fit
The raw data of the experiment can be exported for further analysis using the Mean ROI display
mode and, within this dialogue, the table display of the results.
Note that this modeling is also a very basic approach to your experiment. It offers a first hint on
the possible presence of two mobile fractions of the labeled protein within the cell or cell
compartment examined.
Please refer to relevant scientific literature or the website of the EAMNET (http://www.embl.de/eamnet)
for further information on how to set up and perform FRAP experiments. A schematic curve marking the
data points that are calculated performing the Kinetic Analysis is shown in Fig. 4-143. Please note, that
the naming of the data points is not consistent with the information on the website.
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Fig. 4-143
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OPERATION
Process Menu
Carl Zeiss
Schematic FRAP curve with marks at the relevant data points. I0, I1
and I delta which are calculated performing the Kinetic Analysis
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OPERATION
3D View Menu
Carl Zeiss
4.7
LSM 5 LIVE
LSM 5 LIVE DuoScan
3D View Menu
The 3D View functions serve to record and play back series of images for 3D display of microscopic
structures.
• In the Main menu toolbar, click on 3D View.
− This opens another, subordinate toolbar in the Main menu.
Fig. 4-144
3D View menu
4.7.1
3D DepthCod
(Color Coded Depth Map)
By means of the Depth Coding function, the
depth information contained in a sequence can be
colored with the colors of the rainbow, in which
case "blue " stands for front and "red" stands for
rear.
A stack of images must be available.
4.7.1.1
Open / Close the Depth Coding
Window
• Click on the DepthCod button in the 3D View
subordinate toolbar of the Main menu.
Fig. 4-145
Depth Coding window
− This opens the Depth Coding window.
• Click on the Close button to quit the window.
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4.7.1.2
OPERATION
3D View Menu
Carl Zeiss
Source Panel
In the Source panel, the image source is selected. The currently selected image is displayed in the display
box of the image selection box. Proceed as follows to select an image via the image selection box:
• Click on the arrow button. The image selection box will be opened and all the currently loaded images
are displayed in a minimized form.
• Click on the required image, which will then be shown in the display field of the image selection box
and be available for the following operation.
The Click into window button enables you to select the opened image directly:
• Click on the Click into window button first and then double-click on the relevant Image Display
window. The selected image will then be shown in the display box of the image selection box.
Select the channels to be processed via the Channel selection box:
• Click on the arrow button to open the selection box. Click on the required channel to activate it.
4.7.1.3
Depth Coding Panel
• On the Depth Coding panel you can set the
desired parameters. Activate the Scale Bar
if you want a color scale to be
check box
shown.
Fig. 4-146
(1)
Depth Coding tab
Depth Coding tab
Mode Front View:
The image is viewed from the front / above when this option is activated.
Mode Rear View:
The image is viewed from the rear / below when this option is activated.
Threshold:
All brightness values below the Threshold (range: 0 to 255) are ignored or
treated like 0 when determining the depth and the display.
Contrast:
Defines the factor with which the contrast of the overlaid series affects the
contrast of the depth-coded color.
Brightness:
Defines the factor with which the brightness of the overlaid series affects the
brightness of the depth-coded color.
Display Scale Bar:
Displays a colored scale in the image.
Display Grey level:
The depth information is displayed in gray levels.
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OPERATION
3D View Menu
Carl Zeiss
(2)
LSM 5 LIVE
LSM 5 LIVE DuoScan
Transparency tab
Mode Maximum:
The color is defined by the z position of the brightness value.
Mode Transparent:
The transparent projection is built up from the rear to the front. The color is
defined by the Z position at which the original was last higher than or equal to
Threshold.
Mode
Keep Maximum:
Activating this option modifies the specification governing calculation of the
projection.
Threshold:
Pixel value at which the ramp rises (variable from 0 to 100 %).
Ramp:
Slope of the ramp (variable from 0 to 100 %; 0 % corresponds to a vertical rise).
Maximum Opacity:
Degree of visibility at the top corner of the ramp (variable from 0 to 100 %;
0 corresponds to the bottom edge in the diagram).
Fig. 4-147
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4.7.1.4
OPERATION
3D View Menu
Carl Zeiss
Preview Panel
The Preview function permits you to regard the
influence of parameter changes in an Image
Display window.
• After finding the optimum adjustment using the
Preview function, you have to generate the
final version of the image using the Apply
button.
− The system then generates a color-coded
depth map for the selected channel.
4.7.2
Fig. 4-148
Depth Coding image
Fig. 4-149
Projection window
Projection
By means of the Projection function, one single
projection or a series of projections can be
calculated after rotation of the data package about
the X, Y or Z axis.
A stack of images must be available.
4.7.2.1
Open / Close the Projection
Window
• Click on the Projection button in the 3D View
subordinate toolbar of the Main menu.
− This opens the Projection window.
• Click on the Close button to quit the window.
4.7.2.2
Source Panel
• Select the image for the projection operation from the image selection box.
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OPERATION
3D View Menu
Carl Zeiss
4.7.2.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Projection Panel
• On the Projection panel, set the parameters needed for the animation: Turning Axis, First Angle,
Number Projections and Difference Angle in the Projection tab and the Mode parameters in the
Transparency tab.
(1)
Projection tab
Turning Axis X/Y/Z: Selects the axis about which the data package is to be rotated.
First Angle:
Rotation angle in degrees.
Number
Projections:
Number of projections for a sequence (variable from 0 to 100).
Difference Angle:
Angle increment of a sequence.
The number keys permit the direct selection of preset values for Number Images and
Difference Angle. If the Panorama button is pressed, a panorama sequence of the image
series is computed.
Fig. 4-150
4-156
Projection tab
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(2)
OPERATION
3D View Menu
Carl Zeiss
Transparency tab
Mode Maximum:
The color is defined by the z position of the brightness value.
Mode Transparent:
The transparent projection is built up from the rear to the front. The color is
defined by the z position at which the original was last higher than or equal to
Threshold.
Mode
Keep Maximum:
Activating this option modifies the specification governing calculation of the
projection.
Threshold:
Pixel value at which the ramp rises (variable from 0 to 100 %).
Ramp:
Slope of the ramp (variable from 0 to 100 %; 0 % corresponds to a vertical rise).
Maximum Opacity:
Degree of visibility at the top corner of the ramp (variable from 0 to 100 %; 0 %
corresponds to the bottom edge in the diagram).
Brightness:
The image can be brightened again by modifying the value (from 0.2 to 5).
Fig. 4-151
4.7.2.4
Transparency tab
Preview Panel
The Preview function permits you to regard the
influence of parameter changes in an Image
Display window.
Fig. 4-152
Preview panel
The Slice slider enables you to select the slice
which shall be displayed in the Preview Image
Display window.
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OPERATION
3D View Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• After finding the optimum adjustment using the
Preview function, you have to generate the
final version of the image using the Apply
button.
− The projection appears. The computation
can be followed in the image or by the
progress bar.
The computed 3D sequence can be
animated with the Anim button in the Select
toolbar.
In addition, the Animate window appears, in
which you can influence the direction and speed of
3D image rotation (see Animate, page 4-239).
Fig. 4-153
Projection image
You can browse through the rotation sequence
manually with the Slice button in the Select
toolbar and the Slice slider.
• To view the computed 3D sequence as a gallery on the screen, click on the Gallery button in the
Display toolbar.
Fig. 4-154
4-158
Projection image (Gallery)
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4.7.3
OPERATION
3D View Menu
Carl Zeiss
Stereo
Stereoscopic images can be generated in a variety
of ways by means of the Stereo function.
A stack of images must be available.
4.7.3.1
Open / Close the Stereo Images
Window
• Click on the Stereo button in the 3D View
subordinate toolbar of the Main menu.
− This opens the Stereo Images window.
• Click on the Close button to quit the window.
4.7.3.2
Fig. 4-155
Source Panel
Stereo Images window
• Select the image for the projection operation
from the image selection box.
• Select the channel to be used from the
Channel selection box.
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OPERATION
3D View Menu
Carl Zeiss
4.7.3.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Stereo Images Panel
• In the Stereo Images panel, set the parameters needed for stereoscopic viewing: Mode, Basic
Angle, Right Left Angle, Number Images and Difference Angle in the Projection tab and the
Mode parameters in the Transparency tab.
(1)
Projection tab
Mode
This displays a stereo image for red / green anaglyph observation using red /
Red / Green Image: green spectacles.
Mode
Split Images:
This displays a pair of stereo images for observation through a stereoscope.
Colored stereo images are also possible.
Basic Angle:
Direction angle at which the specimen is viewed; 0° from the front, 180° from
the rear.
Right Left Angle:
Angle between right and left (red and green) image.
Number Images:
Number of 3D images (slices).
Difference Angle:
Angle increment of a sequence.
Fig. 4-156
Projection tab
The number keys permit the direct selection of preset values for Number Images and
Difference Angle. If the Panorama button is pressed, a panorama sequence of the image
series is computed.
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(2)
OPERATION
3D View Menu
Carl Zeiss
Transparency tab
Mode Maximum:
The color is defined by the z position of the brightness value.
Mode Transparent:
The transparent projection is built up from the rear to the front. The color is
defined by the z position at which the original was last higher than or equal to
Threshold.
Mode
Keep Maximum:
Activating this option modifies the specification governing calculation of the
projection.
Threshold:
Pixel value at which the ramp rises (variable from 0 to 100 %).
Ramp:
Slope of the ramp (variable from 0 to 100 %; 0 % corresponds to a vertical rise).
Maximum Opacity:
Degree of visibility at the top corner of the ramp (variable from 0 to 100 %;
0 corresponds to the bottom edge in the diagram).
Brightness:
The image can be brightened again by modifying the value (from 0.2 to 5).
Fig. 4-157
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OPERATION
3D View Menu
Carl Zeiss
4.7.3.4
Fig. 4-158
LSM 5 LIVE
LSM 5 LIVE DuoScan
Preview Panel
The Preview function permits you to regard the
influence of parameter changes in an Image
Display window.
Preview panel
The Slice slider enables you to select the slice
which shall be displayed in the Preview Image
Display window.
• To start computation of the stereoscopic image,
click on the Apply button.
− The image is built up twice (once each for
the red and green colors), resulting in a
stereoscopic image.
The stereoscopic effect can only be seen
with the aid of red / green 3D goggles. The red
lens is to be used for the right eye and the green
lens for the left eye.
The presentation can be modified by selecting the
Split Images (Mode) option in the Projection tab
of the Stereo Images panel.
Fig. 4-159
Fig. 4-160
4-162
• By clicking on the Apply button, the two stereo
pairs are presented side by side and can be
viewed without red / green 3D goggles.
Stereoscopic image (red / green)
Stereoscopic image (split)
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4.7.4
OPERATION
3D View Menu
Carl Zeiss
3D DeConVolution (DCV)
The 3D Deconvolution option is used for the resolution enhancement of fluorescence image stacks.
When a three-dimensional object is reproduced by an optical system the resulting image of the object
does not correspond exactly to the object's actual form. The image of the object is "distorted" as it
passes through the optical system. In physical terms the actual object is convolved by the optical system's
Point Spread Function (PSF).
Fig. 4-161
Point Spread Function (PSF)
The Point Spread Function describes how the light of a point object is distorted by the optical system.
This "convolution" makes the image appear grainy and structures in the image seem blurred. This effect
is most prominent in the axial (Z-)direction as each lens is optimized for the two-dimensional image of the
object.
If the PSF is known it is possible to use mathematical algorithms to undo this distortion. The image of the
object is deconvolved using the PSF and the actual form is reconstructed:
Fig. 4-162
Point Spread Function (PSF)
The effect of 3D deconvolution can be demonstrated impressively on objects with a known form. As a
rule fluorescent beads are used for this purpose. The following figure shows the 3D deconvolution of an
image stack with a fluorescent bead with a diameter of 1 µm.
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OPERATION
3D View Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
As the resolution of an optical system is
significantly lower in the axial direction than in the
lateral (X/Y-)direction, the greatest improvement in
resolution can be achieved in the Z-direction.
The Z Stack must meet the following requirements:
− At least two-fold oversampling in xyz (z: half
of optimal interval button)
− High signal-to-noise ratio
− Detector gain < 500 V
Calculation is either made for one channel of the
opened image which must first be selected
accordingly, or for all channels of a stack.
Fig. 4-163
Image of a fluorescent bead with a
diameter of 1µm before deconvolution
(A,B) and after deconvolution (C,D)
Calculation is started via Apply and can be
stopped using the ESC key, if required.
4.7.4.1
Open / Close the 3D Deconvolution
Window
• Click on the DCV button in the Process
subordinate toolbar of the Main menu.
− This opens the 3D Deconvolution window.
• Click on the Close button to quit the window.
Fig. 4-164
4-164
3D Deconvolution window
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4.7.4.2
OPERATION
3D View Menu
Carl Zeiss
Source Panel
The image to be processed is selected in the
Source panel. The currently selected image is
shown in the display box of the image selection
box. Proceed as follows to select a image via the
image selection box:
Fig. 4-165
Source panel
• Click on the arrow button. The image selection box will be opened and all the currently loaded images
will be displayed in a minimized form.
• Click on the required image, which will then appear in the display box of the image selection box and
has been selected for the interpolation procedure.
You can also use the Click into window button for image selection.
4.7.4.3
Deconvolution Panel
The Deconvolution panel contains the two tabs
Method and PSF.
(1)
Method tab
The Method tab permits selection between the
calculation methods Nearest Neighbour, Inverse
and Iterative.
(a)
Fig. 4-166
Nearest Neighbor
Method tab
The Nearest Neighbor method is the simplest and
fastest algorithm which in principle corresponds to
a 3D sharpness filter.
(b)
Inverse Filter
The regularized inverse filter generally achieves better results than the Nearest Neighbor algorithm. It is
well suited to process several image stacks for a pre-selection of images for the use of the iterative highend methods.
(c)
Constrained Iterative
The best image quality is achieved using the Constrained Iterative Maximum Likelihood Algorithm.
Increasing the resolution in the image, especially in the Z-direction, is only possible with this method. Due
to the complex mathematical method, depending on the image size and the PC being used the
calculation can take up to several hours.
In the Inverse method, the Restoration Effect slider permits the noise-to-signal ratio to be selected
between the settings Weak (low noise) and Strong (pronounced noise).
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3D View Menu
Carl Zeiss
LSM 5 LIVE
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Activation of the Auto detect check box will start a routine for the automatic determination of the noise
level in the entire image part of the Z Stack (not available in the Nearest Neighbour method). If Auto
detect is enabled, the Restoration Effect slider is disabled.
The Iterative method permits (in addition to the parameters of the Inverse method) the maximum
number of iterations to be entered between 1 and 200 under Maximum Iterations and the Auto Stop
function to be activated / deactivated. The Auto Stop function interrupts the calculation depending on
the set image improvement (delta between last but one and last cycle in %), no matter whether the value
under Maximum Iterations has been achieved or not.
The Nearest Neighbour method permits entry of the Number of Neighbours and the Sharpness in
Focus value in addition to the Restoration Effect.
(2)
PSF tab
In the 3D Deconvolution option a theoretical point
spread function (PSF) is calculated from the
systems settings (objective data, wavelengths,
aperture size).
The PSF data are displayed in the PSF tab. In the
case of wavelengths above 700 nm, the NLO
button is automatically enabled.
The displayed values are always taken over by the
system data, but can be edited subsequently for
simulation purposes.
Fig. 4-167
4-166
PSF tab
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4.8
OPERATION
Macro Menu
Carl Zeiss
Macro Menu
The macro function permits the recording, running and editing of command sequences and their
allocation to buttons in the Macro menu.
• In the Main menu toolbar, click on Macro.
− This opens another, subordinate toolbar in the Main menu.
Using the Macro Button (optional) opens the dialogue for editing and recording Macros based on VBA
programming.
Using the Visual Button (optional) allows to construct automated work flows using the arrangement of
symbols which depict the single steps within a work flow. See Visual Macro Editor, page 4-189 for
further details.
Fig. 4-168
Macro menu
4.8.1
Macro Language
"Visual Basic for Applications", called VBA in the following, is used as the Macro language. This language
is well known through its widespread use as Macro language in the "Microsoft Word for Windows" and
"Microsoft Excel for Windows" products. Experience with "Microsoft Visual Basic" would also be
beneficial for macro-programming of the LSM 5 LIVE.
An Integrated Development Environment, called IDE in the following, is available for the editing and
debugging of macros. IDE includes an "online help program" where the VBA language is described in
detail.
Macros are stored in project files. One project file can include several macros.
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OPERATION
Macro Menu
Carl Zeiss
LSM 5 LIVE
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4.8.2
Macro Control
4.8.2.1
Open / Close the Macro Control
Window
• Click on the Macro button in the Macro
subordinate toolbar of the Main menu.
− This opens the Macro Control window.
• Click on the Close button to quit the window.
4.8.2.2
Edit Macro Function
This function allows you to manage project data.
Macros can be recorded, stored, performed, edited
and, if required, deleted.
• Press the Edit Macro button to switch to the
Macro and Recording panels.
Fig. 4-169
(1)
Macro Control window
• Click on the Close button to quit the window.
Macros panel
New button:
Creates a new project.
Load button:
Opens an existing project.
Save button:
Stores the project on the hard disk.
Save As button:
Stores an existing project under a new name.
Unload button:
Removes the selected macro from the Macros list.
Edit button:
Allows macros to be edited and debugged. The editor (Microsoft Visual Basic)
is automatically located at the beginning of the relevant macro.
Run button:
Runs a macro.
Step button:
Opens the editor for line-by-line editing / debugging.
Delete button:
Deletes the selected macro.
Editor button:
Opens the editor. Displays the processed area of the macro edited last.
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OPERATION
Macro Menu
Fig. 4-170
Carl Zeiss
Macro panel
Macros are stored and managed in project files (*.lvb). Before you can record or edit a macro, you have
to create a project as follows:
• Press the New button to create a project file.
− A new project is created and displayed in the Project selection box (e.g.: LSM 150503). The project
name is automatically default, but can be edited afterwards.
To activate an existing project, proceed as follows:
• Press the Load button.
− The Open window will be opened.
• Select the relevant project file (data extension:
*.lvb) from the Macros list box. Click on the
Open button.
− The project file will be opened and the
macros contained in it are displayed in the
Macros selection box of the Macro Control
window.
Recorded macros are stored in main memory first.
Before the macros can be assigned to the buttons
in the Macro submenu, the project must be stored
on the hard disk.
Fig. 4-171
Open window
• Press the Save button under the project name
in the Macro Control window and determine
the file name in the Project selection box, if
required.
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OPERATION
Macro Menu
Carl Zeiss
(2)
LSM 5 LIVE
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Recording panel
Before recording a command sequence, you can enter the name for the macro to be created in the Rec
Name input box of the Recording panel.
Start button:
Starts recording.
Cancel button:
Cancels the recording procedure.
Stop button:
Stops recording.
Edit On Stop:
On stopping the recording procedure, the macro editor is automatically opened
at the relevant position.
Proceed as follows to record a macro:
• Enter a name for the macro to be created under Rec Name in the Recording panel.
• Click on the Start button to start recording the macro.
• Then perform the operations to be stored, e.g.:
− Click on the Find button in the Scan Control window. A Find scan will be performed.
− Click on the New button in the Scan Control window. A new Image Display window will be
opened.
− Click on the Single button in the Scan Control window. A Single scan will be performed.
• Then click on the Stop button to end the recording. (Cancel enables you to cancel recording)
− If recording was successful, the entered Rec Name will then also be available in the Macros list
box of the Macro panel. The new macro is automatically assigned to the current project. It is
possible to assign as many macros as required to a project.
• Click on the Save button to store the new macro.
Fig. 4-172
4-170
Recording panel
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LSM 5 LIVE DuoScan
OPERATION
Macro Menu
Carl Zeiss
Proceed as follows to perform a macro:
• Select the required macro from the Macros list box of the Recording panel.
• Click on the Run button to start performing the macro.
Provided that a macro is linked to a button in the Macro subordinate toolbar, you only need to
click on this button to perform the macro.
Proceed as follows to delete a macro:
• Select the required macro from the Macros list box of the Recording panel.
• Click on the Delete button. The macro will be removed from the list.
Proceed as follows to edit a macro:
• Select the required macro from the Macros list box of the Recording panel.
• Click on the Edit button. The Microsoft Visual Basic editing window will be opened.
• Make the required changes (also see the notes on page 4-172).
4.8.2.3
Assign Macro to Button Function
This function permits stored macros to be linked
with one button each in the Macro subordinate
toolbar.
• Press the Assign Macro to Button button to
switch to the Define Buttons panel.
Define Buttons panel
Proceed as follows to link a macro to a button of
the Macro subordinate toolbar:
• Select the button number from the Button
selection box.
• Enter the button labelling in the Text editing
box.
• Select the name of the project file from the
Project box using the ... button
Fig. 4-173
Macro Control window
• Select the macro name from the Macros box.
• Press the Apply button to assign the relevant
macro to the specified button in the Macro
toolbar.
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4-171
Carl Zeiss
OPERATION
Macro Menu
LSM 5 LIVE
LSM 5 LIVE DuoScan
Delete the linking of a button in the Macro subordinate toolbar
Proceed as follows to delete the linking between a button in the Macro subordinate toolbar and a
macro:
• Select the button number from the Button selection box.
• Press the Delete button to delete the linking.
4.8.2.4
Editing and Debugging of Macros
The Edit button activates IDE (Integrated Development Environment) which allows macros to be edited
and debugged. Under the Help - Contents and Index menu item, IDE contains detailed "online" help
on its operation and on the VBA macro language. Therefore, only a few hints are provided in the
following:
You should activate the required toolbars. We would recommend you to activate the Debug toolbar via
the View - Toolbars -Debug menu item.
The following buttons in the toolbar can help you when debugging macros:
Starts running the command lines.
Stops running the command lines.
Interrupts processing of the command lines (pause).
Sets a breakpoint in the line with the text cursor.
Processes a command line and steps into subprocedures.
Processes a command line and steps over subprocedures.
Exits the subprocedure (step out).
Displays the value of the marked expression (Watch). If nothing is marked, the value of
the variable above the text cursor is displayed.
Activates the Watch window in which values of variables and expressions can be
displayed. For this, text is marked in the code window and dragged into the Watch
window. Variables can be modified in the Watch window.
In the left-hand edge of the code window you will find an arrow beside the current command line. A
new current command line can be determined by moving the arrow via the mouse. This makes it possible
to skip command lines or to process command lines several times.
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LSM 5 LIVE DuoScan
4.8.3
OPERATION
Macro Menu
Carl Zeiss
Overview of Available Macros
Documentation files (*.rtf, *.doc) of advanced macros will be located in the macro directory.
Name
Description
AOTFfitlin.lvb
Linearize laser attenuation (AOTF or mechanical)
Autofocus.lvb
Automatic focusing according to a set configuration
Bleach.lvb
Bleaching of a rectangular area or a line; combines old macros
BleachRectangle.lvb, BleachLine.lvb and Spot.lvb
CameraColor.lvb
(also Button in Maintain)
Color balance of Axiocam HRc
Centerv.lvb
Centers the field of view around the position marked with the cross
tool;
ConcatenateImages.lvb
Enables the combination of all kinds of images irregardless of the
size and data depth to be presented in a time series. Imported
images can also be included. Type of concatenation (fitted to the
smallest frame or ROI, fixed size or fitted to the largest frame) can
be defined by the user.
CopyPasteOverlays.lvb
Copies actual overlay drawing into the clipboard and pastes the
drawing into a selected image window
CopyPasteRoi.lvb
Copies drawing element of overlay into clipboard and pastes it into
other selected windows
Distance.lvb
Example macro for measurement
DivideThroughReferenceImage.lvb
- divide complete time series through a single image/part of the
series
- duplicate a single image or part of a time series to this series.
ExcitationFingerprinting.lvb
Automated generation of excitation lambda stacks for generating
excitation spectra which can be used for linear unmixing to
separate overlapping dyes. Any detector can be used, also NDDs.
Description as guide tour on the installation CD-ROM or DVD.
FastModeSwitch.lvb
Store settings from "Scan-Control" and reuse.
FileExport.lvb
Exports one or more selected images according to the set file
format in one go; Exports image intensity values in ASCI format;
FRET plus.lvb
(Option) Enables the user to perform FRET experiments based on
sensitized emission or acceptor photo bleaching (depending on the
type of LSM) including commonly accepted analysis algorithms.
HotKey.lvb
Shift focus with a button and start Single-Scan
KSPlastv.lvb
KS software macro
Kollimatic.lvb
Automatically generates the calibration matrix of a LSM 5 LIVE
system for optimal confocal aperture and collimator settings
Lambdatrans.lvb
Time series alternating between lambda and transmission mode
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Carl Zeiss
Name
OPERATION
Macro Menu
LSM 5 LIVE
LSM 5 LIVE DuoScan
Description
LsmHWAdmin.lvb (also Button in Direct service hardware access (password protected)
Maintain)
LsmHWAdminEx.lvb
Calibration service macro (password protected)
LsmTime.lvb
Triggered Time scan Macro
MetaExport.lvb
Export of META image files including all channels as tif, bmp, ...
ModifySeries.lvb
Modifies Z Stacks and Time Stacks like Rotation of the stacks, being
mirrored, Conversion of time stacks into z-stacks and vice versa;
StitchArtPlus.lvb
StitchArt Plus macro (Option)
MultiTime.lvb
Set up of time series experiments including repeated imaging,
bleaching and autofocusing with defined configurations at multiple
locations and for various views at each location (Software option)
OptimizeGDC.lvb
Optimize the max. peak power of fiber coupled Ti:Sa lasers (Release
3.0/3.2)
OptimizeGDCV3_2.lvb
Optimize_Live.lvb
Temporarily adjusts the confocal aperture position of a LSM 5 LIVE
without changing the general calibration values
Pixel.lvb
Displays and stores the mean intensity values of each line of one or
more channels of one or more images; data from each line are
stored as a txt file in the current folder;
Profile.lvb
Displays the pixel values along a line
RecoveryChameleon.lvb
Imitates recovery of chameleon NLO laser in case the laser is falling
into CW lasing and does not get to mode lock anymore.
Reset_live_servos.lvb
Reinitializes all servo motors of a LSM 5 LIVE to overcome possible
warmup deviations from default positions
Scalebar.lvb
Indication of self defined intensity levels assigned to a ROI as scale
bar in the image; also attaches tick marks and concentration values
to the grayscale/color wedge.
ScanfieldTransform.lvb
Adjusts LSM 510 or LSM 5 LIVE image match relative to each other
or to LSM DuoScan ROI’s
SphericObjectiveCorrection.lvb
Calibrates the spheric component of objective error by automatic
acquisition of a Z stack on a flat surface and a spherical fit of the
topographic data.
TestGrid.lvb
Projects a testgrid into the image window (password protected).
TileScanRotation.lvb
Defines rotation angle of scanners during tile scan
TimeSeriesShutter.Lvb
Close laser shutter in time series on a LSM 5 LIVE
Trigger.lvb
Trigger test
During installation, default macros will be installed according to their type either in AIM\,
AIM\HWT or AIM\Macros\. Self generated Macros will be in AIM\Macros.
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LSM 5 LIVE DuoScan
OPERATION
Macro Menu
Carl Zeiss
In case of a new installation, old macros will be stored in AIM\Macros\BackupMacros or
AIM\Backup\, to avoid problems with identical names of existing and new macros.
4.8.4
Sample Macros
The LSM 5 software package includes e. g. the Distance, Profile, Multiple Time Series and StitchArt
plus sample macros.
They can be easily executed by clicking on the relevant button in the Macros subordinate toolbar.
During the execution of a macro, the Stop Macros window is always displayed on the screen. This
enables a macro to be stopped any time by pressing the Stop button.
The functions of the sample macros are explained below.
4.8.4.1
Distance Macro
This macro permits measurement of the distance of a line created in the scan image.
• Click on the Distance button in the Macro subordinate toolbar.
− An XY scanning image of the specimen is recorded and displayed. At the same time, the Mouse
position test window appears on the screen.
• Then draw a line over the distance to be measured by clicking and holding down the mouse button.
The click of the mouse sets the starting point, releasing the mouse sets the end point of the line.
− After release of the mouse button, the length of the line in the scanning image is displayed (in µm).
− Any required number of lines can be defined in the image. The previous line is deleted.
• A click on the Exit button in the Mouse position test window will end the macro.
4.8.4.2
Profile Macro
This macro permits the gray values of a line created in the scanning image to be determined pixel by
pixel.
• Click on the Profile button in the Macro subordinate toolbar.
− An XY scanning image of the specimen is recorded and displayed. The Profile window is shown on
the screen.
• Then click and hold down the mouse button to draw a line in the scanning image for which the gray
values shall be determined.
− The current numbers of the pixels of the created line to which the relevant gray value is assigned
now appear in the Profile window.
− At the same time, the distance of the created line is displayed in µm for checking.
− Any required number of lines can be defined in the image. The previous line is deleted.
• A click on Cancel will end the macro.
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4-175
OPERATION
Macro Menu
Carl Zeiss
4.8.4.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Multiple Time Series Macro
The Multi Time Series program is designed to
provide flexible programming of automated time
dependent experiments.
− A pre-program Cycle Delay
− A pre-bleaching functionality (not available)
− An autofocus functionality
− A number of different, single time series
(called a block) either at the same position of
a sample or at different positions (if a
motorized stage is available)
− A time series of a tile scan
− A time series of a tiled Z stack
− A Z-Stack with triggered acquisition of each
single image in the stack
The basic programming unit is a single Time Series
Block in line, frame or Z-stack mode. In each block
the user can define a configuration for the data
acquisition (single or multi-track), the number of
images and the time interval/delay between
images.
The user can activate an optional autofocus
function before each block, pre-program a time
interval before each block (from the beginning of
the previous block to the beginning of the current
block), and/or execute a bleach track with
arbitrarily specified bleach ROI's, laser line(s) and
power.
Fig. 4-174
Multiple Time Series main dialog box
The autofocus function can be executed using a
specified single-track configuration, or with the
configuration used for imaging (using the first
channel of the first track).
One can define the z-offset, the distance in the z direction from the position where the autofocus finds
the reference feature in focus (position of maximum intensity - e.g. position of the cover slip reflection in
the reflected light configuration) to the position, which is moved into focus plane when the image
acquisition begins (e.g. into the tissue).
The image acquisition is done with the configuration specified for the given block.
Autofocus search parameters: Z-offset as well as the Z-range (the distance in the z direction over which
the autofocus function searches for the plane of maximum intensity) can be set independently for
different stage locations (on the systems with the motorized stage).
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LSM 5 LIVE
LSM 5 LIVE DuoScan
4.8.5
OPERATION
Macro Menu
Carl Zeiss
Kollimatic
VBA Macro to calibrate the LSM 5 LIVE depending on the current system configuration (e.g. objectives,
filters,…)
Prerequisites:
−
System Warm-Up (at least 2 hours; for procedure please read the User’s Manual) and
environmental conditions according to setup requirements.
−
“OptimizeLIVE.lvb” Macro: ensure both channel sliders set to 0
−
Proper adjustment of collimators and Confocal Slit Aperture (CSA) 1 + 2 (this has to be done by the
local Service Representative)
−
AOTF RGB + V linearized properly with Macro AOTFFitlin.lvb
−
All used objectives have been stored in the database according their exact order in the nose piece.
−
Necessary calibration slides are available
1x Bead Slide 1µm for dry objectives, # 000000-1371-460,
1x Bead Slide 200nm for immersion objectives, # 000000-1371-461);
to be ordered from Carl Zeiss Jena.
Basic Functions of the Macro
The KolliMatic macro features two main parts, Basic Calibration and Calibration Matrix:
A - Basic Calibration
− Needs to be done only once with one of the recommended objectives (should be part of the
shipment in order to be able to repeat the Basic Calibration without calibrating all objectives again
(see objective list down below)
− Is for calibrating both collimators RGB + V
− Is for calibrating the Y-position of the Beam Shift Compensator (BSC) for all present NFT positions
On currently delivered LSM 5 LIVE Systems, the Basic Calibration is done in the factory (This can
be confirmed by checking that “BasicCalibrationLog.txt” is present in C:\AIM\Bin).
Objective list (objectives are listed in order of most to least recommended)
− Plan Apochromat 20x / 0.75
− Plan Neofluar 40x / 1.3 Oil
− C-Apochromat 10x / 0.45W
− C-Apochromat 40x / 1.2W corr
− Plan Apochromat 5x / 0.16
− Plan Neofluar 10x / 0.3
− Plan Apochromat 63x / 1.4 Oil
− C-Apochromat 63x / 1.2W corr
− Plan Neofluar 20x / 0.5
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OPERATION
Macro Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
B - Calibration Matrix
− Is based on the calibration results of the Basic Calibration
− Needs to be performed for each objective separately
− Is for calibrating the collimator position of both collimators RGB + V only for the selected Objective
depending on:
Laser Line, Zoom + EF Filter Position
− Is for calibrating the BSC Y-position depending on:
Laser Line, Zoom + EF Filter Position
On currently delivered LSM 5 LIVE Systems, the Calibration Matrix is set up in the factory for
every objective belonging to the system (This is done together with the Basic Calibration).
Without running Calibration Matrix, the positions of the collimators and BSC Y should be OK. This is due
to the Basic Calibration, but the results are not fully optimized.
Installation of the Macro:
If not already done, copy the required files to the following destinations:
File Name
Destination
KolliMatic.lvb
C:\AIM\Macros
PotentialKollimaticConfig.ini
C:\AIM\Bin
KolliMatic_Instructions.pdf
C:\AIM\Macros
Starting the Macro:
• Start the LSM software and check first, if all
prerequisites
have
been
fulfilled
(see Paragraph 1. Prerequisites)
• Put the appropriate calibration slide (depending
on current objective: immersion or non
immersion) onto the microscope stage,
• Activate all lasers, and using the and focus with
the 488 nm line start scanning and focus onto
the beads
• Put the appropriate calibration slide (depending
on current objective: immersion or non
immersion) on the microscope stage, start single
scan
• Use the Macro Menu of the main LSM software
to Load and Run the macro KolliMatic (See
Fig.4-175)
Fig. 4-175
4-178
Macro Control Window of the main
LSM Software
Please wait a few seconds, the macro will set up
beam path and all necessary settings automatically.
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OPERATION
Macro Menu
Carl Zeiss
Preparations for using the Calibration Slides:
• Follow the steps shown in the macro Sample
and Configuration Setup window
• Use the arrows or the drop down menu in order
to select one track for each available laser line
(no need to select several tracks for the same
laser line), start with a track for the lowest
wavelength laser (i.e. typically the 405 nm laser
line.)
• Select an area of the bead sample that provides
as many beads as possible, at least 4 (fit into
the green rectangle)
• In Fig. 4-176, the Basic Calibration has been
selected (Check box Basic Calibration:
Requires selecting preferred objective lens),
therefore some more tracks are available
• Use the Laser Power as a first option in order
to set the intensity, second option is
Integration Time
• In order to start / stop the scan, just select /
deselect the check box Continuous Scan
• Focus on the beads
• Defocus with the Collimator slider until the
beads become line shaped
• Focus with the microscope focus drive to get
the vertical lines as thin as possible (Fig. 4-177)
Fig. 4-176
Figure Sample and Configuration Setup
Window of the KolliMatic Macro
• Now use the Collimator slider to get a round
spot out of the line (Fig. 4-178)
• Repeat these steps for a track with the 488 nm
laser line
• After one track for each laser line has been
adjusted, click Next.
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4-179
OPERATION
Macro Menu
Carl Zeiss
4-180
Fig. 4-177
Beads focused for maximal vertical thinness
Fig. 4-178
Beads optimally focused
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LSM 5 LIVE DuoScan
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LSM 5 LIVE
LSM 5 LIVE DuoScan
OPERATION
Macro Menu
Carl Zeiss
Running the Basic Calibration
If you have selected the check box for Basic Calibration, please follow these instructions. In
order to run the Calibration Matrix only, move on to paragraph 7.
• Make sure that both check boxes have been
selected (Fig. 4-179)
• Double check if the objective settings (shown at
the bottom of the macro window) match the
currently used objective
• Click the button Calibrate
• In order to follow the calibration process, just
click into the Message window; a scroll bar will
appear
• The Basic Calibration takes about 15 min
• The displayed curve should resemble a Gaussian
profile
• After the macro has finished, a message will
pop up with the request to restart the LSM
software to make the changes effective.
Fig. 4-179
Collimator Calibration Window
• Press Exit and restart the LSM software
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4-181
OPERATION
Macro Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Running the Calibration Matrix
If you haven’t selected the check box for Basic Calibration, the “Calibration Matrix” will be
selected automatically. In this case, when the Calibration Matrix is running , the Basic Calibration
is disabled.
• This window provides three check boxes (Fig.
4-180)
• The recommended setting is to select Create
new calibration file. All previous files will be
deleted.
Exceptions:
No check box checked
− Especially after an unexpected interruption
of the program, it is possible to continue
from the position where the process stopped
(saving time).
Overwrite Collimator values
− The values for the chosen objective will be
added or updated.
Fig. 4-180
Running the Calibration Matrix
− After selecting the Collimator option, the
Pinhole Y option will be selected
automatically.
• Double check if the objective settings (shown at
the bottom of the Macro window) match the
currently used objective
• Click the button Calibrate
Meaning of the Graph (Figure):
Brown:
Focal plane of the CCD Line Detector
Red:
Focal plane of the collimator
Orange:
Estimated focal plane
The brown and red graphs should be close to this
curve.
Fig. 4-181
4-182
Graph of Focus Function
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LSM 5 LIVE
LSM 5 LIVE DuoScan
OPERATION
Macro Menu
Carl Zeiss
• In order to follow the calibration process, just
simply click into the Message window and; a
scroll bar will appear (Fig. 4-182)
• The Calibration Matrix takes about 25 min for
each objective
• After the macro has finished, a message will
pop up with a the request to switch the
objective lens in order to make the changes
effective
• If necessary, repeat the procedure for the other
objectives
• Use the Update Diagrams button in order to
check, if an objective has already been calibrated
− Select an objective, press the Update
Diagrams button
Fig. 4-182
− If no graphs appear, this particular objective
has yet needs to be calibrated
Collimator Calibration window after
calibration
• Click Exit to finish
Meaning of the Graph (Figure):
− Each coloured line represents an individual laser line.
− Each coloured calibration point represents an emission filter.
− Standard graphs are displayed with black points.
Meaning of the Colours:
− <420 nm:
Æ Pink
− 421…475 nm: Æ Blue
− 476…495 nm: Æ Turquoise
− 496…550 nm: Æ Green
− 551…590 nm: Æ Yellow
− >590 nm:
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Æ Red
B 45-0019 e
4-183
OPERATION
Macro Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Important Information!
If the calibration has been completed successfully
with the Calibration Matrix,
DO NO LONGER USE THE PINHOLE AND
COLLIMATOR FUNCTIONALITY OF THE LSM
SOFTWARE IN THE MAINTAIN MENU.
The use of the pinhole sliders to raise the quality of
the images is well known from the other LSM
Systems.
Fig. 4-183
On the LSM 5 LIVE, only the Optimize sliders of
the “OptimizeLIVE.lvb” macro have to be used
(Fig. 4-183) to compensate for misalignments (e.g.
temperature influences).
Optimize Macro
After the Calibration Matrix has optimized a particular objective, the results will be stored automatically in
a file as shown below. These files can be found in C:\AIM\BIN.
DO NOT CHANGE ANY OF TH VALUES!
Fig. 4-184
4-184
Calibration file
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LSM 5 LIVE
LSM 5 LIVE DuoScan
OPERATION
Macro Menu
Carl Zeiss
The calibration results of the Basic Calibration will
be
saved
in
a
file
named
“BasicCalibrationLog.txt” (to be found in the
BIN-Folder as well).
Changes won’t effect the stored values.
Fig. 4-185
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BasicCalibrationLog file
4-185
OPERATION
Macro Menu
Carl Zeiss
4.8.6
LSM 5 LIVE
LSM 5 LIVE DuoScan
Optimize Macro
For temporary optimization of the LSM 5 LIVE
beampath, try to avoid a modification of the
general adjustment in the pinhole dialogue. Use
the Optimize slider instead. The general setting of
the confocal aperture might still be correct for
other scan parameters.
Fig. 4-186
Optimize window
The Optimize slider can be set independently for
each channel. The slider is moved to the one or the
other side while scanning continuous. The
brightest image result is the optimal setting. This
setting doesn’t affect the general adjustment of
the LSM 5 LIVE collimator and confocal aperture,
but can enhance image brightness efficiently.
4.8.7
ReInit Macro
To improve the internal adjustment of default
positions of motor driven parts, the Reset Servo
macro (ReInit) is available.
After one hour, we recommend to start the ReInit
macro to reset all default positions for the actual
warm up state of the system. Repeat this whenever
you experience changes in the system
performance. After running the macro, close the
window.
Fig. 4-187
4-186
Reset window
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LSM 5 LIVE DuoScan
4.8.8
OPERATION
Macro Menu
Carl Zeiss
Image Match (Scanfield Transformation) Macro
Adjusts the Image match between two scanning imaging systems or a manipulation and a scanning
imaging system.
To adjust the image match, set up a multitracking that creates one channel for each scanning and/or
manipulation system. In case of the LSM DuoScan, use a T-PMT to create an image with the DuoScan
system.
• Start the Image Match (Scanfield Transformation) macro.
• Use the Image match calibration sample delivered with the system. This sample has a fluorescent layer
and a reflective grid, useful to create images in fluorescent, reflective or transmission light mode.
• Start continuous scan, and set laser power and gain to appropriate levels. The point scanning system
will be the speed limiting system, so choose a fast scanning speed to get a faster image refresh.
• Use unidirectional scan to avoid adjustment errors due to bidirectional image jitters.
• Set the matching zoom factors with one of the Match DuoScan zoom/offset or Match LIVE
zoom/offset buttons relative to each other. It is recommended to adjust the LSM zoom.
Fig. 4-188
Scanfield Transform window
These buttons set the two scanheads at a matching scanfield diameter, according to the following
table (LSM 5 LIVE = Zoom 1.0):
Microscope stand
LSM 5 LIVE DuoScan
LSM 5 DUO
Inverted (port config)
(SP/RP) DuoScan = Zoom 1.4142
(RP/SP) LSM 510 = Zoom 1.33978
Upright (port config)
(Tube/RP) DuoScan = Zoom 1.4142
(RP/Tube) LSM 510 = Zoom 1.25295
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OPERATION
Macro Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• Choose Split image for the image window display. You will see the scanned areas of both scanning
systems and an overlay.
Fig. 4-189
Split image
• Choose LSM (510) or DuoScan to adjust the image match.
• While scanning, move the sliders of the Image match macro to get a perfect overlay of the scanning
and/or manipulation system relative to each other.
• Offset X/Y adjusts the horizontal and vertical image shift
• Amplitude adjusts a size difference between the images
• Rotation compensates for a tilt in one of the images.
Store the final image, and use the ReUse function for future image matching. The Multitrack
setup and the zoom factors will then be set automatically.
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LSM 5 LIVE
LSM 5 LIVE DuoScan
4.8.9
OPERATION
Macro Menu
Carl Zeiss
Visual Macro Editor
The Visual Macro Editor is an optional software module which allows the user to program a variety of
scanning procedures and image calculations as well as the combination of both.
4.8.9.1 Open / Close the Visual Macro Editor Window
• Click on the Visual button in the Macro subordinate toolbar of the Main menu.
− This opens the Visual Macro Editor window.
• Click on the
Fig. 4-190
03/06
button in the upper right side of the window to quit[amy362].
Visual Macro Editor window
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4-189
Carl Zeiss
OPERATION
Macro Menu
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.8.9.2 Function Description
The Editor uses predefined action blocks. The action blocks for setting up the work flow of the Macro are
categorized according to their properties. The following categories are displayed on the left-hand side of
the Visual Macro Editor control window.
− Acquisition
− Windows
− Load/Save
− Program Flow
− Process
− Hardware Control
• Click on one of the Category selection buttons will display the available action blocks.
The action blocks can be positioned according to their function either in the Program Flow column or
the Data Flow column. The color of the connection arrows of the action block defines the possibility
to position the block in the program flow column or the data flow column(s).
• Click at the action block with the left mouse button (hold down the mouse button) and drag the
mouse cursor into the flow column. Release the mouse button.
− Action blocks for the program flow show red connection arrows ▼ as well as blue output arrows
▼ to connect to the data flow.
− Action blocks for the data flow show blue connection arrows ▼ only.
It is not possible to drag an action block for program flow into the data flow or an action button
for data flow into the program flow.
Action blocks can be removed using the Delete key of the keyboard.
Without using the Image Display action block in the data flow column, the result of the scan
will not be displayed (see also description of Scan and Time Series action blocks).
The work flow is generated by connecting the action blocks using the mouse cursor.
• Click onto an output arrow with the left mouse button (hold down the mouse button) and drag the
mouse cursor away.
−
A connecting line appears.
• Drag the line to the input arrow of another action block. Only arrows of the same color can be
connected.
According to the action blocks a set of properties is listed which can be assigned with free or predefined
values. A set of defined properties is assigned to one block only. If this block is used more than once the
properties can be set differently.
Clicking the Read Back button will take over the current settings from the LSM main program for the
displayed properties list.
• Mark the check boxes next to the parameters individually or, click Use all or Use none to check or
uncheck all parameters with one click.
• For changing a values click into the Value column.
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OPERATION
Macro Menu
Carl Zeiss
It is possible to define variables. A variable has a (property) name and a mathematical value. The
Assignment action block can be used to assign a new value to a variable when inserted into the
program flow using mathematical algorithms. The new value is then used for any subsequent calculation
in the program flow where the variable is part of the calculation.
• Click the Variable button on the toolbar to display the variable list. Click once more to display the
parameter list again.
• New variables can be added by typing in name and value in the next free line.
Toolbar buttons
Run
Starts the Macro starting with the firs block in the program flow column.
Stop
Stops the Macro at any time:
Break
Interrupts the Macro. Clicking the button again will resume the actual program flow of
the Macro:
Step
Allows to go through the program flow of the Macro stepwise starting with the first block
in the program flow column. Each click will perform the action of the actually highlighted
block, then jump to the next block which will then be highlighted.
Load
Use to open a specific Macro and Save to store the Macro. SaveAs allows to store the
same Macro under a different name.
Variables
By clicking the button the list of defined variables is displayed. New ones can be added by
typing in name and value in the next free line. Variables are assigned to Macros. For each
new Macro a new list of Variables has to be generated.
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OPERATION
Macro Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Acquisition Action Blocks
Loads a Beam Path Configuration form the Configuration list. Use the pull
down menu in the Property/Value list to select the Configuration that should
be used for imaging. Changing between Single Track and Multi Track allows to
chose between the different Configuration lists. Multi Track also lists tracks that
are used for Emission Fingerprinting or Online Fingerprinting. Depending on the
type of track different settings can be loaded optionally. If they are not loaded
and the parameters list of the Scan Parameter block does not offer them as
being editable, for example detector gain and offset, default values will be
used.
The list of properties lists all parameters that can be adjusted for the scan
procedure. The value for each parameter can be typed in individually. Clicking
the Read Back button will take over all current settings from the main
software. The check boxes next to the parameters can be marked individually
or, clicking Use all or Use none they can be all checked or unchecked with
one click. Only the parameters with a mark in the check box will be taken into
account and adjusted when the Scan Parameter block is set in the program
flow column. This allows to change only some of the parameters, for example
the zoom, at a certain position within the program flow as for each block the
check boxes can be set.
This block performs the scanning of the sample according to the settings
chosen with Beam Path and Scan Parameters. It can be linked to the Data flow
column. If no connection to the data flow is made (for example: Display Image)
the result of the scan will not be displayed.
The parameters for bleaching the sample can be loaded from the list of bleach
settings. Bleach settings are assembled and stored in the Bleach control window
of the main software.
This block performs the bleaching of the sample according to the bleach
settings that have been defined and stored in the Bleach control of the main
software. Use the pull down menu to select the bleach setting from the list.
The list of properties lists all parameters that can be adjusted for the acquisition
of the time series.The value for each parameter can be typed in individually.
Clicking the Read Back button will take over all current settings from the main
software. The check boxes next to the parameters can be marked individually
or, clicking Use all or Use none they can be all checked or unchecked with
one click. Only the parameters with a mark in the check box will be taken into
account and adjusted when the Time series block is set in the program flow
column. This allows to change only some of the parameters, for example the
used trigger, at a certain position within the program flow as for each block the
check boxes can be set. This block can be connected to the data flow column.
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LSM 5 LIVE DuoScan
OPERATION
Macro Menu
Carl Zeiss
Windows Action Blocks
This block has to be used if the actually scanned image(s) should be displayed.
For each Image Display block a new image window is opened. If not saved, the
image will be overwritten when the program flow is passing by again.
Save / Load Action Blocks
The image(s) are stored to a image database. The database taken is either the
last one, that was open or the current open one. Clicking Read back updates
the database to be used for storing the image(s). In addition some other
parameters can be set according to the list of properties displayed.
Within the program flow already existing images can be loaded.
The database taken is either the last one, that was open or the current open
one. Clicking Read back updates the database to be used to load the image(s)
from. The images can be loaded by Index or by the name of the image.
It is possible to export images choosing a certain file format from the pull down
list that appears when highlighting the line File Format in the properties list.
Specific parameters are assigned to each file format which can be set for the
image export.
Images can be imported within the program flow. The image to be loaded can
be selected by typing in the file path. The import of single images or a image
series is possible. The images to be imported as series must have the same
name and must be numbered in ascending order.
Program Flow Action Blocks
If any action(s) should be repeated a defined number of times, the Repeat block
should be set into the loop of the program flow. The number of repeats is set in
the properties list. The repeat is only performed using the connection of the
right arrow. After finishing the repeats, the program flow connected to the
arrow pointing down is performed.
Using this block in the program flow allows to assign new values to a variable
using mathematical calculations. By passing this block the variable will be
transiently changed and the new value can for example be used to assign
different numbers to the image name or to increase the time delay during a
time series acquisition.
The two blocks are used as decision makers. Depending on the value of a
predefined variable, the program flow can be directed to one of the two
possible directions. The expression of the variable’s value which should be used
to decide on the program flow is set for each block in the properties list. It
takes the actual transient value of the variable into account.
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Carl Zeiss
OPERATION
Macro Menu
LSM 5 LIVE
LSM 5 LIVE DuoScan
This block introduces a delay in the program flow. The delay time is started
when the block is reached within the program flow.
The block allows to assign a potential error message with a textbox and to
decide on the Error where to direct the program flow. If no textbox is assigned
the program flow will just continue according to the presence of an error
message. Choosing a textbox requires the input of the user to continue with
the program flow.
With the Release 3.5 this works only for loading images from a
database when the images cannot be found because the Index
number is not present. In all other cases of an error, the error message
will be displayed and the program stopped.
Process Action Blocks
This block allows to do image calculation using the pixel intensities of the
image. The operators for the formulas that can be used are described in the
Ratio Dialogue within the Process Menu. The formulas set up in the Image
calculation window can be copied and pasted into the properties list of the
Visual Macro Editor.
For calculations using two different sources the Calculate block with two
possible inputs is used. The calculation starts when both inputs have arrived.
Within program loops the next calculation will start when the inputs have
arrived again in the same sequence as the first time. The operators for the
formulas that can be used are described in the Ratio Dialogue within the
Process Menu. The formulas set up in the Image calculation window can be
copied and pasted into the properties list of the Visual Macro Editor.
The input image will be copied to a new image format that can be defined by
changing the values of the listed properties.
Using this block enables to copy one image into another with the coordinates
and settings put into the properties list. The destination image is connected to
the left input, the source image is connected to the right input.
The subsequently arriving images will be concatenated according to the
coordinate setting and treated as one image. A subsequent image display will
be updated each time a new image arrives.
The second concatenate action block allows to concatenate images from two
different acquisition blocks.
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LSM 5 LIVE DuoScan
OPERATION
Macro Menu
Carl Zeiss
Hardware Control Action Blocks
If a motorized stage is available the movement of the stage can be set up as
part of the program flow. The stage can be moved to an absolute position
which can also be taken from the actual stage position using the Read back
function or typed in individually. Using a relative position will use the current
stage position as relating position. The values have to be typed in.
The movement of the focus can be set up as part of the program flow. The
focus can be moved to an absolute position which can also be taken from the
actual focus position using the Read back function or typed in individually.
Using a relative position will use the current focus position as relating position.
The values have to be typed in. For large travel ranges which exceed half of the
objectives working distance a warning can be displayed.
4.8.9.3
Editing a Macro
Following Example shows a macro which acquires
five subsequent images and moves the stage 10
microns in X and Y following each image. The
images are stored and numbered in ascending
order.
• Arrange and connect the action blocks (Fig.
4-191).
Fig. 4-191
Arrangement of action blocks
Fig. 4-192
Assignment of the variable
• Assign the value 1 to a variable named Index
(Fig. 4-192).
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OPERATION
Macro Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• Click the single action blocks and assign the values to the listed properties (beam path and scan
parameters can be chosen according to the sample).
Fig. 4-193
Fig. 4-194
Fig. 4-195
Fig. 4-196
4-196
Property list of the Beam Path
action block
Property list of the Scan Parameter
action block
Property list of the Append to Database
action block
Property list of the Move stage action
block.
• The database the images will be appended to is
automatically displayed when highlighting the
Append to Database action block. The last
one used will be taken. To update this, open
the desired database and use the Read back
function. Type in a name for the images with or
without quotation marks. All images will get the
same name then. For numbering the images
use quotation marks for the name and add the
variable Index, which has been assigned with
the value 1. This value or the transiently
changed value of the variable will be added to
the name of the image.
• The movement of the stage is defined
highlighting the Move stage action block,
choosing the Mode Relative Position in the
Property list and typing in the value 10 for X
and Y which then will move the stage 10
microns into X and Y direction starting with the
current stage position on passing that action
block.
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OPERATION
Macro Menu
• Now the first complete scan has been
performed and the image is stored. For the next
cycle the variable Index has to get another
value to make sure the numbering of the
images works. Using the Assignments action
block, the Variable Index is chosen and the
value assigned is Index + 1. Such the variable
will get the transient value 2 for the next cycle,
the value 3 for the over next cycle and so on.
• The number of repeats can be defined using a
Decision block and use the value of the
variable as base for the decision. By defining
that if the Expression of the variable is larger
than 5 and not connecting the output arrow
marked with ‘yes’ to any other block, the macro
will stop after the acquisition of five images.
Alternatively one can use at this part of the
program flow the Repeat block and define the
number of repeats to be performed, which
would be 5 in this case (The first scan is already
counted!)
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Carl Zeiss
Fig. 4-197
Property list of the Assignments
action block
Fig. 4-198
Property list of the Decision
action block
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OPERATION
Options Menu
Carl Zeiss
4.9
LSM 5 LIVE
LSM 5 LIVE DuoScan
Options Menu
The Options menu permits performance of the following functions:
− Display of a current list of dyes with preferred wavelengths for the scanning procedure.
− Display / modification of the user-accessible program Settings of the LSM 5 software.
• In the Main menu toolbar, click on Options.
− This opens another, subordinate toolbar in the Main menu.
Fig. 4-199
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4.9.1
OPERATION
Options Menu
Carl Zeiss
Dye DB Function
The Dye DB function is for information only and permits access to the database contained in the system,
including a list of suitable dyes for fluorescence microscopy.
The database contains a comparison of tables of dyes, optimum excitation wavelengths and maxima of
emission wavelengths.
• Click on the Dye DB button in the Options subordinate toolbar.
− The Dye database will be opened and displayed on the screen.
• Click on the Close button to exit the Dye database.
Fig. 4-200
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OPERATION
Options Menu
Carl Zeiss
4.9.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Settings Function
The Settings function permits the individual setting and matching of software settings with regard to the
following points:
Autosave
Recording / Reuse
Database General
Timeseries
Database Table Viewer
Scan Mean of ROIs
Database Gallery Viewer
Temporary Files
Import / Export
Program Start
Scan Information
Hardware
Image Status Display
Image Display Toolbars
Print Status Display
Save
4.9.2.1
Open / Close the Settings for user : ... Window
• Click on the Settings button in the Options subordinate toolbar of the Main menu.
− This opens the Settings for user : ... window.
• Click on the OK button to quit the window. The last settings will be taken over. Cancel enables you
to cancel the procedure, with any changes you made not being taken over.
Fig. 4-201
4-200
Settings for user : ... window
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4.9.2.2
OPERATION
Options Menu
Carl Zeiss
Autosave
This tab permits activation or deactivation of
automatic data storage. Only one option can be
selected at a time.
(1)
No Autosave
On activation of this option, the Autosave
function is switched off. Save and Save As give
the same dialogues.
(2)
Fig. 4-202
Autosave tab
Use LSM image database and auto increment image name
On activation of this option, newly recorded or modified images are stored by Save automatically and
assigned to the name or defined in this function. The image name is automatically created using a base
name and a serial number. For this, a base name must be entered in the Base image name input box,
and a starting value for the serial number in the Counter value input box. The Database selection box
permits selection of the directory in which the data will be stored.
(3)
Export to Attofluor format
On activation of this option, newly recorded or modified images are stored by Save in the Attofluor
format. The displayed Experimental directory selection box permits selection of the directory in which
the data will be stored.
(4)
Export to Metafluor format
On activation of this option, newly recorded or modified images are stored by Save in a subdirectory in
the MetaFluor format. An existing higher layer of folders must be selected for the subdirectory from the
Base directory selection box. Furthermore, a name for the subdirectory must be entered in the
Subdirectory base name input box. The starting value for the images then created, to which a
continuous number is automatically assigned, is set in the Subdirectory counter input box.
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OPERATION
Options Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.9.2.3
Database General
This tab permits the basic starting settings for the
use of databases.
(1)
Fig. 4-203
(2)
Start with "Form"
On opening of a database, the Form option is
displayed first.
Database General tab
Start with "List"
On opening of a database, the List option is displayed first.
(3)
Start with "Gallery"
On opening of a database, the Gallery option is displayed first.
(4)
Show first record set at opening of database
On opening of a database, the first record set is displayed.
(5)
Show first record set at opening of database
On opening of a database, the middle record set is displayed.
(6)
Show first record set at opening of database
On opening of a database, the last record set is displayed.
(7)
Use separate path for "Create" and "Open"
This option permits the path to be changed when the Open or New database function is used.
(a) Save most recently used path at exit and reuse at next program start
On activation of this option, the path setting last used is automatically selected again in the Open
Database or Create New Database window.
(b) Use the following path at program start
On activation of this option, the path for the Open Database or Create New Database window can
be entered directly in the relevant selection box, or selected by clicking on the ... button in the
Choose Directory window. This path will then always be set when a database is opened or created.
Clicking on the User default path button firmly sets the C:\users\default\ path.
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4.9.2.4
OPERATION
Options Menu
Carl Zeiss
Database Table Viewer
The Database Table Viewer tab permits the
definition of the columns for the table display of a
database. This only requires the relevant check box
to be activated with a click of the mouse.
On activation of the Automatic column width
calculation option, the column width is calculated
automatically.
Fig. 4-204
Database Table Viewer tab
Fig. 4-205
Database Gallery Viewer tab
Fig. 4-206
Import / Export tab
On activation of Save and use interactive
column width setting, the column width in the
database can be matched as required. The
individual setting will be retained when the
database is closed.
4.9.2.5
Database Gallery Viewer
The Database Gallery Viewer tab permits the
image information to be displayed in the Gallery
mode of the database to be activated by clicking
on the relevant check box.
4.9.2.6
Import / Export
Use separate path for "Import" or "Export"
This option permits the change of the path setting
for use of the Import or Export function (File
menu).
(1)
Save most recently used path at exit and
reuse at next program start
On activation of this option, the path used last is
automatically selected again in the Import Images
or Export Images and Data window.
(2)
Use the following path at program start
On activation of this option, the path for the Import Images or Export Images and Data window can
be entered directly in the relevant selection box, or selected by clicking on the ... button in the Choose
Directory window. This path will then always be set when the Import / Export function is used.
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OPERATION
Options Menu
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Clicking on the User default path button firmly sets the C:\users\default\ path.
4.9.2.7
Scan Information
This tab permits the setting of which scan
information shall be displayed in the Scan
Information window (see Window pulldown
menu of the Main menu, page 4-222f).
Activation / deactivation of the information to be
displayed is performed with a click of the mouse.
Fig. 4-207
Scan Information tab
4.9.2.8
Image Status Display
This tab permits selection of which image
information is displayed on opening of an image or
on activation of the Info button of the Image
Display window. Furthermore, you can determine
which information will be displayed in the Image
status bar.
Fig. 4-208
Image Status Display tab
On activation of the Show status display upon
opening of a new image display check box, the
image information is automatically displayed
immediately after opening of the Image Display
window (Info button is activated).
4.9.2.9
Print Status Display
This tab permits selection of which information is
displayed in print preview.
On activation of the Print Status Information
check box, the status information will be printed.
Fig. 4-209
4-204
Print Status Display tab
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LSM 5 LIVE DuoScan
4.9.2.10
OPERATION
Options Menu
Carl Zeiss
Recording / Reuse
The parameters to be taken into consideration for
the use or load of a recording configuration are set
in this tab.
As an option, you can also determine whether the
objective setting shall be taken over when the
Reuse function is used.
4.9.2.11
Fig. 4-210
Recording / Reuse tab
Fig. 4-211
Timeseries tab
Fig. 4-212
Scan Mean of ROIs tab
Time Series
In the Time series tab, you can determine
whether the time for the recording of a time series
is set as Cycle Delay or as Time Interval.
Cycle Delay is the interval between the end of
one scan process and the beginning of the next.
Time Interval is the interval between the
beginning of one scan process and the beginning
of the next.
You can select the unit for Mean of ROIs
diagrams.
4.9.2.12
Mean of ROIs
The Mean of ROIs tab permits the presetting of
the Image Display window for the optional
MeanROI function (time series) to be changed
with regard to scaling and display mode of the
intensity time diagrams.
(1)
Diagram Scaling
The following settings are possible by activating
one of the option buttons:
− Automatic diagram scaling
− Fixed time range for diagram time scale; input of the time range in seconds via input box
− Fixed number of cycles for diagram time scale; input of the time range in number of cycles via
input box
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OPERATION
Options Menu
Carl Zeiss
(2)
LSM 5 LIVE
LSM 5 LIVE DuoScan
Initial diagram types
The following settings are possible by activating the relevant option button:
− One diagram
− Channels diagram
− ROIs diagram
On activation of the Black graphs
black (monochrome).
(3)
check box, the intensity profiles in the diagram are displayed in
Live image
(a) Scan the whole image
If you activate this check box, the complete live image will be scanned; only the defined ROIs will be
scanned if the check box is deactivated.
(b) Save the whole time series
If you activate this check box, the complete Time Series will be scanned; only the Mean of ROI series
will be scanned if the check box is deactivated.
4.9.2.13
Temporary Files
The Temporary Files tab permits determination of
the directory in which temporary files are stored.
(1)
Fig. 4-213
Temporary Files tab
Use "TEMP" environment variable
Temporary files are stored in the TEMP standard
directory of the computer's hard disk.
(2)
Use the following path
The directory for temporary files can be selected by
clicking on the ... button in the Choose Directory
window.
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4.9.2.14
OPERATION
Options Menu
Carl Zeiss
Program Start
The Program Start tab permits selection of a track
configuration via the Startup configuration
selection box, which will be loaded automatically
when the software program is started.
check
On activation of the Don't show logo
box, the initial screen with the Zeiss logo will not
be displayed after the start of the LSM 5 software.
4.9.2.15
Fig. 4-214
Program Start tab
Fig. 4-215
Hardware tab
Hardware
The Hardware tab allows you to set several
hardware defaults at the shutdown of the system.
The Lasers off on Exit determine, by activation of
check box, that the
the Lasers off on Exit
lasers are automatically switched off when the
LSM 5 software is exited.
Allow lasers to cool for five minutes before
switching off the system.
Light manager activates or deactivates the light
microscope stands light manager.
Shutter filter wheel activates or deactivates additional laser attenuation by grey filters (LSM 510 only).
Simultaneous grab and bleach activates or deactivates the simultaneous grab and bleach option in the
bleach menu. This function can create fluorescence artifacts depending on sample properties and
confocal aperture setting (it is recommended to close the confocal aperture to 1 airy unit).
4.9.2.16
Image Display
The Image Display tab enables you to determine
the window toolbars which shall be automatically
displayed when an Image Display window is
opened.
Furthermore, the color mode (color / mono), to
which the image display will switch when the
Color Palette function is opened / closed, can be
determined.
(1)
Fig. 4-216
Image Display tab
Display Windows Toolbars
On activation of the relevant check box, the following window toolbars are automatically displayed when
an Image Display window is opened: Channels, Zoom, Slice, Overlay.
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OPERATION
Options Menu
Carl Zeiss
(2)
LSM 5 LIVE
LSM 5 LIVE DuoScan
Palettes / Color
(a) Switch to "mono" on activation of a palette
If this check box is activated, the Mono(chrome) image display mode is switched automatically when
a palette is selected in the Color Palette window.
(b)
Switch to "color" on deactivation of a palette
If this check box is activated, the Color image display mode is switched automatically when a palette is
deactivated in the Color Palette window.
(c)
Remember color Palette
If this check box
previous image.
(d)
is activated, the consecutive image will be displayed using the same palette as the
The background color of images and diagrams can be chosen when clicking on the respective
color bar
.
4.9.2.17
Save
The Save tab permits the presetting for the
storage of scanned or processed images to be
changed.
Activation of one of the three option buttons
enables you to determine the database directories
to which stored images are assigned:
− Image files to subdirectory of the database
− Database and image files to the same
directory
− At "Create Database" automatically create a subdirectory with the same name as the specified
database and create the database and image files in that subdirectory
Fig. 4-217
Save tab
If the Save prompt at closing modified windows check box is activated, you are automatically asked
on closing a changed Image Display window whether the image shall be stored.
If the Warning before overwrite existing recordsets check box is activated, this question is asked
automatically on storing an image under a new name if an image file with this name already exists in the
database.
If the Remember "Name", "Description" and "Notes" in the save dialog check box is activated, the
Name, Description and Notes text boxes of the Save Image and Parameter As window show the text
for the image last saved. You can edit the text boxes as required for the new image to be saved.
If the Remember "Name", "Description" and "Notes" in the save dialog check box is deactivated,
the three text boxes are blank when the Save Image and Parameter As window is opened.
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OPERATION
Options Menu
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4-209
OPERATION
Maintain Menu
Carl Zeiss
4.10
LSM 5 LIVE
LSM 5 LIVE DuoScan
Maintain Menu
The Maintain menu contains additional modules to check and guarantee the interference-free operation
of all the software and hardware components of the LSM 5 LIVE.
• In the Main menu toolbar, click on Maintain. This opens another subordinate toolbar in the Main
menu.
Fig. 4-218
Maintain menu
4.10.1
Scanner
The Scanner function is used for scanner
calibration.
The following type of calibrations are available:
− Electrical calibration with
(unidirectional / bidirectional)
− Electrical calibration
(unidirectional)
Fig. 4-219
Scanner Calibration window
4.10.1.1
Electrical Calibration
with
Speed
Speed
1-10
10-11
Electrical Calibration with Speed 1-10 can be performed unidirectional / bidirectional, but with
Speed 11-13 only unidirectional
(1)
Preliminary notes
The electrical calibration has to be performed every 2-3 months. For electrical calibration no laser
scanning is performed and for that reason no calibration sample is needed.
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LSM 5 LIVE DuoScan
(2)
OPERATION
Maintain Menu
Carl Zeiss
Calibration conditions
Before the calibration process can be started, the system has to be in operation for at least one hour.
(3)
Calibration procedure
• Click on the Scanner button in the Maintain subordinate toolbar of the Main menu.
− This opens the Scanner Calibration window.
• Click on the Speed 1-10 (electr.) or Speed 11-13 (electr.) button respectively.
• Activate the Display Graphics check box enables to check the progress of the calibration process on
the Progress Status bar.
− During successful calibration process, the status button is of green color, in case of an error it
switches to red. The progress of the calibration process is indicated by the Progress Status bar.
The calibration process is completed, when the indicator button is grayed.
• Click on the Calibrate button to start the automatic scanner calibration.
• Confirm warning information with OK.
• Click on the Close button to close the Scanner calibration window.
The More function is for servicing purposes only and can only be performed by authorized personnel. Its
access is therefore password-protected.
4.10.1.2
Important Notes and Hints
The tuning procedure runs automatically to a large extent without any problems. However, several errors
can occur. That’s why it is strongly recommended to observe the complete calibration process.
If a status error message occurs or the calibration procedure is not finished properly, this can have the
following reasons:
Non-regularities of the scanner feedback.
The ticks on the outer sides of the grid vary about more than 1 tick width between consecutive
images (in the middle of the calibration process, the linearity is optimized and the problem starts
to occur).
• Stop the calibration procedure.
• Call the LSM service hotline.
If optical calibration comes not to a successful end, please contact your service hotline.
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OPERATION
Maintain Menu
Carl Zeiss
4.10.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Objective
This function permits changed objectives to be
activated and the parfocality to be set without
having to exit the software.
4.10.2.1
Objective Change
• Change the required objective in the nosepiece.
• Click on the Objective button in the Maintain
subordinate toolbar of the Main menu.
− The Objective Control window appears on
the screen. The Objective button is
activated in accordance with the presetting,
and the Objective panel is displayed in the
Objective Control window.
Fig. 4-220
Objective Control window
• Click on the graphical button of the relevant
nosepiece mount (Position).
− The Change Objective window appears.
All available objectives are listed in the Potential
Objectives directory of the Change Objective
window.
• Select the new objective by double clicking from
the list in the Potential Objectives directory.
• Click on Close to exit the Change Objective
window.
(1)
Add Objective
This function permits new objectives to be added
to the database.
For this, proceed as follows:
• Click on the Add Objective button on the
Change Objective window.
Fig. 4-221
4-212
Change Objective window
− The Create new Objective window is
opened.
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OPERATION
Maintain Menu
Carl Zeiss
• Enter the data of the new objective in the
Create new Objective window, then click on
the Apply button.
The new objective is stored in the database and
included in the Change Objectives window. You
can now activate the new objective as a favorite
objective using the procedure described above.
If you have activated the Non Zeiss
check box, objectives from other
manufacturers can also be included in the
database.
(2)
Fig. 4-222
Create new Objective window
Remove Objective
You can only remove objectives in the User Defined Objectives directory.
• To remove an objective from the database, select it with a click of the mouse in the Change
Objective panel and then click on Remove Objective. The objective is removed from the list.
• Click on Close to close the Remove Objective window.
(3)
Edit Objective
You can only edit objectives in the User Defined
Objectives directories.
• To edit an objective from the database, select it
with a click of the mouse in the Change
Objective panel and then click on Edit
Objective. The Edit user defined Objective
panel will open and the parameters of the
Objective can be edited.
• Click on Close to close the Edit user defined
Objective window.
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Fig. 4-223
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Edit user defined Objective window
4-213
OPERATION
Maintain Menu
Carl Zeiss
4.10.2.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Focus Speed Change
• Change the required objective in the nosepiece.
• Click on the Objective button in the Maintain
subordinate toolbar of the main menu.
− The Objective Control window appears on
the screen. The Focus Speed has to be
activated in the Objective Control window.
− The focusing speed of the relevant objective
can be selected by using either the slider or
the input box in 40 steps.
Fig. 4-224
Focus Speed window
4.10.2.3
Parfocality Correction
The parfocal setting is performed via screen dialogs
in successive panels.
• Click on the Parfocal Correction button.
− The Parfocal Adjustment panel appears.
• Start the setting with the objective of the
highest
magnification
factor
(reference
objective). Proceed in accordance with the
displayed instructions.
• Click on Start.
− The next dialog is displayed in the Parfocal
Adjustment panel.
• Focus on your slide object.
• Click on the Next step button.
Fig. 4-225
4-214
Objective Control window
• Perform these steps for each objective.
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OPERATION
Maintain Menu
Carl Zeiss
• Click on the Close button to exit the Objective
Control window and accept the settings.
4.10.3
Fig. 4-226
Objective Control window
Fig. 4-227
Pinhole and Collimator window
Pinhole Adjustment
In the Pinhole and Collimator window, the
aperture are optimally aligned and adjusted to the
used beam path (configuration).
The position of the aperture (pixel shift and Ycoordinate) in relation to the detector makes a
major contribution to image optimization.
In all existing standard configurations, the
apertures have already been adjusted at the
factory. These settings are taken over for active
operation when a standard configuration is loaded.
If you want to create a setting that differs from the
standard configurations, adjust the aperture as
follows.
The X-position does not influence
brightness, but shifts the whole image
position in X-direction.
Use the Optimize and Kollimatic functions
(see chapter Macro Menu) first, the
Pinhole Adjustment is not recommended
on the LSM 5 LIVE as a general tool.
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OPERATION
Maintain Menu
Carl Zeiss
4.10.3.1
LSM 5 LIVE
LSM 5 LIVE DuoScan
Open / Close the Pinhole and Collimator Window
• Click on the Pinhole button in the Maintain subordinate toolbar of the Main menu.
− This opens the Pinhole and Collimator window.
• Click on the Close button to quit the window.
4.10.3.2
Pinhole Panel
No further software function can be activated and executed during aperture adjustment.
Pinhole / Description
field:
Selection of apertures (PH1 to PH2) to be adjusted via the Pinhole selection
box, display of the relevant channel in the Description field.
Diameter; Pixel Shift;
Position Y slider:
Setting of size, pixel shift and Y-position of the aperture in relation to the
beam path using the slider or arrow buttons, status display for setting
procedure: green for ready and red for busy.
Store current Position
button:
Storage of the current aperture setting.
Move to stored Position Aperture setting is reset to the position last stored.
button:
Adjust Automatically
button:
Automatic aperture adjustment.
Fast Adjust mode check
box:
If this check box is activated, the aperture adjustment is only performed in a
limited area. Used for readjustment.
Fig. 4-228
4-216
Pinhole panel
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4.10.3.3
OPERATION
Maintain Menu
Carl Zeiss
Collimator panel
Collimator Description
field:
Selection of the collimator (IR / VIS or UV / VIS) to be adjusted via Collimator
selection box, display of selected collimator in the Description field.
Positions field:
Setting of collimator position using the slider or arrow buttons; the display to
the right of the slider indicates the current position, status display for setting
procedure: green for ready and red for busy.
Store Current Position
button:
Stores the current collimator position.
Move To Stored
Position button:
Sets the collimator to the stored value.
Move to Optimal
Position button:
Starts the automatic collimator adjustment. Available for most common
objectives.
Fig. 4-229
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Collimator panel
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OPERATION
Maintain Menu
Carl Zeiss
4.10.3.4
LSM 5 LIVE
LSM 5 LIVE DuoScan
Aperture and collimator Adjustment
Adjustment of the LSM 5 LIVE apertures can be performed manually or automatically.
If several channels are used to produce the image, all the used apertures must be adjusted separately.
(1)
Manual aperture and collimator adjustment
The position of the aperture relative to the detector in terms of Y coordinate contributes substantially to
image optimization.
Requirements to make aperture position changes visible immediately:
− The image must be scanned by the continuous scan method.
− Select a fast scanning speed.
− Measurement with Average Number 1 only (no averaging of several measurements).
− On the Channel Settings panel (click on Channels button in the Scan Control window), select
the aperture size so as to have the best possible image contrast.
• Click on the Pinhole button in the Maintain
subordinate toolbar.
• Select the aperture (pinhole) to be adjusted
from the Description list box.
• Use the Diameter slider to set the smallest
possible size which produces a good, highcontrast image.
− This setting changes the aperture size.
− The Z Slice display box simultaneously
displays the depth resolution corresponding
to the aperture size.
Image optimization can be effected with
the Range Indicator or in the Line-Scan mode.
Fig. 4-230
Pinhole panel
• Optimize the pixel shift and the aperture
position in Y relative to the PMT using the Pixel
Shift and Position Y sliders to maximum image
brightness.
• Click on the Save Current Position button to save the aperture adjustment.
• Removing the Position slider in the Collimator panel allows the collimator to be adjusted to
maximum image brightness. Optimum collimator adjustment obtained in this way can be stored by
clicking on the Save Current Position button.
• Click on the Stop button to stop the continuous scan.
Please do not make any program manipulations while the automatic aperture adjustment is
running (status display is red - busy).
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(2)
Automatic aperture
adjustment
OPERATION
Maintain Menu
and
Carl Zeiss
collimator
The automatic adjustment allows the LSM 5 LIVE
apertures to be used with any combination of
beam splitters.
• Click on the Adjust Automatically button.
− The Requirements for
window will then appear.
Adjustment
• Meet the requirements listed in the
Requirements for Adjustment window and
press the OK button.
− Aperture adjustment will then run
automatically. The adjusting procedure takes
approx. 3 min.
Fig. 4-231
Requirements for Adjustment window
− The
determined
data
are
stored
automatically and will be available for all
further examinations using the same
configuration.
• The Move To Stored Position button enables the collimator to be set back to the factoryadjustment.
• Activate the Fast Adjust Mode check box for a faster readjustment.
A change of the aperture size made manually in the Pinhole panel will not be activated in the
Scan Control window. Therefore, changes must always be made in the Channel Settings
panel of the Scan Control window.
A filter change in Autoadjust is not displayed in the Config. Control window.
Please do not make any program manipulations while the automatic aperture adjustment is
running (status display is red - busy).
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OPERATION
Maintain Menu
Carl Zeiss
4.10.4
LSM 5 LIVE
LSM 5 LIVE DuoScan
Camera
In the Camera Color Adjustment window the
user can adjust the white balance and color
balance of a connected camera.
Clicking the Pic button allows to set the white
balance using the mouse cursor in the camera
image.
Using the arrow buttons to adjust the color
balance of the camera.
Fig. 4-232
Camera Color Adjustment window
4.10.5
Reboot
The Reboot function is for servicing purposes only and may only be performed by authorized personnel.
Its access is therefore password-protected.
4.10.6
HW/Admin
The HW/Admin function is for servicing purposes and may only be used by authorized service personnel.
Its access is therefore password-protected.
4.10.7
Test Grid
The TestGrid function is for servicing purposes only and may only be performed by authorized personnel.
Its access is therefore password-protected.
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4.11
OPERATION
Window Menu
Carl Zeiss
Window Menu
The Window menu includes the additional
functions Full Screen, Close All Image
Windows, Toolbar and Scan Information which
are not available from a toolbar.
Fig. 4-233
4.11.1
Window menu
Full Screen
This function shows the active Image Display window in full screen size.
• Activate the image to be shown in full size by clicking on the image content.
• Click on the term Window in the menu bar of the Main menu.
− The Window menu (pull-down) will be opened.
• Click on the Full Screen line.
− The image will be displayed in full screen size.
• Click in the image to show it again as an Image Display window in normal size.
4.11.2
Close All Image Display Windows
This function closes all the opened Image Display windows.
• Open the Window menu.
• Click on the Close All Image Windows line.
− All the opened Image Display windows will be closed.
In the Options menu in the function Settings in tab Save at position Save prompt at closing
modified windows it can be determined whether a prompt is shown on Closing of All Image Display
Windows or not.
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OPERATION
Window Menu
Carl Zeiss
4.11.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Scan and System Information
This function opens the Scan Information
window, in which the current scan data are
displayed.
The extent of the data displayed in the Scan and
System Information window depends on the
settings made in the Options menu under
Settings (see page 4-200).
• Open the Window menu.
• Click on the Scan Information line.
− The Scan and System
window will be displayed.
Fig. 4-234
4-222
Scan Information window
Information
button to close the Scan
• Click on the
Information window.
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4.12
OPERATION
Help Menu
Carl Zeiss
Help Menu
The Help menu permits activation of the Help function and of a window containing information on the
installed software version.
4.12.1
Help
• Open the Help menu.
• Click on the Help line to open the online help.
4.12.2
About
• Open the Help menu.
• Click on the About line to open the About
window.
The
About
window
includes
important
information about the software, such as the
software version number, copyright, version
numbers of the various program components and
firmware, and the Dongle number.
• Click on the Close button to close the About
window.
Fig. 4-235
4.12.3
About LSM 5 LIVE window
FRAP Guide
FRAP Guide opens up a SW integrated Guide for FRAP experiments. Use the buttons in the Guide to
set up your experiment. Follow the steps and the descriptions. You are also referred to relevant literature
and the website of the EAMNET (see also Kinetic Analysis Tool (optional), on page 4-146).
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OPERATION
Display and Analysis of Images
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.13
Display and Analysis of Images
4.13.1
Structure of the Image Display
Window
The Image Display window shows the image or
images when they are
− scanned by any scanning function (see Scan
control and Time series control) or
− loaded from the image database (see Open
database-Load) or
− imported
Import).
Fig. 4-236
Image Display window
single frame image
showing
a
by
the
import
function
(see
In addition to show images the Image Display
window offers two toolbars for
− changing the display parameters and save an
image or images (see Select toolbar below)
− generating new ways of displaying the data
as well as analysis tools (see Display toolbar
below).
The Image Display window of the LSM 5 software corresponds to the basic structure of other
Microsoft ® WINDOWS applications. The Image Display window can be moved as required within the
screen, and its vertical, horizontal and diagonal size can be matched to the current requirements
(identical to Microsoft ® WINDOWS).
The caption at the top of the Image Display window contains the control menu for the Image Display
window (identical to Microsoft ® WINDOWS), the name of the displayed image, and the Minimize,
Maximize and Close buttons.
In the status line at the bottom of the Image Display window, the progress bar of a current scanning
procedure and the parameters used for image display are shown and updated when changed.
On the left-hand side of the Image Display window, an overview of the scan parameter is displayed,
provided that the Info button of the Display toolbar is activated.
The Settings function of the Options subordinate toolbar with the Image Display tab some of the
functions of the Image Display window toolbars can be activated at the opening of a new Image
Display window.
It is possible to display the Chan, Zoom, Slice and Overlay image display toolbars immediately on
opening an Image Display window. The relevant check boxes to be activated in the Image Display
Toolbars tab under Settings (see Options menu).
It is also possible to display the scan parameter of an image (Info button) immediately when an Image
Display window is opened. The data to be displayed can be defined (see Image Status Display tab
under Settings in the Options menu.
The set of functions available at the Image Display window toolbars depends on the type of image
shown. The LSM 5 software handles the following formats:
− frame (single image and Z Stack of images)
− frame time series (time series of images and time series with Z Stack of images)
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OPERATION
Display and Analysis of Images
Carl Zeiss
− line time series (time series of lines and time series with Z Stack of lines)
− point time series (time series of points)
4.13.2
Display Modes
The following display modes are available for the different acquisition modes:
Image type
Frame
Series type
Frame
Frame
Z Stack
Time
Line
Line
Time
Display functions
xy
•
•
•
xt
•
•
tx
•**
•
•
•
•**
•
•
•
•
•
•**
•
•
•
•
•
Split xy
•*
•*
•*
Split tx
Coded
•
Ortho
Max
Cut
•
Gallery
•
•
Histo
•
•
•
Profile
•
•
•
Diagr
•
Mean
t***
Select
•
•
•
3D
•**
•***
•**
Topo
•**
•***
•**
Prev
•
•
•
•
•
Info
•
•
•
•
•
2.5 D
* only active in case of multi channel images
** inactive
*** optional
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OPERATION
Display and Analysis of Images
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
All display functions are exclusive functions. Only one can be active at a given time. To generate different
views of the same image set use the Duplicate function in the Process menu.
During image acquisition all active display functions can be used.
4.13.3
Select - Chan
This function permits to
− change the color assignment of channels of images
− switch individual channels of a multi channel image on/off
− switch to monochrome display of the image instead of color display
• Click on the Chan button in the Select toolbar.
− The Channels toolbar will be displayed on the right-hand side of the Image Display window.
Any changes done with this toolbar are effective immediately.
• Click on the Chan button again to remove the Channels toolbar.
Fig. 4-237
4-226
Image Display window; Select - Chan
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4.13.3.1
OPERATION
Display and Analysis of Images
Carl Zeiss
Assigning Another Color to a
Channel
• Click on one of the channels button in the
Channels toolbar (e.g.: Ch1).
Fig. 4-238
Color selection box
− The color selection box with all the currently
defined colors will appear.
• Click on the required color.
− The selected color will be assigned to the current channel, the color selection box is closed and the
displayed image is updated. The control box of the channel button (e.g.: Ch1) also shows the
selected color.
4.13.3.2
Switching a Channel of a Multi Channel Image off or on
• Click on one of the channel buttons in the Channels toolbar (e.g.:Ch1).
− The color selection box will appear.
• Click on OFF to deactivate the display of the relevant channel.
A newly assigned color or a channel switched off is not taken into consideration during the
following scanning procedure, since the setting in the Configuration Control window always
applies here.
4.13.3.3
Switching to Monochrome Image
Display
• Click on the Mono button in the Channels
toolbar.
− The image will then be displayed in shades
of gray exclusively. If you click on the button
again, the channels will be displayed in color
again.
If you want to view the channels
individually, select the split display by
clicking on Split xy button in the
Display toolbar.
4.13.3.4
Defining a New Color
• Click on the Colors button to open the
Channel Colors window.
• Define a new color in the same way as in the
Configuration Control window (see page
4-51).
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Fig. 4-239
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Channel Colors window
4-227
OPERATION
Display and Analysis of Images
Carl Zeiss
4.13.4
LSM 5 LIVE
LSM 5 LIVE DuoScan
Select - Zoom
This function allows to change the zoom factor of an image displayed.
Click on Zoom will display the Zoom toolbar. Any changes done with this toolbar are effective
immediately.
The image can be zoomed by various methods. The zoom function can be performed online.
• Click on the Zoom button in the Select toolbar.
− The Zoom toolbar will be displayed on the right-hand side of the Image Display window.
• Clicking on the Zoom button again will remove the Zoom toolbar.
Fig. 4-240
Image Display window; Select - Zoom
Zoom-Auto
The image is fitted automatically to size of the Image Display window.
Zoom-Resize
Restores the image to its initial size.
Zoom +
Enlarges the image by factor 2.
Zoom –
Reduces the image by factor 2.
Zoom 1:1
Restores an image zoomed in any way to its original size.
Zoom-Mouse
Allows you to enlarge / reduce the zoom factor of an image using the
left / right mouse button, provided that the cursor is inside the image.
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OPERATION
Display and Analysis of Images
Carl Zeiss
When active it allows you to zoom all images of a gallery to the same extent
without changing the display image format.
All
Zoom-+, Zoom-–, Zoom 1:1, Zoom-Mouse and All can only be
defined when the Zoom-Auto function is deactivated.
Slider with display box
4.13.5
The zoom factor can be set by moving the slider. The display box below
displays the current zoom factor. Factor 1 corresponds to the original size.
Select - Slice
This function allows to select and view individual slices from a Z Stack or a time series, when images
where acquired in frame mode.
The button is grayed, when these conditions are not true.
Click on Slice will display the Slice toolbar. Any changes done with this toolbar are effective immediately.
• Click on the Slice button in the Select toolbar.
− The Slice toolbar is displayed on the right-hand side of the Image Display window.
• If you click on the Slice button again, the Slice toolbar is removed.
Fig. 4-241
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Image Display window; Select - Slice
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OPERATION
Display and Analysis of Images
Carl Zeiss
4.13.6
LSM 5 LIVE
LSM 5 LIVE DuoScan
Select - Overlay
This function allows to
− select from a set of drawing functions such as rectangles and arrows
− add a scale bar to the image
− use a set of interactive measurement functions for length, angle and size
4.13.6.1
Functional Description
The overlay function uses a plane separate from the image plane (the graphics plane) and does therefore
not change the content of the image(s).
The button is only available if the XY or Split XY Display functions are selected. Otherwise it is grayed.
Some of the Display functions such as Ortho or Cut turn the overlay graphics off temporarily.
Any changes done with this function are effective immediately.
The overlay graphics can be stored together with images and can be retrieved from the LSM 5 image
database.
• Click on the Overlay button in the Select toolbar.
− The Overlay toolbar will be displayed on the right-hand side of the Image Display window.
• If you click on the Overlay button again, the Overlay toolbar will be removed.
Provided that the display of the overlay elements has not been deactivated by clicking on the Off button,
the created elements will still be displayed in the Image Display window even after closing of the
Overlay toolbar.
Fig. 4-242
4-230
Image Display window; Select - Overlay
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4.13.6.2
OPERATION
Display and Analysis of Images
Carl Zeiss
Buttons in the Overlay Toolbar
The following functions can be used on activation of the buttons in the Overlay toolbar:
Arrow (selection) button: Activation of the mouse button for selection, resizing or
movement of an overlay element in the Image Display window.
Resizing: Click on the handle and hold down the mouse button, drag the handle, release
the mouse button.
Movement: Click on the line and hold down the mouse button, move the entire element,
release the mouse button.
Line button: Creation of a straight line in the Image Display window.
Click and hold down the mouse button, draw a line in any required direction, release the
mouse button to end the procedure.
Rectangle button: Creation of a rectangle in the Image Display window.
Click and hold down the mouse button, draw a rectangle in any required direction,
release the mouse button to end the procedure.
Closed polyline button: Creation of a closed polyline figure in the Image Display
window.
The first click sets the starting point, each additional click adds a further line, a click with
the right mouse button closes the figure and ends the procedure.
Open polyline button: Creation of an open polyline figure in the Image Display
window.
The first click sets the starting point, each additional click adds a further line, a click with
the right mouse button ends the procedure.
Ellipse button: Creation of an ellipse in the Image Display window.
The first click sets the center point, the displayed line permits the determination of the
first dimension, the second click sets the first dimension, the second dimension and
rotation direction can then be determined, the third click sets the second dimension and
direction and ends the procedure.
Closed free-shape curve button: Creation of a closed Bezier figure in the Image
Display window.
The first click sets the starting point, each additional click adds a further line, a click with
the right mouse button closes the figure and ends the procedure.
Open free-shape curve button: Creation of an open Bezier figure in the Image Display
window.
The first click sets the starting point, each additional click adds a further line, a click with
the right mouse button closes the figure and ends the procedure.
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Carl Zeiss
OPERATION
Display and Analysis of Images
LSM 5 LIVE
LSM 5 LIVE DuoScan
Circle button: Creation of a circle in the Image Display window.
Clicking and holding down the mouse button sets the center point, drag the diameter
and release the mouse button to end the procedure.
Line with arrow button: Creation of a line with arrow in the Image Display window.
Click and hold down the mouse button, drag the line in any required direction, release
the mouse button to end the procedure.
Scale button: Creation of a horizontal or vertical scale with default increments in the
Image Display window. Click and hold the mouse button for the starting point, drag
horizontal or vertical scale, release the mouse button to end the procedure.
Gray tones / color shades button: Generates a rectangle with a display of gray tones or
color shades in the image. Color shades are displayed if a palette has been loaded, with
different colors being assigned to the gray tones.
A (Text) button: Creation of a text box in the Image Display window.
After clicking on A, the Text window will be displayed, and text can be entered via the
keyboard. The Font ... button enables you to select the font style and size in the Font
window. The entered text will be displayed in the left upper corner of the Image Display
window after clicking on OK and can be moved to the required position using the
mouse.
The Text window can also be activated with a double-click on a created text box, and the
entered text can be edited subsequently.
Insert opens up a further window which allows you to annotate coordinates, time and
Z-position with either automatic or user definable units and precision. This annotation is
updated during image acquisition and can be exported with the image. The annotation
can be stamped into already existing images.
Recycle bin button: All the overlay elements and dimensions dragged to the scanned
image are deleted. If one overlay element was marked before, this element is now
deleted from the scanned image.
Multiple button: On activation of this button, the overlay function subsequently selected
is performed several times in succession, without the need to activate the function button
again. This function remains selected until the Multiple button is deactivated again.
Measure button: Measurement of the overlay element in the Image Display window.
On activation of the Measure button, the selected overlay element and all the elements
created afterwards are measured and assigned with a measuring value. The measuring
value can be shifted without regard to the overlay element. If of importance, the length
and perimeter of a line figure, the area of a closed figure and the inclination angle of a
single line will be displayed. On deactivation of the Measure button, the measuring value
of the selected element is no longer displayed, and all the elements created afterwards
will not be assigned with a measuring value.
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OPERATION
Display and Analysis of Images
Carl Zeiss
Off button: Deactivation of the display of overlay elements in the Image Display
window (hide overlay). Deactivation of the overlay functions.
Line button:
This button allows you to determine the line thickness of the area outline.
Cut button: The image contents of an overlay element are cut out, and the area will then
appear in black.
Copy button: The image contents of a closed overlay element are copied to the
clipboard.
Paste button: The image contents of an overlay element copied to the clipboard are
inserted in the current Image Display window and can be positioned anywhere in the
image using the mouse.
Undo button: The last Cut or Paste action can be undone by clicking on the Undo
button.
Extract Region button: The region of a Z Stack or 4D-image surrounded by an Overlay
element is extracted and can be displayed and stored separately in a new Image Display
window. This function is only active if an Overlay element is used, that generates a
closed contour.
Color selection box: The colors displayed in the Color selection box can be assigned to
the overlay elements with a click of the mouse. The currently selected color is displayed in
the larger rectangle (left top) of the selection box. A selected color is automatically
assigned to the currently selected overlay element and then to all the elements created
afterwards.
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Carl Zeiss
OPERATION
Display and Analysis of Images
4.13.7
LSM 5 LIVE
LSM 5 LIVE DuoScan
Select - Contr
This function allows to
− change the contrast and brightness of an
image
− change the contrast and brightness of a
channel of an image
− define interactively a new relationship
between the intensities of pixels in the image
memory and the displayed values of this
pixel intensities on the computer screen
Click on Contr will display the Contrast toolbar.
Any changes done with this toolbar are effective
immediately.
Fig. 4-243
Modification done by this function are for display
purposes only. To permanently change the contrast
and brightness of an image use the function
Contrast in the Process menu.
Image Display window; Select - Contr
• Click on the Contr button in the Select toolbar.
− The Brightness and Contrast window will be displayed.
• Change brightness and contrast via the sliders in the Brightness and Contrast window. You can
adjust each channel individually by activating the channel button (e.g.: Ch1), or influence all channels
simultaneously by clicking on All.
• Clicking on the Reset button will reset the
original setting of brightness and contrast.
• Clicking on the Close button will close the
Brightness and Contrast window.
Further contrast and brightness parameters can be
activated or deactivated alternately using the More
and Less buttons.
Fig. 4-244
Brightness and Contrast window
• Click on the More button to display the additional functions.
− The Brightness and Contrast window will be enlarged, the labeling of the button changes from
More to Less. If you click on Less, the additional functions are no longer displayed.
Simultaneously with the setting of brightness and contrast, the intensity values of the image can be set
directly in the Intensity Screen via the Ramp, PolyLine, Spline and Gamma functions.
The intensity values can also be set either for all channels together or individually.
If the image has already been changed using the Contrast and Brightness sliders, this setting difference
is displayed in the Intensity Screen by means of the Shape and Result lines.
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4.13.7.1
OPERATION
Display and Analysis of Images
Carl Zeiss
Ramp
The intensity is set via two knots in the Intensity
Screen, which allows an intensity line to be
created in the form of a ramp.
The original line form is reset via Reset.
The line form will be retained even when the
additional functions are no longer displayed, and
on closing the Brightness and Control window.
4.13.7.2
Fig. 4-245
Brightness and Contrast window with
activated Ramp function
Fig. 4-246
Brightness and Contrast window with
activated PolyLine function
PolyLine
The intensity is set in the Intensity Screen via a
freely selectable number of knots, which permits
the creation of an intensity line in the form of a
polyline. The number of knots can be selected
from the Number of Knots selection box.
The original line form is reset via Reset.
The line form will be retained even when the
additional functions are no longer displayed or
when the Brightness and Control window is
closed.
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Carl Zeiss
OPERATION
Display and Analysis of Images
4.13.7.3
LSM 5 LIVE
LSM 5 LIVE DuoScan
Spline
The intensity is set in the Intensity Screen via a
freely selectable number of knots, which permits
the creation of an intensity line in the form of a
spline. The number of knots can be selected from
the Number of Knots selection box.
The original line form is reset via Reset.
The line form will be retained even when the
additional functions are no longer displayed or
when the Brightness and Control window is
closed.
Fig. 4-247
Brightness and Contrast window with
activated Spline function
4.13.7.4
Gamma
The intensity is set in the Intensity Screen by
varying the gamma curve (clicking and dragging
with the mouse) or by moving the Gamma slider.
It is possible to set gamma values between 0.1 and
2.0.
The original line form is reset via Reset.
The line form will be retained even when the
additional functions are no longer displayed or
when the Brightness and Control window is
closed.
Fig. 4-248
4-236
Brightness and Contrast window with
activated Gamma function
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4.13.8
OPERATION
Display and Analysis of Images
Carl Zeiss
Select - Palette
This function allows to
− change the palette used for displaying the image(s)
− define and save new palettes
− delete palettes by removing them
Click on Palette will display the Palette toolbar. Any changes done with this toolbar are effective
immediately.
The standard palettes No palette, Range indicator, Glow Scale and Rainbow are system palettes and
can not be deleted.
The Range indicator palette is useful to optimize the gain and offset setting of images in the Scan
control window before scanning.
Palettes are stored and retrieved together with the images when archived in the Image Database.
• Click on the Palette button in the Select toolbar.
− The Color Palette window will be displayed.
• Select the required palette from the Color Palette List panel by clicking on the relevant name.
• If you want to deactivate a palette selected before, click on No Palette in the Color Palette List
panel.
• Click on the Close button to close the Color Palette window.
• A changed image can be stored via the Save As function.
In the Options menu in the function Settings it is possible to switch to Mono automatically when a
palette is activated and to Colour on deactivation of a palette.
In addition it is possible to activate / deactivate Mono in the Channel toolbar.
Some of the handling functions of the Image Display window toolbars can be activated at the opening
of a new Image Display window.
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OPERATION
Display and Analysis of Images
Carl Zeiss
Fig. 4-249
LSM 5 LIVE
LSM 5 LIVE DuoScan
Image Display window, Select - Palette; Color Palette and Add Palette to List window
4.13.8.1
Editing and Storing a Palette
A palette is edited by moving the knots in the
Ramp, Polyline and Spline functions (identical to
the setting in the Contrast and Brightness
window, see page 4-234f).
The palette can be set for all colors together or
separately for each color.
• Activate the relevant button: Red, Green, Blue
or All.
Proceed as follows to store an edited palette under
a new name:
• Click on the Add To List button: the Add
Palette To List window will be displayed.
• Enter a name for the palette and click on Ok.
− The palette will be stored and the name
included in the Color Palette List panel.
Fig. 4-250
4-238
Color Palette window
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4.13.8.2
OPERATION
Display and Analysis of Images
Carl Zeiss
Delete a Palette
Proceed as follows to delete a palette:
• Click on the name of the palette to be deleted in the Color Palette List panel and then on the
Remove button.
− The palette will be removed from the list.
The standard settings (No Palette, Range Indicator, Glow Scale and Rainbow) cannot be
deleted.
4.13.8.3
Import a Palette
Proceed as follows to import a palette:
• Click on the Import button. The Import Palette window will be opened.
• Select the required palette (file extension: *.lut) from the relevant directory and click on Open.
− The palette will be imported and displayed in the Color Palette List panel.
File with the extension *.lut are LSM 310 / 410 palette files.
4.13.9
Select - Anim
This function allows to
− animate frames of a Z Stack or a time series
− specify animation parameters such as range
and animation speed
Click on Anim will display the Animate toolbar.
Any changes done with this toolbar are effective
immediately.
When the image(s) displayed in the Image Display
window is neither a Z Stack nor a time series this
button is grayed and not accessible.
Fig. 4-251
Animate window
• Click on the Anim button in the Select toolbar
of the Image Display window of a stack.
− The Animate window will be displayed and
the animation started immediately.
• Click on the Close button to close the Animate window and to stop the animation.
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OPERATION
Display and Analysis of Images
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
The animation is controlled via the following function elements:
Current Slice slider: Manual movement through the individual slices of a stack by
moving the slider, or by entering the slice number in the input box. Slider can be accessed
only, when the automatic animation is off.
Start slider: The setting of the Start sliders limits the number of slices to be used for the
animation. Previous slices are not taken into consideration for the animation. Can be
changed during automatic animation.
End slider: The setting of the End slider limits the number of slices to be used for the
animation. Subsequent slices are not taken into consideration for the animation. Can be
changed during automatic animation.
Starts the forward motion of the automatic animation. After the last slice has been
passed, restart is made at the first slice.
Starts backward motion of the automatic animation. After the first slice has been passed,
restart is made at the last slice.
Starts the combined forward / backward motion of the automatic animation, i.e. when
the last slice has been reached, the backward motion is activated, and the forward
motion is activated again on reaching the first slice.
Stops the automatic animation.
Move to the first slice.
After each click on this button, backward motion is made by the number of slices set
under Increment.
After each click on this button, forward motion is made by the number of slices set under
Increment.
Move to the last slice.
Speed1 /Speed2 buttons / sliders: Selection between two speeds, change of the
relevant speed via slider or input box.
Increment slider: Reduction of the slices to be displayed by selecting an increment n
(step width) of slices to be taken into consideration for the animation. If n = 3, for
example, only every third slice of the stack will be displayed during the animation.
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4.13.10
OPERATION
Display and Analysis of Images
Carl Zeiss
Select - Reuse
This function allows to
− transfer acquisition parameters of an image from the image data base to the Microscope control,
Configuration Control, Scan Control and Time Series Control windows and applies those
parameters directly on the system.
The acquisition parameters of an image are displayed in the Image Display window and can be viewed
by using the Info function. In the tab Image Status Display in the Settings function of the Options
subordinate toolbar it can be determined what parameters to view with the Info function.
The parameters include the following:
Frame Size, Speed, Data Depth, Scan Direction, Average, Zoom Rotation, Offset, Aperture Size (confocal
aperture), Detector Gain, Amplifier Offset, Amplifier Gain, Excitation, Beam Path and Scan Mode (Line,
Frame, Stack, Time Series).
However, the required objective must be selected by the user.
• Click on the Reuse button. The acquisition parameters of the active image (stack) are applied
immediately to the system.
In the Options menu in the function Settings with the Recording/Reuse tab, it can be determined
whether the objective should also be transferred and set. Setting the microscope objective only works in
microscopes with motorized objective revolvers.
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OPERATION
Display and Analysis of Images
Carl Zeiss
4.13.11
LSM 5 LIVE
LSM 5 LIVE DuoScan
Select - Crop
This function allows to
− interactively define the size and orientation
of a rectangular scan area on the image
displayed in the Image Display window.
− The defined area is displayed together with
the Zoom and Offset parameters in the
Scan Control window in the Mode
submenu.
Fig. 4-252
Image Display window; Select - Crop
Click on Crop will display the Crop Rectangle in
the Image Display window. Any changes done
with the Crop Rectangle are setting the
parameters immediately. On the next execution of
a scan (Find, Fast xy, Single, Continuous in
Scan Control or Start T or Start B in Time Series
Control) these new scan parameters will be used.
To reset the crop function and use default values
set Zoom=1 and Offset=0 in the Scan Control
window in the Mode submenu.
• Click on the Crop button.
− The Crop Rectangle will appear on the Image Display window.
The Crop Rectangle is controlled via the following functional elements:
Offset
• Click into the crop rectangle, keep the left mouse button pressed and drag the crop
rectangle to the required position. Release the mouse button.
Zoom
• Click on a corner of the crop rectangle, keep the left mouse button pressed and set
the required size. Release the mouse button.
Side ratio
• Click on any of the intersection points between crossline and crop rectangle, keep
the left mouse button pressed and change the side ratio as required. Release the
mouse button.
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4.13.12
OPERATION
Display and Analysis of Images
Carl Zeiss
Select - Copy
This function allows to
− copy the current displayed image into the clipboard.
Click on Copy will be immediately effective.
From the clipboard images can be incorporated into other programs such as MS Excel, MS Powerpoint or
MS Word.
To export image series, use the Export function in the File menu.
• Click on the Copy button.
− The content of the Image Display window is copied to the clipboard.
• Start the clipboard application of WINDOWS.
• Select Paste in the Edit menu of the Clipboard application.
4.13.13
Select - Save
This function allows to
− save the image(s) of the Image Display window into an Image Database
− by not showing a dialogue and using the automatic assigned and incremented image name and a
predefined existing Image Database
− Prerequisite: Autosave is checked in the Settings function with the Autosave tab
Click on Save will be immediately effective.
When the prerequisite is not met, the Save As dialogue is displayed.
In the Options menu in the function Settings with the Autosave tab parameters such as an
automatically incremented filename can be determined and the Autosave activated/deactivated.
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OPERATION
Display and Analysis of Images
Carl Zeiss
4.13.14
LSM 5 LIVE
LSM 5 LIVE DuoScan
Select - Save As
This function allows to
− save the image(s) of the Image Display window into an Image Database
− by showing a dialogue
− use the defaults as defined in the Settings function with the Save tab
Click on the Save As button displays the Save Image and Parameter As window. Changes will be
effective on closing this dialogue.
In the Options menu in the function Settings with the tab Save default parameters such as Name,
Description and Notes can be set.
• Click on the Save button.
− The Save Image and Parameter As window appears
• Enter text for the image name, description, notes or change the user name.
• Select the Image Database from the list of databases (MDB) or
• Open other Image Databases by selecting Open MDB or
• Create new Image Databases by selecting New MDB.
4.13.15
Display - xy
This function allows to
− display a single image in frame mode
− display multi channel images in superimposed mode
The settings of Chan, Zoom, Slice, Overlay, Contr and Palette are applied.
Click on xy will be immediately effective.
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4.13.16
OPERATION
Display and Analysis of Images
Carl Zeiss
Display - Split xy
This function allows to
− display the individual channels of a multi channel image as well as the superimposed image
The settings of Chan, Zoom, Slice, Overlay, Contr and Palette apply.
Click on Split xy will be immediately effective.
Fig. 4-253
Image Display window, Split xy display
This function is useful to optimize the individual channels in a multi channel image acquisition
together with the Range Indicator palette .
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OPERATION
Display and Analysis of Images
Carl Zeiss
4.13.17
LSM 5 LIVE
LSM 5 LIVE DuoScan
Display - Ortho
This function allows to
− display a Z Stack of images in an orthogonal view
− measure distances in three dimensions
− generate 2D deconvolution views of the yz and xz plane
The settings of Chan, Zoom, Slice, Overlay, Contr and Palette apply.
Click on Ortho will be immediately effective.
• By clicking on the Ortho button section lines appear in the Display toolbar together with orthogonal
projections in the image. On the right-hand side, the Orthogonal Sections toolbar is shown.
Fig. 4-254
4-246
Image Display window, Ortho display
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OPERATION
Display and Analysis of Images
Carl Zeiss
4.13.17.1 Ortho - Select Function
• By changing the parameters X, Y and Z in the Orthogonal Sections toolbar, the section plane can be
positioned at any XYZ coordinate of the Z Stack.
The position of section planes can be changed in various ways:
• By moving the sliders on the Orthogonal Sections toolbar.
− X and Y settings may range from 1 up to the maximum number of pixels scanned (in the example
shown: 512).
− Z settings may range from 1 to a maximum of n, with n standing for the number of slices produced
in the stack.
• By directly entering the relevant number value in the X-, Y- or Z-input box and pressing the Enter key.
. By
• If you move the cursor into the Image Display window, it changes into a crosslines symbol
dragging this symbol with the mouse you can position the XZ and YZ section planes to any point of
intersection with the XY plane. A click with the left mouse button places the intersection to the
desired position.
• If you move the crosslines symbol
onto the intersection of the red and green section planes, it
symbol. If you now press the left mouse button and keep it pressed you can
changes into the:
reposition both section planes simultaneously.
• If you move the crosslines symbol
onto the green section plane, it changes into the
symbol. If
you now press the left mouse button and keep it pressed, you can reposition the (green) XZ section
plane.
• You can reposition the (red) YZ plane in the same way using the
symbol.
The result of an orthogonal section is visible at the image margin.
− Section of the XZ plane (green line) through the stack: above the XY image.
− Section of the YZ plane (red line) through the stack: right of the XY image.
− Section of the XY plane (blue, slice plane of the stack): center image.
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Carl Zeiss
OPERATION
Display and Analysis of Images
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.13.17.2 Ortho - Distance Function
• Activating the Dist. button permits length measurements in 3D space.
• Click on the Mark button to set the first XYZ-point for the measurement of the spatial distance.
• Set the second XYZ-point for measurement by moving the X-, Y-, Z-sliders or by moving the green, red
and blue lines in the image.
− The projections of the spatial distance are shown in the image planes by yellow lines. The actual
spatial distance is calculated and shown in µm below the Select, Dist. and Mark buttons, e.g. 3D
Distance: 55.60 μm.
Fig. 4-255
4-248
Image Display window, Ortho Distance display
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OPERATION
Display and Analysis of Images
Carl Zeiss
4.13.17.3 Ortho - 2D DeConVolution Function
The 2D deconvolution function causes orthogonal projection enhancement through the computed
correction of the resolution in the Z-coordinate.
Image enhancement is only effective for the two projections of a fluorescence stack in the Ortho display
or for an XZ-scan in fluorescence, and is also only computed for this.
• Activate the 2D DCV button in the Orthogonal Sections toolbar.
If the Fast button is activated, calculation of the 2D-deconvolution (inverse DCV mode) is performed
immediately.
The 2D DCV settings button can only be activated if a licence for the 3D DCV option has been purchased.
Otherwise this button is grayed.
• Click on the Settings button. The 2D Deconvolution window is opened.
The 2D Deconvolution window contains the Deconvolution panel with the two tabs Method and
PSF.
(1)
Method tab
The Method tab enables you to choose between
the calculation methods Inverse and Iterative.
For more details of explanation of deconvolution
and
the
calculation
methods
see
3D
DeConVolution (page 4-163).
In the Inverse method, the Restoration Effect
slider can be used to set the signal-to-noise ratio
between Weak (low noise) and Strong
(pronounced noise).
Fig. 4-256
Method tab
Activation of the Auto detect check box starts a
routine for the automatic determination of the
noise level in the entire image part of the Z Stack.
If Auto detect is enabled, the Restoration Effect
slider is disabled.
The Iterative method permits (in addition to the parameters of the Inverse method) the maximum
number of iterations to be entered between 1 and 200 under Maximum Iterations and the Auto Stop
function to be activated / deactivated. The Auto Stop function interrupts the calculation depending on
the set image improvement (delta between last but one and last cycle in %), no matter whether the value
under Maximum Iterations has been achieved or not.
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OPERATION
Display and Analysis of Images
Carl Zeiss
(2)
LSM 5 LIVE
LSM 5 LIVE DuoScan
PSF tab (optional with 3D DCV)
The objective data are displayed in the PSF tab. In
the case of wavelengths above 700 nm, the NLO
button is automatically enabled.
The displayed values are always taken over by the
system data, but can be edited subsequently for
simulation purposes.
• Select the required method and determine the
relevant parameters.
Fig. 4-257
PSF tab
The deconvolution calculation is performed
immediately after the 2D Deconvolution window
has been closed, and the image display is updated
(on-line).
4.13.18
Display - Cut
This function allows to
− display a Z Stack of images at a user defined
section plane (= cut plane)
− improve the image of the section plane by
trilinear interpolation
The settings of Chan, Zoom, Slice, Contr and
Palette are applied.
• Clicking on the Cut button in the Display
toolbar opens the Cut to the right of the Image
Display window.
Any changes done with this toolbar are effective
immediately. The content of the overlay plane is
temporarily deleted while the toolbar is displayed.
• By varying the parameters X, Y, Z, Pitch and
Yaw, you can position a section plane of any
orientation within the stack volume.
• The resulting position of the section plane is
shown as a red area below the Trilinear
Interpolation button. At the same time, the
result is shown in the Image Display window.
• A click on the Reset All button restores the
original position.
Fig. 4-258
4-250
Image Display window, Cut display
• A click on the Trilinear Interpolation button
will improve the quality of the image by
performing a 3D interpolation of the image.
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4.13.19
OPERATION
Display and Analysis of Images
Carl Zeiss
Display - Gallery
This function allows to
− display images (Z Stack, time series, combination of both) side by side in tiled fashion
− add data relevant to the images displayed (Z Stack slice distance, time of acquisition or wavelength)
− extract a subset of images from the original stack and store the result as a new image
The settings of Chan, Zoom, Slice, Contr and Palette apply.
Click on Gallery will display the Gallery toolbar. Any changes done with this toolbar are effective
immediately.
• A click on the Gallery button in the Display toolbar not only produces the gallery itself but also the
Gallery toolbar with two buttons: Data button and Subset button.
• Clicking on the Data button shows the Z Slice distance, the acquisition time or the wavelength or
combinations.
• Clicking on the color selection button (below the Data button) will open a color selection window
allowing you to choose - at a click of the mouse - in which color the data will be shown in the gallery
display.
• Clicking on the Subset button opens another
window entitled Subset, in which you can
select images of the set of images displayed.
− A stack consisting of the selected images
only is generated and displayed.
• Select Start Slice and End Slice via the sliders,
the input box or the Click (into window)
button.
• Enter a value for n in the Every n-th Slice
panel.
Fig. 4-259
Subset window
• If required, activate the Convert 12 bit to 8 bit
check box .
• Clicking on the Ok button will generate a new
subset of images.
• Cancel will stop the procedure.
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Carl Zeiss
Fig. 4-260
4-252
OPERATION
Display and Analysis of Images
LSM 5 LIVE
LSM 5 LIVE DuoScan
Image Display window, Gallery display
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4.13.20
OPERATION
Display and Analysis of Images
Carl Zeiss
Display - Histo
4.13.20.1 Display - Histo - Overview
This function allows to
− display a histogram (distribution of pixel intensities) of an image
− show the histogram values in table form
− copy table to clipboard or save as text file
− analyze the colocalization between two channels
− measure area and mean gray value and standard distribution in an area
− show separate histograms for each channel in a multi channel image
Colocalization is only available in case of a two or multi channel image.
The settings of Chan, Zoom, Slice, Contr and Palette are applied.
Click on Histo will display the Histogram toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
• Click on the Histo button. The Histogram toolbar will be displayed on the right.
Fig. 4-261
Image Display window, Histogram display
The Histo button can also be used online during scanning.
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Carl Zeiss
OPERATION
Display and Analysis of Images
LSM 5 LIVE
LSM 5 LIVE DuoScan
Histogram functions
Skip Black button
Ignore black pixels (gray value 0) in the histogram.
Skip White button
Ignore white pixels (gray value 255 or 4096) in the histogram.
Statistics
Displays statistical parameters (Mean Intensity, Standard Deviation, Number
of Pixels and Size of Area) in a additional table. Area measurements of very
small areas (< 10 pixels) give only approximate values
Step input box
Set the number of intensity steps which shall be displayed in the diagram.
Step 1 corresponds to 256 intensity steps, Step 64 to 4 intensity steps in case
of 8 bit images. Reduction is made by averaging.
Show Image button
Shows the image in the Image Display window beside the histogram.
Show Table button
The histogram is shown as a table at the bottom left of the Image Display
window.
Copy Table button
The histogram table is copied to the clipboard.
Save Table button
The histogram table can be stored as a text file (extension *.txt).
Area functions
Area button
Interactive definition of area for size and intensity measurement.
Save Values button
Copies area values to the clipboard (only available if the Area button is
activated).
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Display and Analysis of Images
Carl Zeiss
4.13.20.2 Area Function
• Click on the Area button in the Histogram toolbar.
− The function elements for Area measurement are displayed at the bottom right of the Histogram
toolbar.
Fig. 4-262
Image Display window, Area Measure display
The following function elements are available:
Set the number of intensity steps which shall be displayed in the diagram. Step 1
corresponds to 256 intensity steps, Step 64 to 4 intensity steps in case of 8 bit images.
Reduction is made by averaging.
Threshold low slider with Color selection button: The intensity values below threshold
are not displayed. The removed areas are masked in the color selected in the Color
selection button.
Threshold high slider with Color selection button: The intensity values above threshold
are not displayed. The removed areas are masked in the color selected in the Color
selection button.
Ch1, Ch3 ... buttons: Selection of the channel for which the area measurement is to be
performed.
Arrow (selection) button: Activation of the mouse button for selection, resizing, or
movement of a mask element in the Image Display window.
Resize: Click on handle
Movement: Click on line and hold the mouse button, move the entire figure, release
mouse button. and hold down the mouse button, drag the handle, release mouse
button.
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OPERATION
Display and Analysis of Images
LSM 5 LIVE
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Closed polyline button: Creation of a polyline figure in the image. The first click sets the
starting point, each additional click adds a further line, a click with the right mouse
button closes the figure and ends the procedure.
Recycle bin button: All the mask elements are deleted. If one element was marked
before, this element is now deleted from the image.
Mask button: Enables the Mask Mode, where the region can be defined with ink.
Rectangle button: Creation of a rectangle in the image. Click and hold down the mouse
button, drag a rectangle in any direction, release the mouse button to end the procedure.
Ellipse button: Creation of an ellipse in the image. The first click sets the center point,
the displayed line permits the determination of the first dimension, the second click sets
the first dimension, the second dimension and the rotation direction can then be
determined; the third click sets the second dimension and the direction and ends the
procedure.
Line button: Determines the line thickness of the area outline.
Flood fill button: Fills the overlay element or the scatter diagram with the color selected
under Mask.
Closed free-shape curve button: Creation of a closed Bezier figure in the scatter
diagram. The first click sets the starting point, each additional click adds a further line, a
click with the right mouse button closes the figure and ends the procedure.
Circle button: Creation of a circle in the scatter diagram. Clicking and holding down the
mouse button sets the center point; drag the diameter and release the mouse button to
end the procedure.
Color selection button: The colors displayed in the selection box can be assigned to the
mask elements with a click of the mouse. The currently selected color is always displayed
in the larger rectangle (top left) of the selection box.
Clear Mask button: Removes the color filling from an overlay element or from the scatter
diagram.
• The function can be activated by clicking on one of the geometry buttons, e.g.
(polyline).
• The figure of interest can be marked in the image by cursor control in conjunction with a mouse click.
• If more than one Region is drawn either the mean histogram values for all regions is displayed or only
the values of the activated region if it is marked by clicking on the drawing line with the mouse.
• Areas can also be excluded from the analysis. By clicking on the Flood fill button (paint jar) and
moving the cursor to the area to be excluded causes the remaining area to be computed and the
result indicated under Area Measure.
• If you specify a top and bottom intensity threshold, the area lying within this intensity interval can be
computed.
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OPERATION
Display and Analysis of Images
Carl Zeiss
• Specify the thresholds either with the Threshold low and Threshold high sliders, or with the ▲▼
buttons.
4.13.20.3 Colocalisation Function
The Colocalisation function permits interactive analysis of two channels of an image by computing a
scatter diagram (colocalisation).
• Click on the Colocalisation button. The scatter diagram is created and displayed beside the image.
• Click on the xy button of the Display toolbar if you want to return to the original image.
How a scatter diagram is generated
All pixels having the same positions in both images are considered a pair. Of every pair of pixels (P1, P2)
from the two source images, the intensity level of pixel P1 is interpreted as X coordinate, and that of pixel
P2 as Y coordinate of the scatter diagram. The value of the pixel thus addressed is increased by one every
time, up to the maximum number of pixels used. This way, each pixel of the scatter diagram is a value
that shows how often a particular pair of pixels has occurred.
Differences between the images cause irregular spots in the scatter diagram.
Identical images produce a clean diagonal line running from bottom left to top right, because only pixel
pairs (0,0), (1,1), (2,2) with the same intensity can occur. Differences between the images cause an
irregular distribution in the scatter diagram.
Fig. 4-263
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Image Display window, Colocalization display
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OPERATION
Display and Analysis of Images
LSM 5 LIVE
LSM 5 LIVE DuoScan
Function elements
The following function elements are available:
Displays the scatter Histogram of the two image channels
Adds display of the according data table
Adds display of the image
Displays a movable crosshair for different areas in the scatter histogram
Opens the Intensity Threshold window to sets threshold for colocalisation
in the scatter histogram.
Set from Image ROIs button: Sets background threshold from ROI
(Threshold button)
Cut Mask button.
Channel Toggle button.
Invert Mask button: Inverts the mask or the scatter diagram.
Source 1 selection box with Color selection box: Selection of the first
channel to be selected via the selection box, assignment of a defined color
via the Color selection box.
Source 2 selection box with Color selection box: Selection of the second
channel to be selected via the selection box, assignment of a defined color
via the Color selection box.
Mask selection box with Color selection box: Selection of RGB or Overlay
display of the mask, assignment of a defined color via the Color selection
box.
Drawing tools
Allows the selection of ROIs in the Histogram and the Image.
Save at drawing tools
Stores ROIs and threshold settings.
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Carl Zeiss
Enhanced colocalisation
Scatter diagram image display and data table are interactively linked.
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Fig. 4-264
Image Display window, Colocalization display
Fig. 4-265
Scatter diagram and threshold with crosshair
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OPERATION
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Quantitative Parameters
− No. of pixels in image ROI or scatter region
− Area / relative area of image ROI or scatter region
− Mean intensities / SD within image ROI or scatter region
− Colocalization coefficients
− Weighted colocalization coefficients
− Overlap coefficient after Manders
2
− Correlation coefficients (R and R )
Colocalization coefficients
c1 =
pixelsCh1,coloc
c2 =
pixelsCh1,total
pixelsCh 2,coloc
pixelsCh 2,total
− Relative number of colocalizing pixels in channel 1 or 2, respectively, as compared to the total
number of pixels above threshold.
− Value range 0 – 1 (0: no colocalization, 1: all pixels colocalize)
− All pixels above background count irrespective of their intensity.
Weighted colocalization coefficients
∑ Ch1
=
∑ Ch1
i,coloc
M1
∑ Ch2
=
∑ Ch2
i,coloc
i
M2
i,total
i
i
i,total
i
− Sum of intensities of colocalizing pixels in channel 1 or 2, respectively, as compared to the overall
sum of pixel intensities above threshold and in this channel.
− Value range 0 – 1 (0: no colocalization, 1: all pixels colocalize)
− Bright pixels contribute more than faint pixels
Correlation coefficient, Pearson´s correlation coefficient
Rp =
∑ (Ch1
i
− Ch1aver ) * (Ch 2 i − Ch2 aver )
i
∑ (Ch1
i
i
− Ch1aver ) 2 * ∑ (Ch 2 i − Ch 2 aver ) 2
i
− Provides information on the intensity distribution within the colocalizing region
− Value range -1 to +1
-1,+1: all pixels are found on straight line in the scatter diagram
0:
pixels in scattergram distribute in a cloud with no preferential direction
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Display and Analysis of Images
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Overlap coefficient, overlap coefficient after Manders
(Manders, Verbeek and Aten, J. Microscopy 169:375-382, 1993)
r=
∑ Ch1 * Ch2
i
i
i
∑ (Ch1 ) * ∑ (Ch2 )
2
i
i
2
i
i
− Another parameter used to quantify colocalization in image pairs
− Insensitive to differences in signal intensities between the two channels, photo-bleaching or
amplifier settings
− Value range 0 – 1 (0: no colocalization, 1:all pixels colocalize)
4.13.21
Display - Profile
4.13.21.1 Function
This function allows to
− display the intensity distribution of an image along a straight or curved line
− show the intensity values in table form and copy table to clipboard or save as text file
− show separate profiles for each channel in a multi channel image
The settings of Chan, Zoom, Slice, Contr and Palette are applied.
Click on Profile will display the Profile toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
• Click on the Profile button. The Profile toolbar will be displayed.
− The intensity curves are shown in a graph below the image(s).
• On the Profile toolbar you can select the width and color of the profile line.
• You can place two markers on the profile line to measure differences in intensities and distances in the
XY plane.
The Profile button can also be used online during scanning.
• Click on the Diagr. in Image button to see an intensity graph superimposed on the image.
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OPERATION
Display and Analysis of Images
Fig. 4-266
Image Display window, Profile display
Fig. 4-267
Image Display window, Profile display
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Display and Analysis of Images
Carl Zeiss
4.13.21.2 Function Elements
The Profile toolbar contains the following function elements:
Arrow (selection) button: Activates the mouse button for selection, resizing or movement
of the profile line in the Image Display window.
Resize: Click on handle and hold down the mouse button, move the handle, release
mouse button.
Movement: Click on line and hold down the mouse button, move the entire line, release
mouse button.
Line with arrow button :(open arrow): Activates the straight profile drawing mode.
Click into the image and hold the mouse button, drag a line in any direction and release
the mouse button to end the procedure.
Open polyline button: Activates the open polyline drawing mode.
The first click into the image sets the starting point, each additional click adds a further
line, right mouse click ends the procedure.
Open free-shape curve button: Activates the Bezier figure drawing mode.
The first click into the image sets the starting point, each additional click adds a point,
right mouse click ends the procedure.
Line button: This button allows you to determine the line thickness of the profile line. It
has no influence on the way the intensity profile is generated.
Color selection box: The colors displayed in the Color selection box can be assigned to
the overlay profile line with a click of the mouse. The currently selected color is displayed
in the larger rectangle (top left) of the selection box.
Diagr. In Image button: The profile diagram is displayed in the image along the drawn
profile line.
Marker 1 button (red): Activates the red marker for movement in the profile diagram;
the marker shown as a red line in the diagram can now be moved to the right or left of
the diagram using the mouse. The marker in the image display (red circle) follows
accordingly.
Marker 2 button (blue): Activates the blue marker for movement in the profile diagram;
the marker shown as a blue line in the diagram can now be moved to the right or left of
the diagram using the mouse. The marker in the image display (blue circle) follows
accordingly.
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OPERATION
Display and Analysis of Images
LSM 5 LIVE
LSM 5 LIVE DuoScan
Zoom button: Zoom function for profile diagram. Click and drag a rectangle over the
area to be enlarged in the profile diagram; the selected area is enlarged on release of the
mouse button. The zoom function can be performed several times. A click with the right
mouse button will reset the original size.
Reset Zoom button: Resets the zoom factor of the profile diagram to the original size.
Show Table button: The profile diagram is displayed as a table at the bottom of the
Image Display window.
Copy Table button: The profile table is copied to the clipboard.
Save Table button: The profile table can be stored as a text file (extension *.txt).
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4.13.21.3 Display - Mean
This function allows to
− display the intensity time diagram (mean intensity in user defined ROIs over time)
− display the intensity time diagram for volumes (3D ROIs) within a Z-Stack over time
− use frame time series and frame Z Stack time series as input
− show the intensity values in table form and copy table to clipboard or save as text file
− show separate diagrams for each channel in a multi channel image
The settings of Chan, Zoom, Slice, Contr and Palette apply.
Click on Mean will display the Mean of ROIs toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
To use a similar functionality while scanning use the optional Mean of ROI function with the Time
series control.
• Click on the Mean ROI button.
− The Mean of ROIs image display toolbar will be displayed on the right. The used ROIs become
visible in the image, and the Intensity-Time diagram is shown on the left of the Image Display
window.
Fig. 4-268
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Image Display window, Mean ROI display for time series in single
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OPERATION
Display and Analysis of Images
Carl Zeiss
Fig. 4-269
4.13.22
LSM 5 LIVE
LSM 5 LIVE DuoScan
Image Display window, Mean ROI display for time series of Z Stack
Display - 2.5 D
This function allows to
− display the two-dimensional intensity distribution of an image in an pseudo 3D mode
− show the intensity values in profile, grid or filled mode
− show separate distribution for each channel in a multi channel image
The 2.5 D button can also be used online during scanning.
• Click on the 2.5 D button.
− The Pseudo 3D toolbar is displayed.
The settings of Slice apply. The settings of Chan, Zoom, Contr and Palette are not applied.
Click on 2.5 D will display the Pseudo 3D toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
The viewing plane of the Image Display window can be rotated, tilted either directly with the mouse or
by the scroll bars on the right-hand side and the bottom of the Image Display window.
4.13.22.1 Direct Setting in the Image
• Click in the image and hold down the mouse button. The perspective is changed by moving the
mouse button in horizontal or vertical direction.
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4.13.22.2 Setting via Scrollbars
• Move the
horizontal scrollbar to rotate the image around the vertical axis. The rotation angle is
displayed in the yellow display box.
• Move the left vertical scrollbar to rotate the image around the horizontal axis. The rotation angle is
displayed in the yellow display box.
The intensity scale can be varied by the scroll bar on the right-hand side of the Image Display window:
• Moving the
right vertical scrollbar enables you to expand or to compress the intensity scale of the
image, while the expansion of this intensity axis ranges between 10 % and 100 % of the X-scale size.
Fig. 4-270
Image Display window, 2.5 D display
The Pseudo 3D toolbar contains the following function elements:
Profile button
Profile display (vertical polygon display). Setting of the Profile Distance
between 1 and 20 using the slider.
Grid button
Grid display (horizontal grid display). Setting of the Grid Distance between
1 and 20 using the slider.
Filled button
Color diagram (filled 3D diagram). Selection between the Mono, Rainbow
and Six Step color palettes.
Channel list box
Permits the selection of a Channel in a multi channel image.
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4.13.23
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LSM 5 LIVE DuoScan
Display - 3D (Image VisArt)
This optional function allows to
− reconstruct and display 3 D fluorescence image stacks or time series of frames and stacks from the
Image Display window
− select from a variety of reconstruction modes
The settings of Chan are applied. The settings of Zoom, Slice, Contr and Palette are not applied.
Click on 3D will display the 3D toolbar. Any changes done with this toolbar are effective immediately.
The content of the overlay plane is temporarily deleted while the toolbar is displayed.
The 3D button can also be used online during scanning.
• Click on the 3D button. The 3D toolbar will be displayed.
Fig. 4-271
Image Display window, 3D display
The 3D toolbar contains the following function elements:
Shadow projection
Front button
Shadow rendering front view
Back button
Shadow rendering back view
Any View button
Shadow rendering with user defined view
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Transparency projection
Basic button
Transparency rendering (voxel based)
Advanced button
Transparency rendering (voxel based) with textures
Surface projection
Basic button
Surface rendering (voxel based)
Advanced button
Surface rendering (triangle based)
Full Resolution button
High accurate surface rendering (triangle based)
Appearance button
Opens the render properties dialog
Series button
Renders a series of 3D image stack or 3D / 4D time series, opens the Series
render dialog
4.13.23.1 Shadow Projection
With a click on Front, the 3D reconstructed image is displayed in a shadow projection where it is
illuminated at a 45° angle from the front left.
A click on the Back button creates the same projection with illumination from back left.
Fig. 4-272
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Image Display window, 3D display, Shadow projection, Front view
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The zoom wheel to the left of the Image Display window allows continuous zooming of the 3D
reconstructed image.
A click on the Any View button displays the 3D reconstruction image in a shadow projection where the
viewing point can be defined. In addition to the zoom setting, the image can be rotated around the three
orthogonal axes via the relevant setting wheels.
However, the 3D orientation can also be set directly in the Image Display window by clicking, holding
and dragging the 3D reconstructed image with the mouse.
Fig. 4-273
Image Display window, 3D display, Shadow projection, Any View
The following additional buttons are available in the Any View shadow projection mode:
• After activation of the Frame button (below the image), a bounding box is drawn around the 3D
reconstructed image.
Depending on the used mode and hardware configuration, it can take several seconds until the
3D reconstruction is refreshed on the monitor after reorientation.
• A click on the Coordinate System button displays a colored coordinate system in the Image Display
window, where the X axis is displayed in red color, the Y axis in blue and the Z axis in green.
• A click on the Scale button display an X-,Y- and Z-scale in the Image Display window.
• A click on the Home button resets the display parameters to the default values.
A click on the Any View button displays the 3D reconstruction image in a shadow projection where the
viewing point can be defined. In addition to the zoom setting, the image can be rotated around the three
orthogonal axes via the relevant setting wheels.
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However, the 3D orientation can also be set directly in the Image Display window by clicking, holding
and dragging the 3D reconstructed image with the mouse.
The following additional buttons are available in the Any View shadow projection mode:
• After activation of the Frame button (below the image), a bounding box is drawn around the 3D
reconstructed image.
Depending on the used mode and hardware configuration, it can take several seconds until the
3D reconstruction is refreshed on the monitor after reorientation.
• A click on the Coordinate System button displays a colored coordinate system in the Image Display
window, where the X axis is displayed in red color, the Y axis in blue and the Z axis in green.
• A click on the Scale button display an X-,Y- and Z-scale in the Image Display window.
• A click on the Home button resets the display parameters to the default values.
• A click on the Animation button activates the animation mode. The object can be pushed by
dragging in the Image Display window and rotates continuously. Any new push with pressed left
mouse button changes the rotation direction and speed of the animation.
The fastest animation results can be achieved with the advanced surface rendering mode (even
without additional graphics cards).
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4.13.23.2 Transparency Projection
The transparency projection generates a transparent 3D reconstructed image.
The elements for image display (zoom, 3D rotation, home, coordinate system, scale, frame and animation
function) are identical to those of the Any View function of the shadow projection and are operated in
the same way.
The transparency projections Basic and Advanced are perspective-type 3D reconstructions, with the
Advanced projection permitting the perspective impression being varied between parallel and centric
projection by changing the View angle. The Advanced projection also offers the possibility of selecting
between fast and precise calculation via the Precise / Fast slider (at the bottom right in the 3D toolbar).
Of course, the precise calculation method is more time consuming.
Fig. 4-274
4-272
Image Display
Advanced
window,
3D
display,
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4.13.23.3 Surface Rendering
The surface rendering generates surface rendered 3D reconstructed images.
The elements for image display (zoom, 3D rotation, home, coordinate system, scale, frame and animation
function) are identical to those of the transparency projection and are operated in the same way.
The surface projections Basic and Advanced are perspective-type reconstructions of the surface and
differ in the fact that the calculation of the 3D information is based on voxels or triangles.
The Advanced projection permits the View angle to be varied in order to enhance the perspective
impression. It is also possible to select between fast and precise calculation via the Precise / Fast slider
(at the bottom right in the 3D toolbar). Of course, the precise calculation method is more time
consuming.
The Full resolution projection is based on a high precision calculation method for 3D information on the
basis of triangles with maximum resolution.
Depending on the hardware configuration, it can take several seconds until the surface
projection is refreshed on the screen.
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Display and Analysis of Images
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4.13.23.4 Appearance (Settings)
The Appearance button opens the 3D Rendering
window.
This window allows settings for Light, Material,
Background and Projection properties to be
defined for all 3D projection modes.
Depending on the selected 3D projection mode,
different setting parameters are displayed.
In the Shadow projection, the parameter
Roughness is also available and can be set
between 0 and 1.
A default setting is permanently available for all
modes.
If individual settings for 3D rendering are to be
used again, they can be stored and loaded when
required.
See also Show Processing Parameters,
page 4-289.
Proceed as follows to save individual settings:
• Click on the Add to List button.
• Enter a name in the opening Add Shading
Model to list window.
• Click on OK.
Fig. 4-275
3D Rendering window (e.g. Surface
Advanced rendering mode)
− The settings are saved and the entered name
appears in the Shading Model List
selection box.
• To activate the settings, double-click on the
relevant name in the Shading Model List
selection box.
Settings which are no longer required can be
removed.
• Select the name of the setting to be deleted
with a click of the mouse in the selection box
and then click on the Remove button.
− The setting is deleted.
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4.13.23.5 Series
The Series button opens the Render Series
window. This window allows settings for the axis
to be used for rotation of the 3D reconstructed
images.
• Click on the Series button to open the Render
Series window.
• Select the requested projection mode by
clicking on the relevant option button under
Mode.
• Depending on the activated mode, directly set
the parameters for animation in the Render
Series window and the position of the 3D
image in the Image Display window (zoom,
rotation axes, rendering parameters).
• Click on Apply to start the animation
The animation is performed in a separate Image
Display window, which permits the animation to
be saved afterwards.
(1)
Fig. 4-276
Render Series window
(e.g. Turn around X mode)
Turn around X and Turn around Y mode
In this mode, the image is turned around the X-axis or the Y-axis exclusively.
The values for Number of Views, Difference Angle and First Angle can be selected accordingly (see
Projection, page 4-155).
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(2)
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Start and End mode
In the Start and End mode, the image is
reconstructed between a start position and an end
position.
The rotation angles for X, Y and Z and the distance
(zoom) can be determined using the sliders.
The value for Number of View can be varied.
Fig. 4-277
Render Series window
(e.g. Start and End mode)
(3)
Position List mode
In the Position List mode, the image is
reconstructed between any required number of
interim positions to be determined individually.
The rotation angles for X, Y and Z and the zoom
can be determined directly in the image.
Every required interim position is included in the
list of the Render Series window with a click on
the Add Position button.
Clear List permits the contents of the list to be
deleted.
The value for Number of View can be varied.
• Click on the Apply button calculates a spline
along all the defined positions from the list and
starts an animation along this spline track in
space.
Fig. 4-278
4-276
Render Series window
(e.g. Position List mode)
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(4)
OPERATION
Display and Analysis of Images
Carl Zeiss
Time series
When the input images is a Z Stack time series, the
reconstructed images are generated for each time
point.
• If you click on the right mouse button in the
main display window of Image VisArt two
options appear:
− Render properties
− Renderer
This functionality helps to find the optimal
performance when working with different
hardware renderers and to speed up some
advanced rendering modes.}
Fig. 4-279
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3D Renderer window
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4.13.24
LSM 5 LIVE
LSM 5 LIVE DuoScan
Display - Topography for LSM
This optional function allows to
− process, display and measure topographic information.
− use frame Z Stacks
− and frame Z Stacks over time
The Topo function is mainly used for applications in material research and industry.
The settings of Chan and Zoom are applied. The settings of Slice and Contr are not applied. The
Palette settings are applied in some 3D display modes.
Click on Topo will display the Topography toolbar. All changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
• Click on the Topo button. The Topography toolbar is displayed.
− The topography of a Z Stack is displayed in the Image Display box of the Image Display window.
The parameter used at the last exit of the Topo function are applied.
Fig. 4-280
4-278
Image Display window, Topography display
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The Topography toolbar contains the following function elements:
Channels buttons
The selection of a channel to be used.
Generate buttons
The selection of the mode of calculation of the topography image
(maximum, center of gravity, First /Last)
Threshold buttons
The selection of thresholds (intensity, height, Auto Z) to be used.
Filter buttons
The definition of filter procedures (geometrical, frequency cut-off filters) for
smoothing, separation of roughness or waviness.
Geometry buttons
Inversion, various fit procedures, hole filling.
Display buttons
2D (Intensity, Height, Gradient); Iso-Lines (z Map, Intensity, Black)
3D (Profiles, Grid, Filled, Shaded).
Measure buttons
Diagrams (Profile, z Distribution, Bearing area ratio plot, Slope distribution);
Roughness parameters ; Volume parameters.
4.13.24.1 Channel Selection
• Select the channel to be viewed using the relevant button (e. g.: Ch1).
4.13.24.2 Generate Topography
The three buttons provided in the Generate button bar allow you to generate the topography in
different ways:
Maximum
• Click on the Maximum button to calculate the topography surface by finding the maximum intensity
value. If the optical section with the highest intensity value is found, the intensity values of both
neighboring slices are also taken into account, so that a 3 point maximum fit is calculated.
• In case it happens, that the maximum possible intensity value is present in more than one optical slice
for a given pixel (saturation), the mid section of all saturated intensity slices is chosen as a reasonable
approach.
Center
• Click on the Center button to calculate the topography surface by using the center of gravity of all
summed up intensities of the stack for a given xy print.
This mode provides better result for smooth surfaces of low intensity or nearly transparent
surfaces. The receiver gain and offset has to be properly tuned and MarkFirst- MarkLastpositions of the stack should be located approximately in the same distance from the real
surface.
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First / Last
• Click on the First button to calculate the topography surface by using the first slice coming from the
top, where the intensity reaches the value defined by the lower intensity threshold.
This mode provides better result for surfaces of semitransparent materials with inclusions of
higher reflectivity or transparent multilayers with subsurface layers of higher signal intensity.
Extended First / Last Mode
1. Definition of an intensity (I) threshold.
2. Starting from top / bottom of a stack to find I = 400.
3. Search of a local maximum one FWHM of actual Z PSF forwards / backwards.
4. Search of the next local maximum one FWHM forwards / backwards from the last max until you have
not found any new local maximum.
5. Last local maximum is taken as surface point.
Fill Holes procedure
• Intensity of a missing pixel of a hole has to be interpolated by the distance-weighted intensity of all
surrounding pixels.
• Fill hole algorithm is optimized for short calculation times.
4.13.24.3 Topography Thresholds
Intensity threshold
Click on the Intensity button to calculate the topography surface by using the lower and the upper
intensity thresholds for image display. Use of this function is recommended to find the real surface in the
case of images with pronounced noise. All image pixels with intensity less or higher than the thresholds
set are ignored for the surface calculation.
• Click on the Intensity button to select the
intensity thresholds for the surface generation.
The Intensity Threshold window appears.
• Set the lower and upper intensity thresholds
using the appropriate sliders.
Fig. 4-281
4-280
Intensity Threshold window
• Click on Close to
Threshold window.
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Height threshold
Click on the Height button to calculate the
topography surface by using the lower and the
upper height thresholds for image display. Use of
this function is recommended to get rid of
unwanted peaks and valleys taken into account for
parameter calculation. All topographic data with
height values less or higher than the thresholds set
are ignored for the display and parameter
calculation. This threshold applies both for 2D as
well as for 3D topography display modes.
Fig. 4-282
Z Threshold window
• Click on the Height button to select the intensity thresholds for the surface generation. The
Z Threshold window appears.
• Set the lower and upper intensity thresholds using the appropriate sliders.
• Click on Close to close the Z Threshold window.
Auto Z
By clicking on the Auto Z button the surface topography is displayed in the Image Display window in
that way that it is automatically normalized to the lowest and highest Z value of the 3D topography.
• Click on the Auto Z button. The topography is automatically normalized with respect to the highest
and lowest Z value.
4.13.24.4 Processing by Filtering
(1)
Topography smoothing
The three buttons in the Filter button bar allow activation / deactivation of the filter functions for surface
smoothing.
None button
No filter for input data.
Median / Gauss / Aver.
button
Smoothing of z data using a low-pass Median, Gauss or average filter.
Clicking on this button opens a selection box, where the number of
neighboring pixels to be used for filtering can be specified:
1st row: small smoothing via Median/Gauss filter (Median; 3 x 3; 5 x 5; 7 x 7)
2nd row: medium smoothing via Average (9 x 9; 11 x 11; 15 x 15)
3rd row: pronounced smoothing via Average (25 x 25; 35 x 35; 45 x 45)
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OPERATION
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• To investigate the effects of various filter modes, select one of the 3D display modes (Profiles, Grid,
Filled or Shaded) from the Display button bar.
• Click on the Median sub button to set the smoothing of the integrated Median filter.
Or
• Click on a Gauss or Average sub button and select the required degree of smoothing from the
selection box with a click of the mouse.
FFT button:
This function performs a Fast Fourier Transformation (FFT) in the frequency
range, applies highpass or lowpass filtering in the frequency range and
performs the inverse FFT.
• Click on the FFT button, the FFT Filter window opens.
• Click on the arrow in the filter Type select box to choose an adequate filter function:
− Gauss Lowpass
− Gauss Highpass
− Butterworth Lowpass
− Butterworth Highpass
• Select a position of the Cut off slider to display either the lower frequencies (waviness) with the
lowpass filters or the higher frequencies (roughness) with the highpass filters.
• The Cut off frequencies ranges from 1/1000 of the X dimension of the stack to four times of the X
dimension of the stack. The dimensions of the filtering is given in units of μm.
• Select a position of the Degree slider. The filter functions can be calculated from 1st order to 5th order
accuracy.
• Click on the Close button closes the FFT Filter window.
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OPERATION
Display and Analysis of Images
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Changing the topography geometry
The three buttons in the Geometry button bar allow the surface geometry to be changed.
Inverse button
Inverse surface. Depths change to heights, and vice versa.
Fit button
The following fit modes can be set:
1) No Fit
2) Plane
The topography is tilted in such a way that the mean deviation value
plane is calculated.
3) Manu Tilt
4) 3Point Tilt
5) Sphere fit correction
A spherical form is eliminated, determination of micro roughness on
spherical surfaces can be performed.
6) Cylinder fit correction
A cylinder form is eliminated, determination of micro roughness on
cylindrical surfaces can be performed.
You can display the exact values of the Cylinder / Sphere fit by
opening a context menu in the Image Display box with a click of
the right mouse button and selecting the Show processing
parameter function.
Manu Tilt button
Clicking on the Manu Tilt button opens the Manual Tilt window
(Fig. 4-283) for manual tilt correction.
In the manual tilt window three types of arrow buttons are present:
− One arrow changes pitch and yaw in 0.001° steps
− Two arrow changes pitch and yaw in 0.01° steps
− Three arrow changes pitch and yaw in 0.1° steps
• Click on the Inverse button for the inverse display of the topography. Clicking again will reset the
normal display.
• Correct the tilt via the Fit or Manu Tilt functions.
Fig. 4-283
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Manual Tilt window
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4.13.24.5 Display Modes
The three buttons in the Display button bar allow stacks to be displayed in the 2D, Iso-Lines or 3D
display mode.
(1)
2D modes
The following 2D modes can be set:
Intensity button: Display of projection of all intensities of the stack (black-and-white
display).
Height button: Height coded color map with color bar in a separate parameter window
on the right hand side.
Gradient button: Display of height gradient (slope), averaged pixelwise over all neighbors
(black-and-white display).
• Click on the 2D button in the Display button bar.
− The 2D display mode selected last is activated. At the same time, an additional button bar is
displayed beside the 2D button permitting selection of the required 2D display mode.
• Select the required 2D display mode with a click of the mouse.
(2)
2D Iso-Lines display mode
Iso-Lines are lines which connect points of equal height on the topography.
The following 2D Iso-Lines display modes can be set:
Intensity button: Intensity projection superimposed with colored iso-lines (lines of equal
height).
Height button: Height function with black iso-lines.
Black button: White iso-lines on a black background.
• Click on the Iso-Lines button in the Display button bar.
− The Iso-Lines display mode selected last is activated. At the same time, an additional button bar is
displayed beside the Iso-Lines button permitting selection of the required Iso-Lines display mode.
− Below the Measure button bar, the Line Dist. and Line Offset sliders / input boxes are displayed.
• Select the required Iso-Line display mode by clicking the left mouse button.
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The additional function elements of the Iso-Lines display mode have the following meaning:
Line Dist. slider: Changes the distance of the iso-lines.
Line Offset slider: Setting of the height level where the Iso-Lines display starts.
To apply the topography functions to a small portion of the Z Stack image use the Overlay
function (Overlay button) and cut out and store as new topographic evaluation via the Extract
Region function.
(3)
3D display
Topo animations are possible. The following 3D modes can be set:
Profiles button: Profile display.
Grid button: Grid display.
Filled button: Display of color shades.
Shaded button: Surface rendering. Can be combined with LUT. Topo animations are
possible.
• Click on the 3D button in the Display button bar.
− The 3D display mode selected last is activated. At the same time, an additional button bar appears
beside the 3D button to permit selection of the required 3D display mode.
− Below the Measure button bar, the Scaling button bar and the Profile Dist. and Fill Level
sliders / input boxes are displayed.
− The Image Display box contains one horizontal and two vertical scrollbars for the setting of the
image viewing angle.
• Select the required 3D display mode with a click of the mouse.
The additional function elements of the 3D display mode have the following meaning:
Profile Dist. slider: Setting of the distance of profiles and the mesh value of the grid.
Fill Level slider: Used to push through a color LUT Look Up Table (e.g.: if the Rainbow
palette is used) in the Profiles / Filled display mode. In combination with the Volume
button, the filling of the flood function level of the topography can be varied for volume
measurements (see the Measurement functions paragraph).
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OPERATION
Display and Analysis of Images
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The image viewing angle is set as follows:
Setting directly in the image
• Click in the image and hold down the mouse button. The perspective is changed by moving the
mouse button in horizontal or vertical direction.
Setting via scrollbars
• Move the
horizontal scrollbar to rotate the image around the vertical axis. The rotation angle is
displayed in the yellow display box.
• Move the left vertical scrollbar to rotate the image around the horizontal axis. The rotation angle is
displayed in the yellow display box.
right vertical scrollbar enables you to expand the image in height or to compress it,
• Moving the
while the Z-range between 10 % and 100 % of the X-range is scaled.
You can set the x, y and Z scales to an identical ratio by opening a context menu in the Image
Display box with a click of the right mouse button and selecting the Metric equal ratio
function.
The displayed boxes for rotation angle and relative scaling percentage value z : x ratio permit the setting
of identical perspectives for different images (e.g.: the plot of several topographies).
The Profiles and Filled 3D display modes permit a color palette (e.g.: Glowscale, Rainbow or
User defined) to be loaded or redefined by pressing the Palette button (see page 4-237).
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4.13.24.6 Context Menu of the 3D Display
Mode
• Click in the Image Display box with the right
mouse button to open the context menu.
The context menu for the 3D mode currently
selected is displayed.
• Click on the required option with the left
mouse button to execute the function.
(1)
Metric equal ratio item
This option is available in all of the 3D display
modes.
Fig. 4-284
Context menu of the
3D display mode (Profiles)
Fig. 4-285
Save As window
Fig. 4-286
Topography matrix
After activation of the function, the x, y and z
scales are set to an identical ratio.
(2)
Export profiles item
This option is available in the Profiles and Grid 3D
display modes.
Use the function to save the Profiles or Grid data
as a text file.
• Open the context menu with a click of the right
mouse button, then click on the option
Export ... with the left mouse button.
− The Save As window is opened.
• Select the directory where you want the text file
to be stored, enter a file name and click on
Save.
A text file containing the topography in the form
of an XYZ matrix is generated.
(3)
Copy profiles to clipboard item
All 3D parameters shown right from the main 3D topo display window are copied in the clipboard.
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(4)
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Export x,y,z- triples item
This option is available in the Profiles and Grid 3D
display modes.
Use the function to save the Profiles or Grid data
as a text file.
• Open the context menu with a click of the right
mouse button, then click on the option
Export ... with the left mouse button.
− The Save As window is opened.
Fig. 4-287
Save As window
• Select the directory where you want the text file
to be stored, enter a file name and click on
Save.
A text file containing the topography in the form
of an XYZ table is generated.
Fig. 4-288
(5)
Topography table
Copy x,y,z- triples to clipboard item
This option is available in the Profiles and Grid 3D display modes.
After selection of this option, the Profiles or Grid data are copied as an XYZ table to the clipboard and
can be inserted in other programs using the Paste command.
Please make sure that the amount of exportable data is adequate to the maximum importing
size of the following software package. To lower the amount of data points, use the profile
distance slider.
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(6)
OPERATION
Display and Analysis of Images
Carl Zeiss
Show processing parameters
After selection of the Show processing parameters function, a reporting of the following applied topo
processing functionality is displayed on the right-hand side of the Image Display window:
− Mode (calculation mode: Max, Center, First)
− Threshold (applied intensity threshold)
− Filter
− Fit (plane, cylinder / sphere parameters)
Fig. 4-289
(7)
Show processing parameters
Ratio of valid data points
The ratio of valid data points (means signal intensities within a given intensity threshold) is displayed.
(8)
Load Calculation Parameters
Topo routines can be loaded as tgp-files (TopoGraphic Parameters).
(9)
Save Calculation Parameters
Topo routines can be saved and reloaded as tgp-files. These files include settings for:
− reconstruction mode
− intensity threshold
− filters (including FFT)
− tilt angles (manual, 3 point fit)
− fit procedures (plane, cylinder, sphere)
− inverse
− fill holes
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(10) Render properties item
This option is only available in the Shaded 3D
display mode.
Fig. 4-290
Context menu of the 3D display mode
(Shaded)
Use this function to vary the illumination
conditions, reflection properties and projection
settings of the topography. You can either select
preset Shading Models or use parameters
specifically defined as required.
The
specifically
defined
parameters
can
subsequently be stored as a Shading Model and
are then available at any time for further use.
Shading Models can also be deleted if no longer
needed.
Load a Shading Model
• Open the context menu with a click of the right
mouse button, then click on the option Render
properties with the left mouse button.
− The 3D Rendering window is opened.
Fig. 4-291
3D Rendering window
• Click on the name of the required model in the
Shading Model List. The parameters are
immediately set for the current topography.
• Click on Close to close the 3D Rendering
window again.
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Defining a specific Shading Model
• Open the 3D Rendering window.
• Click on the Define button.
• Change the parameters of the topography
using the appropriate sliders.
• Save the settings by clicking on the Add to List
button. The Add Shading Model to List
window is displayed.
• Enter a name for the model and click on OK.
The model is included in the Shading Model
List.
See also Appearance (Settings), page
4-274.
Fig. 4-292
3D Rendering window
Light panel
Determines the properties of illumination on a sample.
Distance
Goes for diffuse and specular, see visualization.
Azimuth
See visualization. Rise angle of the "sun".
Elongation
See visualization. North-south / east-west direction of the "sun".
Background
Choose background color.
Material panel
Determines the reflective properties of a sample.
Ambient Specular
Material properties; how many % of the light component are projected by
the material into which spectral ranges.
Shininess
Goes together with specular light. Shininess equal to 25 % determines
diffuse light.
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Projection panel
Determines the reflective properties of a sample.
View angle
Determines the perspective, 0.0 parallel projection, central projection
Distance
Zoom function, zoom in, zoom out
Fig. 4-293
Render function visualization aid
A zoomed rendering setting permits the zoomed section to be moved via the cursor keys after a click on
the 3D window.
If a change of the 3D image angle follows, centration is made on the center again.
Deleting a Shading Model
• Select the model to be deleted in the Shading Model List, then click on the Remove button. The
model is deleted.
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(11) Renderer item
This option is only available in the Shaded 3D
display mode.
After selection of the Renderer item, the 3D
Renderer window appears. It allows the selection
of the hardware and software option which shall
be used for the 3D graphics calculation.
OpenGI - Software
The graphics calculation is performed using the
installed software.
OpenGI - Hardware
The graphics calculation is accelerated by using the
installed graphics processor.
Direct3D – Software / Hardware Rasterizer /
Hardware
These options can be used for offline versions of
the LSM 5 software for PC's with the WINDOWS ®
2000 or XP operating system (not for WINDOWS ®
NT).
Fig. 4-294
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OPERATION
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4.13.24.7 Measurement Functions
The topography measurement functions are activated via the Measure button bar. The measurement
functions can be performed in the 2D or 3D display mode.
Automated convention in height statistics analysis:
Topo Filters
None, median,
<9x9
FFT High
FFT Low
>9x9
Data formats
Primary profile
Roughness
Waviness
2D profile
Pxx
Rxx
Wxx
n.a.
3D topography
PSxx
RSxx
SWxx
n.a.
The following measurement functions are available:
Diagram button: Diagram display. The Profile, z Histo, Curve of tp
and Grad. Histo diagram display modes can be activated via the
Diagram button and deactivated via the Off Diagram button. By
activation of the Diagram button, an additional button bar is
displayed for the selection of the required diagram or for deactivation.
The labeling of the Diagram button changes depending on which
diagram display mode has been activated.
Roughness button: Calculation of the roughness parameters.
Volume button: Calculation of the volume parameters.
(1)
Profile measurement mode in 2D display
• Select the required 2D display of the stack via the 2D button.
• Click on the Diagram button in the Measure button bar. Click on the Profile button in the button
bar displayed afterwards.
− The Table and Profile button bars are displayed below the Measure button bar.
− A colored arrow (intersection line of the profile) is displayed in the image and the profile diagram
appears below the image.
• If required, match the size of the Image Display window in order to obtain the complete display of
the profile diagram.
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Fig. 4-295
OPERATION
Display and Analysis of Images
Carl Zeiss
Topography display: 2D - Profile
Additional Table / Profile buttons
The additional Table / Profile buttons have the following functions:
Show button: The profile is displayed in the form of a table at the bottom below of the
Image Display window.
Copy button: The profile table is copied to the clipboard and can be transferred to other
programs (MS Word or MS Excel) via the Paste function.
Save button: The profile table can be stored as a text file (ASCII).
Arrow (selection) button: Activation of the mouse button for selection, resizing or
movement of the intersection line in the image.
Click on the handle and hold down the mouse button, drag the handle,
Resizing:
release the mouse button.
Movement:
Click on the line and hold down the mouse button, move the entire
intersection line, release the mouse button.
Line with arrow button (open arrow): Creation of the intersection line to define the
position of the profile to be produced in the image. Click and hold the mouse button,
drag the line in any required direction, release the mouse button to end the procedure.
The profile diagram changes online.
Line button: This button allows you to determine the line thickness of the intersection
line.
Measure button: Activates the Profile measurement mode in the profile diagram. The
required tools are displayed to the right of the profile diagram (see Profile
measurement mode, page 4-294).
z/x=1 button: Sets the z/x ratio in the profile diagram to the value 1. Check: the
following creation of a circle using the relevant tool really results in a circle in the profile
display. Measured angle values correspond to the actual slope of the line displayed.
Color button: Clicking on the Color button opens a color selection box in which the
color for the intersection line can be selected with a click of the mouse.
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OPERATION
Display and Analysis of Images
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Activating the Profile measurement mode
button, the Profile
If you click on the
window with the tools of the profile measurement
mode appears.
This window can be moved as required over the
entire screen.
Fig. 4-296
Tools of the Profile measurement mode
Tools of the Profile measurement mode
The tools of the Profile measurement mode have the following functions:
Zoom button: Zooming of a section of the profile diagram. Click and drag a rectangle
over the area to be enlarged in the profile diagram, release the mouse button to enlarge
the selected area. The zoom function can be performed several times. A click with the
right mouse button resizes the profile.
Marker button: Activation of the marker functions for the intersection line. The red and
blue marker lines in the profile diagram can now be moved using the mouse. After
movement of a marker line in the profile diagram, the relevant marker (red or blue circle)
follows along the intersection line in the 2D and Iso-Lines mode.
Arrow (selection) button: Activation of the mouse button for selection, resizing or
movement of one of the following drawing elements in the profile diagram.
Resizing: Click on the handle and hold down the mouse button, move the handle, release
the mouse button.
Movement: Click on the line and hold down the mouse button, move the entire drawing
element, release the mouse button.
Inclined Line button: Creation of a straight line in the profile diagram. Display of
distance, inclination angle, dxdy and dz. Click and hold down the mouse button, drag the
line in any required direction, release the mouse button to end the procedure.
Free angle button: Creation of a free angle in the profile diagram. Display of the
enclosed angle (max. 180 °). The first click sets the starting point, the second and third
clicks define the angle and the end point.
Rectangle button: Creation of a rectangle in the profile diagram. Display of distance,
area, height and width. Click and hold down the mouse button, drag the rectangle in any
required direction, release the mouse button to end the procedure.
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Open Polyline button: Creation of an open polyline figure in the profile diagram. Display
of the length of the line figure. First click sets the starting point, any further click adds
another line, click with the right mouse button ends the procedure.
Closed Polyline button: Creation of a closed polyline figure in the profile diagram.
Display of the perimeter of the figure. First click sets the starting point, each further click
adds another line, a click with the right mouse button closes the figure and ends the
procedure.
Open free-hand curve button: Creation of an open Bezier figure in the profile diagram.
Display of the length of the line figure. First click sets the starting point, each further click
adds another line, a click with the right mouse button ends the procedure.
Closed free-hand curve button: Creation of a closed Bezier figure in the profile
diagram. Display of the length of the line figure. First click sets the starting point, each
further click adds another line, a click with the right mouse button closes the figure and
ends the procedure.
Ellipse button: Creation of an ellipse in the profile diagram. Display of the area. First click
sets the center point, the displayed line permits the determination of the first dimension,
second click sets the first dimension, the second dimension and rotation direction can
now be determined, third click sets the second dimension and direction and ends the
procedure.
Circle button: Creation of a circle in the profile diagram. Display of radius and area.
Clicking three times to define 3 points on the profile. A circle fit is automatically applied
on the profile.
Recycle bin button: Deletes all drawing elements or the one just selected.
Line width button: Change of the line width of the drawing elements.
Color button: Clicking on the Color button opens a color selection box where the color
of the drawing element can be selected with a click of the mouse.
x1- button: Resets the zoom factor of the profile diagram to its original size.
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(2)
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z Histo measurement mode in 2D display
• Click on the Diagram button in the Measure button bar. Click on the z Histo button in the
additional button bar now displayed.
The lower part of the Image Display box shows the 3D height distribution of the topography.
Fig. 4-297
(3)
Image Display window, Topography display: 2D - Histo
Curve of tp measurement mode in 2D display
• Click on the Diagram button in the Measure button bar. Click on the Curve of tp button in the
additional button bar now displayed.
− The curve of the bearing area ratio as a function of the height is displayed below the image (also
see 3D measurement functions, page 4-304).
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(4)
OPERATION
Display and Analysis of Images
Carl Zeiss
Grad. Histo measurement mode in 2D display
• Click on the Diagram button in the Measure button bar. Click on the Grad. Histo button in the
additional button bar now displayed.
The lower part of the Image Display box shows the gradient distribution of the topography. Before
creation of the slope diagram, the image should be filtered at least once using a low-pass filter, since
otherwise the rough height gradation of the image will result in a comb-shaped histogram. The RootMean-Square Slope (RMS Slope) parameter is calculated and displayed below the chart. The following
formula is used for calculation:
RDQ =
(5)
(
) (
N x N y ⎧⎡
1
⎪ z xi , y j − z xi −1 , y j
⋅ ∑ ⋅∑ ⋅ ⎨⎢
( N x − 1) ⋅ ( N y − 1) i =1 j =1 ⎪⎢
Δx
⎩⎣
) ⎤⎥
2
(
) (
⎡z x , y − x , y
i
j
i
j −1
+⎢
Δy
⎥ ⎢
⎦ ⎣
) ⎤⎥ ⎫⎪⎬
2
⎥ ⎪
⎦ ⎭
Roughness measurement mode in 2D display (Profile display)
• Click on the Profile button in the Measure button bar.
• Click on the Roughn. button in the Measure button bar.
− The roughness parameters are calculated and displayed on the left below the image. All roughness
parameters calculated from a 2D profile are named with R.
− The Copy button is displayed below the right-hand side of the image. This button permits the
roughness parameters to be copied to the clipboard and imported to another program (e.g.: MS
Word or MS Excel) via the Paste function.
2D Amplitude parameters (Profile Roughness):
Mean height z
Rc
Pc
Wc
Arithmetic mean deviation
Ra
Pa
Wa
Root mean square deviation
Rq
Pq
Wq
Asymmetry
Skewness
Rsk
Psk
Wsk
Sharpness
Kurtosis
Rku
Pku
Wku
Extremes
Highest peak
Rp
Pp
Wp
Lowest valley
Rv
Pv
Wv
Absolute peak to valley
Rt
Pt
Wt
Averaged peak to valley
Rz
Pz
Wz
Maximum peak to valley
Rmax
Pmax
Wmax
FFT High
No, M
FFT L
Dispersion
If chosen filters are
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4-299
OPERATION
Display and Analysis of Images
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Calculation of roughness parameters
The following roughness parameters are calculated (e.g. for a Y-section)
− Mean height of all profile height values Rc
y
1
− Rc =
⋅ ∑ ⋅ z (x, y j )
N y j =1
N
Nx, Ny ... number of pixels in X- or Y-direction
Arithmetic mean deviation of all profile height values Ra
[
y
1
− Ra =
⋅ ∑ ⋅ z (x, y j ) − Rc
N y j =1
N
]
− Quadratic mean deviation of all profile height values Rq
[
y
1
⋅ ∑ ⋅ z (x, y j ) − Rc
N y j =1
N
− Rq =
]
2
− Skewness of the distribution of all profile height values RSK
N
RSK
y
1
=
⋅
∑ ⋅ z 3 ( x, y j )
N y ⋅ Rq3 j =1
− Kurtosis of the distribution of all profile height values RKU
N
R KU
y
1
=
⋅
⋅ z 4 ( x, y j )
∑
4
N y ⋅ Rq j =1
− Maximum peak height RP
RP = z max − Rc
− Maximum valley depth RV
RV = Rc − z min
− Maximum roughness depth Rt (= Peak to Valley / PV)
− S t = z max − z min
maximum height difference of the overall topography along a profile.
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OPERATION
Display and Analysis of Images
Carl Zeiss
Classification of topography in 5 equal area elements (rectangles in the 2D mode)
− average roughness depth Rz:
− Rz =
zmax 1 − zmin 1 + zmax 2 − zmin 2 + zmax 3 − zmin 3 + zmax 4 − zmin 4 + zmax 5 − zmin 5
5
Averaging of Rt-values of all the 5 single area elements. When combined, both parameters provide
information about the homogeneity of the surface. Big differences are indicative of pronounced
inclination of the overall area or of spikes.
Developed Surface Area Ratio: Σ (surface areaij) / Σ (projected areaij)
The percentage of the 3D surface area (sum off all triangles formed by adjacent data points) to
the 2D surface area produced by projecting the 3D surface onto the threshold plane.
− maximum roughness depth Rmax:
− Rmax = Max ( zmax 1 − zmin 1 , zmax 2 − zmin 2 , zmax 3 − zmin 3 , zmax 4 − zmin 4 , zmax 5 − zmin 5 )
maximum of Rt-values of all the 25 single area elements.
Both the roughness parameters and the z-histogram can be changed by using filters!
(6)
Roughness measurement mode in 3D display
• Click on the Roughn. button in the Measure button bar.
− The roughness parameters are calculated and displayed on the left below the image. All roughness
parameters calculated from a 3D topography are named with S.
− The Copy button is displayed below the right-hand side of the image. This button permits the
roughness parameters to be copied to the clipboard and imported to another program (e.g.: MS
Word or MS Excel) via the Paste function.
3D Amplitude parameters (Topography Roughness):
Mean height z
RSc
PSc
WSc
Arithmetic mean deviation
RSa
PSa
WSa
Root mean square deviation
RSq
PSq
WSq
Asymmetry
Skewness
RSsk
PSsk
WSsk
Sharpness
Kurtosis
RSku
PSku
WSku
Extremes
Highest peak
RSp
PSp
WSp
Lowest valley
RSv
PSv
WSv
Absolute peak to valley
RSt
PSt
WSt
Averaged peak to valley
RSz
PSz
WSz
Maximum peak to valley
RSmax
PSmax
WSmax
FFT High
No, M
FFT L
Dispersion
If chosen filters are:
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OPERATION
Display and Analysis of Images
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Calculation of roughness parameters
The following roughness parameters are calculated:
− Mean height of all surface height values Sc
− RS c =
1
⋅
Nx ⋅ Ny
Nx
Ny
∑ ∑ ⋅ z (x , y )
⋅
i =1
i
Nx, Ny ... number of pixels in X- or Y-direction
j
j =1
− Arithmetic mean deviation of all surface height values RSa
− RS a =
1
⋅
Nx ⋅ Ny
Ny
∑ ∑ ⋅ [z (x , y ) − RS ]
Nx
⋅
i =1
i
j
c
j =1
− Quadratic mean deviation of all surface height values RSq
− RS q =
1
⋅
Nx ⋅ Ny
Ny
∑ ∑ ⋅ [z (x , y ) − RS ]
Nx
i =1
⋅
2
i
j
c
j =1
− Skewness of the distribution of all surface height values RSSK
RS SK
1
=
⋅
N x ⋅ N y ⋅ RS q3
Nx
Ny
i =1
j =1
∑ ⋅∑ ⋅ z (x , y )
3
i
j
− Kurtosis of the distribution of all surface height values SKU
RS KU =
1
⋅
N x ⋅ N y ⋅ RS q4
Nx
Ny
∑ ∑ ⋅ z (x , y )
i =1
⋅
4
i
j
j =1
− Maximum peak height RSP
RS P = z max − RS c
− Maximum valley depth SV
RSV = RS c − z min
− Maximum roughness depth RSt (= Peak to Valley / PV)
− RS t = z max − z min
maximum height difference of the overall topography.
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OPERATION
Display and Analysis of Images
Carl Zeiss
Classification of topography in 25 equal area elements (rectangles in the 2D mode)
− average roughness depth Sz:
− RS z =
z max 1 − z min1 + z max 2 − z min 2 + ⋅ ⋅ ⋅ + z max 25 − z min 25
25
Averaging of Rt-values of all the 25 single area elements. When combined, both parameters provide
information about the homogeneity of the surface. Big differences are indicative of pronounced
inclination of the overall area or of spikes.
− maximum roughness depth RSmax:
− RS max = Max ( z max 1 − z min1 , z max 2 − z min 2 , ⋅ ⋅ ⋅ , z max 25 − z min 25 )
maximum of Rt-values of all the 25 single area elements.
Both the roughness parameters and the z-histogram will be influenced by the use of filters!
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4-303
OPERATION
Display and Analysis of Images
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.13.24.8 3D Measurement Functions
(1)
Volume measurement mode (Flood function)
• Use the 3D button to select the required 3D display of the stack.
• Click on the Volume button in the Measure button bar.
− The volume parameters are calculated and displayed below the image.
− The Copy button is displayed below the right-hand side of the image. This button permits the
volume values to be copied to the clipboard and imported to other programs (e.g.: MS Word or MS
Excel) via the Paste function.
• Setting the Fill Level slider enables you to change the height level of the topography. The portion of
the topography lying below the set height level is filled with "water" (blue color) and the volume
parameters are calculated online only for the projecting part of the topography.
To use the Fill Level function, load the Profiles 3D display mode containing the Glowscale
palette, or activate No Palette to obtain optimum display.
• If the Diagram function Curve of tp is also activated, a red marker line shows the position of the
height level in the percentage of contact area curve.
Fig. 4-298
4-304
Image Display window, Topography display: 3D - Volume
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OPERATION
Display and Analysis of Images
Carl Zeiss
Parameters
The following parameters are calculated:
Z:
height level (selectable with the Z-Threshold and Fill Level sliders). The setting of this
value influences the following parameters.
Vm (z):
material volume above chosen height level
Vv (z):
void volume below chosen height level
Smr (z):
material volume ratio
S mr ( z ) =
Svr (z):
Vm ( z )
Vm ( zmin )
void volume ratio
Svr ( z ) =
Vv ( z )
Vv ( zmax )
Au:
surface bearing area of the topography at Z (= projection area of those parts which are
situated above chosen height level)
Smr:
surface bearing area ratio of the topography at Z
percentage of contact area (= Au / (x * y) * 100 %)
Sda:
true surface = sum of all triangles formed by adjacent data points of the surface
reconstruction
Sdr:
developed surface area ratio:
Σ (surface areaij) - Σ (projected areaij) / Σ (projected areaij) * 100 %
projected area = x * y
The percentage of the 3D surface area (sum of all triangles formed by adjacent data
points of the surface reconstruction) to the 2D surface area produced by projecting the
3D surface onto the threshold plane.
absolute flat surface ⇒ is equal to base plane (Sdr = 0 %)
The increase by which the 3D surface is larger than the basic plane (e. g. 625 % is a 3D
surface which is about 6.25 times larger than the projected basic plane)
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4-305
OPERATION
Display and Analysis of Images
Carl Zeiss
(2)
LSM 5 LIVE
LSM 5 LIVE DuoScan
Profile measurement mode in 3D display
This function is performed in the same way as in the 2D display mode, with the following exceptions:
The buttons
and
and
. Furthermore, the Position slider and
are replaced with the buttons
the input box (information of the position of the intersection line in pixels) are displayed below the Table
and Profile button bar. Changing the Z-Threshold also results in a change in the profile. In the 3D image,
a red marker line shows the y- and x-position of the displayed profile diagram.
Fig. 4-299
Image Display window, Topography display: 3D - Profile
• The position of the marker line (profile intersection line) can be changed by moving the Position slider
in x or y.
• Press the x- or y-button to select the required intersection plane.
(3)
z Histo measurement mode in 3D display
This function is performed in the same way as in the 2D display mode.
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LSM 5 LIVE DuoScan
(4)
OPERATION
Display and Analysis of Images
Carl Zeiss
Curve of tp measurement mode in 3D display
This function is performed in the same way as in the 2D display mode.
Before determination of the tp bearing portion, individual peaks (noise, steep slopes) must be eliminated.
The Median filter or a 3x3 longpass filter can be used for this purpose.
Fig. 4-300
Image Display window, Topography display: 3D – Curve of tp
Shifting the two cursor crosses permits two bearing portions to be given in percent (e.g. Smr1 = 10 %;
Smr2 = 90 %) as default values for which the height difference Rdc is determined automatically.
(5)
Grad. Histo measurement mode in 3D display
This function is performed in the same way as in the 2D display mode.
(6)
Roughness measurement mode in 3D display
This function is performed in the same way as in the 2D display mode.
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Carl Zeiss
OPERATION
Display and Analysis of Images
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.13.24.9 Export Data
− multiple profiles (Rel. 3.2)
− single profile
− parameters
− topography as matrix
− topography as triples
4.13.24.10 Topo ReUse
Topo routines can be saved and reloaded as tgp-files (TopoGraphic Parameters).
These files include settings for:
− reconstruction mode,
− intensity threshold,
− filters (including FFT),
− tilt angles (manual, 3 point fit),
− fit procedures (plane, cylinder, sphere),
− inverse and
− fill holes.
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4.13.25
OPERATION
Display and Analysis of Images
Carl Zeiss
Display - Prev.
This function allows to
− compose images, graphs and text for printing
− use any image format
− change fonts and line width in graphs via context sensitive menus
The settings of Chan, Zoom, Slice, Contr and Palette apply.
In the Options menu in the function Settings with the tab Print Status Display parameters are
determined and the Print Status Information is activated/deactivated.
Click on Prev will display the Preview window and the Print toolbar. Any changes done with this
toolbar are effective immediately. The content of the overlay plane is temporarily deleted while the
toolbar is displayed.
Fig. 4-301
03/06
Image Display window, Prev display with Assembly of image,
intensity profile and scan info
B 45-0019 e
4-309
OPERATION
Display and Analysis of Images
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.13.25.1 Context Menu for Scan Information Text
The context menu (right mouse button) allows to vary the output of the scan info.
• Click with the right mouse button. A context menu with the options Color and Font is displayed.
• In the Color menu, you can select a different type color for the scan info, in the Font menu a
different type font and type style.
4.13.25.2 Context Menu for Topography Images
When transferring a topography to the print preview, you can change the size and shape of type and
scale lines for the 3D graphics and profile measurement results.
(1)
Context menu for 3D graphics
• Click on the right mouse button. A context menu with the options Font enlargement and Line
width enlargement is displayed.
• You can change the type size in the Font enlargement menu and the line width of the scales in the
Line width enlargement menu.
(2)
Context menu for Profile measurement function
• Click on the right mouse button. A context menu with the options Scaling font enlargement,
Marker font enlargement and Overlay font enlargement is displayed.
• You can change the type font in the Scaling font enlargement menu, the size of the marker table in
the Marker font enlargement menu and the type size of the red measurement results in the
Overlay font enlargement.
4.13.25.3 Arranging and Printing the Print
Preview
• Click on the Arrange button for optimum
layout of image size and position relative to the
textual information.
• A layout generated with Prev. (Preview) can be
printed by clicking on the Print button in the
Print toolbar.
• Clicking on the Setup button opens the Print
Setup window, in which you can specify print
parameters.
Fig. 4-302
4-310
Print Setup window
• Click on the slider to change the zoom value of
the selected items.
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4.13.26
OPERATION
Display and Analysis of Images
Carl Zeiss
Display - Info
This function allows to
− display the parameters used during image acquisition of the image(s) displayed in the Image
Display window
− use any image format
− remove the info display
The settings of Chan, Zoom, Slice, Contr and Palette are not relevant for this function.
In the Options menu in the function Settings with the tab Image Status Display parameters to shown
are determined.
Click on Info will show the parameters. Click again to hide the info display.
Fig. 4-303
03/06
Image Display window, Info display
B 45-0019 e
4-311
OPERATION
Display and Analysis of Images
Carl Zeiss
4.13.27
LSM 5 LIVE
LSM 5 LIVE DuoScan
Additional Display Mode in Time Series
4.13.27.1 Display - Mean
This function allows to
− display the intensity time diagram (mean intensity in user defined ROIs over time)
− display the intensity time diagram for volumes (3D ROIs) within a Z-Stack over time
− use frame time series and frame Z Stack time series as input
− show the intensity values in table form and copy table to clipboard or save as text file
− show separate diagrams for each channel in a multi channel image
The settings of Chan, Zoom, Slice, Contr and Palette apply.
Click on Mean will display the Mean of ROIs toolbar. Any changes done with this toolbar are effective
immediately. The content of the overlay plane is temporarily deleted while the toolbar is displayed.
To use a similar functionality while scanning use the optional Mean of ROI function with the Time series
control.
• Click on the Mean ROI button.
− The Mean of ROIs image display toolbar will be displayed on the right. The used ROIs become
visible in the image, and the Intensity-Time diagram is shown on the left of the Image Display
window.
Fig. 4-304
4-312
Image Display window, Mean ROI display for time series in single
plane
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OPERATION
Display and Analysis of Images
Carl Zeiss
The Mean of ROIs toolbar contains the following function elements:
ROIs selection box: Display of the ROIs used during scanning of the time
series and of the other ROIs available in the system.
Add button: Opens the Add ROI List window for the storage of
changed or newly defined ROIs under a new name.
Remove button: Deletes the selected ROI from the ROIs selection box.
ROI data: Display of the data of the ROI selected from the ROIs selection
box. On deactivation of the check box of a ROI, its intensity values from
the Intensity-Time diagram are not displayed.
Arrow button: Activation of the mouse button for resizing or
movement of the ROI in the Image Display window.
Bezier button: Activates the Bezier figure drawing mode. The first click
sets the starting point, each additional click adds a further line, a
double-click on the starting point closes the figure and ends the
procedure.
Circle button: Activates the circle drawing mode. Clicking and holding
down the mouse button sets the center point; drag the diameter and
release the mouse button to end the procedure.
Recycle bin button: All the ROIs to the image are deleted.
Rectangle button: Activates the rectangle drawing mode. Click and
hold down the mouse button, drag the rectangle in any direction,
release the mouse button to end the procedure.
Ellipse button: Activates the ellipse drawing mode. The first click sets
the center point, the displayed line permits the determination of the first
dimension, the second click sets the first dimension, the second
dimension and the rotation direction can then be determined; the third
click sets the second dimension and the direction and ends the
procedure.
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Carl Zeiss
OPERATION
Display and Analysis of Images
LSM 5 LIVE
LSM 5 LIVE DuoScan
Polyline button: Activates polyline drawing mode. The first click sets
the starting point, each additional click adds a further line, a doubleclick on the starting point closes the figure and ends the procedure.
Line button: This button allows you to determine the line thickness of
the ROI outline.
Color / Auto button: One color from the list of colors can be assigned
to all ROIs. When Auto is pressed, the outlines of all ROIs are
automatically colored differently.
Buttons for diagram display:
− 1 button: Intensity values for ROIs and channels are shown in one
diagram.
− Chan button: Intensity values are shown separately for each
channel.
− ROI button: Intensity values are shown separately for each ROI.
− Mono button: Change between color and monochrome display
of the intensity time diagrams.
− Area button: Display of the area of the ROI in the intensity time
diagram, depending on the set threshold values. Area
measurements of very small areas (< 10 pixels) give only
approximate values.
− Mean button: Display of the mean values of the relevant ROI in
the intensity time diagram.
Ch1 / Ch2 / Ch4 button: Selection of the channel to be used.
Threshold low slider: The intensity values below threshold are not
displayed for the Area function.
Threshold high slider: The intensity values above threshold are not
displayed for the Area function.
Buttons for Table functions:
− Copy Table button: The table of intensity values is copied to the
clipboard.
− Show Table button: The table of intensity values is displayed on
the bottom left of the Image Display window.
− Save Table button: The table of intensity values can be stored as
a text file.
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4.14
Image Optimization
4.14.1
Single Channel
OPERATION
Image Optimization
Carl Zeiss
Described below is the example of the acquisition of an image, using an excitation wavelength of 532 nm
and a fluorescence emission range above 550 nm.
Let the specimen be a thin section through a stem of Convallaria majalis (Lily-of-the-Valley). The
description applies to the use of the Axio Imager.Z1 microscope, and analogously also to the
Axiovert 200 M.
4.14.1.1
Requirements
The suitable laser is switched on.
The specimen has been positioned and focused for
scanning.
• The LSM button has to be activated either in
the LSM software or on the LSM button on the
LSM tube itself.
• Click on the Config button in the Acquire
subordinate toolbar of the Main menu.
− This opens the Configuration Control
window.
• Click on the Single Track button.
ChL1 icon and assign a color to
• Click on the
Channel 1 in the Channel Color Selection
window. Activate channel 1 via check box.
• Click on the
icon of emission filter 1 (before
ChL1) and select the LP 550 filter.
• If required, deactivate all the other channels
(ChL2) via check box.
03/06
Fig. 4-305
B 45-0019 e
Configuration Control window
4-315
OPERATION
Image Optimization
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
icon, activate the 532 nm
• Click on the
laser line by clicking on Line Active . If
required, deactivate other laser lines which are
not needed.
• Use the Transmission slider to set the laser
intensity to approx. 30 % at first.
The Beam Path and Channel Assignment panel
displays the current configuration loaded.
Fig. 4-306
The set laser intensity must be
subsequently optimized for the current
situation via the Transmission slider.
Excitation panel
For overlaying fluorescence and transmitted-light images, activate a second channel without a laser line
but the Halogen illumination of the light microscope as the transmitted image light source.
Choose the BP 675-725 in the emission filter menu and swing in the according red filter at the
condensor
Of course, all other transmitted light applications like
− phase contrast
− differential interference contrast (DIC)
− polarization contrast (Pol)
− darkfield
can also be performed.
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OPERATION
Image Optimization
Carl Zeiss
• In the Main menu click on the Scan button in
the Acquire subordinate toolbar.
− This opens the Scan Control window.
• Click on the Mode button.
• For a frame scan, click on the Frame button.
• On the Objective Lens Image Size & Line
Step Factor panel, select Objective and Frame
size for the scan (e.g. X 512 / Y 512 scan).
• Enter a Exposure Time of 5 to 10 frames per
second, for example, to start with.
• Start with the following settings on the Pixel
Depth, Scan Direction & Scan Average
panel:
Data depth:
Scan direction:
Average:
8 bits
unidirectional
Number: 1
• On the Zoom, Rotation & Offset panel, set a
zoom of 1.
Using the Fast XY button is a convenient
way of creating an overview scan.
Fig. 4-307
03/06
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Scan Control window (Mode)
4-317
OPERATION
Image Optimization
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• Click on the Channels button.
− This displays the preset parameters of the
configuration loaded.
• Click on the Find button. Make sure to position
the slider correctly. Then scan while the slider is
in the LSM position.
− This starts the scanning process.
− The image is seen to build up gradually in a
new window.
Function Find produces images of
different brightness for different scan
speeds.
As a rule, the first scanned image (Pre-Scan) is not
ideal, since the photomultiplier is not matched to
the light output. More often than not, the screen
image is dull and needs subsequent optimization.
Fig. 4-308
Scan Control window (Channels)
Fig. 4-309
4-318
Image Display window
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4.14.1.2
OPERATION
Image Optimization
Carl Zeiss
Confocal Aperture / Detector Gain /
Ampl. Offset
• In the Scan Control window, click on the
Cont. button (see Fig. 4-308).
− This starts a continuous scan.
• Use the Pinhole slider (confocal aperture) to set
the aperture size in the Scan Control window
under Channels.
− The aperture size should be so small that
there is still enough variation for the setting
of the detector gain and that sufficient
image information is still available. 1 Airy is a
good value to enable a confocal fluorescence
XY-image to be obtained.
− A small aperture size will increase the depth
of focus, but reduce the light intensity
received by the CCD detector.
Fig. 4-310
Image Display window with confocal
ROI
Fig. 4-311
Color Palette window
− The influence of the aperture size on image
creation is shown by the example in Fig.
4-310. The entire image was first scanned
with too large a aperture size. The aperture
size was then optimized for a defined ROI.
This considerably improved the display of the
specimen structures.
• Click on the Palette button in the Select image
processing toolbar.
− This opens the Color Palette window.
• In the Color Palette List panel, click on the
Range Indicator item.
− The scanned image appears in a false-color
presentation.
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4-319
OPERATION
Image Optimization
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
If the image is too bright, it appears red on the
screen.
Fig. 4-312
Image Display window
If the image is not bright enough, it appears blue
on the screen.
• On the Channel Settings panel of the Scan
Control window, set the CCD detector gain
with the Detector Gain slider.
− The image should not have more than a
trace of red.
− This adjustment is very sensitive. Try using
the left and right arrows to make the
adjustment instead of dragging the slider
bar.
Fig. 4-313
4-320
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OPERATION
Image Optimization
Carl Zeiss
• To adjust the black level (background), use the
Ampl. Offset slider so that areas without
picture content just show a trace of blue.
• In the Color Palette List panel of the Color
Palette window, click on No Palette.
− This deselects the Range Indicator and
activates the new presentation.
• In the Scan Control window, click on the Stop
button.
− This stops the continuous scan.
If you use the Range Indicator for image
optimization, it may happen that the
ranges marked in the Range Indicator
will vary when the channel color is
changed.
4.14.1.3
Fig. 4-314
Image Display window
Fig. 4-315
Scan Control window
Exposure Time, Scan Average and
Pixel Depth
The signal-to-noise ratio can be substantially
improved by reducing the scanning speed to an
acceptable level and averaging over several scans
(i.e. with an average Number greater than 1 for
the Mean average Method in the Scan Control
window).
• Use the Exposure Time slider in the Objective
Lens, Image Size and Line & Step Factor
panel to set the slowest acceptable exposure
time speed.
− The corresponding value for Frames per
Second is shown below the slider.
• In the Number text box of the Pixel Depth,
Scan Direction & Scan Average panel enter
the number of measurements to be averaged.
Image optimization can be effected much
faster if you select a smaller frame, since
less data have to be processed.
The greater the number of averages
selected for Mean average Method, the
better the image quality will be; the
scanning time will be prolonged
accordingly.
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OPERATION
Image Optimization
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
4.14.2
Multiple-channel
4.14.2.1
Requirements
• The suitable lasers are on.
• The specimen has been positioned and focused
for scanning.
• The LSM button has to be activated either in
the LSM software or on the LSM button on the
LSM tube itself.
In the following example, 2 Channels shall be
activated for the scanning procedure: one for
488 nm using emission filter BP 500-525 and one
for 532 nm with LP 550. NFT 532 is used as the
secondary dichroic beam splitter.
• In the Acquire subordinate toolbar, click on the
Config button.
− This opens the Configuration Control
window.
Fig. 4-316
Configuration Control window
• Click on the Single Track button.
• Activate (in the same way as for the single channel, see page 4-315) channel 2 (ChL2), the indicated
emission filters and the main and secondary dichroic beam splitter for the scanning procedure.
− The configuration loaded is displayed in the Beam Path and Channel Assignment panel.
• Click on the Scan button in the Acquire subordinate toolbar of the Main menu.
− This opens the Scan Control window.
• In the Scan Control window, set the parameters in the same way as described for single-channel
presentation.
• Click on the Find button in the Scan Control window.
− This starts the scanning process. The scanned image appears in a separate window.
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Fig. 4-317
OPERATION
Image Optimization
Carl Zeiss
Scan control and Image Display windows
As a rule, the first scanned image (Pre-Scan) is not ideal, since the photomultiplier is not matched to the
light output. More often than not, the screen image is dull and needs subsequent optimization.
• Click on the Channels button in the Scan Control window.
− This opens the Channel Settings and Excitation of Track panels.
− The channels used are color-highlighted.
4.14.2.2
Image Optimization
The image optimization processes
− setting of aperture size
− Detector Gain / Ampl. Offset
− Scanning speed and Average
must be carried out separately for each channel used (see Single Channel, page 4-315).
For the optimum setting of the single channels, Split xy-display must be selected in the Image Display
window to enable the direct viewing of the separate images of the relevant channels.
• Click on the Cont. button in the Scan Control window.
− This starts a continuous scan.
• Click on the Split xy button in the Image Display window toolbar.
− This displays the separate images scanned in the channels and the composite (overlay) image.
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Image Optimization
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
• Now click on the ChL1 button in the Channel Settings panel to optimize Channel 1. Optimization is
performed in the same way as for the single channel and can be monitored online in the relevant
separate image of the channel.
• Then optimize the second channel by clicking on the relevant button (ChL2) in the Channel Settings
panel of the Scan Control window.
Fig. 4-318
Scan Control and Image Display windows
Now effect image optimization as explained for the single-channel mode, but separately for each
channel.
• Now click on the xy button of the Display toolbar.
− The composite scan image of two channels is presented in a common window.
Image optimization can be effected much faster if you select a smaller frame, since less data
have to be processed.
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4.15
OPERATION
Shut-Down Procedure
Carl Zeiss
Shut-Down Procedure
Never shut down the computer by its main switch while your LSM program is still active, or else
you will lose the currently set operating parameters and the images just scanned.
In the Settings for user dialog window, which can be activated with the Options / Settings
buttons, activate Laser off or Exit in the Shutdown tab. The lasers will then automatically be
switched off when you exit the LSM program.
4.15.1
Exiting the LSM Program
• Close all open windows of the LSM program by clicking on the closing icon
of each window.
in the top right corner
− This closes the respective window and removes the respective icons from the taskbar.
− After all dialog windows have been closed, the LSM 5 LIVE Switchboard window appears.
Fig. 4-319
LSM 5 LIVE Switchboard menu
• Click on the Exit button.
− This terminates the LSM program.
− The monitor screen shows the desktop of the WINDOWS XP operating system.
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OPERATION
Shut-Down Procedure
Carl Zeiss
4.15.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
Shut Down the WINDOWS Operating System
• Move the cursor to the bottom margin of the screen.
− This opens the taskbar containing the Start button.
• Click on the Start button of the taskbar.
− This opens a pop-up menu.
• Click on the Shut Down item.
− This opens the Shut Down Windows window, in which you can select between Shut down,
Restart and Login.
• Unless already set by default, click on Shut down the computer?
• Click on the Yes button.
About 20 seconds after WINDOWS XP has been run down, and your computer turns off.
4.15.3
Turning Power Off
Please bear in mind that a cooling phase of at least 5 minutes is required between switching off
of the laser via the software and switching off of the entire system via the REMOTE CONTROL
main switch or the Power Supply switch of the Enterprise UV laser.
Set the REMOTE CONTROL switches “System/PC” and “Components” or the Main switch on
the System Electronic Rack and the power supply switch of the Ar Laser to position "OFF" after
5 minutes.
This puts your LSM 510 microscope system, including the computer, off power.
4.15.4
Turning off the HBO 100
• Switch off the HBO 103 via the toggle switch of the HBO 100 power supply.
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4.16
OPERATION
Software and Hardware Options
Carl Zeiss
Software and Hardware Options
This section describes optional software and hardware configurations. Depending on your configuration,
the content of dialogue and function may differ.
4.16.1
Software
The following software packages for Release 4.0 are available:
− Software "Physiology"
− Software "Topography"
− Software "Macro Recorder and Editor"
− Software "3D for LSM"
− Software "Multiple Time Series"
− Software "Image VisArt"
− Software "Deconvolution"
− Software "StitchArt plus"
− Software “Visual Macro Editor”
− Software “FRAP”
If your configuration does not include any of the SW packages "Physiology", “FRAP” or FRET, the
following functions are not available:
− Mean of ROI button in the Image Display window
If your configuration does not include the "Physiology " software package, the following functions are
not available:
− Mean of ROI scan button in Time Series Control
− Ion Concentration button in the Process Menu
If your configuration does not include the “Visual Macro Editor” software package, the following
functions are not available:
− VME button in the Macro Menu
If your configuration does not include the "Topography " software package, the following functions are
not available:
− Topo button in the Image Display window after acquisition of image stacks
If your configuration does not include the "Macro Recorder and Editor" software package, the following
functions are not available:
− New, Save and Save as buttons in the Macro Control window
− Edit, Step, Delete, Editor buttons in the Macro Control window
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Carl Zeiss
OPERATION
Software and Hardware Options
LSM 5 LIVE
LSM 5 LIVE DuoScan
If your configuration does not include the "3D for LSM" software package, the following separate
application is not available:
− 3D for LSM
If your configuration does not include the "Multiple Time Series" software package, the following
function is not available:
− Macro: "Advanced Time Series"
If your configuration does not include the "Image VisArt" software package, the following function is not
available:
− 3D button in the Image Display window
If your configuration does not include the "Deconvolution" software package, the following functions
are not available
− DCV Settings button in the Ortho function of the Image Display window
− DCV button in the Process menu
If your configuration does not include the "StitchArt plus" software package, the following function is
not available:
− Macro: "StitchArt plus"
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4.16.2
OPERATION
Software and Hardware Options
Carl Zeiss
Hardware
Depending on whether the following hardware components are available or not, the content of the
screens may differ:
− Piezo objective focusing device
− X-Y scanning stage DC 4 × 4 or DC 100 × 90, each with MCU 28
− Stands: Axio Imager.Z1, Axiovert 200 M
− Depending on the configuration the scan head equipment may differ in filters, beam splitters and
the number of photomultiplier
− Transmitted-light PMT
− Monitor diode
If your configuration does not include the Piezo objective focusing device, the following functions are not
available:
− Hyperfine Z Sectioning in the Z Stack function in the Scan Control window
− HRZ parameters in the Stage and Focus Control window
If your configuration does not include the X-Y scanning stages DC 4 × 4 or DC 100 × 90, each with
MCU 28, the following functions are not available:
− Stage Position and Tile Scan functions in the Stage and Focus Control window
Depending on the used microscope stand: Axio Imager.Z1 or Axiovert 200 M, the following dialogue and
available functions may differ:
− Context and accessibility of the Microscope Control window
If your configuration does not include an AxioCam, the following functions are not available:
− Camera in the Config Control window, Scan Control window
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Carl Zeiss
4.17
OPERATION
LSM 5 LIVE
Courses on "How to Operate the System in an Optimized Way" LSM 5 LIVE DuoScan
Courses on "How to Operate the System in an Optimized Way"
Carl Zeiss is offering training courses on how to operate the system in an optimized way.
− Courses are held in our application center in Jena, Germany.
− Courses are held in English or German language, respectively.
Check out:
www.zeiss.de/lsm
for the latest dates and ask your Zeiss representative for a quotation on courses.
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CHAPTER 5
TOOLS
Contents
Carl Zeiss
TOOLS
CONTENTS
Page
5
Tools.................................................................................................................................5-2
5.1
Change Filters ....................................................................................................................5-2
5.2
Stand Select .......................................................................................................................5-4
5.3
LSM Image Browser ...........................................................................................................5-5
5.4
LSM Image Examiner..........................................................................................................5-6
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5-1
TOOLS
Change Filters
Carl Zeiss
5
TOOLS
5.1
Change Filters
LSM 5 LIVE
LSM 5 LIVE Duo Scan
The Change Filters tool is used to update the filter data in the software after a change of filters in the
reflector turret of the microscope, the emission filters and the beam splitters of the LSM 5 LIVE.
All filter data are updated in the Emission Filters & Beam Splitter Control window. After activation of
the appropriate button (Emission Filters, Filter Cubes Stand or Beam Splitters LSM), the special input
mask for the selected filter type is displayed. Since the procedures of updating or entering a new filter
type are identical for all types, only the updating of filters in the reflector turret (Filter Cubes Stand) will
be described in the following.
• Close the LSM 5 LIVE software program.
• Insert the new filter module in the reflector turret.
• Double-click on the Change Filters icon on the desktop.
− The Emission Filter & Beam Splitter Control window appears on the screen. The name of the
currently used database is displayed in the System Database box, with the filter type being indicated
below for checking purposes.
• Click on the Filter Cubes Stand button. The Filter Cubes Stand panel is displayed.
− The Filter Cubes Stand panel shows the Filter-Wheel No. and the filter positions available. Use the
Name and ID selection boxes to enter the filters installed in the individual positions of the filter wheel.
• Open the Name (or ID) selection box of the relevant filter position and select the new filter set from
the list.
• Click on the Store button to accept the new settings.
• Click on the Close button to close the Emission Filter & Beam Splitter Control window.
All available filter sets have to registered in the filter list (see Edit Filter List function, next
page).
Edit Filter List
The Edit Filter List function permits updating of
the filter data in the software after a change of
filters on the stand.
• Close the LSM 5 LIVE software program.
• Double-click on the Change Filters icon on the
desktop.
Fig. 5-1
Edit Filter/Beam Splitter List
window
• Click on the Edit Filter List button in the
Emission Filter & Beam Splitter Control
window.
− The Edit Filter/Beam Splitter List window
is opened.
This window permits a list of the most frequently used filter sets to be compiled.
• Click on the arrow button in the Filtername list box to open it.
• Select the filter set which shall be included in the list.
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TOOLS
Change Filters
Carl Zeiss
• Click on the Apply button.
The selected filter set is included and displayed in the list (below the Sumary list box).
This filter set is now also available in the Name selection boxes of the Filter Cubes Stand panel and can
be assigned to a filter wheel position.
To remove a filter set which is no longer needed from the list, proceed as follows:
• Click on the name of the filter set concerned in the list box of the Edit Filter/Beam Splitter List
window.
• Click on the Remove button. The filter set is deleted from the list and is then no longer available in
the Filter Cubes Stand panel of the Emission Filter & Beam Splitter Control window.
Add New
This function permits new filter sets to be added to
the database.
For this, proceed as follows:
• Click on the Add New button on the Edit
Filter/Beam Splitter List window.
− The Add New
window is opened.
Filter/Beam
Splitter
• Enter the data of the new filter set in the Filter
Cubes Stand Description panel, then click on
the Apply button.
The new filter set is stored in the database and
included in the New Filter Cubes Stand panel.
You can now activate the filter for a filter wheel
position using the procedure described above.
If you have activated the Non Zeiss check
box, filter sets from other manufacturers
can also be included in the database.
Fig. 5-2
Edit Filter/Beam Splitter List
window
• To remove an new filter set from the database, select it with a click of the mouse in the New Filter
Cubes Stand panel and then click on Remove.
• Click on Close to close the Add New Filter/Beam Splitter window.
• Click on Close to close the Edit Filter/Beam Splitter List window.
• Click on the Store button to accept the new settings.
• Click on the Close button to close the Emission Filter & Beam Splitter Control window.
When you start the LSM 5 LIVE software, the filter data are updated.
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5-3
TOOLS
Stand Select
Carl Zeiss
5.2
Fig. 5-3
Select Stand Database ... window
LSM 5 LIVE
LSM 5 LIVE Duo Scan
Stand Select
The Stand Select tool permits a new or updated
database to be assigned to the LSM 5 LIVE
software program. This function should preferably
be performed by authorized service personnel.
If this is not possible, proceed as follows:
• Close the LSM 5 LIVE software program and double-click on the Stand Select icon on the desktop.
− The Select Stand Database window appears on the screen. The currently used database is
displayed in the Database box.
• Click on the Browse button to activate the new
database.
− The Open window appears on the screen.
• Select the directory where the new database is
stored.
• Click on the name of the database (file
extension: *.mdb) and then on the Open
button.
Fig. 5-4
Open window
− The Open window is closed and the name
of the new database appears in the
Database box.
• Click on the Permanent button. The Select
Name window appears.
• Select the relevant stand icon from the Icon list
box and click on OK. The Select Name window
is closed and the desktop icon is updated.
• Then click on the OK button in the Select
Stand Database ... window to accept the new
settings and to close the window. (Clicking on
Cancel will cancel the procedure.)
Fig. 5-5
5-4
Select Name window
− After the next restart of the LSM 5 LIVE
software program, the new database will be
automatically read in.
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5.3
TOOLS
LSM Image Browser
Carl Zeiss
LSM Image Browser
The LSM Image Browser permits images to be loaded, imported, exported and printed quickly without
having to open the LSM 5 LIVE software. The LSM Image Browser can be used without dongle.
When images are opened, image processing functions of the LSM 5 LIVE software are available to a
limited extent (Chan, Zoom, Contr, Palette, Copy, Save, Save As, xy, Split xy, Prev, Info).
• Click on the LSM Image Browser icon on the desktop of the PC. The Zeiss LSM Image Browser
main menu is opened.
Fig. 5-6
Zeiss LSM Image Browser main menu
The following function buttons are available:
New button
Opens a new database.
Open button
Opens an existing database.
Save button
Saves the current image.
Save As button
Saves the current image under a new name.
Import button
Imports images.
Export button
Exports images.
Full Screen button
The current image is displayed on the full screen. Deactivation of the
function with a click of the mouse.
Multi Print button
Several images are printed on one page.
RAM button
Use of the RAM memory for image display.
DISK button
Use of the hard disk as storage medium for image display.
Exit button
The Zeiss LSM Image Browser main menu is closed.
The functions New, Open, Save, Save As, Import, Export and Multi Print correspond to those of the
Expert Mode of the LSM 5 LIVE software and have already been described in chapter 4.
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5-5
TOOLS
LSM Image Examiner
Carl Zeiss
5.4
LSM 5 LIVE
LSM 5 LIVE Duo Scan
LSM Image Examiner
The LSM Image Examiner can be used without having to open the LSM 5 LIVE software. However, this
requires the installation of the relevant dongle. The LSM Image Examiner provides all the functions of
the LSM Image Browser, plus the 3D functions and selected Process functions of the Expert Mode of
the LSM 5 LIVE.
When images are opened, a large scope of the image processing functions of the LSM 5 LIVE software is
available (for further details see chapter 4).
• Click on the LSM Image Examiner icon on the desktop of the PC. The Zeiss LSM Image Examiner
main menu is opened.
Fig. 5-7
Zeiss LSM Image Examiner main menu
In addition to the buttons of the LSM Image Browser mentioned above, the following function buttons
are available in the lower row of the Zeiss LSM Image Examiner main menu:
Projection button
One single projection or a series of projections can be calculated after
rotation of the data package about the X, Y or Z axis.
DepthCod button
The depth information contained in a sequence can be colored with the
colors of the rainbow.
Stereo button
Stereoscopic images can be generated.
Ratio button
Permits two channels to be linked into a new channel by the creation of a
ratio.
Copy button
Permits one channel each of an existing image to be copied and stored as
a new image.
Filter button
Permits the subsequent processing of scanned images via the integrated
filters.
Interpolate button
Permits the continuous contrast and brightness change in a stack or time
series through interpolation between the starting and end values.
Shift button
Produces a congruent image with relation to the pixels of the various
channels.
Duplicate button
Permits images (including Z Stacks and Time Series) to be duplicated
completely.
Contrast button
Permits the subsequent modification of contrast and brightness of the
stored image.
These functions correspond to those of the Expert Mode of the LSM 5 LIVE software and have already
been described in chapter 4.
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CHAPTER 6
3D FOR LSM
Contents
Carl Zeiss
3D FOR LSM
CONTENTS
Page
6
3D for LSM .......................................................................................................................6-2
6.1
6.1.1
6.1.2
6.1.3
Overview and Explanations.................................................................................................6-2
The Image Sequence ..........................................................................................................6-2
The Image Properties..........................................................................................................6-3
Memory Usage...................................................................................................................6-3
6.2
6.2.1
6.2.2
6.2.3
User Interface.....................................................................................................................6-3
Introduction .......................................................................................................................6-3
Main Window ....................................................................................................................6-4
Display Window .................................................................................................................6-7
6.3
6.3.1
6.3.2
6.3.3
6.3.4
6.3.5
Functions .........................................................................................................................6-11
Functions in the File Menu ...............................................................................................6-11
Functions in the Edit Menu...............................................................................................6-14
Functions in the Process Menu .........................................................................................6-17
Functions in the View Menu.............................................................................................6-43
Functions in the Measurement Menu ...............................................................................6-50
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3D FOR LSM
Overview and Explanations
Carl Zeiss
6
3D FOR LSM
6.1
Overview and Explanations
(0, 0, 0)
6.1.1
Voxel
Single slice with
single channel
Single slice with
multiple channels
Image sequence
Multichannel
Positive rotation directions
of the axes
Z
X
LSM 5 LIVE
LSM 5 LIVE DuoScan
The Image Sequence
The "3D for LSM" handles image sequences
generated by the Zeiss LSM software. This can be
three-dimensional image data or a time sequence
of two-dimensional images (slices). Each slice (as
well as the sequence) can consist of up to eight
channels. An image sequence consists of a series
of individual (2D) images and has a name that
designates the entire sequence. In general an
image sequence is handled as a single object in the
system. Individual channels or slices can be
addressed.
The following terms and definitions apply for the
"3D for LSM" software.
− An image sequence is a number of individual
sequential images (usually called slices in the
dialog boxes), the spacing between which is
equal.
− Image sequences can contain up to 12 bit of
image data (per channel).
Y
Fig. 6-1
− A sequence (slice) can consist of up to eight
channels.
− The maximum size of an image sequence is
limited by the provided memory of the
operating system.
− A voxel is the smallest element of an image sequence (the equivalent of a pixel in a 2D image). All
voxels in a given image sequence are the same size.
− The coordinate system originates in the left upper front corner of the image sequence. This point has
the coordinates 0, 0, 0.
− All angles are positive for rotations to the right in the direction of the positive coordinate axis (righthanded coordinate system).
− A slice is an individual image in a sequence of images. The numbering of the slices starts with "1".
Image sequences can consist of several channels. Most functions and the Display window are providing
buttons to select all or a subset of channels stored in the selected image sequence. The Output image
sequence will only get those channels which are selected on the input side. The button selects all
channels in the image sequence to be used clicking with the left mouse button on it.
Clicking with the left mouse button on any of the number buttons toggles the state of this single
channel.
Clicking with the right mouse button on any of the number buttons selects this single channel exclusively.
All other channels are deselected.
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6.1.2
3D FOR LSM
User Interface
Carl Zeiss
The Image Properties
Every image sequence has its own set of properties. They contain the scaling and the scaling units. The
scaling and its units are required for 3D reconstruction and measurement. If a sequence of LSM-TIFF
images is read in, the image properties are loaded automatically from the file header and allocated to the
image properties of the new image sequence.
6.1.3
Memory Usage
All images shown in the Gallery are currently loaded in the system memory of the operating system.
Some functions need additional temporarily used memory during their execution.
If the memory is running low delete some images from the Gallery. If the images are needed afterwards
they must be saved to disk first. Normally all functions produce a new result (output) image sequence. In
order to save some memory, other image sequences currently presented in the Gallery can be selected
as result position. The output image is overwritten by entry execution of a function.
6.2
User Interface
6.2.1
Introduction
This section describes the following main components of the system:
Main window
Main window with the Menu, the Tool bar and Gallery. All general system
functions are located here.
Gallery
Normally several images are required in order to accomplish a particular task.
These images are displayed in reduced size to provide an overview and facilitate
selection. This area is located just below the Tool bar.
Fig. 6-2
Tool bar
03/06
This menu shows all image processing functions.
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3D FOR LSM
User Interface
Carl Zeiss
Display window
LSM 5 LIVE
LSM 5 LIVE DuoScan
This window is used to display image sequences.
Display window
Fig. 6-3
Dialog boxes
6.2.2
Display window
All dialog boxes provide three buttons. Pressing the OK button executes the
function with the defined parameters and closes the dialog window. Selecting the
Cancel button does not execute the function, restores the parameters, and closes
the dialog window. Pressing the Apply button executes the function with the
defined parameters; the dialog window will stay opened.
Main Window
The Main window includes:
the Menu
the Tool bar
and the Gallery
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3D FOR LSM
User Interface
Carl Zeiss
File Menu
Open Image
Opens a file selector dialog to load an image sequence.
Save Image As
Opens a file selector to save an image or image sequence.
Save Display As
Saves the currently shown contents of the Display window as a single
colour image.
Print
The printer parameters can be set with this tool. The standard Windows
printer dialog is opened.
Exit
Terminates the application.
Edit Menu
Copy
Copies the contents of the Display window to the clipboard.
Edit Channels
Allows to add or to remove channels to a single or multichannel image.
Delete All Images
Deletes all images and image sequences from the memory.
Process Menu
03/06
Arithmetics
Adds or subtracts the grey values of two image sequences
(Add, Subtract).
Contrast
Enhances the contrast and brightness of an image sequence
(Interactive, Automatic, Linearize).
Smooth
Smoothes an image sequence.
Morphology
Performs morphological operations on image sequences
(Erode, Dilate, Open, Close).
Segment
Segmentates an image sequence to propose measurement
(Interactive, Automatic).
Boolean
Combines two image sequences by Boolean operations
(And, Or, Not, Xor, Mask).
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Scrap
Selects or deletes objects of a defined size.
Fill Holes
Fills holes in objects.
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View Menu
Set Channel Colour The colour and the weight of the single channels can be defined.
Properties
The properties of the image (e.g. scaling, use laser etc.) are displayed.
Render
Calculates 3D reconstructions of an image sequence (Surface, Alpha).
Measurement Menu
Automatic Object
Measures geometrical and densitometrical features (General, Object
Features, Volume Features, Condition).
Windows Menu
Arrange All
Arranges the windows automatically.
Display
The current image is displayed in this window.
Help Menu
Content
Opens the help for the software.
About 3D for LSM
Displays status and release message of the software.
Tool Bar
This bar provides buttons with iconized images of nearly all functions. Clicking on one of the buttons will
open a dialog window to define the function parameters. Selecting an entry from the menu alternatively
can activate the same functions. Placing the cursor on a tool bar button will show a short description, if
the window is activated.
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Gallery
The Gallery is used as an overview of the images available in memory and their contents. It is located just
below the Tool bar. Each small image represents a sequence. The middle slice of each image sequence is
shown. The status bar of each image shows the name. The name might be a number or a string.
Every image sequence has its own channel colour assignment (see Display window). When an image is
copied the channel colour assignment is copied too. Drag and drop techniques can be applied to copy
images or define the function parameters Input and Output using the Gallery thumbnails.
• Position the cursor on an image in the Gallery.
• Press the left mouse button.
• Hold the mouse button down and move the mouse to the destination position.
• At the destination release the left mouse button, the destination image will be overwritten.
To delete an image, drag it, move it to the wastebasket, and drop it.
6.2.3
Display Window
This window is used to display an image sequence, regardless of size or type. To show multiple channel
sequences each channel could have its own base colour. The user can set these colours and the
weighting for each channel by pressing the corresponding button
at the bottom of the window. To
display a different image or image sequence, it can be dragged from the Gallery and dropped to the
Display window.
The image can be displayed in full size (one pixel on the screen represents one pixel of the image) or in a
zoomed size. To zoom the display view click and hold down the right mouse button on the window
border and resize the window. The aspect ratio of the image will not be changed. Clicking on the button
resets the Display window to a full size view of the image (see above).
The title bar shows the currently displayed sequence name. The status bar displays the size of the current
sequence and the selected slice on the left. On the right the cursor position within the window and the
corresponding intensity (grey) value of each channel is shown.
The Display window can be closed without any effect to the image processing functions. If no Display
window is opened select the entry Display in the Window menu.
The scroll bar at the lower right of the window enables to show the images in a sequence. The range
reaches from one to the maximum slice provided by the current sequence.
To start the automatic animation of an image sequence start the Player tool by clicking on the button
. The colour selection for the channels can be activated by clicking on the button
image can be displayed as a grey value image by clicking on the button
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Player
This function plays back the sequential images of an image sequence.
Fig. 6-4
The image sequence is displayed in the Display window. The display process is working as a
background task; other functions can be executed while the player is running. There are several ways to
stop the player:
by closing the player window
by pushing the red Stop button of the player window (the window remains open)
by closing the image window.
The Increment parameter specifies whether each sequential image (1) should be displayed or whether
some sequential images should be skipped during display. The value 2 skips one image for every
sequential image displayed, in other words, it displays only every second image.
The parameter Wait Time states the delay in milliseconds between two successive sequential images.
The maximum display speed depends mainly on the hardware. The sequential images are always
displayed in their entirety, regardless of the set delay.
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Control Element of the Player
The three arrow shaped controls on the scale show the start slice and the currently displayed sequential
image. The values (positions) can be changed using the mouse. Press and hold the left mouse button and
move the pointer to the desired position. The set values are shown in the numerical windows at right.
Start slice
Currently displayed sequential image
End slice
The buttons in the left group start and stop playback of an image sequence.
Reverse playback
Forward playback
Play forward and then backward again (jojo)
Stop playback
Pause playback
The buttons in the middle group control the settings of the current sequential image.
Reset to start slice.
Single step backward (1 sequential image each regardless of
Increment).
Single step forward (1 sequential image each regardless of
Increment).
Set to end slice.
Increment
Image increment.
Wait Time
Displays delay between two images (in milliseconds).
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Set Channel Colour
This function sets the colour and weight for the channels.
Fig. 6-5
Each image sequence can get its own colour definitions. All functions will inherit the colour definition
from the Input sequence to the Output sequence. By default the colours are set to 100 % weighting
and the pure base colours (red, green, blue) are defined.
The weight can be any value between 0 % and 200 %. The colour can be redefined by clicking on the
coloured button on the right of the dialog. The standard Windows colour selection dialog is opened. The
solution is done by clicking on one of the colours or by entering appropriate numbers in the
corresponding edit boxes.
Pressing the OK button will close the colour selection dialog and update the Display window
immediately.
Only those channels, which are available in the image sequence, can be defined.
Parameters:
6-10
Image
Image sequence to edit
Weight
Colour weighting for each channel
Colour
Base colour for each channel
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6.3
Functions
6.3.1
Functions in the File Menu
Carl Zeiss
Open Image
This function reads a Zeiss LSM (*.lsm), Zeiss LSM TIFF (*000.tif) or Carl Zeiss Vision (*0.img) image
sequence from a disk or network drive.
Fig. 6-6
The individual files of a Zeiss TIFF image sequence are read and saved as an image sequence in image
memory. In addition, the image properties are read out of the TIFF files and allocated to the image
sequence Input.
The directories of the current drive are listed in the Directories list box. Use the Drives list box to choose
a different drive.
In case of choosing the TIFF-format in the Files of Type box, three number characters are always
expected before the dot in the filename extension. The first number must be 000 at the end of the
filename. From a complete sequence only this file is listed in the dialog, if "LSM TIF Images (*000.tif)" is
selected in the Files of Type box. To view all TIFF files "All TIF Images (*.tif)" in the Files of Type box
must be selected. This selection enables to start with a different file than with the very first (named
*000.tif) at the end of the filenames three number digits.
Currently the Carl Zeiss Vision file format "KE Images (*0.img)" is supported. Two files per channel are
saved.
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Carl Zeiss Vision image sequences must have a number digit at the end of the base filename. They are
used to indicate the different channels in a multichannel sequence. The numbering starts with zero (0). If
a sequence is saved in the Carl Zeiss Vision format the numbers are generated automatically. To load
such an image sequence "KE Images (*0.img)" in the Files of Type box must be selected.
The window incorporates the usual file selection controls. The bottom half displays a selection of the
image properties that are stored in the image sequence.
Parameters:
BaseName
Base name of the TIFF files (image sequence) to be loaded. Only the letters before
the first number are stated.
Input
Name of the resulting image in which the image sequence will be saved.
Save Image As
This function saves an image or image sequence to disk or network drive.
Fig. 6-7
All the files in the current directory that have the selected image format are listed in the File Name list
box.
The directories of the current drive are listed in the Directories list box. Use the Drives list box to choose
a different drive.
Use the list box Files of Type to select the image format. Currently the LSM image format (*.lsm) and
the Carl Zeiss Vision file format "KE Images (*0.img)" is supported.
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By choosing the Carl Zeiss Vision file format "KE Images (*0.img)", two files per channel are saved. On
one hand the Carl Zeiss Vision type image sequence file, on the other hand the file with the image
properties. One pair of files is written per channel. They are numbered automatically, starting with zero.
A one number digit is added to the end of the filenames. The two files share the same filename but have
different filename extensions (*.img and *.3d).
The content of the Gallery is shown in the Input section. The selection of the sequence to save is done
by highlighting one of the provided names or by drag and drop from the Gallery.
Parameters:
Input
Name of the image sequence to be saved
Filename Name of the file to be used on disk
Save Display As
This function saves the current Display window contents to a disk or network drive.
Fig. 6-8
Before the execution of this function any image or image sequence can be selected to be displayed. From
a multichannel sequence any channel status (on or off) combination can be defined. The colours of the
shown channels can be set with the function Set Channel Colour.
The current zoom factor of the Display window is not taken into account, the image is saved without
any zoom.
The image is saved as a true colour image with 24-bit resolution. From the Save as Type list box one of
the provided formats can be selected.
Parameters:
None
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Print
This function prints the current Display window contents.
The standard Windows print dialog is opened.
Before the execution of this function any image or image sequence can be selected to be displayed. From
a multichannel sequence any channel status (on or off) combination can be defined. The colours of the
shown channels can be set with the function Set Channel Colour.
Parameters:
None
Exit
This function terminates the application completely.
All images and image sequences shown in the Gallery will be deleted from the memory. Save those
images which might be used for any further processing.
Parameters:
None
6.3.2
Functions in the Edit Menu
Copy
This function copies the current Display window contents to the clipboard. No dialog is shown.
Before the execution of this function any image or image sequence can be selected to be displayed. From
a multichannel sequence any channel status (on or off) combination can be defined. The colours of the
shown channels can be set with the function Set Channel Colour.
The current zoom factor of the Display window is not taken into account; the image is copied without
any zoom.
The image is copied as a true colour image with 24-bit resolution. Afterwards the contents can be pasted
to any other Windows application.
Parameters:
None
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Edit Channels
This function allows to add or to remove channels to a single or multichannel image.
On the Add Channel tab sheet the channels of (different) Input sequences can be defined to add
(combine) channels to an Output sequence.
Fig. 6-9
This operation is useful to add a segmented channel (or any other result of a function) to the original
image sequence. The selected channels of Input 1 and Input 2 are copied to Output. The maximum
number of channels in an image sequence is eight.
If the image sequences do not have the same extents Output Size defines which input is taken as a
reference. This selection also defines the properties for scaling and units in the output image sequences.
Parameters:
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Input 1
First input image sequence
Input 2
Second input image sequence
Output
Output image sequence
Output size
Defines source image sequence for size, scaling, and units
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On the Delete Channel tab sheet channels of the Input 1 image sequence can be selected to delete
channels.
Fig. 6-10
This operation might save time and memory for further processing if not all channels are needed.
Only the selected channels of Input 1 are copied to Output.
Parameters:
Input 1
Input image sequence
Output
Output image sequence
Delete All Images
This function deletes all images and image sequences from the memory (Gallery).
The function is used whenever a completely new image sequence should be processed. In order to drop
the images item by item to the wastebasket all of them can be deleted by a single function.
If any image or image sequence is needed for further use save them first.
Parameters:
None
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Functions in the Process Menu
Arithmetics - Add
This function adds two image sequences.
Fig. 6-11
The Add tab sheet of the Arithmetics dialog window must be selected.
If one or both input sequences are multichannel sequence, any number or combination can be selected.
The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from
left to right.
This function adds the two image sequences Input 1 and Input 2 voxel by voxel and generates the
image sequence Output. Note that a resulting grey value may be greater than 255 (4095). The
parameter Mode determines how a range overflow is handled:
1 - Wrap
No normalization - the grey values are displayed modulo 256 (4096). If the result is
greater than 255 (4095), the value 256 (4096) is subtracted from it.
2 - Clip
Grey values which exceed 255 (4095) are replaced with 255 (4095).
3 - Normalize
The resulting grey value range is scaled to the range 0...255 (0...4095).
Parameters:
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Input 1
First input image sequence
Input 2
Second input image sequence
Output
Output image sequence
Mode
1 - Wrap
2 - Clip
3 - Normalize
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Arithmetics - Subtract
This function subtracts two image sequences.
Fig. 6-12
The Subtract tab sheet of the Arithmetics dialog window must be selected.
If one or both input sequences are multichannel sequence, any number or combination can be selected.
The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from
left to right.
This function subtracts the two image sequences Input 1 and Input 2 voxel by voxel and generates the
image sequence Output. Note that a resulting grey value may be less than 0. The parameter Mode
determines how a range overflow (negative values) is handled.
1 - Wrap
No normalization - the grey values are displayed modulo 256 (4096). If the result is
less than 0, the value 256 (4096) is added to it.
2 - Clip
Negative values are set to 0.
3 - Normalize
The resulting grey value range is scaled to the range 0...255 (0...4095).
4 - Shift/Clip
128 (2048) is added to the difference, then negative values are set to 0. Values
greater than 255 (4095) are set to 255 (4095).
Parameters:
6-18
Input 1
First input image sequence
Input 2
Second input image sequence
Output
Output image sequence
Mode
1 - Wrap
2 - Clip
3 - Normalize
4 - Shift/Clip
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Contrast - Interactive
This function allows interactive changes of the contrast of an image sequence.
Fig. 6-13
The Interactive tab sheet of the Contrast dialog window must be selected.
A grey value range of the Input image sequence is scaled to another range in the Output image
sequence. Both ranges can be edited interactively. This function is used to achieve a better view of an
image sequence, or to scale a range of grey values to single value for a special coding in an image
sequence. The function does not improve the result of the linear segmentation function Segment.
Input indicates the sequence to enhance. If it is a multichannel sequence, a single channel, all channels,
or any number can be selected. The Input histogram shows the grey value distribution of the selected
channels of the Input image sequence.
Output defines the name of the result sequence. It will get only those channels which are chosen by the
Input parameter. The buttons labeled with 8 and 12 define the grey value (intensity) resolution in bit.
Normally the result will get the same resolution as the Input sequence. A change will be needed if image
sequences with different resolutions should be combined. Rising the grey value range to 12 bit will not
enhance the display quality or measurement accuracy. The smooth and morphology functions will
produce results with finer gradations.
If Clip Grey Values is selected, the output grey values are clipped to the Low (L) and High (H) values. If
Clip Grey Values is not selected, output grey values beyond the Low and High value range are possible.
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The Output histogram shows the resulting histogram. The horizontal axis represents the grey values from
0 to the maximum, which is either 255 or 4095, depending whether the input is 8 bit or 12 bit. The
vertical axis represents the pixel count. The selected range is marked by the borderlines in the histogram.
The blue line or L indicates the lower boundary, the red line or H the upper one, C indicates the center of
the range.
There are three ways to change the range: clicking and dragging the borderlines with the mouse.
or using the arrow keys
Entering a new value in the appropriate text boxes, clicking on the buttons
from the keyboard. To alter the values within the histogram move the mouse pointer over one of the
three coloured lines until the shape changes. Press and hold the left mouse button to move the line to a
new position. To change the values with the arrow keys click once into the histogram. Using the left or
right arrow key by its own will move the whole range. Pressing the Shift key additionally moves the
lower boundary, the Control key the upper boundary.
The vertical scale of the histogram is set using the scroll bar. The units are percents of the maximum grey
value distribution. This setting has no influence on the function.
Parameters:
6-20
Input
Input image sequence
Output
Output image sequence
Channel
Selection of the channel numbers for the Output image after contrast
enhancement
Clip Grey Values
Clipping of grey values to the Low (L) and High (H) output grey values
boundaries
Input L
Lower boundary of grey value range Input
Input C
Center of grey value range Input
Input H
Upper boundary of grey value range Input
Output L
Lower boundary of grey value range Output
Output C
Center of grey value range Output
Output H
Upper boundary of grey value range Output
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Contrast - Automatic
This function scales the grey values of an image sequence to the maximum possible range.
Fig. 6-14
The Automatic tab sheet of the Contrast dialog window must be selected.
This function enhances the contrast of an image sequence by spreading the grey value distribution over
the maximum possible range. This function is used to achieve a better view of an image.
The light and dark grey value ranges with a low share of pixels are excluded from the operation by the
parameter Threshold. The Threshold units are in thousandths of the total number of voxels. Using a
value of 10 means that the scale interval is set so that 5/1000 of the total number of voxels on the light
side, and 5/1000 of the total number of voxels on the dark side of the grey value distribution are
excluded.
Input indicates the sequence to enhance. If it is a multichannel sequence, a single channel, all channels,
or any number can be selected. The Input histogram shows the grey value distribution of the selected
channels of the Input image sequence.
Output defines the name of the result sequence. It will get only those channels which are chosen by the
Input parameter. The buttons labeled with 8 and 12 define the grey value (intensity) resolution in bit.
Normally the result will get the same resolution as the Input sequence. A change will be needed if image
sequences with different resolutions should be combined. Rising the grey value range to 12 bit will not
enhance the display quality or measurement accuracy. The smooth and morphology functions will
produce results with finer gradations.
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The Output histogram shows the resulting histogram. They are not editable. The horizontal axis
represents the grey values from 0 to the maximum, which is either 255 or 4095, depending whether the
input is 8 bit or 12 bit. The vertical axis represents the pixel count. The vertical scale of the histogram is
set using the scroll bar. The units are percentages of the grey value distribution maximum. This setting
has no influence on the function.
Parameters:
6-22
Input
Input image sequence
Output
Output image sequence
Threshold
Exclusion value - 0...1000
Input L
Lower boundary of grey value range Input
Input C
Center of grey value range Input
Input H
Upper boundary of grey value range Input
Output L
Lower boundary of grey value range Output
Output C
Center of grey value range Output
Output H
Upper boundary of grey value range Output
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Contrast – Linearize
This function scales a range of grey values of an image sequence to equal area fractions in the histogram.
Fig. 6-15
The Linearize tab sheet of the Contrast dialog window must be selected.
This function enhances the contrast by linearizing the histogram of the image sequence to equal area
fractions in the histogram. The areas (voxel count multiplied by grey value range) of all grey values in the
Output histogram are the same. This function is used to achieve a better view of an image sequence.
When Skip Black is checked the grey value 0 will not be taken into account for linearization.
Input indicates the sequence to enhance. If it is a multichannel sequence, a single channel, all channels,
or any number can be selected. The Input histogram shows the grey value distribution of the selected
channels of the Input image sequence.
Output defines the range of the result sequence. It will get only these channels which are chosen by the
Input parameter. The grey value (intensity) resolution will be the same as the one from Input.
The Output histogram shows the resulting histogram. The horizontal axis represents the grey values from
0 to 255. The vertical axis represents the pixel count. The vertical scale of the histogram is set using the
scroll bar. The units are percentages of the grey value distribution maximum. This setting has no influence
to the function.
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Parameters:
Image
Input image sequence
Output
Output image sequence
SkipBlack
0 - Grey value black is ignored
1 - Grey value black is taken into account
Input L
Lower boundary of grey value range Input
Input C
Center of grey value range Input
Input H
Upper boundary of grey value range Input
Output L
Lower boundary of grey value range Output
Output C
Center of grey value range Output
Output H
Upper boundary of grey value range Output
Smooth (Gauss)
This function performs a Gauss filter.
Fig. 6-16
The noise in the image sequence is reduced, the edge shape is nearly unchanged, local maxima are
leveled, the dynamic range is reduced.
Image sequences should be smoothed before they are reconstructed or segmented. For most sequences a
Size value of 3 is sufficient enough. If Input is a multichannel sequence, any number and combination of
channels can be selected. Output will only get the selected channels as results.
The grey value of every pixel is substituted by a weighted average of its surrounding neighbors. The
neighbors are defined by a cube. The affected pixel is the central pixel of the filter cube. The weighted
filter cube is approximated by a binomial distribution. The size of the filter cube is set using the Size scroll
bar. Even numbers are set to the next odd value. The Size defines the strength of the smoothing.
Parameters:
6-24
Input
Input image sequence
Output
Output image sequence
Size
Filter size (3...31, only odd numbers)
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Morphology
The following four functions perform basic operations of mathematical morphology on image sequences.
Fig. 6-17
As generalization of the morphology of two-dimensional images to three dimensions the structural
elements are small volumina.
Literature
Bomans, M.; Höhne, K.-H.; Tiede, U.; Riemer, M.:
3D-Segmentation of MR Images of the Head for 3-D Display
IEEE Transactions on Medical Imaging 9, 1990, 177-183
Schiemann, T.; Bomans, M.; Tiede, U.; Höhne, K.-H.:
Interactive 3D-Segmentation of Tomographic Image Volumes
14. DAGM-Symposium Mustererkennung, Springer-Verlag 1992, 73-80
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The input image sequence is analyzed voxel by voxel with a selected shape (Shape). The voxel to be
analyzed is always the central voxel of the shape. The shape type determines which neighboring voxels
are used to compute the resulting voxel.
The following structural elements are available for all morphological operations. They represent
approximated spheres with an increasing radius.
Sequential image:
Volume view:
Cross shape
Volume view:
Cross shape
Z-1
Z
Z+1
Sequential image:
Z-1
Z
Z+1
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Sequential image:
Volume view:
Carl Zeiss
Cube cross shape: created
through application of "cube"
and "cross" one after the other.
Z-2
Z-1
Z
Z+1
Z+2
For regions (voxels) that are at the edge of the image sequence, it assumed for erosion that there are
white voxels with a grey value of 255 (4095) outside the edge. For dilation, it is assumed that there are
black voxels with the grey value 0 outside the image sequence.
If the Grey Morphology tickbox is activated, erosion sets the grey value of the central voxel to the
minimum of all neighboring voxels affected by the structural element; dilation sets the grey value of the
central voxel to the maximum.
If the Grey Morphology tickbox is not activated, the neighboring voxels are only distinguished by grey
value 0 and non-0. For erosion the central voxel is set to 0 if any of the neighbors is 0. It is set to 255
(4095) if any neighbor is not 0. For dilation the central voxel is set to 255 (4095) if any of the neighbors is
not 0. It is set to 0 if all neighbors are 0.
Erosion reduces the size of bright regions, separates thin connections between them, and makes small
regions disappear. Dilation, on the other hand, makes bright regions of the image grow in size, fills gaps,
and smoothes small contour details.
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The result of erosion and dilation is called opening. On the one hand, this maintains to some extent the
original size of the regions while not losing the smoothing effect of erosion on the image. This name
stands for the operation of reducing convex bulges in the contour of the region. Thin connections
between regions are eliminated, broken borders between regions are connected, and small regions
disappear.
The opposite operation (first dilation, then erosion) is called closing. Concave bulges in the contours of
regions are filled in; connections are formed between adjacent regions.
The following example illustrates the operations "Open" and "Close" in two dimensions:
Open = Erosion + Dilation
Fig. 6-18
Close = Dilation + Erosion
Fig. 6-19
The "cube cross" shape was used for the operations shown.
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Morphology - Erode
This function erodes structures in an image sequence.
Fig. 6-20
In the Morphology dialog window, the tab sheet Erode must be selected.
Erosion makes bright regions smaller on a dark background. It also results in separation of thin
connections between regions. Small regions disappear entirely.
If Input is a multichannel sequence any number and combination of channels can be selected. Output
will only get the selected channels as results. The Input image sequence is eroded Count times with the
shape Shape. The Count scroll bar determines the number of recursive operations.
The following shapes (numbered 1 to 3 from left to right) are available:
If Grey Morphology is selected the function will respect all grey value shades of the sequence Input. If
Grey Morphology is not selected the function will distinguish between 0 and non-0 only. The result
Output will be a binary sequence.
Parameters:
Input
Input image sequence
Output
Resulting image sequence
Shape
Shape used
1 - cross
2 - cube
3 - cube cross
Count
Number of recursive operations
Grey Morphology 0 - Distinguish between 0 and non 0 only
1 - All grey value shades are taken into account
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Morphology - Dilate
This function dilates structures in an image sequence.
Fig. 6-21
In the Morphology dialog window, the tab sheet Dilate must be selected.
Dilation makes bright regions larger on a dark background. It also results in the filling of gaps and
smoothing of small contour details.
If Input is a multichannel sequence any number and combination of channels can be selected. Output
will only get the selected channels as results.
The Input sequential image is dilated Count times with the shape Shape. The Count scroll bar
determines the number of recursive operations.
The following shapes (numbered 1 to 3 from left to right) are available:
If Grey Morphology is selected the function will respect all grey value shades of the sequence Input. If
Grey Morphology is not selected the function will distinguish between 0 and non-0 only. The result
Output will be a binary sequence.
Parameters:
Input
Input image sequence
Output
Resulting image sequence
Shape
Shape used
1 - cross
2 - cube
3 - cube cross
Count
Number of recursive operations
Grey Morphology 0 - Distinguish between 0 and non 0 only
1 - All grey value shades are taken into account
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Morphology - Open
This function carries out an opening.
Fig. 6-22
In the Morphology dialog window, the tab sheet Open must be selected.
This function carries out an erosion followed by a dilation. For the most part, the opening maintains the
original size of the regions. Thin connections between regions and small regions themselves disappear.
Convex bulges in the contours of the regions are reduced. The opening is applied to the grey value image
sequence Input Count times with the shape Shape. If Input is a multichannel sequence any number
and combination of channels can be selected. Output will only get the selected channels as results.
The Count scroll bar determines the number of recursive operations.
The following shapes (numbered 1 to 3 from left to right) are available:
If Grey Morphology is selected the function will respect all grey value shades of the sequence Input. If
Grey Morphology is not selected the function will distinguish between 0 and non-0 only. The result
Output will be a binary sequence.
Parameters:
Input
Input image sequence
Output
Resulting image sequence
Shape
Shape used
1 - cross
2 - cube
3 - cube cross
Count
Number of recursive operations
Grey Morphology 0 - Distinguish between 0 and non 0 only
1 - All grey value shades are taken into account
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Morphology - Close
This function carries out a closing.
Fig. 6-23
In the Morphology dialog window, the tab sheet Close must be selected.
This function carries out a dilation followed by an erosion. For the most part, the closing maintains the
original size of the regions. Connections are formed between adjacent regions; gaps and bright concave
bulges in the contours of regions are filled in. The closing is applied Count times to the grey value image
sequence Input with the shape Shape. If Input is a multichannel sequence any number and combination
of channels can be selected. Output will only get the selected channels as results.
The Count scroll bar determines the number of recursive operations.
The following shapes (numbered 1 to 3 from left to right) are available:
If Grey Morphology is selected the function will respect all grey value shades of the sequence Input. If
Grey Morphology is not selected the function will distinguish between 0 and non-0 only. The result
Output will be a binary sequence.
Parameters:
Input
Input image sequence
Output
Resulting image sequence
Shape
Shape used
1 - cross
2 - cube
3 - cube cross
Count
Number of recursive operations
Grey Morphology 0 - Distinguish between 0 and non 0 only
1 - All grey value shades are taken into account
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Segment - Interactive
This function carries out a grey value segmentation by means of thresholding.
Fig. 6-24
The Interactive tab sheet of Segment dialog window must be selected.
Segmentation is especially used to generate binary regions. These are required for the measurement.
Two threshold values determine which grey value range of the Input image sequence is preserved and/or
deleted in the Output image sequence. Only one channel of a multichannel sequence can be selected as
Input. Output will always be a single channel sequence.
The vertical scaling of the histogram can be adjusted with the scroll bar at the right edge of the
histogram. This setting has no influence on the function.
The thresholds Low and High are determined either by moving the borderlines in the grey value
histogram or by the scroll bars underneath. Furthermore, the values for Low, Center and High can be set
through entry in the corresponding fields.
To move the lower (L) and upper (H) thresholds at the same time, move the vertical line in the grey value
histogram or set the scroll bar (C).
The Green and Blue/Red option buttons of the parameter Colour determine whether the voxels within
(Green) or outside (Blue/Red) of the grey value interval [L, H] are displayed with the corresponding
colour.
If Green is selected, the voxels within the selected interval are highlighted in green. The rest of the image
retains its original grey values. The voxels with the grey values Low and Low+1 are displayed in blue. The
voxels with the grey values High and High-1 are displayed in red.
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If Blue/Red is selected, the voxels with grey values within the interval Low, High remain unchanged.
Voxels with grey values less than Low are highlighted in blue; those with grey values higher than High
are highlighted in red.
If the Invert option is selected, the grey values outside the defined interval will be segmented.
If the option Binary is selected, then all grey values in the range from Low to High will be set to white
(grey value 255) in the Output image sequence, while all others will be set to black (grey value 0). If the
option is not selected, the grey values within the selected interval remain unchanged, while those outside
the range will be set to black. The measurement function accepts both results without any difference in
the results.
Parameters:
6-34
Input
Input image sequence
Output
Resulting image sequence
Colour
Green - Selected interval is displayed in green
Blue/Red
Grey values below the selected interval are displayed in blue, grey values
above in red
Binary
0 - Selected voxels retain the original grey value
1 - Selected voxels are set to grey value 255, the rest to grey value 0
Invert
0 - Grey values inside the selected interval are segmented
1 - Grey values outside the selected interval are segmented
L
Low grey value threshold
C
Center of threshold interval
H
High grey value threshold
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Segment - Automatic
The function carries out an automatic grey value segmentation by means of thresholding.
Fig. 6-25
The Automatic tab sheet of the Segment dialog window must be selected. Segmentation is especially
used to generate binary regions. These are required for the measurement.
The function calculates the two strongest local minimums in the histogram of the Input image sequence.
These values are used for the discrimination. Only one channel of a multichannel sequence can be
selected as Input. Output will always be a single channel sequence. The vertical scaling of the histogram
can be adjusted with the scroll bar at the right edge of the histogram. This setting has no influence on
the function.
The Green and Blue/Red option buttons of the parameter Colour determine whether the voxels within
(Green) or outside (Blue/Red) of the grey value interval [L, H] are displayed with the corresponding
colour.
If Green is selected, the voxels within the selected interval are highlighted in green. The rest of the image
retains its original grey values. The voxels with the grey values Low and Low+1 are displayed in blue. The
voxels with the grey values High and High-1 are displayed in red.
If Blue/Red is selected, the voxels with grey values within the interval Low, High remain unchanged.
Voxels with grey values less than Low are highlighted in blue; those with grey values higher than High
are highlighted in red.
If the Invert option is selected, the grey values outside the defined interval will be segmented.
If the option Binary is selected, then all grey values in the range from Low to High will be set to white
(grey value 255 (4095)) in the Output image sequence, while all others will be set to black (grey value 0).
If the option is not selected, the grey values within the selected interval remain unchanged, while those
outside the range will be set to black.
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Parameters:
6-36
Input
Input image sequence
Output
Resulting image sequence
Colour
Green - Selected interval is displayed in green
Blue/Red - Grey values below the selected interval are displayed in blue, grey
values above in red
Binary
0 - Selected voxels retain the original grey value
1 - Selected voxels are set to grey value 255 (4095), the rest to grey value 0
Invert
0 - Grey values inside the selected interval are segmented
1 - Grey values outside the selected interval are segmented
L
Low grey value threshold
C
Center of threshold interval
H
High grey value threshold
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Boolean - And
This function carries out a bit-by-bit And calculation for the image sequences Input 1 and Input 2.
Fig. 6-26
The And tab sheet of the Boolean dialog window must be selected.
This function is especially well suited for masking images.
If one or both input sequences are multichannel sequences, any number or combination can be selected.
The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from
left to right.
Parameters:
Input 1
First input image sequence
Input 2
Second input image sequence
Output
Resulting image sequence
Boolean - Or
This function carries out a bit-by-bit Or calculation for the images Input 1 and Input 2.
Fig. 6-27
The Or tab sheet of the Boolean dialog window must be selected.
This function can be used to combine binary masks or regions.
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If one or both input sequences are multichannel sequences, any number or combination can be selected.
The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from
left to right.
Parameters:
Input 1
First input image sequence
Input 2
Second input image sequence
Output
Resulting image sequence
Boolean - Xor
This function carries out a bit-by-bit Xor calculation for the images Input 1 and Input 2.
Fig. 6-28
The Xor option button of the Function option group in the Boolean dialog window must be selected.
This function can be used to combine binary masks or regions.
If one or both input sequences are multichannel sequences, any number or combination can be selected.
The number of selected channels for Input 1 and Input 2 must be the same. They will be combined from
left to right.
Parameters:
6-38
Input 1
First input image sequence
Input 2
Second input image sequence
Output
Resulting image sequence
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Boolean - Not
This function carries out a bit-by-bit negation of an image.
Fig. 6-29
The Not tab sheet of the Boolean dialog window must be selected.
If Input is a multichannel sequence any number or combination can be selected.
Parameters:
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Input
Input image sequence
Output
Resulting image sequence
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Boolean - Mask
This function masks a grey value image sequence.
Fig. 6-30
The Mask tab sheet of the Boolean dialog window must be selected.
This function modifies the Output image sequence depending on the mask image sequence used.
If the grey value in Input 2 is higher than 0, then the voxel values are copied from Input 1 to the image
sequence Output. If the grey value of the voxel is 0, then the voxel value of the Output image sequence
is taken over.
If one or both input sequences are multichannel sequences, any number or combination can be selected.
The number of selected channels for Input 2 must be 1 or the same as for Input 2. They will be
combined from left to right.
Parameters:
6-40
Input 1
First input image sequence
Input 2
Second input image sequence
Output
Resulting image sequence
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Scrap
This function deletes or selects objects in a specified size range.
Fig. 6-31
The operation deletes or selects objects on the basis of their total volume in voxels. Objects with a volume
within the range MinVolume to MaxVolume are effected.
To delete objects outside the range, the parameter Select must be active. If the parameter is not
activated objects outside the defined volume range are deleted.
Parameters:
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Input
Input image sequence
Output
Output image sequence
MinVolume
Minimum object size
MaxVolume
Maximum object size
Select
0 - Select the objects outside the size range
1 - Select the regions within the size range
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Fill Holes
This function fills holes in all objects.
Fig. 6-32
All holes in objects are filled by this operation. Holes are structures, which have a grey value of 0 and are
surrounded completely by voxels with a grey value not equal to 0. It is assumed that regions outside the
image are black. Holes, which touch the image border, are retained.
Parameters:
6-42
Input
Input image sequence
Output
Output image sequence
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3D FOR LSM
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Carl Zeiss
Functions in the View Menu
Render - Surface
This function displays an image sequence according to the gradient shading method.
Fig. 6-33
The Surface tab sheet of the Render dialog window must be selected.
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Method
The Input sequence defines the data to be reconstructed. If it is a multichannel sequence one or all
channels can be selected for the reconstruction.
Output sets the name of the result image (sequence). If the sequence exists it is overwritten. Pressing the
button New will generate a new name (number). The size of the sequential images in Output is
determined by the size of the sequential images in Input.
Number of Views determines the number of reconstructions which should be computed. The radio
buttons Start and End define which angle settings are currently shown. A definition for the angle End is
only necessary if Number of Views is higher than 1. If this is true the result sequence will get views from
the Start to the End angle definition. The other reconstructions are determined through the linearly
interpolated intermediate angles. The direction of view is determined from the angles as follows:
The angle Angle Z determines the rotation of the direction of view on the Z-axis. The angle Angle Y
determines the rotation of the direction of view on the Y-axis that has been rotated by the angle
Angle Z. The angle Angle X determines the rotation of the direction of view on an X-axis that is rotated
by Angle Z and Angle Y.
Channel defines if the following parameters are valid for All or just for one. Defining the thresholds for
the channels independently is useful if the grey value boundaries of the objects differ too much in the
different channels. The thresholds Grey Low and Grey High define the grey value range of the objects.
The parameter Aperture is a measure of the size of the highlights. Small values generate large
highlights. Large values generate small highlights (similar to a spot).
Use the parameter Reflection to control the ratio of diffuse and reflective brightness components, i.e.,
the overall basic brightness compared with the highlights. When the value of Reflection is low, the
highlights predominate; when the values are high, the region appears to be uniformly illuminated and
the highlights are not so pronounced. When Auto Update is selected, the reconstruction is updated
automatically whenever a parameter is modified (except Input, Output, or Number of Views). Show
Cube defines whether a wire frame cube is shown in the Display window or not.
Application
This method can be applied, if the structures in the Input sequence can be segmented by grey value
thresholding. Because the gradient is calculated for every pixel, the Output appears in very fine detail.
Noisy Input sequences must be smoothed (function Smooth) before rendering, otherwise the surface
appears rough.
Parameters:
Input
Input image sequence
Output
Resulting image sequence
Number of Views Number of reconstructions to be calculated
6-44
Angle X
Angle of rotation on the X-axis, start position
Angle Y
Angle of rotation on the Y-axis, start position
Angle Z
Angle of rotation on the Z-axis, start position
Channel
All - The following parameters are valid for all channels
X - The following parameters are valid for the selected channel only
Grey Low
Low grey value threshold of the region to be displayed
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Grey High
High grey value threshold of the region to be displayed
Aperture
Measure of the extent of the highlights
Reflection
Weight of the defuse brightness components in comparison to the highlights
Auto Update
0 - Function execution is performed on OK or Apply
1 - Function execution for the current angle is performed on any parameter
change
Show Cube
0 - The wire frame cube is not shown
1 - The wire frame cube is shown in the Display window
Render - Surface: Method Description
This method displays the surface of structures in the Input sequence shaded as if a light illuminated it.
The position of the light is behind the view point with parallel rays in the direction of the sequence.
The input sequence is segmented into object and background by grey value thresholding: object voxels
are within the grey value range Grey Low to Grey High.
Each Output pixel corresponds to a point at the surface at which the ray in view direction through the
Output pixels hits the surface. All rays are parallel.
The surface normal required for shading in this gradient renderer is the grey value gradient in the Input
volume at the surface voxel position. It is not the geometric surface normal. The grey value gradient is
determined from the grey values in a 3x3x3 cube around the surface voxel by averaging e.g. the xgradient in y- and z-direction [4].
There is no depth cueing (far objects would appear darker).
The illumination model is a Phong model [1] (surface normal is determined for each Output pixel) with
diffuse reflection and specular reflection. Diffuse reflection means that the surface reflects light with
equal intensity in all directions. The brightness of a given surface patch depends not on the viewdirection, but only on the angle between light and surface normal. Specular reflection is observed on
shiny surfaces as a highlight. The light is reflected as from a mirror. The maximum intensity is observed
when the view direction is the one of the mirrored light direction.
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Render - Alpha
This function displays an image sequence according to the alpha rendering method.
Fig. 6-34
The Alpha tab sheet of the Render dialog window must be selected.
One or more reconstructions of the input image sequence are computed according to the alpha
rendering method. This type of reconstruction should be used if there is no possibility to segment the
structures in the image sequence and also if the objective is to make deeply layered structures visible.
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Method
The Input sequence defines the data to be reconstructed. If it is a multichannel sequence one or all
channels can be selected for the reconstruction.
Output sets the name of the result image (sequence). If the sequence exists it is overwritten. Pressing the
button New will generate a new name (number). The size of the sequential images in Output is
determined by the size of the sequential images in Input.
Number of Views determines the number of reconstructions which should be computed. The radio
buttons Start and End define which angle settings are currently shown. A definition for the angle End is
only necessary if Number of Views is higher than 1. If this is true the result sequence will get views from
the Start to the End angle definition. The other reconstructions are determined through the linearly
interpolated intermediate angles.
The direction of view is determined from the angles as follows:
The angle Angle Z determines the rotation of the direction of view on the Z-axis. The angle Angle Y
determines the rotation of the direction of view on the Y-axis that has been rotated by the angle
Angle Z. The angle Angle X determines the rotation of the direction of view on an X-axis that is rotated
by Angle Z and Angle Y.
Channel defines if the following parameters are valid for All or just for one. Defining the opacity for the
channels independently is useful when the brightness and contrast of the channels differ too much.
Threshold defines the range with no opacity. It is completely transparent. The range starts at grey
value 0.
The length of slope is defined by Ramp. The maximum opacity value is set with the parameter Max.
Opacity. This range ends at the maximum grey value. The Opacity Table shows the grey value
histogram of Input with the opacity definition as a red line.
When Auto Update is selected, the reconstruction is updated automatically whenever a parameter is
modified (except Input, Output, or Number of Views). Show Cube defines whether a wire frame cube
is shown in the Display window or not.
Application
1. This method can be applied, if the structures in the Input sequence are unsharp so that objects are
poorly defined by their grey value.
2. In this case, the Opacity Table is defined as a ramp. Low grey values have weight 0 to suppress the
background voxels. The opacity rises with increasing grey values, depending on the parameter Ramp.
The value of Max. Opacity defines the weight of the high grey values. High grey values above a
threshold have weight 255 to show the "object" voxels unsuppressed. Of course a smooth step can
be used.
3. The result is a display with inside structures shining through. A 3D impression can be obtained by
rendering with several view directions.
4. In contrast to this, a voxel renderer like the gradient renderer would display only the surface of objects
that are defined by grey value-thresholds. This surface would appear shaded as if illuminated by a
light.
5. The method can also be applied to visualize pronounced structures within other enclosing structures, if
the structures have different grey value ranges.
6. In this case, the Opacity Table is defined as a step. Low grey values (background) have weight 0. High
grey values (inside structures) have maximum weight.
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Parameters:
Input
Input image sequence
Output
Resulting image sequence
Number of Views Number of reconstructions to be calculated
6-48
Angle X
Angle of rotation on the X-axis, start position
Angle Y
Angle of rotation on the Y-axis, start position
Angle Z
Angle of rotation on the Z-axis, start position
Channel
All - The following parameters are valid for all channels
X - The following parameters are valid for the selected channel only
Threshold
Grey value where the opacity starts rising
Ramp
Length of the opacity slope
Max. Opacity
Maximum opacity value
Opacity Table
Maximum opacity value
Auto Update
0 - Function execution is performed on OK or Apply
1 - Function execution is performed on any parameter change
Show Cube
0 - The wire frame cube is not shown
1 - The wire frame cube is shown in the Display window
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Render - Alpha: Method Description
Each Output pixel is a weighted sum of the Input voxels along a ray in view direction through the Input
sequence. Each Input voxel has an opacity value, dependent only on its grey value. The opacity values are
defined by the parameters Threshold, Ramp, and Max. Opacity.
Accumulation of pixels proceeds along the ray from back to front, i.e. from far pixels to near pixels. If a
new pixel is added, it increases the result intensity by its grey value weighted by the opacity value, and
attenuates the previously accumulated intensity according to the opacity value. Full intensity stops
accumulation.
This calculation must be repeated for each pixel of the ray to generate one Output pixel. Then for each
Output pixel to produce a 2D Output image for the selected view-angle. Then for each view-angle to
produce an output sequence for Number of Views different view angles.
Render - References
[1] J.D. Foley,A.van Dam, S. K. Feiner, J.F.Hughes, Computer Graphics: Principles and Practice, Addison
Wesley, Reading, MA, 1990.
[2] M. Levoy, Display of Surfaces from Volume Data, IEEE Computer Graphics & Applications, May 1988,
29-37.
[3] J. Ylä-Jääski, F.Klein, O. Kübler, Fast Direct Display of Volume Data for Medical Diagnosis,
VGIP:Graphical Models and Image Processing 53,1991,7-18.
[4] K.H. Höhne, R. Bernstein, Shading 3D-Images from CT Using Gray-Level Gradients, IEEE Transactions
on Medical Imaging, 5, 1986, 45-47.
[5] D.Gordon, R.A. Reynolds, Image Space Shading of 3-Dimensional Objects, CVGIP 29, 1985, 361-376.
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6.3.5
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Functions in the Measurement Menu
Measurement Concept
Measurement is based on regions (objects) in three-dimensional space. Segmenting an image sequence
generates these. The image segmentation process produces a mask image that defines the region.
A region is a group of voxels that touch at the surfaces or at the edges, but not at the corners (18 voxel
neighborhood).
This is illustrated by the following example. The voxels marked black in sequential image Z-1, Z, Z+1 all
belong to the same region as the grey central voxel in sequential image Z. The volume view shows the
neighborhood interrelationships as a 3D projection.
Sequential image:
Volume view:
Z-1
Z
Z+1
Fig. 6-35
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Measurement Process
The measurement process consists of three steps: region definition, checking of the validity of the
regions, and feature calculation.
Region definition:
- Automatically from the mask image
Region validation check depends on:
- Minimum volume
- Measurement condition
Feature calculation depends on
- Shape of the region
- Densitometric value distribution of the region
- Feature parameters
Image
Region
generator
Region
Region
filter
Valid
region
Data
Measurement
Image sequence
Minimum volume
Feature name
Measurement condition Feature parameter
Fig. 6-36
All regions found are checked according to certain conditions. The voxel volume of each region must be
equal to or greater than MinVolume. The measurement condition must be fulfilled. Only those regions
that meet all the conditions are valid for the measurement. The region can be measured or labeled.
Measurement is a process that produces data. Labeling is a process that generates an image volume.
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Automatic Object Measurement – Object Features
A measurement feature describes a region characterized by a number (e.g. volume, area or a
densitometrical statistic). The features can be selected on the Object Features and Volume Features
tab sheets.
Fig. 6-37
The scalings and units are taken automatically from the assigned sequence.
The measurement features can be selected individually for each measurement. The object features
generate a result value for every single object.
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The dialog shows two lists. One shows the Available Features as groups (on the left). The other one
shows the Selected Features. Double-clicking on items of the left list will add the Selected Features to
the right list. Double-clicking on an item of the right list will remove this item from the list. Selected
Features can also be transferred by clicking on the button in the middle (<< / >>) of the dialog.
The combo box above the right list represents predefined feature lists. Selecting one of the entries will fill
the right list with these features; previously selected features will be overwritten.
The button Select All will copy all features to the list of selected features.
The button Remove All will clear the list of selected features.
Clicking on the Apply button will execute the measurement process and switch to the General tab sheet
of the dialog.
Parameters:
Available Features
List of available object features
Selected Features
List of selected object features
Select All
Select all available object features for measurement
Remove All
Remove all object features from the selected features list
The following sections describe all measurement features which are defined in the system.
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Object Features (geometric)
If Object Features are selected, one set of measurement data is calculated for each object.
Group Name
Name
Description
Volume
Volume
Volume of the object.
Volume Filled
VolumeF
Volume of the filled object.
Ellipsoid
EllipseA
Length of the main axis of the ellipsoid with the same
geometrical moment of inertia as the object.
EllipseB
Length of the middle axis of the ellipsoid with the same
geometrical moment of inertia as the object.
EllipseC
Length of the minor axis of the ellipsoid with the same
geometrical moment of inertia as the object.
EllipseAF
Length of the main axis of the ellipse with the same geometric
moment of inertia as the filled object.
EllipseBF
Length of the middle axis of the ellipse with the same geometric
moment of inertia as the filled object.
EllipseCF
Length of the minor axis of the ellipse with the same geometric
moment of inertia as the filled object.
Surface Area
SurfArea
Surface area of the object.
Surface Area Filled
SurfAreaF
Surface area of the filled object.
Sphere Diameter
Dsphere
Diameter of the sphere with the same volume.
Ellipsoid filled
6 * VOLUMEF / π
Sphere Form Factor
Fsphere
Form factor of the object.
6⋅ π ⋅
Number of Holes
6-54
Nparts
VOLUMEF
SURFAREAF3
Number of holes within an object.
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Carl Zeiss
Object Features (densitometric)
Group Name
Name
Description
Mean Densitometric
MeanD
Densitometric mean value of an object.
Standard Deviation
Densitometric
StdD
Standard deviation of the densitometric values of an object.
Minimum
Densitometric
MinD
Minimum grey value of an object.
Maximum
Densitometric
MaxD
Maximum grey value of an object.
Automatic Object Measurement - Volume Features
A measurement feature describes a region characterized by a number (e.g. volume, area, or a
densitometrical statistic). The features can be selected on the Object Features and Volume Features
tab sheets.
Fig. 6-38
The measurement features can be selected individually for each measurement. The object features
generate a result value for every single object.
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Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
The dialog shows two lists. One shows the Available Features as groups (on the left). The other one
shows the Selected Features. Double-clicking on items of the left list will add the Selected Features to
the right list. Double-clicking on an item of the right list will remove this item from the list. Selected
Features can also be transferred by clicking on the button in the middle (<< / >>) of the dialog.
The combo box above the right list represents predefined feature lists. Selecting one of the entries will fill
the right list with these features; previously selected features will be overwritten.
The button Select All will copy all features to the list of selected features.
The button Remove All will clear the list of selected features.
Clicking on the Apply button will execute the measurement process and switch to the General tab sheet
of the dialog.
Parameters:
Available Features
List of available object features
Selected Features
List of selected object features
Select All
Select all available object features for measurement
Remove All
Remove all object features from the selected features list
Volume Features (geometric)
The volume-related measurement generates one measured value per image sequence. The following
table contains the predefined volume characteristics.
Group Name
Name
Description
Count
VolCount
Number of regions measured.
Volume
VolVolume
Total volume of all regions.
Volume Percentage
VolVolumeP
Total volume of all regions, in relation to the volume of the
image sequence.
Volume Features (densitometric)
Group Name
Name
Description
Surface Area
VolSurfArea
Total surface area of all regions.
Mean Densitometric
VolMeanD
Mean grey value of all regions.
Standard
Deviation VolStdD
Densitometric
Grey value standard deviation of all regions.
Minimum
Densitometric
VolMinD
Minimum grey value in the image sequence.
Maximum
Densitometric
VolMaxD
Maximum grey value in the image sequence.
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Automatic Object Measurement - Condition
The measurement conditions are used to limit the objects to be evaluated (e.g. only objects with defined
minimum value). All objects are tested against the defined conditions. If the conditions are fulfilled the
feature values are written to the data table.
Fig. 6-39
To define the following parameter select the Condition tab sheet of the Automatic Object
Measurement dialog window.
The list on the very left at the dialog shows all the measurement Features. The second list provides the
comparison Operators and the next Numbers to define a value. This gives the possibility to compose an
expression to test a feature value against a constant value. The fields above the lists will show the
composed (selected) string. Clicking on the desired list entry does the selection. The button with the
„>>„ characters adds this string to the List of Conditions. All lines of the List of conditions are
combined with the AND expression automatically. To remove a condition line double-click on it.
The parameter Minimum Volume defines the minimum voxel volume for the measurement. This is an
easy way to eliminate very small regions caused by noisy sequences and segmentation process.
The button Remove All will clear the list of defined conditions.
Clicking on the Apply button will execute the measurement process and switch to the General tab sheet
of the dialog.
Parameters:
Feature
Operator
Number
List of conditions
Remove All
Minimum Volume
03/06
List of available object features
List of available condition operators
List of numbers to compose the value
Defined condition list
Remove all entries from the List of conditions
Minimum object volume in voxel
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Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Automatic Object Measurement - General
This function carries out an automatic measurement and labeling.
Measured
Object Features
Measured
Volume Features
Fig. 6-40
The regions must be defined by an image sequence Mask Image (the objects must be separated from
one another by black voxels with the grey value 0). This sequence is generated with the function
Segment. If it is a multichannel sequence a single channel has to be chosen.
The image Dens Image is needed for the measurement of the densitometric features. Image sequence
properties like scaling and unit are taken from Dens Image. A single channel of this sequence (if it is
multichannel) must be selected with the buttons to the right of the parameter.
The measurement results can be stored to database files. These files are tab delimited ASCII files which
can be easily imported to major Windows programs like text processing or spreat sheet application.
Writing database files are independently supported for object and volume features. Activating the
corresponding check boxes enables it. The name of the database is defined with the field Database. The
files will be located in the subdirectory DATA of the main installation directory. The filename extension
TXT will be added automatically.
If the check box Label is activated a single channel sequence will be generated. It contains all the
measured objects, each object is coloured homogeneous but in different colours. To copy all
measurement values to the clipboard activate the check box Clipboard.
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Carl Zeiss
A single object of interest can be visualized. Clicking on a specific row in the data grid chooses the
object. By selecting a row in the data grid a new image is created with the object of interest visualized.
The visualization depends on the settings in the Object Visualisation field. If Render is chosen, the
object of interest is displayed with the Surface Rendering method. If Mask is chosen, the object is
labelled in a pseudo colour in a new image stack.
Parameters:
03/06
Mask Image
Single channel mask image sequence that defines the objects
Dens Image
Image sequence for densitometric measurement and property source
Object
Stores measurement values of objects, including database filename
Volume
Stores volume measurement values of objects, including database filename
Label
Generates an image sequence with all objects labelled in different pseudo
colours
Clipboard
Measurement values are automatically written to the clipboard
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CHAPTER 7
ANNEX
Contents
Carl Zeiss
ANNEX
CONTENTS
Page
7
Annex...............................................................................................................................7-2
7.1
Recommendations for Excitation Laser Lines and Emission Filters of Dyes............................7-2
7.2
7.2.1
7.2.2
Configurations Overview....................................................................................................7-3
LSM 5 LIVE 1 Channel Biomedical Configurations...............................................................7-3
LSM 5 LIVE 2 Channel Biomedical Configurations...............................................................7-4
7.3
Changing Filters in the Scanning Module ...........................................................................7-5
7.4
Detaching / Attaching the Scanning Module from / to Microscope Stands ..........................7-5
7.5
7.5.1
7.5.2
7.5.3
Hints on the Use of the Piezo Fine Focusing Stage ..............................................................7-7
General Description............................................................................................................7-7
Application Fields ...............................................................................................................7-7
Additional Information on the Operation............................................................................7-7
7.6
Piezo Objective Focussing Device ........................................................................................7-9
7.7
Specifications of Trigger-Interface LSM 5 LIVE ..................................................................7-10
7.8
7.8.1
7.8.2
7.8.3
7.8.4
AxioCam High Resolution Digital Cameras .......................................................................7-13
Microscopy Camera AxioCam MRm Rev.2 ........................................................................7-13
High Resolution Microscopy Camera AxioCam HRm Rev.2................................................7-14
High Resolution Microscopy Camera AxioCam HRc ..........................................................7-15
Microscope Camera Port Adapters for the AxioCam .........................................................7-16
7.9
List of Key Words .............................................................................................................7-17
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7-1
ANNEX
Recommendations for excitation laser lines and …
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
7
ANNEX
7.1
Recommendations for Excitation Laser Lines and Emission Filters of Dyes
Dye
Laser line/HFT
Emission/EM
DAPI
405
> 420, max. at 461
EBFP
405
> 420, max. at 447
Hoechst
405
> 420, max. at 440
Fluoro-Gold
405 or 440
> 420/475, max. at 536
ECFP
405 or 440
> 420/475, max. at 501
Lucifer Yellow
440 or 488
> 475, max. at 536
EGFP
488
> 505, max. at 507/516
FM 1-43™
488
> 505, max. at 598
Alexa Fluor 488™
488
> 505, max. at 520
Calcium Green
488
> 505, max. at 531
Cy2™
488
> 505, max. at 508
DiO (DiOC18(3))
488
> 505, max. at 508
Fluo-3
488
> 505, max. at 520
Fluorescein (FITC)
488
> 505, max. at 520
Cy3™
532
> 530, max. at 566
EYFP
532
> 530, max. at 535
Oregon Green
532
> 530, max. at 535
SYTOX Green
532
> 530, max. at 536
FM 4-46
532
> 560, max. at 640
Alexa Fluor 546™
532
> 560, max. at 572
Calcium Orange
532
> 560, max. at 575
DiI (DiIC18(3))
532
> 560, max. at 565
DsRed
532
> 560, max. at 583
Tetramethylrhodamine (TRITC)
532
> 560, max. at 576
Rhodamine B
532
> 560/585, max. at 625
Texas Red™
532
> 560/585, max. at 620
Alexa Fluor 633™
633
> 650, max. at 654
Cy5™
633
> 650, max. at 666
7-2
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ANNEX
Recommendations for excitation laser lines and …
Carl Zeiss
Here you can note your specific combinations:
Dyes
Laser/HFT
EM1
NFT
EM2
Dyes
Laser/HFT
EM1
NFT
EM2
FITC/Cy3
488/532
BP 500-525
532
LP 550
Example:
7.2
Configurations Overview
7.2.1
LSM 5 LIVE 1 Channel Biomedical Configurations
1 channel, FL
VarioOne GB
Laser lines
488, 532 (635) nm
Emissionfilters
channel 1
LP 505
BP 495-525 SK
LP 550
BP 550-615
LP 650 SK
BP 700-750
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7-3
ANNEX
Configurations Overview
Carl Zeiss
7.2.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
LSM 5 LIVE 2 Channel Biomedical Configurations
2 channel, FL
VarioTwo VRGB
Laser lines
(405/440), 488, 532 (635) nm
Secondary
Plate
beam splitter
mirror
NFT 488
NFT 532
NFT 635
Emission-
LP 420
filters
LP 505
channel 1
BP 495-525 SK
LP 550
BP 560-675 SK
LP 650 SK
BP 700-750
Emissionfilters
channel 2
BP 415-480 SK
BP 455-525
BP 495-525 SK
BP 550-615
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7.3
ANNEX
Detaching / Attaching the Scanning Module ...
Carl Zeiss
Changing Filters in the Scanning Module
For optimum investigation of specimens it is useful to employ filter wheels permitting the motorcontrolled change between different filters for narrow-band or broad-band detection depending on the
wavelength. The number of filters is limited by the capacity of the filter wheel. The change of the filter
wheel as a whole involves complete readjustment.
Please ask your LSM service engineer to do this exchange in order to maintain your warranty.
7.4
Detaching / Attaching the Scanning Module from / to Microscope Stands
Tool needed: 3 mm Allen key
The user can remove the Scanning Module from one microscope and attach it to another within
a few minutes. Described below is the change-over from an Axioskop 2 FS MOT to an Axiovert
200 M in sideport configuration.
Before the change-over, shut down the system as described in chapter 4 in order to avoid
damage to the system and loss of data.
• Loosen the three screws (7-1/1) at the Scanning Module (7-1/2) fitted to the Axioskop 2 FS MOT.
• Cautiously pull Scanning Module off the Axioskop 2 FS MOT stand.
• Attach Scanning Module to the left sideport of the Axiovert 200 M, minding the guide pins (7-1/5),
and secure it with the three screws (7-1/1).
As the Scanning Module is heavy, weighing about 19.5 kg, it is easier if the changeover is
carried out by two persons.
• Pull off covering caps (7-1/3) from the CAN-BUS and RS232 interface ports at the rear of the
Axiovert 200 M, remove the two cables 457411-9011 (CAN-BUS) and 457411-9012 (RS232) from the
Axioskop 2 FS MOT, plug them into the Axiovert 200 M and secure them there.
• Switch the LSM 5 LIVE on.
• Click on the Stand select icon to update the system database with the new database of the
Axiovert 200 M microscope.
• Restart the LSM 5 LIVE program.
03/06
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7-5
Carl Zeiss
ANNEX
Detaching / Attaching the Scanning Module ...
LSM 5 LIVE
LSM 5 LIVE DuoScan
04
08
01
07
02
06
03
05
100
90
O
ZER
E
TIV
JEC
OB
OR
CT
FLE
RE
S
CU
FO
Fig. 7-1
7-6
Change-over of the Scanning Module
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ANNEX
Hints on the Use of the Piezo Fine Focusing Stage
7.5
Hints on the Use of the Piezo Fine Focusing Stage
7.5.1
General Description
Carl Zeiss
The Piezo fine focusing stage is a compact attachment for the Axio Imager.Z1 and Axiovert 200 M
microscope stages, which allows the particularly fast and high-precision fine focusing of the object. The
Piezo stage permits fine focusing over a range of 250 µm, with the smallest step width being less than
10 nm, reproducibility better than 40 nm, and the maximum speed amounting to 60 Hz. The stage
allows the use of specimens with a weight of less than 100 g.
The piezo stage is not used if manual coarse focusing is performed. To position the objective in relation
to the optical Z-axis, the standard XY-microscope stage is used.
The piezo stage features a mount for standard object carriers of 76 mm x 26 mm x 1 mm and a milledout receptacle for ∅ 36 mm x 1 mm Petri dishes.
7.5.2
Application Fields
− High-precision fine focusing and translation of the object along the optical axis.
− Fast and high-precision mounting of one-dimensional Z-line sections.
− Fast and high-precision mounting of two-dimensional R-Z-longitudinal sections.
− Fast and high-precision mounting of XY-Z-Stacks for the three-dimensional reconstruction of the
object.
− Exact measurement of Point-Spread-Functions for deconvolution.
7.5.3
Additional Information on the Operation
The piezo fine-focusing stage is a high-precision, sensitive accessory for the LSM 5 LIVE from Carl Zeiss
and must therefore be treated carefully.
High mechanical stress, such as the use of specimens weighing more than 100 g or the application of
pressure or knocks on the movable stage tongue, can result in damage and therefore in failure of the
stage function.
To be able to fully utilize the outstanding precision attainable with the fine focusing stage, anything
which could interfere with its operation, especially mechanical knocks and impact of the LSM 5 LIVE
components, should be avoided. We would recommend you to always use the actively vibration-damped
table.
The specifications of the stage are obtained only after a heating phase of approx. 30 minutes.
Furthermore, the installation conditions for the LSM 5 LIVE system must be observed.
The maximum reproducibility (better than 40 nm) for moving to an absolute position in Z is achieved by
always moving to the required position from below.
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7-7
Carl Zeiss
ANNEX
Hints on the Use of the Piezo Fine Focusing Stage
LSM 5 LIVE
LSM 5 LIVE DuoScan
Fine focusing is performed mechanically via an inclined position of the stage tongue. Therefore, the lifting
range Z at the location of the image field depends on the position of the piezo stage in relation to the
optical axis. This means: if the user shifts the object on the microscope stage to the right via the piezo
stage, the lift will be different from the one in the zero position of the stage (max. 250 µm) and also from
the one after a shift of the stage to the left.
If the LSM 5 LIVE system is equipped with a motorized scanning stage, this shift is read back to •x and
the lift is calibrated automatically if the zero position of the piezo stage has been matched to the zero
position of the scanning stage via an initialization run. For this, activate the Stage button of the Acquire
toolbar. Then position the scanning stage in such a way that the optical axis of the microscope
corresponds to the zero position of the piezo stage, i.e. to the center of the specimen holder in the stage
tongue. Then perform initialization by pressing the piezo Null button. This step must be repeated after
every new start of the system. Also see the notes on the operation of the motorized scanning stages.
If the system is equipped with a manual microscope stage, the user has the option of performing the
calibration by entering the •x shift in mm via the Calibration slider.
The shift is read off from the microscope stages. In the case of the manual AxioImager stage, •x can be
read directly from the scale adhered to the front of the stage. In the case of the manual Axiovert 200 M
stage, a scale is located on the right of the knob, where the 45 mm •x shift relative to the zero position
of the microscope stage can be read off. The •x value is positive for both stages if shift from the zero
position is made to the right and negative if the shift is made to the left.
On account of the inclined position of the stage tongue, the object is also shifted laterally during the fine
focusing motion. This lateral shift is negligibly small if, as recommended by us, specimen carriers with
thickness 1.0 mm are used exclusively. Otherwise, the marked lateral shift of the object during fine
focusing can result in image distortion. For the same reason, Petri dishes without fixation ring must be
used exclusively.
The nosepiece of the Axiovert 200 M stand is moved to the load position prior to switching off the
LSM 5 LIVE system and the piezo stage is then moved to the lowest position to avoid damage of the
objective or object by a possible collision. The user must refocus after start-up of the system. Before an
objective change in the Axiovert 200 M or the Axio Imager.Z1 the nosepiece and the microscope stage
must be moved to the load position by the user, and then back to the work position to prevent the
objectives from hitting the piezo components. This is performed automatically if the objectives are
changed menu-controlled via the relevant buttons of the LSM 5 LIVE program.
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7.6
ANNEX
Piezo Objective Focussing Device
Carl Zeiss
Piezo Objective Focussing Device
For upright stands Axio Imager.Z1, Axio Imager.M1, Axioskop 2 FS MOT
Range:
250 µm
Minimum step size:
5 nm
Speed:
Piezo objective
focussing device
Piezo focussing stage
Slices
Step size [µm]
xz-lines / s
xz-lines / s
20
0.5
60
60
Objectives:
− W0.8/M27
− Modified Achroplan 40x / 0.8 W with reduced
length to compensate for piezo height
Installation:
• Screw in your microscope objective into Piezo
Objective Focusing Device (see Fig. 7-2/1).
• Screw the thread-ring into your microscope (see
Fig. 7-2/2).
• Easy clamp the Piezo Objective Focusing Device
on the thread-ring (see Fig. 7-2/3).
Fig. 7-2
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Installation of the Piezo Objective
Focusing Device
7-9
ANNEX
Specification of Trigger-Interface LSM 5 LIVE
Carl Zeiss
7.7
LSM 5 LIVE
LSM 5 LIVE DuoScan
Specifications of Trigger-Interface LSM 5 LIVE
Application:
With the LSM 5 LIVE you can control various actions externally using Trigger-In or force external devices
to work at a defined time depending on an action using Trigger-Out during time series.
These actions are: Scan-Start / Stop, Bleach, Change of Scan-Interval, end of a countdown, set marker
into image or even a mouse-click on a button.
Interface:
− User Port Adapter on the back of the Real Time Control Unit in the ECU of LSM 5 LIVE:
− Connector 2x high density D-Type plug, 1x coax with outer shield
Number:
− 8x signal IN (all able to generate an interrupt)
− 18x signal OUT (10 synchronous, 8 asynchronous relative scan position)
− 1x clock
Connector:
Coax: Triax Lemosa Serie 00, EPL.00.650NLN
Pin
Signal name
Description
Signal
PCLK_Out
PixelClock Output
Signal shield
GND
Signal ground
Outer shield
Shield
Chassis ground
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ANNEX
Specification of Trigger-Interface LSM 5 LIVE
Carl Zeiss
High density D-Type 15pol.: Plug „A“
Pin
Signal name
Description
1
SyncOUT0
Synchronous output
2
SyncOUT1
Synchronous output
3
SyncOUT2
Synchronous output
4
SyncOUT3
Synchronous output
5
GND
Signal ground
6
AsyncOUT0
Asynchronous output
7
AsyncOUT1
Asynchronous output
8
AsyncOUT2
Asynchronous output
9
AsyncOUT3
Asynchronous output
10
GND
Signal ground
11
AsyncIN0
input
12
AsyncIN1
input
13
AsyncIN2
input
14
AsyncIN3
input
15
Connector detector*
Input / Connector detector
High density D-Type 15pol.: Plug „B“
Pin
Signal name
Description
1
SyncOUT4
Synchronous output
2
SyncOUT5
Synchronous output
3
SyncOUT6 / sync outline **
Synchronous output
4
SyncOUT7 / sync outframe **
Synchronous output
5
GND
Signal ground
6
AsyncOUT4
Asynchronous output
7
AsyncOUT5
Asynchronous output
8
AsyncOUT6
Asynchronous output
9
AsyncOUT7
Asynchronous output
10
GND
Signal ground
11
AsyncIN4
input
12
AsyncIN5
input
13
AsyncIN6
input
14
AsyncIN7
input
15
Connector detector*
Input / Connector detector
*) pull to signal ground
03/06
**) depending on internal switch inside real time cintrol unit
B 45-0019 e
7-11
ANNEX
Specification of Trigger-Interface LSM 5 LIVE
Carl Zeiss
LSM 5 LIVE
LSM 5 LIVE DuoScan
Type/Voltage Range:
− TTL signal level 3.3 V, CMOS low power consumption
−
5.0 V tolerant input/output for interfacing with 5 V logic
Load:
Output:
< 50 mA (internal serial 68 ohm resistor)
Input:
4.7 kOhm input impedance (internal 4.7 kOhm pullup to 3.3 V)
Trigger pulse description:
Signal output:
− Low level < 0.4 V, high level > 2.8 V
− Slew rate 10 ns/V
Signal input:
− Low level < 0.8 V, high level > 2.0 V
− Falling edge force interrupt
− Pulse width to detect signal > 50 ns
Caution:
− Never apply more than 5 V or negative voltages to avoid any damage.
− In and outputs are not galvanically decoupled.
− Therefore proper measures for galvanic decoupling of external devices have to be taken (optocoupler etc.).
Hint:
Use the Zeiss “Trigger Box” (1315-620) for immediate use of the User-Port:
− 4 momentary-on switches, 2 BNC connectors including optional signal inversion for input
− 8 leds and 2 BNC connectors for output
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ANNEX
AxioCam High Resolution Digital Cameras
7.8
AxioCam High Resolution Digital Cameras
7.8.1
Microscopy Camera AxioCam MRm Rev.2
Carl Zeiss
High resolution microscopy camera AxioCam MRm Rev. 2 (D)
Cat. No
000000-0445-554
Mid Range Monochrome
incl. AxioVision AC, digital interface, cable and IR barrier filter BG40 (enclosed)
Number of Pixels:
1388 (H) x 1040 (V) = 1.4 Mega pixel
Pixel size:
6.45 µm x 6.45 µm
Chip size:
8.9 mm x 6.7 mm, equivalent to 2/3"
Spectral range:
With protection glass limited appr. 350 nm to 1000 nm,
With IR barrier filter BG40 appr. 350 nm to 700 nm
NIR-Mode:
Increase of IR sensitivity
Max. Full Well Capacity:
Approx. 17.000 e
Selectable Resolution:
H x
V
276 x
208 (Binning 5x5)
346 x
260 (Binning 4x4)
462 x
346 (Binning 3x3)
694 x
520 (Binning 2x2)
1388 x 1040 (single shot)
Live frame rates (depending on hardware and software configuration):
H x
V
Binning Factor
1388 x 1040
Frame Rate @20ms
1
7
694 x
520
2
13
462 x
346
3
17
Readout of Sensor
Sub Regions("ROI"):
Adjustable
Digitalization:
12 bits / 18 Mhz pixel clock
Dynamic Range:
Typical 1700 : 1 ati 10 e readout noise
Integration time:
1 ms to 20 s
Cooling:
One stage Peltier cooling
Control signals:
TTL output for controlling of external shutters
Interface:
PCI interface card (32 bit / 5 V) with one cable (5m)
for data, control and power supply
Optical Interface:
C-Mount
Thread depth for objectives:
max. 5 mm
03/06
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7-13
Carl Zeiss
ANNEX
AxioCam High Resolution Digital Cameras
Max. file size per image:
About 2.8 MB at 1388 x 1040 @ 1 x 12 bit
Operating Systems:
Win 2000, Win XP
Size / Weight:
About 11 cm x 8 cm x 4.5 cm (2.3” x 3.2” x 1.7”) / 370 g
Housing:
Blue anodized aluminum, with cooling fins,
1/4" photo thread for tripod mount
Registration:
CE, cUL
Power Supply:
Via PC
Environment conditions:
0° ... +35° Celsius, 10% .... 80% relative air huminity,
no condensing, free air circulation required
7.8.2
LSM 5 LIVE
LSM 5 LIVE DuoScan
High Resolution Microscopy Camera AxioCam HRm Rev.2
Cat. No
000000-0445-553, incl. digital interface and cable
High Range Monochrome
Number of Pixels:
1388 (H) x 1040 (V) = 1.4 Mega pixel
Chip size:
8.9 mm x 6.7 mm, equivalent to 2/3"
Spectral range:
With BK-7 protection glass up to 1000 nm, with IR barrier filter BG40
limited to about 350 nm to 700 nm
Selectable Resolution by Binning or Microscanning
H
x
V
694
x
520
1388
x
1040
2776
x
2080
4164
x
3120
Acquisition Time (s) @ 20 ms exposure
0.07 (13 images / s
0.2 (5 images / s)
Dynamic Range:
Better than 2000 : 1 @ 8 e readout noise
Integration Time:
1 ms to several minutes
Cooling:
Single stage Peltier cooling
Optical Interface:
C-Mount
Size:
about 11 cm x 8 cm x 6.5 cm (2.3" x 3.2" x 2.6")
Registration:
GS, CE, cUL
Power Supply:
12 V DC, 1 A, 230 V/110 V, autodetecting
7-14
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
7.8.3
ANNEX
AxioCam High Resolution Digital Cameras
Carl Zeiss
High Resolution Microscopy Camera AxioCam HRc
Cat. No
000000-0412-312, incl. digital interface and cable
High Range Color
Number of Pixels:
1300 (H) x 1030 (V) = 1.3 Mega pixel
Chip size:
8.7 mm x 6.9 mm equivalent to 2/3”
Spectral range:
Limited by IR barrier filter BG40, about 400 nm to 700 nm
Selectable Resolution by Binning or Microscanning
H
x
V
Acquisition Time (s) @ 20 ms exposure
432
x
342
1300
x
1030
0.2
(Color interpolation)
1300
x
1030
0.7
(full resolution for color channels)
2600
x
2060
3900
x
3090
0.07 (Color interpolation)
Dynamic Range:
Typical 2000 : 1 @ 9 e readout noise
Integration Time:
1 ms to several minutes
Cooling:
One stage Peltier cooling
Optical Interface:
C-Mount
Size:
about 11 cm x 8 cm x 6.5 cm (2.3" x 3.2" x 2.6")
Registration:
GS, CE, cUL
Power Supply:
12 V DC, 1 A, 230 V/110 V, autodetecting
03/06
B 45-0019 e
7-15
ANNEX
AxioCam High Resolution Digital Cameras
Carl Zeiss
7.8.4
LSM 5 LIVE
LSM 5 LIVE DuoScan
Microscope Camera Port Adapters for the AxioCam
Adapter Video V200 C 2/3" 0.63x at frontport Axiovert 200 M
Cat. No 000000-1071-171
This adapter is needed for attachment of the high-resolution AxioCam microscope cameras on the
Axiovert 200 M
Adapter Video 60 C 2/3" 0.63x
Cat. No 000000-1069-414
This adapter is needed for attachment of the high-resolution AxioCam microscope cameras on the
Axio Imager.Z1 and Axioskop 2 FS MOT.
For an additional documentation port to be connected to the camera port of the tubes with interface 60:
Double video adapter
Cat. No 000000-1058-640
For connection to interface 60, 2 switching positions for switching to 100% mirror or to interface for
P&C modules.
Adapter Video 44 C 2/3" 0.63x
Cat. No 452997-0000-000
This adapter is needed for attachment of the high-resolution AxioCam microscope cameras on the
Axiovert 200 M SP.
No other cameras are supported by the LSM Software!
7-16
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
7.9
ANNEX
List of Key Words
List of Key Words
Numbers
2.5 D .......................................................4-266
3D ...........................................................4-268
3D for LSM
Terms and definitions...............................6-2
3D view ......................................... 4-15, 4-152
A
About LSM 5 LIVE....................................4-223
Achrogate .................................................4-55
Acquire............................................ 4-15, 4-37
Add image sequences................................6-17
Alpha rendering method............................6-46
And calculation..........................................6-37
Annex..........................................................7-2
Ampl. gain...............................................4-319
Ampl. offset ............................................4-321
Analysis of images ...................................4-224
Animation ...............................................4-239
Area ........................................................4-254
Automatic object measurement .................6-52
Average.....................................................4-73
Axio Imager control ...................................4-41
Axioskop 2 FS control ................................4-50
Axiovert control .........................................4-46
B
Beam path............................. 4-55, 4-57, 4-59,
.......................................... 4-60, 4-316, 4-322
Bleach .....................................................4-110
Bleach parameter.....................................4-112
Bleach regions .........................................4-112
C
Calculation
And .......................................................6-37
Or ..........................................................6-37
Xor ........................................................6-38
Camera color adjustment.........................4-220
Camera detection ......................................4-66
Carl Zeiss vision image sequences ..............6-12
03/06
Carl Zeiss
Channel......................................... 4-75, 4-226
Add, remove .................................. 6-5, 6-15
Delete ................................................... 6-16
Channel assignment ..4-55, 4-60, 4-316, 4-322
Channel mode configuration .................... 4-62
Closing ..................................................... 6-32
Collimator adjustment ............................ 4-218
Color palette .......................................... 4-237
Configuration control ............................... 4-51
Contrast ...................................... 4-132, 4-234
Change contrast ................................... 6-19
Enhance................................................ 6-23
Copy ............................................. 4-25, 4-243
Content of display window ................... 6-14
Copy full resolution ................................ 4-146
Copy window contents........................... 4-145
Crop....................................................... 4-242
Cut......................................................... 4-250
D
Database .................................................. 4-18
Delete function ..................................... 4-28
Export of images ................................... 4-33
Filter function........................................ 4-26
Form mode ........................................... 4-21
Gallery mode ........................................ 4-23
Import of images................................... 4-32
Load function............................... 4-24, 4-25
Multi print............................................. 4-34
New...................................................... 4-18
Open .................................................... 4-19
Saving an image.................................... 4-29
Table mode........................................... 4-24
Deconvolution ........................................ 4-163
Delete
Images .................................................. 6-16
Object in specified size range ................ 6-41
Depth coding.......................................... 4-152
Detaching the Scanning Module ................. 7-5
Detector gain............................... 4-319, 4-320
Dialog boxes............................................... 6-4
Dichroic beam splitters.............................. 4-56
Dilation..........................6-28, 6-30, 6-31, 6-32
Display of images.................................... 4-224
Display window ................................... 6-4, 6-7
Dyes ........................................................... 7-2
B 45-0019 e
7-17
ANNEX
LSM 5 LIVE
LSM 5 LIVE DuoScan
Carl Zeiss
E
Edit menu....................................................6-5
Emission filter .................................... 4-55, 7-2
Erosion .................6-27, 6-28, 6-29, 6-31, 6-32
Excitation ..................................................4-59
Excitation laser lines.....................................7-2
Excitation of bleach track.........................4-113
Exiting the LSM program .........................4-325
Exposure time....................... 4-75, 4-89, 4-317
Render-Surface ......................................6-43
Save Display As ......................................6-13
Save Image As........................................6-12
Scrap .....................................................6-41
Segment-Automatic ...............................6-35
Segment-Interactive ...............................6-33
Set Channel Colour................................6-10
Smooth (Gauss)......................................6-24
Volume Features ....................................6-55
Full screen ...............................................4-221
G
F
File .................................................. 4-15, 4-17
File menu ....................................................6-5
Fills holes...................................................6-42
Filter.............................................. 4-57, 4-130
Find............................................. 4-318, 4-322
Frame .............................................. 4-68, 4-71
creation of a ..........................................4-78
FRAP Guide .............................................4-223
Functions
Add Channel .........................................6-15
Arithmetics-Add.....................................6-17
Arithmetics-Subtract ..............................6-18
Automatic Object Measurement, General6-58
Boolean-And..........................................6-37
Boolean-Mask........................................6-40
Boolean-Not ..........................................6-39
Boolean-Or ............................................6-37
Boolean-Xor...........................................6-38
Condition ..............................................6-57
Contrast-Automatic ...............................6-21
Contrast-Interactive ...............................6-19
Contrast-Linearize ..................................6-23
Copy......................................................6-14
Delete All Images ...................................6-16
Delete Channel ......................................6-16
Edit Channels.........................................6-15
Exit ........................................................6-14
Fill Holes ................................................6-42
Morphology...........................................6-25
Morphology-Close .................................6-32
Morphology-Dilate .................................6-30
Morphology-Erode .................................6-29
Morphology-Open .................................6-31
Object Features ......................................6-52
Open Image ...........................................6-11
Print.......................................................6-14
Render-Alpha.........................................6-46
7-18
Gallery...................................... 4-251, 6-3, 6-7
Gauss filter ................................................6-24
Gradient shading method ..........................6-43
Grey value segmentation ................. 6-33, 6-35
H
Help ........................................................4-223
Help menu...................................................6-6
Histogram................................................4-253
Holes to fill ................................................6-42
Hyperfine Z sectioning ...............................4-84
I
Image
Delete function ......................................4-28
Display and analysis..............................4-224
Export of an ...........................................4-33
Import of an...........................................4-32
Information..........................................4-311
Load an..................................................4-19
Saving an ...............................................4-29
Image acquisition.......................................4-37
Image optimization...................... 4-315, 4-323
Image sequence...........................................6-2
Channels..................................................6-2
Information of the image.........................4-311
Interpolation............................................4-134
Ion concentration ....................................4-141
K
Kinetic .....................................................4-146
L
Laser attenuation.......................................4-59
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
ANNEX
List of Key Words
Laser control..............................................4-38
Line ...........................................................4-68
Creation of a line ...................................4-87
Lowpass filter ..........................................4-130
LSM 5 LIVE switchboard...........................4-325
M
Macro................................. 4-16, 4-167, 4-215
editing and debugging.........................4-172
Main components of the system ..................6-3
Main menu................................................4-15
Main window ..............................................6-3
Maintain........................................ 4-16, 4-210
Admin..................................................4-220
Collimator adjustment..........................4-218
Objectives ............................................4-212
Pinhole adjustment ..............................4-215
Reboot.................................................4-220
Test grid...............................................4-220
Mathematical morphology.........................6-25
Mark first/last ............................................4-83
Marker ........................................ 4-100, 4-103
Mean........................................... 4-265, 4-312
Mean ROI ................................................4-106
Measurement concept ...............................6-50
Measurement menu ....................................6-6
Measurement process................................6-51
Median filter............................................4-131
Microscope control ....................................4-40
Mode ........................................................4-71
monochrome image display .....................4-227
Multi track.................................................4-51
Multiple-channel......................................4-322
multitracking ................................... 4-60, 4-61
N
Negation of image.....................................6-39
NFT............................................................4-55
Non Descanned .........................................4-67
Opening ................................................... 6-31
Options ......................................... 4-16, 4-198
Dye database ...................................... 4-199
Hardware............................................ 4-329
Settings............................................... 4-200
Software ............................................. 4-327
Or calculation ........................................... 6-37
Orthogonal sections................................ 4-246
Overlay ................................................... 4-230
P
Palette .............................. 4-237, 4-319, 4-321
Parfocality............................................... 4-214
Paste ........................................................ 4-25
Paste bitmap........................................... 4-146
Piezo fine focusing stage ............................ 7-7
Piezo objective focussing device.................. 7-9
Pinhole ................................................... 4-319
Pinhole adjustment ................................. 4-215
Pixel depth.............................................. 4-321
Player ......................................................... 6-8
control elements ..................................... 6-9
Preview print .......................................... 4-309
Print .............................................. 4-34, 4-309
Content of display window ................... 6-14
Process .......................................... 4-15, 4-121
add ..................................................... 4-121
channel shift ....................................... 4-135
contrast .............................................. 4-132
copy.................................................... 4-127
duplication.......................................... 4-128
filter.................................................... 4-129
interpolate .......................................... 4-133
multiply............................................... 4-125
ratio.................................................... 4-126
subtract .............................................. 4-124
Process menu ............................................. 6-5
Profile..................................................... 4-261
Projection ............................................... 4-155
Properties ................................................... 6-3
R
O
Object features
Densitometric.........................................6-55
Geometric..............................................6-54
Objective .................................................4-212
03/06
Carl Zeiss
Range....................................................... 4-82
Range indicator ........................... 4-319, 4-321
Ratio....................................................... 4-126
Ratio settings................................... 4-64, 4-65
B 45-0019 e
7-19
ANNEX
LSM 5 LIVE
LSM 5 LIVE DuoScan
Carl Zeiss
Recording configuration .................. 4-51, 4-60
Refresh......................................................4-25
Region of interest
Edit........................................................4-90
Reuse ......................................................4-241
ROI
Edit............................................ 4-90, 4-112
Running down the operating system .......4-326
S
Save ........................................................4-243
As........................................................4-244
Content of display window ....................6-13
Image .......................................... 4-29, 6-12
Image sequence .....................................6-12
Scaling image ..........................................4-145
Scan average ...........................................4-321
Scan control ..............................................4-68
Scan speed .................................... 4-71, 4-321
Scatter diagram .......................................4-257
Segmentation
Automatic..............................................6-35
Interactive ..............................................6-33
Select
Object in specified size range .................6-41
Settings ...................................................4-200
Sharpness filter........................................4-130
Shut-down procedure..............................4-325
Single channel .........................................4-315
Single Track...............................................4-51
single tracking ...........................................4-61
Single tracking...........................................4-55
Slice ................................................ 4-229, 6-2
Split xy ............................... 4-78, 4-245, 4-323
Stage.......................................................4-114
Starting the LSM 5 program ......................4-13
Stereo .....................................................4-159
Structures
Dilate.....................................................6-30
Erode.....................................................6-29
Subset .....................................................4-251
Subtract image sequences .........................6-18
Switching on the enterprise UV laser .........4-38
Switching on the system ............................4-12
7-20
T
Tile scan ..................................................4-118
Time delay .................................................4-98
Time interval..............................................4-98
Time series.................................................4-94
Of a frame ...........................................4-102
Of a Z stack..........................................4-104
With mean ROI ....................................4-106
Toolbar....................................... 4-15, 6-3, 6-6
Topography .............................................4-278
3D measurement functions ..................4-304
Data processing ....................... 4-279, 4-281
Display modes.......................... 4-284, 4-287
Measurement functions .......................4-294
Track .........................................................4-51
Track configuration.......................... 4-51, 4-53
Tracks panel, list of ....................................4-61
Trigger-interface LSM 5 LIVE ......................7-10
Turning power off ...................................4-326
U
Unmix......................................................4-137
User-defined filter....................................4-131
V
Value
Enter new value .....................................6-20
Mask grey value .....................................6-40
Scale a range of grey values ...................6-23
Scale grey value .....................................6-21
Vertical scale of histogram .........................6-20
View menu ..................................................6-6
Visual macro editor...................... 4-177, 4-189
Volume features
Densitometric.........................................6-56
Geometric..............................................6-56
W
Window ..................................................4-221
full screen ............................................4-221
Windows menu ...........................................6-6
X
Xor calculation...........................................6-38
Xy display ................................................4-244
B 45-0019 e
03/06
LSM 5 LIVE
LSM 5 LIVE DuoScan
ANNEX
List of Key Words
Carl Zeiss
Z
Zeiss TIFF image sequence..........................6-11
Z sectioning ...............................................4-81
Z settings.................................................. 4-79
Z stack.......................................................4-79
creation of a ..........................................4-86
Zoom.......................................................4-228
03/06
B 45-0019 e
7-21
LSM 5 LIVE
LSM 5 LIVE DuoScan
Laser Scanning Microscope
Brief Operating Manual
Release 4.0
March 2006
Contents
Page
Starting the System ................................................................................................................3
Setting the microscope...........................................................................................................6
Configuring the beam path and lasers..................................................................................8
Scanning an image ...............................................................................................................11
Storing an image ..................................................................................................................15
Switching off the system .....................................................................................................15
Introduction
For your safety!
Observe the following instructions:
2
−
The LSM 5 LIVE / LSM 5 LIVE DuoScan laser scanning microscope, including its original
accessories and compatible accessories from other manufacturers, may only be used for
the purposes and microscopy techniques described in this manual (intended use).
−
In the Operating Manual, read the chapter Safety Instructions carefully before starting
operation.
−
Follow the safety instructions described in the operating manual of the microscope and
HBO 100 mercury lamp.
03/06
Starting the System
Switching on the LSM system
• Set the MAIN SWITCH on the system rack to
ON position (Fig. 1).
• When set to ON the System/PC switch
provides power to the microscope and the
computer. This allows to use the microscope
and the computer without running the LSM
Software (Fig. 1).
• To switch on the system completely put the
Components switch to ON. Insert the key into
the key-operated LASER switch and switch also
to ON.
Now the complete system is ready to be initialized
with the LSM Software.
Fig. 1
System rack
Fig. 2
HBO 100 power supply
Switching on the HBO 100 mercury lamp
• Switch on the HBO 100 mercury lamp via the
switch of the power supply, see operating
manual of the mercury lamp or microscope.
03/06
3
Starting the LSM 5 software program
LSM 5 Live
• Double click the LSM 5 LIVE icon on the desktop of WINDOWS to start the LSM 5
software program.
The LSM 5 LIVE Switchboard window appears on the screen (Fig. 3).
Fig. 3
LSM 5 LIVE Switchboard menu
• Click on the Scan New Images button in the LSM 5 LIVE Switchboard window.
Clicking on this button activates the complete LSM hardware (on-line mode).
• Click on the Start button in the LSM 5 LIVE Switchboard window.
The LSM 5 LIVE - Main menu appears on the screen.
Use of this mode requires to be thoroughly familiar with the exact microscope procedures
and interrelations.
Main menu (pull-down)
Main menu toolbar
Subordinate toolbar
Fig. 4
4
Main menu
03/06
Creating a database for acquired images
• Click on the File button in the Main menu toolbar.
The File subordinate toolbar appears in the Main menu.
Fig. 5
Main menu with File subordinate toolbar
• Click on the New button in the File subordinate toolbar.
The Create New Database window appears.
• Select drive C: or D: from pull down menu.
• Create a new directory if needed.
Fig. 6
Fig. 7
Create New Database window
Create New Database window
Turning on the lasers
• Click on the Acquire button in the Main menu to open the Acquire subordinate
toolbar.
Fig. 8
03/06
Acquire subordinate toolbar
5
• Click on the Laser button to open the Laser Control window.
• Select the appropriate Laser Unit by clicking on
the name of it.
• Click on the On button to switch required
laser(s) to on (or standby if required).
Fig. 9
Laser Control window
Setting the microscope
Changing between direct observation or laser scanning
The VIS, TV and LSM buttons switch the beam path and indicate which beam path has been set in the
binocular tube of the microscope:
• Click on the VIS button to set the microscope for direct observation via the eyepieces of
the binocular tube, lasers are off.
• Click on the TV button to set the microscope camera observation (if connected) via camera
adapter of the binocular tube.
• Click on the LSM button to set the microscope screen observation via laser excitation using
the LSM 5 LIVE and software evaluation.
Setting the microscope and storing the settings
• Click on the VIS button for direct observation.
• Click on the Micro button in the Acquire subordinate toolbar to open the
Microscope Control window of the used microscope.
The Microscope Control window appears (Fig. 10).
6
03/06
Selecting an objective
• Open the graphical pop-up menu by clicking on
the Objective button (Fig. 10).
• Click on the objective you want to select. The
selected objective will automatically move into
the beam path.
Focussing the microscope for transmitted light
• Open the graphical pop-up menu by clicking on
the Transmitted Light button (Fig. 11).
• Click on the On button. Set the intensity of the
Halogen illuminator using the slider.
• Click on Close to close the pop-up menu.
• Place specimen on microscope stage. The cover
slip must be facing up.
• Use the focusing drive of the microscope to
focus the required object plane.
• Select specimen detail by moving the stage in X
and Y using the XY stage fine motion control.
Fig. 10
Microscope Control window,
e.g.: Axiovert 200 M
Fig. 11
Microscope Control window with
Transmitted Light pop-up menu
Setting the microscope for reflected light
• Click on the Reflected Light button to open
the shutter of the HBO 100 mercury lamp.
• Click on the Reflector button and select the
desired filter set by clicking on it.
Storing the microscope settings
Microscope settings can be stored and up to 8
buttons assigned for fast retrieval and adjustment
using the Microscope Settings panel.
The Store button permits existing microscope
configurations to be stored under any name.
The Apply button permits existing
microscope configurations to be loaded.
stored
The Delete button permits existing microscope
configurations to be deleted.
The Assign button permits the assignment of a
microscope configuration to a button.
Note: Depending
on
the
microscope
configuration, settings must be done manually if
necessary.
03/06
7
Configuring the beam path and lasers
• Click on the LSM button in the Acquire
subordinate toolbar for laser scanning.
Choosing the configuration
Single Track
− Use for single, double and triple labeling;
simultaneous scanning only
− Advantage: faster image acquisition
− Disadvantage: cross talk between channels
Multi Track
− Use for double and triple labeling; sequential
scanning, line by line or frame by frame
− Advantage: when one track is active, only
one detector and one laser is switched on.
This reduces cross talk.
− Disadvantage: slower image acquisition
• Click on the Config button in the
Acquire subordinate toolbar to
open the Configuration Control
window.
Fig. 12
Configuration Control window for
Single Track
The Configuration Control window
appears (Fig. 12).
Setting for single track configuration in Channel Mode
• Select Channel Mode if necessary (Fig. 12).
• Click on the Single Track button in the Configuration Control window (Fig. 12).
The Beam Path and Channel Assignment panel of the Configuration Control window displays the
selected track configuration which is used for the scan procedure.
• You can change the settings of this panel using the following function elements:
Activation / deactivation of the excitation wavelengths (check box) and setting of
excitation intensities (slider). Open the Laser Control window via the Laser button.
Selection of the secondary dichroic beam splitter (NFT) position through selection from
the relevant list box.
Selection of an emission filter through selection from the relevant list box.
Activation / deactivation (via check box) of the selected channel (ChL 1, ChL 2) for the
scanning procedure by assigning an existing color icon or defining a new one.
8
03/06
• Select the appropriate filters and activate the channels.
• Click the Excitation button to select the laser lines and set the attenuation values (transmission in %)
in the displayed window.
For the configuration of the beam path, please refer to the application-specific configurations depending
on the used dyes and markers and the existing instrument configuration.
• Clicking on the Config button
opens the Track Configurations
window (Fig. 13) to load, store or
delete track configurations.
• For storing a new track configuration enter a
desired name in the first line of the
Configurations list box and click an Store.
• For loading an existing configuration select it in
the list box and click on Apply.
Fig. 13
Track Configurations window
• For deleting an existing configuration select it in
the list box and click on Delete.
Setting for multi track configuration in Channel Mode
The Multi Track function permit several tracks to
be defined as one configuration (Channel Mode
Configuration) for the scan procedure, to be
stored under any name, reloaded or deleted.
The maximum of four tracks with up to 8 channels
can be defined simultaneously and then scanned
one after the other. Each track is a separate unit
and can be configured independently of the other
tracks with regard to channels, Acousto-Optical
Tunable Filters (AOTF), emission filters and dichroic
beam splitters.
• Select Channel Mode if necessary (Fig. 14).
Fig. 14
03/06
Configuration Control window for
Multi Track
9
• Click on the Multi Track button in the Configuration Control window (Fig. 14).
The following functions are available in the List of Tracks panel (Fig. 14).
Add Track button
An additional track is added to the configuration list. The maximum of
four tracks can be added. One track each with basic configuration is
added, i.e.: one ChL 1 channel is activated, all laser lines are switched
off, emission filters and dichroic beam splitters are set in accordance with
the configuration last used.
Remove button
The track previously marked in the List of Tracks panel in the Name
column is deleted.
Store/Apply Single Track
button
Opens the Track Configurations window. A selected track defined in a
Recording Configuration can also be stored as a single track for single
tracking applications. Also, it's possible to load a single track in a
multitracking configuration
A click on this arrow button will move the selected track (highlighted in
blue) one position upwards in the list box.
A click on this arrow button will move the selected track (highlighted in
blue) one position downwards in the list box.
• Configure each desired track for Multi Track function as described for Single Track.
• For storing/applying or deleting a Channel Mode Configuration use the Config button.
10
03/06
Scanning an image
• Click on the Scan button in the Acquire subordinate toolbar to open the Scan
Control window.
The Scan Control window appears (Fig. 15).
Setting the parameters for scanning
• Select Mode in the Scan Control window.
• Select the Frame Size as predefined number of
pixels or enter your own values (e.g. 300 x 600)
in the Objective Lens, Image Size & Line
Step Factor panel.
The number of pixels influences the image
resolution!
Note: When using an Axioskop 2 FS MOT, indicate
the objective that is in use in the Scan Control
window. This ensures correct calculation of
pinhole, Z stack optimization etc.
Adjusting the exposure time
• Use the Exposure Time slider in the Objective
Lens, Image Size & Line Step Factor panel
(Fig. 15) to adjust the exposure time.
Fig. 15
Scan Control window, Mode settings
Below the slider is shown the number Frames per Second. Start with 2 to 4 frames per second.
Choosing the dynamic range
• Select the dynamic range 8 or 12 Bit (per pixel) in the Pixel Depth, Scan Direction & Scan Average
panel (Fig. 15).
8 Bit will give 256 gray levels, 12 Bit will give 4096 levels. Publication quality images should be acquired
using 12 Bit data depth. 12 Bit is also recommended when doing quantitative measurements or when
imaging low fluorescence intensities.
Setting the scan averaging
Averaging improves the image by increasing the signal : noise ratio. It can be achieved line by line or
frame by frame. Frame averaging helps reduce photobleaching, but does not give quite such a smooth
image.
• Select the Line or Frame mode for averaging.
• Select the desired scan average method Mean or Sum in the Method selection box.
If you are using the Mean method, the image information is generated by adding up all scans pixel by
pixel and then calculating the mean value.
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In the Sum method, the pixel values of all scans are only added up, without a mean value being
calculated.
• Select the Number for averaging.
Continuous averaging is possible in the Frame mode. In this case a Finish button for ending continuous
averaging is displayed instead of the Cont. button.
Adjusting the pinhole
• Select Channels in the Scan Control window.
• Set the Pinhole size to 1 (Airy unit) for best
compromise between depth discrimination and
efficiency.
Pinhole adjustment changes the Optical Slice.
When collecting multi channel images, adjust the
pinholes so that each channel has the same
Optical Size. This is important for colocalization
studies.
Image acquisition
Fig. 16
Scan Control window, Channel settings
Once you have set up your parameter as defined in
the above section, you can acquire a frame image
of your specimen.
• Use one of the Find, Fast XY, Single or Cont.
buttons to start the scanning procedure and
acquire an image.
Scanned images are shown in separate windows.
• Click on the Stop button to stop the current
scan procedure if necessary.
Select Find for automatically preadjustment of detector sensitivity.
Select Fast for continuous fast
scanning – useful for finding and
changing the focus.
Select Single for recording a single
image.
Fig. 17
Image window
Select Cont. for continuous scanning
with the selected scan speed.
Select Stop for stopping the current
scan procedure.
12
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Image optimization
Choosing a lookup table
• Select Palette in the Image window of the
scanned image (Fig. 18).
The Color Palette window appears.
• In the Color Palette List panel, click on the
Range Indicator item (Fig. 19).
The scanned image appears in a false-color
presentation (Fig. 18).
If the image is too bright, it appears red on the
screen. Red = saturation (maximum).
If the image is not bright enough, it appears blue
on the screen. Blue = zero (minimum).
Fig. 18
Image window
Fig. 19
Color Palette window
Fig. 20
Scan Control window, Channel settings
Adjusting the laser intensity
• Set the Pinhole to 1 Airy Unit (Fig. 20).
• Set the Detector Gain slider to a middle
position.
• When the image is saturated, reduce AOTF
transmission in the Excitation panel (Fig. 20)
using the slider to reduce the intensity of the
laser light at the specimen.
Adjusting gain and offset
• Increase the Amplifier Offset until all blue
pixels disappear, and then make it slightly
positive (Fig. 20).
• Reduce or increase the Detector Gain until the
red pixels only just disappear.
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Scanning a Z stack
• Select Z Stack in the Scan Control window.
• Select Frame if necessary.
The Z Settings panel appears.
• Select Mark First/Last on the Z Settings
panel.
• Click on the XY cont button.
A continuous XY-scan of the set focus position will
be performed.
• Use the focusing drive of the microscope to
focus on the upper position of the specimen
area where the Z Stack is to start.
• Click on the Mark First button to set the upper
position of the Z Stack.
• Then focus on the lower specimen area where
the recording of the Z Stack is to end.
• Click on the Mark Last button to set this lower
position.
• Click on X:Y:Z=1:1:1 button to set the Zinterval in such a way that the voxel has
identical dimensions in the X-, Y- and Zdirections (cube).
Fig. 21
14
Scan Control window, Z Stack settings
• Click on the Start button to start the recording
of the Z Stack.
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Storing an image
• Click on the Save or Save As button in the Image window or in the File subordinate toolbar of the
Main menu.
The Save Image and Parameter As window appears.
Fig. 22
Save Image and Parameter As window
• Enter file name, description and notes in the appropriate text boxes.
• Click on the OK button.
Switching off the system
• Click on the File button in the Main menu and then click on the Exit button to leave LSM 5 software
program (Fig. 5).
• Shut down the computer.
• Put the LASER key switch to OFF position. Remove the key from the system rack.
• Put the COMPONENTS switch and the System/PC switch to OFF position (Fig. 1).
• Put the MAIN SWITCH on the system rack to OFF position (Fig. 1).
• Switch off the HBO 100 mercury lamp (Fig. 2).
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