True Confocal Scanner Leica TCS SP2. User Manual

True Confocal Scanner Leica TCS SP2 User Manual Table of Contents 1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...
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True Confocal Scanner Leica TCS SP2 User Manual

Table of Contents

1. General . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 1 1.1. About these operating instructions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 1

2. Legal Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 2 3. Intended use/purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 4 4. Explanation of Document Symbols . . . . . . . . . . . . . . . . . . . . . . . Page 5 5. Dimensions Leica TCS SP2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 6 5.1.Technical Specifications and connection requirements for TCS SP2 . . Page 7

6. General Safety Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 15 6.1. Safety Notes for the User . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 15 6.2. Which standards does this product meet? . . . . . . . . . . . . . . . . . . . . . . . Page 15 6.3. What should the user of the laser scanning microscope observe? . . . Page 16 6.4. Operational reliability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 17 6.4.1. De−energizing the system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 17 6.4.2. Maximum current load of the power outlet strip at the supply unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 17 6.5. Laser safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 18 6.5.1. Which laser class does this product have? . . . . . . . . . . . . . . . . . . Page 18 6.5.2. Safety notes for operating the laser scanning microscope . . . . . Page 19 6.5.3. Transmitted light lamp housing at upright stands . . . . . . . . . . . . . Page 21 6.5.4. Eye protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 22 6.6. What laser safety devices does the laser scanning microscope have? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 23 6.6.1. Shielding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 23 6.6.2. Detachable−key switch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 23 6.6.3. Emissions warning indicators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 24 6.6.4. Remote interlock connector on the supply unit of the TCS systemPage 25 6.6.5. Remote interlock connector for 405−nm laser (optional) . . . . . . . Page 26 6.6.6. Safety beam guiding and beam shield at inverted microscope stands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 27 6.6.7. Function and position of safety switches . . . . . . . . . . . . . . . . . . . . Page 28 6.7. Which safety labels are used? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 29

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 Englisch Art. No.: 159330053 / Vers.: 20112003

Table of Contents

6.7.1. On the upright microscope stand . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.2. On the inverted microscope stand . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.3. On the scan head . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.4. On the supply unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.7.5. On an external 405−nm diode laser (optional) . . . . . . . . . . . . . . . . 6.8. Overview of usable lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Page 29 Page 34 Page 35 Page 37 Page 38 Page 39

6.9. Which requirements are placed upon the installation/storage site for safety reasons? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 41 6.10. What must be observed when moving the installation site of a Leica laser scanning microscope? . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 42

7. Confocal Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 43 7.1. What is confocal imaging? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 43 7.2. Optical resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 44 7.3. Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 44 7.4. Image processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 45 7.5. Light source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 46 7.6. Integration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 46

8. Starting−up the confocal system . . . . . . . . . . . . . . . . . . . . . . . . Page 47 9. Starting the Operating System . . . . . . . . . . . . . . . . . . . . . . . . . . Page 58 9.1. Setting Up Users . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 59

10. What is the Köhler illumination? . . . . . . . . . . . . . . . . . . . . . . . Page 60 10.1. Setting the Köhler illumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 61

11. Switching off the laser scanning microscope . . . . . . . . . . . Page 63 12. Changing the Scanner to another Leica Microscope Stand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 65 13. The Leica Confocal Software . . . . . . . . . . . . . . . . . . . . . . . . . . Page 68 13.1. Starting the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 68 13.1.1. Requirements for starting the software . . . . . . . . . . . . . . . . . . . . Page 68

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English Art. No.: 159330053 / Vers.: 20112003

Table of Contents

13.1.2. Starting the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 68 13.1.3. The Experiment Software Concept . . . . . . . . . . . . . . . . . . . . . . . . Page 69 13.1.4. The Basic Structure of the User Interface . . . . . . . . . . . . . . . . . . Page 69 13.2. Opening and saving data sets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 71 13.2.1. Readable File Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 71 13.2.2. Automatically Applying the Study Parameters of an Experiment Page 71 13.2.3. Saving Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 72 13.2.4. Data Organization by Grouping Experiments . . . . . . . . . . . . . . . Page 72 13.2.5. Compiling Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 72 13.3. Keyboard Shortcuts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 73 13.4. Menu Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4.1. File Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4.2. The View Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4.3. The Macro Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4.4. The Tools Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4.5. The Window Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.4.6. The Help Menu . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5. LCS File Formats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.5.1. Formats of user−specific and device−dependent data . . . . . . . . 13.5.2. Fixed Leica−specific file formats . . . . . . . . . . . . . . . . . . . . . . . . . .

Page 74 Page 74 Page 75 Page 75 Page 76 Page 77 Page 77 Page 78 Page 78 Page 80

14. Specification of the "Lei" file format (version beta 2.000) Page 81 14.1. Structure of description file . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 81

15. Introduction to the Leica Confocal Software Help . . . . . . . Page 93 15.1. Quick Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 93 15.2. Context−sensitive Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 93 15.3. Retrieving help topics using the table of contents . . . . . . . . . . . . . . . . Page 94 15.4. Retrieving help topics using a keyword . . . . . . . . . . . . . . . . . . . . . . . . . Page 94 15.5. Retrieving help topics using the full−text−search . . . . . . . . . . . . . . . . . Page 94 15.6. Favorites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 95 15.7. Documentation Conventions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 95 15.8. Performing a task . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 96

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 Englisch Art. No.: 159330053 / Vers.: 20112003

Table of Contents

16. Help via the Internet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 97 17. Maintenance and Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 98 17.1. Selecting an installation site . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 98 17.2. Cleaning the optical system of the microscope . . . . . . . . . . . . . . . . . . Page 98 17.3. Cleaning the microscope surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 99

18. Optional trigger control panel . . . . . . . . . . . . . . . . . . . . . . . Page 100 18.1. Description of Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 100 18.2. Installation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 102 18.3. Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 102

19. Declaration of Conformity . . . . . . . . . . . . . . . . . . . . . . . . . . . Page 103

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English Art. No.: 159330053 / Vers.: 20112003

General

1. General 1.1. About these operating instructions These operating instructions direct their main area of concentration to the safety notes which must be strictly observed while working with the laser scanning microscope. In addition, these operating instructions provide the user with a rough overview of the operating principle of laser scanning microscopes, present the first steps for activating and starting up the system and provide a description of the Leica Confocal Software. The Leica TCS SP2 is supplied with the latest version of the licensed Leica Confocal Software. To always keep the information up to date, the description of software functions was intentionally omitted from these operating instructions. Instead, reference is made to the online help of the Leica Confocal Software which presents the most up−to−date explanations and instructions to the corresponding software functions. First, read the chapter EIntroduction to the Leica Confocal Software help" in these operating instructions to familiarize yourself with its design and operation. Additional information about particular functions can subsequently be viewed directly on the screen in electronic form.

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English Art. no.: 159330053 / Vers.: 20112003

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Legal Notes

2. Legal Notes Made in Germany. © Copyright 2001−2004, Leica Microsystems Heidelberg GmbH. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or storing in a retrieval system, or translating into any language in any form without the express written permission of Leica Microsystems Heidelberg GmbH. DISCLOSURE This document contains Leica Microsystems Heidelberg GmbH proprietary data and is provided solely to its customers for their express benefit of safe, efficient operation and maintenance of the product described herein. Use or disclosure of Leica Microsystems Heidelberg GmbH proprietary data for the purpose of manufacture or reproduction of the item described herein, or any similar item, is prohibited, and delivery of this document shall not constitute any license or implied authorization to do so. REVISIONS AND CHANGES Leica Microsystems Heidelberg GmbH reserves the right to revise this document and/or improve products described herein at any time without notice or obligation. Information and specifications in this manual are subject to change without notice. WARRANTY Leica Microsystems Heidelberg GmbH provides this publication "as is" without warranty of any kind, either expressed or implied, including but not limited to the implied warranties of merchantability or fitness for a particular purpose. All reasonable precautions have been taken in the preparation of this document, including both technical and non−technical proofing. Leica Microsystems Heidelberg GmbH assumes no responsibility for any errors or omissions. Leica Microsystems Heidelberg GmbH shall not be responsible for any direct, incidental or consequential damages arising from the use of any material contained in this document. TRADEMARKS Throughout this manual, trademarked names may be used. Rather than including a trademark (I) symbol at every occurrence of a trademarked name, we state that we are using the names only in an editorial fashion, and to the benefit of the trademark owner, with no intention of infringement. SAFETY The terms used of the laser safety class refer to the standard EN 60825−1. Within the scope of the CDRH, the laser class 4 should be replaced by IV in the text, the laser class 3B by IIIb, the laser class 3A by IIIA, the laser class 2 by II and the laser class 1 by I. This instrument is designed and manufactured to comply with applicable performance standards for Class 3B laser products as defined by USHHS, CDRH/FDA, OSHA and EN standards and regulations known to be effective at the date of manufacture.

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Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English Art. no.: 159330053 / Vers.: 20112003

Legal Notes

Every hazardous situation cannot be anticipated, therefore, the user must exercise care, common sense, and observe all appropriate safety precautions applicable to Class 3B lasers and high−voltage electrical equipment during installation, operation and maintenance. Deviating from published operating or maintenance procedures is not recommended. Operation and maintenance procedure changes are performed entirely at the user’s risk. SOFTWARE LICENSE The software described in this document is furnished under a License Agreement which is included with the product. This agreement specifies the permitted and prohibited uses of the product.

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English Art. no.: 159330053 / Vers.: 20112003

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Intended use

3. Intended use/purpose The system was designed for confocal recording (laser scanning images) of fluorescence− labelled living and fixed specimens as well as for quantitative measurements in the area of life science. This device was designed for use in a lab. Use for in−vitro diag−nostics for medicinal purposes is not included in the intended use. The manufacturer assumes no responsibility or liability for any use outside of the intended use or use outside of the specifications from Leica Microsystems Heidelberg GmbH or any risks resulting from such use. In such cases, the certificate of conformity is invalid.

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Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English Art. no.: 159330053 / Vers.: 20112003

Explanation of Document Symbols

4. Explanation of Document Symbols

Such a warning alerts you of an operating procedure, practice, condition, or statement that must be strictly observed to avoid death or serious injury to the persons working on the equipment. Such a safety caution alerts you to an operating procedure, practice, condition, or statement that must be strictly observed to prevent equipment damage or destruction, or corruption or loss of data.

Notes are statements that either provide extra information about a topic or contain special instructions for handling a particular condition or set of circumstances.

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English Art. no.: 159330053 / Vers.: 20112003

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Specifications

5. Dimensions Leica TCS SP2

Figure 1: Dimensions of the Leica TCS SL confocal system (all measurements in mm)

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Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English Art. no.: 159330053 / Vers.: 20112003

Specifications

5.1.Technical Specifications and connection requirements for TCS SP2 The Leica TCS SP2 is classified as Over−voltage Class II / Pollution Degree 2

SP2 VIS

+Kr 568 nm

various laser combinations possible

Melles−Griot

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 Englisch No. art..: 159330053 / Vers.: 20112003

+UV +LP5 351/364 nm heat exchanger Coherent Coherent Enterprise

+LP20 heat exchanger Coherent

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Specifications

Electrical con- 2 x 230 V AC, 50/60 Hz, independent phanection requi- ses, 12 A rements or 2 x 208 VAC, 50/60 Hz, independent phases, to be protected on the customer end with 22−25 A fuses (depending on country− specific regulations) (for use with two transformers, 180−220 V primary/230 V secondary)

Dimensions LxWxH [mm]

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System with System with upright micro- inverse miscope croscope

System with upright and inverse microscope

230 VAC, 50/60 Hz, to be protected on the customer end with 13−16 A fuses (depending on country− specific regulations) or 208 VAC, 50/60 Hz, to be protected on the customer end with 13−16 A fuses (depending on country− specific regulations)

900x270x600

208−240 VAC, 50/60 Hz, 7 kVA (34 A for 208 V, 31 A for 230 V)

94−106 208−240 VAC/6.2 A VAC/10 A, or 50/60 Hz 107−127 VAC/5.5 A or 207−233 VAC/3.0 A or 234−255 VAC/2.75 A

Connector type: in Europe CEE 32 A, 5−phase, 400 V; (all 50/60 outside of Hz) Europe: NEMA 6−50

Laser head: 435x675x 765x510x 1200x240x4 535 610 50 Power pack: 610x450x20 0

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English No. art.: 159330053 / Vers.: 20112003

Specifications

2090x1100x1 2340x1100x1 3030x1100x1 500 400 500

A minimum lateral distance of 900 mm must be kept around the heat exchanger. Input and output of the heat exchanger must be kept clear.

A minimum of 300 mm must be left free around all sides of the system. Weight [kg]

maximum 320

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 Englisch No. art..: 159330053 / Vers.: 20112003

36

71

50

90

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Specifications

Heat emission maximum 5 kW (system)/

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maximum 2.5 kW

max. 4.5 kW max. 5 with water kW with cooling (LP5 air cooling or LP20); 300 W with air cooling

5 kW with external water cooling; water flow of 8−30 liter per minute at max. 7 bar.

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English No. art.: 159330053 / Vers.: 20112003

Specifications

Laser air cooling of 2.5 kW using a vacuum cooling system. If the waste heat is not extracted from the room, the room temperature rises!

The perfect optical behavior of the system with the use of standard objectives and standard immersions is only reached at 22ºC +/− 1ºC. Diameter of adapter piece: 150 mm. Air− flow rate: 350 m3/h

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 Englisch No. art..: 159330053 / Vers.: 20112003

Laser air cooling of 2.5 kW using a vacuum cooling system. If the waste heat is not extracted from the room, the room temperature rises!

Diameter of adapter piece min. 5/8 inch (15.88 mm); the temperature of the coolant The perfect optical behavior must be of the system with the use above the of standard objectives and condensastandard immersions is tion point of the ambient only reached at 22ºC +/− air. 1ºC. Diameter of adapter piece: 125 mm. Air−flow rate: 350 m3/h

It is recommended to install the heat exchanger at a different location than the installation site of the confocal system and the laser itself. Maximum permissible distance: 30 m horizontal, 5.5 m vertical

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Specifications

Environmen18 – 25°C tal/ climatic re- Relative humidity 20 − 90%, condensation− quirements free Avoid the immediate vicinity of air conditioners; avoid draft; protect against dust. Room−darkening measures are recommended. Vibrations should be avoided. Permissible vibration speeds: Frequency range [5 Hz....30 Hz]: less than 30µm/s rms (rms= root mean square error) Frequency range > 30 Hz: less than 60µm/s rms To ensure maximum system stability, the room temperature should be kept stable at 22°C +/− 1°C , even if the microscope system is temporarily not being used. Laser Class

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IIIb/3B

maximum 40°C

IIIb/3B

maximum 40°C

IIIb/3B

Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 English No. art.: 159330053 / Vers.: 20112003

Specifications

+Diode 405 +Diode 430 +HeCd 442 nm nm nm Vioflame Melles−Griot Kimmon IK4153R−C

Electrical connec- 100−240 90−240 tion requirements VAC, 0.7 A, VAC,