LSM 510 ConfoCor Manual

LSM 510 ConfoCor Manual
Operating Manual
ConfoCor 3
Release 4.0
March 2006
Carl Zeiss
Copyright
ConfoCor 3
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
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 MicroImaging GmbH
07740 Jena, Germany
Phone:
+49 (0) 3641 64 3400
Fax:
+49 (0) 3641 64 3144
E-mail:
[email protected]
www.zeiss.de/lsm
II
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NOTES ON DEVICE SAFETY
General
ConfoCor 3
CHAPTER 1
Carl Zeiss
NOTES ON DEVICE SAFETY
CONTENTS
Page
1
NOTES ON DEVICE SAFETY .............................................................................................1-2
1.1
General..............................................................................................................................1-2
1.2
Warning and Information Labels ........................................................................................1-3
1.3
Regulations ........................................................................................................................1-8
1.4
Notes on Setting up the Microscope and Spectroscope Systems .........................................1-9
1.5
Power Requirements ........................................................................................................1-11
1.6
Notes on Handling the Laser Components........................................................................1-12
1.7
Physical Dimensions .........................................................................................................1-14
1.8
Environmental Requirements............................................................................................1-14
1.9
Notes on Handling the Computer and Data Media ...........................................................1-15
1.10
Notes on Care, Maintenance and Service .........................................................................1-16
1.11
1.11.1
1.11.2
User Interface...................................................................................................................1-17
Changing Filter Wheels in the ConfoCor 3 .......................................................................1-17
Using External Connectors ...............................................................................................1-19
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1-1
NOTES ON DEVICE SAFETY
General
Carl Zeiss
1
ConfoCor 3
NOTES ON DEVICE SAFETY
Information on safety issues for the ConfoCor 3 that deviate from those for the LSM 510 and
LSM 510 META replace the latter. Apart from that all other information on the safety for the LSM 510
and LSM 510 META remain unrestricted valid.
1.1
General
The LSM 510 and LSM 510 META laser scanning microscopes as well as the ConfoCor 3, including their
original accessories and compatible accessories from other manufacturers, may only be used for the
purpose of microscopic and spectroscopic techniques.
Laser Scanning Microscopes (LSM) are intended for high resolution imaging of biological or material
samples, whereby in contrast to wide field microscopy the specimen is illuminated raster-fashion with a
focused laser beam and the optical arrangement prevents light from out-of-focus regions of the
specimen contributing to image formation.
The ConfoCor 3 spectroscope is used for fluorescence correlation spectroscopy, whereas the beam is
parked in a sample, which might consist of a solution or a cell, fluorescence fluctuations recorded and
analyzed by the so-called correlation function.
Installation and commissioning of the LSM 510, LSM 510 META and the ConfoCor 3 systems
must be performed by authorized Carl Zeiss service staff. The system should not be used prior
to instruction by a Carl Zeiss representative.
The manufacturer will not assume liability for any malfunction or damage caused by anything
other than the intended use of the LSM 510, LSM 510 META or ConfoCor 3 or individual
modules or parts of it, nor by any repair or other service operation performed or attempted by
persons other than duly authorized service staff. Any such action will invalidate any claim under
warranty, including parts not directly affected by such action. This also includes the
modification of the system computer with new cards, etc. by the user. The use of a camera at
the base port of Axiovert 200 M Combi stands is not allowed for reasons of laser safety. Any
manipulation will result in the loss of warranty of laser safety.
Please read also the notes on device safety and manuals of the LSM 510, the LSM 510 META, the
microscope, the HBO, the HAL and additional optional devices, if ordered, as the UV Laser, the piezo
focusing device, the heating inserts and the Ti:Sa Laser.
⇒ As the system is largely operated via menus on a computer, you should be familiar with the
principles of the operating system and its WINDOWS, WINDOWS 2000 or Windows XP graphical
user interface. The respective manuals are supplied together with the programs.
The LSM 510, LSM 510 META and ConfoCor 3 are devices that belong to laser hazard class 3B.
The systems are equipped with safety interlocks that comply with laser hazard class 3B and 4. If
equipped with a Ti:Sa Laser (see list in section 1.6), the LSM 510, LSM 510 META and
ConfoCor 3 are devices that belong to laser hazard class 4. WHO recommendations concerning
health and industrial protection when handling laser devices must be observed. The operator of
the unit must also observe all and any relevant statutory accident prevention regulations. The
user is referred to the safety data sheet provided together with the manual.
1-2
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NOTES ON DEVICE SAFETY
Warning and Information Labels
ConfoCor 3
1.2
Carl Zeiss
Warning and Information Labels
The warning and information labels attached on the LSM 510, LSM 510 META and ConfoCor 3
must be observed. Check whether all of the labels shown below are provided on your
instrument, and contact Carl Zeiss Germany or one of the service agencies if you should
discover that any of the labels should be missing. You will receive a free replacement.
Description of labels
Caution: Faults and hazards that might arise during operation which might cause damage to
the unit or injury to the user.
Attention: Laser irradiation hazards possible when operating the system.
Attention: High voltage.
Pull the mains plug before opening the device housing.
Caution: Hot surface.
Caution: UV radiation.
Caution: Fingers can be caught.
The arrow points to the opening where laser light comes out during operation of the system.
Other labels on the system include one of the above depicted symbols and a detailed
description of the handling instructions. See also the following drawings of the system parts.
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1-3
Carl Zeiss
NOTES ON DEVICE SAFETY
Warning and Information Labels
ConfoCor 3
E
TIV
OB
O
ZER
JEC
TO
EC
R
FL
RE
S
CU
FO
Fig. 1-1
1-4
Warning and information labels on the Axiovert 200 M microscope with the LSM 510 META
scanning module and the ConfoCor 3 detection module
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03/06
ConfoCor 3
Fig. 1-2
03/06
NOTES ON DEVICE SAFETY
Warning and Information Labels
Carl Zeiss
Warning and information labels on the system electronic rack
B 45-0015 e
1-5
Carl Zeiss
Fig. 1-3
1-6
NOTES ON DEVICE SAFETY
Warning and Information Labels
ConfoCor 3
Warning and information labels on laser components
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ConfoCor 3
NOTES ON DEVICE SAFETY
Warning and Information Labels
CO
HE
RE
Carl Zeiss
nT
En
TE
RP
RIS
E
AK
PE
CH
AR
SE
E4
TE
7
RP
R
J3
ISE
E5
CO
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Fig. 1-4
03/06
um
xim
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wa ure
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pre p.s.I.
60
T
OU
ter
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J6
Warning and information labels on laser components
B 45-0015 e
1-7
NOTES ON DEVICE SAFETY
Regulations
Carl Zeiss
1.3
ConfoCor 3
Regulations
Extensive knowledge of the hardware/the system is indispensable for safe operation of the LSM 510,
LSM 510 META and the ConfoCor 3.
Read these operating instructions and all device publications belonging to the system
conscientiously before operating the LSM 510, LSM 510 META or the ConfoCor 3! You can
obtain additional information on the hardware configuration delivered and on optional system
extensions from the manufacturer or via the service hotline.
⇒ The LSM 510, LSM 510 META and ConfoCor 3 have been designed, built and tested in conformity
with the following regulations and guidelines:
DIN EN 61010-1 (IEC 601010-1) "Safety requirements for electrical equipment for measurement,
control and laboratory use"
DIN EN 60825-1 (IEC publication 60825-1) "Safety of laser equipment", taking relevant CSA and UL
specifications into account
DIN EN 61326: "Electrical equipment for control technology and laboratory use – EMCrequirements"
Low voltage directive: 73/23/EWG
EMC directive: 89/336/EWG
⇒ The company works according to a certified Environment Management System according to
ISO 14001.
The Product was developed, tested and produced in accordance with the valid regulations and
guidelines for environmental law of the European Union.
The products and their accessories have been classified as instrument category 9 (laboratory
equipment or comparable standard). The product and its accessories agree with the EU-regulations
2002/95/EG (RoHS) and 2002/96/EG (WEEE), if applicable for the products.
Carl Zeiss has installed a process for taking back and recycling the instruments within the member
states of the European Union, which takes care of the appropriate utilization according to the said
EU guidelines.
For details on the disposal and recycling please refer to your relevant Carl Zeiss sales or service
organization.
The product must not be disposed in the household waste or through the municipal disposal
organizations. In case of resale the seller is obliged to inform the buyer, that the product has to be
disposed according to the said regulations.
1-8
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ConfoCor 3
1.4
NOTES ON DEVICE SAFETY
Notes on Setting up the Microscope and …
Carl Zeiss
Notes on Setting up the Microscope and Spectroscope Systems
Installation and commissioning of the LSM 510, LSM 510 META and the ConfoCor 3 systems
must be performed by authorized Carl Zeiss service staff. The system should not be used prior
to instruction by a Carl Zeiss representative.
The LSM 510, LSM 510 META laser scanning microscope and the ConfoCor 3 spectroscope are delivered
in several crates.
The LSM 510, LSM 510 META and ConfoCor 3 must be set up so as to ensure that the
minimum clearance between the wall and the rear of the system is no less than 0.5 m. This
clearance is needed for adjustment and maintenance operations.
Do not set up the unit in the proximity of heat sources such as radiators or direct sunlight. To avoid heat
build-ups, the ventilation slots on the microscope system must not be covered up.
The system must not be set up in areas with potential danger by explosives.
The unit must be connected to a properly installed socket outlet with earthing contact by means of the
mains cables supplied. Continuity of PE connection must not be affected by the use of extension leads.
Please note the following for the ConfoCor 3:
− The ConfoCor3 requires an Axiovert 200 microscope and is attached to channel 4 of the
LSM 510 (META). Note that only system tables with sufficient anti-vibration functionality
should be used.
− A scanning stage is recommended for automatic positioning for solution measurements.
For cell measurements the scanning mirrors are recommended.
− For FCS measurements with VIS and UV light the C-Apochromat 40 x water objective NA
1.2 is recommended. For measurements with NLO light the C-Apochromat 63 x water
objective NA 1.2 is recommended.
The system contains components with dangerous voltage. The system must not be opened by
anybody else than authorized Carl Zeiss Service staff. Before opening the main plug has to be
disconnected.
Before connecting the mains cables, please check whether your mains voltage corresponds to
the voltage specified on the rating plate of the laser module.
For reasons of laser safety, all ports must either be equipped with the corresponding device
(scan head, camera, HBO lamp etc.) or covered with the counterpart of the laser safety kit
provided.
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1-9
Carl Zeiss
NOTES ON DEVICE SAFETY
Notes on Setting up the Microscope and …
ConfoCor 3
Maintenance, repair, modification, removal or exchange of components, or other interference
with the equipment beyond the operations described in this manual may only be carried out by
the manufacturer Carl Zeiss or by persons expressly authorized by Carl Zeiss to do so.
This applies especially to the microscope system, the laser scanning module, lasers, the PC
system, the power supply units, cable connections and other system components.
Please note that the LSM 510, LSM 510 META and ConfoCor 3 are high-precision optoelectronic instruments. Inexpert handling may easily impair their function or even damage
them.
The openings for ventilation must not be covered.
There are hot surfaces on the HBO and HAL lamp.
When sliding the compact Laser module V in and out of the System electronic rack take care
not to catch your fingers.
After installation or conversion of the LSM system, authorized specialized staff must carefully check that it
is in a proper condition and, particularly, that covers protecting against laser radiation are provided.
Tube openings or other unused mounts should always be protected against dust and moisture with the
corresponding device components or with termination covers/blind plugs.
By establishing a corresponding workplace environment, please ensure that the formation of electrostatic
charges of electronic components is avoided.
To avoid vibrations during operation, the LSM 510, LSM 510 META and ConfoCor 3 should only be
operated in conjunction with the system table (vibration damping).
1-10
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NOTES ON DEVICE SAFETY
Power Requirements
ConfoCor 3
1.5
Carl Zeiss
Power Requirements
The LSM 510, LSM 510 META and ConfoCor 3 come with a mains power supply cord and
plug, either CEE red (3/N/PE 400/230V/16A), or NEMA L 14-30P (2/N/Ground 120/240V/30A),
and with the matching mains socket outlet.
A ground wire (AWG10 green/yellow) is supplied because it is necessary to ground the system.
The connecting part on both ends of the cable is a cable eye with 8 mm inner diameter.
A suitable grounding point must be installed in the room.
For systems (220 ... 240 V AC) equipped with X-Cite 120 the mains socket outlet must be
equipped with a fuse having minimum tripping characteristic C according to IEC/EN 60898.
Line voltage
220 ... 240 V AC (±10 %)
100 ... 125 V AC (±10 %)
Line frequency
50...60 Hz
50...60 Hz
− Max. current
3 phases at 16 A
2 phases at 25 A
− Power
Phase 1 = 1.9 kVA max.
Phase 1 = 3.2 kVA max.
Phase 2 = 1.5 kVA max.
Phase 2 = 2.8 kVA max.
LSM/ConfoCor incl. VIS
laser
Phase 3 = 2.6 kVA max.
− Power consumption
5000 VA max.
5000 VA max.
208...240 V AC
208...240 VAC
(±10 %) 50 / 60 Hz
(±10 %) 50 / 60 Hz
1 phase at 63 A
1 phase at:
Argon UV laser
− Line Voltage
− Max. current
208 V: 34 Amps
Note: For Line Voltage 220 V the
connector and power plug are rated
for 63 Amps, However wiring and
fuse should be rated for 32 Amps.
230 V : 31 Amps
240 V : 29 Amps
− Power consumption
7000 VA max.
7000 VA max.
Class of protection
I
I
Type of protection
IP 20
IP 20
Overvoltage category
II
II
Pollution degree
2
2
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NOTES ON DEVICE SAFETY
Notes on Handling the Laser Components
Carl Zeiss
1.6
ConfoCor 3
Notes on Handling the Laser Components
The LSM 510, LSM 510 META and ConfoCor 3 are laser hazard class 3B instruments. If
equipped with a Ti:Sa Laser, the LSM 510, LSM 510 META and ConfoCor 3 are devices that
belong to laser hazard class 4.
This moderate and high-risk class embrace medium-power and high power lasers. You must
take care not to expose yourself to the radiation of such lasers. In particular, never look into the
laser beam! Only personnel which has been instructed on laser safety is allowed to operate the
system.
The following laser types are currently intended for use in the LSM 510 and LSM 510 META. The use of
any other lasers as the ones listed below is not authorized.
Laser
Class
Power
1 Ar 351/364 nm (UV)
4*
80 mW
2 Diode laser 405 nm
3B
30 mW
3 Ar/ML 458/477/488/514 nm
3B
30 mW
4 HeNe 543 nm
3B
1 mW
5 DPSS 561 nm
3B
10 mW
6 HeNe 594 nm
3B
2 mW
7 HeNe 633 nm
3B
5 mW
8 Titanium:Sapphire Laser Mai Tai (Spectra Physics)
710-990 nm (depending on the model)
4
2W
9 Titanium:Sapphire Laser Chameleon (Coherent)
710-980 (depending on the model)
4
2W
10 Diode laser 405 nm
3B
50 mW
11 OPSS laser 488 nm
3B
100 mW
12 DPSS laser 532 nm
3B
75 mW
* Laser type class 4, if mounted on laser module with fiber output class 3B.
Please note that for the maintenance of the UV Laser it is recommended to run the laser at
maximum power once a day if the laser is not used frequently or only at low power levels. This
enables the Autofill process which keeps up the correct tube gas pressure. This operation
prolongs the life time of the tube and prevents complete tube failure if the laser is not used
for a prolonged period of time. For details please refer to the Operator’s Manual of the UV
laser.
Please contact Carl Zeiss if you intend to use a different laser other than the ones above.
1-12
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ConfoCor 3
NOTES ON DEVICE SAFETY
Notes on Handling the Laser Components
Carl Zeiss
If used properly, the LSM 510, LSM 510 META and ConfoCor 3 will not pose any laser radiation risks for
operating staff. Nevertheless, you should observe the following warnings:
• If necessary – insofar as specified by law – inform the laser protection officer before
commissioning the laser.
• The laser modules are equipped with a key-interlock.
• Always store keys for laser key switches and, if applicable, keys for further laser power
supply units, where they are inaccessible to persons not authorized to operate the laser.
• A red LED on the front of the scan head lights up when one or all of the lasers are switched
on.
• Do not place any reflecting objects into the beam path.
• Never open any covers or panels.
• Never look into the laser beam, not even to simply view the specimen, whether with the aid
of optical instruments or without. Otherwise you risk going blind!
• Do not leave any empty objective positions of the nosepiece uncovered.
• If a class 4 laser is attached to the system, already stray light can impose danger to the
operator.
• With class 4 lasers take special care of fire protection requirements. Do not use or store
flammable or explosive solids, fluids or gases in the vicinity of the system.
• Class 4 lasers can inflame also flammable materials like cloth or paper. Do not put such
materials into the beam path.
• Do not reach into the process beam inside the sample area whenever the Class 4 laser is
active!
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.
For NLO systems equipped with a specific push and click filter for NDD imaging be aware that
the NDD reflector cube in the reflector turret leads to a strong back reflection of HBO light into
the specimen plane and the eyepiece. When observing the specimen through the ocular lens
the use of the NDD reflector cube should be avoided. The light flash is not harmful but
unpleasant. The reflex of closing the eyelid is sufficiently protective. To completely avoid this
situation an additional filter (#1261-345) can be mounted into the NDD reflector cube which
prevents the back reflection of the HBO light in the ocular plane.
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NOTES ON DEVICE SAFETY
Physical Dimensions
Carl Zeiss
1.7
ConfoCor 3
Physical Dimensions
Length (cm)
Width (cm)
Height (cm)
Weight (kg)
Large system table
150
80
78
100
Small system table
80
65
78
60
Passively damped anti-vibration table
130
100
75
137
Active anti-vibration table (NLO)
for Mai Tai Laser or Chameleon
120
140
75
200
Active anti-vibration table (NLO)
for Verdi Mira or Millenia Tsunami
Laser
180
140
75
400
Scanning Module LSM 510
25
20
25
15
Scanning Module LSM 510 META
28
27
30.5
13
Module ConfoCor 3
49
27
18
25
Microscope
50
35
50
20
Laser Module RGB
110
70
28
95
Laser Module, UV
140
20
20
60
Laser Module V (405 nm)
66
52
22
30
Plug-in unit external laser
66
52
22
9
110
70
58
90
Power supply for Ar (UV)
50
50
30
30
Cooling unit for Ar (UV)
80
45
50
30
System Electronic Rack
1.8
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 30 °C, any conditions different from 1. and 5.
3. Storage, less than 16 h
T = -40 °C to 55 °C
4. Storage, less than 6 h
T = -55 °C to 70 °C
5. Temperature gradient
± 0.5 °C/h
6. Warm up time
1 h, for high-precision and/or long-term measurements ≥ 3 h
7. Relative humidity
< 65 % at 30 °C
8. Operation altitude
max. 2000 m
9. Loss of heat
4 kW
These requirements do not include the requirements for high precision measurements. Please
refer to the Operator’s Manual of the microscope for these requirements.
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ConfoCor 3
1.9
NOTES ON DEVICE SAFETY
Notes on Handling the Computer and Data Media
Carl Zeiss
Notes on Handling the Computer and Data Media
The computer used as standard in your LSM system is an IBM-compatible high-end Pentium computer
with WINDOWS XP operating system.
Do make sure, though, that you receive your LSM-ConfoCor 3 system with the operating
system installed, with initialization and start files set up and with the LSM-ConfoCor program
also installed.
When working with the hard disk, it is important to know that the more data it contains, the
slower its operation will become. Therefore, data that you do not need permanently should be
stored on other external devices.
When handling diskettes and USB sticks, avoid data losses by protecting them against extreme
temperatures, moisture and magnetic fields. The data on a diskette is stored in the form of
magnetic signals. To some extent, monitors, telephones or even lamps generate magnetic fields
that might destroy this data. Also, never open the metal cover on diskette cases. A diskette’s
surface can also be destroyed by touching it.
When handling CDs, CD ROMs or DVDs, do not touch the data side of the disc (the side of the
disc with no label or printing).
Do not apply paper labels or write on any part of the disc, data side or label side. If dust or
fingerprints get on the disc, wipe it with a soft cloth from the center to the edge, but do not
use benzine, paint thinner, record cleaner, or static repellent. This can damage the disc.
Do not place the disc in any place where it is exposed to direct sunlight or high temperatures.
Backup your data on a regular basis.
Do not install any other software without talking to your Carl Zeiss representative.
Never turn your computer off before you have terminated the LSM program and run down the
WINDOWS XP operating system. Otherwise, the program and/or data files may get lost.
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Carl Zeiss
1.10
NOTES ON DEVICE SAFETY
Notes on Care, Maintenance and Service
ConfoCor 3
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 render all warranty claims null and void and also laser safety is no longer warranted.
You are well advised to arrange a service agreement with your nearest Carl Zeiss representative to
guarantee perfect functioning of the microscope system in the long term.
Modifications and conversion work on the components of the system must only be carried out by the
manufacturer, by the service agency or by persons authorized and trained for this purpose by the
manufacturer.
Damaged units or parts may only be repaired or maintained by the responsible service agency.
During maintenance or repair carried out by the service personnel the customer is requested to stand
aside and wear a pair of laser safety goggles if needed.
Before opening the housing of the halogen lamp switch off all laser units.
Care operations that may be carried out by operating staff are limited to cleaning painted and glass
surfaces.
• Before cleaning the instrument make sure the main power supply is disconnected.
• Cleaning painted surfaces
To do this, use a clean cloth that has been moistened in a mixture of water and some detergent; do
not use any solvent, however. Dry with a lint-free cloth.
• Cleaning glass surfaces
Glass surfaces that have become soiled or which are marked with fingerprints may be rubbed with a
clean optical cleaning cloth.
If soiling is persistent, dip the optical cleaning cloth into a mixture of distilled water and a small
quantity of detergent.
To complete cleaning, lightly breathe on the glass surface and rub it dry with a clean cloth. Lint or dust
is best removed with a clean brush.
• Make sure that no cleaning liquid penetrates into the system.
• Dust filters in the ventilation entries of the system electronic rack have to be replaced every 6 month.
For replacement please contact your local service representative.
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NOTES ON DEVICE SAFETY
User Interface
ConfoCor 3
1.11
Carl Zeiss
User Interface
All user interface ports are equipped with a safety interlock system which warrants laser safety.
These interlock devices must not be manipulated. Other interfaces which are not described here
are service interfaces and are only to be operated by authorized Carl Zeiss service personnel.
The following devices can be mounted and dismounted by the user or are accessible by the
user:
−
ConfoCor 3 filter wheels
−
ConfoCor 3 external connectors
The ConfoCor 3 module must not be detached from the LSM 510 or LSM 510 META scan
modules!
1.11.1
Changing Filter Wheels in the ConfoCor 3
By opening the lid (see Fig. 1-5 and Fig. 1-6) you have access to all the filter wheels in the ConfoCor 3.
You can remove and replace filter wheels by push and click.
Be careful not to disrupt any connectors in doing this.
Please note that the data base has to be exchanged if filter wheels are replaced with another
filter set.
When opening the lid, the safety regulations will switch off the laser light.
Do not remove the ConfoCor 3 detection head from its attachment to the LSM 510 or
LSM 510 META.
Laser light can escape the system through the external port used as the attachment site for the
ConfoCor 3 that can lead to bodily damage.
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1-17
Carl Zeiss
NOTES ON DEVICE SAFETY
User Interface
ConfoCor 3
Fig. 1-5
Top view APD module. The location of exchangeable filter wheels are indicated (BF = block
filter; NFT = secondary beam splitter; EF 1 = emission filter 1; EF-2 = emission filter 2)
Fig. 1-6
Top view GaAsP module. The location of exchangeable filter wheels are indicated (BF = block
filter; NFT = secondary beam splitter; EF 1 = emission filter 1; EF-2 = emission filter 2)
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ConfoCor 3
1.11.2
NOTES ON DEVICE SAFETY
User Interface
Carl Zeiss
Using External Connectors
In the rear of the GaAsP and APD modules, you
have external access to the TTL pulses of both
channels delivered at the APD1 and APD2 plugs
for the APD module (see Fig. 1-7) or at the
FCS1 and FCS2 plugs of the GaAsP module (see
Fig. 1-8).
In the GaAsP module you have also access to the
NIM pulse at the FLIM1and FLIM2 plugs.
Fig. 1-7
Rear view APD module
Fig. 1-8
Rear view GaAsP module
Please note that either plug is of BNT
type and BNT triax cables should used.
If using BNC connectors and coax
cables the signal quality can suffer
dramatically.
03/06
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1-19
NOTES ON DEVICE SAFETY
User Interface
Carl Zeiss
ConfoCor 3
The assignment of the connectors is as follows:
Name
APD
GaAsP
Meaning
LVDS
14pin MDR
14pin MDR
Main communication link
14pin MDR
Communication to optional module
Sub-LVDS
Safety
8pin LEMO EXJ-1B
8pin LEMO EXJ-1B
Laser safety
Power
16pin LEMO ECJ2B
16pin LEMO ECJ-2B
Power consumption: less than 20W
CAN
15pin HD Sub-D
female
15pin HD Sub-D
female
Optional CAN Port / Service Port
9pin Sub-D male
Service Port
RS232
APD 1 / 2
Triax BNT
FCS 1 / 2
Gate 1 / 2
Photon pulse- level: 2,[email protected]Ω, 30 nsek long
Triax LEMO ERN-00
Triax BNT
Photon pulse- level: 2,[email protected]Ω, 25 nsek long
Gate APD- TTL level
FLIM 1 / 2
Triax LEMO ERN-00
Double amplified PMT signal (up to –2V)
CH 1 / 2
4pin Binder (not
implemented)
No connector
To connect the ConfoCor 3 to an optional correlator card, use the connectors APD 1 /2 or FCS 1 / 2,
respectively. As long as the maximum count rate is not reached, each TTL Pulse corresponds to one
photon. Take care to use 50Ω termination to avoid reflections and use double shielded ‘triax’ cable to
avoid heavy distortions of the signal. To connect to BNC equipment, connect inner shield to ground and
leave outer shield open.
For gating the APD use TTL signals.
To connect the ConfoCor 3 to an optional FLIM card, use the connectors FLIM 1 / 2 or APD 1 / 2
respectively. APD 1 / 2 delivers directly the APD signal (jitter 350psec), whereas FLIM 1 / 2 delivers an
unshaped and amplified negative pulse of the PMT signal ranging from -0,9V of up to -2V (jitter
280psec).
1-20
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SETUP REQUIREMENTS
Contents
ConfoCor 3
CHAPTER 2
Carl Zeiss
SETUP REQUIREMENTS
CONTENTS
Page
2
SETUP REQUIREMENTS ...................................................................................................2-2
2.1
2.1.1
2.1.2
2.1.3
2.1.4
2.1.5
Space Requirements...........................................................................................................2-2
ConfoCor 3 and LSM on large system table: 2 m × 3,50 m.................................................2-2
ConfoCor 3 and LSM on small system table: 2 m x 3,5 m ...................................................2-3
ConfoCor 3 and LSM on passive anti vibration table: 2,2 m x 4,2 m ...................................2-4
ConfoCor 3, LSM and Ar UV Laser .....................................................................................2-5
ConfoCor 3 and LSM prepared for Two Photon Laser (NLO) ...............................................2-6
2.2
Power Requirements ..........................................................................................................2-8
2.3
Physical Dimensions .........................................................................................................2-12
2.4
Dimension of Shipment Crates .........................................................................................2-13
2.5
Environmental Requirements............................................................................................2-13
2.6
Vibrations ........................................................................................................................2-14
2.7
Microscopes.....................................................................................................................2-14
2.8
Scanning Module LSM 510 and LSM 510 META...............................................................2-15
2.9
Detection Module ConfoCor 3 .........................................................................................2-15
2.10
Laser Module RGB (458, 477, 488, 514, 543 or 561,594, 633 nm) ..................................2-16
2.11
Laser Module V (405 nm) .................................................................................................2-16
2.12
Laser Module UV (351, 364 nm).......................................................................................2-16
2.13
System Overview ConfoCor 3 and LSM 510 / LSM 510 META...........................................2-17
03/06
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2-1
SETUP REQUIREMENTS
Space Requirements
Carl Zeiss
2
SETUP REQUIREMENTS
2.1
Space Requirements
2.1.1
ConfoCor 3 and LSM on large system table: 2 m × 3,50 m
Fig. 2-1
ConfoCor 3
Space requirements for ConfoCor 3 and LSM on large system table
(measurements in mm)
The System Electronic Rack contains all electronics for control of the hardware components of the
LSM system, the power supply for the microscope, the scanning unit, the Laser Module V and the
Laser Module RGB equipped with the choice of lasers. The Laser Module RGB is set on top of the System
Electronic Rack.
2-2
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03/06
SETUP REQUIREMENTS
Space Requirements
ConfoCor 3
2.1.2
Carl Zeiss
ConfoCor 3 and LSM on small system table: 2 m x 3,5 m
Fig. 2-2
Space requirements for ConfoCor 3 and LSM on small system table
(measurements in mm)
The System Electronic Rack contains all electronics for control of the hardware components of the
LSM system, the power supply for the microscope, the scanning unit, the Laser Module V and the
Laser Module RGB equipped with the choice of lasers. The Laser Module RGB is set on top of the System
Electronic Rack.
03/06
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2-3
SETUP REQUIREMENTS
Space Requirements
Carl Zeiss
2.1.3
ConfoCor 3
ConfoCor 3 and LSM on passive anti vibration table: 2,2 m x 4,2 m
Fig. 2-3
Space requirements for ConfoCor 3 and LSM on Passively Damped Anti Vibration
Table (system table with breadboard).
Depending on the ordered table it might be turned by 90°.
(measurements in mm)
The System Electronic Rack contains all electronics for control of the hardware components of the
LSM system, the power supply for the microscope, the scanning unit, the Laser Module V and the
Laser Module RGB equipped with the choice of lasers. The Laser Module RGB is set on top of the System
Electronic Rack.
2-4
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SETUP REQUIREMENTS
Space Requirements
ConfoCor 3
2.1.4
Carl Zeiss
ConfoCor 3, LSM and Ar UV Laser
We recommend placing the cooling unit of the Ar laser (UV) in a separate room to prevent heat
accumulation and vibration. Length of the water hose: 400 cm
Fig. 2-4
Space requirements for ConfoCor 3, LSM
and AR UV Laser (measurements in mm)
The System Electronic Rack contains all electronics for control of the hardware components of the
system, the power supply for the microscope, the scanning unit and the Laser Module RGB equipped
with the choice of lasers. The Laser Module RGB is set on top of the System Electronic Rack. The lab cart
holds the power supply for the UV laser and the UV Laser module.
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2-5
SETUP REQUIREMENTS
Space Requirements
Carl Zeiss
ConfoCor 3
2.1.5
ConfoCor 3 and LSM prepared for Two Photon Laser (NLO)
2.1.5.1
Coherent “Chameleon” or Spectra Physics “Mai Tai”. Directly-coupled to inverted
Microscope: 2,5 m x 4 m
Fig. 2-5
Space requirements for ConfoCor 3, LSM and Two Photon Laser and
Small NLO System Table (measurements in mm)
The System Electronic Rack contains all electronics for control of the hardware components of the
system, the power supply for the microscope, the scanning unit and the Laser Module RGB equipped
with the choice of lasers. The Laser Module RGB is set on top of the System Electronic Rack. Power supply
and cooling unit of the NLO Laser can be stored under the system table. The electronics for the NLO laser
have to be set aside the System Electronic Rack. An additional lab cart (000000-0465-515) is
recommended. The NLO laser incoupling unit will be placed will be conducted below the ConfoCor 3
unit.
2-6
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SETUP REQUIREMENTS
Space Requirements
ConfoCor 3
2.1.5.2
Carl Zeiss
Coherent “Verdi Mira” or Spectra Physics “Millenia Tsunami”. Directly-coupled to
inverted Microscope: 3,5 m x 3,5 m
Fig. 2-6
Space requirements for Two Photon Laser and
Large NLO System Table. Set up of ConfoCor 3, LSM and
inverted microscope (measurements in mm)
The System Electronic Rack contains all electronics for control of the hardware components of the
system, the power supply for the microscope, the scanning unit and the Laser Module RGB equipped
with the choice of lasers. The Laser Module RGB is set on top of the System Electronic Rack. Power supply
and cooling unit of the NLO Laser can be stored under the system table. Coherent “Chameleon” or
Spectra Physics “Mai Tai” lasers can also be used. The electronics for the NLO laser have to be set aside
the System Electronic Rack. An additional lab cart (000000-0465-515) is recommended.
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2-7
SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
2.2
ConfoCor 3
Power Requirements
The LSM 510, LSM 510 META and ConfoCor 3 come with a mains power supply cord and
plug, either CEE red (3/N/PE 400/230V/16A), or NEMA L 14-30P (2/N/Ground 120/240V/30A),
and with the matching mains socket outlet.
A ground wire (AWG10 green/yellow) is supplied because it is necessary to ground the
system. The connecting part on both ends of the cable is a cable eye with 8 mm inner
diameter.
A suitable grounding point must be installed in the room. For systems (220 ... 240 V AC)
equipped with X-Cite 120 the mains socket outlet must be equipped with a fuse having
minimum tripping characteristic C according to IEC/EN 60898.
Line voltage
220 … 240 V AC (±10 %)
100 … 125 V AC (±10 %)
Line frequency
50...60 Hz
50...60 Hz
− Max. current
3 phases at 16 A
2 phases at 25 A
− Power
Phase 1 = 1.9 kVA max.
Phase 1 = 3.2 kVA max.
Phase 2 = 1.5 kVA max.
Phase 2 = 2.8 kVA max.
LSM/ConfoCor
incl. VIS laser
Phase 3 = 2.6 kVA max.
− Power consumption
5000 VA max.
5000 VA max.
208...240 V AC
208...240 VAC
(±10 %) 50 / 60 Hz
(±10 %) 50 / 60 Hz
1 phase at 63 A
1 phase at:
Argon UV laser
− Line Voltage
− Max. current
208 V: 34 Amps
Note: For Line Voltage 220 V the
connector and power plug are rated
for 63 Amps, However wiring and
fuse should be rated for 32 Amps.
230 V : 31 Amps
240 V : 29 Amps
− Power consumption
7000 VA max.
7000 VA max.
Class of protection
I
I
Type of protection
IP 20
IP 20
Overvoltage category
II
II
Pollution degree
2
2
2-8
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ConfoCor 3
Fig. 2-7
03/06
SETUP REQUIREMENTS
Power Requirements
Carl Zeiss
Power connector for LSM 510 / LSM 510 META and Laser Module V. Free/reserve outlets may
be used to supply power to additional equipment. No more than 1 A can be provided by
each outlet. (Scheme is turned 90° to real system.)
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2-9
Carl Zeiss
Fig. 2-8
2-10
SETUP REQUIREMENTS
Power Requirements
ConfoCor 3
Power connector for Laser Module RGB
(Scheme is turned 90° to real system.)
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SETUP REQUIREMENTS
Power Requirements
ConfoCor 3
1
2
Carl Zeiss
Key-interlock Laser ON/OFF
Door interlock interface
Fig. 2-9
Key-interlock Laser ON/OFF and interface for connection of door interlock
The door interlock interface is covered with a green plug to bypass a door interlock.
• To use the interface remove the top of the green plug and the bypass wire.
• Then connect the wires of the door interlock at the same position.
Two door interlocks can be connected.
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2-11
SETUP REQUIREMENTS
Physical Dimensions
Carl Zeiss
2.3
ConfoCor 3
Physical Dimensions
Length (cm)
Width (cm)
Height (cm)
Weight (kg)
Large system table
150
80
78
100
Small system table
80
65
78
60
Passively damped anti-vibration table
130
100
75
137
Active anti-vibration table (NLO)
for Mai Tai Laser or Chameleon
120
140
75
200
Active anti-vibration table (NLO) for
Verdi Mira or Millenia Tsunami Laser
180
140
75
400
Scanning Module LSM 510
25
20
25
15
Scanning Module LSM 510 META
28
27
30.5
13
Module ConfoCor 3
49
27
18
25
Microscope
50
35
50
20
Laser Module RGB
110
70
28
95
Laser Module, UV
140
20
20
60
Laser Module V (405 nm)
66
52
22
30
Plug-in unit external laser
66
52
22
9
110
70
58
90
Power supply for Ar (UV)
50
50
30
30
Cooling unit for Ar (UV)
80
45
50
30
Water hose for Ar (UV)
700
Fiber optic cable, VIS(ible)
300
Fiber optic cable, UV
300
Fiber optic cables at NLO systems
400
Cables
350
SCSI cable
350
2-12
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System Electronic Rack
03/06
SETUP REQUIREMENTS
Dimensions ... / Environmental …
ConfoCor 3
2.4
Carl Zeiss
Dimension of Shipment Crates
Crate containing
Length (cm)
Width (cm)
Height (cm)
Weight (kg)
Large system table
160
85
95
120
Small system table
90
75
80
80
Passively damped anti-vibration
table
145
115
115
150
Active anti-vibration table (NLO)
for Mai Tai Laser or Chameleon
145
160
110
330
Active anti-vibration table (NLO)
for Mira or Tsunami Laser
200
160
110
460
System Electronic Rack and Laser
module
135
90
100
300
LSM, Microscope, Computer
135
90
100
150
Module ConfoCor 3
67
42
31
27
Support ConfoCor 3
67
42
31
10
Upgrade package containing
module and support ConfoCor 3
80
58
83
40
Additional Hardware
Components
135
90
61
100
UV laser unit
125
55
50
100
UV cooling unit
120
60
90
50
2.5
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 30 °C, any conditions different from
1. and 5.
3. Storage, less than 16 h
T = -40 °C to 55 °C
4. Storage, less than 6 h
T = -55 °C to 70 °C
5. Temperature gradient
± 0.5 °C/h
6. Warm up time
1 h, for high-precision and/or long-term measurements ≥ 2 h
7. Relative humidity
< 65 % at 30 °C
8. Operation altitude
max. 2000 m
9. Loss of heat
4 kW
03/06
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2-13
SETUP REQUIREMENTS
Vibrations / Microscopes
Carl Zeiss
2.6
ConfoCor 3
Vibrations
Vibrations under operation conditions
(with system table)
Shipping shock (LSM 510 box)
5 µm pp at 5 Hz
3g
10 µm pp at 10 Hz
10 µm pp at 20 Hz
2.7
Microscopes
Inverted Axiovert 200 M BP or SP
All ICS objectives from Carl Zeiss and their accessories can be
accommodated.
Z motor
DC servomotor, opto-electronically coded
Least Z interval:
Piezo Objective focus
50 nm (Axiovert 200 M BP or SP)
Piezo-driven single objective drive
Max. travel 250 µm; resolution 10 nm
In the unlikely case of extreme fluctuations of the external power net or
electromagnetic radiation, the piezo crystal will vary and disturbance in
the image is visible. Note that this is not a defect and the piezo drive will
not be damaged.
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SETUP REQUIREMENTS
Scanning Modules / Detection Module
ConfoCor 3
2.8
Carl Zeiss
Scanning Module LSM 510 and LSM 510 META
Scanners
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 confocal reflection/fluorescence channels (PMT) simultaneously
or
b) 2 confocal reflection/fluorescence channels (PMT) and 1 META detector
1 transmitted light channel (PMT) and 2 NDD
or
3 - 4 NDD (Non descanned detectors, PMT)
1 reference monitor diode
Fiber-optic adaptation of external detectors
Dynamic range
12-bit DAC for each detection channel
Pinholes
4 individual variable pinholes (one per confocal channel or META detector)
Computer controlled automatic alignment
2.9
Detection Module ConfoCor 3
Channels
a) 2 channels (APD) or
b) 2 channels (GaAsP PMT)
Dynamic range
12-bit DAC for each detection channel
Pinholes
1 individual pinhole (channel 4 of LSM 510 or LSM 510 META)
Computer controlled automatic alignment
03/06
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2-15
SETUP REQUIREMENTS
Laser Modules
Carl Zeiss
2.10
ConfoCor 3
Laser Module RGB (458, 477, 488, 514, 543 or 561,594, 633 nm)
Single-mode polarization preserving fiber
Laser beam attenuation for all lasers by VIS-AOTF
HeNe laser (543 nm, 1 mW)
HeNe laser (594 nm, 2 mW)
HeNe laser (633 nm, 5 mW)
DPSS laser (561 nm, 10 mW)
Ar laser (458, 477, 488, 514 nm, 30 mW)
2.11
Laser Module V (405 nm)
Single-mode polarization preserving fiber
Laser beam attenuation by UV-AOTF
Diode laser (405 nm, 30 mW)
2.12
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
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ConfoCor 3
2.13
03/06
SETUP REQUIREMENTS
System Overview ConfoCor 3 and LSM 510 / LSM 510 META
Carl Zeiss
System Overview ConfoCor 3 and LSM 510 / LSM 510 META
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2-17
Carl Zeiss
2-18
SETUP REQUIREMENTS
System Overview ConfoCor 3 and LSM 510 / LSM 510 META
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ConfoCor 3
03/06
INTRODUCTION …
Contents
ConfoCor 3
CHAPTER 3
Carl Zeiss
INTRODUCTION TO
LASER SCANNING MICROSCOPY
AND
FLUORESCENCE CORRELATION
SPECTROSCOPY
CONTENTS
Page
3
INTRODUCTION TO LASER SCANNING MICROSCOPY AND FLUORESCENCE
CORRELATION SPECTROSCOPY ......................................................................................3-2
3.1
Principle of Laser Scanning Microscopy...............................................................................3-2
3.2
Principle of Fluorescence Correlation Spectroscopy .............................................................3-3
3.3
Optical Diagram of the ConfoCor 3 and the LSM 510 and LSM 510 META (Schematic) ......3-4
3.4
3.4.1
3.4.2
3.4.3
Performance Features of the ConfoCor 3 and the LSM 510 or LSM 510 META ...................3-5
Optical and Mechanical Aspects .........................................................................................3-5
Microscope Equipment of the ConfoCor 3 and the LSM 510 or LSM 510 META System .....3-6
Computer Hardware and Software.....................................................................................3-8
03/06
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3-1
Carl Zeiss
INTRODUCTION …
Principle of Laser Scanning Microscopy
ConfoCor 3
3
INTRODUCTION TO LASER SCANNING MICROSCOPY AND FLUORESCENCE
CORRELATION SPECTROSCOPY
3.1
Principle of Laser Scanning Microscopy
To yield information on their inner structure by conventional transmitted-light microscopy, specimens
have to be very thin and translucent; otherwise image definition will be poor. In many cases it is a
problem to satisfy these requirements.
The essential considerations have led to trailblazing changes in conventional microscopy and supplied a
successful solution to the above problem.
• Unlike the practice of even illumination in conventional microscopy, the LSM technique projects the
light of a point light source (a laser) through a high-NA objective onto a certain object plane of
interest as a nearly diffraction-limited focus. However, if not for another "trick", the stray light
produced outside the object plane, or the fluorescence of fluorescent specimens, would disturb the infocus image of object point of interest, resulting in a blurred image of poor contrast. The problem
therefore is 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.
• In order to obtain an image of the selected
object plane as a whole, it is necessary to scan
the object plane in a point-by-point, line-by-line
Fig 3-1
Principle of confocal imaging
raster by means of an XY light deflection
system. The detectors - as a rule,
photomultipliers - convert the optical information into electric signals. This allows the image of any
object plane to be generated and stored within less than a second. By a defined focusing (Z axis)
movement it is possible to look at any object plane of interest. By scanning a succession of object
planes in a specimen, a stack of slice images can be produced.
This way, the LSM technique in conjunction with ICS optics (Infinity Color-Corrected System) has brought
decisive improvements over conventional microscopy in terms of resolving power and confocal depth
contrast:
Object features in the order of 0.2 μm can be resolved, and height differences of less than
0.1 μm made visible, without the use of interference methods.
3-2
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INTRODUCTION …
Principle of Fluorescence Correlation Spectroscopy
ConfoCor 3
3.2
Carl Zeiss
Principle of Fluorescence Correlation Spectroscopy
Fluorescence Correlation Spectroscopy (FCS) analyzes the diffusion time of molecules and their
differences if they have bound together. This is done by fluctuation analysis of fluorescence-labeled
molecules within a well defined volume element. In most experiments, Brownian motion drives the
fluctuation. The volume element is the confocal volume defined by the excitation spot of a well focused
laser beam and the selected emission region defined by the properly aligned pinhole of the detection
optics (see Fig 3-1).
As such, the setup is the same as for a Laser-Scanning Microscope (LSM), but in the latter we are not
interested in the fluctuations but in the average intensity. As a matter of fact, what is the signal in FCS is
noise in the LSM. Since the fluctuations are more pronounced, if less molecules are in the volume, FCS
requires little molecule numbers (1-10). Whereas LSM is a scanning technique, FCS uses the beam is
parked in one spot.
The fluctuations are analyzed by treating the measured photon counts with mathematical methods called
correlation functions (see Fig. 3-2). The amplitude of the function is inverse proportional to the molecule
number and the decay time gives the residence time of the molecule in the confocal volume and hence
its diffusion time. If the two interacting molecules are of different size, only the smaller one has to be
labeled using fluorescent dyes. This method is called auto-correlation. In this case the total autocorrelation is the sum of the two different species. If the diffusion constants of both partners are similar,
they are both labeled with different dyes and cross-correlation is used. Often, photophysical processes
like triplet states impinge on the correlation function, but can be accommodated in the model. Then the
total correlation is the product of the single processes.
Fig. 3-2
Correlation functions
Figure 3-2 shows a correlation curve for a two component translational diffusion with triplet. The two
components can be separated on behalf of their diffusion time (circles). Note that the contribution of the
two components add up, whereas the contribution of the total diffusion process and the triplet multiply
to obtain the total correlation.
Because of the tiny size of the confocal volume and its nature, the measurement can be carried out, in
principle, in every area that is reachable by light and that is not smaller than an Escherichia coli bacterium
(approximately 0.2 fl). In particular, measurements can be done inside living cells or on cell membranes.
In order to be able to place the measurement volume at its proper place, it is advantageous to combine
FCS with powerful light microscopy, particularly a confocal LSM.
03/06
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3-3
INTRODUCTION …
Optical Diagram of …
Carl Zeiss
ConfoCor 3
3.3
Optical Diagram of the ConfoCor 3 and the LSM 510 and LSM 510 META (Schematic)
Fig. 3-3
Optical path, schematic (3-channel configuration)
AOTF
APD
CO
DBC
DBS
EF
HAL
HBO
Acousto Optical Tunable Filter
Avalanche photodiode
Collimation Optics
Dichroic Beam Combiner
Dichroic Beam Splitter
Emission Filter
Halogen Lamp
Mercury Vapor Short-Arc Lamp
IBF
LSF
MDBS
NDF
VP
PMT
T-PMT
ZO
Infrared Blocking Filter
Line Selection Filter
Main Dichroic Beam Splitter
Neutral Density Filter
Variable Pinhole
Photomultiplier
Transmission-Photomultiplier
Zoom Optics
The diagram above is a schematic representation of the LSM 510-ConfoCor 3 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 PMT3) or via Channel 4 into the ConfoCor 3 module.
Here light passes though block filters for NLO light (if applicable), and is directed via a dichroic beam
splitter (DBS 5) to avalanche photodiodes (APD1 ... maximally APD2) or GaAsP (GaAsP1 … maximally
GaAsP2) detectors. The pinholes (VP) block all out of focus light, whereas emission filters (EF) are used to
select a specific spectrum of the emitted light and to suppress any remaining excitation light.
3-4
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ConfoCor 3
INTRODUCTION …
Performance Features of the ConfoCor 3 …
3.4
Performance Features of the ConfoCor 3 and the LSM 510 or LSM 510 META
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) or upright (Axio Imager.Z1, Axio Imager.M1 or Axioskop 2 FS MOT) microscope in less than three
minutes. On the Axiovert 200 M, the scanning module may be mounted either to the base port directly
below the microscope or to the side port. Please note that the Axiovert is required for attachment of the
ConfoCor 3.
The spectral range available extends from the UV to the IR region.
For the VIS (visible-light) Laser Module, the user can select from up to six lasers with wavelengths of 633,
594, 561, 543, 514, 488, 477, 458 and 405 nm. The UV Laser Module provides wavelengths of 351 and
364 nm. A Ti:Sa Laser provides pulsed laser light from 690 to 1040 nm for Multiphoton imaging (NLO).
Coupling of the laser light is through polarization-preserving single-mode optical fibers. One variable
beam collimator each for the UV or NLO 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 up to 8 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 three internal image acquisition channels, usable for reflection or fluorescence, and an additional
transmitted-light channel are ideal for the investigation of multiple fluorescence specimens. The
ConfoCor 3 is attached to the fourth channel. The ConfoCor 3 comes with two detector types:
Gallenium Arsenide Phosphide detectors (GaAsP) or avalanche photodiodes (APD). The APDs will deliver
a TTL pulse, the GaAsP detectors deliver TTL pulse, NIM pulse and an analogue signal. 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 fluorescence,
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 not
available if the ConfoCor 3 is attached.
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. In the ConfoCor 3
all filter wheels can be exchanged by push and click.
The high-NA C-APOCHROMAT objectives specially developed for the LSM technique reach the physical
limit in resolving power, and can be used throughout the 350...800 nm spectral range with the same
high quality, producing brilliant images. Note, for fluorescence correlation spectroscopy the CAPOCHROMAT objectives are highly recommended.
A two-mirror scanner system, controlled by a real time electronics, 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.
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INTRODUCTION …
Performance Features of the ConfoCor 3 …
Carl Zeiss
ConfoCor 3
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.
For ConfoCor 3 operation, the mirrors will be parked.
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 leftto-right, and wavelength 2 for right-to-left scanning), two fluorochrome dyes can be viewed and
documented in a quasi-simultaneous mode. This will prevent cross talk between detection channels.
3.4.2
Microscope Equipment of the ConfoCor 3 and the LSM 510 or LSM 510 META System
The LSM 510 or LSM 510 META system is equipped either with the inverted Axiovert 200 M BP or SP
microscope required in combination with the ConfoCor 3.
Only the differences from the delivered operating manual "Axiovert 200 M" will be explained here.
(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 performed via LSM 5 software control. The "Restriction
of the nosepiece height to protect the objectives during motorized objective change" is inactivated. The
nosepiece will be moved down automatically before each motorized objective change.
b) The reflector mount is motorized and provided with the Axiovert 200 M reflector turret. The reflector
turret has 5 positions: One transmitting light position, which is identical to the LSM position, and four
further positions for fluorescence filter sets (reflector modules). If you want to use more than five
conventional fluorescence filter sets, it is advisable to use a further reflector turret. When changing the
reflector turret position you must make sure that the turret will click into position, since otherwise the
image area will be cut. c) The stand has a motorized focusing drive (fine coarse). Sensitivity of the
focusing drive is adjusted to the delivered objectives by the manufacturer. If you want to use other
objectives, sensitivity and parfocality can be adjusted via the Axioset program.
d) The stand features an integrated power supply for the internal motors and stand electronics. The
power supply can be switched on at the right side of the stand. External power supply units will be used
for the mercury vapor short arc lamp.
e) The analyzer slider for conventional DIC methods will be operated from the right side and is located
just below the nosepiece.
When the rod is pushed in, the analyzer is located in the beam path. In the LSM-mode, the analyzer must
not be located in the beam path, and the analyzer rod must be pulled out.
(2) Specimen stages and fine focus drives
a) Mechanical stage
The stage with coaxial drive must be mounted on the right side of the stand.
b) Scanning stage
c) Piezo objective focus drive
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ConfoCor 3
INTRODUCTION …
Performance Features of the ConfoCor 3 …
Carl Zeiss
(3) Transmitted-light illumination
a) The illuminator support contains a security circuit which activates a shutter preventing laser light from
reaching the stand when the support is moved to the back. A complementary shutter built in the stand
prevents laser light from reaching the eyepieces during the scanning mode.
b) The illuminator support is equipped with a rotary polarizer. The Axiovert 200 M description contains
the adjustment for the DIC mode during conventional observation.
For scanning in the 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 must not be located in the beam path because the laser
light is already polarized.
c) A fully motorized, LSM 5 software-controlled switching mirror is mounted on the illuminator support.
Alternatively, the light is directed to the LSM 5 transmitted-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 on the halogen lamp and the condensers is provided in the Axiovert 200 M
operating manual.
(4) Reflected light fluorescence
With the exception of the reflector slider, all the Axiovert 200 M fluorescence accessories can be used.
Further information is provided in the Axiovert 200 M operation manual.
(5) Imaging optics
Optovar sliders cannot be used.
The analyzer for the 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 does not feature an integrated SLR-port, but microscope cameras as described in the
Axiovert 200 M and LSM 510 / LSM 510 META operation manual can be used.
(7) TV adaptation
The TV port at the side and the tubes can be used as described in the Axiovert 200 M operation manual.
The TV interface side port can be used with TV adapters 60 N or LSM adapters.
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Carl Zeiss
3.4.3
INTRODUCTION …
Performance Features of the ConfoCor 3 …
ConfoCor 3
Computer Hardware and Software
The LSM 510- and LSM 510 META-ConfoCor 3 are controlled via a standard high-end Pentium PC.
Linking to the electronic control system is made via Gigabit Ethernet interface. The PC comes with the
WINDOWS XP operating system.
The instrument is fully motorized, permitting fast change-over between methods as well as automatic
operation. Parameters once set or complex examination sequences once established can be saved and
reproduced; therefore, complete application programs can be loaded and performed by pushbutton
control.
The software of the LSM 510, LSM 510 META and ConfoCor 3 have 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 an enormously wide range of image processing functions, including all standard
2D/3D (stereo, projection) functions identical to sophisticated 3D reconstruction capabilities (surface and
alpha rendering), digital processing of voxels and 3D measurement functions (surface areas, volumes).
As all files and images are recorded in MS Access databases, elegant image database editing is just as
easy as transferring the records to other programs.
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Contents
ConfoCor 3
CHAPTER 4
Carl Zeiss
CONFOCOR 3 IN COMBINATION
WITH LSM 510 AND
LSM 510 META
CONTENTS
Page
4
OPERATION OF CONFOCOR 3 IN EXPERT MODE .............................................. 4-4
4.1
Main Menu for ConfoCor 3 ........................................................................................... 4-4
4.2
File Menu for ConfoCor 3 .............................................................................................. 4-6
4.3
Acquire Menu for ConfoCor 3 ....................................................................................... 4-7
4.3.1
4.3.1.1
4.3.1.2
4.3.2
Micro Button..................................................................................................................... 4-8
Reflected-light Observation (Epi-fluorescence) ................................................................... 4-9
Transmitted-light Observation ........................................................................................... 4-9
VIS, TV, LSM Buttons......................................................................................................... 4-9
4.4
ConfoCor Menu for ConfoCor 3 .................................................................................. 4-10
4.4.1
4.4.2
4.4.3
4.4.4
4.4.4.1
4.4.4.2
4.4.4.3
4.4.4.4
4.4.5
4.4.6
4.4.7
4.4.7.1
4.4.8
4.4.8.1
4.4.8.2
4.4.8.3
4.4.8.4
4.4.9
4.4.10
4.4.10.1
4.4.10.2
Open File ........................................................................................................................ 4-10
Laser Control for ConfoCor 3.......................................................................................... 4-11
Methods ......................................................................................................................... 4-11
Adjust ............................................................................................................................. 4-13
Define a New Sample Carrier .......................................................................................... 4-13
Save a New Sample Carrier ............................................................................................. 4-13
Delete a Carrier Configuration ........................................................................................ 4-14
Z (Focus) Panel ................................................................................................................ 4-14
Orienting the Carrier in X and Y ...................................................................................... 4-18
Measure.......................................................................................................................... 4-19
System Configuration...................................................................................................... 4-25
Beam Path Panel ............................................................................................................. 4-25
Adjust Pinhole Coordinate Panel ..................................................................................... 4-34
Beam Path Panel ............................................................................................................. 4-34
Adjust Coordinates Panel ................................................................................................ 4-35
Excitation Panel............................................................................................................... 4-36
Carrier Position Panel ...................................................................................................... 4-36
Adjust Collimator Panel................................................................................................... 4-38
Acquisition...................................................................................................................... 4-39
Times Panel ................................................................................................................ 4-39
Kinetics Panel ............................................................................................................. 4-40
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
Contents
ConfoCor 3
4.4.10.3
4.4.10.4
4.4.10.5
4.4.10.6
4.4.10.7
4.4.10.8
4.4.11
4.4.12
4.4.13
4.4.13.1
4.4.13.2
4.4.14
4.4.15
4.4.15.1
4.4.15.2
4.4.16
4.4.16.1
4.4.16.2
4.4.16.3
4.4.16.4
4.4.16.5
4.4.16.6
4.4.16.7
4.4.17
4.4.17.1
4.4.17.2
4.4.17.3
4.4.17.4
4.4.17.5
4.4.18
4.4.18.1
4.4.18.2
4.4.18.3
4.4.19
4.4.19.1
4.4.19.2
Positions Panel ............................................................................................................4-42
Moving the Scan Mirrors .............................................................................................4-42
Moving the Microscope Stage .....................................................................................4-43
Current Position ..........................................................................................................4-44
Using the Crosshair Function .......................................................................................4-44
Using the Carrier .........................................................................................................4-44
Sample Carrier .................................................................................................................4-45
Loading and Configuring Sample Carriers ........................................................................4-45
Selecting a Chamber at the Sample Carrier ......................................................................4-47
Single Position .............................................................................................................4-47
Multiple Positions ........................................................................................................4-48
LSM Image.......................................................................................................................4-49
Processing........................................................................................................................4-51
Fit Panel ......................................................................................................................4-51
Data Handling Panel ....................................................................................................4-52
Fitting the Correlated Data to Model Equations................................................................4-54
The Correlation Function .............................................................................................4-54
Model Equations .........................................................................................................4-55
Amplitudes..................................................................................................................4-56
Anti-bunching Terms ...................................................................................................4-59
Bunching Terms ..........................................................................................................4-61
Diffusion Terms ...........................................................................................................4-65
Photon Counting Histogram (PCH) ..............................................................................4-70
Models.............................................................................................................................4-71
Close...........................................................................................................................4-71
New ............................................................................................................................4-71
Modify ........................................................................................................................4-71
Delete .........................................................................................................................4-71
Defining a Model ........................................................................................................4-72
Settings............................................................................................................................4-80
Measurement ..............................................................................................................4-80
Auto Save ...................................................................................................................4-83
LSM + ConfoCor .........................................................................................................4-83
Compensating Offsets between FCS and LSM ..................................................................4-84
Determination of the Offset between LSM Scanner and FCS Stage Positioning ............4-84
Determination of the Offset between LSM Scanner and FCS Scanner Positioning.........4-92
4.5
Data Evaluation and Result Presentation for FCS Measurements.............................4-94
4.5.1
4.5.2
4.5.3
4.5.3.1
4.5.3.2
4.5.3.3
4.5.4
4.5.5
4.5.6
4.5.7
Structure of the Data Evaluation Window.........................................................................4-94
Open / Close the Data Evaluation Window .......................................................................4-95
Description of the Raw Data Format.................................................................................4-96
ConfoCor 1......................................................................................................................4-96
ConfoCor 2......................................................................................................................4-96
ConfoCor 3......................................................................................................................4-98
Display - Correlation.........................................................................................................4-99
Display - Fit ....................................................................................................................4-105
Display - Coincidence .....................................................................................................4-111
Display - Preview ............................................................................................................4-112
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
Contents
Carl Zeiss
4.5.8
4.5.9
4.5.10
4.5.11
4.5.12
4.5.13
4.5.14
4.5.14.1
4.5.14.2
4.5.14.3
4.5.14.4
4.5.15
Display - Save Data........................................................................................................ 4-114
Display - Copy Table...................................................................................................... 4-114
Display - Save Table....................................................................................................... 4-114
Display - Copy Graphs................................................................................................... 4-114
Display - Export ............................................................................................................. 4-115
Reuse............................................................................................................................ 4-115
Reload .......................................................................................................................... 4-115
Automatic Dust Filter................................................................................................ 4-116
Correlator Settings ................................................................................................... 4-116
Count Rate Settings.................................................................................................. 4-116
Photon Counting Histogram Settings........................................................................ 4-117
Info............................................................................................................................... 4-117
4.6
Combined Application of LSM Scanning and FCS Measurement Procedure ......... 4-118
4.6.1
4.6.1.1
4.6.1.2
4.6.1.3
Taking FCS Measurements within a Cell ........................................................................ 4-118
Using the Current Position ............................................................................................ 4-118
Using the LSM Image Window ...................................................................................... 4-119
Performing FCS Measurements on the Cell Membrane .................................................. 4-120
4.7
Index ........................................................................................................................... 4-122
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
Main Menu for ConfoCor 3
ConfoCor 3
4
OPERATION OF CONFOCOR 3 IN
EXPERT MODE
4.1
Main Menu for ConfoCor 3
The major functions can be selected in the Main
menu of the Expert Mode either via the pull-down
menus in the menu bar or via the Main menu
toolbar which can be displayed or removed as
required.
Further subordinate toolbars are available below
this toolbar, depending on which button has just
been pressed (File, Acquire, etc.).
Fig. 4-1
LSM 510-ConfoCor 3
Switchboard window
In the standard setting of the LSM 510ConfoCor 3 software, the toolbars are automatically displayed after the start of the Expert
Mode.
However, since the LSM 510-ConfoCor 3 software is operated more conveniently with the help
of the toolbars, only this method of function activation will be described in the following.
• Click on the Start Expert Mode button in the LSM 510-ConfoCor 3 Switchboard window.
− The LSM FCS - Expert Mode Main menu appears on the screen.
The Acquire button is active automatically, and the submenus selectable in it are shown in the second
(bottom) toolbar. Fig. 4-1
Fig. 4-2
4-4
LSM FCS- Expert Mode Main menu
File button
Open, save, import and export of image data. Printing
individual or several images on one page. Ending (Exit) the
Expert Mode.
Acquire button
Calling up and setting the necessary operating parameters.
During the preparation for and execution of laser scan image
acquisition, this menu item is used as the working dialog
between the computer and the microscope.
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ConfoCor 3
OPERATION OF CONFOCOR 3 IN EXPERT MODE
Main Menu for ConfoCor 3
Carl Zeiss
Process button
Used for processing of acquired images.
3D View button
Used for three-dimensional reconstruction.
Macro button
Makes it possible for the user to store frequently used processes
(Macro recorder) and to run them automatically (Macro play). It
is possible to write new macros or to edit existing ones.
ConfoCor button
Loading FCS data files and methods; defining and selecting
models; define settings; executing measurement and data
analysis
Options button
For custom-configuration of software and hardware options,
and for exporting system operating sequences to the Routine
Mode.
This menu item enables access to the coloring table.
In the Settings window you can specify essential operating
modes and informative help, organized by tabs, which have an
effect on the user interface.
Maintain button
03/06
Service mode for adjustment and setting of other parameters
(e.g. objectives).
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Carl Zeiss
4.2
OPERATION OF CONFOCOR 3 IN EXPERT MODE
File Menu for ConfoCor 3
ConfoCor 3
File Menu for ConfoCor 3
The functions of the File menu permit images and the relevant information to be managed and handled
completely in a database system. You can also create your own databases. The databases allow images to
be stored, loaded and deleted. The additional functions Import and Export permit images from other
systems to be made available to the LSM 510-ConfoCor 3 software, or the export of images to other
software packages. The Print function allows individual or several images to be arranged on a print page
for printout. The Expert Mode can be ended via the Exit function.
• In the Main menu toolbar, click on File.
− This opens another, subordinate toolbar in the Main menu.
Fig. 4-3
LSM-FCS - File menu
The New button allows you to create a new database
The Open button allows you to open an existing database
The Save button allows you to save an existing database with the name and file location unchanged
The Save As button allows you to save an existing database under another name and different file
location.
The Import button allows you to import an existing image. You have the option of different file formats.
The Export button allows you to exort the highlighted image in different file formats.
The Multi Print button allows you to print a highlighted image.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
Acquire Menu for ConfoCor 3
ConfoCor 3
4.3
Carl Zeiss
Acquire Menu for ConfoCor 3
• In the Main menu toolbar, click on Acquire.
− This opens another, subordinate toolbar in the Main menu.
Fig. 4-4
LSM-FCS - Acquire menu
For preparing and acquiring a scanning image, it is recommended to call up and use the tools of the
subordinate toolbar in the following order:
− Conventional microscope setting.
− Laser setting.
− Configuring the optical system for the Scanning Mode.
− Setting of scan parameters.
− EditROI permits up to 99 regions within a frame to be defined and scanned.
− TimeSeries permits user-specific time series to be selected for the scan procedure.
− The EditBleach function is used to bleach a defined, freely selectable area within the scanning
field.
− Upon selecting Stage you can set the focus (Z coordinate) and the Z step size between successive
slices. If the optional, motorized X/Y-stage is connected, the X and Y-positions of the sample can
also be selected.
− The VIS, FCS and LSM buttons switch the beam path and indicate which beam path has been set
in the binocular tube of the microscope (VIS for viewing, FCS for FCS measurements via laser
excitation and monitor observation, LSM for laser scanning operation with monitor observation).
• For the scanning process, the LSM button in the toolbar subordinate to the Acquire item must be
activated.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
Acquire Menu for ConfoCor 3
Carl Zeiss
4.3.1
ConfoCor 3
Micro Button
The Microscope Control (Micro button) window
permits motorized functions (objective and
reflector change settings) and the illumination
mode (transmitted light) of the connected
microscope to be controlled via the software (see
Fig. 4-5).
Without any difference to software control, these
microscope functions can also be operated directly
on the stand via the relevant controls. In that case,
any changes are recorded by the software and
displayed in the relevant windows / panels.
• Click on the Micro button.
− This opens the Microscope Control window
on the screen.
After conclusion of the conventional setting of the
connected microscope, the Microscope Control
window can be closed again.
• Click on the Close button in the Microscope
Control window.
Fig. 4-5
Microscope Control window
− The Microscope Control window will be
closed.
The Microscope Control window contains the following functions:
Transmitted Light
button
Transmitted light is switched on / off via ON button in the Transmitted Light
frame, setting of light intensity can be varied via input box or slider. 3200 K
color temperature for photo documentation can be switched on via 3200 K
button in the Transmitted Light frame. The transmission light control
potentiometer on the stand is disabled via the Remote button. By clicking on
the Close button the Transmitted Light frame is closed.
Condensor button
Numerical aperture of the condensor is set via input box or slider. Turret
position selected from graphical pop-up menu (only for motorized
condensors). By clicking on the Close button the Condensor frame is closed.
Objective button
Objective can be selected via graphical pop-up menu. Please note, that for FCS
only the C-Apoochromat 40x/60x W N1.2 are specified
Reflector button
Push and click, reflector cube can be selected via graphical pop-up menu.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
Acquire Menu for ConfoCor 3
ConfoCor 3
Tube Lens button
Push and click, tube lens can be selected via graphical pop-up menu.
Reflected Light button
The shutter is switched on and off.
4.3.1.1
Carl Zeiss
Reflected-light Observation (Epi-fluorescence)
• Turn on the HBO 50 power supply switch.
• Click on the check box for Reflected Light
On.
• In the Reflector Turret list box, select the desired filter set by clicking on it.
− The filter is automatically moved into the beam path to enable observation in epi-fluorescence.
• In the Tubelens list box, select the desired tube lens by clicking on it.
• Swing the required objective for FCS measurements into the working position. This is performed by
selecting the objective in the Objective selection box in the Axiovert Control window.
We recommend to use the C-Apochromat 40x/1.2 W corr on account of its optimized optics.
4.3.1.2
Transmitted-light Observation
• Click on the check box for Transmitted Light
On.
• Activate the condensor function in the Condensor panel.
• Swing the required objective for FCS measurements into the working position. This is performed by
selecting the objective in the Objective selection box in the Axiovert Control window.
• Select Light Remote or 3200 K or set the transmitted light intensity via slider.
4.3.2
VIS, TV, LSM Buttons
The VIS, TV and LSM buttons switch the beam path of the microscope (VIS for viewing, TV for camera,
LSM for LSM laser operation with monitor observation).
For the measuring process, the LSM button in the toolbar subordinate to the ConfoCor item must be
activated.
Please note, in the LSM 510 – ConfoCor 3 the major beam splitter is shared between the
systems. A change in its setting will only affect the system, where the change was made.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
4.4
ConfoCor 3
ConfoCor Menu for ConfoCor 3
• In the Main menu toolbar, click on ConfoCor.
− This opens another, subordinate toolbar in the Main menu.
Fig. 4-6
ConfoCor menu
For preparing and measuring, it is recommended to call up and use the tools of the subordinate toolbar
in the following order:
− Open File
− Laser
− Methods
− Adjust
− Measure
− Models
− Settings
4.4.1
Open File
This function is intended for use of already measured data in the FCS mode. It also allows you to import
data stored with the ConfoCor 1, ConfoCor 2 or the ConfoCor 3. In addition, raw data stored with the
ConfoCor 2 and ConfoCor 3 can be loaded.
• To open previously stored data, click on the
Open File button in the ConfoCor subordinate
toolbar of the Main menu.
− This opens the Open FCS file window for
the selection of drives, directories and
subdirectories in which data files have been
stored.
• If you want to load a data file in another folder
(drive / directory), click on the arrow button to
the right of the Look in box.
Fig. 4-7
Open FCS file window
− This opens a drop-down list box in which
you can select from all available folders.
• Select the appropriate data file(s) via mouse click and click on the Open button.
− This opens the FCS data file in a separate window from saved data or opens FCS data files in one
window of exported data.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
4.4.2
Carl Zeiss
Laser Control for ConfoCor 3
The Laser function allows you to switch on and off
the lasers and set the tube currents if applicable.
• Click on the Laser button in the ConfoCor
subordinate toolbar of the Main menu.
− This opens the Laser Control window (see
Fig. 4-8), which consist of two panels. The
upper Lasers panel lists all the available
lasers, that can be selected within the display
box. The selected laser can than be
controlled then in the lower panel that bears
the name of the selected channel.
Fig. 4-8
Laser Control window
− The Argon laser is first switched to Standby, until it is warmed up. Then the laser can be switched
on by pressing the On button and the tube current set by the Output (%) slider. A good value is
setting the tube current to 50% output. The laser can be switched off by pressing the Off button
or brought to standby by pressing the Standby button. Whenever the laser is required, he will
automatically go from Standby to On and does not need to be switched on in this menu. Other
lasers with a constant tube current can be either switched On or Off.
4.4.3
Methods
The Methods function shows all existing measure methods for selection or deletion.
• Click on the Methods button in the ConfoCor subordinate toolbar of the Main menu.
− This opens the Select Method window (see Fig. 4-9). The name of the currently selected method
will appear in the status bar of the window.
Fig. 4-9
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Select Method window
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4-11
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
ConfoCor 3
• You can select the analysis method you want to use by double-clicking on the corresponding list entry.
After the double click the analysis method is selected and the Select Method... window will be closed
automatically.
• Alternatively you can select the analysis method by a single click – which will highlight the selected
method – and a subsequent click on the OK button. The window will then be closed automatically.
• The Cancel button of the Select Method... window allows you to leave the dialog without any
action.
• The Print button opens the Print Setup window. Set the print parameters and start the print function
by clicking OK. The Print Setup window is closed automatically.
• The Delete button allows you to delete a method from the database. To do so, click on the method
entry in the list and then click the Delete button. A window will pop up requesting confirmation of
this action. If you confirm, the method will really be deleted from the database.
Use this function with extreme care! You might lose valuable data!
• After a click on the Info button the Method Information window will pop up which shows a
detailed description of the analysis method (see Fig. 4-10).
All parameters will load with this method.
Fig. 4-10
Method Information window
• Click on the Copy All button to copy the details to the clipboard. Afterwards you can insert the
details in a WINDOWS application (e.g. Winword) and save it.
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ConfoCor 3
Carl Zeiss
• Click the Print button to print the details table. This opens the Print Setup window. Set the print
parameters and start the print function by clicking OK. The Print Setup window is closed
automatically.
• Click the Close button to close the Method Details window.
4.4.4
Adjust
The Adjust button opens the Carrier Position
window (see Fig. 4-11), which is used to define
and orient the sample carrier.
4.4.4.1
Define a New Sample Carrier
• In the Carrier Position window, use the select
dropdown menu box, if you want to edit a preexisting carrier than press the Properties
button. If you want to create a new carrier, just
press the Properties button. The selected
Carrier configuration will be displayed. Enter the
number of chambers and the distance (mm)
between the chambers in the appropriate
Column and Row input boxes.
4.4.4.2
Fig. 4-11
Carrier Position window
Fig. 4-12
Save Sample Carrier window
Save a New Sample Carrier
• Click on the Save button to save the new
sample carrier.
− The Save Carrier window appears on the
screen.
• Choose a memorable name for the new sample
carrier which you are likely to remember.
• Click on OK to save the new sample carrier.
• Click on Cancel if you don’t want to save.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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Carl Zeiss
4.4.4.3
ConfoCor 3
Delete a Carrier Configuration
A no longer required sample carrier can be deleted as follows:
• Select the sample carrier to be deleted from the Sample Carrier selection box.
• Click on the Delete button. Confirm the deleting in the following window by clicking OK.
• When you are finished, close the Carrier Definition window by clicking on Close.
4.4.4.4
Z (Focus) Panel
The functions of the Z (Focus) panel allows you to
position the focus in Z direction (see Fig. 4-13).
Following function elements are available:
Laser On/Off button
Switches selected laser lines on/off
Fig. 4-13
Z (Focus) panel
Z-position display field
Displays the current Z position.
Store button
Stores the current Z position.
Reuse button
Moves the z drive to the stored Z position.
Work button
Moves the stage / nosepiece back to the Work position. This is the position last set before the Load
button was pressed.
Load button
Lowers the stage / nosepiece to make it easier for you to change the sample carrier (or objective).
+200 µm button
Moves the Z drive for +200 µm per mouse click (upwards).
-200 µm button
Moves the Z drive for –200 µm per mouse click (downwards).
The Z-position stored by activation of the Store button is a relative parameter and is only valid
during the current session. Accordingly, this position can be approached in a defined way only
during this session via Reuse.
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. The position of the glass surfaces is detected most conveniently via line scanning.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
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.
• Choose a chamber by clicking on one in the
sample carrier drawing in the X, Y (Stage)
panel of the Carrier Position window.
• Set up a beam path in Configuration Control
(see Fig. 4-14). Convenient settings are 488 line
at 2 % AOTF, 80/20 splitter in the major beam
splitter and an appropriate channel and
emission filter, for example channel 3 with a
KP680 id available.
Fig. 4-14
Beam path configuration for finding
glass reflection
Fig. 4-15
Image settings for line scan mode
• Set up scan mode (see Fig. 4-15). Choose Line
scan, highest Scan speed, 512 x 512 and Zoom
1 for convenience.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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Carl Zeiss
ConfoCor 3
• Set up scan control (see Fig. 4-16). Open
Pinhole to maximum. Set detector gain between
250 and 300, and laser power to 2 %.
Fig. 4-16
Channel settings
reflection
for
finding
glass
• Start scan and press Diagr button (see
Fig. 4-17).
• The diagram shows a line with a hill, which is
the result of reflected light not focused yet on
the lower glass surface. You might adjust the
line to lower or higher values by altering the
gain.
Fig. 4-17
4-16
Line Scan diagram far away from the
lower cover slip surface
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
• 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 position of the line will shift
to higher values (see Fig. 4-18).
Fig. 4-18
Line diagram near the lower cover slip
surface
Fig. 4-19
Line diagram at lower cover slip surface
• Continue cautiously moving upwards. The line
position will reach its maximum (see Fig. 4-19).
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
ConfoCor 3
• Having cautiously moved over a short distance
the line position will decline again to a
minimum and than rise again to a second
maximum (see Fig. 4-20), which corresponds to
the upper cover slip surface has been found.
• Store the reached position using the Store
button.
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.
Fig. 4-20
Line diagram at upper cover slip surface
4.4.5
Orienting the Carrier in X and Y
• Click on the Orientation button in the X, Y
(Stage) panel
Fig. 4-21
Help window
• Select one of the chambers of the sample for
orientation in x and y.
• Position the center of one 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 the Orientation automatically deactivates. The stage is now oriented.
• Now you can move any chamber into the measurement position by clicking the appropriate field on
the sample carrier.
The selected chamber is then automatically approached via the motorized microscope stage. When the
defined position has been reached, the position of the chamber is displayed in the lower cleft corner in
the Positions panel and is indicated in green.
By pressing the ? button, a Help window appears (see Fig. 4-21), explaining the work flow for
orienting the Carrier.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
4.4.6
Carl Zeiss
Measure
The Measure function opens the Measurement
window. It is used for optimizing an existing
method or for creating a new one.
• Click on the Methods button in the ConfoCor
subordinate toolbar of the Main menu
(see Fig. 4-6).
− The Measurement window will appear (see
Fig. 4-22).
A measurement has three major sections:
− System Configuration. It is used for
defining beam paths and methods and
adjusting the pinholes and collimators.
− Acquisition. It is used for setting the
measurement parameters and positioning
the laser beam in respect to the sample via
the stage or the scanners.
− Processing. It is used for analyzing the data
and how data are handled.
− These major sections are available in
separate subwindows by clicking the
appropriate button in the Method
Optimization window.
The following functions are available on the righthand side of the Method Measurement window:
Fig. 4-22
(1)
Measurement window
Close button
The Measurement window is closed.
(2)
Open button
The Select Method window (see Fig. 4-23) will open that allows you to select a predefined method.
Fig. 4-23
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Select Method window
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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Carl Zeiss
(3)
ConfoCor 3
Save button
The Save Method window will appear. Saves the optimized or new created measurement method.
(4)
Info button
The current Method Information window will appear that inform you on the actual settings of the
method (see Fig. 4-24).
Display - Info
• The Info button is used to open the current Method Information window in which all the relevant
parameters of the method are displayed.
Fig. 4-24
Method Information window
• With Copy All, the complete parameter set will be copied to the clipboard.
A click on the Print button opens the Print Setup window and allows the parameters of the method to
be printed in the form of a table.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
(5)
Carl Zeiss
New button
Opens a new Data Display Window.
(6)
Start button
Starts the measuring procedure.
Measurement procedure
After setting the parameters the measurement can be run.
• Click on the Start button on the right-hand side of the Measurement window to start the
measurement and data analysis process.
− The FCS data evaluation window will appear.
Fig. 4-25
FCS data evaluation window
The further procedure will be explained in section 4.5, Data Evaluation and Result Presentation
for FCS Measurements.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
(7)
ConfoCor 3
Single button
Starts a single measurement with the current settings.
(8)
Stop button
Ends the measuring procedure.
(9)
Count rate button
Opens the Count rate window (see Fig. 4-26).
(a)
Count rate window
• Click on the Count rate button on the righthand side to open the Count rate window.
In two Channel and one Correlation panels, the
Count rate window shows the values for Count
rate, Correlation or Counts / Molecule depending
on the activated Display button.
Fig. 4-26
Count rate window
The Channel panels and the Correlation panel
feature a big display field for the count rate and, at
the underside, a scale allowing the measuring
range for the displayed count rate to be read from
a colored bar. A click on one of the four arrow
buttons permits the measuring range display to be
narrowed or widened.
The buttons in the lower range of the Count rate
window have the following functions:
(b)
Unit
Selecting the unit Hz or kHz.
(c)
Average
Averaging after 1 s, 10 s or Off.
(d)
Display
Selecting one of the display modes Count rate, Correlation or Counts / Molecule. The arrow buttons
allow to adjust the scale bar range.
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ConfoCor 3
Carl Zeiss
(10) X,Y,Z buttons
Opens the X,Y,Z-Scan window (see Fig. 4-27) for performing a X,Y,Z-scan.
(a)
X,Y,Z-Scan for positioning the focus within structured samples
To make the positioning of the detection volume easier, the ConfoCor 3 is provided with a X,Y,Z-Scan
possibility.
This means, that the focus is moved in either the X, Y, or Z direction over a preselected distance while the
count rate in one or both of the detection channels as defined by the channel check boxes is recorded. A
maximum (or minimum) in the resulting curve (Fig. 4-28) usually indicates a region of interest. The focus
can be positioned to this region by a simple mouse click.
• Click on the X,Y,Z-Scan button on the right-hand side of the Method Optimization window.
− The X,Y,Z-Scan operating window appears on the screen (Fig. 4-27).
Fig. 4-27
(b)
X,Y,Z-Scan operating window
For X,Y,Z-Scanning proceed as follows:
• Press the VIS button.
• Position the object in X and Y.
• Find the upper cover slip surface (interface between the glass and the cell) by carefully focusing the
objective.
• Switch to the FCS mode by clicking on the FCS button in the Analysis subordinate toolbar.
The next steps depend on whether you already have a functional measurement method for your
particular problem or not.
• If you already have a functional method: Select the method first using the Select Method button in
the Analyse FCS subordinate toolbar of the Main menu.
• If you do not have a functional method: Create a new method.
• Select the Channel, Start and End position and the Number of positions.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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Carl Zeiss
ConfoCor 3
• Clicking on Start opens a new window showing a count rate graph (Fig. 4-28).
Fig. 4-28
Z-Scan result window
When the scan has been finished, a vertical red line appears at the current position. This line can be
dragged with the mouse when the left button is held down.
• Position the vertical red line where the focus should be placed by using the mouse in case of a z-Scan,
or where the sample should be positioned in x or y.
• Then close the window with Close. The Refresh Positions button will actualize the current position if
pressed.
Please note, that X, Y scans are performed by the stage regardless if the Scanner button is
pressed or not.
The data of the X,Y,Z-Scan result window can be
copied to the clipboard via the context menu or
saved directly as an ASCII file.
Fig. 4-29
Context menu of the
X,Y,Z-Scan result window
• Click in the X,Y,Z-Scan result window with the
right mouse button to open the context menu.
• Select the line Copy data to clipboard with a
click of the mouse if you want to insert the data
into other WINDOWS programs directly via the
clipboard. The Paste function enables you to
insert these data directly into the required
program.
• Select Export data to file to save the data in
an external ASCII file (.txt).
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
4.4.7
Carl Zeiss
System Configuration
• Click on the System Configuration button on top of the Measurement window (Fig. 4-22).
− The System Configuration subwindow appears on the screen.
4.4.7.1
Beam Path Panel
The Beam Path panel allows new / existing beam path configurations to be created / edited and saved.
The Delete function enables you to delete existing beam paths.
• Click on the System Configuration button in the Measurement window
− The Beam Path window will be displayed as the upper submenu.
• When the beam path definition is finished, you can use the buttons on the right to start a task.
Fig. 4-30
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ConfoCor - Beam Path panel
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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Carl Zeiss
(1)
ConfoCor 3
Loading a predefined beam path
In the Beam Path panel you can select a saved beam path configuration by the drop down menu. The
selected beam path will be shown as a drawing. The delete button will delete the selected beam path.
If a two-channel beam path is selected, you can activate / deactivate both the channels to be used and
the calculation of the cross correlation. You can obtain cross-correlation curves by either shifting channel
1 versus channel 2 (Ch1 ->2) or channel 2 versus channel 1 (Ch2->1).
• Select a corresponding beam path from the Beam Path list box.
• Activate / deactivate the channels and the cross correlation function via the appropriate check boxes.
It is possible to define a new beam path or edit a loaded beam path in this menu. If a new
settings are required, please select the correct filters and beam splitters, press Save, select
Only beam path, make the appropriate entries for Name and press Ok. Once a new beam
path is ready, it can be immediately used in the Measurement window.
(2)
Define a beam path
(a)
Light path definition
The following filter and dichroics can be set.
button
Activation / deactivation of a main dichroic beam splitter (HFT) through selection from the relevant list
box (see Fig. 4-30). The HFT reflects the specified laser lines and allows the resulting fluorescence
spectrum to pass through. Note that the HFT is from the LSM module.
button
Activation / deactivation of secondary dichroic beam splitter (NFT) through selection from the relevant list
box (see Fig. 4-30). The NFT splits the fluorescence spectrum onto the various detection channels.
button
Activation / deactivation of an infrared blocking filter (IBF) through selection from the relevant list box
(see Fig. 4-30). The IBF is used to block any IR excitation light in NLO applications.
button
Activation / deactivation of an emission filter (EF) or infrared blocking filter (IBF) through selection from
the relevant list box (Fig. 4-30). The EF is used to narrow the fluorescence spectrum. The bandpass (BP)
allows the range within the specified border wavelengths to pass through. The longpass (LP) allows the
range above the specified border wavelength to pass through. TH short pass (KP) allows the range below
the specified border wavelength to pass through.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
Channel check boxes
Selection of the channels to be used:
−
Ch1 and Ch2 are physical channels for auto-correlation
−
Ch2 → Ch1 and Ch1 → Ch2 are software channels for cross-correlation
Ch1 / Ch2 input boxes
For entering of a special comment (e.g. used dye).
Save button
For saving the new or edited configuration.
Delete button
Deletion of an existing configuration.
(b)
Laser definition
Press the Excitation button and the laser control
window will appear. It consists of two panels, one
for the laser settings that will be used for the
measurement and pinhole adjustment and the
second for those used for the pre-bleach (see
Fig. 4-31).
Note, that lasers for measurements and
pre-bleach can be selected independently
from each other.
The Excitation panel allows to activate / deactivate the wavelengths via check boxes and to set
the requested laser attenuation (%) using the
sliders and by choosing the power attenuation in
the selection box.
In the AOTF Dampening Factor (%) box the
AOTF can be set to suppress the laser light over all
by a factor of 1, 10, 100 and 1000 fold by moving
the slider to the respective dampening factor of a
100, 10, 1 an 0.1, respectively.
Fig. 4-31
Laser attenuation window
If Automatic laser dampening is activated under FCS Settings, the range of available
attenuation is increased to lower power settings. In this case the AOTF Dampening Factor (%)
box is not displayed. If Automatic laser dampening is not activated under FCS Settings, the
AOTF Dampening Factor (%) box is displayed. Any change in one of the dampening boxes is
updated in the other.
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
Furthermore you can open the Laser Control
window via Laser button to switch on / off the
necessary lasers.
• Activate / deactivate the laser line via mouse
click.
• If necessary switch on / off the lasers using the
Laser button. When finished, close the Laser
Control window.
• Set the laser attenuation using the sliders. For
this purpose, open the Count rate window by
clicking the Count rate button on the righthand side of the Measurement window.
Fig. 4-32
Laser control window
A good starting point is to set the intensity in such
a way that a count rate between 50 kHz and
200 kHz is obtained.
• For most dyes, the Counts/Molecule setting
should be optimized in a second step to a value
just under its maximum by changing the laser
power.
If carriers of different slide thickness are employed
the Counts / Molecule setting should be optimized
by using the correction ring on the lens.
The correction ring is turned counterclockwise or
clockwise until a maximum value is obtained. The
correction ring should also be used for adjusting
the Counts / Molecule setting whenever the
immersion media is changed. This is especially
important in cases where the refractive index of
the immersion media is different from that of the
sample.
• When finished, close the Count Rate window
using the Close button.
Fig. 4-33
4-28
Count rate window
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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Carl Zeiss
The Close button will close the Laser attenuation
window and returns to the Beam Path panel.
The Laser button will open the Laser control
window (see Fig. 4-32). The Lasers panel shows
the types, excitation wavelengths and operating
statuses of the lasers available.
The subordinate laser settings panel shows the
relevant and currently set Maximum Power,
Wavelength, Status, Tube Current and
Output [%] values of the current laser. The
buttons On, Off and Standby permit the current
laser to be set in the required status, and the laser
intensity (Output) can be set using the slider or
the input box. The name of the selected laser
(Argon, HeNe1 or HeNe2) is displayed in the
headline of this setting panel for checking.
• Click on the Laser button in the ConfoCor 3
subordinate toolbar.
− This opens the Laser Control window,
which shows all lasers connected to the
system.
When the setting of the required lasers has been
finished, the Laser Control window can be closed
again.
• Click on the Close button to close the Laser
Control window.
Fig. 4-34
Measurement window with System
Configuration panel activated
− The Laser Control window will be closed.
Lasers panel (upper)
List of available lasers, including the display of relevant wavelengths and switching status. Selection of the
laser to be switched on / off and setting of the laser output is performed in the subordinate setting panel.
Laser settings panel (lower)
Switch on / off the required laser or set Standby operation. Display Maximum Power, Wavelength, Status
and Tube Current (only Enterprise and Argon) of the relevant laser. Set the laser output for Argon.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
(c)
ConfoCor 3
Settings
• Click on the desired laser on the (upper) Lasers panel.
− This highlights the selected laser.
On the lower panel of the Laser Control window, activate the laser as follows:
This applies to the Ar-multiline laser:
• Click on the Standby button.
− Wait for the laser to heat up, until the Status ready - Standby message appears.
• Click on the On button.
− Status ready - On appears.
• Use the Output [%] slider to set the laser power which is ideal for the measurement job.
Thus, the laser needed for image acquisition is available.
• Set output between 25 and 100 % of the maximum tube current.
− Optimum operation is at 8 A (lowest laser noise). However, the laser life is reduced if the laser is
constantly operated at 8 A. Therefore, 8 A should be used only if this is absolutely necessary.
This applies to HeNe lasers:
• After selecting the laser, click on the On button.
− The required laser for image acquisition is now available.
• You can switch on an off lasers and set the tube current if applicable.
Note that the Argon laser must be first set into standby and after warming can be switched on.
The Close button returns to the Laser attenuation window.
(d)
Procedure for defining a new beam path configuration
• First tick the laser or lasers (in case of cross -correlation) you need for excitation of your sample.
• Then choose a (main) dichroic beam splitter which should correspond to the chosen laser(s). Click on
the Beam Splitter symbol
and a list box will appear. Then click on the beam splitter you need.
• Select an infrared block filter in case you need one with NLO applications by clicking on the
corresponding
button.
Please note that you can use IR lasers for FCS, there is, however, no specifications for the
performance of the instrument for these excitation.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
• Select the second beam splitter in a similar way
to the first one.
− For cross correlation, choose an appropriate
filter like the NFT 488/633 beam splitter.
− For auto correlation measurements in
detection channel 1select Plate in the
second beam splitter position.
− All auto correlation measurements in
detection channel 2select Mirror in the
second beam splitter position.
03/06
Fig. 4-35
IBF Infrared Filter list box open
Fig. 4-36
NFT Secondary Beam Splitter list box
open
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
• Click on the Emission Filter symbol(s)
to
get the list box allowing you to select the
emission filters you need.
Fig. 4-37
Emission Filter 1 list box open
• It is recommended to give the channel a name
which is easy to remember.
Fig. 4-38
4-32
Emission Filter 2 list box open
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ConfoCor 3
(e)
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
Save a new beam path configuration
• Click the Save button to save the new beam
path.
− The Save Beam Path window appears.
− Activate Only Beam Path
• Choose a memorable name for the new beam
path which you are likely to remember.
Fig. 4-39
Save Beam Path window
• Click on OK to save the new beam path.
Before you can use a newly defined beam path, you have to adjust the pinhole(s) for this
particular beam path.
(f)
Delete a beam path configuration
A no longer required beam path can be deleted as follows:
• Select the configuration to be deleted from the Beam Path selection box.
• Click on the Delete button. Confirm the deleting in the following window by clicking OK.
• A beam path can only be deleted if it is not used by any method. If you intend to delete such a beam
path, you will have to delete the corresponding method first.
(g)
Pinhole panel
The Pinhole panel (Fig. 4-40) allows you to set the
pinhole diameter. It also displays the current
Collimator position. The wavelength window for
the respective Collimator is indicated to the left of
the position window.
(h)
Pinhole Diameter
In the Pinhole panel you can set the pinhole
diameters for channel 4 of the LSM using the slider
or the input box (see Fig. 4-40).
Fig. 4-40
Pinhole panel for ConfoCor 3
• It is recommended to set the pinhole diameter to a size corresponding to the used excitation
wavelength that corresponds to 1 AIRY unit:
458 nm
66 µm
488 nm
70 µm
514 nm
74 µm
543 nm
78 µm
633 nm
90 µm
Pinhole sizes are given in μm and AIRY units.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
(i)
Pinhole
Settings
Adjustment
ConfoCor 3
and
Collimator
Pressing the Adjust button will open the Pinhole
and Collimator Adjustment window. The
window has two panels, one for pinhole
adjustment (upper) and one for the collimator
settings (lower) (see Fig. 4-41).
Fig. 4-41
Pinhole and
window
Collimator
Adjustment
4.4.8
Adjust Pinhole Coordinate Panel
The pinhole is adjusted using a dye solution. The general approach is to move the pinhole cyclically in x
and y until the intensity maximum is found. For each excitation wavelength a suitable dye must be used.
We recommend:
− Rhodamine 6G for 458, 488 and 514 nm
− Tetra-Methyl-Rhodamine (TMR) for 543 nm and
− Cy 5 for 633 nm.
It is also recommended to work with a relatively concentrated solution (10-5 mol) and low laser power to
achieve smooth intensity curves.
• Click on the Adjust button in the Pinhole
panel of the Measurement window.
− This opens the Pinholes and Collimator
Adjustment window.
• Click on the Close button to quit the window.
4.4.8.1
Beam Path Panel
Here you can select the beam path for pinhole
adjustment. The date of the last pinhole
adjustment is shown in the Last Adjustment
display box.
• Select the beam path in the selection box.
− The selected beam path appears in a
drawing.
Fig. 4-42
4-34
Beam Path panel
− For the definition of a new beam path, see
section 4.4.7 System Configuration.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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ConfoCor 3
4.4.8.2
Carl Zeiss
Adjust Coordinates Panel
The Adjust Coordinates panel is used to adjust
the pinhole of channel 4 of the LSM. Adjustment
can be performed manually or automatically.
Manual adjustment is not recommended, since the
relevant procedure is very complex.
Please note that there is only 1 pinhole
for both channels. You can adjust the
pinhole to either channel. The values for
the pinhole alignment done with the last
channel will be stored and used.
Fig. 4-43
Adjust Coordinates panel
Following functions are available:
Pinhole
Selection of the channel for adjustment.
Diameter
Setting of pinhole diameter to the used wavelength (see Fig. 4-43).
Auto Adjust buttons:
− X fine / X coarse
Starts the automatic coarse or fine adjustment for X direction depending on the
activation / deactivation of the Coarse check box.
− Y fine / Y coarse
Starts the automatic coarse or fine adjustment for Y direction depending on the
activation / deactivation of the Coarse check box.
Z-positions can be changed by altering the Collimator position
Coarse check box
If ticked, the coarse adjustment for X or Y is performed, if not ticked, the fine adjustment for X or Y is
performed.
Position input boxes
Display / input of the pinhole position in X and Y.
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
4.4.8.3
ConfoCor 3
Excitation Panel
The
Excitation
panel
allows
you
to
activate / deactivate the wavelengths via check
boxes and to set the laser attenuation (%) using
the sliders. Furthermore you can open the Laser
Control window via Excitation button to switch
on / off the necessary lasers (see Fig. 4-42).
Fig. 4-44
Laser control window for measurements and pinhole adjustment
The AOTF Dampening Factor (%) display box
allows you to reduce the overall power by factors
of 1, 10, 100 and 1000.
4.4.8.4
Carrier Position Panel
In the Carrier and the Position display boxes, the
carrier and the currently selected position are
shown.
Fig. 4-45
Carrier Position panel
If the carrier or the position have to be changed for
adjustment use, the Carrier button to open the
Carrier Position panel (see Fig. 4-6).
To adjust the pinhole proceed as follows:
• Mount a carrier with a suitable dye solution and orient the sample stage as described in section 4.4.1
Open File.
• Click the Adjust button in the Pinhole panel of the Measurement window. The Measurement /
Pinhole Adjustment window will appear (Fig. 4-44).
In the Beam Path panel of the Measurement window you will see a drawing of the current beam path.
Select the beam path you want to adjust in the Beam Path selection box in the Beam Path panel of the
Measurement window.
• Select the test sample. Click on the Sample Carrier button in the Positions panel of the Acquire
window. Then select the sample by mouse click.
• Use the slider in the Laser Control window to set the laser intensity to minimum.
• Click on the Count Rate button at the right-hand side. This will open the Count Rate window.
• Then cautiously increase the laser intensity until a count rate of about 100 kHz is indicated.
• Close the Count Rate window.
• Set the pinhole diameter to a size corresponding to the used excitation wavelength:
458 nm
66 µm
488 nm
70 µm
514 nm
74 µm
543 nm
78 µm
633 nm
90 µm
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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Carl Zeiss
• Select the pinhole you want to adjust by clicking on the corresponding Ch1 or Ch2 radio button in the
Adjust Pinholes Coordinates panel of the window.
In the case of a cross correlation beam path, adjust the one pinhole to either of the two
channels.
Each of the pinhole axes must be adjusted separately. If the Coarse check box is activated, the
corresponding axis buttons will be labeled X Coarse and Y Coarse respectively and the pinhole will travel
over the maximum range for each axis.
If the Coarse check box is deactivated, the buttons will be labeled X Fine and Y Fine respectively. In this
case the pinhole travel will be limited to speed up adjustment.
If the pinholes are adjusted for the first time, the coarse adjustment must be performed first and
then the fine adjustment, each time for X, Y and Z (only coarse). For subsequent readjustments,
the fine adjustment is normally sufficient.
• Perform the pinhole adjustment in the following sequence:
X fine; Y fine
If one of the above buttons, e.g. X Coarse or X Fine etc., is clicked, the instrument will behave as
follows:
− The Automatic Pinhole Adjustment window will appear (Fig. 4-46). It is possible to exit the
process using the Cancel button. When the process is finished, quit with OK.
− In the Count Rate/Position diagram, the black line corresponds to the measured intensity (count
rate). The red line corresponds to the intensity curve fitted from it to find the optimum pinhole
position.
Fig. 4-46
Automatic Pinhole Adjustment
• Repeat the procedure for all axes.
• You will find peaks for x-Coarse, and y-coarse, in any case.
• A click with the right mouse button on the diagram opens the context menu. Selection of the option
Copy to clipboard or Export data to file permits the data to be copied to the clipboard or saved
directly in an ASCII file.
When the whole pinhole adjustment is completed, close the dialog with Close.
You might be able to increase count rate by adjusting the collimator in the Adjust Collimator
The position of the collimator influence the z-settings.
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
4.4.9
ConfoCor 3
Adjust Collimator Panel
The Adjust Collimator panel permits the setting
of the collimator for FCS measurements.
Fig. 4-47
Adjust Collimator panel
Under normal conditions, the collimator settings
should not be changed.
In some cases when the pinhole is close to the limit
of the pinhole range for a certain wavelength it
might be useful to alter the collimator settings
carefully.
This function should be used with extreme care. An unsuitable collimator setting can render the
pinhole adjustment impossible.
• To change the collimator setting, click the Adjust button in the Pinhole panel of the Measurement
window (see Fig. 4-49).
• The Collimator tab will appear in the Adjust Collimator panel.
Now the new collimator settings for the Collimator VIS or Collimator IR (if applicable) can be entered
either by drawing the slider, clicking on the arrows or directly entering a new number.
If the collimator setting is changed, an information is given indicating the previous position as
long as the x and y coordinates of the pinholes are not changed.
• Set the laser attenuation using the sliders. For
this purpose, open the Count rate window by
clicking the Count rate button on the righthand side of the Count rate window.
A good starting point is to set the intensity in such
a way that a count rate between 50 kHz and
200 kHz is obtained.
• For most dyes, the Counts/Molecule setting
should be optimized in a second step to a value
just under its maximum by changing the laser
power.
If carriers of different slide thickness are employed
the Counts / Molecule setting should be optimized
by using the correction ring on the lens.
Fig. 4-48
Count rate window
The correction ring is turned counterclockwise or
clockwise until a maximum value is obtained. The
correction ring should also be used for adjusting
the Counts / Molecule setting whenever the
immersion media is changed. This is especially
important in cases where the refractive index of
the immersion media is different from that of the
sample.
• When finished, close the Count Rate window
using the Close button.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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ConfoCor 3
4.4.10
Carl Zeiss
Acquisition
• Click on the Acquisition button on top of the
Measurement window.
− The Acquisition subwindow appears on the
screen.
4.4.10.1
Times Panel
In the Times panel you can set the values for the
Bleach Time, Measure Time and Repeat Count
(see Fig. 4-50).
Bleach Time:
Bleach Time is the time prior to the measuring
procedure during which the laser already has an
effect on the sample. The Bleach Time is taken
into consideration only once (at the beginning of a
measuring cycle).
Measure Time:
Measure Time is the period of one measurement.
Fig. 4-49
Measurement window, Application
Fig. 4-50
Times panel
Repeat Count:
The Repeat Count value determines the number
of measurements, i. e. how often Measure Time
is to be performed in a row.
• Set the Bleach Time.
Start with the Bleach Time of 0 s. If the signal
decreases during the subsequent measurement,
this might be caused by bleaching, as a remedy,
you can prebleach the sample before
measurement by setting the Bleach Time to a
non-zero value. If no laser check box was
activated, no bleaching occurs, but the
measurement will be delayed for the bleach
time. Bleaching occurs with the lasers checked
and the power defined by the AOTF setting
under
System
configuration
in
the
Measurement. If the box is checked, the laser
power defined by the slider is used for each
selected laser.
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Carl Zeiss
ConfoCor 3
• Set the Measure Time.
If you do not have any idea about the behavior of your sample, start with a measurement time of
about 20 seconds. Depending on the signal-to-noise ratio of your correlation curve, decrease or
increase the measurement time. Apart from that, set the measurement time to the correct value right
from the beginning. As a rule of thumb, the measurement time should be a 1000 fold the diffusion
time of your molecule under investigation.
• Set the Repeat Count.
Start with Repeat Count set to 1. If you want to obtain information about the variation of your fitted
values later on, increase the repeat count to produce a measurement series. 10 times is a good value
to start with
4.4.10.2
Kinetics Panel
In the Kinetics panel you can set kinetics
parameters.
Fig. 4-51
Kinetics panel
• If a kinetics measurement is required, tick the
Kinetics check box.
The input boxes Point to Point time, Number, Shape and Void (const. Shape) / Summand (linear
shape) / Factor (exponential shape) permit entry of the time spacing between consecutive and the
number of measurements.
Please note that the hierarchy in which measurements are performed is channel – repetition –
position – kinetics. For example all the selected chambers or positions are measured once in one
cycle in the determined order. We will refer to a cycle as the period between the start and end
of a measurement including all positions.
Depending on these four parameters, The total measurement time, including the time the system needs
to change position, will be displayed in the Calculated Duration display box.
Note, if the Point to Point time is chosen too short compared to the needed measurement
time, for example only 1 s if the measurement time is 10 s with 1 repetition selected, than the
system will automatically calculate the minimal needed Point to Point time, in the example
10 s.
The Number box allows you to select, how many cycles you want to perform. One cycle consists of the
measurement protocol defined in the times window and the repeats for all positions.
The Shape and Void / Summand / Factor selection boxes allow you to choose from three possibilities
of distributing the time spacing between single consecutive cycles.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
const:
The period between two successive measuring cycles is constant and will be exactly the time selected in
the Point to Point time selection box. For example, if 20 is selected, the start of each measurement
cycle is delayed by 20 s from the end of the previous one. In this case the Void / Summand / Factor box
is void and inactive.
Definition of variables:
Point to Point time of the measurement = pi (given start value=p0)
Start time of the measurement: pt,i
Number of cycles: n
The cycle count = i (note: i starts with 0; so for first cycle i = 0; or general i = n-1)
Summand/factor = f
With the definition of variables for const: pi = p0; pt,I = i x p0; i > 0
lin:
The period between two successive measuring cycles as specified in the Point to Point time selection
box is increased linearly by a summand of which the initial value is defined in the Summand selection
box. With the definitions of variables: pi=p0+(i-1) x f; pt,i= i x p0 + (i-1) x f; i>0
exp:
The period between two successive measuring cycles as specified in the Point to Point time selection
box is increased by an exponential factor of which the initial base is defined in the Factor selection box.
Note, that the first measurement is therefore assigned a 0 for the exponent.
i-1
i-1
With the definitions of variables: pi = p0 x f ; pt,i = p0 x (Σf ); i > 0
• Enter the time spacing you want to start with in the Point to Point time input box of your kinetics
measurement.
• Enter the number of repeated cycles in the Number box
• In the Shape selection box, set one of the shape options const, lin or exp.
• Enter a number in the Summand/Factor selection box.
Note, if const was selected, the Summand/Factor selection box is inactivated (void).
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
4.4.10.3
ConfoCor 3
Positions Panel
In the Positions panel you can select the carrier
and the sample position by the stage or by the
scanners.
Fig. 4-52
Positions panel with LSM image mode
activated
4.4.10.4
Moving the Scan Mirrors
To activate scanner positioning press the Scanner
button at the bottom of the panel. The Scanner
Control options will appear (see Fig. 4-53).
Fig. 4-53
Positions panel, Scanner
• Move the scanning mirrors in the appropriate
position by clicking on the arrow keys.
Each mouse click moves the scanners in the
appropriate direction by one step.
• Set the required step width via the Step slider
or the relevant input box.
The position of the scanner is indicated in the
lower X,Y display box.
If the Scanner button is activated, all positioning will be done via the scanning mirrors.
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4.4.10.5
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
Moving the Microscope Stage
To activate stage positioning click on the XY Stage
button. In this case the stage control options will
be available (see Fig. 4-54).
• Move the stage in the appropriate position by
clicking on the arrow keys.
Fig. 4-54
Positions panel, XY Stage
Each mouse click moves the microscope stage in
the appropriate direction by one step.
• Set the required step width via the Step slider or the relevant input box.
The travel speed of the microscope stage can be set to steps 1, 2 and 3.
Step 2 is preferably used for standard positioning.
Step 1 (slow speed) should only be used for very precise positioning, since positioning for longer paths
requires more time.
Step 3 is suitable if large vessels are used, since precise center positioning only plays a minor role in such
cases.
• Select the required travel speed of the microscope stage via the Speed selection box.
When 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.
If the Piezo Stage Option has been chosen the Piezo Stage instead of the normal XY stage will
be used.
If the XY Stage button is activated, all positioning will be done via the scanning stage.
There are three ways to position the laser beam with respect to the sample by pressing either of the
following buttons:
•
Current Position: no automated positioning possible. Sample has to be manually positioned in
respect to the laser beam
•
Sample Carrier: automated positioning used for maneuvering between wells of a carrier
•
LSM image: automated positioning using an LSM image
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Carl Zeiss
4.4.10.6
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
Current Position
− If the Current Position is pressed, The Positions panel changes to allow to activate the Crosshair
function. If you press the Crosshair button, a crosshair will be displayed in a scanned image. You
can position the crosshair by clicking into the image with the left mouse button. You can inactivate
the crosshair if you press the Crosshair button again. You can lock that position by pressing the
Lock button. Pressing the Lock button again, will unlock the crosshair.
− When using this method, FCS measurements are performed on a fixed position with parked
scanners and without automatic table movement. In this case the structure of interest must be
moved to the position manually under LSM control prior to FCS measurements.
The crosshair serves only the function to mark a site of interest. But the laser will stay with the
scanning mirrors parked. If you want to measure with stage movement, the crosshair can be
used to indicate the position of the laser, and the site of interest can be moved beneath it.
4.4.10.7
Using the Crosshair Function
In this mode you have to position your sample
manually.
• Activate the current pos button via mouse click
and click into the image.
Fig. 4-55
Current position panel with activated
crosshair
− The crosshair appears in the LSM scan
image. Place the cross at the position, where
the Laser beam is positioned as determined
in section 4.4.7 System Configuration. We
recommend that having set the crosshair you
do not close that Scan window. Then the
crosshair will be preserved within the new
scan. The crosshair will be positioned on the
site were you click with the left mouse
button. If you have activated the Lock
button, than the position will be fixed.
• Scan the image continuously and position the
site of interest under the crosshair.
4.4.10.8
Using the Carrier
If you press the Adjust button, the Carrier Position window will open. If you adjust the carrier (see
section 4.4.12 Loading and Configuring Sample Carriers), than the carrier will be displayed in the current
position window. You can now approach any well by clicking on it.
The wells will be approached by the scanning stage. Even if the Scanner button is activated,
selecting a well will automatically deactivate the Scanner and activates the X,Y-Stage mode. If
you take an image of the well, than the position can be changed in Scanner mode within the
well.
This mode correspond to the Sample Carrier mode, if single position is selected. Regardless
which well is selected, in the result table of the FCS results window the position is indicated as
“1”.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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ConfoCor 3
4.4.11
Carl Zeiss
Sample Carrier
The Sample Carrier function opens the Carrier
Position options. It is used for selecting the carrier
and the sample, for orienting the sample in x- and
y-direction and for setting in Z position.
• Click on the Sample Carrier button in the
Positions panel.
− The sample select options pop up (Fig. 4-56).
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.
Fig. 4-56
Positions panel, sample carrier adjusted
Fig. 4-57
Carrier Position window
• Close the Sample Carrier window by clicking
one of the other positioning options.
Note, the carrier is only visible, if it was
adjusted in the Carrier Position window
that opens by pressing the Adjust
button.
4.4.12
Loading and Configuring Sample
Carriers
Press the Adjust button. This opens the Carrier
Position window (see Fig. 4-57).
Window contains two panels:
− The (X,Y) Stage panel
− The Z (Focus) panel
On the right hand side you have the following
buttons:
Close Pressing the Close button will close the
Carrier Position window.
Laser Pressing the Laser button will open the
Laser Control window.
Micro Pressing the Micro button will open the
Microscope Control window.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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Carl Zeiss
(1)
ConfoCor 3
X, Y (Stage) panel
In the X, Y (Stage) panel you can select the
sample carrier and orient the sample carrier in xand y-direction.
(a)
Selecting the sample carrier
• Select the sample carrier from already stored
ones in the Sample Carrier selection drop
down menu. This will load in the defined
configuration.
• You can delete stored carrier configuration by
pressing the Delete button. This will delete the
currently selected carrier configuration.
(b)
Fig. 4-58
Defining a new Carrier
• Click the Properties button.
Properties window will open.
X, Y (Stage) panel
The
Carrier
The Properties windows allows new / existing
sample carriers to be created / edited and saved.
• Click on the Properties button in the Carrier Position window.
− The Carrier Definition window is opened.
Columns - Number input box
Setting of the number of columns for the sample carrier.
Columns - Distance input box
Setting of the distance between the chambers of the column.
Rows - Number input box
Setting of the number of rows for the sample carrier.
Rows - Distance input box
Setting of the distance between the chambers of the row.
Close button
The Carrier Definition window is closed.
Save button
For saving the new or edited sample carrier.
Delete button
Deletion of an existing sample carrier.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
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ConfoCor 3
4.4.13
Carl Zeiss
Selecting a Chamber at the Sample
Carrier
A scheme of the selected sample carrier is shown
in the center part of the panel. The lines of the
chambers are marked with letters, and the
columns with numbers.
You have different options to select chambers in a
loaded carrier by choosing an option from the
positions drop down menu (see Fig. 4-59).
4.4.13.1
Single Position
Select from the drop down menu Single Position.
Fig. 4-59
Positions drop down menu
Fig. 4-60
Current position panel
In the Positions panel (see Fig. 4-60) you can
select the chamber you want to take the
measurement in (only one is selectable).
• Click on the chamber you want to use. The
motorized stage moves the selected chamber to
the defined position under the objective.
• If you want to move to other positions within a
chamber, click onto the respective chamber.
The chamber to be used for the X and Y alignment
of the stage can be selected at a click of the
mouse.
Note, that before clicking on a chamber
you have to orient the carrier. For a nonoriented carrier, all chambers are
displayed in white. Please orient first
before you select a position. After
orientation, the selected well will appear
green. The position of the current well is
displayed in the left lower corner (left
field, e.g. A - 2). The current position of
the mouse pointer is shown in the right
lower corner.
• Select the chamber by clicking on it. The stage
moves to that chamber of which the
dimensions must be specified earlier. This well
becomes green when the stage has finished
movement.
You can also use the arrow buttons or the joy stick to move the microscope stage, but in those
cases, the orientation gets lost. The selected chamber will not any more be highlighted in green in
this case. You can set the size of the steps to be performed via the Step slider (or the input box).
Regardless which well is selected, in the result table of the FCS results window the position is
indicated as “1”.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
The travel speed of the microscope can be set to steps 1, 2 and 3.
Step 1 (slow speed) should only be used for very precise positioning, since positioning for longer paths
requires more time.
Step 2 is preferably used for standard positioning.
Step 3 is suitable if large vessels are used, since precious center positioning only plays a minor role in such
cases.
• Select one of the three speeds from the Speed selection box.
• Select the step size from the Step selection box with the slider or numerically.
• The current coordinate position of the stage is indicated in the lower right corner in X and Y.
4.4.13.2
Multiple Positions
Select from the drop down menu other than
Single Position.
In the Positions panel you can select the chambers
you want to take the measurement in (all are
selectable, see Fig. 4-61).
The samples to be measured can be selected in the
schematic drawing of the sample carrier. Activated
chambers will be highlighted in the drawing in
green. In the Sample Carrier display box, the
selected sample carrier is shown for information.
• Select the chambers and the work direction for
the measuring procedure by loading the
respective routine from the drop down menu.
Individual chambers can be selected / deselected by
clicking on them with the mouse.
Fig. 4-61
Sample Carrier panel
Alternatively, a whole block of chambers can be
selected / deselected by drawing a frame which
contains the centers of the selected or deselected
chambers or by pressing the Select All / Deselect
All buttons
If multiple samples are selected, the work direction can be chosen as well.
Following routines can be loaded:
Row by row. Measurement is performed row by row. Each row starts from the left site.
Row by row meander. Measurement is performed row by row. Rows start alternatively from the left
and right. First row starts from the left.
Column by column. Measurement is performed column by column. Each column starts from the top.
Column by column meander. Measurement is performed column by column. Columns start
alternatively from the top and bottom. First column starts from the top.
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ConfoCor 3
Carl Zeiss
Following functions are available:
Select All button
Selects all chambers of the sample carrier.
Deselect All button
Deselects all chambers of the sample carrier.
Adjust button
Opens the Carrier Position window.
4.4.14
LSM Image
If LSM Image is activated, you can define positions
for FCS measurements in a just scanned LSM
image (Fig. 4-62).
The Positions list shows the numbers and
coordinates of the selected positions in the LSM
scan image.
Select button
Activates the cursor (crossline) to allow the
definition of positions in the scan image. Shows or
hides just selected positions if activated or not
activated.
Please note, if the Scanner button is
pressed, the scanner will be positioned to
the high-lighted position. If the XY Stage
button is pressed, the stage will not be
positioned to the high-lighted position.
Only after triggering a measurement the
stage will move to the indicated position
and will go back to the initial position
after the end of the measurement. If you
want to obtain the count rate by pressing
the Count Rate button at the indicated
position, you have to start a measurement
beforehand.
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Fig. 4-62
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LSM image panel
4-49
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
Add Pos button
Adds the position of the cursor to the Positions
list. A crossline is set at the selected cursor position
in the image. To select further positions click at the
appropriate position in the image and then on
Add Pos. If more than one position is selected the
crosslines are getting current numbers.
Del Pos button
Deletes the selected (highlighted) position in the
Positions list and the corresponding crossline in
the scan image.
Del List button
Deletes all positions in the Positions list and all
crosslines in the scan image.
Mark Pos button
Fig. 4-63
Sample panel for defining positions
Marks the selected positions as overlay elements in
the scan image. The crosslines are also visible in
the scan image if the Select button is deactivated
or the Method Measurement window is closed.
Please note, positioning is by scanning
mirrors if the Scanner button or by stage, if the
XY Stage button is pressed. If more than one
position was selected, measurement is only taken
at the highlighted position. Using the crosshair the
mirrors do not move and positioning has to be
done manually with the stage.
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ConfoCor Menu for ConfoCor 3
ConfoCor 3
4.4.15
Carl Zeiss
Processing
• Click on the Processing button on top of the
Measurement window.
− The Processing subwindow appears on the
screen (see Fig. 4-64).
4.4.15.1
Fit Panel
The Fit panel of the menu allows you to load in a
fit model and define, how the parameters are to
be fitted.
The channel is selected by pressing either the Ch1,
Ch2 or Cross Correlation button, if applicable. To
each channel a different Fit model can be assigned.
By pressing the List button, the Model list window
will appear. You can close the window by pressing
the Close button. You can delete the highlighted
model by pressing the Delete button. You can
modify the existing highlighted model by pressing
the Modify button or you can define a new model
by pressing the New button.
Load a predefined model by selecting the
appropriate one from the Model drop down
menu.
You can select and deselect parameters by
checking / de-checking the check boxes of the
parameter column.
Fig. 4-64
Processing window
You can type in values for the Fit range start and Fit range end values.
You can type in values for the parameters under the value column.
You can define the values of the various model parameters as Start values, or you can leave them free or
fix them by selecting the appropriate routine from the pull down menu under the type column.
You can set upper and lower limits by typing the limits into the boxes under the upper limit and
lower limit columns.
You can globally link parameters for different setting by selecting the appropriate routine from the pull
down menu under the global column.
It is generally accepted that non-linear fitting procedures yield more reliable results when the number of
free parameters is low. It is recommended to fix parameters which are known from independent
measurements. Good candidates for fixing are diffusion times of the free dye and the free (i. e. not
bound) partner, which had been determined in previous measurements and the structural parameter,
that is an instrumental parameter.
If you press the Save button and select All settings, the next time the method is called up data
will be fitted to the settings defined in the Fit panel.
You can reset all manipulations by pressing the Reset button. The current status is displayed in the
Status display window.
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Carl Zeiss
4.4.15.2
ConfoCor 3
Data Handling Panel
The Data Handling panel allows you to select
how certain measurements should be recorded. All
changed settings can be reset to the default
settings by pressing the Default button (see Fig.
4-65). You have different options.
Fig. 4-65
(1)
Data Handling panel, Correlation
Correlation
Pressing the Correlation button will activate the correlation settings (see Fig. 4-65). Here you can set the
initial binning time, the initial tau channels and the maximum tau time by adjusting the number in the
Binning, Maximum correlation time and Tau Channels display boxes. You can either directly enter
values or use the arrows. The units are indicated at the right site of the display boxes. Default values can
be entered by pressing the 0.2, 1000 and 8 buttons. Please note that with the default settings, the
algorithm works the fastest.
(2)
Fig. 4-66
4-52
Data Handling panel, Count rate
Count-Rate
Pressing the Count-Rate button will activate the
count-rate settings (see Fig. 4-66). You can either
check the Automatic check-box, in which case
dynamic binning will be applied, or you can deactivate the check box and enter a number of a
constant binning time in the Binning display
window. You can either type in values or use the
arrows. Units are displayed at the right of the
display box. The default value of 1 ms can be
selected by pressing the 1 button.
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Carl Zeiss
PCH
Pressing the PCH button will activate the photon
counting histogram settings (see Fig. 4-67). You
can either check the Automatic check-box, in
which case 32 different binning times will be used,
or you can de-activate the check box and enter a
number of a constant binning time in the Binning
display window. You can either type in values or
use the arrows. Units are displayed at the right of
the display box. The default value of 10 μs can be
selected by pressing the 10 button.
Fig. 4-67
Data Handling panel, PCH
In automatic binning mode, binning starts with a value of 50 ns, which is doubled 32 times. So
n
binning times are 50 x 2 , with n=1 to 32. The histogram with the best dynamic range will be
selected and displayed.
(4)
Dust-filter
Pressing the Dust-filter button will activate the
dust-filter settings (see Fig. 4-68). The Dust-Filter
selection box allows you to activate an electronic
dust filter that will be active in operation during
the measurement. The threshold in % is set by
typing or by using the arrows. All measurement
points within a binned count rate time window
having a deviation of more than the specified value
from the average count rate will be cut out and
not used for the correlation analysis. The default
value of 10% can be activated by pressing the 10
button.
Fig. 4-68
Data Handling panel, Dust-Filter
Please note that the cut off count rate is defined as the exceeding of the average count rate
during a certain measurement period. Thus, the consecutive fast succession of low peaks might
accumulate the same count rate as one high peak within a certain period of time and hence, the
cut off is not defined by the peak height but rather by the counts / binning time.
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4.4.16
ConfoCor 3
Fitting the Correlated Data to Model Equations
In the following, the mathematical equations of the correlation functions and the fit equations will be
more detailed. The acquired correlation functions must be fitted to models in order to retrieve
meaningful parameters. It depends on the process, which model is the most appropriate. If the
underlying process is known, the model can be chosen a priori. For example, if one studies diffusion in a
membrane, a 2-D diffusion model should be applied. In other cases, the process is not known, for
example, if one deals with free or anomalous diffusion. In this case, one can screen different potential
2
models and look for the best fit taken into account the Χ value. Often two models work nearly the
same, for example, a two component free diffusion model can give you as satisfactorily a fit as a one
component anomalous diffusion model and without prior knowledge on the system it will be impossible
to decide, which is the better one. In principle,. models can be ruled out, if the fit does not work,
however, a working model is only a potential candidate but does not signify it to be the correct one. Care
should be taken to minimize the free parameters as much a possible to improve on the fit quality. It does
not make too much sense to fit to three components without fixing parameters of at least one. If , for
example, the diffusion time of a free ligand can be determined in a pre-experiment, that value should be
fixed to reduce the number of floating parameters for the evaluation of the binding experiment to its
receptor.
The software of the ConfoCor was designed to be flexible. That means that the user can define or
assemble equations that do not make sense. Care should therefore be exerted and used formulas
matched with the ones from literature to obtain meaningful results. Also, the presence of a model does
not automatically mean, that the recorded data are of a quality that allows it usage. For example, antibunching requires a lot of care in data acquisition like long measurement times and cross-correlation to
reduce dead times of the detectors and elimination of after-pulsing artefacts. It is in the responsibility of
the user to set up his experiments accordingly.
4.4.16.1
The Correlation Function
The auto-correlation function is defined as follows:
δI (t ) ⋅ δI (t + τ )
G δI (τ ) =
2
δI (t )
=
1 T
⋅ (δI (t ) ⋅ δI (t + τ ))dt
T ∫0
1
T2
T
⋅ ∫ (δI (t )) dt
T
=
T ⋅ ∫ (δI (t ) ⋅ δI (t + τ ))dt
0
T
∫ (δI (t )) dt
2
0
(1a)
2
0
or
G I (τ ) =
I (t ) ⋅ I (t + τ )
I (t )
2
=
1 T
⋅ (I (t ) ⋅ I (t + τ ))dt
T ∫0
1
T2
where
intensity.
T
⋅ ∫ (I (t )) dt
T
=
2
0
denotes the time average and
T ⋅ ∫ (I (t ) ⋅ I (t + τ ))dt
0
T
∫ (I (t ))
2
(1b)
dt
0
δI (t ) = I (t ) − I (t )
describes the fluctuations around the mean
For long time average of I (no bleaching) the following relation exists:
G I (τ ) = 1 + G δI (τ )
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(1c)
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Carl Zeiss
Definition of the cross-correlation function
The formalism for the cross-correlation function is identical to the auto-correlation function, with the
exception that the signal in one channel is not compared to itself, but to a signal in a second channel.
Lets assign the indices “r” and “b” for the red and blue channel, respectively, than the cross-correlation
function would read as follows:
G δXI (τ ) =
G XI (τ ) =
δI b (t ) ⋅ δI r (t + τ )
I b (t ) ⋅ I r (t )
I b (t ) ⋅ I r (t + τ )
I b (t ) ⋅ I r (t )
=
δI r (t ) ⋅ δI b (t + τ )
(1d)
I b (t ) ⋅ I r (t )
I r (t ) ⋅ I b (t + τ )
=
(1e)
I b (t ) ⋅ I r (t )
I
Note that the ConfoCor 3 calculated G functions. The acquired correlation functions are than compared
to model equations.
4.4.16.2
Model Equations
In the following available equations used for the fits are given that define the accessible parameters. For
some equations useful conversions to other parameters are listed as well. The total correlation is given by
equation 2:
I
(τ ) = 1 + d + B + A ⋅ ∏ ∑ G k ,l (τ )
G tot
k
(2)
l
where d is the offset, B the background correction, A the amplitude and Gk,l (τ) the correlation for a single
process. The suffixes k and l signify correlation terms for dependent and independent processes,
respectively, that are multiplied with or added to each other.
The total correlation is therefore an amplitude corrected for background, which is multiplied to the
product of the single correlation terms that are dependent and hence convolute each other. Any offset
can beaded to the correlation values. In cases, when the processes are independent from each other, the
single correlations terms add up, for example in cases where there are more than one component all
bearing the same label or of bunching terms that are independent from each other. If independent and
dependent processes are present, all independent terms will add up and are multiplied with the
dependent terms.
One can distinguish between different classes of processes: anti-bunching, bunching and diffusion. In
addition, the corrected amplitude has to be added.
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4.4.16.3
ConfoCor 3
Amplitudes
The Amplitude of the correlation function is influenced by the offset, background and the number of
particles in dependence of the geometric factor. The amplitude is also influenced by the process of
correlation.
1)
The “1”
In a normal correlation, the curve converges to 1, in case intensities I are correlated as is the case with the
ConfoCor 3 software. Note that in other cases, if fluctuations δI are correlated, the correlation function
converges to 0. If no bleaching occurs
G ∂I (τ ) = 1 + G I (τ )
(2)
You can therefore easily convert G (τ) to Gδ (t) values by adding a fixed offset of –1.
I
2)
I
Offset d
d =c
(3)
c is any rational number that can be negative or positive.
In the ConfoCor software the offset can be a fit parameter or a fixed value.
In some cases, especially if very slow or immobile components are present, there can be a positive offset
from 1. This offset can be taken into account by fitting to d. On the other hand, if the offset is known, it
can be fixed. The offset will be added to each correlation value.
3)
Background B
B = (1 −
Ib 2
)
It
(4)
where Ib is the background intensity and It is the total intensity.
The background in the ConfoCor Software is always a fixed value and never a fit parameter. This means
that the background must be user defined.
Note that the background in this case refers to a non-correlating background. If there is no background
intensity, B = 1, otherwise B < 1. The background can be determined by measuring an unlabeled solution
or cell at the same settings than the real experiment and recording the count rate Ib. The real experiment
with the labelled species will give It. A non-correlating background will result in a lower amplitude and
hence overestimation of molecule numbers, if not corrected for. Note the squared correction term.
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Carl Zeiss
Amplitude A
A=
γ
N
= G ( 0) − 1
(5a)
where γ is the geometric factor accounting for the point spread function (PSF) and N the number of
particles.
In the ConfoCor software γ can be a fit or a predefined fixed value. In case γ is a fit value N must be fixed
in the fit procedure. N is normally a fit parameter.
Please note that γ takes different values for different fitting models depending on the assumed intensity
distribution of the points spread function (PSF):
γC=1.000 (cylindrical),
γ2DG=0.500 (2-D Gaussian),
γ3DG=0.350 (3-D Gaussian),
γGL=0.076 (Gaussian-Lorentzian).
γ can also be calibrated, if a known concentration c of a dye is measured. In this case N can be fixed and
γ fitted. The obtained number can be entered as the calibrated fixed number. N can be calculated from
equation
c=
N
V ⋅ LA
(5c)
23
-1
with V being the confocal volume and LA = 6.023 x 10 mol the Avogadro number)
The volume V is calculated from equation
V =π
3
2
⋅ ω r2 ⋅ ω z
(5d)
with ωz axial focus radius and ωr the lateral focus radius. The radii themselves have to be determined by a
calibration measurement using a dye with a high quantum yield and a known diffusion coefficient D from
the fitted diffusion time τd and the structural parameter S employing a free diffusion model with triplet
state.
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The following relations exists:
τd =
τd =
S=
ω r2
4⋅ D
ω r2
8⋅ D
for 1 photon excitation
(5e)
for 2 photon excitation
(5f)
ωz
ωr
(5g)
Equation 5e or 5f, dependent on the excitation source, can be used to retrieve ωr; with its knowledge ωz
can be calculated from equation 5g.
Please note, that N can have different meanings in different fit models. For biology, normally the number
of diffusing particles is of interest. In this case, if photo-physical processes (triplet, blinking, stretched
exponentials) are involved, it is recommended to use their normalized forms, since then, the number of
molecules correspond directly to the number of diffusing particles. If photo-physical terms are not
normalized, the number measured is the total number of diffusing particles an those undergoing photophysical processes.
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4.4.16.4
Carl Zeiss
Anti-bunching Terms
Anti-bunching is the phenomenon that a molecule cannot produce emitted photons as long as it stays in
the excited state. Hence during the transition time required to drop back to the ground state, which
corresponds in most of the cases to the lifetime if no other photo-physical processes are involved, no
photon can be expected, which results in auto-correlation and hence a drop of the correlation function
below 1.
1)
Dependent to other terms
G a (τ ) = (1 − C − C ⋅ e
−τ
τa
) not normalized
(6a)
−τ
C ⋅ e τa
) normalized
G a (τ ) = (1 −
1−C
(6b)
where C is the amplitude and τa the transition time, also referred to as the lifetime.
2)
Independent in combination with other terms
G a (τ ) = ( −C ⋅ e
−τ
τa
)
(6c)
C in this case is either a fit parameter or a fixed value and often takes the value 9/5.
There are two cases to be distinguished: First, if the anti-bunching is dependent with other processes,
than equations 6a and 6b in the non-normalized or normalized form must be used and the terms are
multiplied with other correlation terms. In case the anti-bunching is treated independent to other
processes, than equation 6c is the correct one to use and the term is added to other correlation terms.
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3)
ConfoCor 3
Stretched exponential – anti-bunching
This is a more general term adding frequency and stretched factors to the exponent.
G k (t ) = 1 − K 1 − K 1 ⋅ e
( − k1 ⋅ τ
( − k1 ⋅ τ
τ k1
K ⋅e
G k (t ) = 1 − 1
1− K1
τ k1
)κ 1
not normalized
(6d)
)κ 1
normalized
(6e)
where K1 is the fraction of molecule, and τk1 the exponential decay time, k1 the frequency factor and κ1
the stretch factor.
K1 and τk1 are fit parameters; k1 is a fixed parameter and must be user defined; κ1 is either a fit parameter
or can be fixed.
Note, fixing k1 and κ1 to “1” result s in a simple anti-bunching term.
4)
Double stretched exponential – anti-bunching
This is a double exponential function, where the exponentials are substracted.
G k (t ) = 1 − K 1 − K 1 ⋅ e
G k (t ) = 1 −
K1 ⋅ e
( − k1 ⋅ τ
( − k1 ⋅ τ
τ k1
)κ 1
τ k1
)κ 1
− K2 − K2 ⋅e
+ K2 ⋅e
1− K1 − K 2
( − k2 ⋅ τ
τk2
( − k2 ⋅ τ
τk2
)κ 2
not-normalized
(6f)
)κ 2
normalized
(6g)
where K1 and K2 are the fractions of molecules, and τk1 and τk2 the exponential decay times, k1 and k2 the
frequency factors and κ1 and κ2 the stretch factors.
K1, K2, τk1 and τk2 are fit parameters; k1 and k2 are fixed parameters and must be user defined; κ1 and κ2
are fit parameters or can be fixed.
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4.4.16.5
Carl Zeiss
Bunching Terms
Bunching is the phenomenon of a burst of photons during a certain time interval, the duration of which
is determined by photo-physical processes including triplet, blinking, flickering and protonation. This
terms are exponential decay functions. Formally, they look the same, only the exponential decay might be
different.
1)
Triplet
G t (τ ) = (1 − Tt + Tt ⋅ e
−τ
τt
) not normalized
(7a)
−τ
T ⋅e τt
G t (τ ) = (1 + t
) normalized
1 − Tt
(7b)
where Tt is the triplet fraction, that is the number of molecules undergoing triplet states and τt the triplet
decay time.
Tt and τt are fitted parameters.
Triplet is based on an un-allowed intersystem crossing from the excited to the so-called triplet state. This
state lasts for 1 – 5 μs. If the electron drops back to the ground state, no photon is emitted and hence
during the triplet state the molecule is in a dark state. Triplet is indicated as a rise in the correlation
amplitude, which is indicated as a deviation from the flattening curve at shorter correlation times. If not
normalized, the triplet fraction contributes to the total number of molecules.
2)
Blinking
G b (τ ) = (1 − Tb + Tb e
Gb (τ ) = (1 +
Tb ⋅ e
−τ
1 − Tb
−τ
τb
τb
) non-normalized
(7c)
) normalized
(7d)
where Tb is the blinking fraction, that is the number of molecules in the dimmer state and τb the blinking
decay time of the dimmer state. Note, if the blinking term is not normalized, the number of blinking
molecules will influence the total number of molecules.
Tb and τb are fitted parameters.
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ConfoCor 3
Blinking is based on the phenomenon that the electron distribution over conjugated systems can change
in dependence on the local environment, for example changes in the pH, which will lead to molecules in
a bright and dim or dark states. It is therefore a kinetic process that can be described in the following
way with the following relations:
k F ;k R
B ←⎯
⎯
⎯→ D
τb =
Tb =
(7f)
1
kF + kR
(7g)
k F ⋅ k R ⋅ (η B − η D )
2
(
( k F + k R ) ⋅ k F ⋅ η B2 + k R ⋅ η D2
with the constraint η B > η D
)
(7h)
with B and D representing the brighter and darker states, kF and kR the forward and backward reaction
rates and ηB and ηD the emission yields or molecular brightness of molecule species (D or B) in Hz or the
relative dimensionless brightness. In case, where darker state is completely dark (ηD=0), equation 7hn
simplifies to
Tb =
kR
kF + kR
(7i)
Note that Blinking is referred to a process that does not lead to a covalent modification in the chemical
bonds. If covalent changes occur the process is referred to as Flickering, which is formally treated in the
same way.
3)
Independent triplet and blinking
In this case the terms are just representatives for two independent bunching terms that are linked by
addition (double exponential term). Note that the triplet fraction, if present, could be potentially fitted to
either of the terms.
G t (τ ) = (1 − T1 + T1 ⋅ e
G t (τ ) = (1 +
T1 ⋅ e
−τ
−τ
τ t1
− T2 + T2 ⋅ e
+ T2 ⋅ e
1 − T1 − T2
τ t1
−τ
τt2
−τ
τt2
) not-normalized
) normalized
(7j)
(7k)
where T1 and T2 are the fractions of molecules in the triplet state, and τt1 and τt2 the triplet exponential
decay times.
T1, T2, τt1 and τt2 are all fitted parameters.
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4)
Carl Zeiss
Dependent triplet and blinking
In this case the terms are just representatives for two dependent bunching terms that are linked by
multiplication. Note that the triplet fraction, if present, could be fitted to either of the terms.
G t (τ ) = (1 − T1 + T1 ⋅ e
−τ
τ t1
)(1 − T2 + T2 ⋅ e
−τ
−τ
T ⋅ e τ t 1 1 + T2 ⋅ e
)(
G t (τ ) = (1 + 1
1 − T1
1 − T2
τt2
−τ
τt2
) not normalized
) normalized
(7l)
(7m)
where T1 and T2 are the fractions of molecules in the triplet state, and τt1 and τt2 the triplet exponential
decay times.
T1, T2, τt1 and τt2 are all fitted parameters.
5)
Stretched exponential - bunching
In some reactions like, the kinetics cannot be fitted to simple exponential functions but require stretched
exponentials.
G k (t ) = 1 − K 1 + K 1 ⋅ e
( − k1 ⋅ τ
( − k1 ⋅ τ
τ k1
K ⋅e
G k (t ) = 1 + 1
1− K1
τ k1
)κ 1
not normalized
(7n)
)κ 1
normalized
(7o)
where K1 is the fraction of molecule, and τk1 the exponential decay time, k1 the frequency factor and κ1
the stretch factor.
K1 and τk1 are fit parameters; k1 is a fixed parameters and must be user defined; κ1 is either a fit
parameter or can be fixed.
Note, fixing k1 and κ1 to “1” result s in a simple bunching term.
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6)
ConfoCor 3
Double stretched exponential - bunching
This is a double exponential function, where the exponentials are added.
G k (t ) = 1 − K 1 + K 1 ⋅ e
G k (t ) = 1 +
K1 ⋅e
( − k1 ⋅ τ
( − k1 ⋅ τ
τ k1
)κ 1
τ k1
)κ 1
− K2 + K2 ⋅e
+ K2 ⋅e
1− K1 − K 2
( − k2 ⋅ τ
τk2
( − k2 ⋅ τ
τk2
)κ 2
not-normalized
(7p)
)κ 2
normalized
(7q)
where K1 and K2 are the fractions of molecules, and τk1 and τk2 the exponential decay times, k1 and k2 the
frequency factors and κ1 and κ2 the stretch factors.
K1, K2, τk1 and τk2 are fit parameters; k1 and k2 are fixed parameters and must be user defined; κ1 and κ2
are fit parameters or can be fixed.
This term is often required to fit protonation, with the second stretch factor and the frequency factors are
set to “1”.
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4.4.16.6
Carl Zeiss
Diffusion Terms
Diffusion is driven by Brownian motion. We can distinguish translational, rotational and flow.
1)
Rotational diffusion
In the most general form, rotation can be described as the sum of 5 exponential terms
g r (τ ) = 1 − Ra + Ra ⋅
5
∑ cm ⋅ e
− rm ⋅τ / τ r , m
(8a)
m =1
with Ra being the amplitude, cm the relative amplitude, rm the frequency factor and τr,m the rotational
diffusion time.
However, there are special cases that are of more use.
In symmetric rotation, the general formula reduces to:
G r (τ ) = 1 − Ra + Ra ⋅ e
−τ
τr
not normalized
(8b)
−τ
R ⋅ e τr
normalized
G r (τ ) = 1 + a
1 − Ra
(8c)
with Ra being the rotational amplitude and τr the rotational diffusion time.
Ra and τr are both fit parameters.
If rotation occurs independent from other processes, the formula used as an additive term is defined as:
G r (τ ) = Ra ⋅ e
−τ
τr
(8d)
Ra in this case is either a fit parameter or a fixed value and often takes the value 4/5.
In case of asymmetric rotation, the term is as follows:
G r (τ ) = 1 − Ra + Ra ⋅ (c 1 ⋅ e
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τ r ,2
) not normalized
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G r (τ ) = 1 +
Ra ⋅ (c 1 ⋅ e
−
r1τ
τ r ,1
+ c2 ⋅ e
1 − Ra
−
r2τ
τ r ,2
)
normalized
ConfoCor 3
(8f)
with Ra being the amplitude, c1 and c2 relative amplitudes, r1 and r2 frequency factors and τr,1 and τr,2 the
rotational diffusion times.
Ra, τr,1 and τr,2 are fitted parameters; c1 and c2 as well as r1 and r2 are fixed values and must be user
defined.
Rotational frequencies take often the following values:
r1
1
r2
10/3
The relative amplitudes dependent on the polarisation of the excitation light and the analyser in the
emission beam path and are as follows:
Ex
lin.
pol.
lin. pol.
lin. pol. unpol.
unpol.
unpol.
Em parallel perpendicular all
parallel perpendicular all
c1
80
20/9
860/9
5/9
215/9
20
c2
64/9
4
4
16/9
1
1
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
2)
Carl Zeiss
Translational diffusion
In its general form, translational diffusion is defined as:
⎛
⎜
⎜
⎜
⎜ 3
G d (τ ) = ⎜ ∑
⎜ i =1
α
⎞ i
⎜ ⎛⎜ ⎛ τ
1
+
⎜
⎟
⎜ ⎜ ⎝ τ d ,i ⎠
⎜ ⎝
⎝
Φi
⎞
⎟
⎟
⎠
ed 1
1 ⎞
⎛
αi
⎞ 2⎟
⎜⎛ ⎛
⎞
⋅ ⎜ ⎜1 + ⎜ τ
⋅ 1 2⎟ ⎟
τ d ,i ⎟⎠
S ⎟ ⎟
⎜ ⎜⎝ ⎝
⎠
⎠
⎝
ed 2
⎞
⎟
⎟
⎟
⎟
⎟ with the constraint
⎟
⎟
⎟
⎟
⎠
∑Φ
i =1
(8g)
i
with τd,i representing the diffusional correlation time of molecule species i , S the structural parameter
that is the ratio of axial to lateral focus radii, αi the anomaly parameter or temporal component of
molecule species I, ed1, ed2 fixed exponentials to define dimensionality of diffusion (1-D: ed1=1/2; ed2=0; 2D: ed1=1; ed2=0; 3-D: ed1=1; ed2=1)
ed1 and ed2 are fixed values and have to be user defined. The following values define 1-, 2-and 3-D
diffusion:
ed1
ed2
dimensionality
1/2
0
1-D
1
0
2-D
1
1
3-D
Note that in the ConfoCor 3 software this values are automatically selected with the choice of
dimensionality.
S is either a fit parameter or a fixed value. It is an instrumental parameter and can be determined by a
calibration experiment using a dye solution with a known diffusion as a fit result.
αi is either a fitted value for anomalous diffusion or a fixed value (set to “1) for free diffusion. The
following relation exists
α
Diffusion process
=1 Free diffusion
<1 Anomalous sub-diffusion
<1 Anomalous super-diffusion
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
ConfoCor 3
Note that αi is set automatically to “1”, if free diffusion is selected. If anaomalous diffusion is selected,
the parameter will float.
τd,i are fitted parameters. They can be converted to diffusion coefficients Di using formulas 5e or 5f. The
ConfoCor software allows you to directly fit to Di values, but in this case the lateral radius ωr has to be
specified as a fixed value.
Please note that in the case of anomalous diffusion The following relations exist:
τ α d ,i =
τ α d ,i =
ω r2
Γ2 , i
1 photon excitation
ω r2
2 ⋅ Γ2 , i
(8h)
2 photon excitation
(8i)
with Γ representing the transport coefficient of the fractional time dimension.
Please note the following relation between D and G :
D ( t ) = Γ ⋅ t α −1
(8j)
If activating the fitting to the diffusion coefficient in the ConfoCor 3 the Γ values have to be calculated
from the D values by the following conversion:
Γ = 4⋅ D
(8k)
The Φi values are fit parameters. They account for different brightness of different components. In
principle, if molecules of different brightness are present, the apparent molecular brightness is defined as
3
η=
∑ f i ⋅η i2
i =1
3
∑
i =1
(8l)
f i ⋅η i
with ηi being the brightness of the molecules in kHz or the dimensionless relative brightness values. The
brightness of the species have to be determined beforehand in control experiments.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
Carl Zeiss
Note that the brightness contributes as the square to the correlation function, in other words a double as
bright molecule will contribute 4 fold more. Therefore, the fitted number of molecules must be corrected
for to obtain the real number Ndiff of diffusing particles; please note that to obtain the diffusing particle
number directly, other terms should be used in their normalized form :
3
N diff = N ⋅
(∑ f i ⋅η i ) 2
i =1
3
∑
i =1
(8m)
f i ⋅ η i2
If one wants to know the true fraction fi of each species, with the known brightness values those can be
retrieved from the relation
Φi =
f i ⋅ η i2
3
∑
i =1
with the constraints
f i ⋅ η i2
∑Φ
i =1
and
∑ fi
=1
(8n)
i
i
If there is no brightness difference between the components, Φi will become to fi.
In the ConfoCor software you can fit directly to the fractions even in case of different brightness values.
In this case, the brightness values have to be fixed parameters and defined by the user. The fit formula
converts from equation 5a in combination with 8g taking into account the corrected amplitude to
equation 8o:
⎛
⎜
⎜
2
∑ f i ⋅η i ⎜⎜ 3
γ
G tot (τ ) =
⋅ i3=1
⋅⎜∑
N
α
( ∑ f i ⋅ η i ) 2 ⎜ i =1 ⎛
⎞ i
⎜ ⎜ ⎛τ
i =1
⎜ ⎜ 1 + ⎜⎝ τ d , i ⎟⎠
⎜ ⎝
⎝
f 1 ⋅ η 12
3
3
∑ f i ⋅η i
2
i =1
⎞
⎟
⎟
⎠
ed 1
1 ⎞
⎛
α
2
⎜⎛ ⎛
⎞ i 1 ⎞⎟ ⎟
⎜
τ
⋅⎜ 1+ ⎜
⋅
⎟
⎟
2
τ d ,i ⎠
S ⎟ ⎟
⎜ ⎝⎜ ⎝
⎠
⎝
⎠
ed 2
⎛
⎞
⎜
⎟
⎜
⎟
⎜
⎟
⎟ γ ⎜ 3
⋅⎜∑
⎟=
⎟ N ⎜ i =1
α
⎞ i
⎜ ⎛⎜ ⎛ τ
⎟
⎜ ⎜ 1 + ⎜⎝ τ d , i ⎟⎠
⎟
⎜ ⎝
⎟
⎝
⎠
f 1 ⋅ η 12
3
(∑ f i ⋅η i ) 2
i =1
⎞
⎟
⎟
⎠
ed 1
1 ⎞
⎛
α
2
⎜⎛ ⎛
⎞ i 1 ⎞⎟ ⎟
⎜
τ
⋅⎜ 1+ ⎜
⋅
⎟
⎟
2
τ d ,i ⎠
S ⎟ ⎟
⎜ ⎜⎝ ⎝
⎠
⎝
⎠
ed 2
⎞
⎟
⎟
⎟
⎟
⎟
⎟
⎟
⎟
⎟
⎠
(8o)
with fixed brightness values ηi.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
3)
ConfoCor 3
Flow
Flow signifies active transport either via cytoplasmic movement or directed transport.
If flow occurs in the absence of translational diffusion, the term is defined as follows:
G f (τ ) =
⎛
⎞
− ⎜⎜ τ
τ f ⎟⎟⎠
⎝
e
2
(8m)
In the presence of translational diffusion, the term alters to:
(τ
−
G f (τ ) = e
)2
τf
1+ ( τ )
τd
(8n)
with τf representing the average residence time for flow and τd the diffusion correlation time.
Note, in the ConfoCor 3 software, the correct term is automatically loaded in dependence on the
absence or presence of a translational term.
With the knowledge of the lateral radius ωr given as a fixed value, the software allows to fit directly to
the velocity v instead of the average residence time. The following relation exists:
v=
ωr
τf
4.4.16.7
(8o).
Photon Counting Histogram (PCH)
In the photon counting histogram (PCH) no closed formulas exist, to which the data can be fitted. Hence
in this case, fitting is done numerically.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
4.4.17
Carl Zeiss
Models
If you press the Models button in the ConfoCor
submenu toolbar, the Model List window will
open (see Fig. 4-69).
The window lists all the stored models with the
Model name and the Type. You can select a
model by highlighting it.
The type is of either Assemble, PCH or User
defined depending where the model was created,
in Correlation, PCH or Formula, respectively.
You have the following options:
4.4.17.1
Close
Pressing the Close button will close the Model List
window.
4.4.17.2
New
Pressing the New button will open the Model
window, where you can define a model by
predefined terms or user defined.
4.4.17.3
Fig. 4-69
Model List window
Modify
Pressing the Modify button will open the Model window, where you can alter the highlighted preexisting method.
4.4.17.4
Delete
Pressing the Delete button will delete the highlighted method. You will be prompted if you want to
delete the model. Press yes or no I you want to delete or keep the model, respectively.
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
4.4.17.5
ConfoCor 3
Defining a Model
Press the New or Modify button and the Model
window will appear (see Fig. 4-69).
Note, the Modify button will load the
highlighted configuration and either the
Correlation, PCH, or Formula is available (see Fig.
4-83); in New all options are available and can be
selected by pressing the Correlation, PCH or
Formula button (see Fig. 4-70).
You have three options to define a model:
− Correlation: assemble a fluorescence
correlation spectroscopy (FCS) model
− PCH: assemble a photon counting histogram
model
− Formula: program a user defined model
Fig. 4-70
Model window, new model
(1)
Correlation
You can assemble a with predefined terms.
• Press the Correlation button to activate the
options for assembling a model.
You have the following options (see Fig. 4-71):
• Press the Close button to exit the Correlation
window.
• Press the Save button to save a defined model.
The Save Model window will appear. You can
type in a name. Pressing Ok will save the model
in a database. Pressing Cancel will close the
Save Model window without saving the
model.
• To define a model, just activate the respective
terms that are available.
• You can choose from:
Fig. 4-71
Model window, predefined
− Offset: Deviation from 1
− Background:
correction
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
− Amplitude: Number of molecules and geometric factor
− Antibunching: Antibunching term
− Triplet: Exponential term for triplet state, blinking, flickering or other bunching terms
− Rotation: Rotational diffusion term
− Translation: Translational diffusion term
− Flow: Flow term
− Stretched exponential: Stretched exponential term for protonation and other kinetics
All highlighted terms are assembled in a way that is displayed in G(τ)= display box.
For all terms specific settings can be applied. These settings are accessible if the respective Settings
button is pressed. All Settings windows can be closed by pressing the Close button. The settings
windows are bipartite: Above you can select parameters and define values, below is a description box
that display the formula in its most general form, explains the parameters and gives useful conversions of
the fitted parameter to other interesting parameters. Press the close button to exit the Setting window.
The following settings are available:
Offset
• You can either set the offset (see Fig. 4-72) to 0
by activating the Normalized option.
• Or you set an offset by activating the
Calibrated option. This enables you to type in a
value in the Calibrated selection box, or by
setting a value using the arrows.
Fig. 4-72
Settings Offset panel
Fig. 4-73
Settings Background panel
Note, the offset can have positive or
negative numbers and the value specified
will be added to all correlation values.
The description box gives you information on the
offset.
Background
• You can set an offset by setting a value in the
Background selection box (see Fig. 4-73).
Either type in a value or use the arrows.
The description box gives you information on the
background.
If there is no background, the background correction factor will be set to 1.
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
Amplitude
• You can set a value for the geometric factor γ,
which describes the point-spread function (see
Fig. 4-74).
• By activating the corresponding option you can
select between a cylindrical, 2 dimensional
Gaussian (2DG), 3 dimensional Gaussian (3DG)
and Gaussian-Lorentzian (GL) PSFs. If you
activate the Calibrate selection box, you can
type in a user defined number or use the arrows
to set a value.
The description box gives you information on the
amplitude
Fig. 4-74
Settings Amplitude panel
At least one the γ factor or the number of
molecules N have to be fixed in the fit procedure.
Antibunching
You can select the anti-bunching term in its
normalized form by checking the Normalized
check box, or in its non-normalized form by dechecking the box (see Fig. 4-75).
You can select the Independend antibunching,
rotation and translation form by checking the
respective box. This equation will be used as an
additiv term to rotational and translational
diffusion terms.
You have the option to leave the amplitude value
to Free or to 9/5 by activating the corresponding
option.
Fig. 4-75
4-74
Settings Antibunching panel
The description box gives you information on the
anti-bunching.
B 45-0015 e
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ConfoCor 3
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
Triplet
The triplet represents bunching terms, that are
exponential decay functions.
You can select the triplet term(s) in its/their
normalized form by checking the Normalized
check box, or in its/their non-normalized form by
de-checking the box (see Fig. 4-76).
You have several options for the bunching terms
which you can select by the Components drop
down menu (see Fig. 4-77):
Triplet: 1 exponential function
Blinking: 1 exponential function
Independent Triplet and Blinking: Sum of 2
exponential functions
Fig. 4-76
Settings Triplet panel
Fig. 4-77
Components drop down menu
Dependent Triplet and Blinking: Product of 2
exponential functions
The description box gives information on the
bunching terms.
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
Rotation
You can select between symmetric rotation (1
exponential term) and asymmetric rotation (double
exponential term) by selecting / deselecting the
Asymmetric check box (see Fig. 4-78).
You can select between normalized and nonnormalized rotation functions by selecting /
deselecting the Normalized check box.
If the Asymmetric box is checked, you can define
relative amplitudes and rotational frequencies
(note, these parameters are no fit values and have
to be defined by the user).
Relative amplitudes are defined by selecting the
excitation polarization (linear polarized or
unpolarized) and the emission detection
(parallel, perpendicular or all), which will result
in the corresponding values displayed in the c1 and
c2 display boxes. Alternatively, if others is
activated, you can type in user defined values.
Fig. 4-78
Settings Rotation panel
Rotational frequencies can be selected by
activating the Default settings with the
corresponding values will be shown in the r1 and
r2 display boxes, or by user defined values that can
be entered when others is activated.
You can select the Independend antibunching,
rotation and translation by checking the
respective box. This equation will be used as an
additiv term to anti-bunching and translational
diffusion terms. You have the option to leave the
amplitude value to Free or to 4/5 by activating the
corresponding option.
The description box gives information on the
rotational diffusion terms.
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ConfoCor 3
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
Translation
• You have the following possibilities for setting
parameters (see Fig. 4-79):
• You can either fit to fractions (used normally
when no brightness differences are observed
between different components) or to fractional
intensities, when the Fractional Intensities
option is selected. In this case, you have to
provide the absolute or relative brightness
values of the components into the Molecular
brightness selection boxes.
• You can either fit to the diffusion time, or you
can directly fit to the Diffusion coefficient by
activating the Diffusion coefficients option. In
the latter case you have to provide the
dimension of the radius of the confocal volume
in the ωr selection box. Either type in a value or
use the arrow keys.
• You must also specify if you use two photon
excitation by checking the 2 Photon check box,
since that will influence the fit formula in the
case the Diffusion coefficients option was
chosen.
• You can select the numbers of components (1,
2 and 3) by pressing the Components 1, 2 or 3
buttons.
Fig. 4-79
Settings Translation panel
• For each component you can select free or
anomalous diffusion in the pull down menus of
the Free/anomalous selection box.
• For each component you can set the dimensionality of diffusion in the pull down menus of the
Dimension selection boxes. You can toggle between 1-D, 2-D and 3-D.
• For each component you can enter brightness values in the Brightness select boxes. This values are
only displayed, if the Fractional Intensities option is selected. You can type in absolute values (that
must have the same units) or relative values.
The description box gives information on the translational diffusion terms.
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
Flow
You can determine, if you want to fit to the
velocity directly instead of the diffusion time by
checking the Velocity check box (see Fig. 4-80). In
this case you have to provide the radial dimension
of the confocal volume in the ωr selection box.
Type in the number or use the arrow keys.
The description box gives you information on the
flow terms. Note, that the system automatically
toggles between the pure flow and the one that is
used when the Translation term is activated.
Stretched exponential
You can select between bunching and antibunching terms by activating the Bunching or
Antibunching option (see Fig. 4-81).
You can select between 1 (mono exponential) or 2
(double exponential) stretched exponential terms
by pressing the Components 1 or 2 button.
You can select between normalized and nonnormalized terms by selecting / deselecting the
Normalized check box.
Fig. 4-80
Settings Flow panel
Note, the frequencies and stretch factors
are no fit parameters and must be defined in the
Frequencies k1 and k2 as well as the Stretch
factors κ1 and κ2 display boxes. Either enter a
value or press the 1 button for the default setting.
The description box will give information on the
stretched exponential terms.
Fig. 4-81
4-78
Stretched exponential panel
B 45-0015 e
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ConfoCor 3
(2)
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
PCH
PCH = Photon Counting Histogram allows you to
determine concentrations and the molecular
brightness of molecules.
You can choose between 1, 2 or 3 components by
activating the Components 1, 2 and 3 buttons,
respectively (see Fig. 4-82). For each activated
component you can enter a brightness value in the
Specific brightness enter boxes.
You can also enter Instrumental parameters
values in the respective square (a1), cubic (a2)
and power (a3) enter boxes. These values have to
be determined in independent calibration
experiments using a defined dye solution and
correct for the deviation of the confocal volume
from a true Gaussian distribution.
(3)
Formula
Defining a model with user defined terms
Fig. 4-82
Model window, PCH
Fig. 4-83
Model window, Formula
• Press the Formula button for the user defined
model options (see Fig. 4-83).
This opens up a calculator, that you can use to edit
your formula. You can also type in directly in the
Selection box.
All variables that are defined at the beginning, are
taken as fixed variables, all others as fit
parameters.
The formula is continuously parsed and any
expected operation indicated in the description box
at the bottom.
• Press the Save button to open the Save Model
window. The formula will be automatically
parsed or compiled and any syntax errors will be
displayed. Give the model a name and press Ok
to save it. Press Cancel to close the window
without saving.
• The description box at the bottom will inform
you on the parameters and operations.
• Press the Close button to leave the User
defined Window.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
4.4.18
ConfoCor 3
Settings
Pressing the Settings button of the ConfoCor submenu bar opens the Options for ConfoCor window
(see Fig. 4-84). You can close the window by pressing the Close button. The menu has three submenus:
− Measurement
− Auto Save
− LSM+ConfoCor
4.4.18.1
(1)
Measurement
Raw data
If the Save raw data during measurement
check box is activated (see Fig. 4-84), raw data will
be saved in a specified directory.
If the check-box is de-activated, raw data
will be saved in the Temp file. If the FCS results
window is closed, the raw data will be lost in this
case.
Fig. 4-84
Options for ConfoCor window,
Measurement
When you tick the Save raw data during
measurement check box, the raw data (photon
trace) will be stored on disk. This option is used
when access to the raw data is required to analyze
the data in a different way than by calculating the
correlation functions.
The raw data file structure is described in section
4.5.3 Description of the Raw Data Format.
• In the Directory field, the directory will be set
where the data should be saved.
button is clicked, a WINDOWS
• When the
directory selection dialog will open (see Fig.
4-85).
• The maximum number of files can be set (up to
100).
Each single measurement is stored in one
raw data file. For two channel experiments, each
channel will be stored in a separate file.
Fig. 4-85
4-80
Save raw data
B 45-0015 e
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ConfoCor 3
(2)
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
Stage movement
If the Move stage during measure check box is activated, the stage will move during an measurement
This option allows you to move the stage during measurement. In this case the stage will be moved forth
and back during the entire measurement time. This option is useful during rare event detection when
large, slowly moving objects in extremely small concentrations have to be registered. Since these
aggregates are diffusing very slowly without such movement the measurement times would be
prohibitively long. It should be taken into account that, in this case, the data stream is analyzed with the
coincidence analysis rather than by correlation analysis.
If the Use Piezo Stage if available check box is checked, the Piezo Stage will be used whenever the
Piezo button is pressed in the measurement window.
(3)
Activating the Piezo Stage
You have the option to mount a Piezo Stage onto your normal stage. Be sure to select on the control unit
of the Piezo Stage the closed loop operation. If closed loop is selected the corresponding LCD will light
up. To work with the Piezo Stage, it should be activated under FCS Options in Measure tab by pressing
the Piezo Stage button.
If the Move stage during measure check box is checked, the following settings are possible in the
dialog:
− Travel distance of the stage in millimeters: Only integer numbers of millimeters are possible.
− Speed of the stage: Only the three preset stage speeds 0.48 mm/s, 4.81 mm/s and 24.01 mm/s are
possible.
− Travel direction: The stage can be moved along the x- or y-axis of the stage.
If you haven't chosen the closed loop operation mode, the Piezo Stage will not work properly in
the LSM 510-ConfoCor 3 Software.
The Piezo Stage allows for 2D translational motion in the x and y direction with a maximum motion of
100 μm in each direction. The resolution is 1 nm. Positioning accuracy using the Piezo Stage is 10 nm and
less.
03/06
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4-81
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
(4)
ConfoCor 3
Working with the Piezo Stage
Open the Measurement window (see Fig. 4-86)
and press the Piezo Stage button.
Any stage movement done with the stage control
will address the Piezo Stage.
Movement by the joystick will overrule the Piezo
Stage and the normal stage will be addressed.
The position of the Piezo Stage will be centered in
respect to the normal stage whenever the Piezo
Stage to 0 button was pressed.
The position in x and y are displayed in the Stage
Position display box.
If the Piezo Stage is activated the x, and y-Scans in
the X,Y,Z-Menu will be nevertheless done by the
scanning mirrors.
Note, the numbers displayed will not
correspond to the positions in the LSM mode, if
the Piezo Stage is activated. Position numbers
correspond to the numbers displayed on the Piezo
Stage unit. Zeroing will display 40.000 in x and y. If
the standard stage is used, the numbers will be the
same.
Fig. 4-86
(5)
Measurement window
Shutters
If additional shutters (before the HeNe lasers and DSSP lasers and a line suppression filter wheel for the
Argon laser) are available in the laser module, they can be activated by checking the Enable laser
shutter filter wheel check box.
In this case, if the HeNe and DSSP filters are not used, a shutter will be placed in front of them; a line
selection filter will be used for the laser line activated for the Argon laser.
Note, that activating two lines of the Argon, only in the case of 458/514 a filter be in place,
otherwise a non-position.
This function is recommended when measuring close (within 0.2 mm) close to the cover slip
glass to suppress any residual excitation light.
4-82
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
(6)
Carl Zeiss
AOTF dampening
If the AOTF control automatically check box is activated, the laser AOTF setting will be extended for
the dynamic range in the lower range. Please note that in case two activated laser are set at different
ranges, the one in the lower range will be set to the lowest available AOTF of the higher range.
If the box is unchecked, the AOTF can be suppressed overall by 1, 10, 100 or a 1000 fold. By moving the
slider of the AOTF Power (%) selection box, the respective dampening factor can be set (100%, 10%,
1%, 0.1%).
4.4.18.2
Auto Save
If the Auto Save check box is activated (see Fig.
4-87), all curves will be saved automatically in the
directory, which is specified Directory selection
box (see figure settings-auto-save).
The Auto Save panel of the window enables you
to instruct the program to save the results
automatically during the measurement process
without human intervention.
• Activate the Auto Save check box via mouse
click.
• The directory for the automatically saved files
can be chosen as well in the Directory
selection box.
Fig. 4-87
Option for ConfoCor window, Auto
Save
The Task Name specified in the Task Description panel is normally used as file name. For checking
purposes, it is then displayed in the Filename display box.
4.4.18.3
LSM + ConfoCor
The LSM + ConfoCor function (see Fig. 4-88) can
be used to type in offset values between LSM
scanners and the stage, if this is used for FCS
measurements. The offset can be determined by
bleaching holes into a dye layer and determining
the offset between the crosses and actual bleach
spot. Type in the corresponding numbers.
If the scanners are uses, offsets has to be
corrected with the Bidirectional sliders.
The numbers typed inhere are then
meaningless.
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Fig. 4-88
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Option for ConfoCor window,
LSM+ConfoCor
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
ConfoCor 3
4.4.19
Compensating Offsets between FCS and LSM
4.4.19.1
Determination of the Offset between LSM Scanner and FCS Stage Positioning
(1)
Alignment in X, Y
You can compensate manually or by using a Macro.
(a)
Manual Adjustment
Do the manual adjustment according to the
following procedure:
• Obtain an LSM image of the Rh6G layer.
• Set the following LSM parameters in the
Configuration Control (see Fig. 4-89):
− Channel 1: select
− Excitation: 488 nm at 2.1%, tube current 6.1
Amps
− Main beam splitter: HFT 488
− Emission filter of channel 1: LP505
Fig. 4-89
4-84
LSM beam path for imaging the test
layer of Rh6G
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
• Set the following LSM parameters in the Scan
Control (see Fig. 4-90):
− Frame Size: X=512, Y=512
− Scan Speed: 8
− Data Depth: 8 Bit
− Scan direction: ->
− Mode: Line
− Method: Mean
− Number: 4
− Zoom: 5
− Rotation: 0
Fig. 4-90
Settings at the LSM control
Fig. 4-91
LSM image of the Rh6G layer before
bleaching. Usually some texture is
observed.
• Optimize focus position and Detector Gain
value for a good LSM image (see Fig. 4-91).
• Leave the LSM image window and LSM control
window on the computer screen.
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4-85
Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
• Switch to FCS.
− In the main toolbar press the FCS button.
− Click Measurement button.
− Select a suitable beam path.
− Press LSM Image button.
− Mark the points in the LSM image that you
want to bleach by positioning the cross and
pressing the Add button. The marked points
will be listed. Click Mark Pos to overlay the
markers in the LSM image (see Fig. 4-92 and
Fig. 4-93).
Fig. 4-92
The Method Measurement window in
the LSM mode
• Bleach the marked sites.
− Click Start. The usual FCS measurement
window appears.
− Once finished, close the FCS measurement
window without saving.
Fig. 4-93
4-86
LSM image of the Rh6G layer with
markers for FCS-LSM adjustment
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
• Rescan the Rh6G layer.
− Switch to LSM mode.
− Click Single in the Scan Control.
− Once the scan is finished you see the
bleached spots (see Fig. 4-94).
Fig. 4-94
LSM image of the Rh6G layer after
bleaching. Deviations between the
markers and the bleached spots are
noticeable.
Fig. 4-95
LSM image after bleaching
overlaid distance markers
• Measure the distance between the burned spots
and the markers (see Fig. 4-95).
− Click on Overlay.
− Click on the 1 µm button.
− Draw a line between the center of the
bleached spot to the vertical and horizontal
lines of the marker cross.
• Determine the average X and Y deviation values
between the markers and bleached spots.
Depending on the direction of these deviations
increase or decrease the X and Y values by the
measured mean deviation. Type these X and Y
values into Settings / LSM+ConfoCor.
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Carl Zeiss
OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
ConfoCor 3
• Type the measured distance correction values
under Settings in the LSM + ConfoCor button
(see Fig. 4-96).
− If the bleached point is left to the marker:
type distance in X as positive value
− If the bleached point is right to the marker:
type distance in X as negative value
− If the bleached point is below the marker:
type distance in Y as positive value
Fig. 4-96
− If the bleached point is right to the marker:
type distance in Y as negative value
The Settings for user window with the
LSM+Confocor activated
− Offset Z: no entry
• Check the correctness of your settings by
repeating the adjustment procedure (see
Fig. 4-97).
If the deviations between bleached areas and
markers is marginal, the LSM/FCS superposition
alignment is sufficient to guarantee a precision of
1 µm.
Please note, that precision is higher if you
decide to make a measurement in one section of
the image rather than all over it. In this case only
deviations in that sector should be taken into
account for calculating the offset values.
Fig. 4-97
LSM image after bleaching
correction of offset values
and
(b)
Adjustment Using the LSM/FCS Macro
• Before running the Macro choose a suitable
method in the Method file menu in FCS and
set the beam path for the LSM. Please, focus on
the Rhodamine 6 Green layer.
• Activate the Macro toolbar by clicking the
Macro button.
• Choose the Lsm-Fcs macro by clicking the
corresponding button.
Fig. 4-98
The Macro "Fcs-Lsm" display before
offset compensation
• The Requirements for Adjustment window
will be displayed.
• Click Ok to run the Macro, Cancel to abort.
• Once Ok was chosen, the Lsm-Fcs macro window will be displayed (see Fig. 4-98).
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ConfoCor Menu for ConfoCor 3
Carl Zeiss
• Set the Pinhole diameter to 10 and the Bleach power to 1%. If holes are too weak or getting to
big you should increase or decrease the Bleach power, respectively.
• Choose the numbers of Bleach spots by checking the corresponding number.
• You have now two possibilities to continue: Stepwise or automatic.
• Stepwise Procedure:
− Press the Scan Ref button which is highlighted and labeled by a red 1. A scan is performed. Once
finished the Bleach button will be highlighted and labeled with a red 2. The 1 will turn to black.
− Press the Bleach button. The bleach is performed. The FCS evaluation window will appear. Once
ready, the Rescan button will be highlighted and labeled with a red 3. The 2 will turn to black The
FCS evaluation window will disappear.
− Press the Rescan button. The image is rescanned. You will see a number of bleached spots
according to your settings which corresponds to the positions defined by the rigid FCS beam path.
You will also see yellow crosses which are the positions of the laser beam for the LSM. Because
there might be an offset between both, crosses do not necessarily correspond with the bleached
spots. Once ready, the Autoscan button is highlighted and labeled with a red 4. The 3 will turn to
black.
− Press the Autoscan button. Once finished,
the 4 will turn black. The program will find
the burned spots which will turn red and
label them by green circles. The distance
between the green circles and yellow crosses
is indicated by the green line and the value
displayed as the R value (see Fig. 4-99). The
program also calculates the deviation in X
and Y, the values which are also displayed
under X and Y offsets (see Fig. 4-100). The
program also calculates the scan drift during
the procedure and gives a warning if the
scan drift was to great.
− Press the Save button to store the offset
values. Deviations between LSM and FCS will
than be automatically compensated with the
corresponding offsets.
Fig. 4-99
LSM image after performance of the
"Fcs-Lsm" Macro
Please note that you can do a
readjustment without leaving the Lsm-Fcs
Macro. However, all buttons will be left
highlighted and numbers will stay black.
That means that you have to watch for
yourself when one step is finished and
you have to remember, which is the next
step you have to activate.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
ConfoCor 3
• Press the Help button if you wish to view the
Requirements for adjustment display. Press
Close to leave the Macro.
By activating the Macro a Stop Macro
window will appear. You can leave the Macro at
any time by pressing the Stop Macro button.
Automatic Procedure:
Fig. 4-100
The "Fcs-Lsm"
compensation
Macro
after
offset
− Press the Auto Adjust button. The Macro
will automatically perform Steps 1. to 4. The
corresponding submenu button will be
highlighted.
− Once finished, offsets are displayed. Press
Save to store the offsets, Help to view the
Requirements for adjustment display or
Close to leave the Macro.
(c) Determination of the Fixed X,Y Position
of the Laser Beam in the Measurement...
Window in the Current Position mode
• Get a LSM image of a Rh6G layer.
− Go to Current Position mode
− Click the Crosshair button (see Fig. 4-101).
• Perform a bleach at the position of the fixed
laser beam.
− Click Start. The usual FCS measurement
window appears.
− Once finished, close the FCS measurement
window without saving.
• Rescan the Rh6G layer.
− Click Single in the Scan Control.
Fig. 4-101
4-90
Positions panel, Current Position
− Once the scan is finished you see a bleached
spot corresponding to the position of the
rigid laser beam (see Fig. 4-102).
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
Since the scanners are parked, the spot
should lie in the center of the image, if no
crop function and offsets were used.
Fig. 4-102
LSM image after bleaching in the
Method Optimization
Fig. 4-103
LSM image after bleaching with
crosshair
• Record the position of the spot.
− With the crosshair. Place crosshair above the
bleached spot (see Fig. 4-103). We
recommend to use the crosshair for
positioning your cell.
− With the cursor. Make sure that under
Options / LSM Settings in the Image
Status Display tab the Pixel Intensity box
is checked to activate the display of the
coordinates. The coordinates will than be
displayed at the lower bar of the image.
− With an overlay arrow. Click Overlay and
activate the Arrow button. Place the
arrowhead in the middle of the spot.
If the Position box is checked, the
Crosshair function is inactivated.
(2)
Alignment in Z
No compensation in z is necessary
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
Carl Zeiss
4.4.19.2
ConfoCor 3
Determination of the Offset between LSM Scanner and FCS Scanner Positioning
There is no need to compensate between defined positions and the actual measurement positions
because of scanning mirror positioning. However, the precise arrest position of the mirrors depend on the
used scan zoom and speed. To adjust for differences, the compensation sliders of the bidirectional Scan
can be adjusted. For the Method Optimization in the Crosshair mode you locate the position of the
beam and place the structure of interest at the defined site. For Z adjustment you have to use the
Method Optimization window. In all cases you need a thin layer of a fluorescent dye, preferentially
Rhodamine 6 Green, dried on a glass bottom of for example a Labtec 8-well chamber.
Alignment in X, Y in the Positions Mode
No compensation is necessary. A defined site is positioned by the scanning mirrors with pixel precision
(see Fig. 4-104).
Fig. 4-104
4-92
Bleaching of a spot in a dye layer in Method Optimize via Position list.
Note the correspondence of the defined (cross) and actual (bleached
spot) positions.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
ConfoCor Menu for ConfoCor 3
To compensate for Scan Zoom and Scan Speed go
to Scan Control under Mode. Use a sample with
high contrast, like "Convolaria", and focus.
Activate Bidirectional Scan (bended arrow) under
Scan direction (see Fig. 4-105). During scanning
press the Auto button. The system will adjust
automatically the Scan Corr X and Y values.
Alternatively you can do the compensation
manually.
Carl Zeiss
Fig. 4-105
The Pixel Depth, Scan Direction & Scan
Average Window
Fig. 4-106
Image of a line before scan correction
with pixel shift in
Fig. 4-107
Image of a line
compensation in X
To this end use an edge of a structure or a line. If
the Scan Corr is not adjusted, you will see a pixel
shift (see Fig. 4-106).
Move the Scan Corr X slider until the pixel shift is
at the minimum (see Fig. 4-107). Rotate the image
by 90 degrees and redo the same adjustment for
Scan Corr Y.
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after
pixel
shift
4-93
OPERATION OF CONFOCOR 3 IN EXPERT MODE
Data Evaluation and Result Presentation …
Carl Zeiss
4.5
Data Evaluation and Result Presentation for FCS Measurements
4.5.1
Structure of the Data Evaluation Window
ConfoCor 3
The data evaluation window (Fig. 4-108) of the FCS software part corresponds to the basic structure of
other Microsoft ® WINDOWS applications.
The control line at the top of the data evaluation window contains the control menu for the data
evaluation window (identical to Microsoft ® WINDOWS), the name of the displayed data file, and the
Minimize, Maximize and Close buttons (identical to Microsoft ® WINDOWS).
In the status line at the bottom of the data evaluation window, the progress of a current measuring
procedure are shown online. During the measurement you can stop the procedure by clicking the Stop
button.
The major part of the window contents the measuring results (graph and table) corresponding to the
selected display modes.
To its right, the Display toolbar is always shown in the standard setting. Depending on whether some
buttons in the Display toolbar are activated / deactivated, further toolbars (e.g.: Correlation, Print, Fit)
are displayed / not displayed on the right-hand side of the data evaluation window.
The data evaluation window can be moved as required within the screen, and its vertical, horizontal and
diagonal size can be matched to the current requirements (identical to Microsoft ® WINDOWS).
Fig. 4-108
4-94
Data evaluation window
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ConfoCor 3
4.5.2
OPERATION OF CONFOCOR 3 IN EXPERT MODE
Data Evaluation and Result Presentation …
Carl Zeiss
Open / Close the Data Evaluation Window
• Click Start either from the Measure window to trigger a new measurement, or open an existing data
file from the database by pressing the Open FCS button.
− The Data Evaluation window will appear (Fig. 4-108).
• Click the Close button to leave the Data evaluation window.
Formats that will be recognized by pressing the Open FCS button include:
− ConfoCor1 data files. If more than 1 file is selected they will be opened in the same Data
Evaluation window. According to the setting at the Average selection box in the Table
Properties window, which is obtained by clicking the right mouse button within the table and
selecting Properties, the average of the correlation or the average of the fit results are shown in
the last row.
− ConfoCor 2 and 3 data files (*.fcs). Each data file will be opened in a separate Data evaluation
window.
Note, that exported data files will be opened within the same window. ConfoCor 2 raw data
files: Each file will be opened in a different Data evaluation window.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
Data Evaluation and Result Presentation …
Carl Zeiss
4.5.3
Description of the Raw Data Format
4.5.3.1
ConfoCor 1
ConfoCor 3
For the ConfoCor 1 no raw data format was available
4.5.3.2
ConfoCor 2
This raw data format is the one for ConfoCor 2 (and ConfoCor 3) data files of Rel 3.5 and lower.
The data format is an exact representation of the dual channel photon trace within the limits given by the
digitalization. This means that the data are recorded without losses within these limits.
The basic idea is to record the time between subsequent pulses from the detector in units of elapsed
clock cycles (run length encoding). However, the format is modified to conserve space at high count rates
and to be capable to handle dual channel data.
Data will be recorded in 16 bit words. Whenever a pulse is detected or the counter counting the clock
cycles overruns 255 (FF hex), a word will be recorded. The word will contain information how many clock
cycles elapsed (1 ... 255) since the last word had been recorded and additionally what happened in the
four cycles of data generation bt1 ... bt4.
The recorded word has the following structure:
Bit
0 (LSB) ... 7
Table 1
Meaning
clock counter value (starting at 1) during the triggering event
trigger events are pulse recordings or counter overruns
zero is reserved and only transmitted at the end of the measurement
8
1, if pulse recorded in channel 1 during cycle bt1; else 0
9
1, if pulse recorded in channel 2 during cycle bt1; else 0
10
1, if pulse recorded in channel 1 during cycle bt2; else 0
11
1, if pulse recorded in channel 2 during cycle bt2; else 0
12
1, if pulse recorded in channel 1 during cycle bt3; else 0
13
1, if pulse recorded in channel 2 during cycle bt3; else 0
14
1, if pulse recorded in channel 1 during cycle bt4; else 0
15 (MSB)
1, if pulse recorded in channel 2 during cycle bt4; else 0
Structure of the recorded word
We hope the two examples will make these statements clearer. The tables show part of the running pulse
train (from left to right) divided into clock cycles. "1" in the corresponding box indicates that a pulse
arrived in this cycle. The "counter" row shows the counter readings. The lowest row indicates when the
word has been recorded.
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Data Evaluation and Result Presentation …
ConfoCor 3
Carl Zeiss
Example 1
The following words will be recorded:
− at W1:
high byte: 00011001(bin) = 19 (hex); low byte: 123 (dec) = 7B (hex); resulting word = 7B19 (hex)
− at W2:
high byte: 00000000(bin) = 00 (hex); low byte: 255 (dec) = FF (hex); resulting word = FF00 (hex)
bt1
CH 1
1
CH 2
counter
bt2
bt3
bt4
bt1
1
0
bt3
bt4
0
0
0
...
1
120 121 122 123
bt2
...
0
0
1
2
3
4
...
254 255
⇑
W1
Table 2
1
2
⇑
W2
Example 1
Example 2
The following words will be recorded:
− at W3:
high byte: 00010001(bin) = 11 (hex); low byte: 123 (dec) = 7B (hex); resulting word = 7B11 (hex)
− at W4:
high byte: 00100100(bin) = 24 (hex); low byte: 255 (dec) = FF (hex); resulting word = FF24 (hex)
bt1
CH 1
bt2
1
bt3
bt4
bt1
1
...
CH 2
counter
120 121 122 123
0
0
0
1
2
3
4
...
bt3
bt4
1
...
⇑
W3
Table 3
bt2
1
254 255
0
0
0
1
2
⇑
W4
Example 2
The clock runs at a clock rate of 20 MHz. This means a maximum data rate of 10 Mbyte/s. If no pulses
are recorded, the clock rate drops to approx. 155 Kbyte/s according to counter overflows.
The first 30 bytes of the raw data file contain the comment "ConfoCor_2_-_Raw_data_file_1.0" and
have to be ignored.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
Data Evaluation and Result Presentation …
Carl Zeiss
4.5.3.3
ConfoCor 3
ConfoCor 3
This raw data format is for ConfoCor 3 (and ConfoCor 2) Rel 4.0 and higher.
In the ConfoCor 3 the format is already processed into one that describes the time distance
between photons.
Raw data files can be opened directly. They are also opened automatically if linked to an .fcs
measurement. In this case all raw data files associated with the .fcs file will be opened in the same
window automatically.
The format is explained in a figure (Fig. 4-109). The figure displays the raw data in an editor that shows
the raw data in file offset, hexadecimal and as ASCII.
Fig. 4-109
ConfoCor 3 format
The first 52 bytes (bytes 1-52) represent the File identifier with the channel number. The identifier is a
randomly created number that will be assigned to all repetitions of the same measurement.
The next 16 bytes (bytes 53-68) is the measurement identifier. This will be assigned to all channels of the
same repetition. Hence, in a cross-correlation experiment, the two auto-correlation pairs that belong
together can be identified.
The next 4 bytes (bytes 69-72) encode the position of the measurement and are zero based.
The next 4 bytes (bytes 73-76) encode the kinetic index and are zero based.
The next 4 bytes (bytes 77-80) encode the repetition number and are zero based.
The next 4 bytes (bytes 81-84) are reserved for comments an set to 0.
The next 4 bytes (bytes 85 -corresponding to a file offset of 5E (hex)=94 (dec)-to 88) code for the first
pulse distance in detector clocks.
The next bytes (starting from byte 89) code for follow up pulse distances.
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4.5.4
OPERATION OF CONFOCOR 3 IN EXPERT MODE
Data Evaluation and Result Presentation …
Carl Zeiss
Display - Correlation
• If not selected, click on the Correlation button to activate the Correlation display mode.
− The Correlation display toolbar will appear to the right of the Data Evaluation window
(Fig. 4-108).
Up to four windows with different graphs become visible in the upper left part of the window depending
on the activated Diagrams buttons. By clicking on the appropriate button, the diagram can be toggled
between ON and OFF. The size of the diagrams can be matched as required by moving the border lines.
Click on the border line, hold down the mouse button, move the line in the required direction; and
release the mouse button.
The measuring results are displayed in a table below the diagrams. The width of the columns can also be
changed by moving the border lines. The order of the columns can also be changed. For this purpose,
click on the head line of the relevant column, hold down the mouse button and move the column to the
required position. When the mouse button is released, the column is inserted in the new position.
If more than one line of the table is selected, an appropriate legend can be added to the diagrams by
activating the required Diagram Legend check boxes.
To select a line in the table, click on it with the mouse (multiple choice is possible by additionally pressing
the Shift or Ctrl key). Selected lines are highlighted in color.
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.
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.
Pressing the right mouse button, when the cursor
is within the table, will open a menu (see Fig.
4-110) offering different options (see next page):
Fig. 4-110
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Table menu
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
Data Evaluation and Result Presentation …
Carl Zeiss
ConfoCor 3
Select all
All the channels from the different measurement and the average channels will be
selected.
Select all channels
All channels belonging to the currently selected one and to one measurement of
repetitive measurements will be selected and highlighted.
Select all
repetitions
All channels belonging to the currently highlighted one of repetitive
measurements will be selected and highlighted.
Select all positions
All channels belonging to the same measurement position than the highlighted
channel will be selected.
Select all kinetic
indices
All channels belonging to the same kinetic time point than the highlighted
channel will be selected.
Delete
The highlighted columns will be deleted.
Cut
The highlighted columns will be stored in the clipboard. Only if the data are
pasted in a new window, the data are deleted from the old one.
Paste
Will paste columns currently stored in the clipboard into the table.
Copy text to
clipboard
The table will be copied into the clipboard and can be opened in other Text
programs.
Write text to file
Will store the table in a .txt file. You will be prompted to choose a name and a
folder.
Properties
Choosing Properties will open the Table Properties window (see Fig. 4-111).
If the Show standard deviation box is checked,
all parameters will be displayed with their values
and the calculated standard deviation from
different measurements.
In the Sort order display window you can choose,
how different measurements are grouped
together. The hierarchy of grouping of categories
is determined by their listed sequence.
Fig. 4-111
Table properties window
In the Average display window you can select,
how the data are averaged. You can either select
Fit Results, in which case the single values of the
parameters are averaged, or Correlation results,
where the single data points of each correlation
functions are averaged and an average correlation
function is calculated.
Please note, only in the latter case the displayed average correlation function can be
meaningfully fitted.
The Displayed columns selection window allows you to change the appearance of the table with regard
to columns to be displayed. All parameters with their boxes checked will be displayed.
The table of measuring rows in the lower part of the Data Evaluation window contains a check box on
the left-hand side of each row. Deactivation of these check boxes will exclude the relevant rows from all
subsequent evaluation procedures. These settings will be stored by saving the data.
However, immediate reactivation is possible via the check box, if required.
The order of the displayed columns can be adjusted using drag and drop.
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Data Evaluation and Result Presentation …
ConfoCor 3
Carl Zeiss
The Correlation toolbar contains the following function elements for different displays:
Count Rate Trace button
Shows 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. If you have set an electronic dust filter
in Processing or loaded Raw data files with an electronic dust filter activated the cut off regions will be
displayed as grayed out areas. Correlation analysis is than performed separately with all sections of the
correlation curves that are disrupted by cut off regions and the calculated curves are averaged.
You can zoom in the image by pressing the left mouse button and drawing a rectangle of the area you
want to zoom in. If the button is disengaged, the zoom image is displayed.
By clicking the right mouse button you obtain the
count rate context menu (see Fig. 4-112).
If you have zoomed in, you can go to the original
image size by choosing the Reset diagram zoom
option.
If you have a raw data file available, you can define
cut regions by choosing the New cut region
option. The cut region size can be adapted by two
sliders. The cut out region is thereby displayed as a
matted box (see Fig. 4-113). You can select
independent cut regions for different channels of a
cross correlation experiments. In cross-correlation
calculations, a cut region in one channel will
automatically define the same cut region in the
other channel. You can select more cut regions by
repeatedly choosing this option.
Fig. 4-113
Fig. 4-112
Count Rate menu
Cut Out Regions
These regions can overlap. Remove cut region will remove the last cut region. By repeatedly choosing
this option, the cut regions in the reverse order of their creation will be removed. Remove all cut
regions will remove all cut regions simultaneously.
Note, that the remaining parts of the count rate will be separately correlated and the average
will be calculated and displayed. Copy text to clipboard will copy the diagram coordinates into
the clipboard, from which they can be pasted into other programs like Excel. Write text to file
will store the diagram coordinates in a .txt file. You will be prompted to choose a name and a
folder before saving.
• Choosing Abscissa logarithmic or Ordinate logarithmic will display the abscissa or ordinate in a
logarithmic scaling.
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Correlation Curve button
Shows 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 zoom function can be used to display certain diagram areas of interest in an enlarged form: Use the
left mouse button to click on the margin of the area of interest in the diagram, keep the mouse button
pressed and draw a rectangular above the area of interest in any required direction. On release of the
mouse button, the selected area is displayed in an enlarged form in the diagram. The scaling of axes is
matched automatically.
By clicking the right mouse button you can choose between different options in the context menu (see
correlation-menu).
The context menu of the relevant diagram can be used to reset the zoom to its original value.
• Click on the diagram with the right mouse button.
− The context menu is opened.
• Click on the line Reset Diagram Zoom with the left mouse button.
− The zoom value is reset.
The data (measuring values) of each diagram can be copied to the clipboard or stored directly as an ASCII
file via the context menu.
• Click on the diagram with the right mouse button to open the context menu.
• Select the line Copy data to clipboard with a click of the mouse if you want to insert the data
directly into other WINDOWS programs via the clipboard. The Paste function permits direct insertion
of these data into the required program.
• Select Write text to file to store the data in an external ASCII file (.txt). Normalized values will also be
exported in this way.
Note that the Export button in the Display Correlation window will only export the original
values.
The diagram display can be manipulated with the context menu as follows:
• Click on the diagram with the right mouse button to open the context menu.
− Selection of Abscissa logarithmic will scale the x-axis logarithmically.
− Selection of Ordinate logarithmic will scale the y-axis logarithmically.
− Selection of Normalize will normalize the curves to 2 in the following way (see Fig. 4-115):
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The G(0) value corresponding to the number of diffusing particles will be normalized to 2, not the total
correlation to which also other processes like triplet state can contribute. All other values (subtracted by
1) will than be multiplied by the correction factor defined by the ratio of 1 to [G(τn2)-1] and increased by1,
hence G (τ)normalized=[1/ (G(0)-1) * (G(τ)-1)]+1. Deactivating Normalize will display the normal correlation
curve (see Fig. 4-114).
Fig. 4-114
The Correlation Curve window in normal display
Fig. 4-115
The Correlation Curve window in normalized display
Count Rate Histogram button
Shows 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. You can
determine the binning time in Processing under PCH or iIf you load a raw data file with the Reload
button.
You can zoom in the image by pressing the left mouse button and drawing a rectangle of the area you
want to zoom in. If the button is disengaged, the zoom image is displayed.
By clicking the right mouse button you can choose between different options in the context menu.
If you have zoomed in, you can go to the original image size by choosing the Reset diagram zoom
option.
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Copy text to clipboard will copy the diagram coordinates into the clipboard, from which they can be
pasted into other programs like Excel. Write text to file will store the diagram coordinates in a .txt file.
You will be prompted to choose a name and a folder before saving.
Choosing Abscissa logarithmic or Ordinate logarithmic will display the abscissa or ordinate in a
logarithmic scaling.
Pulse Density button
Shows 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.
You can zoom in the image by pressing the left mouse button and drawing a rectangle of the area you
want to zoom in. If the button is disengaged, the zoom image is displayed.
By clicking the right mouse button you can choose between different options in the context menu (see
pdh-menu).
Copy text to clipboard will copy the diagram coordinates into the clipboard, from which they can be
pasted into other programs like Excel. Write text to file will store the diagram coordinates in a .txt file.
You will be prompted to choose a name and a folder before saving.
Choosing Abscissa logarithmic or Ordinate logarithmic will display the abscissa or ordinate in a
logarithmic scaling.
Note that, normalization only works for a curve, for which a fit has been conducted in the Fit
window.
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Display - Fit
• Click on the Fit button to activate the Fit display mode.
− A Fit menu subordinate will appear (Fig. 4-116).
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. 4-116
Display fit
The upper graph shows the correlation curve with the overlaid fit graph. It also displays the fit range
defined by the red (start value) and the blue (end value) bars. The lower graph depicts the fit residuals.
• Select one or more channels in the lower table. The last activated channel will be displayed in the
Channel display.
• Use the Model pull down menu to select a predefined model. All the terms and components of the
model will be displayed activated. All parameters to one term are listed as a block in the Parameter
column. The parameters’ values are displayed in the Values column.
• You can deselect and select terms by de-checking / checking the boxes on the left of each term.
• Set the range of the curves to be fitted by setting the red and blue bars in the curve fit window. Press
the Fit button to fit the loaded curves to the model. If you press Fit all the other channels in the
window corresponding to the highlighted ones will be fitted as well.
Note, all other channels will not be fitted and the model has to be selected anew, if such a
channel is selected.
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• An additional button bar (Free, Fix or Start)
will appear if you click at the right-hand side of
the parameter fields in the Type column (see
Fig. 4-117).
− If you select Free, the parameter will be
defined as free. After the fit, the field will
contain the fit result for the parameter. The
start values of the parameter (initial guesses)
will be calculated by an algorithm.
Fig. 4-117
Fit Type selection
− Fix allows you to fix the parameter. This is
useful if you know its value from other
measurements.
− Start will assign a start value to the
parameter and leaves the parameter free to
fit. In this case, no initial guesses will be
made.
• Upper and lower values for the fit parameters can be entered in the fields under the Upper limit and
Lower limit columns. Defaults are the extreme possible values. If fit limits are exceeded, the fit will try
to find another solution or will fail.
• Parameters can be linked globally by an additional button bar (link measurement M, link kinetic
indexes K, link positions P, link repetitions R, link channels C). You can select the type of linkage
by checking the corresponding boxes. After closing the button bar, the linkage type is displayed as a
one letter abbreviations. If a linkage is activated that does not apply for a measurement, it will be just
disregarded. Global fitting will fit all linked parameters of different measurements to the same value.
On-off measurements will interfere with global fitting and should be deactivated. The following
linkages of a parameter is possible:
Link measurement: links a parameter from different measurements
Link kinetic indexes: links a parameter from the same time points
Link positions: links a parameter obtained from measurements at the same site
Link repetitions: links a parameter for all repetitions of one measurement
Link channels: link a parameter for the same channels
• Start Channel and End Channel enable you to determine which part of the correlation curve should
be fitted to the model. Moving the red or blue line with the mouse permits the parameters Start
Channel and End Channel to be determined directly in the Correlation-Time diagram. The start and
end position of the channels will be displayed as the absolute position (1 – 255) or correlation times (in
μsec) and are represented by the red and blue bar, respectively.
Caution: The numbers indicate which channels are used for correlation. If you want to know
the number of channels omitted from the respective end you have to subtract the Start
channel number by 1 and subtract from 255 the End channel number, respectively.
The State display box informs you about the fitting conditions. The quality of the fit is displayed in the
2
2
2
chi display box of the result table. The Χ (chi ) value should approach zero for highest quality.
• When you are finished, click the Fit button and the fit procedure will b e run for the fit settings field
(on the right-hand side of the diagrams).
− 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.
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− The result table will also be updated for the highlighted channels.
• With a click on the Fit all button, the fit procedure is performed for all activated measurements (via
the check box of the row) in the result table of those channels that were of the same type as the
highlighted ones.
• With the Undo button, you can cancel a previous command. Repeatedly pressing will cancel
commands in the reverse of their execution. The Redo button will re-execute the previous undone
command. Repeatedly pressing will re-execute previous undone commands in the order of their
previous execution.
The ToMethod function permits the fitted parameters to be transferred directly to an existing or a new
Measuring Method.
• Open the Measure window by clicking the appropriate button in the ConfoCor subordinate toolbar
of the Main menu and press the Processing button. Choose the same model as used in the fit.
• Then click on the Write to Method button.
The parameters of the selected fitting model are assigned to the method selected in the Processing
window and stored.
• If the parameters shall be assigned to a new method, click on the Save button and enter a new name
for the method in the appearing Save Method window. Confirm your entry by clicking on OK.
All the result table functions in the Fit mode are identical to those in the Correlation mode.
The zoom function can be used to display certain
diagram areas of interest in an enlarged form:
• Use the left mouse button to click on the
margin of an area of interest in the diagram,
keep the mouse button pressed and draw a
rectangular above the area of interest in any
required direction. On release of the mouse
button, the selected area is displayed in an
enlarged form in the diagram. The scaling of
axis is matched automatically.
The context menu can be used to reset the zoom
to its original value.
Fig. 4-118
Context menu of the
Fit diagram
• Click on the diagram with the right mouse button to open the context menu (see Fig. 4-118).
• A click on the Reset Diagram Zoom with the left mouse button will reset the zoom value.
The data of the diagram can be copied to the clipboard or stored directly as an ASCII file via the Context
menu.
• Click on the diagram with the right mouse button to open the context menu.
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• Select the line Copy data to clipboard with a click of the mouse if you want to insert the data
directly into other WINDOWS programs via the clipboard. The Paste function permits direct insertion
of these data into the required program.
• Select Write to file to store the data in an external ASCII file (.txt). Only the coordinates of displayed
curves are exported.
The display of the diagram can be adapted by use of the context menu.
• Click on the diagram with the right mouse button to open the context menu.
• Select Abscissa logarithmic or Ordinate logarithmic to scale the x- or y-axis, respectively,
logarithmically.
Fig. 4-119
Correlation Curve window in normal display
• Select View measured data will display the measured Correlation curve in addition to the Fit curve
(see Fig. 4-119). If this option is not selected, only the Fit curve is displayed (see Fig. 4-121).
• Select Normalize to display the correlation curves in a normalized form (see Fig. 4-120). The
amplitude of the diffusion contribution will be normalized to 2, not the total amplitude.
• Select Show fit range text to display the start and end values of the fitted data defined by the red
and blue bars.
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Fig. 4-120
Correlation Curve window in normalized display
Fig. 4-121
Correlation Curve window in fit curve display ("View measured data"
unchecked)
Carl Zeiss
The zoom function can be used to display certain areas of interest in the Fit deviation diagram see fitdeviation-window) in an enlarged form:
• Use the left mouse button to click on the margin of the area of interest in the Fit deviation diagram,
keep the mouse button pressed and draw a rectangular above the area of interest in any required
direction. On release of the mouse button, the selected area is displayed in the diagram in an enlarged
form. The scaling of axes is matched automatically.
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The context menu of the Fit deviation diagram
(can be used to reset the zoom to its original value
(see fit-deviation-context).
• Click on the diagram with the right mouse
button.
− The context menu (see Fig. 4-122) is opened.
• Click on the line Reset Diagram Zoom with
the left mouse button.
Fig. 4-122
Context menu of the
Fit deviation diagram
− The zoom value is reset.
The scaling of the G(t) axis of the Fit deviation
diagram can be varied in percentage values of the
fit scaling via the context menu.
• Click on the diagram with the right mouse
button to open the context menu.
Fig. 4-123
Diagram scaling window
• Select the line Scaling with a click of the
mouse.
− The Diagram scaling window (see Fig. 4-123) is opened.
• Enter the required percentage value (from 1 to 100) for the scaling and click on OK.
− The scaling is changed accordingly.
− Enter Cancel if you want too close the window without changes
The data of the diagram can be copied to the clipboard or stored directly as an ASCII file via the context
menu.
• Click on the diagram with the right mouse button to open the context menu.
• Select the line Copy data to clipboard with a click of the mouse if you want to insert the data
directly into other WINDOWS programs via the clipboard. The Paste function permits direct insertion
of these data into the required program.
• Select Write to file to store the data in en external ASCII file (.txt).
The table of measuring rows in the lower part of the Data Evaluation window contains a check box on
the left-hand side of each row. Deactivation of these check boxes will exclude the relevant rows from all
subsequent evaluation procedures.
However, immediate reactivation is possible via the check box, if required.
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Display - Coincidence
• Click on the Coincidence button to activate the Coincidence display mode.
− The coincidence analysis generates a two-dimensional color-coded count rate histogram
(Fig. 4-124).
This is an alternative to the standard correlation analysis during rare event detection. If the FCS is used to
look for small amounts of double-labeled objects (molecules, aggregates etc.) in liquids, the
measurement time to generate a correlation curve which can be evaluated by the standard model may be
prohibitively long. As a rule of thumb, approximately 1000 transitions of labeled aggregates are required
to generate a correlation curve of sufficient quality.
Fig. 4-124
Coincidence diagram window
During the measurement in small time windows the count rates in channel 1 (red) and channel 2 (blue)
are registered and the respective frequencies are calculated. The coordinates of the entry respective to
the x- and y-axes of the diagram are given. Once the coordinates of the cell are found, the value will be
incremented.
If double-labeled aggregates are present, the diagonal of the diagram will be populated.
All the table functions in the Coincidence mode are identical to those in the Correlation mode
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4.5.7
ConfoCor 3
Display - Preview
• Click on the Preview button to activate the Preview display mode.
− A print preview and the additional Print subordinate toolbar will appear on the screen (Fig. 4-125).
Fig. 4-125
Preview / Print window
The Preview / Print window shows an overview, as a table and/or a graph, of the results of a
measurements and the relevant measuring parameters of the used measuring method. This result
overview can be printed directly on a connected printer.
For this purpose, the following function buttons are available in the Print subordinate toolbar (at the
right-hand side of the Preview / Print window):
Print button
Starts the print function.
Setup button
Opens the printer setup window to set the print parameters.
Landsc. button
Selects Landscape paper orientation.
Portrait button
Selects Portrait paper orientation.
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Compressed button
Selects the Compressed Report preview without diagrams.
Standard button
Selects the Standard Report preview with correlation and fit curves.
Full button
Selects the Full Report preview with all diagrams.
All button
Prints all measurements.
Selection button
Prints selected measurements.
Current button
Prints currently selected measurements only.
To print a report, proceed as follows:
• Select the Compressed, Standard or Full button.
• Select the All, Selection or Current button.
• Click on Landsc. or Portrait for paper orientation.
• If required, change the printer settings using the printer Setup button.
• Click on Print to start the print procedure.
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4.5.8
ConfoCor 3
Display - Save Data
• Save allows you to save the whole data set
(curves, fitting results, fit parameters, raw data)
to disk. If Save is selected, the Save window
appears (see Fig. 4-126).
The Save menu offers the possibility of entering
file name in the Name field.
Fig. 4-126
Save window
It also offers the possibility of entering a comment
in the Comment field.
The directory can be chosen in the Directory field.
A directory can be selected if the button to the
right is pressed.
It is possible to store the data as ANSI text (space-consuming, but readable) or as a binary file.
If the Save raw data box is checked, the raw data will be stored in a file bearing the same name than
the .fcs file, which contains all channels as separate files.
• OK will confirm the action, Cancel will exit the menu without further action.
4.5.9
Display - Copy Table
• With Copy Table, the whole table will be copied to the WINDOWS NT clipboard. The contents of the
clipboard can be used by most WINDOWS NT applications via the Paste function.
4.5.10
Display - Save Table
• With Save Table, the result table can be written to a white space in a separate text file. If the button
is clicked, a file menu will appear offering the possibility to select a directory and to enter a file name.
4.5.11
Display - Copy Graphs
• Click on the Copy Graphs button to copy the displayed graphs to the clipboard.
− Using the Paste function of Microsoft WINDOWS, you can paste the graphs into other WINDOWS
applications.
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Display - Export
• With Export, the measured data will be
exported in a ConfoCor 1-compatible format
and can be handled using the FCS Access Fit
program. In the Export window (see Fig.
4-127) you can enter a name in the File name
field and a type in the Data type field. In the
Save pull down menu you can select a
directory. You can specify if only the Selected
rows (highlighted), the Current row (the current
selected one) or all rows are exported. Exported
files can be opened again with the Open FCS
button. All selected files will be loaded in one
Correlation display window.
Fig. 4-127
4.5.13
Export window
Reuse
• With a click on the Reuse button, the configuration used to record the of the present fit curve will be
loaded.
4.5.14
Reload
The Reload can be used for already opened raw
data files. The Correlate window will appear (see
Fig. 4-128).
You have the following options:
− Automatic dust filter
− Correlator settings
− Count rate settings
− Photon Counting Histogram settings
Fig. 4-128
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4.5.14.1
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Automatic Dust Filter
If the Automatic cut box is checked, you can type in a threshold in % in the Automatic cut ratio
display box. This sets a threshold intensity for an electronic dust filter. The value (0 … 100) will define the
threshold in percentage. A default value of 10% can be selected by pressing the button on the right.
If the integrated count rate over a certain count interval will exceed the average count rate by that
threshold, this special interval is discarded for correlation analysis. For example, if the system detects a
count rate in a certain time interval that exceeds the average count rate by over 30 % and the threshold
was set at 30, this interval is discarded for correlation analysis. The time intervals before and after the
discarded region are separately correlated and the results averaged. These holds also true, if more than
one region is discarded.
The single regions taken into account and disrupted by discarded regions are separately correlated and
the resulting average will be displayed.
Note, that due to built up of an average, this kind of dust filter does not work for count rates
exceeding the average that will come at the beginning of the measurement time before the
system had the possibility to calculate the average count rate. Also, due to the necessity to
average signals over a certain integration time, more than only the peak area will be discarded.
Another outcome of the necessity to average the count rate signal is that several small peaks
following close to each other will be treated as a huge peak and might be cut out. This means,
in the Automatic cut mode accumulated count rates rather than peaks are cut out. For crosscorrelation experiments, any of the regions discarded in either autocorrelation function will not
be used. Cut off regions are framed by stippled boxes and appear matted in the Count Rate
window.
4.5.14.2
Correlator Settings
You can determine the start value of the Correlator binning time in μs, the Maximum correlator
time in s and the start value for the Correlator tau channels by typing the values in the respective
selection boxes. Default values (0.2 μs, 1000 s and 8) can be loaded by pressing the corresponding
default buttons on the right.
4.5.14.3
Count Rate Settings
You can either check the Automatic count rate binning box, in which case a dynamic binning is
chosen, or deactivate the box and type in a value in ms in the Count rate binning selection box for a
constant binning with the defined value. The default value of 1 ms can be loaded by pressing the default
button to the right.
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Photon Counting Histogram Settings
You can either check the Automatic photon counting histogram binning box, in which case 32
histograms with different binning are calculated and the one with the best dynamic range (3 standard
deviations) is chosen, or deactivate the box and type in a value in μs in the Photon count histogram
binning selection box for a constant binning with the defined value. The default value of 10 μs can be
loaded by pressing the default button to the right.
4.5.15
Info
Pressing the Info button will open the Method Information window (see method-information). With
Copy All, the complete parameter set will be copied to the clipboard.
A click on the Print button opens the Print Setup window and allows the parameters of the method to
be printed in the form of a table.
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4.6
Combined Application of LSM Scanning and FCS Measurement Procedure
4.6.1
Taking FCS Measurements within a Cell
ConfoCor 3
The simultaneous installation of the ConfoCor 3 onto the channel 4 of the LSM 510 permits examination
of points selected within a single LSM image with the ConfoCor 3.
For combined application, proceed as follows:
• Click on the ConfoCor button in the of the Main menu to select the FCS mode.
• Click on the Measure button in the ConfoCor subordinate toolbar to open the Measurement
window. Select the FCS measurement method by clicking the methods button and select a method.
Close the window.
• Open the Adjust window. Initialize stage and focus. Switch on the lasers using the Laser button.
Close the Laser Control and the Adjust windows.
• Click on the VIS button in the ConfoCor subordinate toolbar
• Select the specimen area to be examined by moving the stage and focus.
• Click on the LSM button in the ConfoCor subordinate toolbar
• Select the appropriate beam path configuration in the Configuration Control window.
• Open the Scan Control window by clicking the Scan button in the Acquire subordinate toolbar. Set
the scan parameters and click on Single to scan an image of the sample.
• When finished, select the Acquisition mode in the Measurement window.
There are two ways of defining the positions, where FCS measurements should be performed in an
image: Current Position and LSM Image.
4.6.1.1
Using the Current Position
When using this method, FCS measurements are performed on a fixed position without scanner or stage.
In this case the structure of interest must be moved to the position manually under LSM control prior to
FCS measurements. In either case, f If Scanner or XY Stage are used and no rotation was defined, than
the laser position with the parked mirrors is the centre of the image. In case of rotation the position hof
the laser beam with this rotation within the image has to be determined by a bleach experiment. You can
use the Crosshair in any case to mark the position of the laser beam.
• Open the Measurement window by clicking on the Measurement button in the ConfoCor
subordinate toolbar. Press the Current Position button in the Position panel via mouse click. The
Position panel changes to allow the activation of the Crosshair function.
• Activate the Crosshair button via mouse click and click into the image.
− The crosshair appears in the LSM scan image. Place the cross at the position, where the Laser beam
is positioned. If you have activated the Lock button, than the position will be fixed.
• Scan the image continuously and position the site of interest under the crosshair.
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Using the LSM Image Window
With this method you can conveniently choose several points of interest at which FCS measurements will
be performed. You can approach this positions either with the scanning stage is the XY stage button is
activated, or by the mirrors, if the Scanning button is activated.
• Open the Measurement window by clicking the Measure button in the ConfoCor subordinate
toolbar. Press LSM Image and select between Scanner, XY stage or Piezo stage.
− The Positions panel changes to allow the
definition of positions in the LSM scan
image.
• Activate the Select button via mouse click.
− The cursor (crossline) appears in the LSM
scan image.
• Move the cursor to the desired position in the
scan image (drag and drop). Click on the Add
Pos button to select this position. The
coordinates of the position appears in the
Positions list of the Sample panel.
• In a Z Stack, you can also vary the Z-plane in
which measurement is to be performed. For this
purpose, select the required Z slice in the stack
or set the Z value via the focusing drive of the
microscope.
• Select further positions to be measured in the
same way.
Fig. 4-129
LSM scan image with cursor (crossline)
and 3 selected positions
• Click on the Mark Pos button to fix the
selected positions as overlay elements in the
image.
The X-, Y- and Z- coordinates of the selected positions are only stored in the Positions table and
not in the scanned image (stack). Accordingly, all the marked positions in all the Z-planes are
displayed in the scanned image (Z Stack), no matter in which Z-plane they lie.
• When finished, click on the Start button in the Measurement window to start the FCS
measurements of the selected positions.
− The measurement procedure starts and the data and evaluation window appears on the screen.
Please note that the positions selected for FCS measurements in the Measurement window
using the ConfoCor 3 are approached by the scanning stage, Piezo stage or scanning mirrors,
whichever was chosen. In case of scanning mirrors, the stage does not move. All positions will
be approached sequentially.
In case of x and y scan, it is the scanning mirrors that are used. Hence there is no offset between
defined positions and actual positions. However, a scan correction in x and y can be required,
which is done by changing the Scan Corr X and Scan Corr Y positions in the Scan window, if
the bidirectional scan was chosen. If stages are used, the offsets have to be entered in the
Settings menu under LSM + ConfoCor.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
Data Evaluation and Result Presentation …
Carl Zeiss
4.6.1.3
ConfoCor 3
Performing FCS Measurements on the Cell Membrane
Due to the shape of the confocal volume, the membrane should not be approached from the side but
instead rather from the top of the cell. Please note that it is better to use the upper membrane for
measurement, since the lower membrane might be too close to the glass bottom surface resulting in
disturbing reflections. You can home in on the membrane either manually or using the Z Scan feature in
the Method Optimization ... window.
(1)
Manual Focusing on the Membrane
• Acquire an image of the cell.
• Place the focus above the cell and scan continuously.
• Focus slowly down until you see a fluorescence signal.
• Stop focusing.
Due to its high sensitivity ConfoCor 3 can still detect signals that cannot be imaged by the LSM. If the
signal is too low for imaging, the membrane can still be detected using the Z Scan feature in the
Measurement window.
(2)
Focusing on the Membrane by a Z Scan
The Z Scan measures the count rate at previously defined Z positions. It is performed without table
movement, that is at the fixed position of the laser beam.
• Position the region of interest at the site of the fixed laser beam path as described in chapter 14.6.1.
• Optional: Perform a Z Stack that includes the membrane of the cell with the LSM.
• Open the Measurement window by clicking on the Measurement button in the ConfoCor
subordinate toolbar. Press the Current Position check box or the LSM Image button in the Position
panel via mouse click.
− The Position panel changes and shows the Adjust and the Crosshair buttons for Current
Position or the Position list for LSM Image.
• Activate the Crosshair button via mouse click or define a position.
− The crosshair or cross appears in the LSM scan image.
• Move the cursor to the appropriate position in case of Current Position or select by pressing the Add
button in LSM Image followed by Mark Pos to fix the position as an overlay element
• Press the Z Scan button. The Z Scan display shows the current Z position of the laser beam. Check the
corresponding channel if not yet activated.
• Select the start and end position of the Z Stack. If you have acquired a Z Stack in the LSM mode, use
the values defined by the Mark First/Last button. Note that the values defined by the Z Stack in the
LSM mode are not automatically used in the Z Scan of the FCS mode.
• Enter the number of positions. If you have acquired a Z Stack, use the value defined in the
Z Sectioning tab.
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ConfoCor 3
OPERATION OF CONFOCOR 3 IN EXPERT MODE
Data Evaluation and Result Presentation …
Carl Zeiss
• Press the Start button to perform a Z scan. Press the Cancel button if you want to leave the Z Scan
settings.
• After pressing the Start button a Z scan is
performed and the intensity displayed in
dependence of the Z position (see Fig. 4-130).
The red line in the diagram shows the actual
Z position. Peaks show Z levels of high signal
intensity and may correlate to labeled
membranes. Note, that the glass surface will
also give a peak. To determine its position, just
perform a Z Scan at a position where there is no
cell. Place the red line at the peak that
corresponds to the membrane to select this
Z position.
Fig. 4-130
Z-Scan image
• If no clear signal can be detected, or the peak of interest lies too close to the border, close the Scan
window and perform a new Z scan. Choose a different Z range by modifying start and end positions
to values that lie closer to the position at which the signal should be expected.
• If the Z position has been selected, close the Image Display window by clicking on the Close button.
• Press the Start button in the Measurement window to perform an FCS measurement.
− The FCS measurement is performed at the same X/Y position as the Z scan.
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OPERATION OF CONFOCOR 3 IN EXPERT MODE
Index
Carl Zeiss
4.7
Index
3
3D View ........................................................4-5
A
Acquire ................................................. 4-4, 4-7
Acquisition ..................................................4-39
Adjust .........................................................4-13
Adjust Pinhole .............................................4-34
Amplitudes..................................................4-56
Anti-bunching Terms ...................................4-59
Auto Save....................................................4-83
Automatic Dust Filter .................................4-116
B
Beam path................................ 4-25, 4-26, 4-34
Bunching Terms...........................................4-61
C
Carrier...................................... 4-18, 4-45, 4-46
Carrier Configuration ..................................4-14
Carrier Position............................................4-36
Chamber .....................................................4-47
Coincidence ..............................................4-111
Collimator, adjust ........................................4-38
Combined Application of LSM and FCS......4-118
ConfoCor menu ..........................................4-10
Context menu .............................................4-24
Context menu ............................... 4-102, 4-110
Copy Graphs .............................................4-114
Copy Table ................................................4-114
Correlation ..................................................4-99
Correlation Function ....................................4-54
Correlator Settings ....................................4-116
Count rate Settings ...................................4-116
Count rate window .....................................4-22
Cross-correlation function, definition ...........4-55
Crosshair Function .......................................4-44
Current Position ..........................................4-44
D
Data Evaluation ................................. 4-21, 4-94
Data Handling .............................................4-52
Defining a Model.........................................4-72
Diffusion Terms ...........................................4-65
Display - Coincidence ................................4-111
Display – Copy Graphs...............................4-114
Display – Copy Table .................................4-114
Display - Correlation ....................................4-99
Display – Export............................... 4-20, 4-115
Display - Fit................................................4-105
Display – Preview .......................................4-112
4-122
ConfoCor 3
Display - Save Data ....................................4-114
Display – Save Table...................................4-114
E
Excitation.....................................................4-36
Export.............................................. 4-20, 4-115
F
File ............................................... 4-4, 4-6, 4-10
Fit.................................................... 4-51, 4-105
Fitting the Correlated Data ..........................4-54
I
Info ...........................................................4-117
K
Kinetics........................................................4-40
L
Laser Control ..................................... 4-11, 4-29
LSM + ConfoCor .........................................4-83
LSM Image ..................................................4-49
M
Macro............................................................4-5
Main Menu....................................................4-4
Maintain........................................................4-5
Measure ......................................................4-19
Measurement ..............................................4-80
Measurement methods................................4-11
Measurement procedure..............................4-21
Method Details ............................................4-13
Methods......................................................4-11
Micro Button .................................................4-8
Microscope Stage ........................................4-43
Model Equations................................ 4-54, 4-55
Models ........................................................4-71
Multiple Positions ........................................4-48
O
Offsets............................................... 4-84, 4-92
Options .........................................................4-5
P
Photon Counting histogram (PCH) ...............4-70
Photon Counting Histogram Settings .........4-117
Pinhole adjustment ......................................4-34
Positions ......................................................4-42
Preview......................................................4-112
Print ..........................................................4-112
Process ..........................................................4-5
Processing ...................................................4-51
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ConfoCor 3
OPERATION OF CONFOCOR 3 IN EXPERT MODE
Index
R
Reflected-light Observation............................4-9
Reload .......................................................4-115
Result Presentation ............................ 4-21, 4-94
Reuse ........................................................4-115
S
Sample Carrier.......................... 4-13, 4-45, 4-47
Save Data ..................................................4-114
Save Table .................................................4-114
Scan Mirrors ................................................4-42
03/06
Carl Zeiss
Settings ...................................................... 4-80
Single Position ............................................ 4-47
System Configuration ................................. 4-25
T
Times.......................................................... 4-39
Toolbar......................................................... 4-4
Transmitted-light Observation....................... 4-9
Z
Z (Focus) Panel............................................ 4-14
Zoom function............................... 4-102, 4-109
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Carl Zeiss
4-124
OPERATION OF CONFOCOR 3 IN EXPERT MODE
Index
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ConfoCor 3
03/06
LSM 510 / LSM 510 META
and ConfoCor 3
Laser Scanning Microscopes
Brief Operating Manual
Release 4.0
March 2006
Contents
Page
Starting the System ................................................................................................................3
Setting the microscope...........................................................................................................6
Configuring the beam path and lasers..................................................................................8
Scanning an image ...............................................................................................................11
Storing an image ..................................................................................................................15
Using the ConfoCor ..............................................................................................................15
Switching off the system .....................................................................................................20
Introduction
For your safety!
Observe the following instructions:
2
−
The LSM 510- and LSM 510 META-ConfoCor 3 laser scanning microscope, including its
original accessories and compatible accessories from other manufacturers, may only be
used for the purposes and microscopy techniques described in this manual (intended use).
−
In the Operating Manual, read the chapter Safety Instructions carefully before starting
operation.
−
Follow the safety instructions described in the operating manual of the microscope and
HBO 100 mercury lamp.
03/06
Starting the System
Switching on the LSM system
• When set to ON the REMOTE CONTROL switch
labeled System/PC provides power to the
microscope and the computer. This allows to
use the microscope and the computer without
running the LSM Software (Fig. 1).
• To switch on the system completely put the
Components switch also to ON. Now the
complete system is ready to be initialized with
the LSM Software.
Switching on the HBO 100 mercury lamp
• Switch on the HBO 100 mercury lamp via the
switch of the power supply, see operating
manual of the mercury lamp or microscope.
Fig. 1
REMOTE CONTROL switch
Fig. 2
Power supply of UV-Ar laser
Switching on the Enterprise UV-Ar Laser
• If the UV laser is required, switch it on via the
toggle switch (Fig. 2/1) of the power supply.
− It will be ready for operation after a few
seconds.
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3
Starting the LSM 5 software program
• Double click the LSM 510-FCS icon on the desktop of WINDOWS to start the LSM 5
software program.
The LSM 510 / ConfoCor Switchboard window appears on the screen (Fig. 3).
Fig. 3
LSM 510-ConfoCor Switchboard menu
• Click on the Scan New Images button in the LSM 510-ConfoCor 3 Switchboard
window.
Clicking on this button activates the complete LSM hardware (on-line mode).
• Click on the Start Expert Mode button in the LSM 510-ConfoCor 3 Switchboard
window.
The LSM 510 - Expert Mode Main menu appears on the screen.
Use of this mode requires to be thoroughly familiar with the exact microscope procedures
and interrelations.
Fig. 4
4
Main menu for Expert Mode
03/06
Creating a database for acquired images
• Click on the File button in the Main menu toolbar.
The File subordinate toolbar appears in the Main menu.
Fig. 5
Main menu with File subordinate toolbar
• Click on the New button in the File subordinate toolbar.
The Create New Database window appears.
• Select drive C: or D: from pull down menu.
• Create a new directory if needed.
Fig. 6
Fig. 7
Create New Database window
Create New Database window
Turning on the lasers
• Click on the Acquire button in the Main menu to open the Acquire subordinate
toolbar.
Fig. 8
03/06
Acquire subordinate toolbar
5
• Click on the Laser button to open the Laser Control window.
• Select the appropriate Laser Unit by clicking on
the name of it.
• Click on the Standby button to switch required
laser(s) to Standby.
• When status is Ready click on On button.
• Set Output [%] so that the Tube Current is
about 4 A (50% of the output power).
Fig. 9
Laser Control window
Setting the microscope
Changing between direct observation or laser scanning
The VIS, TV and LSM buttons switch the beam path and indicate which beam path has been set in the
binocular tube of the microscope:
• Click on the VIS button to set the microscope for direct observation via the eyepieces
of the binocular tube, lasers are off.
• Click on the TV button to set the microscope camera observation (if connected) via
camera adapter of the binocular tube.
• Click on the LSM button to set the microscope screen observation via laser excitation
using the LSM 510 and software evaluation.
Setting the microscope and storing the settings
• Click on the VIS button for direct observation.
• Click on the Micro button in the Acquire subordinate toolbar to open the
Microscope Control window of the used microscope.
The Microscope Control window appears (Fig. 10).
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03/06
Selecting an objective
• Open the graphical pop-up menu by clicking on
the Objective button (Fig. 10).
• Click on the objective you want to select. The
selected objective will automatically move into
the beam path.
Focussing the microscope for transmitted light
• Open the graphical pop-up menu by clicking on
the Transmitted Light button (Fig. 11).
• Click on the On button. Set the intensity of the
Halogen illuminator using the slider.
• Click on Close to close the pop-up menu.
• Place specimen on microscope stage. The cover
slip must be facing up.
• Use the focusing drive of the microscope to
focus the required object plane.
• Select specimen detail by moving the stage in X
and Y using the XY stage fine motion control.
Fig. 10
Microscope Control window,
e.g.: Axiovert 200 M
Fig. 11
Microscope Control window with
Transmitted Light pop-up menu
Setting the microscope for reflected light
• Click on the Reflected Light button to open
the shutter of the HBO 100 mercury lamp.
• Click on the Reflector button and select the
desired filter set by clicking on it.
Storing the microscope settings
Microscope settings can be stored and up to 8
buttons assigned for fast retrieval and adjustment
using the Microscope Settings panel.
The Store button permits existing microscope
configurations to be stored under any name.
The Apply button permits existing
microscope configurations to be loaded.
stored
The Delete button permits existing microscope
configurations to be deleted.
The Assign button permits the assignment of a
microscope configuration to a button.
Note: Depending
on
the
microscope
configuration, settings must be done manually if
necessary.
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7
Configuring the beam path and lasers
• Click on the LSM button in the Acquire
subordinate toolbar for laser scanning.
Choosing the configuration
Single Track
− Use for single, double and triple labeling;
simultaneous scanning only
− Advantage: faster image acquisition
− Disadvantage: cross talk between channels
Multi Track
− Use for double and triple labeling; sequential
scanning, line by line or frame by frame
− Advantage: when one track is active, only
one detector and one laser is switched on.
This reduces cross talk.
− Disadvantage: slower image acquisition
Fig. 12
Configuration Control window for
Single Track
• Click on the Config button in the
Acquire subordinate toolbar to
open the Configuration Control
window.
The Configuration Control window
appears (Fig. 12).
Setting for single track configuration in Channel Mode
• Select Channel Mode if necessary (Fig. 12).
• Click on the Single Track button in the Configuration Control window (Fig. 12).
• Click on the Descanned button if necessary.
The Beam Path and Channel Assignment panel of the Configuration Control window displays the
selected track configuration which is used for the scan procedure.
• You can change the settings of this panel using the following function elements:
Activation / deactivation of the excitation wavelengths (check box) and setting of
excitation intensities (slider). Open the Laser Control window via the Laser button.
Selection of the main dichroic beam splitter (HFT) or secondary dichroic beam splitter
(NFT) position through selection from the relevant list box.
Selection of an emission filter through selection from the relevant list box.
Activation / deactivation (via check box) of the selected channel (Ch 1-4, monitor diode
ChM, META detectors ChS1-8, transmission ChD) for the scanning procedure by
assigning an existing color icon or defining a new one.
8
03/06
• Select the appropriate filters and activate the
channels.
• Click the Excitation button to select the laser
lines and set the attenuation values
(transmission in %) in the displayed window.
For the configuration of the beam path, please
refer to the application-specific configurations
depending on the used dyes and markers and the
existing instrument configuration.
• Clicking on the Spectr button
opens the Detection Spectra &
Laser Lines … window (Fig. 13) to
display the activated laser lines for
excitation (colored vertical lines) and
channels (colored horizontal bars).
Fig. 13
Detection Spectra & Laser Lines …
window
Fig. 14
Track Configurations window
Fig. 15
Configuration Control window for
Multi Track
• Clicking on the Config button
opens the Track Configurations
window (Fig. 14) to load, store or
delete track configurations.
• For storing a new track configuration enter a
desired name in the first line of the
Configurations list box and click an Store.
• For loading an existing configuration select it in
the list box and click on Apply.
• For deleting an existing configuration select it in
the list box and click on Delete.
Setting for multi track configuration in
Channel Mode
The Multi Track function permit several tracks to
be defined as one configuration (Channel Mode
Configuration) for the scan procedure, to be
stored under any name, reloaded or deleted.
The maximum of four tracks with up to 8 channels
can be defined simultaneously and then scanned
one after the other. Each track is a separate unit
and can be configured independently of the other
tracks with regard to channels, Acousto-Optical
Tunable Filters (AOTF), emission filters and dichroic
beam splitters.
• Select Channel Mode if necessary (Fig. 15).
03/06
9
• Click on the Multi Track button in the Configuration Control window (Fig. 15).
• Click on the Descanned button if necessary.
The following functions are available in the List of Tracks panel (Fig. 15).
Modes
Switch tracks after each
Line button
Tracks are switched during scanning line by line. The following settings
can be changed between tracks: Laser line and intensity and channels
incl. settings for gain and offset.
Switch tracks after each
Frame button
Tracks are switched during scanning frame by frame. The following
settings can be changed between tracks: Laser line and intensity, all
filters and beam splitters, the channels incl. settings for gain and offset
and the pinhole position and diameter.
Frame Fast button
The scanning procedure can be made faster. Only and the laser line and
intensity and the Amplifier Offset are switched, but no other hardware
components. The tracks are all matched to the current track with regard
to emission filter, dichroic beam splitter, setting of Detector Gain,
pinhole position and diameter. When Line button is selected, the same
rules apply as for Frame Fast.
Settings
Add Track button
An additional track is added to the configuration list. The maximum of
four tracks can used. One track each with basic configuration is added,
i.e.: one Ch 1 channel is activated, all laser lines are switched off,
emission filters and dichroic beam splitters are set in accordance with the
configuration last used.
Remove button
The single track previously marked in the List of Tracks panel in the
Name column is deleted.
Store/Apply Single Track
button
Opens the Track Configurations window. A selected track defined in a
Channel Mode Configuration can also be stored as a single track for
single tracking applications. Also, it's possible to load a single track in a
multi tracking configuration.
A click on this arrow button will move the selected track (highlighted in
blue) one position upwards in the list box.
A click on this arrow button will move the selected track (highlighted in
blue) one position downwards in the list box.
• Configure each desired track for Multi Track function as described for Single Track.
• For storing/applying or deleting a Channel Mode Configuration use the Config button.
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03/06
Scanning an image
• Click on the Scan button in the Acquire subordinate toolbar to open the Scan
Control window.
The Scan Control window appears (Fig. 16).
Setting the parameters for scanning
• Select Mode in the Scan Control window.
• Select the Frame Size as predefined number of
pixels or enter your own values (e.g. 300 x 600)
in the Objective Lens, Image Size & Line
Step Factor panel. Click on the Optimal
button for calculation of appropriate number of
pixels depending on N.A. and λ.
The number of pixels influences the image
resolution!
Note: When using an Axioskop 2 FS MOT, indicate
the objective that is in use in the Scan Control
window. This ensures correct calculation of
pinhole, Z stack optimization etc.
Fig. 16
Scan Control window, Mode settings
Adjusting the scan speed
• Use the slider in the Speed panel (Fig. 16) to adjust the scan speed.
A higher speed with averaging results in the best signal to noise ratio. Scan speed 8 usually produces
good results. Us speed 6 or 7 for superior images.
Choosing the dynamic range
• Select the dynamic range 8 or 12 Bit (per pixel) in the Pixel Depth, Scan Direction & Scan Average
panel (Fig. 16).
8 Bit will give 256 gray levels, 12 Bit will give 4096 levels. Publication quality images should be acquired
using 12 Bit data depth. 12 Bit is also recommended when doing quantitative measurements or when
imaging low fluorescence intensities.
Setting the scan averaging
Averaging improves the image by increasing the signal : noise ratio. It can be achieved line by line or
frame by frame. Frame averaging helps reduce photobleaching, but does not give quite such a smooth
image.
• Select the Line or Frame mode for averaging.
• Select the desired scan average method Mean or Sum in the Method selection box.
If you are using the Mean method, the image information is generated by adding up all scans pixel by
pixel and then calculating the mean value.
03/06
11
In the Sum method, the pixel values of all scans are only added up, without a mean value being
calculated.
• Select the Number for averaging.
Continuous averaging is possible in the Frame mode. In this case a Finish button for ending continuous
averaging is displayed instead of the Cont. button.
Adjusting the pinhole
• Select Channels in the Scan Control window.
• Set the Pinhole size to 1 (Airy unit) for best
compromise between depth discrimination and
efficiency.
Pinhole adjustment changes the Optical Slice.
When collecting multi channel images, adjust the
pinholes so that each channel has the same
Optical Size. This is important for colocalization
studies.
Image acquisition
Fig. 17
Scan Control window, Channel settings
Once you have set up your parameter as defined in
the above section, you can acquire a frame image
of your specimen.
• Use one of the Find, Fast XY, Single or Cont.
buttons to start the scanning procedure and
acquire an image.
Scanned images are shown in separate windows.
• Click on the Stop button to stop the current
scan procedure if necessary.
Select Find for automatically preadjustment of detector sensitivity.
Select Fast for continuous fast
scanning – useful for finding and
changing the focus.
Select Single for recording a single
image.
Fig. 18
Image window
Select Cont. for continuous scanning
with the selected scan speed.
Select Stop for stopping the current
scan procedure.
12
03/06
Image optimization
Choosing a lookup table
• Select Palette in the Image window of the
scanned image (Fig. 19).
The Color Palette window appears.
• In the Color Palette List panel, click on the
Range Indicator item (Fig. 20).
The scanned image appears in a false-color
presentation (Fig. 19).
If the image is too bright, it appears red on the
screen. Red = saturation (maximum).
If the image is not bright enough, it appears blue
on the screen. Blue = zero (minimum).
Fig. 19
Image window
Fig. 20
Color Palette window
Fig. 21
Scan Control window, Channel settings
Adjusting the laser intensity
• Set the Pinhole to 1 Airy Unit (Fig. 21).
• Set the Detector Gain high.
• When the image is saturated, reduce AOTF
transmission in the Excitation panel (Fig. 21)
using the slider to reduce the intensity of the
laser light at the specimen.
Adjusting gain and offset
• Increase the Amplifier Offset until all blue
pixels disappear, and then make it slightly
positive (Fig. 21).
• Reduce the Detector Gain until the red pixels
only just disappear.
03/06
13
Scanning a Z stack
• Select Z Stack in the Scan Control window.
• Select Frame if necessary.
The Z Settings panel appears.
• Select Mark First/Last on the Z Settings
panel.
• Click on the XY cont button.
A continuous XY-scan of the set focus position will
be performed.
• Use the focusing drive of the microscope to
focus on the upper position of the specimen
area where the Z Stack is to start.
• Click on the Mark First button to set the upper
position of the Z Stack.
• Then focus on the lower specimen area where
the recording of the Z Stack is to end.
• Click on the Mark Last button to set this lower
position.
• Click on X:Y:Z=1:1:1 button to set the Zinterval in such a way that the voxel has
identical dimensions in the X-, Y- and Zdirections (cube).
Fig. 22
14
Scan Control window, Z Stack settings
• Click on the Start button to start the recording
of the Z Stack.
03/06
Storing an image
• Click on the Save or Save As button in the Image window or in the File subordinate toolbar of the
Main menu.
The Save Image and Parameter As window appears.
Fig. 23
Save Image and Parameter As window
• Enter file name, description and notes in the appropriate text boxes.
• Click on the OK button.
Using the ConfoCor
• Click on the ConfoCor button in the Main menu toolbar.
The ConfoCor subordinate toolbar appears in the Main menu.
Fig. 24
03/06
ConfoCor subordinate toolbar
15
Setting up the configuration
• Click on the Measure button to open the Measurement window.
• Press the System Configuration
Configuration panel.
button
to
activate
the
System
The Beam Path and Pinhole panels of the measurement window display the selected track
configuration which is used for the FCS procedure and the pinhole size.
• You can change the settings of this panel using the following function elements:
Activation / deactivation of the excitation wavelengths (check box) and setting of
excitation intensities (slider). Open the Laser Control window via the Laser button.
Selection of the main dichroic beam splitter (HFT) or secondary dichroic beam splitter
(NFT) position through selection from the relevant list box.
Selection of a block filter.
Selection of an emission filter through selection from the relevant list box.
Activation / deactivation (via check box) of the selected channel (Ch 1-2 for the FCS
procedure.
Pinhole slider
Setting of the Pinhole diameter.
Press the Count rate button to open the Count rate window. In Excitation set the
laser power to obtain a satisfactorily count rate.
Pinhole Alignment for a new defined beam path. After adjusting the sample carrier,
align the pinhole in x and y by first conducting a coarse and then a fine alignment.
Taking a measurement
• Click on the Measure button to open the Measurement window.
• Press the Acquisition button to activate the Acquisition panels.
The Times, Kinetics and Position panels of the Measurement window display the selected
measurement configuration and the positions which is used for the FCS procedure.
16
03/06
• You can change the settings of this panel using the following function elements:
Times panel
enter boxes
Enter the bleach time, measurement time and repletion number.
Kinetics check
box
Activate / deactivate if you want to conduct kinetics or a 1 time point measurement.
Position panels
Select a carrier or position in an LSM image.
Press the Methods button to open the Select Method window. You can switch the
method to the highlighted one by just clicking the Ok button. If your current method is
not stored, all settings will be lost.
Press the Save button to open the Save Method window. You can save either only the
beam path configuration or the whole method (including (including configuration and
processing settings).
Press the Info button Current Method Information window, which displays
information on the system set up and the measurement procedure.
Press the New button to open the FCS results … - ConfoCor window. If a
measurement is triggered, all data are displayed in that window if highlighted.
Press the Start button to trigger a measurement. If no FCS results … - ConfoCor
window a new one will be opened. If FCS results … - ConfoCor windows are open,
data will be written to the highlighted one. All defined positions will be approached
consecutively.
Press the Single button to trigger one measurement at the highlighted position. Note,
this button is only available in the LSM Image panel.
Press the Stop button to end a measurement. All data accumulated so far will be
available and can be stored.
Press the Count rate button to open the Count rate window. This allows you to
optimize your experiment by changing the laser power and the pinhole size.
Press the XYZ button to open X,Y,Z-Scan window. The current coordinates will be
displayed. You can define boundaries where a scan is performed with simultaneous
acquisition of the count rate. This allows you, for example, to identify labeled molecules
accumulated in the membrane.
Press XY Stage if positioning should be accomplished by the motorized stage.
Press Scanner if the positioning should be accomplished by the scanning mirrors
After the end of a measurement, the data are displayed FCS results … - ConfoCor window (see Fig. 25).
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Fig. 25
FCS results … - ConfoCor window with Correlation activated
You have the following function elements:
Press the Save Data button to open the Save window. You can save the whole data
set in an ANSI text format. Optionally you can save the raw data trace.
Pressing the Reuse button will set the system configuration to exactly the same values,
as used in the experiment.
Pressing the Reload button will open the current measurement, if stored raw data are
available
Press the Fit button to open the Fit panel, which allows you to analyze your data.
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Analyzing the data
Data can be analyzed in the Fit display of the FCS results … - ConfoCor window (see Fig. 26).
Fig. 26
FCS results … - ConfoCor window with Fit activated
You have the following options:
Curve window
Set the red and blue bars to define the start and end points of the fit.
Model scroll
bar
Load a predefined model. You can assemble a model by pressing the Model button in
the ConfoCor submenu.
Table
You can define the conditions of the fit by activating / deactivating terms, setting the
type of a parameter (fixed, free or start value), defining limits and globally link
parameters.
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Pressing the Fit button will fit the current loaded correlation functions to the defined
model. The fitted data will be displayed.
Pressing the Fit all button will fit to all measurements of the same channels than the
selected ones.
Pressing the Undo button will cancel the last operation, or previous ones as well, if the
button is pressed repeatedly.
Pressing Redo will redo the last cancelled operation, or previous ones, if the button is
pressed repeatedly.
Pressing the Write to Method button will write back the settings to the method. If the
method is stored, the settings will be active when the method is selected the next time.
Switching off the system
• Click on the File button in the Main menu and then click on the Exit button to leave LSM 5 software
program (Fig. 5).
• If any lasers are still running you should shut them off now in the pop up window indicating the lasers
still in use.
• Shut down the computer.
• Wait until fan of Argon laser has switched off.
• On the REMOT CONTROL switch turn off the Components switch and the System/PC switch (Fig. 1).
• Switch off the HBO 100 mercury lamp.
• Switch the UV-Ar laser of via the toggle switch of the power supply (Fig. 2).
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