LSM 510, LSM 510 DuoScan Operation Manual April 2006

INTRODUCTION Carl Zeiss

LSM 510 DuoScan LSM 510 META DuoScan

Knowledge of this manual is required for the operation of the instrument. Would you therefore please make yourself familiar with the contents of this manual and pay special attention to hints concerning the safe operation of the instrument. The specifications are subject to change; the manual is not covered by an update service. ©

Unless expressly authorized, forwarding and duplication of this document, and the utilization and communication of its contents are not permitted. Violations will entail an obligation to pay compensation. All rights reserved in the event of granting of patents or registration of a utility model.

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

B 45-0021 e

03/06

LSM 510 LSM 510 META

INTRODUCTION Carl Zeiss

How to make best use of the LSM 510 and LSM 510 META operating instructions: This operating manual also includes the instructions for the LSM 510 META DuoScan and the LSM 510 DuoScan 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 problem, these operating instructions will supply you with various possibilities: • If you want to know where to find certain general areas of information, refer to the following outline of sections to get a general overview. • You will find a detailed table of contents at the start of every chapter. There you will see at a glance what topics are dealt with in detail. Always remember:

03/06

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

B 45-0021 e

III

INTRODUCTION Carl Zeiss

LSM 510 DuoScan LSM 510 META DuoScan

Contents

1

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

2

LSM 510 and LSM 510 META - Setup Requirements The Setup Requirements section outlines the installation and supply requirements of the LSM 510 and LSM 510 META Microscope Systems, together with the relevant specifications.

3

Introduction to Laser Scanning Microscopy Here you will find an introduction to Laser Scanning Microscopy, with an explanation of the principles of confocal imaging. The section also outlines the ways to present LSM image series in three dimensions, and introduces you to the performance features of your LSM 510 or LSM 510 META.

4

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

5

VBA Programming for LSM

6

Routine Mode and Tools This section contains a description of the Routine Mode for scanning images using the LSM scanning module and the use of the tools for setting the microscope.

7

3D for LSM 510 and LSM 510 META This section contains a description of the LSM 3D software package (basic program and addons). At the same time, all functions and settings are presented in a systematic form and in the order in which they can be reached from the basic menu via sub-menus and dialog boxes.

8

Annex The annex contains the Application-specific Configurations, special notes and information for using the LSM microscope and the brochure The confocal Laser Scanning Microscopy.

9

Multiphoton Laser Scanning Microscopy - Using the Zeiss LSM 510 META NLO

10

Certification

11

Brief Operating Manual This section contains the brief instructions of the LSM 510 and LSM 510 META Microscope Systems.

12

IV

Laser safety warning labels

B 45-0021 e

03/06

LSM 510 LSM 510 META

NOTES ON DEVICE SAFETY General

CHAPTER 1

NOTES ON DEVICE SAFETY

Carl Zeiss

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 ...1-11

1.4

Notes on Setting up the Microscope System ...1-12

1.5

Power Requirements ...1-14

1.6

Notes on Handling the Laser Components...1-15

1.7

Physical Dimensions ...1-17

1.8

Environmental Requirements...1-17

1.9

Notes on Handling the Computer and Data Media ...1-18

1.10

Notes on Care, Maintenance and Service ...1-19

1.11 1.11.1 1.11.2

User Interface...1-20 Mounting and Dismounting Lamps, TPMT and Switching Mirror ...1-20 Mounting and Dismounting the Scan Head ...1-21

03/06

B 45-0021 e

1-1

NOTES ON DEVICE SAFETY General

Carl Zeiss

1

NOTES ON DEVICE SAFETY

1.1

General

LSM 510 DuoScan LSM 510 META DuoScan

The LSM 510 and LSM 510 META laser scanning microscope, including its original accessories and compatible accessories from other manufacturers, may only be used for the purpose of microscopic techniques. Laser Scanning Microscopes (LSM) are intended for high resolution imaging of biological or material samples, whereby in contrast to wide field microscopy the specimen is illuminated raster-fashion with a focused laser beam and the optical arrangement prevents light from out-of-focus regions of the specimen contributing to image formation.

Installation and commissioning of the LSM 510 and LSM 510 META system must be performed by authorized Carl Zeiss service staff. The system should not be used prior to instruction by a Carl Zeiss representative. The manufacturer will not assume liability for any malfunction or damage caused by anything other than the intended use of the LSM 510 or LSM 510 META or individual modules or parts of it, nor by any repair or other service operation performed or attempted by persons other than duly authorized service staff. Any such action will invalidate any claim under warranty, including parts not directly affected by such action. This also includes the modification of the system computer with new cards, etc. by the user. The use of a camera at the base port of Axiovert 200 M Combi stands is not allowed for reasons of laser safety. Any manipulation will result in the loss of warranty of laser safety. Please read also the notes on device safety and manuals of the microscope, the HBO, the HAL and additional optional devices, if ordered, as the UV Laser, the piezo focusing device, 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 and LSM 510 META 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 and LSM 510 META 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

B 45-0021 e

03/06

LSM 510 LSM 510 META

1.2

NOTES ON DEVICE SAFETY Warning and Information Labels

Carl Zeiss

Warning and Information Labels The warning and information labels attached on the LSM 510 and LSM 510 META must be observed. Check whether all of the labels shown below are provided on your instrument, and contact Carl Zeiss Germany or one of the service agencies if you should discover that any of the labels should be missing. You will receive a free replacement.

Description of labels

Caution: Faults and hazards that might arise during operation which might cause damage to the unit or injury to the user. Attention: Laser irradiation hazards possible when operating the system.

Attention: High voltage.

Pull the mains plug before opening the device housing.

Caution: Hot surface.

Caution: UV radiation.

Caution: Fingers can be caught.

The arrow points to the opening where laser light comes out during operation of the system. Other labels on the system include one of the above depicted symbols and a detailed description of the handling instructions. See also the following drawings of the system parts.

03/06

B 45-0021 e

1-3

Carl Zeiss

NOTES ON DEVICE SAFETY Warning and Information Labels

LSM 510 DuoScan LSM 510 META DuoScan

1:1

ZE

OB

RO

JEC

TIV

RE FO

Fig. 1-1

1-4

CU

E

FLE

CT

OR

S

Warning and information labels on the Axiovert 200 M microscope with the LSM 510 META scanning module

B 45-0021 e

03/06

LSM 510 LSM 510 META

Fig. 1-2

03/06

NOTES ON DEVICE SAFETY Warning and Information Labels

Carl Zeiss

Warning and information labels on the Axio Imager.Z1 microscope with LSM 510 META scanning module

B 45-0021 e

1-5

Carl Zeiss

NOTES ON DEVICE SAFETY Warning and Information Labels

LSM 510 DuoScan LSM 510 META DuoScan

04

01

07

02

06

03

05

08

0 10

90

Fig. 1-3

1-6

Warning and information labels on the Axioskop 2 FS MOT microscope with the LSM 510 META scanning module

B 45-0021 e

03/06

LSM 510 LSM 510 META

NOTES ON DEVICE SAFETY Warning and Information Labels

Fig. 1-4

Warning and information labels on LSM DuoScan (systems LSM 510 DuoScan or LSM 510 META DuoScan only)

Fig. 1-5

Warning and information labels on the system electronic rack

03/06

B 45-0021 e

Carl Zeiss

1-7

Carl Zeiss

Fig. 1-6

1-8

NOTES ON DEVICE SAFETY Warning and Information Labels

LSM 510 DuoScan LSM 510 META DuoScan

Warning and information labels on laser components (page 1)

B 45-0021 e

03/06

LSM 510 LSM 510 META

Fig. 1-7

03/06

NOTES ON DEVICE SAFETY Warning and Information Labels

Carl Zeiss

Warning and information labels on laser module LSM 5 LIVE

B 45-0021 e

1-9

Carl Zeiss

NOTES ON DEVICE SAFETY Warning and Information Labels

CO HE RE

LSM 510 DuoScan LSM 510 META DuoScan

nT

En

TE

RP RIS E

PE

AK

CH

AR

SE

E4

TE RP

7

J3

RIS E

E5

CO HE RE

nT

Fig. 1-8

1-10

um xim ma ter wa ure ss pre p.s.I. 60 T OU

ter wa

En

E4 J6

Warning and information labels on laser components (page 2)

B 45-0021 e

03/06

LSM 510 LSM 510 META

1.3

NOTES ON DEVICE SAFETY Regulations

Carl Zeiss

Regulations

Extensive knowledge of the hardware/the system is indispensable for safe operation of the LSM 510 and LSM 510 META.

Read these operating instructions and all device publications belonging to the system conscientiously before operating the LSM 510 or LSM 510 META! 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 and LSM 510 META 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 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.

03/06

B 45-0021 e

1-11

Carl Zeiss

1.4

NOTES ON DEVICE SAFETY Notes on Setting up the Microscope System

LSM 510 DuoScan LSM 510 META DuoScan

Notes on Setting up the Microscope System Installation and commissioning of the LSM 510 and LSM 510 META system must be performed by authorized Carl Zeiss service staff. The system should not be used prior to instruction by a Carl Zeiss representative.

The LSM 510 or LSM 510 META laser scanning microscope is delivered in several crates. The LSM 510 and LSM 510 META must be set up so as to ensure that the minimum clearance between the wall and the rear of the system is no less than 0.5 m. This clearance is needed for adjustment and maintenance operations. Do not set up the unit in the proximity of heat sources such as radiators or direct sunlight. To avoid heat build-ups, the ventilation slots on the microscope system must not be covered up. The system must not be set up in areas with potential danger by explosives. The unit must be connected to a properly installed socket outlet with earthing contact by means of the mains cables supplied. Continuity of PE connection must not be affected by the use of extension leads. The system contains components with dangerous voltage. The system must not be opened by anybody else than authorized Carl Zeiss Service staff. Before opening the main plug has to be disconnected.

Before connecting the mains cables, please check whether your mains voltage corresponds to the voltage specified on the rating plate of the laser module. For reasons of laser safety, all ports must either be equipped with the corresponding device (scan head, camera, HBO lamp etc.) or covered with the counterpart of the laser safety kit provided. 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 and LSM 510 META are high-precision opto-electronic instruments. Inexpert handling may easily impair their function or even damage them. The openings for ventilation must not be covered.

There are hot surfaces on the HBO and HAL lamp.

When sliding the compact Lasermodule V in and out of the System electronic rack take care not to catch your fingers.

1-12

B 45-0021 e

03/06

LSM 510 LSM 510 META

NOTES ON DEVICE SAFETY Notes on Setting up the Microscope System

Carl Zeiss

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 and LSM 510 META should only be operated in conjunction with the system table (vibration damping).

03/06

B 45-0021 e

1-13

NOTES ON DEVICE SAFETY Power Requirements

Carl Zeiss

1.5

LSM 510 DuoScan LSM 510 META DuoScan

Power Requirements The LSM 510 and LSM 510 META comes with a mains power supply cord and plug, either CEE red (3/N/PE 400/230V/16A), or NEMA L 14-30P (2/N/Ground 120/240V/30A), and with the matching mains socket outlet. A ground wire (AWG10 green/yellow) is supplied because it is necessary to ground the system. The connecting part on both ends of the cable is a cable eye with 8 mm inner diameter. A suitable grounding point must be installed in the room. For systems (220 ... 240 V AC) equipped with X-Cite 120 the mains socket outlet must be equipped with a fuse having minimum tripping characteristic C according to IEC/EN 60898.

Line voltage

220 ... 240 V AC (±10 %)

100 ... 125 V AC (±10 %)

Line frequency

50...60 Hz

50...60 Hz

− Max. current

3 phases at 16 A

2 phases at 25 A

− Power

Phase 1 = 1.9 kVA max.

Phase 1 = 3.2 kVA max.

Phase 2 = 1.5 kVA max.

Phase 2 = 2.8 kVA max.

LSM incl. VIS laser

Phase 3 = 2.6 kVA max. − Power consumption

5000 VA max.

5000 VA max.

208...240 V AC

208...240 VAC

(±10 %) 50 / 60 Hz

(±10 %) 50 / 60 Hz

1 phase at 63 A

1 phase at:

Note: For Line Voltage 220 V the connector and power plug are rated for 63 Amps, However wiring and fuse should be rated for 32 Amps.

208 V: 34 Amps

− 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

Argon UV laser − Line Voltage − Max. current

1-14

B 45-0021 e

230 V: 31 Amps 240 V: 29 Amps

03/06

LSM 510 LSM 510 META

1.6

NOTES ON DEVICE SAFETY Notes on Handling the Laser Components

Carl Zeiss

Notes on Handling the Laser Components The LSM 510 and LSM 510 META are laser hazard class 3B instruments. If equipped with a Ti:Sa Laser, the LSM 510 and LSM 510 META 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.

If used properly, the LSM 510 and LSM 510 META will not pose any laser radiation risks for operating staff. Nevertheless, you should observe the following warnings:

03/06

B 45-0021 e

1-15

Carl Zeiss

NOTES ON DEVICE SAFETY Notes on Handling the Laser Components

LSM 510 DuoScan LSM 510 META DuoScan

• If necessary - insofar as specified by law - inform the laser protection officer before commissioning the laser. • The laser modules are equipped with a key-interlock. • Always store keys for laser key switches and, if applicable, keys for further laser power supply units, where they are inaccessible to persons not authorized to operate the laser. • A red LED on the front of the scan head lights up when one or all of the lasers are switched on. • Do not place any reflecting objects into the beam path. • Never open any covers or panels. • Never look into the laser beam, not even to simply view the specimen, whether with the aid of optical instruments or without. Otherwise you risk going blind! • Do not leave any empty objective positions of the nosepiece uncovered. • 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.

1-16

B 45-0021 e

03/06