Zeiss TIRF 3 User manual

Laser TIRF 3 Imaging System
Operating Manual

Carl Zeiss Copyright Laser TIRF 3
2 423683-7144-001 02/2014
Knowledge of this manual is required for the operation of the instrument. You should therefore
familiarize yourself with the contents of this manual and pay special attention to instructions concerning
the safe operation of the instrument.
Subject to alterations in the interest of technical progress. The manual is not covered by an update
service.
© Unless explicitly authorized, dissemination or duplication of this document and commercial
exploitation or communication of its contents are not permitted. Persons in contravention of this
copyright shall be liable to pay compensation for damages.
All rights reserved in the event of a patent grant or registration of a utility patent.
Published by: Carl Zeiss Microscopy GmbH
Carl-Zeiss-Promenade 10
07745 Jena, Germany
[e-mail]
www.zeiss.com/microscopy
Carl Zeiss Microscopy GmbH
Königsallee 9-21
37081 Göttingen, Germany
Order number: 423683-7144-001
Date of publication: Version 14, 07/02/2014

Laser TIRF 3 Contents Carl Zeiss
02/2014 423683-7144-001 3
CONTENTS
Page
1DEVICE SAFETY...................................................................................................... 6
1.1 General Notes .................................................................................................................... 6
1.2 Safety Instructions .............................................................................................................. 8
1.2.1 Laser Radiation................................................................................................................... 8
1.2.2 Installation Instructions ....................................................................................................... 9
1.2.3 Notes on Computer and Software..................................................................................... 11
1.2.4 Service and Customer Interfaces ....................................................................................... 12
1.2.5 Warning and Information Labels on the Instrument ........................................................... 18
1.3 Notes on Warranty ........................................................................................................... 22
2INSTRUMENT DESCRIPTION ................................................................................ 23
2.1 Intended Use.................................................................................................................... 23
2.2 Operating Principle ........................................................................................................... 24
2.3 Instrument Description and Main Features......................................................................... 26
2.3.1 Slider Laser TIRF, man. / mot. ............................................................................................ 26
2.3.2 Laser Bench Tower and Laser Bench Rack or Laser Bench Rack 3F ...................................... 28
2.3.3 Signal Distribution Box...................................................................................................... 29
2.3.4 TIRF Controller ................................................................................................................. 29
2.4 Laser Safety for the Sample Compartment......................................................................... 30
2.5 Basic kit laser safety Rev. 2................................................................................................ 31
2.6 Remote switch for laser mode........................................................................................... 32
2.7 Laser Safety Concept ........................................................................................................ 33
2.8 Configuration Overview of the Laser TIRF Imaging System ................................................. 34
2.9 System Overview Laser TIRF Imaging System...................................................................... 35
2.9.1 Compatibility of Stages and Laser Safety Devices ............................................................... 39
2.9.2 Compatibility of XLmulti Incubators and Accessories.......................................................... 40
2.9.3 Overview of Stage Versions for TIRF in Axio Observer Program........................................... 41
2.10 Objectives to Be Used with the Laser TIRF Imaging System ................................................. 42
2.11 Fluorescence Filter Sets to Be Used with the Laser TIRF Imaging System .............................. 43
2.12 Combination of Laser TIRF Imaging System and Colibri.2 Illumination System ..................... 46
2.13 Optical Fibers to Be Used with the Laser TIRF Imaging System ............................................ 46
2.14 Technical Data.................................................................................................................. 47
3START-UP ............................................................................................................. 52
3.1 Installation Requirements.................................................................................................. 52
3.1.1 Space Requirements ......................................................................................................... 52
3.1.2 Power Requirements......................................................................................................... 53
3.1.3Ambient Conditions ......................................................................................................... 55
3.2 Requirements for the Microscope Stand ............................................................................ 55
3.3 Basic Microscope Settings ................................................................................................. 56

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3.4 Mounting the Laser Safety Cover for Reflected Light Provided for Mechanical and
Scanning Stages................................................................................................................57
3.5 Mounting the S TIRF S1/S TIRF DARK S1 Incubator .............................................................58
3.6 Incubator XLmulti S1 RED LS and Incubator XLmulti S1 DARK LS.........................................60
3.6.1 Laser Safety: Position of the Reed Contacts ........................................................................62
3.6.2 Mounting .........................................................................................................................62
3.7 PM S1 LS Incubators..........................................................................................................65
3.7.1 Mounting .........................................................................................................................66
3.7.2 Assembly of the Magnet Screw .........................................................................................68
3.8 S TIRF S1 and S TIRF DARK S1 Incubators ...........................................................................69
3.9 Insert Slider Laser TIRF .......................................................................................................70
4OPERATION ......................................................................................................... 71
4.1 Control and Functional Elements .......................................................................................71
4.1.1 Manual Slider Laser TIRF / mot. ..........................................................................................71
4.1.2 Laser Bench Tower ............................................................................................................72
4.1.3 Laser Bench Rack ..............................................................................................................73
4.2 Switching on.....................................................................................................................74
4.3 Switching off ....................................................................................................................75
4.4 ZEN Software (blue edition) ...............................................................................................75
4.5 Checking and Adjusting the Laser Beam Divergence and the TIRF Angle .............................75
4.5.1 Delivery Scope of the TIRF Adjustment Tool Kit...................................................................75
4.5.2 Adjusting the Laser Beam Divergence Using the Adjustment Tool .......................................77
4.5.3 Adjusting and Checking the TIRF Angle Using the Adjustment Tool ....................................78
4.6 Adjusting the TIRF Effect on the TIRF Slider man.................................................................79
4.7 Controlling the Laser TIRF Imaging System Using ZEN 2012 (Blue Edition) ...........................81
4.8 Calibration of the Angle Using the Motorized Slider Laser TIRF ...........................................82
4.8.1 Measurement of Penetration Depth...................................................................................82
4.8.2 Snell's Law........................................................................................................................82
4.8.3 The Angle-Voltage Relationship of the Slider Laser TIRF ......................................................83
4.8.4 Determining the Critical Angle...........................................................................................84
4.8.5 Determination of the Refractive Index of the Specimen.......................................................84
5CARE, MAINTENANCE, TROUBLESHOOTING AND SERVICE.............................. 85
5.1 Instrument Care................................................................................................................85
5.2 Instrument Maintenance ...................................................................................................85
5.3 Troubleshooting................................................................................................................86
5.4 Fuse Replacement .............................................................................................................87
5.4.1 Signal Distribution Box ......................................................................................................87
5.4.2 Laser Bench Tower ............................................................................................................87
5.4.3Laser Bench Tower coupling module..................................................................................88
5.5 Changing the HAL Halogen Lamp......................................................................................88
5.6 Retrofitting the Laser Units ................................................................................................88

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5.7 Mounting the Camera ...................................................................................................... 89
5.8 Connecting the HXP 120 Illumination Unit ........................................................................ 90
5.9 Service Request ................................................................................................................ 91
6USING THE SLIDER LASER TIRF WITH CUSTOMERS' OWN LASERS................... 92
6.1 Laser Safety Concept ........................................................................................................ 93
6.2 Safety Kit for Customer-Provided Laser Module ................................................................. 95
6.3 Remote switch for laser mode........................................................................................... 96
6.4 Connecting the Remote Switch for Laser Mode ................................................................. 97
6.5 Basic kit laser safety Rev. 2................................................................................................ 98
6.6 Connecting the Slider Laser TIRF mot. ............................................................................. 101
6.7 Coupling a Laser to the Laser TIRF Imaging System .......................................................... 101
6.8 Safety Check Prior to First Start-up.................................................................................. 103
6.9 Power Requirements....................................................................................................... 105
7APPENDIX .......................................................................................................... 107
7.1 List of Abbreviations....................................................................................................... 107
7.2 Index.............................................................................................................................. 108

DEVICE SAFETY
Carl Zeiss General Notes Laser TIRF 3
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1DEVICE SAFETY
1.1 General Notes
The Laser TIRF imaging system has been designed, produced and tested in compliance with the standards
DIN EN 61010-1 (IEC 61010-1) "Safety requirements for electrical equipment for measurement, control
and laboratory use", DIN EN 60825-1 (IEC 60825-1) "Safety of laser equipment", and DIN EN 61326-1
"Electrical equipment for measurement, control and laboratory use – EMC requirements".
In combination with the Axio Observer, the instrument meets the requirements of the European Directive
98/79/EC (In Vitro Diagnostics Directive) and carries the marking.
The Laser TIRF imaging system has to be disposed of in compliance with the WEEE Directive 2012/19/EG.
The warning and information symbols explained below will help you recognize at a glance the type and
purpose of the notes included in this operating manual:
CAUTION
This symbol indicates a potential hazard to the user.
CAUTION
Disconnect the instrument from the power supply before opening!
CAUTION Laser radiation
Laser class 3B, max. 500 mW on Laser Bench Tower, Rack or microscope outputs.
Do not expose yourself to the beam. Avoid radiation exposure to the skin!
CAUTION High energy UV radiation!
Do not expose yourself to the beam. Avoid eye or skin exposure to the laser beam!
CAUTION
Keep combustible and highly flammable materials away from the laser beam.
CAUTION
Hot surface!
CAUTION
Dangerous electrical potential!
ATTENTION
This symbol indicates a potential hazard to the instrument or system.
NOTE
This symbol indicates a note containing special instructions.
Always remember: Time invested in familiarizing yourself with the equipment will pay off
many times in your work.

DEVICE SAFETY
Laser TIRF 3 General Notes Carl Zeiss
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In addition to this manual of the Laser TIRF imaging system, please observe the detailed information given
in the following operating manuals:
−Operating manual for the Axio Observer microscope (B 46-0111)
−ZEN software description (blue edition) (Online version)
−Operating manual for the installation of peripherals (B 40-650 d)
−Operating manual for the computer system
−Operating manual for HBO 100 (B 46-0063 d) reflected light fluorescence illumination
−Operating Manual for HXP 120 V
−Operating manual for control computer
−Operating manual for monitor
−Operating manuals for individual incubation systems
−Operating manual for the SVB 1 signal distribution box (M60-2-0032)
−Operating manual for Definite Focus (434533-7144-001)

DEVICE SAFETY
Carl Zeiss Safety Instructions Laser TIRF 3
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1.2 Safety Instructions
1.2.1 Laser Radiation
The Laser TIRF system is a class 3B laser system and labeled accordingly (total output power on
the microscope: max. 500
mW). If you use laser instruments, adhere to the provisions
concerning health protection and industrial safety. Users should also ensure that they observe
all relevant legal requirements.
When used properly, the Laser TIRF imaging system does not constitute a laser radiation hazard to the
operators. Nevertheless, you should observe the following warnings:
Inform the laser safety officer about the intended first start-up of the Laser
TIRF imaging
system.
•The system is equipped with a safety key switch.
•Always keep the key for the laser key switch and, where applic
able, the keys for any other
laser starter switches in places which are not accessible to persons who are not authorized to
handle the laser.
•Do not expose yourself to the laser beam!
•Do not place any reflecting objects into the beam path!
•Never open any covers on the microscope system while the laser is in operation!
•Never look into the laser beam, either with or without optical instrument. Nor should you do
so if you just want to view the specimen. Non-
observance involves the risk of eye
damage!
•Cover all objective nosepiece mounts and camera ports not currently used with the dust-
protective caps provided!
•
Take suitable precautions if the laser radiation produces noxious gases, dust and vapors,
secondary radiation or explosive substances on the specimen.
Observe the additional national laser protection requirements!
Keep combustible and highly flammable materials away from the laser beam.

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Laser TIRF 3 Safety Instructions Carl Zeiss
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1.2.2 Installation Instructions
The Laser TIRF imaging system must be installed and commissioned by technicians trained in
laser safety and authorized by Carl Zeiss. The client is liable to pay the costs of services
rendered by the Carl Zeiss Microscopy GmbH.
To install the Laser TIRF imaging system, ensure a minimum dist
ance of 0.5 m between the
wall and the rear of the system. This distance is necessary for adjustment and maintenance.
Do not set up the equipment near heat sources, such as heaters or direct sunlight. Do not
cover ventilation slots on the microscope system to avoid heat build-up.
The power plugs must be inserted in outlets with a grounding (earth) contact. The protective
capacity must not be rendered ineffective by using an extension cable without a ground wire.
Before connecting the power cables, verify that the available line voltage conforms to the
voltage specified on the type label of the laser bench tower or rack.
Always disconnect the power cable before opening the device and replacing fuses.
Only use fuses for the given rated current which comply with the specifications on the fuse
carriers and in this manual. The use of makeshift fuses and short-circuiting of fuse holders are
not permitted.
For reasons of laser safety, the C mount outputs of the microscope must be fitted either with
a camera or closed by a protective cap.
If any protective measures are no longer effective, the device must be taken out of service and
secured against inadvertent operation. Please contact a Carl Zeiss service agency or the
Carl Zeiss Microscopy Service department to repair the instrument.
Immersol 518 F®immersion oil irritates the skin. Avoid any contact with your skin, eyes or
clothing.
In case of actual skin contact, wash off with plenty of water and soap. After eye contact,
immediately rinse the eye with plenty of water for at least five minutes. If irritation persists,
consult a physician.
Proper disposal of Immersol 518 F®: Ensure that immersion oil does not enter surface water or
the sewage system.
Do not touch the hot lamp housings and adhere to a cooling time of about 15 minutes.
The device contains electronic components. At the end of its service life, it has to be disposed
of in compliance with the relevant national legislation.

DEVICE SAFETY
Carl Zeiss Safety Instructions Laser TIRF 3
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Defective instruments are not to be disposed of as ordinary domestic waste. They should be
disposed of in accordance with the relevant regulations.
Samples should also be disposed of in compliance with the relevant legal requirements and
internal operating procedures.
The Laser TIRF imaging system must be connected to the power supply using several multiple
sockets connected separately to installation sockets. Connection via a single multiple socket
plugged into a single installation socket would lead to inadmissibly high touch
current levels
(see Section 3.1.2, page 53).
Maintenance or repair work as well as the modification, removal or exchange of components
not described in this manual, or any other interventions in the equipment may only be carried
out by the manufacturer Carl Zeiss Microscopy GmbH or by persons expressly authorized by
this company. This applies in particular to the microscope system, the Slider Laser TIRF
, the
Laser Bench Tower or Laser Bench Rack, the control PC system and the cable connections.
Only trained specialists or service staff are permitted to open the instrument.
Operation of the Laser TIRF imaging system is permitted in closed rooms only.
Please consider that the Laser TIRF imaging system is a high-precision opto-electronic
instrument. Improper handling of the instrument may easily affect its performance or even
damage it and will void all warranty claims.
Microscope tube openings or other ports not used must always be protected against dust and
humidity by mounting the corresponding instrument components or dust caps.
Dirt and dust may impair the performance of the devices. They must therefore be protected
from such influences to the greatest possible extent.
Ensure that the generation of electrostatic charges is avoided by suitable design of the
workplace environment.
The power adaptor for the TIRF Controller is class 1 protected. Disconnect the power unit if
the housing is damaged.
The laser TIRF imaging system should only be used with the power adaptor supplied. The plug-
in power supply should not be brought into contact with moisture.
The instruments are not equipped with any special devices for protection against substances
that are corrosive, potentially infectious, toxic or radioactive, or other substances that could be
hazardous to health. All legal regulations must be observed when handling such substances,
particularly the relevant national accident prevention regulations.
Do not operate the supplied equipment in areas in which there is a danger of explosion, in the
presence of volatile anesthetics or flammable solvents, such as alcohol, benzene, etc.
Keep combustible and highly flammable materials away from the laser beam.

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Laser TIRF 3 Safety Instructions Carl Zeiss
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Do not replace detachable power cables by power cables with inadequate specifications.
Only the specified power cables should be used.
Portable multiple sockets may not be used. Cables are to be laid such that there is no risk of
tripping, or they are to be covered.
Never disconnect the power cable while the device is in use. Use the power switch to turn the
devices off.
To ensure proper functioning of the Laser TIRF imaging system, it should be subjected to an
annual safety inspection. The safety checks must be carried out by an authorized Carl Zeiss
service technician. All national safety checks should be carried out.
Blocked or covered ventilation slots may lead to heat accumulation that may damage the
device and, in extreme cases, cause a fire. Always keep the ventilation slots clear and make
sure that no objects are pushed into or fall through the ventilation slots.
Health and safety protection regulations should be observed when handling the HXP 120 V
UV radiation source. Relevant national laws should also be complied with.
−
Follow the operating instructions for the HXP 120V illumination unit provided by the
manufacturer.
−Follow the manufacturer's instructions for changing t
he lamp. Otherwise lamp changes
carry a risk of burns or explosion.
−
Users should not expose themselves to the UV radiation of the HXP 120 V illumination
unit.
−
Never look directly into the optical waveguide when the HXP 120 V illumination unit is
turned on. Non-observance carries the risk of irreversible damage to the eye.
In order to prevent vibrations during operation, the Laser TIRF imaging system should be used
together with the Axio Observer anti-vibration plate.
1.2.3 Notes on Computer and Software
For TIRF microscopy, the use of a monitor is imperative. That means that you need a control PC with a
monitor connected to it. Additionally, a software program for the digital camera, for image recording and
image display on the monitor must be installed on the control PC.

DEVICE SAFETY
Carl Zeiss Safety Instructions Laser TIRF 3
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1.2.4 Service and Customer Interfaces
Fig. 1-1 Service interfaces between Laser Bench Tower and Slider Laser TIRF
CAUTION
The fiber mounts (Fig. 1-1/1 and 2) may be detached by Carl Zeiss Service staff only.
If one fiber end is not firmly connected to the fiber mount, laser class 3B radiation will emerge!
Non-observance involves the risk of eye damage!

DEVICE SAFETY
Laser TIRF 3 Safety Instructions Carl Zeiss
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Fig. 1-2 Service interfaces between Laser Bench Rack 3F - Slider Laser TIRF (optional),
Cell Observer SD (optional) and DirectFRAP (optional)
CAUTION
The fiber mountings in the Laser Bench Rack (Fig. 1-2/1
), on the laser manipulation slider
(Fig. 1-2/2) and on the optional Laser TIRF (Fig. 1-2/3) and Cell Observer SD (Fig. 1-2/4) slider
units may only be loosened by Carl Zeiss service technicians.
If one fiber end is not firmly connected to the fiber mount, laser class 3B radiation will emerge!
Non-observance involves the risk of eye damage!

DEVICE SAFETY
Carl Zeiss Safety Instructions Laser TIRF 3
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Fig. 1-3 Retaining ring - camera adapter (with protective cover) service interface on the sideport of
the microscope
CAUTION
The screws (Fig. 1-3/1) on the retaining ring (Fig. 1-3/2) for the camera adapter should only be
unscrewed by Carl Zeiss service technicians!
Non-observance involves the risk of eye damage!
If no camera is mounted on the sideport, it should be sealed by the protective cover
(Fig. 1-3/3), otherwise it is not possible to open the laser shutter.

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Fig. 1-4 Camera adapter - camera / protective cover customer interface
The camera adapter (Fig. 1-4/1) is equipped with an interlock connection (two reflected light
barriers) which automatically closes the laser shutter as soon as the camera (Fig. 1-4/2) or the
protective cover (Fig. 1-3/3) is removed from the camera adapter.

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Carl Zeiss Safety Instructions Laser TIRF 3
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Fig. 1-5 Microscope illumination connector - HXP 120 coupling adapter service interface;
Service interfaces for HXP 120 illumination unit;
Sensor ring - HXP 120 coupling adapter / protective cap
A) Microscope illumination connector - HXP 120 coupling adapter service interface
CAUTION
The screws (Fig. 1-5/1) on the sensor ring (Fig. 1-5/2) and the screw (Fig. 1-5/6) on the contact
ring (Fig. 1-5/4) may only be unscrewed by Carl Zeiss service technicians!
Non-observance involves the risk of eye damage!
B) Service interfaces for HXP 120 illumination unit
1) HXP 120 optical waveguide - HXP 120 coupling adapter
2) HXP 120 optical waveguide - HXP 120 illumination unit
CAUTION
The screws (Fig. 1-5/8) of the safety clamp (Fig. 1-5/9
) for the optical waveguide on the
coupling adapter and the same safety clamp on the illumination unit HXP 120 may only be
unscrewed by Carl Zeiss service technicians!
Non-observance involves the risk of eye damage!
C) Microscope reflected light port – HXP 120 coupling adapter service interface
The sensor ring is equipped with interlock contacts which automatically close the laser shutter
unless the HXP 120 is connected using the coupling adapter (Fig. 1-5/5) and the contact ring
(Fig. 1-5/4). If the HXP 120 is not used, the sensor ring (Fig. 1-5/2
) must be sealed with the
protective cap (Fig. 1-5/3) to prevent the laser from being used. Only the screw (Fig. 1-5/7) on
the illuminat
ion connector of the microscope may be tightened or loosened for assembly /
dismantling of the HXP 120.

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Laser TIRF 3 Safety Instructions Carl Zeiss
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Fig. 1-6 Transmitted light carrier - HAL 100 Illuminator service interface
CAUTION
The clamp screw (Fig. 1-6/1
) is a safety screw and may only be unscrewed by Carl Zeiss Service
technicians.
If the lamp housing is removed, laser class 3B radiation may emerge!
Non-observance involves the risk of eye damage!
CAUTION
For changing the halogen lamp of the HAL 100 Illuminator, the laser must be switched off!
Non-observance involves the risk of eye damage!

DEVICE SAFETY
Carl Zeiss Safety Instructions Laser TIRF 3
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1.2.5 Warning and Information Labels on the Instrument
Observe the warning and information labels affixed to the Laser TIRF imaging system and Axio Observer
microscope. Please check that all labels shown below are actually in place. Contact Carl Zeiss Microscopy
GmbH if a sign or label is found to be missing. You will receive replacements free of charge.
Fig. 1-7 Warning and information labels on the Laser TIRF imaging system

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Fig. 1-8 Warning and information labels on the Laser TIRF imaging system with laser safety cover (for
labels on the microscope stand see Fig. 1-7)
Fig. 1-9 Warning and information labels on the Slider Laser TIRF

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Carl Zeiss Safety Instructions Laser TIRF 3
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Fig. 1-10 Warning and information labels on the Laser Bench Tower