Leica TCS SP8 SMD User manual

10
Living up to Life
User Manual
Leica TCS SP8 SMD
for FCS, FLIM and FLCS

Published by:
Leica Microsystems CMS GmbH
Am Friedensplatz 3
D-68165 Mannheim (Germany)
http://www.leica-microsystems.com
http://www.confocal-microscopy.com
Responsible for contents:
Leica Microsystems CMS GmbH
Copyright © Leica Microsystems CMS GmbH.
All rights reserved.
This manual was created in cooperation with
PicoQuant GmbH:
PicoQuant GmbH
Rudower Chaussee 29
12489 Berlin (Germany)
http://www.picoquant.com

3
Copyright
Copyright
All rights to this document are held by Leica Microsystems CMS GmbH. Adaptation,
translation and reproduction of text or illustrations (in whole or in part) by print, photocopy,
microfilm or other method (including electronic systems) is not allowed without express
written permission from Leica Microsystems CMS GmbH.
Programs such as LAS and LAS AF are protected by copyright laws. All rights reserved.
Reproduction, adaptation or translation of these programs is prohibited without prior written
permission from Leica Microsystems CMS GmbH.
This User Manual specifies names of products or services that are trademarks or registered
trademarks of the respective trademark owners. Rather than including a trademark (TM or
®) symbol at every occurrence of a trademarked name, we state that we are using the
names only in an editorial fashion, and to the benefit of the trademark owner, with no
intention of infringement.
Made in Germany.
© Copyright Leica Microsystems CMS GmbH.
All rights reserved.

4
Copyright

5
Contents
Contents
Copyright. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
1 About this User Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .15
1.1 Additional Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .16
2 Intended Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .17
3 Liability and Warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
3.1 Important Information for Operators and Users. . . . . . . . . . . . . . . . . . . . . . . . . . . . .19
4 Meaning of the warning messages in the manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21
5 General Safety Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
5.1 Commissioning and Use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .23
5.2 Modifications to the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
5.3 Safety Devices and Safety Labels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
5.4 Laser Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .24
5.5 Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
5.6 Contact with Liquids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
5.7 Malfunction of the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .26
6 Additional Notes on Handling the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
6.1 Location . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
6.2 Using the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29
6.3 Protecting the System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
6.3.1 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
7 System Overview and Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
7.1 TCS SP8 SMD System Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
7.1.1 TCS SP8 SMD System Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .33
7.1.2 TCS SP8 SMD with Upright Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
7.1.3 TCS SP8 SMD with Inverted Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .34
7.2 TCS SP8 SMD System Variants with White Light Laser . . . . . . . . . . . . . . . . . . . . . .35
7.2.1 TCS SP8 SMD System Components with White Light Laser. . . . . . . . . . . . . . . .35
7.2.2 TCS SP8 SMD with White Light Laser and Upright Microscope . . . . . . . . . . . .36
7.2.3 TCS SP8 SMD with White Light Laser and Inverted Microscope . . . . . . . . . . .37
7.3 TCS SP8 SMD System Variants with MP Configuration. . . . . . . . . . . . . . . . . . . . . .38
7.4 Controls on the Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40

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Contents
7.4.1 Main Switch Board on the Flexible Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . .40
7.4.2 Control Panel Field on the Compact Supply Unit (Only for FLIM). . . . . . . . . . . .40
7.5 Technical Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
7.5.1 Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
7.5.2 Weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41
7.5.3 Electrical Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
7.6 "Electromagnetic Compatibility". . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .42
7.7 Serial Number. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .43
8 Ambient Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
8.1 General Requirements Regarding Ambient Conditions. . . . . . . . . . . . . . . . . . . . . . .45
8.2 Vibrations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
8.3 Room Dimensions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
8.4 Electrical Connection Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
8.4.1 System with Flexible Supply Unit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .47
8.4.2 System with Compact Supply Unit (Only with FLIM) . . . . . . . . . . . . . . . . . . . . . .48
8.4.3 External Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .48
8.5 Load capacity of the multiple socket outlet on the flexible supply unit . . . . . . . . .49
8.6 Waste Heat and Cooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
8.6.1 System with Flexible Supply Unit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
8.6.2 System with Compact Supply Unit (Only with FLIM) . . . . . . . . . . . . . . . . . . . . . .50
8.6.3 External Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50
9 SMD Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
9.1 Hardware Components and Software Licenses. . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
9.1.1 FCS/FCCS Application Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
9.1.1.1 Special SMD Hardware. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
9.1.1.2 Software License . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .51
9.1.2 FLIM Application Area. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
9.1.2.1 Special SMD Hardware. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
9.1.2.2 Software License . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52
9.1.3 Application area FCS/FCCS, FLIM, FLCS, gated FCS . . . . . . . . . . . . . . . . . . . . . .53
9.1.3.1 Special SMD Hardware. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
9.1.3.2 Software License . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
9.2 Beam Path. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
9.3 Detection Units from PicoQuant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .54
9.4 Leica APD Detector Unit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
9.4.1 General Precautionary Measures for Using APD Detector Units . . . . . . . . . . .56
9.4.2 Changing the Fuse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
9.4.3 Safety Shutoff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56

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Contents
9.5 Trigger Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .56
9.6 Laser Coupling Unit (LCU). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57
9.6.1 Attenuation Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .58
9.6.2 Other PicoQuant Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .59
10 Laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
10.1 Laser Classes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
10.2 Overview of Usable Lasers for Image Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . .61
10.2.1 VIS/UV Lasers for TCS SP8. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .61
10.2.2 VIS/UV Lasers for TCS SP8 X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62
10.2.3 IR lasers for TCS SP8 MP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62
10.2.3.1 Picosecond laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .62
10.2.3.2 Femtosecond laser. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
10.3 Overview of Usable Lasers for FCS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
10.3.1 VIS/UV Lasers for TCS SP8 SMD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .63
10.4 Overview of usable lasers for FLIM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
10.4.1 VIS/UV Lasers for TCS SP8 SMD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
10.4.2 Infrared Lasers for TCS SP8 SMD with MP Configuration . . . . . . . . . . . . . . . . .64
10.4.2.1 Picosecond laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
10.4.2.2 Femtosecond laser. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .64
10.5 Overview of Usable Lasers for FLCS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66
10.5.1 VIS/UV Lasers for TCS SP8 SMD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .66
11 Safety Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67
11.1 Main Circuit Breaker for Disconnecting the Power Supply . . . . . . . . . . . . . . . . . . .67
11.1.1 Compact Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67
11.1.2 Flexible Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .67
11.1.3 Other Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68
11.2 Key Switch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68
11.2.1 Master Key Switch on the Compact Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . .68
11.2.2 Master Key Switch on the Flexible Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . .68
11.2.3 Key Switch for the White Light Laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69
11.2.4 Key Switch for UV Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69
11.2.5 Key Switches for Other External Lasers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70
11.3 Emission Warning Indicators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .70
11.3.1 Emission Warning Indicator on the Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . .70
11.3.2 Emission Warning Indicator on the White Light Laser . . . . . . . . . . . . . . . . . . . .71
11.3.3 Emission Warning Indicator on UV Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .71
11.3.4 Emission Warning Indicator on Other External Lasers . . . . . . . . . . . . . . . . . . . .72
11.3.5 Malfunction of Emission Warning Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72

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Contents
11.4 Interlock Connectors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72
11.4.1 Interlock Connector on the Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .72
11.4.2 Interlock Connector on the White Light Laser . . . . . . . . . . . . . . . . . . . . . . . . . . .74
11.4.3 Interlock Connector on Other External Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . .74
11.4.4 Interlock Connector on the Scan Head . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75
11.5 Safety Switches on the Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
11.6 Warning Messages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .76
11.7 Special Laser Safety Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .78
11.7.1 Laser Protection Tube and Laser Protection Shield . . . . . . . . . . . . . . . . . . . . . .78
11.7.2 Safety Beam Guide on the MP System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .79
12 Safety Labels on the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81
12.1 Compact Supply Unit (Only for FLIM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .81
12.2 Flexible Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .82
12.3 Inverted Microscope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .83
12.4 Upright Microscope. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .84
12.5 Mirror Housing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
12.6 Cover for Replacement Flange . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .86
12.7 Transmitted Light Detector (TLD)/Reflected Light Detector (RLD) . . . . . . . . . . . . .87
12.8 Scan Head. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .87
12.9 White Light Laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .89
12.10 External UV Laser. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .89
12.11 MP Beam Coupling Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90
12.12 Other External Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .90
13 Switching On the System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91
13.1 Confocal System with Flexible Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .91
13.2 Confocal System with Compact Supply Unit (Only with FLIM). . . . . . . . . . . . . . . . .96
13.3 HyD Reflected Light Detectors (HyD RLDs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .99
13.4 Starting the SMD Hardware and Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .100
14 LAS AF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103
14.1 Starting LAS AF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103
14.2 Structure of the Graphical User Interface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .104
14.3 Design of the FLIM Wizard in LAS AF. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .107
14.4 Design of the FCS Wizard in LAS AF. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .110
14.5 LAS AF Online Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .113
14.5.1 Structure of Online Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .113
14.5.2 Accessing Online Help . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .113

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14.5.3 Selecting the Language for Online Help. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .113
14.5.4 Using Online Help. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .114
14.5.5 Full-text Search with Logically Connected Search Terms . . . . . . . . . . . . . . . .115
15 Selecting the Laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .117
15.1 Activate laser as the excitation source in the configuration menu . . . . . . . . . . .117
15.1.1 Using Continuous Wave VIS Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .118
15.1.2 Using MP lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .119
15.1.3 Using Pulsed VIS Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .121
15.1.4 Using Pulsed UV Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .124
15.1.5 Using a Pulsed White Light Laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .124
16 FLIM Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .127
16.1 Setup Imaging Step – Image Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .127
16.1.1 Selecting Detectors for the Image Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . .127
16.1.1.1 Internal Photomultipliers (Including SP FLIM PMT) . . . . . . . . . . . . . . . . . . .127
16.1.1.2 External APDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .129
16.1.1.3 External FLIM Photomultiplier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130
16.1.2 Selecting Laser Lines as an Excitation Source for Image Acquisition . . . . . .130
16.1.2.1 Using Continuous Wave Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .130
16.1.2.2 Using MP Lasers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .131
16.1.2.3 Using Pulsed VIS Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .131
16.1.2.4 Using Pulsed UV Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .132
16.1.2.5 Using White Light Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .133
16.1.3 Adjusting the Pinhole for Image Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . .133
16.2 Setup FLIM Step – Optimizing the FLIM Measurement Conditions. . . . . . . . . . . .134
16.2.1 Selecting FLIM Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .134
16.2.1.1 FLIM Data Acquisition with Internal SP FLIM Detectors. . . . . . . . . . . . . . .135
16.2.1.2 FLIM Data Acquisition with External MPD APDs . . . . . . . . . . . . . . . . . . . . .136
16.2.1.3 FLIM Data Acquisition with External Photomultiplier. . . . . . . . . . . . . . . . . .137
16.2.1.4 FLIM Data Acquisition with Detectors at NDD Position (HyD RLD). . . . . .138
16.2.2 Selecting Laser Lines for FLIM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .140
16.2.2.1 Do Not Use Continuous Wave VIS Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . .140
16.2.2.2 Using Pulsed Diode Lasers (UV, VIS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .141
16.2.2.3 Using MP Lasers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .141
16.2.2.4 Using White Light Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .142
16.2.3 Adjusting the Fluorifier Disc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .142
16.2.3.1 Setting for SP FLIM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .142
16.2.3.2 Setting for External FLIM or Intensity Image Acquisition . . . . . . . . . . . . . .144
16.2.3.3 Setting for FLIM White Light Laser . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .144

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16.2.4 Changing the Pulse Frequency for Pulsed Diode Lasers
(405, 440, 470, 640 nm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .145
16.2.5 2-Laser PIE (405, 470, 640 nm) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .146
16.2.6 Changing Pulse Frequency for White Light Lasers . . . . . . . . . . . . . . . . . . . . . .147
16.2.7 Setting the Pinhole. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .148
16.2.8 Optimizing FLIM Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .148
16.2.9 Count Rate Monitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .149
16.2.10 Loading and Saving FLIM-specific Instrument Parameter Settings . . . . . . . .149
16.3 Measurements Step – Time Series for FLIM Measurement
at Multiple Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150
16.3.1 FLIM Network Connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150
16.3.2 Definition of the FLIM Measurement File Names Transferred to
SymPhoTime . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .150
16.3.3 Defining a Single FLIM Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .152
16.3.4 Defining an xyz or xzy FLIM Stack. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .153
16.3.5 Defining an FLIM Time Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .154
16.3.6 Defining a Time Series of xyz or xzy FLIM Stacks . . . . . . . . . . . . . . . . . . . . . . .155
16.3.7 Defining an xy or xz FLIM Stack. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .157
16.3.8 Defining a Time Series of xy or xz FLIM Stacks . . . . . . . . . . . . . . . . . . . . . . . . .158
16.3.9 Defining an xy or xz FLIM Stack. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .160
16.3.10 Control of FLIM Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .162
17 Summarized Manual for FLIM Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163
17.1 Prerequisites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163
17.2 Selecting Position for the FLIM Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . .163
17.3 Changing from Continuous to Pulsed Excitation. . . . . . . . . . . . . . . . . . . . . . . . . . . .163
17.4 Changing from Internal Detection on the SP8 to External TCSPC Detectors . . .164
17.5 Using Internal SP FLIM Detection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .164
17.6 Using FLIM Detectors at the NDD Position (HyD RLD) . . . . . . . . . . . . . . . . . . . . . .164
17.7 Setting Suitable Scan Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .165
17.8 Optimizing the Photon Count Rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .165
17.9 Selecting the Correct Laser Repetition Rate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .168
17.10 Starting FLIM Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .170
17.11 Resulting Raw Data File and Documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .170
17.12 Measuring the Instrument Response Function (IRF) . . . . . . . . . . . . . . . . . . . . . . . .172
17.12.1 Preparing IRF Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .172
17.12.1.1 Estimating the IRF. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .172
17.12.1.2 With Reflection Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .172
17.12.1.3 With Fluorescence Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .173
17.12.1.4 With SHG (Second Harmonic Generation – Possible for
MP Lasers Only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .173

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17.12.2 Running IRF Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .174
17.13 Remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .176
17.13.1 Ad-hoc-Inspection of a Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .176
17.13.2 Bidirectional Scanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .176
17.13.3 Setting the Laser Intensity of the Diode Lasers . . . . . . . . . . . . . . . . . . . . . . . . .176
17.13.4 Sensitivity of the Fluorescence Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .176
17.13.5 Optimum Lifetime Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .176
18 F(L)CS Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177
18.1 Preparing the FCS Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177
18.1.1 Selecting an Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .177
18.1.2 Calibrating the Positioning Accuracy of the FCS Measuring Point. . . . . . . . .177
18.1.3 Testing the Positioning Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .181
18.1.4 Adjusting the Correction Ring on the Objective . . . . . . . . . . . . . . . . . . . . . . . . .183
18.1.5 Setting the Reference Position . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .186
18.1.6 Acquiring a Reference Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .186
18.1.6.1 Image Acquisition Using Photomultipliers. . . . . . . . . . . . . . . . . . . . . . . . . . .187
18.1.6.2 Image Acquisition with PE APDs (AQR Type) or MPD APDs
(PDM Type) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .188
18.2 Setup Imaging Step – Image Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .190
18.2.1 Selecting Detectors for the Image Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . .190
18.2.1.1 Photomultiplier (PMT) / Internal Hybrid Detector (HyD) . . . . . . . . . . . . . . .190
18.2.1.2 PE / MPD APDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .190
18.2.2 Selecting Laser Lines as an Excitation Source for the Image Acquisition. . .191
18.2.2.1 Using Continuous Wave Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .191
18.2.2.2 Using MP Lasers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .192
18.2.2.3 Using Pulsed VIS Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .192
18.2.2.4 Using a Pulsed UV Laser. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .193
18.2.2.5 Using White Light Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .194
18.2.3 Adjusting the Pinhole for Image Acquisition. . . . . . . . . . . . . . . . . . . . . . . . . . . .194
18.3 Setup FCS Step – Optimizing FCS Measurement Conditions . . . . . . . . . . . . . . . . .195
18.3.1 Selecting APDs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .195
18.3.2 Selecting Laser Lines for FCS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .195
18.3.2.1 Using Continuous Wave VIS Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .195
18.3.2.2 Using Pulsed VIS Diode Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .196
18.3.2.3 Using White Light Lasers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197
18.3.3 Fluorifier Disc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197
18.3.4 Setting the Pinhole. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .197
18.3.5 Optimizing FCS Settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .198
18.3.6 Count Rate Monitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .199
18.3.7 Loading and Saving FCS-specific Instrument Parameter Settings . . . . . . . . .200

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18.4 Measurements Step – FCS Measurement Time Series at Multiple Points . . . . .201
18.4.1 FCS Network Connection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .201
18.4.2 Definition of Multiple FCS Measuring Points in an Image or Stack. . . . . . . . .202
18.4.3 FCS Time Series at Multiple Measuring Points. . . . . . . . . . . . . . . . . . . . . . . . . .204
18.4.4 Definition of the File Names Transferred to SymPhoTime during the
FCS Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .206
18.4.5 FCS z-Stack . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .207
18.4.6 Operating the FCS Measurement Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .208
19 Summarized manual for FCS or other point measurements . . . . . . . . . . . . . . . . . . . . . . .209
19.1 Prerequisites. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .209
19.1.1 Choosing the Location for the FCS Measurement . . . . . . . . . . . . . . . . . . . . . . .209
19.2 Starting Point/ FCS Data Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .210
20 Changing the Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .213
20.1 Changing the Specimen on an Upright Microscope . . . . . . . . . . . . . . . . . . . . . . . .213
20.2 Changing the Specimen on an Inverted Microscope . . . . . . . . . . . . . . . . . . . . . . .213
21 Changing the Objective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .215
22 Piezo Focus on Upright Microscope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .217
23 Changing the Filter Cube . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .219
24 Changing Detector Cable Connections on the Scan Head and Router When
Using HyD-RLD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .221
24.1 Hardware Trees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .222
24.1.1 MP on FCS FLIM 2 APD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .223
24.1.2 MP off FCS FLIM 2 APD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .223
24.1.3 MP on HyD RLD FLIM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .223
24.2 Connect and Use Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .224
24.2.1 Using SP FLIM PMT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .224
24.2.2 Using MPD APDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .224
24.2.3 Using HyD-RLD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .225
25 Switching Off the System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .229
25.1 System with Flexible Supply Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .229
25.2 System with Compact Supply Unit (Only for FLIM). . . . . . . . . . . . . . . . . . . . . . . . . .233
26 Care and Cleaning. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .239
26.1 Cleaning Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .239
26.2 Cleaning the Optical System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .239
26.3 Cleaning Immersion Lenses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .240

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Contents
26.4 Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .240
27 Repairs and Service Work. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .241
28 Maintenance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .243
28.1 Having Coolant Replaced . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .243
29 Disassembly and Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .245
30 Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .247
31 Troubleshooting. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .249
31.1 Hardware Configuration Gets Lost or Software Needs to be Installed Again. . .249
31.2 The Instrument Is Losing Sensitivity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .249
31.2.1 Causes for Decreased Performance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .250
31.3 No FLIM Image is Displayed During Measurement . . . . . . . . . . . . . . . . . . . . . . . . .250
31.4 How to Handle PQ Error Codes in LAS AF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .252
32 Contact . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .253
33 Recommended literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .255
34 Abbreviations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .257
35 Appendix. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .259
35.1 Patents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .259
35.2 Safety Data Sheets from Third-Party Manufacturers . . . . . . . . . . . . . . . . . . . . . . .259
35.3 Compliance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .265
35.4 People's Republic of China. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .267

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Contents

15
About this User Manual
1 About this User Manual
Prior to commissioning the system, carefully read through this User Manual and be
absolutely certain to follow the safety notes contained in it. So that you can operate the
system safely and react quickly and correctly in the event of an emergency, you must
familiarize yourself with the safety devices before using it for the first time. In this case, read
Chapter "Safety Features" in this manual. Keep this User Manual and the included manuals
for the microscope and other components in a safe place easily accessible for all users.
This Manual gives you important information about safe handling of the system. All
information is intended for the safety of users and trouble-free operation of the system.
Unless the information pertains specifically to certain system variants, the instructions
always apply to the basic system described here.
This User Manual provides you with important information for using the system, the
necessary ambient conditions and the usable lasers. It explains system startup. The system
is assembled and disassembled by service technicians that have been authorized by Leica
Microsystems CMS GmbH. This is why unpacking, assembly and installation of the system
are not described in this manual. You can find an overview of the system and specifications
in the Chapter "System Overview and Properties". For information about special
configurations, such as optional lasers or specific objectives, refer to the respective
included manual. In Chapter 34 you will find a list of abbreviations used in this manual.
This operating manual was created by PicoQuant GmbH and Leica Microsystems CMS
GmbH and is concentrated on specialized knowledge about SMD (Single Molecule
Detection). The basic procedures for acquiring FLIM images and point measurements for
FCS using the TCS SMD system are described here.
This User Manual does not contain any information about basic optical principles or the
operating principle of microscopes, confocal systems and the like. If you are interested in
these topics or certain applications from the area of optics and confocal microscopy, you
can read more about them at the Leica Microsystems CMS GmbH knowledge portal: http://
www.leica-microsystems.com/science-lab/
The system is delivered with the latest version of the licensed "Leica Application Suite
Advanced Fluorescence" (LAS AF) software. Within the Leica LAS AF software, FCS and
FLIM experiments are designed and executed using special wizards. After data acquisition,
the SMD data analysis is carried out within the "SymPhoTime" (SPT) software by PicoQuant.
Read Chapter "LAS AF" in this User Manual in order to familiarize yourself with the design
and basic operation of the software. Additional information about specific functions can be
found in the online help.
The instructions contained in this documentation reflect state-of-the-art technology and
knowledge standards at the time of publication. Leica Microsystems CMS GmbH reserves
the right to revise this documentation and/or to further develop and improve the products
described in this document at any time without prior notice or any other obligation.
If you have any suggestions or improvements for this User Manual, please contact the Leica
branch office in your country.

16
About this User Manual
1.1 Additional Documentation
The system is delivered with additional manuals. These manuals contain detailed
information about the hardware components and the software-based analysis that
absolutely must be observed. Manuals for the following components are provided with the
system:
•Detection unit: This manual varies depending on the detection system. Here you can find
basic information about alignment of the detection path and how to change filters.
No separate manual is provided for the Leica APD detection unit. This detection unit is
described in this User Manual in Chapter 9.4.
•PicoHarp 300: Provides all information about the TCSPC (Time Correlated Single Photon
Counting) unit. This manual also includes an introduction about single photon counting.
•Light sources: The green folder contains detailed information about the features of your
pulsed diode laser.
•Router (PHR800): The router is required in systems with multiple detectors.
•Laser driver (PDL): The manual for the laser driver varies depending on the laser driver
used and includes information for configuring different intensities.
•Software (SymPhoTime): Contains all information about the software, including data
acquisition and analysis. The SymPhoTime software also contains a detailed help
function, which can be accessed by pressing the F1 button.
Aside from this, there is one more important document that must be observed:
•System specifications: The System Specifications appendix contains information about
your specific FLIM/FCS system, including a description of the included parts, information
about filter handling, and a wiring diagram that enables you to restore the configuration
more easily after disassembly.

17
Intended Use
2 Intended Use
This system is intended for use in a lab. The system was designed for confocal scanning
(laser scanning images) of fluorescence-marked living and fixed specimens as well as for
quantitative measurements in the area of life science.
Applications of in-vitro diagnostics in accordance with MPG (German Medical Devices Act)
are excluded from proper intended use.
This system must not be used together with life-support systems such as those found in
intensive-care wards.
The owner/operator and user of this product are responsible for proper and safe operation
and safe maintenance of the system and for following all applicable safety regulations. The
owner/operator and user are fully liable for all consequences resulting from the use of the
system for any purposes other than those listed in the User Manual or the online help.
The manufacturer assumes no liability for damage caused by, or any risks arising from, use
of the microscopes for purposes other than those for which they are intended, or not using
the microscopes within the specifications of Leica Microsystems CMS GmbH. In such
cases, the Declaration of Conformity shall be invalid.

18
Intended Use

19
Liability and Warranty
3 Liability and Warranty
Leica Microsystems CMS GmbH shall not be liable for damages resulting from failure to
observe the information in this User Manual. The information here does not in any way
modify the warranty and liability clauses contained in the general terms and conditions of
Leica Microsystems CMS GmbH.
Repairs and servicing may be performed only by service technicians authorized by Leica
Microsystems CMS GmbH. Opening or working on the system in any way shall void any and
all warranty claims.
The manufacturer assumes no liability for damage caused by, or any risks arising from, use
of the microscopes for purposes other than those for which they are intended, or not using
the microscopes within the specifications of Leica Microsystems CMS GmbH. In such
cases, the Declaration of Conformity shall be invalid.
Leica Microsystems CMS GmbH shall not be liable for any damage caused by incorrect
storage, improper transport or an unsuitable installation location.
Figures are for illustration purposes. The system you purchased can deviate from the
illustrations without Leica Microsystems CMS GmbH explicitly specifying such.
Leica Microsystems CMS GmbH shall not be liable for any injury or property damage caused
by untrained or unauthorized persons.
3.1 Important Information for Operators and Users
• The owner/operator is required to designate a Laser Safety Officer or a Laser Protection
Advisor according to the applicable legal requirements in each country.
• The owner/operator and user of this product are responsible for proper and safe
operation and safe maintenance of the system and for following all applicable safety
regulations.
• The owner/operator and user are fully liable for all consequences resulting from the use
of the system for any purposes other than those listed in the User Manual or the online
help.
• The owner/operator and user are obligated to perform and monitor suitable safety
measures (according to national regulations).
• The owner/operator and user are responsible for observing the laser safety regulations
according to applicable country-specific regulations.
• The owner/operator and user must ensure that this laser product is commissioned and
operated only by persons who have been trained in the use of the system and the
potential dangers of laser radiation.
• The owner/operator and user are fully liable for all consequences resulting from the use
of the system if it is opened, improperly serviced or repaired by persons other than
authorized Leica service representatives.

20
Liability and Warranty