Leica TCS SP5 User Manual - unifr.ch Microsystems CMS GmbH Am Friedensplatz 3 68165 Mannheim Germany...

Leica TCS SP5

User Manual

Leica Microsystems CMS GmbHAm Friedensplatz 368165 MannheimGermany

Telefon: +49 621 7028 0Fax: +49 621 7028 2980E-Mail: [email protected]

Table of Contents

User Manual TCS SP5V: 02 | Document-ID-No.:156500002

1 Notes about this manual page 1. . . . . . . . . . . . .1.1 Copyright page 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2 General page 3. . . . . . . . . . . . . . . . . . . . . . . . . . .2.1 About these operating instructions page 3. . . . . . . . . . . . . . . . . . . . . . . . .

3 Proper intended use page 5. . . . . . . . . . . . . . . . .

4 Legal Notes page 7. . . . . . . . . . . . . . . . . . . . . . . .4.1 DISCLOSURE page 7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.2 REVISIONS AND CHANGES page 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.3 WARRANTY page 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.4 Patents page 8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.5 TRADEMARKS page 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4.6 Software licenses page 10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 TCS SP5 specifications page 11. . . . . . . . . . . . . .5.1 System overview page 11. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2 Dimensions page 12. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.1 System with inverted stand: page 12. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.2.2 System with upright stand page 13. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.3 Installation site requirements page 13. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.4 Permissible ambient conditions page 14. . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.5 Electrical connections page 14. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.6 Required cooling capacity page 16. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.7 Important additional notes page 16. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.8 Required safety measures for VIS and UV systems page 17. . . . . . . . . .

5.9 Required laser safety measures for MP systems page 17. . . . . . . . . . . .

5.10 Important additional notes page 17. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6 Safety Notes page 19. . . . . . . . . . . . . . . . . . . . . . .6.1 Which standards does this product meet? page 19. . . . . . . . . . . . . . . . . .

6.2 Which laser class does the product have? page 21. . . . . . . . . . . . . . . . . .

6.3 Laser class for VIS and UV systems page 21. . . . . . . . . . . . . . . . . . . . . . .

6.4 Laser class for MP systems page 21. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.5 Warnings, Safety Cautions, and Notes page 22. . . . . . . . . . . . . . . . . . . . .

6.6 What should the user of the laser scanning microscope observe? page 23

6.7 Safety Notes for the User page 25. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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6.8 Safety Notes for Operation page 26. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.9 Specific Safety Notes page 30. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.9.1 Operational reliability page 30. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.9.2 De-energizing the system page 30. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.9.3 Maximum current load of the power outlet strip at the supply unit page 31

6.10 Laser safety devices page 32. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.10.1 Eye protection for VIS or UV systems page 32. . . . . . . . . . . . . . . . . . . . . .

6.10.2 Detachable-key switch page 33. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.10.3 Emissions warning indicators page 34. . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.10.4 Remote interlock connector on the supply unit page 36. . . . . . . . . . . . . . .

6.10.5 Remote interlock connector on the external 405nm advanced laser page 37

6.10.6 Remote interlock connectors on additional external lasers page 37. . . .

6.10.7 Interlock connection on scanner page 39. . . . . . . . . . . . . . . . . . . . . . . . . . .

6.10.8 Function and position of safety switches page 40. . . . . . . . . . . . . . . . . . . .

6.10.9 Transmitted-light lamp housing for inverted stands page 40. . . . . . . . . . .

6.10.10 Transmitted-light lamp housing for upright stands page 42. . . . . . . . . . . .

6.10.11 Mirror housing on upright stand page 43. . . . . . . . . . . . . . . . . . . . . . . . . . .

6.10.12 Special laser safety equipment page 44. . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.10.12.1 Safety beam guide and beam collector page 44. . . . . . . . . . . . . . . . . . . . .

6.10.12.2 Eye protection for MP systems page 45. . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.10.12.3 Shielding in MP systems (UV laser) page 46. . . . . . . . . . . . . . . . . . . . . . .

6.11 Overview of usable VIS/UV lasers page 47. . . . . . . . . . . . . . . . . . . . . . . . .

6.12 Overview of usable MP lasers (IR) page 48. . . . . . . . . . . . . . . . . . . . . . . . .

6.13 Safety label on TCS SP5 system page 49. . . . . . . . . . . . . . . . . . . . . . . . . .

6.13.1 Inverted stand DMI 4000/6000 CS page 49. . . . . . . . . . . . . . . . . . . . . . . . .

6.13.2 Upright stand DM 5000/6000 CS page 50. . . . . . . . . . . . . . . . . . . . . . . . . .

6.13.3 Scan head page 53. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.13.4 Supply unit page 54. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.13.5 External 405-nm laser Advance / 405-nm imaging laser page 55. . . . . .

6.13.6 External 488-nm laser page 56. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.13.7 MP beam coupling unit page 57. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.13.8 Cover (for replacement flange) page 58. . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.14 Requirements related to the installation/storage location page 59. . . . . .

6.15 Changing the installation site page 60. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

6.16 Scanner cooling page 61. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

7 Care and cleaning page 63. . . . . . . . . . . . . . . . . .7.1 Cleaning the optical system of the microscope page 63. . . . . . . . . . . . . .

7.2 Cleaning the microscope surface page 64. . . . . . . . . . . . . . . . . . . . . . . . . .

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8 Startup of the system page 67. . . . . . . . . . . . . . . .8.1 Setting Up Users page 70. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

9 Starting the LAS AF page 75. . . . . . . . . . . . . . . .

10 Introduction to LAS AF - Help page 79. . . . . . . .10.1 General page 79. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10.1.1 Calling Online Help page 79. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10.1.2 Structure of the online help page 80. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10.2 Imprint page 81. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10.2.1 In the respective context (context-sensitive) page 82. . . . . . . . . . . . . . . . .

10.2.2 Via the Help menu page 82. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

10.2.3 Full-text search with logically connected search terms page 83. . . . . . . .

10.3 Key combinations page 84. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

11 Structure of the user interface page 85. . . . . . . .11.1 General structure of the user interface page 85. . . . . . . . . . . . . . . . . . . . .

12 What is the Köhler illumination? page 87. . . . . .12.1 Setting the Köhler Illumination page 88. . . . . . . . . . . . . . . . . . . . . . . . . . . .

13 Introduction to confocal work page 91. . . . . . . .13.1 Preparation page 91. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.1.1 The objective page 92. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.1.2 Conventional microscopy page 93. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.1.3 Why scan? page 96. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.1.4 How is an optical section created? page 98. . . . . . . . . . . . . . . . . . . . . . . . .

13.2 Acquiring optical sections page 100. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.2.1 Data acquisition page 101. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.2.2 Illumination page 103. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.2.3 Beam splitting page 104. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.2.4 Emission bands page 104. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.2.5 The pinhole and its effects page 106. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.2.6 Image detail and raster settings page 108. . . . . . . . . . . . . . . . . . . . . . . . . . .

13.2.7 Signal and noise page 113. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.2.8 Profile cuts page 116. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.3 Multiparameter fluorescence page 117. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.3.1 Illumination page 117. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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13.3.2 Beam splitting page 118. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.3.3 Emission bands page 119. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.3.4 Crosstalk page 119. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.3.5 Sequential capture page 121. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.3.6 Unmixing page 121. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4 3D Series page 124. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4.1 z-stack page 124. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4.2 About section thickness... page 124. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4.3 ...and spacing page 125. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4.4 Data volumes page 125. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4.5 Depictions page 127. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4.5.1 Gallery page 127. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4.5.2 Movie page 128. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4.5.3 Orthogonal projections page 128. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.4.5.4 Rotated projections page 129. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.5 Time series page 131. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.5.1 About scan speeds page 131. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.5.2 Points page 132. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.5.3 Lines page 132. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.5.4 Planes page 132. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.5.5 Spaces (time-space) page 133. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.5.6 FRAP measurements page 133. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.6 Spectral series page 134. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.6.1 Data acquisition and utilization page 134. . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.6.2 About spectral resolution page 134. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

13.7 Combinatorial analysis page 135. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5 Switching off the system page 137. . . . . . . . . . . . .

6 Contact page 139. . . . . . . . . . . . . . . . . . . . . . . . . . .

7 Declaration of conformity page 141. . . . . . . . . . . .

8 Glossary page 143. . . . . . . . . . . . . . . . . . . . . . . . . .

18 Safety data sheets page 153. . . . . . . . . . . . . . . . . . .

Notes about this Manual

Page 1User Manual TCS SP5V: 02 | Document-ID-No.: 156500002

1 Notes about this manual

1.1 Copyright

The contents of this manual are current as ofthe time of publication and are subject tochange without notice.

Leica Microsystems CMS GmbH assumesno warranty with respect to this manual, inc-luding but not limited to implicit warranties forsalability and for suitability for a specific pur-pose. Leica Microsystems CMS GmbH is notliable for errors or accidental damages or in-cidental damages in conjunction with equip-ment, performance or usage of this manualor the examples contained therein.

� Leica Microsystems CMS GmbH

This document contains information that isprotected by copyright law. All rights reser-ved. Duplication, adaptation or translation ofthis manual requires the prior written approvalof Leica Microsystems CMS GmbH unlessotherwise permitted through copyright arran-gements.

The programs for controlling this product areprotected by copyright law. All rights reser-ved. Reproduction, adaptation or translationof these programs without prior written ap-proval of Leica Microsystems CMS GmbH isstrictly prohibited.

Microsoft Windows, Windows XP, the Win-dows logo and the Windows CE logo are ei-

Notes about this Manual

Page 2 User Manual TCS SP5V: 02 | Document-ID-No.: 156500002

ther registered trademarks or trademarks ofMicrosoft Corporation in the US and/or othercountries. Microsoft products are licensed forother companies by Microsoft Licensing, Inc.,a subsidiary company that is wholly ownedby Microsoft Corporation.

All other tradenames and product names inthis document are brands, service brands,trademarks or registered trademarks of theirrespective owners.

General


2 General

2.1 About these operating instructions

The main focus of these operating instruc-tions is directed to the safety instructions thatmust be observed while working with the la-ser scanning microscope.In addition, these operating instructions pro-vide a rough overview of the operating princi-ple of laser scanning microscopes. It pre-sents you with the first steps for starting andstartup of the system and provides a descrip-tion of the Leica Application Suite AdvancedFluorescence (LAS AF).The Leica TCS SP5 is supplied with the la-test version of the licensed Leica ApplicationSuite Advanced Fluorescence. To maintaininformation on the most current level, thedescription of software functions was inten-tionally omitted from these operating instruc-tions. Instead, you are referred to the onlinehelp of the Leica Application Suite AdvancedFluorescence in which you can obtain themost up-to-date explanations and instruc-tions about the corresponding software func-tions.Please read the chapter ”Introduction to thehelp of the Leica Application Suite AdvancedFluorescence” in these operating instructionsto familiarize yourself first with setup andoperation. Additional information about spe-cific functions can subsequently be obtainedelectronically at your TCS workstation.

General


Proper Intended Use


3 Proper intended use

The system was designed for confocal recor-ding (laser scanning images) of fluore-scence-marked living and fixed specimensas well as for quantitative measurements inthe area of life science.This system is intended for use in a lab.Applications of in-vitro diagnostics accordingto the medical products law are excludedfrom proper intended use.The manufacturer assumes no liability forany improper intended use and for use out-side the specifications of Leica MicrosystemsCMS GmbH as well as any risks resultingfrom it. In such cases, the declaration of con-formity becomes invalid.

Proper Intended Use


Legal Notes


4 Legal Notes

Manufactured in the Federal Republic ofGermany.© Copyright 2005,Leica Microsystems CMS GmbH.All rights reserved.No part of this publication may be reprodu-ced or transmitted in any form or by anymeans, electronic or mechanical, includingphotocopying, recording, or storing in a re-trieval system, or translating into any lan-guage in any form without the express writ-ten permission of Leica Microsystems CMSGmbH. This also includes photocopying, re-cording or storing on a retrievable system aswell as the translation into another language.

4.1 DISCLOSURE

This document contains Leica MicrosystemsCMS GmbH proprietary data and is providedsolely to its customers for their express be-nefit of safe, efficient operation and mainte-nance of the product described herein.Use or disclosure of Leica MicrosystemsCMS GmbH proprietary data for the purposeof manufacture or reproduction of the itemdescribed herein, or any similar item, is pro-hibited, and delivery of this document shallnot constitute any license or implied authori-zation to do so.

Legal Notes


4.2 REVISIONS AND CHANGES

Leica Microsystems CMS GmbH reservesthe right to revise this document and/or tofurther develop and improve the productsdescribed in this document at any time wi-thout prior notice or any other obligation.Information and specifications in this manualare subject to change without notice.

4.3 WARRANTY

Leica Microsystems CMS GmbH providesthis publication ”as is” without warranty ofany kind, either expressed or implied, inclu-ding but not limited to the implied warrantiesof merchantability or fitness for a particularpurpose.All reasonable precautions have been takenin the preparation of this document, includingboth technical and non-technical proofing.Leica Microsystems CMS GmbH accepts noresponsibility for any errors or omissions. Inaddition, Leica Microsystems CMS GmbH isnot responsible for direct, incidental or con-sequential damages that could result fromthe use of the material described in this do-cument.

4.4 Patents

The product is protected by the following USpatents:5,886,784; 5,903,688; 6,137,627; 6,222,961;

Legal Notes


6,285,019; 6,311,574; 6,355,919; 6,423,960;6,433,814; 6,444,971; 6,466,381; 6,510,001;6,614,526; 6,654,165; 6,657,187; 6,678,443;6,687,035; 6,738,190; 6,754,003; 6,801,359;6,831,780; 6,850,358; 6,867,899.Further patents are pending.

Legal Notes


4.5 TRADEMARKS

Throughout this manual, trademarked namesmay be used. Rather than including a trade-mark (™) symbol at every occurrence of atrademarked name, we state that we areusing the names only in an editorial fashion,and to the benefit of the trademark owner,with no intention of infringement.

4.6 Software licenses

The software described in this document isfurnished under a License Agreement whichis included with the product. This agreementspecifies the permitted and prohibited usesof the product.

Specifications


5 TCS SP5 specifications

5.1 System overview

1 2

3456

figure1. System components (overview)

1 TCS SP5 Scanner2 Control panel3 TCS workstation4 Supply unit5 Panel box6 Stand with scanner

Specifications


5.2 Dimensions

5.2.1 System with inverted stand:

figure2. Dimensions of the TCS SP5 with inverted stand

Specifications


5.2.2 System with upright stand

figure3. Dimensions of the TCS SP5 with upright stand

5.3 Installation site requirements

Do not expose the system to draft.

Ensure that the system is not installed nextto elevators, air conditioners or ventilationsystems. For this reason, the installation siteshould be carefully planned.

Ensure that the environment is as dust-free as possible.

Specifications


Please also read the notes on protectionagainst dust in the chapters on maintenanceand cleaning. Installing the system in darke-ned rooms is also advisable.

For installation, maintenance and trans-port, the TCS SP5 system requires doorswith inside spans of 1.00 m.

With regard to the load-bearing capacity ofthe floor, please note that the system will ap-ply a static load of 200 kg/m@.

5.4 Permissible ambient conditions

Permissible temperature range for operation:18 ... 25q CPermissible relative humidity: 20-80% (non-condensing)Permissible vibration speeds:Frequency range [5 Hz–30 Hz]: less than 30Pm/s (effective value)Frequency range [> 30 Hz]: less than 60Pm/s (effective value)Pollution degree: Class 2

5.5 Electrical connections

Supply voltage (Input 1/Input 2): 100...240 VAC +/- 10%Frequency: 50/60 HzPower consumption: 2 x 1.6kWOvervoltage category: IIThe building installation must feature two se-parate power connections with the followingfuse protection:D 100 V - 120 V AC power: 20 A

Specifications


D 200 V - 240 V AC power: 12-16 AFor the specifications of external lasers suchas UV and MP lasers, please refer to the ma-nufacturer’s documentation.

Specifications


5.6 Required cooling capacity

The TCS SP5 system has a maximum powerconsumption of 3.2kW (VIS system).For the specifications of external lasers suchas UV and MP lasers, please refer to the ma-nufacturer’s documentation.

5.7 Important additional notes

The optimal optical performance of the sy-stem can be achieved using standard objec-tives and standard immersions only at 22qCelsius + 1 Kelvin.

Specifications


5.8 Required safety measures for VIS and UV sy-stems

Please observe valid country-specific regula-tions pertaining to laser safety measures forlaser class IIIb. The operator is responsiblefor observing the laser safety regulations.

5.9 Required laser safety measures for MP sy-stems

Please observe valid country-specific regula-tions pertaining to laser safety measures forlaser class 4/IV. The operator is responsiblefor observing the laser safety regulations.

5.10 Important additional notes

The optimal optical performance of the sy-stem can be achieved using standard objec-tives and standard immersions only at 22qCelsius + 1 °Celsius.

Specifications


Safety Notes


6 Safety Notes

The instrument is Class 3B or 4 laserequipment(depending on the laser used) in accor-dance with IEC/EN 60225-1.This laser equipment may be operatedonly by persons who have been trained inthe use of the systems and the potentialdangers of laser radiation.As it is not possible to anticipate every po-tential hazard, please take care and applycommon sense to the installation, operationand maintenance of this product. Observe allsafety precautions relevant to Class IIIb la-sers and Class IV MP systems.Do not deviate from the operating and main-tenance instructions provided herein.The failure to observe these instructionsshall be exclusively at the user’s own riskand may void the warranty.

6.1 Which standards does this product meet?This device was tested and meets the requi-rements of the following standards:D IEC/EN 61010-1

”Safety requirements for electrical equip-ment for measurement, control and labo-ratory use”

D IEC/EN 60825-1”Safety of laser products, Part1: Equipment classification, require-ments and user’s guide”

D IEC/EN 61326”Electrical equipment for measurement,control and laboratory use - EMC requi-rements” (Class A).This is a Class A device. Operating thisdevice on a public low-voltage grid mayresult in radio interference. The operator

Safety Notes


must take suitable measures should thisoccur.

D IEC/EN 61000-3-2 (EMC)”Electromagnetic Compatibility” Part3-2: Limits - Limits for harmonic currents

D IEC/EN 61000-3-3 (EMC)”Electromagnetic Compatibility” Part3-3: Limits - Limits - Section3: Limits for voltage fluctuations and flik-ker in low-voltage networks.

D For use in the USA:CDRH 21 CFR 1040.10:Laser Products U.S. Food and DrugAdministration (FDA) (”Complies withFDA performance standards for laserproducts except for deviations pursuantto laser notice No. 50, dated 26 July,2001.”)For the scope of the CDRH/FDA(USA), the designation Laser Class3B in the text must be replaced by IIIband Class 4 by IV.

Safety Notes


6.2 Which laser class does the product have?

Lasertype

Wavelength range Configuration Laser class

VIS 400 - 700 nm,(visible laserradiation)

Combination of lasersfrom Chapter 6.11

(without lasers having wave-lengths of 350 - 400 nm)

3B / IIIb

UV 350 - 700 nm,(visible and invisible la-

ser radiation)

Combination of lasersfrom Chapter 6.11(VIS and UV lasers)

3B / IIIb

MP 350 - 1050 nm,(visible and invisible la-

ser radiation)

Combination of lasers fromChapter 6.11 (VIS/UV lasers)and Chapter 6.12 (IR lasers)

4 / IV

6.3 Laser class for VIS and UV systems

According to IEC/EN 60825-1, this laserscanning microscope is a laser device ofClass IIIb.

Avoid exposing eyes or skin to direct ra-diation.

6.4 Laser class for MP systems

According to IEC/EN 60825-1, this laserscanning microscope is a laser device ofClass IV.

Avoid exposing eyes or skin to direct andindirect radiation.

Safety Notes


6.5 Warnings, Safety Cautions, and Notes

Notes either contain additional informationon a specific topic or special instructions onthe handling of the product.

A safety note points out an operation, a pro-cess, a condition or an instruction that must beobserved strictly to prevent severe damage tothe system or loss of data.

A laser warning points out an operation, aprocess, a condition or an instruction thatmust be observed strictly to prevent seriouseye injuries to the persons using the system.

A high-voltage warning points out an ope-ration, a process, a condition or an instruc-tion that must be observed strictly to preventpossible injury or death of the persons usingthe system.

Safety Notes


6.6 What should the user of the laser scanning mi-croscope observe?

The user of this product is responsiblefor proper and safe operation and safemaintenance of the system as well as forfollowing all applicable safety regula-tions. The user isfully liable for all consequences resultingfrom the use of the system for any other pur-poses than those listed in the Operating In-structions or the online help.

The user is obligated to perform and mo-nitor suitable safety measures

according to IEC / EN 60825-1 andthe corresponding national regulations.

Users must have received instructionsconcerning the risk potential associated withthe operation of laser devices.

To assure classification as a3B/IIIb or 4/IV laser product according toIEC/EN60825-1 and electrical safety com-pliance, all safety devices, interlocks, andsafety systems of the laser device mustbe in operational condition.Deactivating or damaging these safety devi-ces or any intervention in any of these safetydevices may lead to serious eye injuries,physical injuries or property damages. Inthese cases, Leica Microsystems CMSGmbH does not assume any liability.

According to IEC/EN 60825-1:”Safety of laser products, Part1: Equipment classification, requirementsand user’s guide,” the user is required to

Safety Notes


designate a Laser Safety Officer or a La-ser Protection Advisor.

Repairs and servicing may only be perfor-med by authorized Leica MicrosystemsCMS GmbH service personnel.

The user is fully liable for allconsequences resulting from the use of thesystem if it is opened, improperly serviced orrepaired by other persons than authorizedLeica customer service representatives.

If repairs or service measures are perfor-med that require opening parts of thehousing, only trained Leica service tech-nicians may occupy the room in whichthe laser scanning microscope is located.

Leica Micro-systems CMS GmbH will not be liable for da-mages resulting from nonobservance of theabove information. The above informationdoes not, in any way, implicitly or explicitly,modify the warranty and liability clauses con-tained in the general terms and conditions ofLeica Microsystems CMS GmbH.

Safety Notes


6.7 Safety Notes for the User

Read and observe the safety notes in theOperating Instructions and the safety la-bels located on the system. Failure toobserve the safety notes may lead to seriousinjuries and to significant damages to the sy-stem and loss of data.

Observe the instructions for operating thesystem located in the Operating Instruc-tions.

Before performing operating steps for thefirst time with the system, read the cor-responding description of the function inthe online help first.You can get an overview of the single func-tions in the contents file of the online help.

Do not connect any external equipment.Connect only those electrical devices to theproduct that are listed in the Operating Instruc-tions. Otherwise, please contact your localLeica service agency or Leica MicrosystemsCMS GmbH.

Safety Notes


6.8 Safety Notes for Operation

figure4. Specimen area of upright and inverted stand

During scanning, the laser radiation isfreely accessible after exiting the objec-tive in the specimen area of the laserscanning microscope.

This circumstance demands special attentionand caution. If the laser radiation comes incontact with the eyes, it may cause seriouseye injuries. For this reason, prudent hand-ling is absolutely necessary as soon as oneor several laser emission warning indicatorsare lit.If the laser scanning microscope is used asprescribed and the safety notes are obser-ved during operation, there are no dangersto the user. Maintain a safety distance of 20cm between your eyes and the eyepieceopening.

Safety Notes


Do not look into the eyepieces during thescan process.

Do not look into the eyepieces when swit-ching the beam path in the stand..

Never look directly into a laser beam or areflection of the laser beam. Avoid allcontact with the laser beam.

Never deactivate the laser protection de-vices. Please read the chapter ”Laser pro-tection devices” to familiarize yourself withthe safety devices of the laser scanning mi-croscope.

Do not introduce any reflective objectsinto the laser beam path.If, for example, micromanipulators areused in the specimen area, you mustensure that no uncontrolled laser lightleaves the safe beam path due to reflec-tion or scattering during the scanningprocess, as it could pose a hazard to thesurrounding area.

Safety Notes


Do not change specimens during scan-ning.

Proceed as follows:

Upright microscope Inverted microscopeFinish the scan process. Finish the scan process.Ensure that no laser radiation is pre-sent in the specimen area.

Ensure that no laser radiation is pre-sent in the specimen area.Tilt the transmitted-light arm back.

Exchange the specimen.Insert the specimen correctly into thespecimen holder.

Exchange the specimen.Insert the specimen correctly into thespecimen holder.Tilt the transmitted-light arm back intothe working position.

Do not change objectives while scanning.

Should it become necessary nevertheless,please follow these procedures:1 Finish the scan process.2 Rotate the objective turret so that the ob-

jective to be changed is swiveled out ofthe beam path and points outward.

3 Exchange the objective.

- All unoccupied positions in the objec-tive turret must be closed using the sup-plied caps.

Safety Notes


Do not change any filter cubes or beamsplitters during scanning.

Proceed as follows:

Upright microscope Inverted microscopeFinish the scan process. Finish the scan process.Remove the cover of the fluorescencemodule(see Microscope Stand operating instruc-tions).

Pull out the fluorescence module.

Remove the filter cube/beam splitter. Remove the filter cube/beam splitter.Insert the desired filter cube/beam split-ter.

Insert the desired filter cube/beam split-ter.

Reattach the cover to the front of thefluorescence module.

Reinsert the fluorescence module.

Never disconnect an optical waveguide.

Never remove the scanner from the mi-croscope stand during operation.Before removing the scanner, the systemmust be completely switched off.

Do not use an S70 microscope conden-ser.The large working distance and thelow numeric aperture of the S70 micro-scope condensers could result in a threatfrom laser radiation. Therefore, only S1and S23 Leica microscope condensersshould be used.

Safety Notes


6.9 Specific Safety Notes

6.9.1 Operational reliability

This instrument must not be used togetherwith life-support systems such as thosefound in intensive-care wards.

This instrument may only be used with agrounded AC power supply.

Contact with liquids or the entry of liquidsinto the housing must be avoided.

6.9.2 De-energizing the system

The main circuit breaker is located on theright rear side of the supply unit. It is used tode-energize the complete system using asingle switch.The main circuit breaker functions as aswitch and as an overcurrent fuse.The main circuit breaker is not to be used asthe regular on/off switch for the system.The supply unit must be set up so that themain circuit breaker is freely accessible atall times.

figure5. Supply unit with main circuit breaker

Safety Notes


6.9.3 Maximum current load of the power outlet strip at the sup-ply unit

The total power consumption of all loadsconnected to the power outlet strip must notexceed

800 VA.The connectors are intended for:D TCS control computerD Monitor 1D Monitor 2D Microscope

figure6. Power outlet strip, rear side of supply unit

Safety Notes


6.10 Laser safety devices

The light of all employed VIS lasers(wavelength range 400-800 nm, visible spec-trum) and UV lasers (wavelength range < 400 nm, invisible) is fedthrough an optical waveguide and, therefore,completely shielded until it leaves the micro-scope objective and reaches the specimen.For MP systems, see the chapter “Shieldingfor MP systems”.

6.10.1 Eye protection for VIS or UV systems

It is not necessary to wear eye protection. Ifthe device is used as prescribed and the sa-fety notes are observed, the limit of the laserradiation is maintained so that eyes are notendangered.

For MP systems, see the chapter “Eyeprotection for MP systems”.

Safety Notes


6.10.2 Detachable-key switch

The detachable-key switch for protectionagainst unauthorized use of the laser devi-ces is located on the control panel.

figure7. Detachable-key switch for the internal lasers

In the case of an optional external 405nm advanced laser, the detachable-keyswitch for the laser is located on its powerpack.

figure8. Detachable-key switch of the external 405-nm Advance laser

For lasers that are not connected as des-cribed above, please refer to the suppliedmanual of the laser manufacturer for theposition of the detachable-key switches.

Safety Notes


6.10.3 Emissions warning indicators

The operational readiness of lasers locatedin the supply unit is signaled by an emissionwarning indicator.The emission warning indicator is locatedabove the detachable-key switch and is yel-low when lit.As soon as the emission warning indicatorof the lasers is lit, it is possible from a func-tional standpoint that laser radiation is pre-sent in the specimen area.

figure9. Emission warning indicators on the control panel

The optional external 405nm advanced laser features the yellowemission warning indicator (1) above the de-tachable-key switch.

figure10. Emission warning indicator of the external 405-nm Advance laser

Immediately disconnect the system fromthepower supply if any of the following oc-cur:

D The emission warning indicator is not litafter being switched on using the keys-witch.

Safety Notes


D The indicator continues to be lit afterbeing switched off using the keyswitch

D Scanning of the specimen is not activa-ted after being switched on properly (la-ser radiation in the specimen area).

Contact Leica Service immediately.

For lasers whose readiness is not indica-ted as described above, please refer tothe supplied manual of the laser manufac-turer for the location of the emission war-ning indicator.

Safety Notes


6.10.4 Remote interlock connector on the supply unit

figure11. Position of the remote interlock connector

The remote interlock connection is locatedon the rear side of the supply unit (12V DC operating voltage). The remote inter-lock connector is plugged into this connec-tion.Remote interlocks that are connected to theroom, the door or other locally fixed safetyinterlocks can be connected to the remoteinterlock connection. The laser beam path isinterrupted if the contact is open.The overall length of the cable between thetwo connecting pins of the remote interlockconnector should not exceed 10 m.

Safety Notes


6.10.5 Remote interlock connector on the external 405nm advan-ced laser

Example/

If the laser scanning microscope is equip-ped with the optional 405-nm laser (non-pul-sed), the “Interlock“ remote interlock con-nection is located on the rear side of the la-ser power supply.The remote interlock connector contains ashorting bridge.Remote interlocks that are connected to theroom, the door or other locally fixed safetyinterlocks can be connected to the remoteinterlock connection.The laser beam path is interrupted if thecontact is open.

figure12. Example of a remote interlock

The supply voltage of the remote inter-lock circuit of the 405 nm Advance laseris 100-240V AC. For this reason, the re-mote interlock circuit of the 405nm Advance laser must never be connec-ted to other remote interlock circuits but,instead, must be securely separated fromthem.

Due to the live voltage of 100-240V, replacing the shorting plug by an exter-nal interrupt circuit (e.g. door interlockswitch)may only be performed by a qualifiedelectrician.

6.10.6 Remote interlock connectors on additional external lasers

Safety Notes


For lasers whose remote interlock con-nector is not indicated in Chapter 6.10.6,please refer to the supplied manual of thelaser manufacturer for the location of theremote interlock connector.

Safety Notes


6.10.7 Interlock connection on scanner

figure13. Position of the interlock connector

The interlock connector (operating voltage12 V DC) is located on the rear side of thescanner.The inverted microscope or, if an uprightmicroscope is used, the mirror housing isconnected to this connector. This ensuresthat the safety switch of the microscope isintegrated in the interlock circuit.

Safety Notes


6.10.8 Function and position of safety switches

When the safety switches are released, thelight path of the laser beam is interrupted.

1

2

figure14. Position of the transmitted-light illumination arm (1) and switching from scan mode toeyepiece (2)

Position Activated by: Type of micro-scope

Activated if: Function

1 Transmitted-light illuminatorarm

Inverted standDMI 4000 CSDMI 6000 CS

The illuminatorarm is tilted(e.g. for workingon the speci-men).

Prevents laser lightwhile working onthe specimen.

2 Motorized chan-geover betweenscanning modeand eyepiece

Inverted standDMI 6000 CSDMI 4000 CS

The deflectionmirror to thescanner is mo-torized.

Prevents stray lightif the user switchesfrom confocal ob-servation to eye-piece observation.

6.10.9 Transmitted-light lamp housing for inverted stands

Safety Notes


During the time when no transmitted-lightlamp housing is connected to the micro-scope stand, the opening must be tightly co-vered with the cap provided with the systemto prevent laser radiation from exiting.

figure15. Cover for replacement flange

If your inverted stand features a transmitted-light housing that you would like to replace,proceed as follows:D Switch off the lasers.D Disconnect the lamp housing from the

power supply.D Remove the lamp housing.D Perform the intended tasks at the lamp

housing.D After finishing the tasks, screw the new

lamp housing back onto the microscopestand.

figure16. Connecting the transmitted light lamp housing

To prevent the emission of laser radia-tion, do not switch the lasers on without alamp housing or cover on the microscopestand.

Safety Notes


6.10.10 Transmitted-light lamp housing for upright stands

During the time when no transmitted-lightlamp housing is connected to the upright mi-croscope stand, the opening must be tightlycovered with the cap provided with the sy-stem to prevent laser radiation from exiting.

figure17. Cover

If your upright stand features a transmitted-light housing that you would like to replace,proceed as follows:D Switch off the lasers.D Disconnect the lamp housing from the

power supply.D Remove the lamp housing.D Perform the intended tasks at the lamp

housing.D After finishing the tasks, screw the new

lamp housing back onto the microscopestand.

figure18. Connecting the transmitted light lamp housing

To prevent the emission of laser radia-tion, do not switch the lasers on without alamp housing or mirror housing connec-ted to the microscope stand, or if a coveris not present.

Safety Notes


6.10.11 Mirror housing on upright stand

If a mirror housing is not connected to theupright microscope stand, the opening mustbe tightly covered using the cap providedwith the system to prevent any laser radia-tion from escaping.

figure19. Cover

1

If your upright microscope stand is equippedwith a mirror housing, note the following:D The interlock connector on the mirror

housing (see arrow) must be connectedto the scan head at all times.

D The unused output on the mirror hou-sing must be covered using the coverprovided (1).

D If the mirror housing is removed, placea cover on the adapter left on the stand.

figure20. Mirror housing on upright stand

To prevent the emission of laser radia-tion, do not switch the lasers on without amirror housing or cover on the micro-scope stand.

Safety Notes


6.10.12 Special laser safety equipment

6.10.12.1Safety beam guide and beam collector

1

2

3The safety beam guiding and beam collectorare used with inverted microscopes to protectagainst laser radiation and are located betweencondenser base and transmitted-light detector.1 Safety beam guide2 Beam collector3 Condenser base

figure21. Inverted stand

When subsequently ordering condenserbases (3), be aware that condenser basesare supplied without beam collectors (2).The existing beam collector (2) must bereinstalled.

When using a condenser base with filterholder, always make sure that unused fil-ter holders are swung out of the beampath, and that thesafety beam guide covers the beam path.When equipping multiple filter holderswith filters, do so from bottom to top sothat the safety beam guide can cover thebeam path to the greatest possible extent.

Do not swing in the filters during thescanning process.

Safety Notes


6.10.12.2 Eye protection for MP systems

System with inverted microscope stand:It is not necessary to wear eye protection.If the device is used as prescribed and thesafety notes are observed, the limit of the la-ser radiation is maintained so that eyes arenot endangered.

System with upright microscope stand:The IR laser beam can be deflected or scat-tered by the specimen or objects moved intothe specimen area.Therefore, it is not possible to completely eli-minate hazards to the eye from IR laser ra-diation.Using protective eyewear(specification: 680-990 DIR L5 / 990-1064DIR L4) is required.Appropriate safety goggles for IR laser radia-tion are provided with the system when deli-vered.The supplied eye protection only provi-des safe protection against the infraredlasers supplied by Leica MicrosystemsCMS GmbH.

Safety Notes


6.10.12.3 Shielding in MP systems (UV laser)

The light of all employed VIS lasers(wavelength range 400-700 nm, visible spec-trum) and UV lasers (wavelength range < 400 nm, invisible) is fedthrough an optical waveguide and, therefore,completely shielded until it leaves the micro-scope objective and reaches the specimen.

For systems with infrared laser (wavelengthrange > 700 nm), the beam is passedthrough a safety beam guide and, if neces-sary, also passed through an optical wave-guide.This shields the laser beam until it leavesthe microscope objective and reaches thespecimen.

figure22. Safety beam guiding (1) and IR laser (2)

Safety Notes


6.11 Overview of usable VIS/UV lasers

The laser scanning microscope features acombination of the lasers listed below.

Laser typeWavelength

[nm]

Maximum lumi-nous power atlaser output

[mW]

Maximum lumi-nous power in

focal plane[mW]

Pulse duration

Ar-UV 351, 364 < 60 < 4 Continuouswave (cw)

Diode 405 405 < 60 < 6 Continuouswave (cw)

Diode 405 405 < 5 (mean power)

< 0.3 (meanpower)

pulsed, 60 ps

DPSS 442 442 < 25 < 5 Continuouswave (cw)

Ar 458, 476,488, 496,514

< 200 < 30 Continuouswave (cw)

Ar, external 458, 476,488, 496,514

< 500 < 125 Continuouswave (cw)

Solid state488

488 < 500 < 150 Continuouswave (cw)

HeNe 543 < 1.5 < 0.5 Continuouswave (cw)

DPSS 561 561 < 12 < 4 Continuouswave (cw)

HeNe 594 < 4 < 1 Continuouswave (cw)

HeNe 633 < 15 < 4 Continuouswave (cw)

Table 1 Table of usable lasers (without MP)

Safety Notes


6.12 Overview of usable MP lasers (IR)

Each MP system contains only one of theMP lasers listed below.In addition, the MP system may also containadditional VIS/UV lasers (see the table forusable VIS/UV lasers).

Laser type Wavelength[nm]

Maximum lumi-nous power atlaser output

[W]

Maximum lumi-nous power infocal plane [W]

Pulse duration

TiSa* 780-920 < 1.2 < 0.6 pulsed, 1.0 -1.5ps

TiSa* 710-920 < 2.5 < 1.2 pulsed1.0-1.5 ps

TiSa* 710-990 < 2.5 < 1.2 pulsed1.0-1.5 ps

TiSa* 720-930 < 2.0 < 1.0 pulsed1.0-1.5 ps

TiSa* 720-980 < 2.5 < 1.2 pulsed1.0-1.5 ps

Table 2 Table of usable MP lasers

* Modified picosecond version

Safety Notes


6.13 Safety label on TCS SP5 systemThe corresponding safety labels are selecteddependent on the laser configuration (VIS,UV, MP) and attached in the following loca-tions.

6.13.1 Inverted stand DMI 4000/6000 CS

Angled rear view of right side of stand

figure23. Safety label for DMI 4000/6000 CS inverted stand

Safety Notes

Page 50 User Manual TCS SP5V: 02 | Document–ID–No.: 156500002

52008–005 Zust.0188100–017 Zust. 00

figure24. Safety label for DMI 4000/6000 CS inverted stand

6.13.2 Upright stand DM 5000/6000 CS

Safety Notes


Angled front view of right side of stand

figure25. Safety label for DM 5000/6000 CS upright stand

Safety Notes


Rear view of stand

figure26. Safety label for DM 5000/6000 CS upright stand

Safety Notes


6.13.3 Scan head

Angled front view of left side of scan head

figure27. Safety label for the scanner

Safety Notes


6.13.4 Supply unit

View of TCS SP5 supply unit.

figure28. Safety label for the supply unit TCS SP 5 (front side)

Safety Notes


6.13.5 External 405-nm laser Advance / 405-nm imaging laser

figure29. Safety label for the external 405-nm laser / safety label for the 405-nm imaging laser

Safety Notes


6.13.6 External 488-nm laser

figure30. Safety label for the external 488-nm laser

Safety Notes


6.13.7 MP beam coupling unit

Angled front view of the right side of thebeam coupling unit MP.

figure31. Safety label for the beam coupling unit MP (top side)

Safety Notes


6.13.8 Cover (for replacement flange)

View from front on the cover.

figure32. Cover for replacement flange

If the replacement flange for transmittedlight is not equipped with a functionalmodule such as a lamp housing, place acover over the opening for laser safetyreasons.

Safety Notes


6.14 Requirements related to the installation/sto-rage location

This device was designed for use in a laband may not be set up in areas with medi-cal devices serving as life-support sy-stems such as intensive-care wards.

This equipment is designed for connec-tion to a grounded (earthed) outlet. Thegrounding type plug is an important sa-fety feature.To avoid the risk of electrical shock or da-mage to the instrument, do not disablethis feature.

To avoid the risk of fire hazard and elec-trical shock, do not expose the unit torain or humidity.Do not open the cabinet. Do not allow anyliquid to enter the system housing orcome into contact with any electricalcomponents.The instrument must be thoroughly drybefore connecting it to the power supplyor turning it on.

Safety Notes


6.15 Changing the installation site

Before moving the laser scanning micro-scope, it should be thoroughly cleaned.The same also applies to the removal ofcomponents. This applies in particular tosystems that are located in biomedical re-search labs.This is necessary to remove a possiblecontamination and, thereby, avoid carry-over of dangerous substances and patho-gens and its accompanying risk of per-sons.Pay not only attention to surfaces, but es-pecially to fans and cooling devices sincedust can frequently accumulate at theselocations.

Safety Notes


6.16 Scanner cooling

The scanner of the TCS SP5 is liquid-cooled.

Observe the attached safety data sheet pro-vided by the manufacturer, Innovatek, regar-ding the coolant used.

In case of a coolant leak, switch thepower off immediately!Inform Leica or a Leica-approved servicefacility immediately.

The coolant contains an irritating sub-stance. Avoid eye and skin contact.

The scanner cooling system must be ser-viced by Leica or a Leica-approved ser-vice facility every two years.

Safety Notes


Care and Cleaning


7 Care and cleaning

Please refer to the corresponding manualsfor information on how to maintain the Leicaresearch microscope.The instructions and additional informationrelating to the components of the confocalsystem are summarized below.

Protect the microscope from dust andgrease.

When not in use, the system should be cove-red with a plastic foil (part of delivery) or apiece of cotton cloth. The system should beoperated in a room which is kept as dust andgrease-free as possible.Dust caps should always be placed over theobjective nosepiece positions when no ob-jective is in place.

Exercise care in the use of aggressivechemicals.

You must be particularly careful if your workinvolves the usage of acids, lyes or other ag-gressive chemicals. Make sure to keep suchsubstances away from optical or mechanicalcomponents.

7.1 Cleaning the optical system of the microscope

Care and Cleaning


The optical system of the microscope mustbe kept clean. Under no circumstancesshould users touch the optical componentswith their fingers or anything which may beardust or grease.Remove dust by using an air puffer (not sol-vent-based) or a fine, dry hair pencil. If thismethod fails, use a piece of lint-free cloth,moistened with distilled water.Persistent dirt can be removed from glasssurfaces by means of pure alcohol or chloro-form.If an objective lens is accidentally contamina-ted by unsuitable immersion oil or by thespecimen, please contact your local Leicabranch office. for advice on the use of certainsolvents for cleaning purposes.Take this seriously, because some solventsmay dissolve the glue which holds the lens inplace.

Do not open objectives for cleaning.

Oil should be removed from oil immersionlenses after use.First, remove the immersion oil using a cleancloth. Once most of the oil has been remo-ved with a clean tissue, a piece of lens tissueshould be placed over the immersion end ofthe lens. A drop of recommended solventshould be applied, and the tissue gentlydrawn across the lens surface. Repeat theprocess until the lens is completely clean.Use a clean piece of lens tissue each time.

7.2 Cleaning the microscope surface

Use a lint-free linen or leather cloth (moiste-ned with alcohol) to clean the surfaces of themicroscope housing or the scanner (varnis-hed parts).

Care and Cleaning


Never use acetone, xylene or nitro thinnersas they attack the varnish.

All LEICA components and systems are ca-refully manufactured using the latest produc-tion methods. If you encounter problems inspite of our efforts, do not try to fix the devi-ces or the accessories yourself, but contactyour Leica representative.

Before moving the confocal system, itshould be thoroughly cleaned. This appliesin particular to systems that are located inbiomedical research labs.

This is necessary to remove any existingcontamination and to prevent any carry-overand endangering of others. Pay not only at-tention to surfaces, but especially to fans andcooling devices since dust can frequently ac-cumulate at these locations.

Care and Cleaning


Startup of the System


8 Startup of the system

Proceed as follows to start your TCS SP5system:1 Switch on the TCS workstation.

figure33. Switching on the workstation

1

2

Check whether the microscope stand isswitched on.If the readiness indicator (Figure 31/1) onthe MIC box is lit, the stand is operating.If the readiness indicator is not lit, activatethe toggle switch (Figure 31/2) of the MICbox.

figure34. Switching on the microscope

You do not have to start the operating sy-stem—it starts automatically when you turnon your PC. You will first see a splashscreen.



1 Next you have to log on to your compu-ter. As you can see from the instructionsin the box, pressing the Ctrl, Alt and De-lete keys at the same time will log youon. After pressing the Ctrl, Alt, and De-lete keys, the Logon information dialogbox appears.

2 Typing your password identifies you as avalid user for this computer.

The default user name for the Leica TCSSP5 system is ”TCS_User”.A standard password was not set. It is re-commended setting up a separate user IDfor each user (system administrator). Thiswill create individual directories that can beviewed only by the respective user. Since theLCS AF software is based on the user admi-nistration of the operating system, separatefiles are created for managing user-specificprofiles of the LCS AF software. For informa-tion about setting up users, please refer tothe chapter ”Setting Up Users” in this ma-nual.3 After logging on with your user ID, you

may change your password by pressingthe keys Ctrl, Alt, and Delete at thesame time.

4 Then click on Change Password. TheChange Password dialog box displays.

5 Type your current password in the ”OldPassword” field (passwords are casesensitive, so be sure you use the rightcase).

6 Then press the Tab key. Pressing theTab key moves the cursor to the nextfield.

7 Type your new password, then press theTab key again. Confirm your new pass-word by re-entering it. This will eliminateany typing errors. This is especially im-portant since the characters you type ap-pear as asterisks on the screen.



8 Then click the OK button. Your newpassword will be in effect the next timeyou log on.



8.1 Setting Up Users

1 Log on as administrator. Us the ID”Administrator” and the password ”Ad-min”

2 Open the User Manager. Select Start /Programs / Administrative Tools / UserManager.

3 Define a new user. Enter at least the fol-lowing information in the open dialogwindow:

D User nameD Password (must be entered again in the

next line for confirmation purposes)4 Select the following two check boxes:a.) ”User must change password at next lo-gon” (this allows the new user to define hisor her own password at logon)b.) ”Password never expires” (this allows adefined password to be valid either until it ischanged in the User Manager or the user isdeleted)5 Select the ”Profiles” option in the bottom

section of the dialog. In the ”Local path”field, enter the following path for storingthe user-specific file: d:\users\username(”username” is a wildcard which must bereplaced by the currently defined username.)

Factory-installed hard disks are providedwith two partitions (C:\ and D:\). The user di-rectory should be set up on partition D:\.



figure35. The ”booted” system

6 Wait until the boot process of the TCSworkstation is completed.

7 If not yet done, create a user and log on.



8 Turn on the TCS SP5 scanner.

figure36. Turning on the scanner

figure37. Turning on the scanner

The Windows status bar must show the ”SP5Scanner Gigabit Interface” as connected!



9 Switch on the lasers.The power supplies and fans of the systemstart first.

figure38. Switching on the laser

10 Activate the detachable key switch.

figure39. Activating the detachable key switch

Laser radiation may be present in thespecimen area as of this time. Follow thesafety instructions given in Chapter 6.

Please follow the instructions in Chapter14 to switch off the TCS SP5 system.

Starting the LAS AF


9 Starting the LAS AF

1 Start the LAS AF by double-clicking theprogram icon.

figure40. Starting the LAS AF

Starting the LAS AF


figure41. Resonant or non-resonant

If you purchased this option, you canstart the system in resonant or non-reso-nant mode at this point.

2 Select whether the TCS SP5 systemshould be operated in resonant or non-resonant mode.

Starting the LAS AF


figure42. LAS AF start window

3 Start the LAS AF by clicking on ”OK”.

Starting the LAS AF


figure43. LAS AF base view

You are now in the base view of the LAS AF.4 Start the LAS AF help with the F1 key.

Introduction to the LAS AF Help


10 Introduction to LAS AF -- Help

10.1 General

The LAS AF software is used to control allsystem functions and acts as the link to theindividual hardware components.The experimental concept of the softwareallows for managing the logically intercon-nected data together. The experiment is dis-played as a tree-structure in the softwareand features export functions to open indivi-dual images (JPEG, TIFF) or animations(AVI) in an external application.

10.1.1 Calling Online Help

The LAS AF software features a context-sen-sitive help system that explains the differentfunctions of the system.The online help can be called in two ways:D In the respective context (context-sensi-

tive)D Via the Help menu



10.1.2 Structure of the online help

The online help is divided into 6 differentbooks:

Books Contents

General This book contains general information about online help andthe contact information of the manufacturer.

Structure of the user inter-face

This book describes the structure of the LAS AF user interfaceand features topics about the various menus, registers, opera-

ting steps and symbols.

Dialog descriptions This book describes each individual dialog window of the userinterface in individual topics.

Learning the basics The fundamental steps for performing an experiment are des-cribed here.

Guidelines This book contains step-by-step guidelines for performing cer-tain applications. They are divided into two categories: for be-

ginners and for advanced users.

Additional information This book contains detailed descriptions about certain topicsof biology, image editing, filter...



10.2 Imprint

Online help LAS AF; Version: 1.0

Address Leica Microsystems CMS GmbHAm Friedensplatz 368165 Mannheim

Phone +49-621-7028-0Fax +49-621-7028-2980

Internet http://www.confocal-microscopy.com.

Copyright � 1997-2005 Leica Microsy-stems CMS GmbH. All rights reserved.The contents of this online help may not bereproduced or transmitted by electronic ormechanical means, in whole or in part, wi-thout the express permission of Leica Micro-systems CMS GmbH. This also includesphotocopying, recording or storing on a re-trievable system as well as the translationinto another language.Leica Microsystems CMS GmbH reservesthe right to revise this document and/or tofurther develop and improve the productsdescribed in this document at any time wi-thout prior notice or any other obligation. Theinformation and technical specifications inthis online help may be changed at any timeand without prior notice.Leica Microsystems CMS GmbH accepts noresponsibility for any errors or omissions. Inaddition, Leica Microsystems CMS GmbH isnot responsible for direct, incidental or con-sequential damages that could result fromthe use of the material described in this do-cument.Throughout this online help, trademarked na-mes may be used. Rather than including atrademark symbol (TM) at every occurrenceof a trademarked name, we state that we areare using the names only in an editorial fa-shion, and to the benefit of the trademarkowner, with no intention of infringement.



10.2.1 In the respective context (context-sensitive)

1 Click on the small question mark locatedin the top right corner of every dialogwindow.

2 Online help opens directly to the descrip-tion for the corresponding function.

10.2.2 Via the Help menu

1 Click on the Help menu on the menubar. The menu drops down and revealsthe following search-based options:

Contents This dialog field contains the table of contents in form of a directorytree that can be expanded or collapsed. Double-click an entry ofthe table of contents to display the corresponding information.

Index Enter the word you want to look up. The online help shows the keyword that represents the closest match to the word you entered.Select a keyword. View the corresponding content pages by

double-clicking the key word or selecting it and then clicking theDisplay button.

Search Enter the term or definition you want to look up and click on theLIST TOPICS button. A hierarchically structured list of topics is re-

turned.



10.2.3 Full-text search with logically connected search terms

Click on the triangle to the right of the inputfield on the Search tab to view the availablelogical operators.1 Select the desired operator.2 Enter the second search term you would

like to associate with the first searchterm behind the operator:

Examples Results

Pinhole and sec-tions

This phrase finds help topics containing both the word ”pinhole” and the word”sections”.

Pinhole or sec-tions

This phrase finds help topics containing either the word ”pinhole” or the word”sections” or both.

Pinhole nearsections

This phrase finds help topics containing the word ”pinhole” and the word ”sec-tions” if they are located within a specific search radius. This method also looks

for words that are similar in spelling to the words specified in the phrase.

Pinhole not sec-tions

This phrase finds help topics containing the word ”pinhole” and not containing theword ”sections”.



10.3 Key combinations

In order to accelerate recurring softwarefunctions, special key combinations havebeen defined:

CTRL + N Opens a new experiment

CTRL + O Starts the Open dialog window to open an existing file.



11 Structure of the user interface

11.1 General structure of the user interface

The user interface of the LAS AF is divided in five areas:

1 Menu bar: The different menus for cal-ling functions are available here

2 Arrow symbols: Operating step with theindividual functions. These operatingsteps mirror the typical sequence of animage recording and subsequent imageprocessing. The functions are arrangedappropriately in these operating steps.

D ConfigurationD AcquireD ProcessD QuantifyD Application3 Tab area: Different tabs belong to every

operating step (arrow symbol) in whichthe settings for the experiment can bemade.



Acquire Experiments: Directory tree of opened files

Setup: Hardware settings for the current experiment

Acquisition: Parameter settings for the image recording

Process Experiments: Directory tree of opened files

Tools: Directory tree with all the functions available in the respective operatingstep

Quantify Experiments: Directory tree of opened files

Tools: Tab with the functions available in this operating step

Graphs: Graphical display of values measured in regions of interest (ROI)

Statistics: Display of statistical values that were determined in the drawn in re-gions of interest (ROI)

4 Working area: This area provides theBeam Path Settings dialog window inwhich the control elements for settingthe recording parameters are located.

5 Viewer window : Displays the recordedimages. In the standard setting, the Vie-wer window consists of the image win-dow in the center and the buttons forimage editing (5a) and channel display(5b).

Koehler Illumination


12 What is the Köhler illumination?

In a microscopic image, only a certain areaof a specimen can be displayed (imagefield). Köhler illumination allows for illumina-ting only this particular area. The technicalbackground for the illumination of the imagefield is described as follows:If the illuminated area is smaller than theimage field, the luminous cone detected bythe objective lens as well as the numericaperture becomes smaller. Since the opticresolution is directly dependent upon the nu-meric aperture, a lower illumination also re-duces the optic resolution—which is not desi-red in most cases.If the illuminated area is larger than theimage field, it leads to increased scatteredlight. This, in turn, leads to a reduction of theimage contrast, possibly resulting in the si-tuation where optically dissolved structuresof the microscopic image can no longer beobserved.Köhler illumination represents a compromisebetween maximum contrast and maximumresolution. The most efficient microscope ob-jectives frequently reach their optimum opticperformance only with exactly adjusted Köh-ler illumination.



12.1 Setting the Köhler Illumination

1 Focusing: Focus an area of the object.Neglect the quality of the illumination forthe time being

2 Opening the aperture diaphragm:Fully open the aperture diaphragm. It willbe closed at a later point in time until thedesired contrast is adjusted.

figure44. DMxxxx (left) / DMIxxxx (right)

3 Closing the field diaphragm.The image field darkens in most areas. Youwill see an unfocused light spot. If the spotdisappears upon closing the field dia-phragm, the field diaphragm must be cente-red. In this case, open the field diaphragmuntil you can just see the light spot at theborder of the image field.

figure45. Field diaphragm



If no light spot is visible, the condensercould be set to the wrong height. Youshould, therefore, adjust the height of thecondenser until the field diaphragm is vi-sible.

4 FocusingFocus the border of the light spot by adju-sting the height of the condenser.5 CenteringTurn the centering screws of the condenseruntil the light spot is centered in the middle ofthe image field. The centering is easier if youslightly open the field diaphragm to enlargethe light spot.6 Opening the field diaphragmOpen the field diaphragm until the light spotjust disappears at the border of the imagefield.7 Closing the aperture diaphragmClose the aperture diaphragm until you haveset the desired image contrast (open to ap-proximately 70% of the maximum diameter).8 If you change the objectiveIt may become necessary to readjust theKöhler illumination after you have changedthe objective.

Introduction to Confocal Work


13 Introduction to confocal work

13.1 Preparation

The following sections describe a number ofbasic procedures that cover most of thetasks related to the instrument.

Upright stand

Inverted stand

StageFocus

LensFocus

Objective

CoverslipSeal

Glass slide

Immersion

Specimen

Embedding

figure46. Arrangement of coverslip and specimen on an upright microscope (top) and invertedmicroscope (bottom). When using objectives with coverslip correction, ensure that thecoverslip (i.e. the top side of embedded specimens) is facing down.

Background information has also been provi-ded to explain the reasons behind varioussettings. These are not descriptions of the



individual functions and controls of the instru-ment and graphical user interface, but an in-formative tour of the essential tasks that isdesigned to remain valid even if future up-grades change the specific details of opera-ting the instrument.The very first step is to place a specimen inthe microscope, of course. When placingspecimens in an inverted microscope, en-sure that fixed specimens on slides are in-serted with the coverslip facing down(Fig.43). Failing to do so is a frequent reasonfor not being able to find the specimen or fo-cus on it in the beginning.

13.1.1 The objective

Select the objective with which you want toexamine the specimen initially.

Medium Refractive Index

Water Imm 1,333PBS Emb 1,335

Glycerol 80% (H2O) Imm 1,451Vectashield Emb 1,452Glycerol Imm 1,462Moviol Emb 1,463

Kaisers Glycerol Gel Emb 1,469Glass Mat 1,517Oil Imm 1,518

Canada Balsam Emb 1,523

Table 3 Immersion media

When using immersion objectives, ensurethat an adequate quantity of immersion me-dium is present between the front lens of theobjective and the specimen. Immersion oil,glycerol 80% and water may be used as im-mersion media (Table 3). Apply the immer-sion medium generously, but be sure that itdoes not flow into the stand of inverted mi-croscopes.



13.1.2 Conventional microscopy

To view the specimen conventionally throughthe eyepieces, select ”VISUAL” operatingmode. ”SCAN” is for use with the laser scanimage process. Select a suitable positionand focus on the specimen.

Specimen

Shutter

Objective lens

LampFilter cube

Eyepiece

figure47. Incident light fluorescence scheme: light from a mercury lamp is collimated, selectedspectrally via an exciter filter and applied to the specimen via a color splitter mirror. Ashutter permits the specimen to be darkened. The emission (longer wavelength thanthe excitation) is visible through the color splitter mirror and emission filter via the eye-piece. The exciter filter, color splitter mirror and emission filter are grouped in a filtercube.

Optical sections are created using a trans-mitted-light process. Your specimen musttherefore reflect or fluoresce. Fluorescentspecimens are most common. In many ca-ses, specimens with multiple stains will beexamined. Reflective specimens can alsoprovide interesting results, however.Filter cubes (Fig. 44) suitable to the fluore-scence must be positioned in the beam when



viewing the specimen via the eyepieces. Formore information on selecting fluorescencefilter cubes, please refer to the Leica fluore-scence brochure or contact your Leica part-ner. For a selection of filter cubes, see Table4 below.As specimen fluorescence can fade quickly,always close the shutter of the mercury lampwhen you are not looking into the micro-scope.To switch to scan mode, press the appro-priate buttons on the stand or use the swit-ching function in the software. The switchingfunction may vary according to the motoriza-tion of the microscope. Please consult helpfor more information.



Filter cube Excitation filter Dichroic mirror Emission filter

A 400A/G 430;500B/G/R 415B/R 435;565

BFP/GFP 420CFP 455D 455E4 455FI/RH 500G/R 505GFP 500H3 510I3 510K3 510L5 505M2 580N2,1 580N3 565Y3 565Y5 560YFP 515

Table 4 Selection of filter cubes for Leica research microscopes and associated filter specifications.



13.1.3 Why scan?

X

Y

Specimens must be illuminated over thesmallest possible area to achieve a trueconfocal image – this is essential to attai-ning truly thin optical sections.That has been achieved when the illumina-tion spot is diffraction limited; i.e. it cannotbe made physically smaller. The diameter ofsuch a diffraction limited spot correspondsto dB=1.22*ë/NA, with ë representing theexcitation wavelength and NA the numericalaperture of the objective used (Fig. 46).

figure48. Illustration of the raster scan. Two mirrors move the illumination spot in x and y direc-tions across the specimen in rows so that the entire image can be reconstructed inparallel.



To create a two-dimensional image, the spotmust be moved over the entire surface andthe associated signal recorded on a point-by-point basis.This is performed in a raster process similarto that of SEM instruments or the cathoderay tubes still used in computer monitorsand televisions (Fig. 45). In a confocal mi-croscope with point scanners, the move-ment is realized by two mirrors mounted onso-called galvanometric scanners. Thesescanners have the same design as electricmotors; their rotors are fixed at their base tothe housing. Applying power to the scannerturns the axis to the point at which the tor-sional force and the electromagnetic forcebalance. The mirror can thus be movedquickly between two angles by applying analternating voltage.

figure49. Smallest possible, diffraction-limited illumination spot (Airy disk). Below: an intensityprofile.

To scan a line, the mirror must travel acrossthe field of view. The y mirror is then moveda small amount, after which the x mirror thenscans the next line. The signals from thespecimen are written to an image memoryand can be displayed on the monitor.



13.1.4 How is an optical section created?

The term ”confocal” is strictly technical anddoes not describe the effects of such an ar-rangement. That will be described in greaterdetail here.As already described in 13.1.3, the illumina-tion of the specimen is focused on the smal-lest possible spot. The confocal design alsoinvolves an observation point. The sensitivitydistribution of the detector is reduced to apoint by focusing light from the specimen ona very small opening, the so-called pinhole.This pinhole cuts off all information not co-ming from the focal plane (Fig. 47).

Pinhole

Out of focus

In focus

figure50. Creating an optical section using an incident-light process. Light not originating fromthe focal plane is cut off by a spatial filter (here, a pinhole). Only information from thefocal plane can reach the detector.

The diaphragm thus acts as a spatial filter,only when used with the correct, i.e. point-shaped illumination.As a rule, the optical section becomes thin-ner when the size of the pinhole is reduced.This effect is reduced near the wavelength ofthe light used, and at a pinhole diameter of



zero one would theoretically receive the thin-nest optical section for the wavelength andnumerical aperture used. A range apparentlyexists at 1 Airy which does not yet offer thethinnest optical sections, but which is nevert-heless very close to the theoretical limit. Asthe intensity of the passing light increasesroughly in proportion to the square of the pin-hole diameter, it is advisable not to close thepinhole too far to avoid excessive imagenoise. A value of 1 Airy is a very good com-promise and is selected automatically by theLeica TCS SP5. A dialog is available to setsmaller or larger diameters if required.Playing with this parameter to study its ef-fects can be very worthwhile when you havethe time.



13.2 Acquiring optical sections

figure51. Use the ”Acquire” arrow button to acquire data in all Leica LAS AF applications.

The Leica TCS SP5 contains many functionsin its user interface that reflect its wide rangeof potential applications. The functions notneeded for a given application are disabled,however, to ensure efficiency and ease ofuse. Select the task at hand from the row ofarrow buttons at the top. The functions requi-red for data acquisition (and that is the focusof this section) are grouped under ”Acquire”(Fig. 48).For descriptions of the individualfunctions, please see the online help.This section will describe the aspects affec-ting the configuration of the most importantacquisition parameters and special pointsthat must be taken into consideration.



13.2.1 Data acquisition

Press the ”Live” button to begin data acqui-sition (Fig. 49). Data will be transferred con-tinuously to video memory and displayed onthe monitor. Initially, the data will not be sto-red in a manner suitable for subsequent re-trieval.

figure52. The ”Live” button starts data acquisition in all Leica LAS AF applications.

This is a preview mode suitable for settingup the instrument. Stopping data acquisitionwill also immediately stop the scan process,even if the image has not been fully rende-red.

z-stack

Time series

Lambda series

Alternatively, a single image can be captu-red. This image is then stored in the experi-ment and can be retrieved later or stored onany data medium. Individual image capturehas the advantage of only exposing the spe-cimen once, but is less convenient if additio-nal setup work is required. Once all parame-ters are correctly set up, an image of the re-sult may be captured. Functions such as ac-cumulation and averaging are supported.The third data acquisition situation is the ac-quisition of a series in which the preselectedparameters are changed incrementally bet-ween the capture of the individual images.Time series, lambda series and z stacks canbe acquired in this manner (Fig. 50).When using the instrument in ”LiveData-Mode”, all captured images are automati-cally stored with the time of capture. A pre-view mode is not available in that case (Fig.51).

figure53. Stack acquisition for 3D, time and lambda series



This method is especially suitable for the ob-servation of living objects over time whilechanging the medium, applying electrical sti-muli or executing changes triggered by light.

I (t)

T

figure54. LiveDataMode supports the continuous acquisition of data while changing setting pa-rameters, manipulating the specimen or performing bleaching sequences between theindividual captures. The clock continues running throughout the experiment and inten-sity changes in interesting areas can be rendered graphically online.

The setting parameters for a simple opticalsection are described and discussed below.These settings are identical for all work withthe instrument. Preconfigured parametersets have been stored in the software for ty-pical specimen situations. You may alsostore and recall custom parameter sets. Thedescription below is based on the assump-tion that you are using a specimen similar tothe included standard specimen. The stan-dardspecimen is a Convallaria majalis rhi-zome section with a histological fluorescentstain. The specimen can be used for a widerange of fundamental problems and has theadvantage that it practically does not bleach.



13.2.2 Illumination

488nm

0

100

Emission

(SP detector)O

(AOTF)Excitation

%In

tens

ity

Laser lines suitable for the excitation of fluo-rescence may be selected as illumination.The intensity of the laser line can be adju-sted continuously using the line’s slider. Mo-ving the slider all the way down disables theline. The intensity setting of the slider is re-alized steplessly via an acousto-optic tuna-ble filter (AOTF). The intensity at which asufficiently noise-free image of the speci-men can be obtained must be determined toreduce deterioration of the specimen. Fac-tors affecting this are the fluorescence stain,the line used, the density of the stain in thespecimen, the location and width of the se-lected emission band, the scanning speedand the diameter of the emission pinhole.

figure55. Selecting the illumination intensity via acousto-optic tunable filter (AOTF, above) andselection of the emission band in the SP detector (below).

When selecting the ”FITC” parameter set,the 488nm argon line and a suitable bandbetween 490nm and 550nm is set.12

The entire beam path is represented graphi-cally on the user interface. A spectral bandwith the settings for the emission bands islocated on the emission side. The laser lineis visible at the appropriate location in thespectrum as soon as a line is activated.When viewing the specimen through the mi-croscope, the light in the selected color willbecome lighter or darker according to the po-sition of the slider. This does not involve anydanger for the viewer’s eyes, despite the factthat this is laser light. As the light is focusedon the specimen through the objective, thebeam is strongly divergent and is harmlessat a distance of a few centimeters. Neverthe-less, please read the safety notes in this ma-nual.If all of the other settings are in order, darkerand lighter images will be visible on the mo-



nitor when moving the slider for the illumina-tion.

13.2.3 Beam splitting

Stokes shift

Em 1Em 2

Exc1

Exc2 The simplest case would involve the selec-

tion of a laser line roughly at the maximumof the excitation spectrum of a given fluore-scence stain. This would achieve the bestyield. In general, however, lasers delivermuch more light than necessary, and atte-nuation to 10% is generally sufficient forgood images (although that depends verystrongly on the specimen’s stain, of course).One can thus also excite the fluorescenceon the blue side of the excitation maximum,which has the advantage of providing abroader band for the collection of the emis-sion (Fig. 53).

figure56. Excitation spectrum of a fluorescence stain (blue) and emission spectrum (red). Anexcitation in the maximum (Exc1) would result in only a narrow band to be collected onthe emission side (Em1). A significantly broader emission band (Em2) is availablefrom an excitation in the blue range, at which point the intensity of the laser can beincreased without detrimental effects.

Experimenting a bit is worthwhile here. Anacousto optical beam splitter (AOBS�), per-mits all available laser lines to be conve-niently added or removed without devotingattention to the beam splitter characteristicsor the spacing of the lines.

13.2.4 Emission bands

Once the excitation light has reached thespecimen via the AOBS� and the objective,an emission is generated in the fluorescentmolecules, the light of which is shifted towardlonger (redder) wavelengths. This is knownas ”Stokes shift”, and its degree depends onthe fluorochrome. As a rule, the excitationand de-excitation spectra overlap, and theStokes shift is the difference between the ex-citation maximum to the emission maximum.



It is, of course, advantageous for good sepa-ration and yield if the Stokes shift is veryhigh. Typical stains have a Stokes shift bet-ween 10nm and 30nm. Shifts of more than100nm also occur, in natural chlorophyll forexample, an excellent stain for curious expe-rimenters.

Em 1 Em 2

Exc2Exc1

Exc

Refl Em

The emission characteristics of stains canbe displayed on the spectral band graphic ofthe user interface. It is therefore very easyto choose where an emission band shouldbegin and end. If an emission curve has notbeen stored, it is possible to record andsave such a curve directly with the system.An adjustable bar under the spectral bandhas been assigned to each confocal detec-tor. The limits to the left and right indicatethe limits for the selected emission band.

figure57. SP detector setting options for two fluorescences with different excitations (above) orfor fluorescence and reflection (below).

It is possible to move the entire bar back andforth to adjust the average frequency, ormove the limits independently. Using the ex-citation lines and displayed emission charac-teristics for orientation and adapting theemission band using the Leica SP� detectionsystem is thus very convenient. This is alsotrue while images are being captured.Theeffects of settings on the images are imme-diately apparent and suitable values can thusbe selected empirically (Fig. 54).The reflected excitation light also appears inthe image as soon as the emission bandcrosses under the excitation line. While thisis naturally undesirable for fluorescence, itdoes provide a very simple way of creating areflection image. The smallest band is 5nm,and such a 5nm band would generally be setunder the excitation line for reflectometry ap-plications.



To suppress interference from reflected exci-tation light, it generally suffices to set thestart of the emission band to around 3 to5nm to the red side of the excitation line. Na-turally, this depends strongly on the reflectiveproperties of the specimen. It is usually alsonecessary to maintain a greater distancewhen focusing close to the glass surface forthis reason. That especially applies to speci-mens embedded in aqueous media. The furt-her the refractive index of the embeddingmaterial deviates from 1.52, the more likelydistracting reflections become. Greater cau-tion would also be required for specimenscontaining a high number of liposomes, forexample.

13.2.5 The pinhole and its effects

The reason for deploying confocal micro-scopy is its ability to create optically thin sec-tions without further mechanical processingof the specimen. The essential component ofthe instrument that creates these sections isa small diaphragm in front of the detector –the so-called pinhole – as already describedin 13.1.4. Ideally, the diameter of this pinholewould be infinitely small, but this would nolonger allow lightto pass, preventing the cap-ture of an image. However, the effect wouldbe lost if the pinhole were too wide, as theimage would contain excessive blurred sha-res of the specimen from above and belowthe focal plane.



SectionThickness

Pinhole Diameter

DiffractionLimit

1 Airy

Geometry

The relationship of the thickness of the opti-cal section to the diameter of the pinhole islinear for large diameters and approaches alimit value at smaller diameters, beingroughly constant near zero (Fig. 55).The li-mit value is dependent on the wavelength ofthe light and the numerical aperture. As thesection thickness changes little when initiallyopening the pinhole, but the passing lightincreases in proportion to the square of thepinhole diameter, it is advisable not to usetoo small a diameter.

figure58. Relation of optical section thickness to pinhole diameter

4 AE

1AE

0.25AE0.25AE

0.25AE

0.5AE1AE

2 AE4 AE

A good compromise is the point where thediffraction limitation(constant dependence) transitions to geo-metric limitation (linear dependence). Whendepicted in the specimen plane at this point,the pinhole has roughly the size of the dif-fraction-limited light disk of a focused beam.This is known as the Airy diameter. The Airydiameter can easily be calculated from theaperture and wavelength. Setting the pin-hole to roughly the size of the diffraction-li-mited spot thus results in sharp optical sec-tions with a good signal-to-noise ratio (S/N)(Fig. 56).Naturally, the instrument can calculate andset this value automatically. The objectiveused is known to fully automatic instrumentsand can be set when working with manualsystems. The excitation lines used are alsoknown to the system.

figure59. Optical sections with a variety of pinhole diameters (63x/1.4 objective). The strong lossof light can be seen clearly with the small diameters, as can the pronounced back-ground in the images with very large diameters.

A pinhole diameter of 1 Airy is therefore thedefault setting. Switching objectives also ad-justs the diameter of the pinhole accordingly.



A larger pinhole may be selected simply byadjusting a slider on the user interface forspecimens with weak fluorescence or highsensitivity against exposure to light. Smallerpinhole diameters may be selected as wellfor very bright specimens. With reflectingspecimens in particular, the pinhole may bereduced to 0.2 or even 0.1 Airy units (AU) forthe thinnest possible sections.

13.2.6 Image detail and raster settings

Depending on the objective used, conventio-nal microscopes show a circular excerpt ofthe specimen. The diameter of the circle,multiplied by the magnification of the objec-tive, is the field number (FOV). The fieldnumber is therefore a microscope valuewhich is independent of the objective, andwhich conversely can be used to calculatethe size of the specimen being observed. Ascanner always acquires square or rectangu-lar excerpts, of course. If such a square isexactly circumscribed by the field of vision,than the diagonal dimension will correspondexactly to the field number, allowing the lar-gest possible image to be displayed on themonitor without restrictions.Unlike eyes or conventional cameras, scan-ners can simply be set to a smaller angle. Afurther-enlarged section of the field of viewwill then be displayed on the monitor. It isthus possible to zoom into details without theneed for additional optics. As the scan anglecan be adjusted very quickly and continu-ously over a wide range, magnification in-creases of around 40x can be achieved sim-ply by moving a slider. As always inmicroscopy, the total



Zoom Pan

fov 15.1

fov 21.2

magnification must be appropriate, i.e. wit-hin a suitable range, to obtain good images.Other scales are important for overviewsand bleaching experiments.As errors can easily be made when interpre-ting captured data, the following is an exam-ple of how an appropriate total magnificationcan be calculated, as well as the informationthat is automatically provided to the user bythe software.

figure60. Fields of view for conventional (21.2mm) and resonant scanners (15,1mm). A smallerscanning angle increases the magnification (zoom), while a scan offset shifts theimage detail (pan) within the field of view.

The edge length of the displayed field of aconventional scanner corresponds to 15mmwithout magnification by the objective (1xscale). A field number of 21.2 is thus alsofully utilized (Fig. 57).That is suitable for most good research mi-croscopes. How many points are now ac-tually resolved optically in this dimension?That depends on the numerical aperture ofthe objective and the wavelength. Accordingto Ernst Abbe’s formula, two points can stillbe distinguished if the distance betweenthem is not smaller than d=ë/2*NA. A linecan thus contain a maximum of 15mm/d re-sels (resolved elements). When using an ac-tual objective such as a plan apochromat10x/0.4, the edge length corresponds to1.5mm (15mm/10) and d=0.625 Pm whenusing blue-green light with a wavelength of500nm. Such an image would thus contain1500/0.625=2400 optically resolved ele-ments along each edge.Rendering this resolution in a digital pixelimage would require working withtwice theresolution to prevent losses (Nyquist theo-rem). That would be an image with4800x4800 pixels. Some purists require 3xoversampling, i.e. 7200x7200 pixels, or 52



megapixels. Image formats for x and y canbe adjusted independently and in very finesteps, with the Leica TCS SP5 supportingcapture formats of up to 64 megapixels(8000x8000 pixels) (Table 5).

Magnification 63 40 10

Numerical Aperture 1,4 1,25 0,4Optical Resolution ( 400nm) Pm 0,14 0,16 0,5Intermediate Image (Edge) mm 15 15 15

Field (Edge) Pm 238 375 1500# Resel (Field / Resolution) 1667 2344 3000

2x Oversampling 3333 4688 60003x Oversampling 5000 7031 9000

Table 5 Table of resolved elements at 400nm for a variety of objectives over the entire scan field. Itbecomes apparent here that a resolution of 64 megapixels (8000x8000 pixels) is appropriatefor quality microscopy applications.

It is thus possible to capture truly all of theimage information resolved by the micro-scope in a single image at that setting. Thisnaturally results in large data volumes, whichare especially undesirable for measurementswith a high temporal resolution. Zoom is thecorrect solution here. When capturing data inthe standard 512x512 format, this means li-miting the image to a field 10-15 times smal-ler to avoid a loss of information. Zoom fac-tors of 10x and higher provide usable data,but from rather small fields of view. The infor-mation required will determine what constitu-tes an acceptable compromise here.Such an image is initially an excerpt of thecenter of the scan field. That is notalwaysdesirable, as it can be difficult to center theinteresting structures with such precision.However, the scan field excerpt can be mo-ved across the entire scan field to the actualpoints of interest, a method called ”panning”.



512 128

256 641024

figure61. The same image detail in a variety of pixel resolutions. Please note that the printingmedium may not be capable of reproducing the full detail of high resolutions. You maytherefore have difficulty detecting the differences between the top two images, despitethe enormous differences in the optical resolution. This must also be taken into consi-deration in publications.

The simplest solution is to combine both me-thods in the so-called ”Zoom In” function.Simply select a square on the monitor thatencloses the structures of interest, and theinstrument will automatically select the ap-propriate zoom and pan values. This functionis very fast and thus easy on the specimen.An ”Undo Zoom” function returns you to yourstarting point – for quickly concentrating on adifferent cell in the field of view, for example.The size of the grid spacing used can befound in the image properties. The spacingof the image points in x, y and z can befound under ”Voxel Size”. At Zoom 1, the pic-tures calculated above would have a gridspacing between 200nm and 300nm. Largerspacings would lead to a loss of resolutionwhen using an objective with an aperture of0.4 (Fig. 58).



Rectangular formats are important for higherimage capture rates. An additional parameteris required here: the rotation of the scanfield. As field rotation is performed opticallyin the Leica TCS SP5, rotation by +/- 100qdoes not have any effect on the speed andpossible grid formats (Fig. 59).Finally, it must be pointed out in this sectionthat a good microscopic image in a scientificcontext must always contain a scale. Such ascale can simply be added to the image andadjusted in its form, color and size as requi-red. Scales were not added to the images inthis document for the sake of clarity.



13.2.7 Signal and noise

The gain of the capture system must bematched to the signal intensity when captu-ring data. Signal strengths can vary by se-veral orders of magnitude, making such anadjustment necessary to ensure a good dy-namic range for the capture. The goal is todistribute the full range of intensity over theavailable range of grayscale values. 256grayscale values (from 0 to 255) are availa-ble for images with 8-bit encoding. If thegain is too low, the actual signal may onlycorrespond to 5 grayscale values, causingthe image to consist solely of those values.If the gain is too high, parts of the signal willbe truncated – i.e. they will always be assi-gned the value 255, regardless of the infor-mation they originally contained. This imageinformation is then lost (Fig. 60).Correctly setting the zero point is also im-portant. This can be accomplished by shut-ting off the illumination via the AOTF andsetting the signal to zero with ”Offset”.

figure62. Zoom, Pan and Rotation combined in one example.

Turn the illumination back on and adjust thegain to prevent distortion.This configuration work is simplified by spe-cial color tables such as ”Glow-over/Glow-under” – a table that initially uses yellow andred for intensities in steps to indicate the si-gnal strengths. The grayscale value zero isalways shown as green, value 255 as blue.Both values can thus be identified immedia-tely. The zero point is set correctly whenaround half the pixels are zero – i.e. green.To be safe, the offset can be set one or twograyscale values higher to ensure that thelower signal values are not truncated. The



loss of dynamic range is negligible (approx.0.4% per grayscale value at 8 bits).

8 Bit approx.2 Bit

The electronic deviations from the zero pointwill generally be negligible; nevertheless,occasional testing is advisable. The actualsignificance of adjusting the offset value isto compensate for unspecific fluorescencein the specimen at the time of capture. Sim-ply set the offset value in such a mannerthat the background fluorescence is no lon-ger visible. Please note that this may alsotruncate signals containing image informa-tion.

figure63. Top left. An 8-bit image (256 gray steps). At the right, the same specimen with a consi-derably smaller dynamic range. Around 6 gray steps can be made out in the false-co-lor image at the bottom. That corresponds to less than 3 bits.

Such settings must always be verified by acareful examination of the results.The amplification of the signal must be per-formed after the offset correction. This ope-ration is quite simple with the described colortable: adjust the high tension at the PMT untilno more blue pixels are visible. We recom-mend focusing to ensure that truly the brigh-test signals in the field of view are used forthe adjustment. This is also the right time tocheck whether the intensity of the excitationlight is correctly set. The intensity of the illu-mination can be increased at the AOTF toreduce image noise. However, it must betaken into consideration here that a higherillumination intensity is detrimental to thespecimen. In the case of extremely sensitivespecimens and in situations in which rapidchanges in intensity in living specimens is ofinterest, images with more noise can be ac-ceptable. This compromise depends on thespecimen and the application, however.The signal-to-noise ratio may be influencedby a number of other factors in addition toillumination intensity: the speed at whichdata is captured. The actual speed of the



scan, which can be adjusted via the horizon-tal frequency (1 Hz - 1,400 Hz, conventionalscanners only), and the averaging processoffer additional options for enhancing the si-gnal. The change of the scan speed itselfleads to averaging in the pixels, as data isrecorded for each pixel over a longer period.When averaging lines, each line is scannedseveral times and the result of the averagingdisplayed. In the case of image averaging,an entire image is captured and then avera-ged with the subsequent image captured atthe same location.All processes have their advantages and di-sadvantages – as always. Temporal correla-tion is important for moving objects, callingfor a slower scan. On the other hand, tripletphenomena speak for longer times betweenaverages, i.e. for the averaging of entireimages. Averaging lines represents a com-promise here. The averaging of completeimages is the gentlest method, but has thedisadvantage of not immediately showing thequality of the results; this can be evaluatedeasier with the other methods. On the otherhand, the averaging operation can also beaborted manually when averaging imagesonce the quality impression is adequate. Ageneral rule of thumb for all application situa-tions thus does not exist. Choosing the bestmethod is a matter of experimentation andexperience.



13.2.8 Profile cuts

So far, we have always assumed thatimages are captured parallel to the focalplane That is both correct and appropriatefor conventional microscopy. A confocalpoint scanning system offers interesting newoptions for capturing data, however. For ex-ample, profile cuts through the specimencan be realized by always moving the lightspot along the same line, and instead of ma-king an incremental y movement, moving itbetween the lines of the focal plane (usingthe fast, precise SuperZ galvanometerstage, for example).

figure64. Profile cut through the Convallaria majalis specimen, indicating a thickness of approx.30mm.

This is similar to slicing through a cake, per-mitting impressions to be gained online ab-out the contents of the specimen.Camera-based systems (also ”confocal” sy-stems) can only compute such profiles out ofcomplete stacks (Fig. 61).



13.3 Multiparameter fluorescence

In many cases today, specimens are usedthat contain more than one fluorescent stain.Multiple stains are achieved using hybridiza-tion of various linked fragments (fluore-scence in situ hybridization, FISH), throughdifferently marked antibodies or with fluore-scence proteins with differing spectral pro-perties. Traditional histological fluorescentstains and autofluorescence are also usableparameters (Fig. 62).

13.3.1 Illumination

Specimens with multiple stains generally re-quire illumination with multiple colors (in thiscase: laser lines). That is not always thecase, however: there are naturally alsostains with differing emissions that can beexcited by the same wavelengths. A distinc-tive example would be a botanical specimenwith a FITC stain and blue excitation. Theemission of FITC would then be visible inthe blue-green range of the spectrum. Thesame excitation can also be applied to chlo-rophyll, however, which would respond withemission in the deep-red range.

figure65. Simultaneous capture of two fluorescences, in this case excited by a single laser line.The depiction in the colors green and red is arbitrary.

Fluorescence and reflection images can alsobe rendered at the same time. Using a singleexcitation, this merely requires observing asecond ”emission band” below the laser line.



Under normal circumstances, stains will beused that require different excitation wave-lengths, however. A variety of lasers areusually installed in the instrument for thispurpose. To activate a second excitationline, simply set the desired slider for the se-cond wavelength as described in 13.2.2 forsimple excitation. Additional excitation wa-velengths can be added just as easily. It isfrequently helpful for the bleaching experi-ments described below to activate multipleAr lines, even if you are not capturing a si-gnal or are using only one channel. Thisprovides additional intensity.Experimenting a bit with laser combinationsis always beneficial. It frequently becomesapparent that one does not need all of thelines initially selected for the stains, or a dif-ferent line turns out to be a better compro-mise. Default configurations for illumination,beam splitting and emission band settingscan be selected from a list for most typicalstain combinations.

figure66. Simultaneous capture of two fluorescences, in this case excited by a single laser line.The depiction in the colors green and red is arbitrary.

13.3.2 Beam splitting

Beam splitting is very easy to describe inAOBS� systems: there is no need to give itany thought. The AOBS automatically swit-ches a narrow band for the selected lines toensure that the excitation is applied to thespecimen. Such bands have a width ofaround 2nm. Everything else is available tocapture the emission.A suitable beam splitter must be selectedwhen using instruments with traditional beamsplitters. In this regard, it is important to



know that not only simple, but also doubleand triple beam splitters are available (DDand TD for double-dichroic and triple-dich-roic).Lines in close proximity to one another can-not be served with dichroic splitters. For ex-ample, no usable splitters are available forthe simultaneous use of 594nm and 633nmHeNe lines. In these cases, an AOBS is asignificant advantage: thanks to the verysmall bands (approx. 1 to 2nm), both linescan be used for excitation, while capturing anemission band of 35nm in between with theSP detector.

13.3.3 Emission bands

Naturally, the same boundary conditions ap-ply for the emission bands as described in13.2.4 – with the difference that two laser li-nes limit the band for all stains except thereddest, and that precautions must be takento ensure that the excitation light does notreach the detector. In addition, the suppres-sion of crosstalk can have a strong effect onthe choice of band limits. The following sec-tion will cover this in greater detail. Settingthe bands is described in Section 13.2.

13.3.4 Crosstalk

The emission spectra of stains (includingthose that are responsible for autofluore-scence) typically have a rather simple cha-racteristic with a maximum emission and ablue flank that drops more steeply than thered side. The emission extends quite far, butwith low amplitude, on either side. Especiallythe red side can be a problem. Crosstalk orbleed-through refers to the fact that the emis-sion of a stain not only contributes to the si-gnal in one channel, but in other detectionchannels as well. This should naturally beavoided, as it leads to the display of incorrect



images and falsifies the determination of cor-relations. The reliability of separation – andthus the avoidance of crosstalk – is thereforean important issue.Several parameters must be considered forthis purpose: illumination intensity, laser se-lection, sequential capture, emission bandsand unmixing methods. Initially, we will becovering illumination and emission parame-ters.Crosstalk is frequently caused by strong dif-ferences in the concentration of the fluoroch-romes used. Even illumination will then resultin a very good signal from the more highlyconcentrated stain, yet it is very likely thatthe signal will also bleed into other channels.This can be compensated by setting the va-rious laser intensities in such a manner thatstains with weak concentrations are excitedwith higher intensities while the higher con-centrations receive less-intense excitation.Balancing in this manner already eliminatesa significant crosstalk problem. Thanks to thecontinuously adjustable intensity via AOTF,the results can be monitored directly on thedisplay and can thus be adjusted online withsuitable feedback.It may be useful to try a variety of laser linesfor excitation in order to obtain sufficient lee-way to adjust the emission bands. This para-meter can also be used for balancing: if astain is very dominant, the selection of a dif-ferent excitation line can reduce the intensityof the stain (and thus improve the separationagainst the other stain) while increasing thespacing to the other excitation, permitting lar-ger emission bands and thus enhancing sen-sitivity. Every improvement in this regard per-mits a reduction of excitation energy, whichin turn reduces bleaching.A further option for the reduction of crosstalkis the selection of suitable emission bands.The emission characteristics of the stainsused can be displayed in the user interface,and a lot can be gained if the emissionbands are restricted to ranges that do not



overlap in the graphic on the monitor. Natu-rally, the stored characteristics are not ne-cessarily identical to the actual emissions, asmany factors (e.g. polarity, metabolic pro-ducts) can affect the spectrum. However, inthis case it is also possible to change andoptimize the settings during data capture.

13.3.5 Sequential capture

A further way to reduce crosstalk is to cap-ture the information for the various stains se-quentially. This has two advantages: Whene-ver different laser lines are used forexcitation (and this is generally the case),sequential capture provides significantly im-proved separation, as only one stain is exci-ted at a time and the emissions are thus so-lely from that stain, regardless of the spectralrange in which the signals are captured. Thisis naturally the ideal state – in practice otherstains may also be excited slightly, but theseparation is nevertheless clearly better thanthat from a simultaneous capture. Generally,crosstalk can be virtually eliminated this way.A further advantage of the sequential methodis that the emission bands of the individualstains can be set rather widely. This impro-ves sensitivity and is thus easier on the spe-cimen.An obvious disadvantage is that the captureof two stains also takes twice as long. In thecase of physiological measurements, this isfrequently not permissible.

13.3.6 Unmixing

As in most cases, a software solution is avai-lable to deal with crosstalk whenever a phy-sical separation is not possible. However, werecommend optimizing separation with themeans provided by the instrument (see 3.4and 3.5) to the greatest extent possible and



to use the software only in those cases inwhich the results are still not satisfactory.The unmixing method determines the shareof a stain’s emissions distributed across thevarious capture channels. This process isapplied to each of the stains. The result is adistribution matrix that can be used to redis-tribute the signal strengths so that they cor-respond to the stains. This is described fortwo stains in the following figures, but it isequally valid for any number of stains. Theprecondition is that the number of channelsused is at least the same as the number ofstains. The shares can then be correctly re-distributed with the simple methods of linearequation systems.The actual objective for effective unmixing isto determine the required coefficients of thematrix. This is also covered by a variety ofmethods available in the Leica software. It isadvisable to experiment a bit to determinethe best method for the task at hand. As allmeasurement data contains certain error andnoise components, there is no perfect recipefor the ultimate truth.The simplest approach for the user is to de-termine the coefficients on the basis of thestatistical data of the captured images. In thisprocess, the coefficients of the scatter dia-grams of both channels are determinedusing statistical methods. ”Hard” and ”soft”separation methods are available, leavingthe degree of separation at the user’s discre-tion.If the coefficients are known from other expe-riments, the data can be entered into a ma-trix manually. This method is also suitable fortrial-and-error work – when manually com-pensating for background interference or au-tofluorescence, for example.The method that delivers the most accurateresults is channel dye separation. In it, thedistribution of stains in the various channelsis determined directly using individual stainreference data. When using this method it isimportant to ensure that the parameter set-



tings of the instrument are not altered, as thelaser intensity and gain at the PMT also af-fect these coefficients.In the spectral dye separation method, theemission spectra of the individual stainsknown from literature or determined by mea-surements directly at the instrument are usedto calculate the relative intensity of thestains. This method is especially suited forsituations in which the stains do not signifi-cantly change their emission in situ and inwhich the related data is well-known.



13.4 3D Series

Altering the position of the focus betweencaptures permits a whole series of opticalsections to be captured and their structurerendered as a 3D data record. Naturally,such a three-dimensional ”image” cannot beobserved directly, but it contains spatial infor-mation related to the observed structures,and – in the case of multiple stains – theirlocal connections.

13.4.1 z-stack

To capture such a 3D series (”z stack”), setthe upper and lower limits simply by travelingto the top of the specimen, marking the loca-tion, and doing the same for the bottom.Next, determine the number of steps to becaptured between the two positions and therest will be handled automatically by the in-strument.

13.4.2 About section thickness...

As described in sections 13.1.4 and 13.2.5,the thickness of the optical section dependson the wavelength, the numerical aperture ofthe objective, and the diameter of the pin-hole. The relationship of these parameters isexpressed by the formula described there.The aperture should be as high as possibleto obtain truly good (thin) sections. Confocalmicroscopes use objectives with large aper-tures for this reason. The wavelength of theemission will generally be between 450nmand 600nm, so 500nm would be a suitablevalue for a rough estimate. Selecting the pin-hole diameter 1 Airy will result in sectionthicknesses between 0.5Pm and 2.5Pm forapertures from 0.7 to 1.4. These are typicalvalues in practice. In the literature – espe-cially in advertising materials – the thickness



is often stated for sections in reflection atpinhole diameter zero. Although this value ismuch smaller and thus looks better, it is notrelevant for practical applications in fluore-scence microscopy.

13.4.3 ...and spacing

The thickness of the optical sections is im-portant when capturing z stacks. If the spa-cing between the sections is too large (grea-ter than the thickness of the section), this willresult in gaps in the data record and a loss ofinformation. A reconstruction then can nolonger be calculated correctly. On one hand,there is little point in taking as many sectionsas possible, as a very tight spacing will resultin reduced differences between the individualsections and an unnecessarily high data vo-lume. This relates to the z axis in the sameway as ”empty magnification” in a conventio-nal microscope. For a dense data record wi-thout gaps, but also without superfluousoversampling, set the spacing between thesections to around one half to one third ofthe optical section thickness. In practice, thiswill be around 0.7 to 0.2 Pm. This meanscapturing between 1 and 5 sections per mi-cron in z, depending mainly on the apertureof the used objective.

13.4.4 Data volumes

A further factor that must be consideredwhen capturing a series is that it may resultin very large volumes of data that in somecases may not be suitable for processing orwhich can only be processed very slowly. A”normal” image with 512x512 pixels, onechannel and a standard 8-bit grayscale reso-lution weighs in at 0.25MB. One hundredsuch images (i.e. a 20Pm thick specimen athigh resolution) already require 25MB, whichonly a couple of years ago amounted to avery cumbersome volume of data. If 5 chan-



nels are captured in parallel with an imageformat of 1000x1000 pixels, the stack willhave a volume of 500MB, almost filling astandard CD. Using 16-bit grayscale and8000x8000 pixels would result in a data re-cord of 64 GB, a volume that most moderncomputers cannot handle with ease. A criti-cal assessment of the data capture parame-ters to be used is definitely called for here.



13.4.5 Depictions

figure67. Gallery of a z stack. This ”stamp collection” is well-suited for monochrome publica-tions.

As mentioned earlier, a three-dimensionalimage cannot truly be displayed on a two-di-mensional monitor. A variety of methods aretherefore available for presenting this infor-mation.

13.4.5.1 Gallery

The simplest of these is to display all of thesections of a series in a gallery – rather like astamp collection (Fig. 66). Changes fromsection to section can thus be analyzed andthe images printed in periodicals.



13.4.5.2 Movie

Many publications today are available via theInternet, making it possible to publish moviespresenting such sequences at a convenientspeed. These movies provide the impressionof focusing directly through the specimen atthe microscope. Both methods are suitablefor monochrome (black and white) and multi-channel captures.

13.4.5.3 Orthogonal projections

A further option for displaying the full rangeof information (with losses) compressed intotwo dimensions is to compute projections ofthe entire series. The most common methodis the so-called maximum projection. Thebrightest value along the z axis is determi-ned for each pixel and entered into the re-sulting image at this point. The result is animage consisting solely of the sharply focu-sed values, but distributed over the entiredistance of the image in the z direction.

figure68. Color-coded relief of the series shown above

The operation also increases the depth offocus over the entire height of the z stack.Such projections are therefore called ”exten-ded depth of focus” images. This method isalso suitable for multichannel captures.Coloring each layer differently, for exampleby mapping the colors of the rainbow to the zaxis, permits the z positions of structures tobe identified immediately in this projection.This is only possible with one channel, ofcourse, as the color is used for the height.This representation is known as ”height-colorcoded extended depth of focus” (Fig.65).The SFP (simulated fluorescence projection)method uses a more complex approach to



achieve impressive images with shadow pro-jections. The quantification must always bechecked with care when using this method,however.

13.4.5.4 Rotated projections

figure69. Stereo image of the same 3D data. A bit of practice is required, but this neverthelessrepresents a worthwhile exercise for any confocal microscope user.

The methods described in 13.4.5.3 initiallyassume that the projection will be performedalong the visual axis. The computer data isavailable in a spatially homogeneous state,however, permitting projections from any di-rection.In the simplest case, two projections fromslightly different angles can be displayednext to one another and superimposed by”unaided fusion”, or squinting. We then men-tally generate a three-dimensional image inthe same way as we would of any other ob-jectviewed with both eyes (Fig. 66).If only one channel is used, it is possible todisplay both views in different colors andview them through spectacles containing fil-ters for those specific colors (red-green ana-glyph). This is simpler for most users, butcannot be applied to multiparameter data.



Like the sections themselves series of pro-jections can be observed with increasing an-gles and presented as movies. 3D movies ofthis type are today the most common andconvincing means of displaying three-dimen-sional data.



13.5 Time series

A confocal scanning microscope recordsimages like a camera. It can therefore alsobe used to record a time series – essentiallya z stack without altering z. Such time lapseexperiments are an important tool in physio-logy and developmental biology, wheneverinterest is focused on dynamic processes.

13.5.1 About scan speeds

Temporal resolution is an important parame-ter in dynamic processes, especially thoserelated to kinetic studies of cellular biophysi-cal processes. Unfortunately, are imposedhere by a number of factors such as the me-chanical speed of the scanner, the bandwidthof the data line, and the simple volume ofphotons that can be expected from the speci-men during the period of observation. Whilemechanical and data bottlenecks can be re-solved in principle and great progress hasbeen made in this regard in recent years, li-mitations related to light are a hurdle thatcannot be overcome. Little light leads to apoor signal-to-noise ratio, and thus to poorresolutions and poor image quality. It is the-refore necessary to verify the parametersthat truly require measurement. A central dif-ference between various measurements isthe dimensionality that attempts to compen-sate for mechanical limits.



13.5.2 Points

The highest temporal resolution can beachieved when the mechanical elements ofthe scanner do not move at all. This amountsto measuring the changes in light intensity ata fixed, preselected point in the Leica TCSSP5 with a temporal resolution of 40MHz(corresponding to 25ns). Naturally, that parti-cular spot in the specimen can be expectedto bleach within a very short time.

13.5.3 Lines

Less fast, but nevertheless suitable for manyhighly dynamic processes, is the restrictionto images consisting of a single line. Thedata can be displayed as an xt image, withone dimension being the location (the selec-ted line) and time as the second dimension.An 8 kHz resonant scanner thus supports aresolution of 16 kHz (63Ps) in bidirectionalmode.

13.5.4 Planes

The standard scenario is the capture of xyimages as a t series. In this case, the tempo-ral resolution depends on the speed of thescanner and the number of lines per image.When limiting the capture to a band-shapedimage of 16 lines, a resonant scanner cancapture up to 200 images per second (5ms).This standard capture process (generally at512x512 pixels) is also used for long-termexperiments in which the image of the speci-men is captured repeatedly over the courseof hours or days, for example when recor-ding the development of embryos or cell cul-tures. In these cases, mechanical and photo-nic limitations play a subordinate role; thesystem must be extremely stable, free of driftand climate controlled, however.



13.5.5 Spaces (time-space)

The three-dimensional development of struc-tures in biology is naturally of great interest.The broad application field of 4D microscopyhas established itself here. This is realizedby recording a series of z stacks and proces-sing them into 3D movies. This is a field inwhich many innovations and exciting resultscan be expected in the future.

13.5.6 FRAP measurements

A completely different field of application forlaser scanning microscopy involves dynamicstudies in which a system is subjected to in-terference to disturb its equilibrium and stu-died as the restoration of its equilibrium pro-gresses. The FRAP method (fluorescencerecovery after photobleaching) is very well-known in this regard.Such experiments can be used to make de-ductions about membrane permeability, diffu-sion speeds and the binding behavior of mo-lecules. The capture of a time series isalways integral to such measurements.



13.6 Spectral series

Section 2.4 described how the Leica SP� de-tector is capable of selecting emission bandsover a continuously variable range. Incre-mental shifts of the emission band can alsobe used as the basis for an image series.The Leica SP� detector was thus the first in-strument with which a spectral image seriescould be captured using a confocal micro-scope. Experience has shown that its tech-nology is the most efficient; all other spectralmicroscopes that have arrived on the marketsince its introduction have significant wea-knesses with regard to their signal-to-noiseratio.

13.6.1 Data acquisition and utilization

The capture of a Lambda series does not dif-fer significantly from that of a z series or atime series. The emission band for the begin-ning and the end of the measurement mustbe specified, as well as the number of stepsfor the spectrometer to cover the specifiedrange.Sections of the image are then chosen inter-actively for evaluation. Their average inten-sity is then graphed as a function of the wa-velength, a spectrum at the selected point.

13.6.2 About spectral resolution

The debate has developed recently in con-junction with spectral series related to thetechnology that offers the best spectral reso-lution, i.e. technology capable of detectingthe finest differences in the spectrum. TheTCS SP5 supports the adjustment of emis-sion bands in 1nm steps,which correspondsto a formal resolution of one nanometer. Theoptical spectral resolution is dependent on



the wavelength, however, and amounts toroughly 0.5nm in the blue and 2nm in the redrange. This resolution is far better than requi-red in practice: in typical specimens that arein a liquid or gel state at room temperature,fluorescent emissions are never sharper thanroughly 20nm.

13.7 Combinatorial analysis

Many of the methods described above canbe combined and deliver new insights in bio-logy, with both fixed and living specimens.The term ”multidimensional microscopy” hasbeen coined to describe this form of combi-natorial analysis. However, a certain inflationin this regard has become apparent recently.Stitching together a large number of dimen-sions (measuring parameters) does not initself make a good experiment, and it is defi-nitely not conducive to sound results. Thesynthesis of a broad range of measurementsis often difficult and always requires a solidintellectual overview to avoid the creation ofdata graveyards and incorrect conclusions.

Switching off the System


14 Switching off the system

1 Save your image dataOn the menu bar, selectFile->Saveto save the data record.2 Close the LAS AFOn the menu bar, selectFile->ExitExit the LAS AF.

3 Switch off the lasers with the key switch(Fig. 67/2).

The emission warning indicator (Fig. 67/1)will go out.4 Next, turn off the switches on the con-

trol panel for the TCS workstation andthe TCS SP5 (Fig. 67/4/5) scanner.

5 The external fan of the argon laser willswitch off automatically after several mi-nutes. Also set the switch for the lasers(Fig. 67/3) to ”O” at this point.

figure70. Control panel

The delayed shutdown of the laser coo-ling system ensures the operational relia-bility of the TCS SP5 system.

Switching off the System


6 Shut down the computerOn the toolbar, selectStart->Shutdownto shut down the TCS Workstation.

figure71. Shutting down the computer

7 Switch off the microscope and any acti-vated fluorescence lamps

If your system features external lasers(IR, UV or others), switch them off in ac-cordance with their respective manuals.

Contact


15 ContactIf you have any further questions related toyour TCS SP5, please contact the Leicabranch office in your country.Please refer to the country list below for con-tact information.If your country is not listed below, please usethe area selector at http://www.confocal-mi-croscopy.com.

Country City Phone Fax

Australia Gladesville +61 2 9879 9700 +61 2 9817 8358

Austria Vienna +43 1 486 80 50 0 +43 1 486 80 50 30

Canada Richmond HillOntario

+1 905 762 2000 +1 905 762 8937

Denmark Herlev +45 4454 0101 +45 4454 0111

France Rueil-Malmaison +33 1 473 285 85 +33 1 473 285 86

Germany Bensheim +49 6251 136 0 +49 6251 136 155

Italy Milan +39 0257 4861 +39 0257 40 3273

Japan Tokyo + 81 3 5421 2800 +81 3 5421 2896

Korea Seoul +82 2 514 65 43 +82 2 514 65 48

Netherlands Rijswijk +31 70 4132 100 +31 70 4132 109

PRC Hong Kong +852 2564 6699 +852 2564 4163

Portugal Lisbon +351 21 388 9112 +351 21 385 4668

Singapore +65 6779 7823 +65 6773 0628

Spain Barcelona +34 93 494 95 30 +34 93 494 95 32

Sweden Sollentuna +46 8 625 45 45 +46 8 625 45 10

Switzerland Glattbrugg +41 1 809 34 34 +41 1 809 34 44United Kingdom Milton Keynes +44 1908 246 246 +44 1908 609 992

USA Bannockburn/lllinois

+1 847 405 0123 +1 847 405 0164

Contact


Declaration of Conformity


16 Declaration of conformity

Declaration of Conformity


Glossary


17 Glossary

AchromaticDescribes a correction class for objectives.The chromatic aberration for two wave-lengths is corrected for objectives of thistype. Usually an objective of this type is cor-rected to a wavelength below 500 nm andabove 600 nm. Furthermore, the sine condi-tion for one wavelength is met. The imagecurvature aberration is not corrected.Airy discThe Airy disc refers to the inner, light circle(surrounded by alternating dark and light dif-fraction rings) of the diffraction pattern of apoint light source. The diffraction discs of twoadjacent object points overlap some or com-pletely, thus limiting the spatial resolution ca-pacity.AliasingAn image distortion caused by a samplingfrequency that is too low in relation to the si-gnal frequency.AOTFThe acousto-optical tunable filter is an optictransparent crystal that can be used to infini-tely vary the intensity and wavelength of ra-diated light. The crystal generates an internalultrasonic wave field, the wavelength ofwhich can be configured to any value. Radia-ted light is diffracted vertically to the ultraso-nic wave field like through a grid.ApochromaticDescribes a correction class for objectives.For objectives of this type, the chromatic ab-erration is corrected for three wavelengths(usually 450 nm, 550 nm and 650 nm) andthe sine condition is met for at least two co-lors. The image curvature aberration is notcorrected.

Glossary


Working distanceThe distance from the front lens of an objec-tive to the focal point. For a variable workingdistance, the gap between the front lens ofthe objective and the cover slip or uncoveredsample is specified. Usually objectives withlarge working distances have low numericalapertures, while high-aperture objectiveshave small working distances. If a high-aper-ture objective with a large working distanceis desired, the diameter of the objective lenshas to be made correspondingly large.These, however, are usually low-correctionoptic systems, because maintaining extremeprocess accuracy through a large lens dia-meter can only be achieved with great effort.Instrument parameter settingAn instrument parameter setting (IPS) con-sists of a file, in which all hardware settingsare stored which are specific to a certain re-cording method. The designation «FITC-TRITC», for example, refers to the settingsfor a two-channel recording with the two fluo-rescent dyes FITC and TRITC. An instru-ment parameter setting enables the user tostore optimum hardware settings in a file andto load them again with a simple double-click. Instrument parameter settings labeledwith the letter «L» are predefined by Leicaand cannot be changed. User-defined, modi-fiable instrument parameter settings are sto-red below «U» in the list box.Image curvature aberrationThe curved surface to which a microscopicimage is to be clearly and distinctly mappedis described as image curvature aberration. Itis conditional on the convex shape of thelens and makes itself apparent as an errordue to the short focal distances of micro-scope objectives. Here, the object image isnot in focus both in the center and at the pe-riphery at the same time. Objectives that arecorrected for image curvature aberration arecalled plane objectives (plane = flat imagefield).

Glossary


Refraction indexThe factor by which the light velocity in anoptical medium is less than in a vacuum.Chromatic aberrationAn optical image distortion conditional on thevarying refraction of light rays of different wa-velengths on a lens. Thus light rays of shor-ter wavelengths have longer focal distancesthan light rays of longer wavelengths.DichroicDichroic filters are interference filters at anangle of incidence of light of 45°. The trans-missivity or reflectivity of dichroites dependson a specific wavelength of light. For exam-ple, with a short-pass filter RSP 510 (reflec-tion short pass), excitation light below 510nm is reflected and above this value it istransmitted. The transmission values are ge-nerally between 80% and 90% and the re-flection values between 90% and 95%.Digital phase-true filterA digital filter consists of a computing ruleused to modify image data. Filters are al-ways applied to remove disturbing imagecomponents. A phase-true filter ensures thatquantifiable image values do not changethrough filtering and remain a requirementfor standardized measuring methods (e.g.,characterization of surfaces in accordancewith ISO).Double dichroiteDouble dichroic filters are interference filtersat an angle of incidence of light of 45q. Thetransmissivity or reflectivity of double dichroi-tes depends on two specific wavelengths oflight. With a double dichroite DD 488/568, forexample, the excitation light at 488 nm and568 nm is reflected and above these valuesit is transmitted. The transmission values aregenerally at 80% and the reflection valuesare between 90% and 95%.Experiment

Glossary


A file with Leica-specific data format (*.lei)that consists of one ore more individualimages or image series. Images recordedwith different scan parameters or resultimages from image processing can be com-bined here.Fluorescent dyeA dye used for analysis that reacts with theemission of light of other wavelengths uponexcitation with light energy (Stokes shift). e.g.Fluorescein, Rhodamin, Eosin, DPA.Fluorescence microscopyA light-optical contrast process for displayingfluorescent structures. Auto-fluorescent sam-ples have a so-called primary fluorescence.They do not need to be enriched with additio-nal, fluorescent substances. Secondary fluo-rescent substances, on the other hand, haveto be treated with appropriate dyes or stainscalled fluorochromes. Specific dyeing me-thods additionally allow the precise localiza-tion of the stained structure elements of anobject. Fluorescence microscopy allows bothpotential morphological examinations andthe ability to carry out dynamic examinationson a molecular level.Fluorite objectivesDescribes a correction class for objectives.Fluorite objectives are semi-apochromatic,i.e.objectives whose degree of correctionfalls between achromatic and apochromatic.FrameA frame corresponds to the acquisition of asingle optical section. For example, if a sin-gle optical section is acquired four times (toaverage the data and to eliminate noise),then frames are created for this optical sec-tion.Immersion objectiveA microscopic objective, developed with therequirements for applying immersion media.The use of incorrect or no immersion me-dium with an immersion objective can lead to

Glossary


resolution loss and impairment of the correc-tion.IR-LaserLaser with a wavelength > 700 nm, invisiblelight. (infrared).Confocal subprocedureMethods for examining microstructures thatare derived from the classical contrast me-thods (bright field, interference contrast,phase contrast, polarization) in conjunctionwith a confocal system. These procedureseach define a certain configuration of opticalelements (filter cubes, ICT prisms, phaserings). In addition, some of them are depen-dent upon the selected objective.ConfocalityWhile the optical design of conventional mi-croscopes allows the uniform detection offocussed and unfocussed image compo-nents, the confocal principle suppresses thestructures found outside of the focal plane ofthe microscope objective. Screens are imple-mented in optically conjugated locations toachieve this. They function as point lightsource (excitation screen) and point detector(detection screen). The optical resolution dia-meter of the detection pinhole, the wave-length and the numerical aperture of the se-lected objective determine the axial range ofan optical section (optical resolution).Reflection short pass filterReflection short pass filters are interferencefilters that transmit short-wave light while re-flecting long-wave light. An optical reflectionshort pass filter is characterized by the rea-ding of the wavelength edge at which the fil-ter changes from transmission to reflection(50% threshold).Lambda seriesStack of individual images of a single opticalplane that were each detected at a specificwavelength.Reflection long pass filter

Glossary


Reflection long pass filters are interferencefilters that reflect short-wave light but aretransparent for long-wave light. An opticalreflection long pass filter is characterized bythe reading of the wavelength edge at whichthe filter changes from reflection to transmis-sion (50% threshold).Empty magnificationA magnification without additional gain of in-formation. Empty magnification is used assoon as distances are displayed that aresmaller than the optical resolution. Magni-fications with a larger scale than that of theempty magnification do not provide any addi-tional information about the object but, in-stead, only diminish the focus and the con-trast.MP laserMulti-photon, the designation for infrared la-sers with a high photon density (generatedby pulsed lasers).Neutral filterNeutral filters are semi-reflective glass pla-tes. They are used to distribute the light pathindependent of wavelength. The incominglight is partially reflected and partially trans-mitted. Neutral filters are usually placed at a45 angle in the path of the beam. The ratingsof a neutral filter are based on its reflectivity-to-transmissivity ratio. For example, for aneutral filter RT 30/70, 30% of the excitationlight is reflected and 70% transmitted.Numerical ApertureAperture is the sine of the aperture angle un-der which light enters the front lens of a mi-croscope objective; Symbol NA. The aper-ture influences both the light intensity andthe resolution capacity of an objective opticalsystem. Since different media can be locatedbetween specimen and objective (e.g. theembedding medium of the specimen), thenumerical aperture (NA = n * sina) is gene-rally used as the unit of measure for the lumi-nous intensity and the resolution capacity.

Glossary


Optical bleachingThe destruction of fluorochromes, by intenselighting. In fluorescence microscopy, fluo-rochromes are excited with laser light to ahigh state of energy, the singlet state. Whenthe excited molecules return to their normalenergy state, a fluorescent signal is emitted.If the intensity of the excitation is too high ho-wever, the color molecules can change viaintercrossing from a singlet state to a tripletstate. Due to the significantly longer life oftriplet states (phosphorescence), these exci-ted molecules can react with triplet-oxide andbe lost for further fluorescence excitation.Phase visualizationThe principle of phase visualization as usedby Leica is an optimized alternative methodto ratiometric displaying. The main area ofapplication is the measurement of ion con-centrations in physiology. In contrast with ra-tiometric procedures, phase visualization ob-tains more information on the specimen. Inaddition, this method allows for adapting thedisplay of physiological data to the dynamicsof the human eye. For detailed informationon phase visualization, please contact LeicaMicrosystems CMS GmbH directly.PixelAn acronym based on the words, picture andelement. A pixel represents the smallest, in-divisible image element in a two-dimensionalsystem. In this documentation, both the sam-pling points of the specimen as well as theimage points are referred to as pixels.Plane objectivesDescribes a correction class for objectives.The image curvature aberration is correctedfor objectives of this type. Correcting this er-ror requires lenses with stronger concavesurfaces and thicker middles. Three types ofplane objectives, plane achromate, planeapochromate and plane fluorite, are basedon the type of additional correction for chro-matic aberration.ROI

Glossary


Abbreviation for ”Region of Interest”. ROIencloses an area for which a measurementanalysis is to be performed. On top of that,an ROI can also designate the area of a spe-cimen to be scanned (ROI scan).Signal-to-noise ratioThe ratio of signals detected in the specimento the unwanted signals that are caused ran-domly by various optic and electronic compo-nents, which are also recorded by the detec-tor.Spherical aberrationAn optical image distortion conditional on thevarying spacing of parallel light rays of thesame wavelength from the optical axis. Lightrays that travel through outer lens zoneshave shorter focal distances than rays thattravel through the lens center (optic axis).Stokes shiftThe Stokes shift is a central term in fluore-scence microscopy. If fluorescent moleculesare excited with light of a specific wave-length, they radiate light of another, largerwavelength. This difference between excita-tion light and fluorescent light is referred toas Stokes shift. Without Stokes shift, separa-ting the high-intensity excitation light from thelow-intensity fluorescence signals in a fluore-scence microscope would not be possible.Triple dichroicTriple dichroic filters are interference filters atan angle of incidence of light of 455t Thetransmissivity or reflectivity of triple dichroitesdepends on three specific wavelengths oflight. With a triple dichroite TD 488/568/647,for example, the excitation light at 488 nm,568 nm and 633 nm is reflected and abovethese values it is transmitted. The transmis-sion values are generally at 80% and the re-flection values are between 90% and 95%.Dry objectiveA microscopic objective used without immer-sion media. Between the objective lens andthe specimen is air.

Glossary


UV-LaserLaser with a wavelength < 400 nm, invisiblelight.VIS-LaserLasers of the wavelength range400 - 700 nm, visible light.VoxelAn acronym based on the words, volumeand pixel. A voxel represents the smallest,indivisible volume element in a three-dimen-sional system. In this documentation, boththe volume elements of the specimen as wellas the 3D image points are referred to as vo-xels.z-stackZ-stacks are comprised of two-dimensionalimages that were recorded on different focalplanes and displayed as three-dimensional.

Glossary


Safety Data Sheets


18 Safety data sheets

The following are safety data sheets fromthird-party manufacturers.

Safety Data Sheets


Leica Microsystems CMS GmbH Tel.: +49 (0)621 7028 - 0 Am Friedensplatz 3 Fax: +49 (0)621 7028 - 1028 D-68165 Mannheim (Germany) http://www.leica-microsystems.com Copyright © Leica Microsystems CMS GmbH x All rights reserved

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