LSM 510 – ConfoCor 2 Combination System

LSM 510 – ConfoCor 2 Combination System
LSM 510 – ConfoCor 2
Combination System
Operating Manual
Rel. 3.2
INTRODUCTION
Carl Zeiss
LSM-FCS
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.
Developed in
Collaboration with the
Issued by
European Molecular Biology Laboratory (EMBL)
PF 102209
Meyerhofstr. 1
69012 Heidelberg
GERMANY
Phone:
++49-6221-387-0
Telefax:
++49-6221-387-306
Carl Zeiss
Advanced Imaging Microscopy
07740 Jena
GERMANY
Phone:
++49-3641 64-34 00
Telefax:
++49-3641 64-31 44
E-mail:
[email protected]
Internet:
www.zeiss.de/lsm
Number of this manual: B 45-0006 e
Date of issue: 12/2002
II
B 45-0006 e
12/02
INTRODUCTION
LSM-FCS
Carl Zeiss
How to Make Best Use of the LSM-FCS 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: The time you invest in getting acquainted with the product will pay
for itself many times over in your application task.
B 45-0006 e
12/02
III
INTRODUCTION
Carl Zeiss
LSM-FCS
Scope of Equipment Supplied
Country:
Order number:
Serial number:
Delivery date:
Custom configuration:
Axiovert 200 M SP
Axiovert 200 M BP
Objectives:
..............................................
..............................................
..............................................
..............................................
..............................................
000000-1028-779
000000-1028-780
..............................................
..............................................
..............................................
Configuration 1
Configuration 2
Configuration 3
Configuration 4
Configuration 5
Configuration 9
Configuration 11
Configuration 12
Configuration 13
Configuration 15
Configuration 16
Configuration 18
Configuration 3 META
Configuration 13 META
Configuration 15 META
Configuration 18 META
000000-1219-975
000000-1219-977
000000-1219-978
000000-1219-979
000000-1219-980
000000-1219-981
000000-1219-982
000000-1219-983
000000-1219-984
000000-1219-985
000000-1219-986
000000-1219-987
000000-1219-995
000000-1219-997
000000-1220-000
000000-1220-004
Control computer
21“ monitors
TFT monitors
000000-0438-360
000000-1060-089
000000-0435-035
The license to the LSM control software is included in each configuration 1...18 (META).
IV
B 45-0006 e
12/02
INTRODUCTION
LSM-FCS
Carl Zeiss
Optional software:
Image VisArt option
3D for LSM option
3D Deconvolution option
Physiology option
Topography option
Multiple Time Series option
Macro Reader and Editor option
StitchArt option
000000-1207-934
000000-1207-919
000000-1207-920
000000-1207-930
000000-1207-933
000000-1207-926
000000-1207-925
000000-1207-932
Canon S 830 D Photo printer
Kodak XLS 8650 PS printer
Transparency exposure device (D)
000000-0445-508
000000-1113-131
412410-9011-000
Large system table
Small system table
System baseplate
453031-0000-000
453032-0000-000
000000-1171-342
Active anti-vibration table 30" x 30"
Active anti-vibration table 1200 mm x 1400 mm
Active anti-vibration table 1800 mm x 1400 mm
000000-1177-841
000000-1198-394
000000-1150-353
UV laser
412410-9015-000
XY scanning stage for Axiovert 200 M BP
XY scanning stage for Axioplan 2 imaging MOT
Piezo objective drive W = 0.8"
000000-1113-509
000000-1027-823
000000-1210-045
High resolution Z stage HRZ 200 for Axiovert 200 M
High resolution Z stage HRZ 200 for Axioplan 2 imaging MOT (ie)
000000-1115-248
000000-1115-245
Set of INDO filters
Set of SNARF filters
447960-0000-000
447961-0000-000
AxioCam HRm
AxioCam HRc
000000-0445-553
000000-0412-312
B 45-0006 e
12/02
V
INTRODUCTION
Carl Zeiss
LSM-FCS
The LSM-FCS in the configuration as checked above
was installed and handed to the customer in functional condition
on
................................................
by
................................................
Phone:
................................................
Fax:
................................................
The customer has been instructed on how to operate and
maintain the equipment.
(Place)......................................................, (date) .....................................
...............................................................
Carl Zeiss Jena GmbH
Microscopy Division
...............................................................
Customer
One copy to be kept by customer
One copy to be kept by Carl Zeiss
VI
B 45-0006 e
12/02
INTRODUCTION
LSM-FCS
1
Carl Zeiss
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-FCS Setup Requirements
The Setup Requirements section outlines the installation and supply requirements of the LSMFCS Microscope System, 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-FCS image
series in three dimensions, and introduces you to the performance features of your LSM-FCS.
4
Quickstart
5
Operation in Expert Mode
In the Operation section you will find the most important steps and procedures of the LSMFCS menu structure. The step-by-step description how to get an image will be shown by
typical application examples including the WINDOWS NT 4.0 graphic user environment.
6
VBA Programming for LSM
7
Routine Mode and Tools
This section contains a description of the Routine Mode for scanning images using the LSM
scanning module and the use of the tools for setting the microscope.
8
3D for LSM 510
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.
9
Annex
The Annex contains the Application-specific Configurations and special notes and information
for using the LSM-FCS microscope.
10
Multiphoton Laser Scanning Microscopy - Using the Zeiss LSM 510 NLO
11
Certification
B 45-0006 e
12/02
VII
INTRODUCTION
Carl Zeiss
VIII
LSM-FCS
B 45-0006 e
12/02
NOTES ON DEVICE SAFETY
Contents
LSM-FCS
CHAPTER 1
Carl Zeiss
NOTES ON DEVICE SAFETY
CONTENTS
Page
1
NOTES ON DEVICE SAFETY ...........................................................................................1-3
1.1
General ...........................................................................................................................1-3
1.2
Regulations .....................................................................................................................1-3
1.3
Notes on Setting up the Microscope System .....................................................................1-4
1.4
Notes on Handling the Computer and Data Media ...........................................................1-5
1.5
Notes on Care, Maintenance and Service .........................................................................1-6
1.6
Notes on Handling the Laser Components........................................................................1-7
1.7
Warning and Information Labels ......................................................................................1-8
B 45-0006 e
12/02
1-1
Carl Zeiss
1-2
NOTES ON DEVICE SAFETY
Contents
LSM-FCS
B 45-0006 e
12/02
NOTES ON DEVICE SAFETY
General / Regulations
LSM-FCS
1
NOTES ON DEVICE SAFETY
1.1
General
Carl Zeiss
The LSM-FCS 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).
The manufacturer will not assume liability for any malfunction or damage caused by any thing
other than the intended use of the LSM-FCS 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.
1.2
Regulations
Extensive knowledge of the hardware/the system is indispensable for safe operation of the LSM-FCS.
☞
⇒
⇒
⇒
Read these operating instructions and all device publications belonging to the system
conscientiously before operating the LSM-FCS!
The LSM-FCS has been designed, built and tested in conformity with the standards DIN EN 61010-1
(IEC 1010-1) "Safety requirements for electrical instrumentation and control and laboratory
apparatus", and DIN EN 60825-1 (IEC publication 825-1) "Safety of laser equipment", and taking
relevant CSA and UL specifications into account.
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 NT 4.0 and WINDOWS 2000 graphical user
interface. The respective manuals are supplied together with the programs.
In accordance with WHO regulations, the LSM-FCS is a device that belongs to laser hazard class 3 B.
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.
B 45-0006 e
12/02
1-3
Carl Zeiss
1.3
☞
NOTES ON DEVICE SAFETY
Notes on Setting up the Microscope System
LSM-FCS
Notes on Setting up the Microscope System
Setting up, assembly on the system base plate and commissioning of the LSM-FCS must be
performed by authorized Carl Zeiss service staff, who are also advised to give the customer's
operators a basic introduction to operation and maintenance.
The LSM-FCS laser scanning microscope is delivered in several crates:
•
•
•
•
•
•
•
•
Crate 1: microscope stand, laser scanning module, control unit
Crate 2: computer
Crate 3: monitor
Crate 4: large system table
Crate 5: second microscope stand
Crate 6: small system table
Crate 7: scan module META
Crate 8: upgrade kit META
The LSM-FCS 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 louvers on the microscope system must not be covered up.
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.
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, the TV port on the microscope must either be equipped with a
camera or covered by a cap.
Maintenance, repairs, modifications, 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 us to do so.
This applies especially to the microscope system, the laser scanning module, lasers, the PC
system, the power supply units, cable connections and other system components.
Please note that the LSM-FCS is a high-precision opto-electronic instrument. Inexpert handling
may easily impair it’s function or even damage it.
After installation or after conversion of the LSM-FCS 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 by electronic components is avoided.
To avoid vibrations during operation, the LSM-FCS should only be operated in conjunction with the
system table (vibration damping).
1-4
B 45-0006 e
12/02
NOTES ON DEVICE SAFETY
Notes on Handling the Computer and Data Media
LSM-FCS
1.4
Carl Zeiss
Notes on Handling the Computer and Data Media
The computer used as standard in your LSM-FCS system is an IBM-compatible high-end pentium
computer with WINDOWS NT 4.0 or WINDOWS 2000 operating system.
As standard, your computer has one hard disk drive, one drive for 1.44 MB diskettes and one CD-ROM
drive. An CD reader/writer is installed.
☞
☞
Do make sure, though, that you receive your LSM-FCS system with the operating system
installed, with initialization and start files set up and with the LSM-FCS 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 a diskette or CD-ROM.
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.
Never turn your computer off before you have exited the LSM-FCS program and run down the
WINDOWS NT operating system. Otherwise, the program and/or data files may get lost.
When handling discs of the CD reader/writer, 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.
B 45-0006 e
12/02
1-5
Carl Zeiss
1.5
NOTES ON DEVICE SAFETY
Notes on Care, Maintenance and Service
LSM-FCS
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.
You are well advised to arrange a service agreement with your nearest 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.
Care operations that may be carried out by operating staff are limited to cleaning painted and glass
surfaces.
• 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 hairbrush.
The air filter mat at the bottom of the LSM-FCS Control Unit must be cleaned every six months.
Filter mats can be ordered from our Service Department.
1-6
B 45-0006 e
12/02
NOTES ON DEVICE SAFETY
Notes on Handling the Laser Components
LSM-FCS
1.6
Carl Zeiss
Notes on Handling the Laser Components
The LSM-FCS is a laser hazard class 3 B instrument and is marked as such.
This moderate-risk class embraces medium-power lasers. You must take care not to expose
yourself to the radiation of such lasers. In particular, never look into the laser beam!
The following laser types are currently recommended for use in the LSM-FCS.
1 Ar 351/364 (UV)
2 Ar/ML 458/477/488/514
3 HeNe 543
4 ArKr 488/568
5 HeNe 633
☞
Please contact Carl Zeiss if you intend to use a laser type with a wavelength other than the
ones above.
If used properly, the LSM-FCS will not pose any laser radiation risks for operating staff. The dangerous
laser radiation area is limited to the beam path and to a distance of up to around 10 cm from the
specimen. Nevertheless, you should observe the following warnings:
• If necessary - insofar as specified by law - inform the laser protection officer before
commissioning the laser.
• Always store 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.
• Do not place any reflecting objects into the beam path.
• Never open any covers or panelings.
• 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 screw 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.
B 45-0006 e
12/02
1-7
Carl Zeiss
1.7
NOTES ON DEVICE SAFETY
Warning and Information Labels
LSM-FCS
Warning and Information Labels
The warning and information labels attached on the LSM-FCS 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.
label means: "Do not remove securing screw as otherwise laser beam will escape. For
The
use by service only!"
HA
L
10
0
1:1
RO
ZE
E
IV
CT
JE
OB
R
TO
EC
FL
RE
S
CU
FO
Fig. 1-1
1-8
Warning and information labels on the Axiovert 200 M microscope with the LSM-FCS
scanning module
B 45-0006 e
12/02
NOTES ON DEVICE SAFETY
Warning and Information Labels
LSM-FCS
Fig. 1-2
Carl Zeiss
Warning and information labels on laser components (page 1)
B 45-0006 e
12/02
1-9
Carl Zeiss
NOTES ON DEVICE SAFETY
Warning and Information Labels
CO
HE
RE
LSM-FCS
nT
En
TE
RP
RIS
E
PE
SE
AK
AR
CH
E4
En
J3
RP
RIS
1-10
7
E5
E
CO
HE
RE
Fig. 1-3
um
im
max terre
wasu
.
presp.s.I
60
T
OU
ter
wa
TE
E4
J6
nT
Warning and information labels on laser components (page 2)
B 45-0006 e
12/02
LSM-FCS - SETUP REQUIREMENTS
Contents
LSM-FCS
CHAPTER 2
Carl Zeiss
LSM-FCS - SETUP REQUIREMENTS
CONTENTS
Page
2
LSM-FCS - SETUP REQUIREMENTS................................................................................2-3
2.1
2.1.1
2.1.2
2.1.3
Space Requirements ........................................................................................................2-3
LSM (one microscope, large system table): 300 × 250 cm .................................................2-3
LSM-FCS with Ar UV Laser ...............................................................................................2-4
LSM-FCS prepared for Two Photon Lasers (NLO)...............................................................2-6
2.2
2.2.1
2.2.2
2.2.3
Power Requirements........................................................................................................2-8
Phase 1 (LSM)..................................................................................................................2-9
Phase 2 (LSM, Power 3) ...................................................................................................2-9
Separate Connection .......................................................................................................2-9
2.3
Physical Dimensions ....................................................................................................... 2-10
2.4
Dimension of Shipment Crates ....................................................................................... 2-11
2.5
Environmental Requirements.......................................................................................... 2-11
2.6
Vibrations...................................................................................................................... 2-12
2.7
2.7.1
2.7.2
2.7.3
2.7.4
2.7.5
2.7.6
2.7.7
Laser Specifications........................................................................................................2-12
Coherent Enterprise 653 II: 352, 364 nm, 80 mW, Laser Class 3 B .................................. 2-12
Point Source i-flex 2000: 405 nm, 25 mW, Laser Class 3 B.............................................. 2-12
LASOS LGK 7786 P / Power supply 7460 A: 543 nm, 1 mW, Laser Class 3 B ................... 2-13
LASOS LGK 7628-1: 633 nm, 5 mW, Laser Class 3 B ...................................................... 2-13
LASOS LGK 7812 ML-4 / LGN 7812: 458, 477, 488, 514 nm, 30 mW, Laser Class 3 B..... 2-13
Melles Griot 643-YB-A02 / Power supply 171B: 488, 568 nm, 30 mW, Laser Class 3 B.... 2-13
AOTF............................................................................................................................. 2-13
2.8
Microscopes .................................................................................................................. 2-14
2.9
Scanning Module........................................................................................................... 2-15
2.10
Laser Module VIS (405, 458, 477, 488, 514, 543, 633 nm)............................................. 2-16
2.11
Laser Module UV (351, 364 nm) .................................................................................... 2-16
2.12
System Overview LSM 510 META................................................................................... 2-17
2.13
System Overview LSM 510 META - NLO ......................................................................... 2-19
B 45-0006 e 12/02
2-1
Carl Zeiss
2-2
LSM-FCS - SETUP REQUIREMENTS
Contents
LSM-FCS
B 45-0006 e 12/02
LSM-FCS - SETUP REQUIREMENTS
Space Requirements
LSM-FCS
2
LSM-FCS - SETUP REQUIREMENTS
2.1
Space Requirements
2.1.1
LSM (one microscope, large system table): 300 × 250 cm
Carl Zeiss
Power Plug
System rack
500
1000
2500
Large system table
800
1500
3000
Fig. 2-1
The system rack contains the laser module (Helium-Neon laser 543 nm, 633 nm and Argon ion laser), the
power supply for the Argon ion laser, for HBO lamp and halogen lamp, the electronic control unit (ECU)
and the MCU28 unit (if a motorized XY stage is applied).
B 45-0006 e 12/02
2-3
LSM-FCS - SETUP REQUIREMENTS
Space Requirements
Carl Zeiss
2.1.2
☞
LSM-FCS
LSM-FCS 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
One microscope:
Power Plugs
To Cooling Unit
LSM
System
Rack I
UV
System
Rack II
1000
500
500
3000
Large system table
800
1500
3000
Fig. 2-2
2-4
B 45-0006 e 12/02
LSM-FCS - SETUP REQUIREMENTS
Space Requirements
LSM-FCS
Carl Zeiss
Two microscopes:
Power Plugs
To Cooling Unit
LSM
System
Rack I
UV
System
Rack II
1000
500
500
3000
Small
system table
Large system table
800
800
650
1500
3500
Fig. 2-3
The system rack I contains the VIS laser module (Helium-Neon laser 543 nm, 633 nm and Argon ion
laser) and the Argon UV laser module. The system rack II contains the power supplies for lasers, for HBO
and halogen lamps, the electronic control unit (ECU) and the MCU28 unit (if a motorized XY stage is
applied).
B 45-0006 e 12/02
2-5
LSM-FCS - SETUP REQUIREMENTS
Space Requirements
Carl Zeiss
LSM-FCS
2.1.3
LSM-FCS prepared for Two Photon Lasers (NLO)
2.1.3.1
Coherent "Mira 900" Direct-coupling with Inverted Stand
(Upright Stand also possible)
Power Plug LM VIS
Power Supplies Cooling Unit
System
Rack
500
1000
1400
Large NLO system table
2000
PC table
1800
800
1200
4000
Fig. 2-4
2.1.3.2
Spectra Physics "MaiTai" Direct-coupling with Upright Stand
(Upright Stand also possible)
Power Plug LM VIS
1000
Cooling Unit
Power Supply
System
Rack
500
1400
2500
Small NLO
system table
PC table
1200
800
1200
3000
Fig. 2-5
2-6
B 45-0006 e 12/02
LSM-FCS - SETUP REQUIREMENTS
Space Requirements
LSM-FCS
2.1.3.3
Carl Zeiss
Coherent "Mira" Fiber-coupling with Inverted Stand (Upright Stand also possible)
Power Plug LM VIS
1400
LSM
System
Rack
1000
Large NLO
system table
1800
500
2500
Large system table
500
1500
3500
Fig. 2-6
2.1.3.4
Coherent "Chameleon" Direct-coupling with Upright Stand
(Inverted Stand also possible)
Power Plug LM VIS
1000
Cooling Unit
Power Supply
System
Rack
500
1400
2500
Small NLO
system table
PC table
1200
800
1200
3000
Fig. 2-7
B 45-0006 e 12/02
2-7
LSM-FCS - SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
2.2
☞
LSM-FCS
Power Requirements
The LSM-FCS comes with a mains power supply cord and plug, either CEE red (230 V, 16 A,
3 phases), or CEE yellow (115 V, 32 A, 3 phases), and with the matching mains socket outlet.
Line voltage
230 V AC: 220...240 V AC (±10 %)
115 V AC: 100...125 V AC (±10 %)
Line frequency
50...60 Hz
50...60 Hz
2 phases at 16 A
2 phases at 25 A
Phase 1 = 1.8 kVA max.
Phase 1 = 1.8 kVA max.
Phase 2 = 2 kVA max.
Phase 2 = 2 kVA max.
− Power consumption
2000 VA per phase
2000 VA per phase
− Power plug
CEE red (230 V, 16 A):
3 phases+N+PE, phases 1 and 2
connected
CEE yellow (115 V, 32 A):
3 phases+N+PE, phases 1 and 2
connected
208...240 V AC
208...240 VAC
(±10 %) 50 / 60 Hz
(±10 %) 50 / 60 Hz
1 phase at 63 A
1 phase at:
LSM incl. VIS laser
− Max. current
Argon UV laser
- Line Voltage
− Max. current
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.
208 V: 34 Amps
230 V: 31 Amps
240 V: 29 Amps
− Power consumption
7000 VA
7000 VA
Class of protection
I
I
Type of protection
IP 20
IP 20
Overvoltage category
II
II
Pollution degree
2
2
☞
2-8
If the line voltage in your country is 115 V AC, you need to order an additional 2.5 kW stepup-transformer, part no. 234.366, to be able to run the ArKr laser. Reason: The ArKr laser
requires a 220 V input.
B 45-0006 e 12/02
LSM-FCS - SETUP REQUIREMENTS
Power Requirements
LSM-FCS
Carl Zeiss
Power distribution inside the Laser Module VIS:
Fig. 2-8
2.2.1
Phase 1 (LSM)
feeds the following units:
2.2.2
Phase 2 (LSM, Power 3)
feeds the following units:
2.2.3
Laser Module
HeNe 2×
via Power 1 (5-socket adapter)
Computer + monitor
Microscope
MCU28
Scanning Module
via Power 2:
HAL lamp
HBO lamp
Ar laser
or ArKr laser
2000 VA
2000 VA
Separate Connection
Ar laser (UV)
B 45-0006 e 12/02
7000 VA
2-9
LSM-FCS - SETUP REQUIREMENTS
Physical Dimensions
Carl Zeiss
2.3
Physical Dimensions
Large system table
Small system table
Active anti-vibration table
Active anti-vibration table (NLO)
For Mai Tai Laser or Chameleon
Active anti-vibration table (NLO)
For Mira or Tsunami Laser
Scanning Module LSM 510
Scanning Module LSM 510 META
Microscope
Laser Module, VIS(ible light)
Laser Module, UV
Electronics box
Power supply for Ar, ArKr
Power supply for Ar (UV)
Cooling unit for Ar (UV)
Water hose for Ar (UV)
Fiber optic cable, VIS(ible)
Fiber optic cable, UV
Cables
SCSI cable
2-10
LSM-FCS
Length (cm)
Width (cm)
Height (cm)
Weight (kg)
150
65
75
120
80
80
75
140
78
78
75
75
100
60
125
200
180
140
75
400
25
28
50
90
140
50
30
50
80
700
200
200
250
200
20
27
35
40
20
30
30
50
45
25
30.5
50
50
20
30
20
30
50
15
13
20
60
60
10
10
30
30
B 45-0006 e 12/02
LSM-FCS - SETUP REQUIREMENTS
Dimensions ... / Environmental Requirements
LSM-FCS
2.4
Carl Zeiss
Dimension of Shipment Crates
Crate containing
Length (cm)
Width (cm)
Large system table
160
85
95
120
Small system table
90
75
80
80
Active anti-vibration table
145
Active anti-vibration table
145
(NLO)
For Mai Tai Laser or Chameleon
Active anti-vibration table
200
(NLO)
For Mira or Tsunami Laser
115
160
115
110
150
330
160
110
460
LSM
190
85
120
350
Monitor, computer
120
80
90
80
UV laser unit
125
55
50
100
UV cooling unit
120
60
90
50
META scan head
52
47
47
13
META upgrade kit
64.5
60.5
42.5
20
2.5
Height (cm)
Weight (kg)
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
B 45-0006 e 12/02
2-11
LSM-FCS - SETUP REQUIREMENTS
Vibrations / Laser Specifications
Carl Zeiss
2.6
LSM-FCS
Vibrations
Vibrations under operation conditions
(with system table)
5 µm pp at 5 Hz
Shipping shock (LSM 510 box)
3g
10 µm pp at 10 Hz
10 µm pp at 20 Hz
2.7
Laser Specifications
2.7.1
Coherent Enterprise 653 II: 352, 364 nm, 80 mW, Laser Class 3 B
Line voltage
208...240 V
Line frequency
50...60 Hz
Max. current
1 phase at:
208 V: 34 Amps
230 V: 31 Amps
240 V: 29 Amps
Power consumption
7000 VA
With heat exchanger LP5:
Water flow
8.0 l/min (max. 16 l/min)
Water pressure
1.4...4.2 kg/cm²
Water temperature
10...60 °C at 8.0 l/min
2.7.2
Point Source i-flex 2000: 405 nm, 25 mW, Laser Class 3 B
Line voltage
100...240 V
Line frequency
50...60 Hz
Power consumption
30 VA
2-12
B 45-0006 e 12/02
LSM-FCS - SETUP REQUIREMENTS
Laser Specifications
LSM-FCS
2.7.3
Carl Zeiss
LASOS LGK 7786 P / Power supply 7460 A: 543 nm, 1 mW, Laser Class 3 B
Line voltage
115/230 V with factory setting
Line frequency
50...60 Hz
Power consumption
20 VA
2.7.4
LASOS LGK 7628-1: 633 nm, 5 mW, Laser Class 3 B
Line voltage
100...240 V with factory setting
Line frequency
50...60 Hz
Power consumption
20 VA
LASOS LGK 7812 ML-4 / LGN 7812: 458, 477, 488, 514 nm, 30 mW, Laser Class 3 B
Line voltage
100...240 V with factory setting
Line frequency
50...60 Hz
Max. current
1 phases at 25 A
Power consumption
2000 VA
Cooling fan
on top of laser head
2.7.6
Melles Griot 643-YB-A02 / Power supply 171B: 488, 568 nm, 30 mW, Laser Class 3 B
Line voltage
100...240 V with factory setting
Line frequency
50...60 Hz
Max. current
1 phase at 16 A
Power consumption
2000 VA
2.7.7
☞
AOTF
In the unlikely case of complete utilization of the acousto-optical tunable filter (100 % intensity
of all AOTF-supported lines) the tolerable limits of the EMV regulations could be slightly
exceeded in the MHz range.
B 45-0006 e 12/02
2-13
LSM-FCS - SETUP REQUIREMENTS
Microscopes
Carl Zeiss
2.8
LSM-FCS
Microscopes
Inverted Axiovert 200 M BP or SP
All Zeiss ICS objectives and accessories can be accommodated.
Z motor
DC servomotor, opto-electronically coded
(Axioplan 2 imaging MOT,
Axiovert 200 M BP or SP)
100 nm (Axioskop 2 FS MOT)
Least Z interval: 50 nm
HRZ 200
Galvanometer-driven precision focusing stage
Max. travel 200 µm; resolution 6 nm; accuracy 40 nm
Allows continuous Z-scan at up to 10 Hz
Piezo Objective focus
Piezo-driven single objective drive
Max. travel 100 µm; resolution 5 nm
Allows continuous Z-scan at up to 20 Hz
2-14
B 45-0006 e 12/02
LSM-FCS - SETUP REQUIREMENTS
Scanning Module
LSM-FCS
2.9
Carl Zeiss
Scanning Module
2 individually driven galvanometric scanners
Scanning speed
Up to ~5 frames/sec (512 × 512 pixels)
Field resolution
Max. 2048 × 2048 pixels (individually adjustable for each axis)
Field of view
10 × 10 mm² with a 1.25× objective
Zoom
1× ... 40×, continuous control
Channels
a) Up to 4 channels simultaneously or
b) 3 traditional confocal channels and 1 META channel
4 confocal reflection/fluorescence channels (PMT) or 3 PMT and 1 META
1 transmitted light channel (PMT) and 3 NDD or 4 NDD
1 reference monitor diode
Cooled PMTs (option, forthcoming)
Fiber-optic adaptation of external detectors (option, forthcoming)
Dynamic range
12-bit DAC for each detection channel
Pinholes
4 individual variable pinholes (one per confocal channel)
Computer controlled automatic alignment
B 45-0006 e 12/02
2-15
LSM-FCS - SETUP REQUIREMENTS
Laser Module VIS / Laser Module UV
Carl Zeiss
2.10
LSM-FCS
Laser Module VIS (405, 458, 477, 488, 514, 543, 633 nm)
Single-mode polarization preserving fiber
Laser beam attenuation for all lasers by VIS-AOTF
HeNe laser (543 nm, 1 mW)
HeNe laser (633 nm, 5 mW)
Diode laser (405 nm, 25 mW)
Ar laser (458, 477, 488, 514 nm, 30 mW)
ArKr laser (488, 568 nm, 30 mW)
Fuses and automatic circuit breakers
for 230 V:
G-type fuse 5 × 20 mm; slow-blow 3.15 A / H / 250 V, acc. to IEC 127
2 circuit breakers; C 10 A
for 110 V:
G-type fuse 5 × 20 mm; slow-blow 3.15 A / H / 250 V, acc. to IEC 127
Circuit breaker; B 25 A
Circuit breaker; C 25 A
Circuit breaker; B 16 A
Circuit breaker; B 10 A
2.11
Laser Module UV (351, 364 nm)
Single-mode polarization preserving fiber
Laser beam attenuation for all lasers by UV-AOTF
Ar laser (351, 364 nm, 80 mW)
2-16
B 45-0006 e 12/02
LSM-FCS - SETUP REQUIREMENTS
System Overview LSM 510 META
LSM-FCS
2.12
Carl Zeiss
System Overview LSM 510 META
B 45-0006 e 12/02
2-17
Carl Zeiss
2-18
LSM-FCS - SETUP REQUIREMENTS
System Overview LSM 510 META
LSM-FCS
B 45-0006 e 12/02
LSM-FCS - SETUP REQUIREMENTS
System Overview LSM 510 META - NLO
LSM-FCS
2.13
Carl Zeiss
System Overview LSM 510 META - NLO
B 45-0006 e 12/02
2-19
Carl Zeiss
2-20
LSM-FCS - SETUP REQUIREMENTS
System Overview LSM 510 META - NLO
LSM-FCS
B 45-0006 e 12/02
INTRODUCTION TO LASER SCANNING MICROSCOPY
Contents
LSM-FCS
CHAPTER 3
Carl Zeiss
INTRODUCTION TO
LASER SCANNING MICROSCOPY
CONTENTS
Page
3
INTRODUCTION TO LASER SCANNING MICROSCOPY .................................................3-3
3.1
Principle of Laser Scanning Microscopy.............................................................................3-3
3.2
Three-Dimensional Presentations of LSM Image Stacks .....................................................3-4
3.3
Optical Diagram of the LSM-FCS (Schematic)....................................................................3-6
3.4
3.4.1
3.4.2
3.4.3
Performance Features of the LSM-FCS..............................................................................3-7
Optical and Mechanical Aspects .......................................................................................3-7
Microscope Equipment of the LSM-FCS System ................................................................3-8
Computer Hardware and Software ................................................................................ 3-10
B 45-0006 e
12/02
3-1
Carl Zeiss
3-2
INTRODUCTION TO LASER SCANNING MICROSCOPY
Contents
LSM-FCS
B 45-0006 e
12/02
INTRODUCTION TO LASER SCANNING MICROSCOPY
Principle of Laser Scanning Microscopy
LSM-FCS
3
INTRODUCTION TO LASER SCANNING MICROSCOPY
3.1
Principle of Laser Scanning Microscopy
Carl Zeiss
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-FCS 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
in-focus 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 pinhole diaphragm
by the same objective and a tube lens. The
focus inside the specimen and the pinhole 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 pinhole and be
registered by a detector. Unwanted light
coming from other specimen areas is focused
outside the pinhole, which passes only a small
fraction of it. The smaller the pinhole, 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.
Fig 3-1
B 45-0006 e
12/02
Principle of confocal imaging
3-3
Carl Zeiss
INTRODUCTION TO LASER SCANNING MICROSCOPY
Three-Dimensional Presentation of LSM Image Stacks
LSM-FCS
• 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 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-FCS 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
Three-Dimensional Presentations of LSM Image Stacks
One of the advantages of the LSM-FCS 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
3-4
B 45-0006 e
12/02
INTRODUCTION TO LASER SCANNING MICROSCOPY
Three-Dimensional Presentation of LSM Image Stacks
LSM-FCS
Carl Zeiss
• 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)
B 45-0006 e
12/02
3-5
Carl Zeiss
INTRODUCTION TO LASER SCANNING MICROSCOPY
Optical Diagram of the LSM-FCS
3.3
Optical Diagram of the LSM-FCS (Schematic)
Fig. 3-2
Optical path, schematic (4-channel configuration)
AOTF
APD
DBC
DBS
EF
HAL
HBO
Acousto Optical Tunable Filter
Avalanche photodiode
Dichroic Beam Combiner
Dichroic Beam Splitter
Emission Filter
Halogen Lamp
Mercury Vapor Short-Arc Lamp
LSF
MDBS
NDF
VP
PMT
T-PMT
LSM-FCS
Line Selection Filter
Main Dichroic Beam Splitter
Neutral Density Filter
Variable Pinhole
Photomultiplier
Transmission-Photomultiplier
The diagram above is a schematic representation of the LSM-FCS 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 XY scanner onto a main dichroic
beam splitter (MDBS), which separates the emissions from the excitation light. The fluorescences are
separated from each other by a series of dichroic beam splitters (DBS1 ... maximally DBS4) and directed
to individual photomultipliers (PMT1 ... maximally PMT4) or avalanche photodiodes (APD1 ... maximally
APD2).
3-6
B 45-0006 e
12/02
INTRODUCTION TO LASER SCANNING MICROSCOPY
Performance Features of the LSM-FCS
LSM-FCS
3.4
Performance Features of the LSM-FCS
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
BP or SP) microscope in less than three minutes. On the Axiovert, the LSM scanning module may be
mounted to the base port directly below the microscope and the ConfoCor 2 detection head to the side
port.
The spectral range available extends from the UV to the IR region (for Scan Head).
For the VIS (visible-light) Laser Module, the user can select from up to five lasers with wavelengths of
633, 568, 543, 514, 488, 477, 458 and 413 nm. The UV Laser Module provides wavelengths of 351 and
364 nm. Coupling of the laser light is through polarization-preserving single-mode optical fibers. One
variable beam collimator each for the UV and visible ranges provides optimum adaptation of the
respective laser wavelength to the objective used and, thus, optimum correction for Z aberrations.
Acousto-optical tunable filters (AOTF) adjust the necessary brightness for all desired laser lines within
microseconds.
A monitor diode permanently registers the laser output; it can be used for the on-line checking of the
intensity of the exciting light. This check is also possible selectively for the different wavelengths if a line
selection filter is inserted.
The four simultaneous image acquisition channels, usable for reflection or fluorescence, and an
additional transmitted-light channel are ideal for the investigation of multiple fluorescence specimens.
Separately in each of the four channels, the diameters of the pinholes and their XY positions can be
optimized, and the desired emission filter placed into the beam path, by servo-motor control. In the case
of pinhole VP1, this adjustment also includes positioning along Z. 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.
The microscope's transmitted-light channel is equipped with a photomultiplier, too. It is therefore
possible to superimpose a multiple fluorescence image on a brightfield, differential interference or phase
image.
A fiber-optic cable connection to external special detectors, such as cooled PMTs or spectrometers, is
under development.
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-FCS 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 two-mirror scanner system, controlled by a digital signal processor (DSP), offers several advantages.
The large deflection angle of the scanning mirrors allows a wide area to be scanned. With a 1.25×
objective, the object area scanned is 10 × 10 mm².
The scanning field size can be freely selected between 4 × 1 and 2048 × 2048 pixels.
B 45-0006 e
12/02
3-7
Carl Zeiss
INTRODUCTION TO LASER SCANNING MICROSCOPY
Performance Features of the LSM-FCS
LSM-FCS
It is possible to rotate the XY scanning field through 360° and carry out 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 approx. 5 frames/sec (at 512 ×
512 pixels); if two different laser wavelengths are used for the two scanning directions (wavelength 1 for
left-to-right, and wavelength 2 for right-to-left scanning), two fluorochrome dyes can be viewed and
documented in a quasi-simultaneous mode. This will absolutely prevent "bleeding".
3.4.2
Microscope Equipment of the LSM-FCS System
The LSM-FCS system is equipped with the Axiovert 200 M microscope (with baseport and sideport.
Referring to the delivered operating manual "Axiovert 200 M" only differences to this manual will be
explained.
(1) Stand
a) The motorized objective nosepiece 5× H DIC is firmly fixed to the stand, where no operating elements
can be found for the nosepiece. Operation will be done LSM-FCS 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 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.
3-8
B 45-0006 e
12/02
INTRODUCTION TO LASER SCANNING MICROSCOPY
Performance Features of the LSM-FCS
LSM-FCS
Carl Zeiss
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 510 scanning module can be mounted to the special interface port. Different camera
systems can be adapted to the TV interface using the TV adapters 452982/83/92/94/95/97/98-0000-000.
(2) Specimen stages
a) Mechanical stage
The stage must be mounted with the coaxial drive on the right side of the stand.
b) Scanning stage
c) HRZ 200 fine focusing stage
(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 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-FCS software controlled switching
mirror fully motorized. Alternatively the light is directed to the LSM-FCS 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 operating manual.
(4) Reflected light fluorescence
All Axiovert fluorescence accessories exceptional the reflector slider can be used.
Further information you will find in the Axiovert operation manual.
B 45-0006 e
12/02
3-9
INTRODUCTION TO LASER SCANNING MICROSCOPY
Performance Features of the LSM-FCS
Carl Zeiss
LSM-FCS
(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 an integrated SLR-port, but microscope cameras, as described in the Axiovert operation
manual, can be used.
(7) TV adaptation
The TV port aside and the tubes can be used as described in the Axiovert operation manual.
The TV interface side port or base port can be used with TV adapters 44 or LSM adapters.
3.4.3
Computer Hardware and Software
The LSM-FCS is controlled through a standard high-end Pentium PC. Linking with the electronic control
system is via an ultrafast SCSi interface. The PC comes with the 32-bit WINDOWS NT 4.0 or WINDOWS
2000 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-FCS has two levels. On the simple operator interface level, a result will be
achieved after a few prompts; graphical prompting of the user in conjunction with automatic setting of
many parameters is an ideal tool for daily routine jobs. The expert level 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.
3-10
B 45-0006 e
12/02
QUICKSTART
Contents
LSM-FCS
CHAPTER 4
Carl Zeiss
QUICKSTART
CONTENTS
Page
4
QUICKSTART ...............................................................................................................4-3
4.1
4.1.1
4.1.2
4.1.3
4.1.4
4.1.5
Purpose of this Section and other Operating Manuals.....................................................4-3
Software .......................................................................................................................4-3
Windows and Window Elements ...................................................................................4-4
Convention for the Text in this Manual ..........................................................................4-5
Backup..........................................................................................................................4-6
Software Operation .......................................................................................................4-6
4.2
4.2.1
4.2.2
4.2.3
Switching on the System ...............................................................................................4-7
Log on to WINDOWS NT ...............................................................................................4-8
Switching on the Enterprise UV Laser ........................................................................... 4-10
Starting the LSM-FCS Program..................................................................................... 4-10
4.3
4.3.1
4.3.2
4.3.3
4.3.4
4.3.5
4.3.6
Quick Start in the LSM Expert Mode ............................................................................ 4-13
Start the Expert Mode ................................................................................................. 4-13
Set the Axiovert 200 M Microscope ............................................................................. 4-14
Turn on the Lasers....................................................................................................... 4-15
Set the Beam Path....................................................................................................... 4-16
Scan an Image............................................................................................................. 4-17
Store the Image........................................................................................................... 4-21
4.4
4.4.1
4.4.2
4.4.2.1
4.4.2.2
4.4.2.3
4.4.2.4
4.4.2.5
4.4.3
4.4.3.1
4.4.3.2
4.4.3.3
Quick Start for FCS Measurements............................................................................... 4-23
Analysis Method.......................................................................................................... 4-23
General Preparations ................................................................................................... 4-23
Setting the Axiovert 200 M Microscope ....................................................................... 4-23
Turning on the Lasers .................................................................................................. 4-25
Selection of a Method ................................................................................................. 4-26
Mounting and Orienting the Sample ............................................................................ 4-27
Running the Measurements ......................................................................................... 4-30
Data Evaluation and Result Presentation....................................................................... 4-31
Display Correlation ...................................................................................................... 4-33
Manual Fit................................................................................................................... 4-33
Data Storage and Export.............................................................................................. 4-34
4.5
4.5.1
4.5.2
4.5.3
Shut-Down Procedure ................................................................................................. 4-35
Exiting the LSM-FCS Program....................................................................................... 4-35
Shut Down the WINDOWS Operating System............................................................... 4-36
Turning Power Off....................................................................................................... 4-37
B 45-0006 e 12/02
4-1
QUICKSTART
Carl Zeiss
4-2
LSM-FCS
B 45-0006 e 12/02
QUICKSTART
Purpose of this Section and other Operating Manuals
LSM-FCS
4
QUICKSTART
4.1
Purpose of this Section and other Operating Manuals
Carl Zeiss
This section describes the operation of the LSM-FCS Combination System exemplified by typical
applications in conjunction with the LSM-FCS software and its graphic user environment.
When starting up and operating the microscope system, mind the operating instruction manual for the
Axiovert 200 M microscope:
–
B 40-080 e
4.1.1
Axiovert 200 M, Operating Manual
Software
The LSM-FCS software, Version 3.2, controls the microscope, the scanning and laser modules, tools
(filters, stand, CLM) and the image acquisition process, and displays and analyzes the images. It is based
on the network-capable graphic 32-bit Microsoft ® WINDOWS NT 4.0 operating system and WINDOWS
2000, respectively.
Portions © Copyright 1996, Microsoft Corporation. All rights reserved.
The installation of the software for the LSM-FCS 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-FCS 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.
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.
B 45-0006 e 12/02
4-3
QUICKSTART
Purpose of this Section and other Operating Manuals
Carl Zeiss
4.1.2
LSM-FCS
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
Expert Mode).
Panel (e.g.: Argon 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.
4-4
B 45-0006 e 12/02
QUICKSTART
Purpose of this Section and other Operating Manuals
LSM-FCS
Window element
Carl Zeiss
Description / Explanation
Check box
– Activates / deactivates setting options.
Button
– Selection / performance of a function via mouse
click.
4.1.3
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.
B 45-0006 e 12/02
4-5
QUICKSTART
Purpose of this Section and other Operating Manuals
Carl Zeiss
4.1.4
LSM-FCS
Backup
System backup
–
A complete backup is contained on the enclosed optical disk.
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
–
Pinhole 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:
–
Parameter file for pinhole setting: AIM \ *.set
–
Parameter file after pinhole adjustment: AIM \ *.adj
–
Scanner files: AIM \ bin \ *.bin
–
Microscope stand database: AIM \ database \ system_configuration_*.mdb
4.1.5
Software Operation
The LSM-FCS 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.
4-6
B 45-0006 e 12/02
QUICKSTART
Switching on the System
LSM-FCS
4.2
Carl Zeiss
Switching on the System
The LSM system is turned on with the REMOTE CONTROL switch. This switches all the system
components on except for the "Enterprise" UV laser.
If the UV laser shall be used, it can be switched on after the start of the WINDOWS ® NT operating
system - but must always be switched on before the LSM-FCS software is started.
If REMOTE CONTROL switch is not used, turn the system on with the "I" button on the laser module;
in addition, the jumper plug supplied must be connected at the POWER REMOTE CONTROL terminal.
• Turn the REMOTE CONTROL switch to "ON"
position (see Fig. 4-1).
–
This switches the entire system on.
–
Microscope and laser will be ready for
operation after a short time.
–
Computer boots up.
–
Computer hardware system test runs.
Drive "A" of the computer must not
contain a diskette.
The monitor shows a dialog box for selection of
the operating system version.
Fig. 4-2
B 45-0006 e 12/02
Fig. 4-1
REMOTE CONTROL switch
Selecting the operating system version
4-7
QUICKSTART
Switching on the System
Carl Zeiss
LSM-FCS
• Confirm the default setting of the "Windows
NT Workstation Version 4.00" by pressing the
Enter key.
Fig. 4-3
–
WINDOWS NT operating system is being
loaded.
–
The Begin Logon window appears on the
screen.
Begin Logon window
4.2.1
Log on to WINDOWS NT
• Press the three keys Ctrl, Alt and Del at the
same time.
–
The Logon Information window appears
on the screen, permitting you to log on to
the WINDOWS NT 4.0 operating system.
• Enter the valid user name into the User name
text box.
Fig. 4-4
4-8
Logon Information window
• Enter your password into the Password text
box.
B 45-0006 e 12/02
QUICKSTART
Switching on the System
LSM-FCS
Carl Zeiss
• After entries, confirm by clicking the OK button or Enter.
–
The WINDOWS NT operating system desktop appears on the screen, showing a number of icons.
Change Filters icon
Stand Select icon
LSM-FCS icon
LSM-5 Image Examiner icon
LSM-5 Image Browser icon
Fig. 4-5
WINDOWS NT operating system desktop
B 45-0006 e 12/02
4-9
QUICKSTART
Switching on the System
Carl Zeiss
4.2.2
LSM-FCS
Switching on the Enterprise UV
Laser
• If the UV laser is required, switch it on via the
toggle switch (4-6/1) of the power supply.
–
It will be ready for operation after a few
seconds.
Only for LSM.
Fig. 4-6
Power supply of UV-Ar laser
4.2.3
Fig. 4-7
Starting the LSM-FCS program
Starting the LSM-FCS Program
The LSM-FCS 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) and no hardware functions are available in
the off-line mode. 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-FCS icon on the desktop of WINDOWS to start the LSM-FCS software
program (see Fig. 4-5).
–
4-10
The LSM-FCS Switchboard menu appears on the screen (see Fig. 4-8).
B 45-0006 e 12/02
QUICKSTART
Switching on the System
LSM-FCS
Fig. 4-8
Carl Zeiss
LSM-FCS Switchboard menu
The LSM-FCS Switchboard menu presents the following items for selection:
–
Scan New Images
Clicking on this button activates the complete LSM hardware (on-line mode).
–
Use Existing Images
This item allows you to process and analyze previously acquired images with the LSM-FCS software. In
this mode, control of the hardware (laser module ...) is not possible (off-line mode).
Please note that the Scan New Images button must be activated before setting up the Routine
Mode or the Expert Mode. Otherwise, the hardware can not be controlled by the LSM-FCS
software.
–
Start Routine Mode
Click on this button if you want to work with pre-configured system settings (typical applications).
In the Routine mode, the FCS measurement functions are not available.
B 45-0006 e 12/02
4-11
QUICKSTART
Switching on the System
Carl Zeiss
–
LSM-FCS
Start Expert Mode
Use of this mode requires to be thoroughly familiar with the exact microscope procedures and
interrelations.
You need to set all parameters and functions upon your own decision; this mode therefore provides
you with the greatest flexibility of operation.
It is also possible, however, to call up stored configurations and to modify the parameters / settings if
necessary.
After the start of the Expert Mode or the
Routine Mode, instrument initialization is
performed and can be monitored in the
Initialization window and interrupted with a click
on the Cancel button, if required.
Fig. 4-9
OFF LINE Initialization window
Depending on the selected option (Scan New
Images or Use existing Images), initialization is
performed in the offline or online mode.
Existing images can only be loaded and processed in the Expert Mode.
If you want to change from the Expert Mode to the Routine Mode and vice versa, close all
the windows first.
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-FCS software.
Don’t switch off the KODAK printer during the scanning process.
4-12
B 45-0006 e 12/02
QUICKSTART
Quick start in the LSM Expert Mode
LSM-FCS
4.3
Carl Zeiss
Quick Start in the LSM Expert Mode
Proceed as follows to generate images in the Expert Mode:
–
start the Expert Mode
–
test / change the microscope setting: objective, fluorescence / attenuation filters, illumination mode,
diaphragms
–
normal setting of the microscope on the specimen with observation in brightfield or fluorescence
contrast (KÖHLER-type illumination)
–
swit to LSM mode
–
setting of lasers: laser type, laser intensity (power)
–
configuration of beam path and channel assignment: tracks, multi-tracks, switching on / off of laser
lines and intensity (excitation)
–
image creation: determine the scan method (line, frame) and scan parameters (image size, scan
speed, pixel depth, scan direction, scan average, zoom, rotation, offset)
–
image optimization and storage
4.3.1
Start the Expert Mode
• Double-click on the LSM-FCS icon on the
WINDOWS NT operating system desktop.
–
The LSM-FCS Switchboard menu appears
on the screen.
• Click on the Scan New Images button and
Start Expert Mode button in the LSM-FCS
Switchboard menu.
–
The LSM-FCS will go through the
initialization and open the Main menu
labeled LSM-FCS Expert Mode. The Main
menu appears on the screen.
Fig. 4-11
B 45-0006 e 12/02
Fig. 4-10
LSM-FCS Switchboard menu
Main menu of LSM-FCS - Expert Mode
4-13
QUICKSTART
Quick Start in the LSM Expert Mode
Carl Zeiss
4.3.2
LSM-FCS
Set the Axiovert 200 M Microscope
This step is used to set:
– microscope objective
–
specimen position
–
specimen focus
• Click on the Acquire button in the toolbar of the Main menu.
• Click on the VIS button in the Acquire subordinate toolbar.
• Click on the Micro button.
–
The Axiovert Control window appears on
the screen.
• Put the specimen on the stage - make sure the
specimen is mounted securely and flat. Use the
supplied Convallaria specimen at first.
You can view the specimen in either fluorescence
(reflected light) or transmitted light.
• To view the specimen in transmitted light, set
the Reflector Turret position to None and
activate the Transmitted Light panel by
clicking on the Transmitted Light button.
Select the On button and control the intensity
by the slider.
• Select an objective with low magnification by
clicking in the Objective panel of the Axiovert
Control window.
• Set the microscope to KÖHLER illumination
manually (see Axiovert 200 M operating
manual).
Fig. 4-12
Axiovert Control window
• Select the specimen area to be examined by
moving the XY-stage and focus exactly on the
selected area.
• Close the Axiovert Control window. Click on
the LSM button.
4-14
B 45-0006 e 12/02
QUICKSTART
Quick Start in the LSM Expert Mode
LSM-FCS
4.3.3
Carl Zeiss
Turn on the Lasers
This step is used to switch the lasers and set the intensity.
• Click on the Laser button in the Acquire
subordinate toolbar of the Main menu.
–
A Laser Control window with a list of
available lasers will appear. Depending on
the lasers available in your system, your
screen may differ from the one displayed.
• Using the mouse, click on the laser(s) featuring
the appropriate wavelength to excite the dyes
used to label your specimen.
• In the case of the argon and UV laser
(Enterprise), click on the Standby button first.
–
Warming Up appears in the line Status.
–
After 2 minutes, when the warming-up
phase is finished, the Ready message
appears.
Fig. 4-13
Laser Control window
• Then click on the On button.
–
The laser is switched on.
• Use Output [%] scrollbar to set the intensity to 50 %.
• In the case of the HeNe laser, click on the On button directly.
• Close the window with the Close button.
• For precise measurements the system should warm up for 2 hours.
• Depending on the lasers available in your system, your screen may differ from the one displayed.
B 45-0006 e 12/02
4-15
Carl Zeiss
QUICKSTART
Quick Start in the LSM Expert Mode
4.3.4
LSM-FCS
Set the Beam Path
This step is used to specify beam path parameters
by using a predefined Track Configuration.
• Click on the Config button in the Acquire
subordinate toolbar of the Main menu.
–
The Configuration Control window
appears on the screen. Your screen may
differ from the one displayed.
• Click on the Single Track button, unless it has
already been activated.
• Click on the Config button
Configuration Control window.
–
Fig. 4-14
Configuration Control window
in
the
The Track Configurations window appears
on the screen.
Stored standard configurations (Tracks) are
available in the Track Configurations window,
which can be used for fast and easy image
acquisition.
• The list of configurations will appear by clicking
button. Choose the FITC/Rhod
on the
configuration from the list.
Fig. 4-15
Track Configurations window
• Click on the Apply button.
All the settings of the selected standard configuration, such as beam path, excitation wavelength and
intensity, AOTF attenuation (Acousto-Optical Tunable Filters), Gain, Offset and Data Depth, are loaded
via the software and displayed in the relevant windows and panels. The Track Configurations window
is closed automatically.
If you click on the Close button, the Track Configurations window will be closed without any
change being made to the Track Configuration.
4-16
B 45-0006 e 12/02
QUICKSTART
Quick Start in the LSM Expert Mode
LSM-FCS
4.3.5
Carl Zeiss
Scan an Image
This step is used to specify parameters and execute
image acquisition.
• Click on the Scan button in the Acquire
subordinate toolbar of the Main menu.
–
The Scan Control window appears on the
screen.
The microscope must be in the LSM mode (press
LSM button and move the relevant slider(s) on the
microscope stand to the LSM position).
On the right-hand side of the Scan Control
window various buttons appear.
We will use the: Find, Single, Cont., (Stop) scan
buttons.
• To scan an XY-image, click on the Frame
button.
• Click on the Find button on the right-hand side
of the Scan Control window.
–
An XY-image with automatically generated
settings for brightness and contrast is
produced.
B 45-0006 e 12/02
Fig. 4-16
Scan Control window
4-17
QUICKSTART
Quick Start in the LSM Expert Mode
Carl Zeiss
LSM-FCS
• A specimen with 2 labels (FITC, Rhod.) with defined channels is easier to view in split screen where
each channel is arranged side by side. You can toggle between xy and Split xy in the Image Display
window.
Fig. 4-17
Image Display window with Split xy mode
The scanned image can now be optimized for contrast, brightness and confocality.
4-18
B 45-0006 e 12/02
LSM-FCS
QUICKSTART
Quick Start in the LSM Expert Mode
Carl Zeiss
Proceed as follows for image optimization:
• Click on the Channels button in the Scan
Control window.
–
The Channel Settings panel and the
Excitation of Track (...) panel are displayed
in the Scan Control window.
• Press the Cont. (continuous scan) button on
the right-hand side of the window. This starts
the continuous image acquisition, which can be
interrupted by pressing the Stop button.
• Under the Channel Settings panel all buttons
for each channel you have set up are displayed.
Click Ch1 (channel 1 of track 1), for example, if
you want to adjust the first image displayed in
the split mode window.
• Use the Pinhole slider to set the pinhole
diameter.
–
The selected pinhole diameter should be
small enough to still allow the Detector Gain
setting and to provide sufficient image
information. 1 Airy unit is a decent value to
obtain a confocal XY-image (use the 1
button).
• If required, adjust the pinhole again (see Main
menu, Maintain subordinate toolbar, Pinhole
button).
Fig. 4-18
Scan Control window
• Use the Detector Gain slider to set image contrast and brightness. This adjustment is very sensitive.
Try using the left and right arrows to make the adjustment instead of dragging the slider bar. Use the
Shift and Crtl keys for changing to coarse and fine steps.
• To adjust the black level (background) use Ampl. Offset.
• Also, try adjusting the microscope by manual focusing. Sometimes you will find that there are other
focal planes within the specimen which are brighter, and therefore the detector gain will need to be
turned down.
• Once you have optimized a particular channel, you can switch to the next channel required and
repeat the optimization.
• As soon as all channels are optimized, click on the Stop button.
B 45-0006 e 12/02
4-19
QUICKSTART
Quick Start in the LSM Expert Mode
Carl Zeiss
LSM-FCS
• To further improve image quality you can slow
down the scan speed, allowing more photons
to integrate on the detector, or apply image
averaging to remove random noise, or a
combination of both. These adjustments are
made by clicking on the Mode button on the
Scan Control window.
• Set the Scan Speed in the Speed panel.
• Select the Line or Frame average Mode, the
Mean or Sum average Method and the
Number of averages in the Depth, Scan
Direction & Scan Average panel accordingly
by observing your image. The setting average of
16 (Number 16) should improve signal / noise
dramatically; however, the image acquisition
rate will be slower.
• When the image optimization has been
finished, click on the Single button to generate
a single image of the specimen.
Fig. 4-19
Scan Control window
• If your specimen is sensitive to photobleaching,
you can attenuate the laser illumination by
clicking on the Channels button in the Scan
Control window. At the bottom of the window
you can set the percentage of laser power
(Transmission [%])
for
each
excitation
wavelength. You will probably have to increase
the Detector Gain if you decrease the laser
power. This setting controls the transmission
degree of the Acousto-Optical Tunable Filters
(AOTF).
Try to use as low laser light intensity as possible to prevent sample bleaching. For that purpose
increase the detector gain to a value of approximately 800 V.
4-20
B 45-0006 e 12/02
QUICKSTART
Quick Start in the LSM Expert Mode
LSM-FCS
4.3.6
Carl Zeiss
Store the Image
This step is used to activate an existing database or to create a new database in which the acquired
image is stored with the used settings and comments.
• To save the image, click on the Save As button on the right-hand side of the Image Display
window.
–
The Save Image and Parameter As window appears on the screen.
Fig. 4-20
Save Image and Parameter As window
Proceed as follows to store images in an existing database:
• To select the required database, click on its name (blue cursor bar) in the Database (MDB) list box of
the Save Image and Parameter As window.
• Enter a suitable name for the image in the Name input box. If required, enter further details on the
image in the Description and Notes input boxes.
• Click on the OK button to add the image to the selected database.
B 45-0006 e 12/02
4-21
QUICKSTART
Quick Start in the LSM Expert Mode
Carl Zeiss
LSM-FCS
Proceed as follows to store images in a new
database:
• Click on the New MDB button in the Save
Image and Parameter As window.
–
The Create New Database window will be
opened.
• Enter a database name in the File name input
box. The name can consist of as many
characters as you like.
Fig. 4-21
Create New Database window
• Before clicking on the Create button in the
Create New Database window, set the
location in which the database will be created
by selecting the drive in the Create in list box,
and double-click on the required folder icon in
the list displayed.
• Click on Create.
–
The new database will be created and displayed on the screen.
• As described above, enter a name and, if required, further image details in the Save Image and
Parameter As window.
• Click on the OK button.
–
4-22
The image will be stored and included in the new database.
B 45-0006 e 12/02
QUICKSTART
Quick Start for FCS Measurements
LSM-FCS
4.4
Quick Start for FCS Measurements
4.4.1
Analysis Method
Carl Zeiss
Central paradigm of the user interface conception of the FCS Measurements is the analysis method.
The analysis method is a complete description of the measurement and data evaluation method. It
includes instrument settings such as choice of laser lines and their respective intensities, choice of filters
and beam splitters, pinhole settings, measurement time(s), repeat counts and so on. Also part of the
measurement method are preset parameters for the fitting procedure.
Once a method is worked out and filed, it can be easily reused. We also provide a convenient way to
develop and optimize methods.
4.4.2
General Preparations
After clicking the Start Expert Mode button in the LSM-FCS Switchboard menu, the instrument is
running and the Main menu appears (Fig. 4-22). From this moment the instrument can be almost
entirely controlled by software.
Fig. 4-22
4.4.2.1
Main menu with activated Analyse FCS subordinate toolbar
Setting the Axiovert 200 M Microscope
This step is used to set:
– microscope objective
–
specimen position
–
specimen focus
B 45-0006 e 12/02
4-23
QUICKSTART
Quick Start for FCS Measurements
Carl Zeiss
LSM-FCS
• The first step is to set up the microscope for FCS measurements. This is done in the Axiovert Control
window (Fig. 4-24) or manually at the microscope stand. This window is reached by clicking the
Micro button in the Config FCS subordinate toolbar of the Main menu (Fig. 4-23).
Fig. 4-23
Main menu with activated Config FCS subordinate toolbar
To set-up the Microscope:
• Swing the required objective for FCS
measurements into the working position. This is
performed by clicking in the Objective panel of
the Axiovert Control window.
We recommend to use the C-Apochromat 40x/1.2 W
on account of its optimized optics.
• Set the Reflector Turret position to None.
• Activate the Transmitted Light panel by
clicking on the Transmitted Light button and
switch off the transmitted light attachment.
• Close the window using the Close button.
• Click on the FCS button on the right-hand side
of the Analyse FCS subordinate toolbar in the
Main menu (Fig. 4-22) to select the FCS
detection head.
Fig. 4-24
4-24
Axiovert Control window
B 45-0006 e 12/02
QUICKSTART
Quick Start for FCS Measurements
LSM-FCS
4.4.2.2
Carl Zeiss
Turning on the Lasers
This step is used to switch the lasers and set the intensity.
• Click on the Laser button in the Config FCS
subordinate toolbar of the Main menu
(Fig. 4-23).
–
You will see a Laser Control window with a
list of available lasers (Fig. 4-25). Your screen
may differ from the one displayed.
• Click on the laser(s) featuring the appropriate
wavelength to excite the dyes in your sample.
• In case of the argon laser, click on the Standby
button first.
–
Warming up appears in the line status.
–
After 2 minutes when the warming-up
phase is finished, the Ready message
appears.
Fig. 4-25
Laser Control
• Then click on the On button.
–
The laser is switched on.
• Use Output [%] scrollbar to set the intensity to approximately 6 to 8 ampere
• In case of the HeNe laser, click on the On button directly.
The Enterprise laser is not available for FCS measurements.
• Close the window using the Close button.
B 45-0006 e 12/02
4-25
QUICKSTART
Quick Start for FCS Measurements
Carl Zeiss
4.4.2.3
LSM-FCS
Selection of a Method
This step is used to select a defined method with prepared parameters.
• Click the Method button in the Analyse FCS toolbar subordinate of the Main menu (Fig. 4-22). The
Method Selection ... window will appear (Fig. 4-26).
Fig. 4-26
Method Selection ... window
• Alternatively you can select the analysis method by a single click – which will highlight the selected
method – and a subsequent click on the Select button. The window will then be closed
automatically.
4-26
B 45-0006 e 12/02
QUICKSTART
Quick Start for FCS Measurements
LSM-FCS
4.4.2.4
Mounting
Sample
and
Orienting
Carl Zeiss
the
This step is used to mount a sample on the
motorized stage and to orient the sample with
respect to the center of the used objective (or with
respect to the optical axis).
• This is done with the Select Carrier button of
the Analyse FCS toolbar subordinate of the
Main menu (Fig. 4-22).
–
The sample select window (Carrier Position
window) pops up (Fig. 4-27).
Before the sample can be positioned,
make sure that the microscope stand is
set up for FCS measurements and that
the appropriate laser(s) is (are) switched
on.
(1)
Selecting Carrier
• Select the sample carrier you are currently using
in the Sample Carrier selection box of the
Carrier Position window (Fig. 4-27).
B 45-0006 e 12/02
Fig. 4-27
Carrier Position window
4-27
Carl Zeiss
QUICKSTART
Quick Start for FCS Measurements
(2)
LSM-FCS
Orienting the Sample in x and y
• Now click on the Orientation button in the
Carrier Position window (Fig. 4-28).
• Select one of the chambers of the sample for
orientation in x and y via mouse click.
–
The selected chamber is displayed in yellow
with a black frame.
• Position the center of the selected chamber
over the center of the objective by clicking the
arrow keys with the mouse or by using the
control panel's joystick (if available).
• When you have finished, click the Orientation
button once more. The stage is now oriented.
• Now you can move any chamber into the
measurement position by clicking the
appropriate field on the sample carrier.
Fig. 4-28
Sample drives unlocked
The selected chamber is then approached
automatically via the motorized microscope stage.
When the defined position has been reached, the
chamber is displayed in the Carrier Position
window in yellow with a black frame.
If the required chamber is approached by
activation of the arrow keys, the relevant
chamber is displayed in white with a
black frame, since normally the defined
position cannot be set in this way.
4-28
B 45-0006 e 12/02
QUICKSTART
Quick Start for FCS Measurements
LSM-FCS
(3)
Carl Zeiss
Positioning the Focus in Z for Solutions
Since the sample is just a solution in many cases and has no structure, we cannot “focus“ the instrument
by looking at sample features. Cover slip reflection has to be used instead to find the glass- / solution
interface.
Proceed as follows:
• Make sure that the front lens of the water
immersion objective is wetted by a drop of
water. Use fluorescence-free double-distilled
water, since otherwise the immersion water will
cause background fluorescence and deteriorate
the correlation signal.
• The camera window shows interference fringes
caused by the laser light (Fig. 4-29).
Fig. 4-29
Camera image far away from the
lower cover slip surface
Fig. 4-30
Camera image near the lower cover
slip surface
Fig. 4-31
Lower cover slip surface
• Move the objective cautiously upward by
turning the Z focus knob of the microscope
stand. A lens moving upward will be indicated
by increasing numbers in the position field to
the left of the camera window.
• If the focus position approaches the lower cover
slip surface, the camera window looks similar to
(Fig. 4-30).
• Continue cautiously moving upwards. If the
camera image looks like (Fig. 4-31), you are
close to the lower cover slip surface.
• Having cautiously moved over a short distance,
the cover slip reflection should look as shown in
(Fig. 4-32). The upper cover slip surface has
been found.
• Store the reached position using the Store
button.
All positions
coordinates.
B 45-0006 e 12/02
are
given
in
relative
4-29
Carl Zeiss
Fig. 4-32
QUICKSTART
Quick Start for FCS Measurements
Upper cover slip surface
LSM-FCS
•
Now you can position the detection volume
into the sample in a well-defined way: if you
use one of the C-Apochromat water
immersion objectives, position the detection
volume 150-200 µm deep into the sample to
get rid of disturbing interface effects. The
easiest way of doing this is by clicking the
+200 µm button.
•
Close the Carrier Position window by clicking
the Close button.
4.4.2.5
Running the Measurements
Now measurements can be run.
• Click the Measure Method button in the
Analyse FCS toolbar subordinate of the Main
menu (Fig. 4-22). The Method Measurement
... window will appear on the screen (Fig. 4-33).
The name of the currently used method will
appear in the status bar of the window.
• Give your measurement a Task Name by
entering a string in the Task Name input box.
Under this name, all the measurement data will
be stored, regardless of whether the
measurement covers only a single sample or a
whole series. It is recommended to choose a
name which is easy to remember.
The Comment input box allows you to store a
comment on the measurement you are
performing.
The samples to be measured can be selected by
mouse in the schematic drawing of the sample
carrier. Activated chambers will be highlighted in
the drawing.
Fig. 4-33
4-30
Method Measurement ... window
The buttons Select All and Clear All select or
deselect all chambers.
B 45-0006 e 12/02
QUICKSTART
Quick Start for FCS Measurements
LSM-FCS
Carl Zeiss
• Individual chambers can be selected / deselected by clicking on them with the mouse.
• Alternatively, a whole block of chambers can be selected / deselected by drawing a frame which
contains the centers of the selected or deselected chambers.
If multiple samples are selected, the work direction (Row, Column, Meander) can be chosen as well.
• Click on the Start button on the right-hand side of the Method Measurement ... window.
4.4.3
Data Evaluation and Result Presentation
• When you click Start either from the Method Measurement ... window, the Data Evaluation
window will appear (Fig. 4-34).
Fig. 4-34
Data Evaluation window
Up to four windows with different graphs become visible in the upper left part of the window.
The Count Rate diagram:
–
Here the count rate(s) vs. running time is displayed. If a cross correlation set-up is used, the count
rate trace for each channel is displayed.
B 45-0006 e 12/02
4-31
Carl Zeiss
QUICKSTART
Quick Start for FCS Measurements
LSM-FCS
The Correlation Curve graph:
–
In case of auto correlation measurement, the developing correlation curve is displayed.
–
In case of cross correlation measurement, three curves will be shown: the two auto correlation
curves for each of the channels and the cross correlation curve.
The Count Rate histogram:
–
To obtain this histogram, the number of pulses (or: photons recorded from the detector) in a
moving time window are recorded and included in a histogram.
The Pulse Density histogram:
–
Here the times elapsed between two subsequent pulses (or: photons recorded from the detector)
are measured and the times are included in a histogram.
After completion of the measurement, the correlation curves are fitted to the model curve using the
parameters provided by the measurement method.
The fitting results are written into the table in the lower part of the window.
–
The current line in the table is marked (preset color: gray).
–
Special situations which occurred during the fit are marked with special colors in the table:
Unsuccessful fit (preset color: red)
Fixed fit parameters (preset color: gray)
Auto cut-off (preset color: yellow)
–
The used colors can be configured in the Columns menu.
–
The displayed columns can be chosen in the Columns menu.
–
The order of the columns in the table can be altered by standard window means: the column
caption can be dragged to the desired position.
–
Any line of the table can be made the current one by clicking on it. This also means that the
corresponding graphs are shown in the displays.
4-32
B 45-0006 e 12/02
QUICKSTART
Quick Start for FCS Measurements
LSM-FCS
4.4.3.1
Carl Zeiss
Display Correlation
If you click on the Correlation button (Fig. 4-34), the Diagrams subordinate at the very right-hand side
of the window will appear. By clicking on the appropriate button the diagram can be toggled between
ON and OFF.
4.4.3.2
Manual Fit
When you click on the Fit button, a Fit menu subordinate will appear (Fig. 4-35). In general this menu
allows you to work with already measured and probably also fitted correlation curves. The intention is to
have the possibility of performing another fit using different fitting parameters if the automatic fit did
not yield optimal results.
Fig. 4-35
Display fit
• Use the 1, 2 and 3 buttons to select a one-, two- or three-component model. If a one- or twocomponent model is selected, the parameters not used are grayed.
• Clicking on the Triplet button allows you to change between taking the triplet fraction into account
or not.
B 45-0006 e 12/02
4-33
QUICKSTART
Quick Start for FCS Measurements
Carl Zeiss
LSM-FCS
• An additional button bar (Free or Fix) will appear if you click on the button at the right-hand side of
the parameter.
–
If you click on Free, the parameter will be defined as free. After the fit, the field will contain the fit
result for the parameter.
–
Fix allows you to fix the parameter. This is useful if you know its value from other measurements.
• With Start Channel and End Channel it is possible to determine which part of the correlation curve
should be fitted to the model.
Caution: The number indicates how many channels are omitted from the respective end.
When you have finished, click the Preview button and the fit procedure will be run. When the fit is
completed, the free parameters will be replaced with the new fitting results and the fit result graph in
the correlation curve diagram will be redrawn.
4.4.3.3
Data Storage and Export
• Save Data allows you to save the whole data set (curves, fitting results, fit parameters). If Save is
selected, the Save menu appears.
–
The Save menu offers the possibility of entering the task name from which the file name is
derived.
–
It also offers the possibility of entering a comment in the comment field.
–
The directory can be chosen.
–
It is possible to store the data as ANSI text (space-consuming, but readable) or as a binary file.
–
OK will confirm the action, Cancel will exit the menu without further action.
4-34
B 45-0006 e 12/02
QUICKSTART
Shut-Down Procedure
LSM-FCS
4.5
Carl Zeiss
Shut-Down Procedure
Never shut down the computer by its main switch while your LSM-FCS 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-FCS program.
4.5.1
Exiting the LSM-FCS Program
• Close all open windows of the LSM-FCS program by clicking on the closing icon
corner of each window.
in the top right
–
This closes the respective window and removes the respective icons from the taskbar.
–
After all dialog windows have been closed, the LSM-FCS Switchboard window appears.
Fig. 4-36
LSM-FCS Switchboard menu
• Click on the Exit button.
–
This terminates the LSM-FCS program.
–
The monitor screen shows the desktop of the WINDOWS NT operating system.
B 45-0006 e 12/02
4-35
QUICKSTART
Shut-Down Procedure
Carl Zeiss
4.5.2
LSM-FCS
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.
Fig. 4-37
Start menu
Taskbar
4-36
B 45-0006 e 12/02
QUICKSTART
Shut-Down Procedure
LSM-FCS
–
Carl Zeiss
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.
The screen now displays the message
Shutdown in Progress - Please wait while the
system writes unsaved data to the disk.
Fig. 4-38
Shut Down window
About 20 seconds after WINDOWS NT has been run down, the Shutdown Computer window appears
which tells you that you can now turn off your computer.
4.5.3
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.
Throw the REMOTE CONTROL main switch and the power supply switch of the Ar Laser to
position "OFF" after 5 minutes.
–
This puts your LSM-FCS microscope system, including the computer, off power.
B 45-0006 e 12/02
4-37
QUICKSTART
Carl Zeiss
4-38
LSM-FCS
B 45-0006 e 12/02
Was this manual useful for you? yes no
Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Download PDF

advertisement