LSM 710 and ConfoCor 3 Operating Manual April 2009

INTRODUCTION Carl Zeiss

LSM 710 and 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.

Issued by

Carl Zeiss MicroImaging GmbH 07740 Jena, Germany Phone: +49 (0) 3641 64 3400 Fax: +49 (0) 3641 64 3144 E-mail: [email protected] www.zeiss.de/lsm

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INTRODUCTION LSM 710 and ConfoCor 3

Carl Zeiss

How to make best use of the LSM 710 operating instructions: This operating manual also includes the instructions for the ConfoCor 3 systems. A few symbols in these operating instructions will help you to recognize the nature and purpose of information immediately: The WARNING symbol warns against hazards for the user that might arise when operating the laser. This WARNING symbol warns against hazards from dangerously high voltages.

The CAUTION symbol warns against faults and hazards that might arise during operation and which might cause damage to the unit. The NOTE symbol will help you to optimally solve your work problem. It represents a practical tip which will help you to find out which settings and methods are capable of improving or accelerating a procedure. The HOT SURFACE symbol warns against hazards for the user that might arise when touching the lamp housing during operation. The MAINS PLUG symbol remembers service personal to pull the mains plug before opening the device housing.

Depending on the request, these operating instructions will supply you with various possibilities: • If you want to know where to find certain general areas of information, refer to the following outline of sections to get a general overview. • You will find a detailed table of contents at the start of every chapter. There you will see at a glance what topics are covered in detail. Always remember:

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The time you invest in getting acquainted with the product will pay for itself many times over in your application task.

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INTRODUCTION Carl Zeiss

LSM 710 and ConfoCor 3

Contents

1

Notes on Device Safety This section contains general notes on device safety, safe operation, and possible hazards caused by failure to observe the instructions.

2

Setup Requirements The Setup Requirements section outlines the installation and supply requirements of the LSM 710 and ConfoCor 3 Microscope Systems, together with the relevant specifications.

3

Introduction to Laser Scanning Microscopy This section contains the "LSM 710 and LSM 710 NLO Quick Guide", the "Confocal Laser Scanning Microcopy Principles", and the "Confocal Laser Scanning Microcopy Methods" brochures. Here you will find an introduction to Laser Scanning Microscopy, with an explanation of the principles of confocal imaging. The section also outlines the ways to present LSM image series in three dimensions, and introduces you to the performance features of your LSM 710.

4

System Operation In this section you will find the most important steps and procedures of the LSM menu structure. The step-by-step description how to get an image will be shown by typical application examples including the WINDOWS VISTA graphic user environment.

5

Macros and Visual Basic This section contains a description of the use of additional functions, e.g. maintenance, macros.

6

Tools, Additional Software This section contains a description of the use of optional software packages and the tools for setting the microscope, e.g. "3D for LSM".

7

Annex The annex contains the Application-specific Configurations, special notes and information for using the LSM microscope.

8

Multiphoton Laser Scanning Microscopy - Using the LSM 710 NLO This section provides detailed back ground information on Multiphoton Microscopy including trouble shooting procedures.

9

ConfoCor 3 This section provides background information on Fluorescence Correlation Spectroscopy (FCS) and Photon Counting Imaging

10

Certification

11

Laser Safety Warning Labels

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CHAPTER 1

NOTES ON DEVICE SAFETY Contents

Carl Zeiss

NOTES ON DEVICE SAFETY

CONTENTS Page

1

NOTES ON DEVICE SAFETY ...1-2

1.1

General... 1-2

1.2

Regulations... 1-3

1.3

Power Requirements ... 1-4

1.4

Physical Dimensions ... 1-8

1.5

Environmental Requirements... 1-9

1.6

Notes on Setting up the Microscope System... 1-9

1.7

Warning and Information Labels ... 1-11

1.8

Notes on Handling the Laser Components and Illumination Systems ... 1-22

1.9

Notes on Handling the Computer and Data Media... 1-25

1.10

Notes on Care, Maintenance and Service ... 1-26

1.11

User Interface ... 1-28

1.11.1

Mounting and Dismounting Lamps, TPMT and Switching Mirror ... 1-28

1.11.2

Mounting and Dismounting the Scan Heads ... 1-30

1.11.3

Changing the Filter Wheel in the LSM 710 ... 1-33

1.11.4

Changing Filter Wheels in the ConfoCor 3 ... 1-34

1.11.5

Using External Connectors ... 1-35

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NOTES ON DEVICE SAFETY General

Carl Zeiss

1

NOTES ON DEVICE SAFETY

1.1

General

LSM 710 and ConfoCor 3

The LSM 710 laser scanning microscope 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 710 and 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 710 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 Axio Observer.Z1 SP stands with motorized beam path switching is not allowed for reasons of laser safety. Any manipulation will result in the loss of warranty of laser safety.

Please read also the notes on device safety and manuals of the microscope, the HBO, the HAL and additional optional devices, if ordered, as the UV Laser, the piezo focusing device, 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 WINDOWS operating system and its graphical user interface. The respective manuals are supplied together with the programs.

The LSM 710 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 1.8), the LSM 710 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. The LSM 710 and ConfoCor 3 meets the EMC requirements for EN 55011 Class A (intended use in industrial environment). If the LSM is operated in a residential area or in a small trade area other devices may be influenced by conducted or radiated disturbance. In this case special EMC measures are required.

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NOTES ON DEVICE SAFETY Regulations

Carl Zeiss

Regulations

Extensive knowledge of the hardware/the system is indispensable for safe operation of the LSM 710 and ConfoCor 3. Read these operating instructions and all device publications belonging to the system conscientiously before operating the LSM 710 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 710 and ConfoCor 3 have been designed, built and tested in conformity with the following regulations and guidelines: − DIN EN 61010-1 (IEC 61010-1) "Safety requirements for electrical equipment for measurement, control and laboratory use", taking relevant CSA and UL specifications into account. − DIN EN 60825-1 (IEC publication 60825-1) "Safety of laser equipment". − 21 CFR §1040.10: "Performance Standards for light emitting products – laser products". − DIN EN 61326: "Electrical equipment for control technology and laboratory use – EMCrequirements", − Low voltage directive: 2006/95/EG, − EMC directive: 2004/108/EG. The company works according to a certified Environment Management System according to ISO 14001. The Product was developed, tested and produced in accordance with the valid regulations and guidelines for environmental law of the European Union. The product and its accessories have been classified as instrument category 9 (laboratory equipment or comparable standard). The product and its accessories agree with the EU-regulations 2002/95/EG (RoHS) and 2002/96/EG (WEEE), if applicable for the product. Carl Zeiss has installed a process for taking back and recycling the instruments within the member states of the European Union, which takes care of the appropriate utilization according to the said EU guidelines. For details on the disposal and recycling please refer to your relevant Carl Zeiss sales or service organization. The product must not be disposed in the household waste or through the municipal disposal organizations. In case of resale the seller is obliged to inform the buyer that the product has to be disposed according to the said regulations.

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NOTES ON DEVICE SAFETY Power Requirements

Carl Zeiss

1.3

LSM 710 and ConfoCor 3

Power Requirements The LSM 710 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. The mains socket outlet must be equipped with a fuse having minimum tripping characteristic C according to IEC/EN 60898.

Line voltage

3/N/PE 400/230 V AC (±10 %)

2/N/Ground 240/120 V AC (±10 %)

Line frequency

50...60 Hz

50...60 Hz

Max. current Power

3 phases at 16 A Phase 1 = 1.9 kVA max. Phase 2 = 1.5 kVA max. Phase 3 = 2.6 kVA max.

2 phases at 25 A Phase 1 = 3.2 kVA max. Phase 2 = 2.8 kVA max.

Power consumption

4000 VA max.

4000 VA max.

Class of protection

I

I

Type of protection

IP 20

IP 20

Overvoltage category

II

II

Pollution degree

2

2

LSM incl. VIS laser:

EMC test according to DIN EN 61326-1 (10/2006) 1. Noise emission according to CISPR 11 / DIN EN 55011 (11/2007) 2. Noise immunity according to table 2 (industrial sector) In the rare case of a voltage surge on the power supply line (e.g. from indirect lightning stroke), a momentary interruption of the real time system's functionality is possible. This is no defect. A possible protective means to further minimize the probability of a functional disruption is a transient voltage protector in your facility's power system.

Fig. 1-1

1-4

Fuses F1/F2 of the external laser module

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Fig. 1-2

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NOTES ON DEVICE SAFETY Power Requirements

Carl Zeiss

Power connector for LSM 710 and components. Free/reserve outlets may be used to supply power to additional equipment. No more than 1 A can be provided by each outlet.

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Carl Zeiss

Fig. 1-3

1-6

NOTES ON DEVICE SAFETY Power Requirements

LSM 710 and ConfoCor 3

Power connector for LSM 710 with Laser In Tune and components. Free/reserve outlets may be used to supply power to additional equipment. No more than 1 A can be provided by each outlet.

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NOTES ON DEVICE SAFETY Power Requirements

Carl Zeiss

The door interlock interface (Fig. 1-4/1) 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.

Fig. 1-4

Door interlock interface (1) on the back of the electronic rack (see Fig. 1-2 and Fig. 1-3, top)

The LSM 710 is controlled by a remote control. This remote control contains the main switch for the system and the key switch for the laser. • To start the system switch the main switch (Fig. 1-5/1) to ON. • To activate the laser turn the key switch (Fig. 1-5/2) to ON position.

1 2

Main switch ON/OFF Laser key switch

Fig. 1-5

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LSM 710 Remote control

1-7

NOTES ON DEVICE SAFETY Physical Dimensions

Carl Zeiss

1.4

LSM 710 and ConfoCor 3

Physical Dimensions Length (cm)

Width (cm)

Height (cm)

Weight (kg)

Small passively damped system table

90

75

77

80

Small actively damped system table

90

75

77

90

Large actively damped system table

120

90

77

120

Active anti-vibration table (NLO) for Mai Tai Laser or Chameleon

180

150

75

200

Active anti-vibration table (NLO) for two-microscope configuration

250

150

75

400

Scanning Module LSM 710

50

45

22

27

Scanning Module LSM DuoScan

40

15

13

8

Module ConfoCor 3

49

27

18

25

Microscope

50

35

50

20

Electronic Rack with laser units 710

80

60

65

80

Plug-in unit external laser

70

55

25

10

Electronic Rack 5 LIVE / 7 DUO

70

60

65

20

Laser Module LIVE

66

52

22

58

Laser Rack In Tune

80

60

45

40

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1.5

NOTES ON DEVICE SAFETY Environmental Requirements

Carl Zeiss

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 = 15 °C to 35 °C, any conditions different from item 1. and 5.

3. Storage, less than 16 h

T = -20 °C to 55 °C

4. Storage, less than 6 h

T = -20 °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.

1.6

Notes on Setting up the Microscope System Installation and commissioning of the LSM 710 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 710 laser scanning microscope and the ConfoCor 3 spectroscope are delivered in several crates.

The LSM 710 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.

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Carl Zeiss

NOTES ON DEVICE SAFETY Notes on Setting up the Microscope System

LSM 710 and ConfoCor 3

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 electrical connection box. 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. 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 710 and the ConfoCor 3 are high-precision opto-electronic instruments. Inexpert handling may easily impair their function or even damage them. The openings for ventilation must not be covered.

There are hot surfaces on the HBO and HAL lamp.

The HBO 50 and 100 W, XBO 75 W and X-cite 120 lamps used on the light microscopes incident light path emit UV light which is harmful to the human eye and skin when observed without appropriate filters. Never remove the lamp and look direct into the emitted light.

After installation or conversion of the LSM system, authorized specialized staff must carefully check that it is in a proper condition and, particularly, that covers protecting against laser radiation are provided. Tube openings or other unused mounts should always be protected against dust and moisture with the corresponding device components or with termination covers/blind plugs. By establishing a corresponding workplace environment, please ensure that the formation of electrostatic charges of electronic components is avoided. To avoid vibrations during operation, the LSM 710 and ConfoCor 3 should only be operated in conjunction with the system table (vibration damping). Please note the following for the ConfoCor 3: − The ConfoCor 3 requires an Axio Observer.Z1 microscope and is attached to channel 4 of the LSM 710. 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.

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1.7

NOTES ON DEVICE SAFETY Warning and Information Labels

Carl Zeiss

Warning and Information Labels The warning and information labels attached on the LSM 710 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 radiation 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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Carl Zeiss

Fig. 1-6

1-12

NOTES ON DEVICE SAFETY Warning and Information Labels

LSM 710 and ConfoCor 3

Warning and information labels on the Axio Observer.Z1 microscope with the LSM 710 scanning module

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Fig. 1-7

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NOTES ON DEVICE SAFETY Warning and Information Labels

Carl Zeiss

Warning and information labels on the Axio Observer.Z1 microscope with the LSM 710 scanning module (US version only)

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Carl Zeiss

Fig. 1-8

1-14

NOTES ON DEVICE SAFETY Warning and Information Labels

LSM 710 and ConfoCor 3

Warning and information labels on the Axio Observer.Z1 microscope with the LSM 710 scanning module and the ConfoCor 3 detection module

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NOTES ON DEVICE SAFETY Warning and Information Labels

Fig. 1-9

Warning and information labels on the Axio Observer.Z1 microscope with the LSM 710 scanning module and the ConfoCor 3 detection module (US version only)

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1-15

Carl Zeiss

Fig. 1-10

1-16

NOTES ON DEVICE SAFETY Warning and Information Labels

LSM 710 and ConfoCor 3

Warning and information labels on the Axio Imager.Z2 microscope with the LSM 710 scanning module

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