Leica TCS SP8 SMD User Manual

Living up to Life

User Manual

Leica TCS SP8 SMD

for FCS, FLIM and FLCS

Published by:

This manual was created in cooperation with PicoQuant GmbH:

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

PicoQuant GmbH Rudower Chaussee 29 12489 Berlin (Germany)

http://www.picoquant.com

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.

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

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

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

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

Contents

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

Contents

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

Contents

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

Contents

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

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

Contents

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 configurations, such as optional lasers or specific objectives, refer to the respective included manual. In

Chapter "System Overview and Properties". For information about special

Chapter 34 you will find a list of abbreviations used in this manual.

About this User 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 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.

AF) software. Within the Leica LAS AF software, FCS and

If you have any suggestions or improvements for this User Manual, please contact the Leica branch office in your country.

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

• 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.

Chapter 9.4.

• 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.

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.

Intended Use

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.

Leica Microsystems CMS GmbH shall not be liable for any damage caused by incorrect storage, improper transport or an unsuitable installation location.

Liability and Warranty

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.

Liability and Warranty

Meaning of the warning messages in the manual

4 Meaning of the warning messages in the manual

WARNING Electric shock

This warns you of hazardous electrical voltage. Following the instructions is mandatory, since otherwise there is a risk of severe or fatal injury.

WARNING Severe injuries from ...

This note warns you of hazards that can cause severe or fatal injuries.

WARNING Permanent eye and skin damage from laser radiation

This note warns you of eye and skin damage that can occur when using lasers if safety precautions are not taken.

WARNING Risk of injuries due to harmful or irritating substances

This note warns you of substances that pose a health hazard.

WARNING Risk of injuries due to biological substances

This note warns you of biological substances that pose a health hazard.

WARNING Risk of burns on hot surfaces

This note warns you of hot surfaces that can cause burns.

D A T A

Meaning of the warning messages in the manual

CAUTION Injuries from…

This note warns you of minor to moderate injuries that can be prevented by following instructions.

NOTICE Risk of damage to the system

This note describes possible material damage that can occur in case of misuse.

NOTICE Loss of data

This note warns you of the potential for losing data.

Observe user manual

This mandatory sign indicates that an additional user manual must be foll

owed.

Wear laser safety glasses

This mandatory sign indicates that laser safety glasses must be worn to

nt eye injuries.

preve

Additional note

This note serves to emphasize important instructions for handling the

uct or contains special instructions about a certain topic.

prod

5 General Safety Notes

You have to follow the instructions listed below to work with the instrument safely and without disturbance. If you do not follow these or other instructions in this User Manual or the included manual, Leica Microsystems CMS GmbH shall not be liable for any resulting injury or property damage.

As it is impossible to anticipate every potential hazard, please be careful and apply common sense when using the system.

5.1 Commissioning and Use

• The system components have been packaged securely for transport in multiple crates.

Do not open these crates. The crates may be opened and unpacked by Leica service technicians or by people who are authorized by Leica Microsystems CMS GmbH only.

• The system may only be set up by Leica service technicians or by people who are

authorized by Leica Microsystems CMS GmbH.

General Safety Notes

• This laser equipment may be operated only by persons who have been trained in the use

of the system and about the potential hazards of laser radiation.

• Have your laser safety officer instruct you about the dangers of laser radiation and about

suitable laser safety precautions, such as wearing suitable laser safety glasses. This applies to all persons present in the room where the system is set up and operated.

• Each user must have read the instructions included and follow the instructions it

contains.

• Specimens must always be securely fixed in place.

• Do not introduce any flammable objects, such as paper, into the specimen area when

you are working with a laser.

• Do not place any flammable or combustible objects on or near the system and do not put

it near hot surfaces.

• During start up and during operation, you have to keep your hands and fingers away from

the specimen area, as otherwise there is a risk of crushing hazards or injury from rotating objectives and the motorized specimen stage.

• Set up the workplace (for example, chair and monitor) on the system so that it

corresponds to your requirements. Observe the national regulations for occupational safety.

• Before each service call by a service technician or whenever you relocate the

instrument, you have to clean it thoroughly. This is necessary to remove any possible contamination, thereby preventing the transfer of dangerous substances and pathogens and avoiding hazards and dangers. The same also applies to the removal of components. This applies in particular to systems that are located in biomedical research labs.

• You must not deviate from the operating and maintenance instructions provided herein.

General Safety Notes

5.2 Modifications to the System

• The system is installed by service technicians from Leica Microsystems CMS GmbH. You

must not change the position of the system components.

• The supply unit must always be set up and operated in an upright position.

• Under no circumstances may you open housing parts.

• Never disconnect a fiber optic cable.

• The cable and fiber optic cable may not be folded, stretched, pinched or rolled up tightly

or damaged in any other way.

• The product has a closed liquid coolant circuit. The cooling liquid hoses may not be

folded, stretched, pinched or rolled up tightly or damaged in any other way.

• Do not connect any external equipment or other components. If you have questions,

please directly contact the Leica branch office in your country.

5.3 Safety Devices and Safety Labels

• 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. Read

• Never remove the safety devices on the system.

• Never deactivate the laser protection devices.

• All safety devices must be ready to operate. Do not carry out any procedures that modify,

disable or damage the functionality of safety features. Unauthorized procedures could result in serious injuries or property damage.

• Safety labels on the system may not be removed. Missing or damaged safety labels must

be attached immediately and at the described location. Observe

on the System".

Chapter "Safety Features" of this User Manual carefully.

5.4 Laser Safety

• The instrument is a Class 3B/IIIb (VIS and UV lasers) or a Class 4/IV (IR lasers) laser

product.

• You must observe all suitable safety measures applicable for this laser class.

Chapter "Safety Labels

• When using an MP system, you must wear laser safety glasses (Order No.: 158002570).

Appropriate laser safety glasses for IR laser radiation are provided with the system when delivered. During the scanning operation, all persons present in the room must wear such laser safety glasses. These laser safety glasses do not provide any protection against visible laser radiation (visible spectrum)!

• It is not necessary to wear protective eyewear when using VIS and UV lasers. When

used as intended and safety notes have been followed, laser radiation is kept within the limit value that eliminates the chance for eye injuries.

• Never look directly into a laser beam or a reflection of the laser beam. Avoid all contact

with the laser beam.

General Safety Notes

• Never expose your eyes or skin to direct or indirect laser radiation. The radiation can

cause irreparable eye and skin injuries.

• During the scanning operation, the laser radiation is accessible in the microscope's

specimen area without obstruction after coming out of the objective. Always maintain a nominal ocular hazard distance of at least 20 cm (8") between your eyes and the opening of the objective.

Figure 1: Specimen area of upright and inverted microscope

• Make sure that the fiber optic cables are not damaged. The system may not be turned on

with damaged fiber optic cables, as laser radiation can escape and lead to irreparable eye and skin injuries.

• It is not necessary to wear protective eyewear when using VIS and UV systems. When

used as intended and safety notes have been followed, laser radiation is kept within the limit value that eliminates the chance for eye injuries.

• Do not use an S70 microscope condenser. The large working distance and the low

numerical aperture of the S70 microscope condenser could result in a hazard from laser radiation.

• Only use S1 and S28 Leica microscope condensers.

• Do not look into the eyepieces during the scanning operation.

• Never change samples during a scanning operation.

• Never change objectives, filter cubes, beam splitters, condensers or other components

during a scanning operation.

• Do not look into the eyepieces when switching the beam path in the microscope.

• Do not introduce any reflective objects or mirrors into the laser beam path or into the

specimen area.

• If there is no lamp housing or mirror housing connected to the microscope, attach the

cover to the replacement flange.

• All unoccupied positions in the objective nosepiece must be closed using the supplied

caps.

• For MP systems, dry objectives (air objectives) may not be used with a numerical

aperture (NA) larger than 0.85. This does not apply to immersion objectives (oil, water).

General Safety Notes

5.5 Electrical Safety

• This system is designed for connection to grounded (earthed) outlets. The grounding

plug performs an important safety function. To avoid the risk of electrical shock or damage to the instrument, do not disable this feature. Operation without grounded sockets is not permitted.

• Make sure that the supply voltage at the system remains in an approved tolerance range

(100 V~ - 240 V~ ±10%).

• The system may be connected to a power supply with ground protection conductor only!

Do not interfere with the grounding function by using an extension cord without a ground wire. Any interruption of the ground wire inside or outside of the system, or release of the ground wire connection, can cause the system to become hazardous. Intentionally disconnecting the ground protection conductor is not permitted.

• Before any cleaning or servicing, de-energize the entire system. To do so, use the power

switches of all components and disconnect all power cables from the power supply.

• Only use the power cable included or provided by your local Leica service technicians

for connecting individual peripheral devices to the power supply.

• Fuses inside the system may be replaced only by authorized Leica service employees. If

you have any further questions, please directly contact the Leica branch office in your country.

• Check that the actual line voltage corresponds to the value configured on the PDL-800-

B or D laser driver.

5.6 Contact with Liquids

• To avoid the risk of electrical shock and fire hazards, never expose the system to rain or

moisture.

• Do not allow any liquid to enter the system housing or come into contact with any

electrical components.

• Avoid condensation.

• The system must be completely dry before connecting it to the power supply or turning

it on.

• Do not operate the system if coolant is leaking or has leaked.

5.7 Malfunction of the System

You must immediately disconnect the system from the power supply if any of the following occur:

• The emission warning indicator is not lit after being switched on using the detachable-

key switch.

• The indicator continues to be lit after being switched off using the keyswitch

• Scanning of the specimen is not activated after being switched on properly (laser

radiation in the specimen area).

General Safety Notes

If any of these occur, immediately notify the Leica branch office in your country or your local contact person.

General Safety Notes

Additional Notes on Handling the System

6 Additional Notes on Handling the System

Follow these instructions to ensure that you handle the system without interference to avoid damage to the instrument and loss of data.

6.1 Location

• You need sufficient space for temporary storage and for unpacking the delivered

components. Always protect the transport crates and their contents from moisture and condensation and store them facing upwards (see the indication on the crate).

• Upon receiving the crates, make sure they are intact. If you find that the crates or seals

have been damaged, have the supplier confirm this; inform your contact person at Leica Microsystems about this immediately.

• Keep the packaging material in case you need to return a defective component.

• Be absolutely certain to observe the ambient conditions applicable for this system.

• You may use the system indoors only.

• The room must be free of dust, oil and chemical vapors.

• After installing the system, you may carry out interior finish work on the room only if the

system is stored in a dust-free location while this work is underway.

• Avoid direct sunlight and vibrations, since these can distort measurements and

micrographic scans.

• We recommend using a room that can be completely darkened.

• Do not expose the system to drafts.

• If the system has to be moved to a new location for any reason, contact the Leica branch

office in your country.

6.2 Using the Software

• Before carrying out operating steps with the system, first read the corresponding

description of the function in LAS functions, refer to the table of contents of the online help.

• Back up your data regularly to a suitable data carrier.

• Do not install any hardware or software on the workstation, as otherwise serious

damage to the system or loss of data can result.

AF Online Help. For an overview of the individual

Additional Notes on Handling the System

• Do not switch the workstation off after a software crash, but restart the LAS AF software

after 15 seconds. No image data are lost in case of a software crash. If the LAS AF software is restarted without restarting the workstation, the data are automatically restored. If the software crash is caused by a crash of the workstation, the image data will be lost.

6.3 Protecting the System

• Observe the maintenance instructions and intervals prescribed in the Chapter

"Maintenance".

• During the update of the firmware, a continuous tone sounds. After the updated

component is automatically restarted, the signal stops. During the automatic update and the automatic restart of the component, you may not switch off or restart the system, since otherwise this can lead to damage to the system.

• Protect the system from dust and grease.

• Make sure to use only one small drop of immersion fluid. The immersion fluid may not

contaminate or enter the microscope.

• Make sure that the specimen carrier is not against the objective and cannot be damaged

by it or cause broken glass.

• Be absolutely certain to prevent the optics and mechanical parts from coming into direct

contact with acids, bases and other aggressive chemicals.

• Never use abrasive products to clean the system and its components. Abrasives can

scratch the surface and thus have a negative effect on the protection of the parts.

• Protect the microscope from excessive temperature fluctuations. Such fluctuations can

lead to the accumulation of condensation, which can damage the electrical and optical components.

• Allow the entire system to cool down to room temperature before covering the system

with a dust cover. This prevents condensation from forming below it, which can enter the system and damage it.

• When used as intended, the HyD reflected light detectors are sufficiently protected from

destruction due to overexposure by measures in LAS AF and by an electronic protective circuit. An audible signal (beep) warns the user if the detector is being operated near the maximum permitted signal level. If the maximum permitted signal level is exceeded, the detector automatically switches off and the red status LED on the detector module (see Chapter 13.3, Figure 81, item 2) lights up.

• APDs are extremely sensitive detectors which can be damaged irreparably by light that

is too intense (such as room lighting). For this reason, APDs are protected by an automatic shut-off. If the light that falls on the APDs is too intense, they are switched off for a few seconds and an audible warning signal is emitted. The APDs are automatically reactivated after a few seconds. Either switch off the APDs or reduce the light intensity (e.g. by reducing the light intensity of the laser).

• To protect the counting units, never connect or disconnect any cable while the data

acquisition and control electronics are activated. Charged signal cables can destroy the instruments.

• Protect the photodetectors (APD or photomultiplier), particularly from excessive light

intensity, such as that from the microscope illumination, unattenuated backscatter

+ 240 hidden pages

Leica TCS SP8 SMD User Manual

Specifications and Main Features

Frequently Asked Questions

User Manual

Copyright

Contents

1 About this User Manual

1.1 Additional Documentation

2 Intended Use

3 Liability and Warranty

3.1 Important Information for Operators and Users

4 Meaning of the warning messages in the manual

5 General Safety Notes

5.1 Commissioning and Use

5.2 Modifications to the System

5.3 Safety Devices and Safety Labels

5.4 Laser Safety

5.5 Electrical Safety

5.6 Contact with Liquids

5.7 Malfunction of the System

6 Additional Notes on Handling the System

6.1 Location

6.2 Using the Software

6.3 Protecting the System