Leica TCS SP5 X User manual

Leica TCS SP5
Leica TCS SP5 X
User Manual

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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.

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Table of Contents
1. General .................................................................................................................9
1.1 Copyright.............................................................................................................9
1.2 About this Operating Manual ............................................................................10
2. Leica TCS SP5 / TCS SP5 X ..............................................................................11
2.1 TCS SP5 system overview ...............................................................................11
2.2 TCS SP5 X system overview ............................................................................12
2.3 Intended Use.....................................................................................................12
2.4 Conformity.........................................................................................................13
2.5 Patents..............................................................................................................14
2.6 Serial Number ...................................................................................................14
2.7 Specifications....................................................................................................15
2.7.1 Dimensions .....................................................................................................15
2.7.1.1 TCS SP5 with inverted microscope ............................................................15
2.7.1.2 TCS SP5 with upright microscope..............................................................15
2.7.1.3 TCS SP5 X with inverted microscope.........................................................16
2.7.1.4 TCS SP5 X with upright microscope ..........................................................16
2.7.2 Electrical Connection Requirements ...............................................................17
2.7.2.1 Electrical connection requirements of supply unit ......................................17
2.7.2.2 Electrical connection requirements of achromatic light laser .....................17
2.7.3 Requirements Regarding Ambient Conditions ................................................18
2.7.4 Permitted Ambient Conditions.........................................................................18
2.7.5 Waste Heat/Required Cooling Performance ...................................................19
2.8 Features............................................................................................................19
2.8.1 Overview of Usable VIS/UV Lasers ................................................................19
2.8.2 Overview of Usable MP Lasers (IR Lasers) ....................................................20
2.8.2.1 Picosecond laser ........................................................................................20
2.8.2.2 Femtosecond laser .....................................................................................21
2.8.3 Overview of Usable VIS/UV Lasers for TCS SP5 X........................................22
2.8.4 Which Laser Class Does the Product Have? ..................................................23
2.8.5 Required Laser Safety Measures....................................................................23
3. Safety Instructions and their Meanings...........................................................25

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4. General Safety Instructions ............................................................................. 27
4.1 Laser Class for VIS and UV Systems............................................................... 27
4.2 Laser Class for MP Systems ............................................................................ 27
4.3 What does the owner/operator have to observe? ............................................ 27
4.4 Safety Instructions for the User........................................................................ 29
4.5 Operational Reliability ...................................................................................... 29
4.6 Maximum Current Load of the Multiple Socket Outlet at the Supply Unit ........ 30
5. Safety Devices................................................................................................... 31
5.1 Disconnecting the Power Supply...................................................................... 31
5.2 Detachable-key Switch..................................................................................... 32
5.3 Emissions Warning Indicators .......................................................................... 34
5.4 Remote Interlock Connection on the Supply Unit............................................. 35
5.5 Remote interlock connection on the achromatic light laser ............................. 36
5.6 Remote Interlock Connections on External Lasers .......................................... 37
5.7 Remote interlock jack/interlock connector on the scanner............................... 37
5.8 Function and Position of Safety Switches ........................................................ 38
5.9 Special Laser Safety Equipment ...................................................................... 39
5.9.1 Laser protection tube and beam stop............................................................. 39
5.9.2 Shielding in MP Systems (IR Lasers)............................................................. 40
5.10 Safety labels on the system ............................................................................. 41
5.10.1 Inverted microscope DMI 6000 CS................................................................. 41
5.10.2 Upright microscope DM 5000/6000 CS.......................................................... 43
5.10.3 Scan Head...................................................................................................... 45
5.10.4 Achromatic light laser .................................................................................... 46
5.10.5 External UV laser ........................................................................................... 47
5.10.6 Supply Unit ..................................................................................................... 48
5.10.7 MP beam coupling unit................................................................................... 49
5.10.8 Cover (for Replacement Flange) .................................................................... 50
5.10.9 Mirror Housing................................................................................................ 51
6. Safety Instructions for Operating the System................................................ 53
6.1 Requirements Related to the Installation/Storage Location ............................. 53
6.2 General Safety Instructions for Operation ........................................................ 53
6.3 Eye Protection.................................................................................................. 54
6.3.1 MP System with Upright Microscope.............................................................. 54
6.3.2 MP System with Inverted Microscope ............................................................ 54

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6.3.3 VIS and UV Systems with Inverted or Upright Microscope .............................54
6.4 Specimen Area .................................................................................................55
6.5 Changing Specimens........................................................................................56
6.6 Changing Objectives.........................................................................................57
6.7 Changing the Transmitted-Light Lamp Housing................................................58
6.8 Mirror housing on upright microscope...............................................................60
6.9 Changing Filter Cubes, Beam Splitters or Condenser ......................................62
6.10 Piezo focus on upright microscope ...................................................................63
6.10.1 Objective Change with Piezo Focus Configuration .........................................64
7. Starting Up the System .....................................................................................65
7.1 Switching On the System..................................................................................65
7.2 Starting the LAS AF ..........................................................................................70
7.3 Setting Up Users...............................................................................................72
8. Switching Off the System .................................................................................73
9. Introduction to LAS AF .....................................................................................75
9.1 General .............................................................................................................75
9.2 Online Help .......................................................................................................75
9.2.1 Structure of the Online Help............................................................................75
9.2.2 Accessing the Online Help ..............................................................................76
9.2.3 Full-text Search with Logically Connected Search Terms...............................76
9.3 Structure of the graphical user interface ...........................................................78
9.3.1 General Structure of the Graphical User Interface..........................................78
9.4 Key Combinations.............................................................................................79
10. Introduction to Confocal Work .........................................................................81
10.1 Preparation .......................................................................................................81
10.1.1 The Objective ..................................................................................................82
10.1.2 Conventional Microscopy ................................................................................82
10.1.3 Why Scan?......................................................................................................85
10.1.4 How Is an Optical Section Created? ...............................................................86
10.2 Acquiring Optical Sections ................................................................................88
10.2.1 Data Acquisition ..............................................................................................88
10.2.2 Illumination ......................................................................................................90
10.2.3 Beam Splitting .................................................................................................91
10.2.4 Emission Bands ..............................................................................................92

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10.2.5 The Pinhole and Its Effects............................................................................. 93
10.2.6 Image Detail and Raster Settings................................................................... 95
10.2.7 Signal and Noise ............................................................................................ 99
10.2.8 Profile Cuts................................................................................................... 101
10.3 Multiparameter Fluorescence......................................................................... 102
10.3.1 Illumination ................................................................................................... 102
10.3.2 Beam Splitting .............................................................................................. 104
10.3.3 Emission Bands............................................................................................ 104
10.3.4 Crosstalk ...................................................................................................... 104
10.3.5 Sequential Scanning .................................................................................... 105
10.3.6 Unmixing ...................................................................................................... 105
10.4 3D Series........................................................................................................ 106
10.4.1 Z-stack.......................................................................................................... 106
10.4.2 Section Thicknesses .................................................................................... 106
10.4.3 Distances...................................................................................................... 107
10.4.4 Data Volumes ............................................................................................... 107
10.4.5 Depictions..................................................................................................... 108
10.4.5.1 Gallery ..................................................................................................... 108
10.4.5.2 Movie ....................................................................................................... 108
10.4.5.3 Orthogonal Projections ............................................................................ 109
10.4.5.4 Rotated Projections ................................................................................. 110
10.5 Time Series .................................................................................................... 110
10.5.1 Scan Speed .................................................................................................. 110
10.5.2 Points ........................................................................................................... 111
10.5.3 Lines............................................................................................................. 111
10.5.4 Planes .......................................................................................................... 111
10.5.5 Spaces (Time-Space)................................................................................... 111
10.5.6 FRAP Measurements ................................................................................... 112
10.6 Spectral Series ............................................................................................... 112
10.6.1 Data Acquisition and Utilization.................................................................... 112
10.6.2 About Spectral Resolution............................................................................ 112
10.7 Combinatorial Analysis................................................................................... 112
11. Care and Maintenance.................................................................................... 115
11.1 General........................................................................................................... 115
11.2 Cleaning the Optical System.......................................................................... 115

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11.3 Cleaning the Microscope Surface ...................................................................116
11.4 Maintaining the Scanner Cooling System .......................................................116
12. Transport and Disposal...................................................................................117
12.1 Changing the Installation Location..................................................................117
12.2 Disposal ..........................................................................................................117
13. Contact .............................................................................................................117
14. Glossary ...........................................................................................................118
15. Appendix ..........................................................................................................124
15.1 Safety Data Sheets from Third-party Manufacturers.......................................124
15.2 Declaration of conformity ................................................................................129
15.3 People´s Republic of China ............................................................................130

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1. General
1.1 Copyright
The instructions contained in the following documentation reflect state-of-the-art technology
and knowledge standards. We have compiled the texts and illustrations as accurately as
possible. Nevertheless, no liability may be assumed for the accuracy of this manual's
contents. If you have any comments on this operating manual or on any of our other
documentation, we would be pleased to hear from you. The information contained in this
operating manual is subject to change without prior notice.
All rights to this documentation 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.
The term "Windows" may be used in the following text without further identification. It is a
registered trademark of the Microsoft Corporation. Otherwise, no inference with regard to the
free usability of product names may be drawn from the use of those names. All other brand
names and product names in this document are brands, service marks, trademarks or
registered trademarks of the respective manufacturers.
Made in Germany.
© Copyright Leica Microsystems CMS GmbH.
All rights reserved.

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1.2 About this Operating Manual
Whenever this manual refers to the "system"or provides no specific information for
one of the systems, then the notes, instructions and information applies to
TCS SP5 and TCS SP5 X.
The main focus of this operating manual is directed to the safety notes that must be strictly
adhered to when working with the Leica TCS SP5 and Leica TCS SP5 X.
In addition, this operating manual provides a rough overview of the operating principle of
laser scanning microscopes. It presents you with the first steps for activating and
commissioning the system and provides important information about the Leica Application
Suite Advanced Fluorescence (LAS AF) software.
The system is delivered with the latest version of the licensed LAS AF. To maintain
information on the most current level, the description of software functions was intentionally
omitted from this operating manual. Instead, reference is made to the online help of the LAS
AF in which you can find up-to-date explanations and instructions for the corresponding
software functions.
Please read the chapter "Introduction to the LAS AF" in this operating manual to familiarize
yourself with its setup and operation. Additional information about specific functions can then
be found in the online help.

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2. Leica TCS SP5 / TCS SP5 X
2.1 TCS SP5 system overview
Figure 1: TCS SP5 system components (overview)
1 TCS SP5 Scanner
2 Main switch board
3 TCS workstation
4 Supply unit
5 Control panel
6 Microscope

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2.2 TCS SP5 X system overview
Figure 2: TCS SP5 X system components (overview)
1 TCS SP5 X scanner
2 Main switch board
3 TCS workstation
4 Supply unit
5 Control panel
6 Microscope
7 Achromatic light laser
2.3 Intended Use
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.
This system is intended for use in a lab.
Applications of in-vitro diagnostics in accordance with MPG (German Medical Devices Act)
are excluded from proper intended use.

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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.
2.4 Conformity
This device has been tested and meets the requirements of the following standards:
IEC/EN 61010-1 "Safety requirements for electrical equipment for measurement, control
and laboratory use"
IEC/EN 60825-1 "Safety of laser products, Part
1: Equipment classification, requirements and user's guide"
IEC/EN 61326
"Electrical Equipment for Measurement, Control and Laboratory Use −
EMC Requirements" (Class A).
This is a Class A instrument for use in buildings that do not include
domestic premises and buildings not directly connected to a low-
voltage power supply network that supplies buildings used for
domestic purposes.
IEC/EN 61000-3-2
"Electromagnetic Compatibility (EMC)"
Part 3-2: Limits — Limits for harmonic currents
IEC/EN 61000-3-3
"Electromagnetic Compatibility (EMC)"
Part 3-3: Limits — Limitation of voltage fluctuations and flicker in low-
voltage supply systems.
The declaration of conformity for the system is located in the appendix of this operating
manual.
For use in the USA:
CDRH 21 CFR 1040.10:
U.S. laser products Food and Drug Administration (FDA)
(”Complies with FDA performance standards for laser products
except for deviations pursuant to laser notice No. 50, dated 26
July, 2001.
For the USA (area of validity of the CDRH/FDA), the designations of the laser class are to be
changed in the text from 3B to IIIb and Class 4 to IV.

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2.5 Patents
The Leica TCS SP5 product is protected by the US patents:
5,886,784; 5,903,688; 6,137,627; 6,222,961; 6,285,019; 6,311,574; 6,355,919; 6,423,960;
6,433,814; 6,444,971; 6,466,381; 6,510,001; 6,614,526; 6,654,165; 6,657,187; 6,678,443;
6,687,035; 6,738,190; 6,754,003; 6,801,359; 6,831,780; 6,850,358; 6,867,899.
Further patents are pending.
The Leica TCS SP5 X protect by the US patents:
5,886,784; 5,903,688; 6,137,627; 6,222,961; 6,285,019; 6,311,574; 6,355,919; 6,423,960;
6,433,814; 6,444,971; 6,466,381; 6,510,001; 6,567,164; 6,611,643; 6,614,526; 6,654,165;
6,654,166; 6,657,187; 6,678,443; 6,687,035; 6,710,918; 6,738,190; 6,754,003; 6,796,699;
6,801,359; 6,806,953; 6,831,780; 6,850,358; 6,867,899; 6,888,674; 6,898,367; 6,958,858;
6,961,124; 7,005,654; 7,092,086; 7,110,645; 7,123,408.
Further patents are pending.
2.6 Serial Number
The serial number of your system is located on the rear side of the scanner:
Figure 3: Rear side of scanner – label with serial number

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2.7 Specifications
2.7.1 Dimensions
2.7.1.1 TCS SP5 with inverted microscope
1400 mm
600 mm 730 mm
1200 mm
2540 mm
Figure 4: Dimensions of TCS SP5 with inverted microscope
2.7.1.2 TCS SP5 with upright microscope
600 mm
730 mm
2400 mm
1490 mm
1200 mm
Figure 5: Dimensions of TCS SP5 with upright microscope

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2.7.1.3 TCS SP5 X with inverted microscope
Figure 6: Dimensions of TCS SP5 X with inverted microscope
2.7.1.4 TCS SP5 X with upright microscope
Figure 7: Dimensions of TCS SP5 X with upright microscope

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2.7.2 Electrical Connection Requirements
The building installation must feature three separate power connections with the following
fuse protection:
•3 x 100 V - 120 V power supply at 20 A
or
•3 x 200 V - 240 V power supply at 12 - 16A
For the specifications of external lasers such as UV and MP lasers, please refer to the
manufacturer's documentation.
2.7.2.1 Electrical connection requirements of supply unit
Supply voltage: 100 - 240 V AC ± 10 %
Frequency: 50/60 Hz
Power consumption: 3200 VA
Overvoltage category: II
2.7.2.2 Electrical connection requirements of achromatic light laser 1
Supply voltage: 100 - 240 V AC ± 10 %
Frequency: 50/60 Hz
Power consumption: 2.2 A
Overvoltage category: II
Fuse 2x T4A, 250V AC
1Applies only to the TCS SP5 X system.

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2.7.3 Requirements Regarding Ambient Conditions
Do not expose the system to drafts.
Ensure that the system is not installed next to air conditioners or ventilation systems. For this
reason, the installation location should be carefully planned.
Ensure that the environment is as dust-free as possible.
Also read the notes on protection against dust in Chapter 11 Care and Maintenance
Installing the system in darkened rooms is also advisable.
The system requires doors with inside widths of 1.0 m for installation, maintenance and
transport.
With regard to the load-bearing capacity of the floor, note that the system will apply a static
load of 200 kg/m².
Ensure that the environment is as vibration-free as possible.
2.7.4 Permitted Ambient Conditions
Permissible temperature range for operation: +18 to +25 ºC
Temperature range for optimum optical
behavior: +22 °C ±1 °C
Permitted relative humidity: 20 - 80% (non-condensing)
Permitted vibrations:
Frequency range [5 Hz–30 Hz]:
Frequency range [> 30 Hz]:
< 30 μm/s (RMS)
< 60 μm/s (RMS)
Pollution degree: Class 2

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2.7.5 Waste Heat/Required Cooling Performance
The TCS SP5 system has a maximum power consumption of 3.2 kW (VIS system) or 6.2 kW
(MP system).
The TCS SP5 X has a maximum power consumption of 3.4 kW.
For the specifications of external lasers such as UV and MP lasers, please refer to the
manufacturer's documentation.
2.8 Features
2.8.1 Overview of Usable VIS/UV Lasers
The Leica TCS SP5 features a combination of the lasers listed below.
Laser type
Wavelength
[nm]
Maximum
luminous power
at laser output
[mW]
Maximum
luminous power
in focal plane
[mW]
Pulse duration
Diode 405 405 < 120 < 7 Continuous wave
(cw)
Diode 405 p 405 < 5 (mean
power)
< 0.3 (mean
power) pulsed, 60 ps
DPSS 445 445 < 75 < 7 Continuous wave
(cw)
Ar 458, 476, 488,
496, 514 < 200 < 50 Continuous wave (cw)
Ar, external 458, 476, 488,
496, 514 < 500 < 125 Continuous wave (cw)
HeNe 543 < 1.5 < 0.5 Continuous wave
(cw)
DPSS 561 561 < 100 < 12 Continuous wave
(cw)
HeNe 594 < 4 < 1 Continuous wave
(cw)
HeNe 633 < 15 < 5 Continuous wave
(cw)
UV, external 355 < 500 < 18 Continuous wave
(cw)
Table 1: Usable lasers for TCS SP5 (without MP)

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The components for laser safety are designed only for the laser variants listed.
2.8.2 Overview of Usable MP Lasers (IR Lasers)
Furthermore, the MP system may contain additional VIS/UV lasers (see the table for usable
VIS/UV lasers).
2.8.2.1 Picosecond laser
Laser type Wavelength
[nm]
Luminous
power at laser
output [W]
Luminous
power in focal
plane [W]
Pulse duration
MaiTai ps 780 - 920 < 1.2 < 0.6 pulsed, 1.0 - 1.5 ps
MaiTai ps
wideband 710 - 950 < 2.5 < 1.2 pulsed, 1.0 - 1.5 ps
MaiTai ps
broadband 710 - 990 < 2.5 < 1.2 pulsed, 1.0 - 1.5 ps
MaiTai ps HP 690 - 1040 < 3.0 < 1.9 pulsed, 1.0 - 1.5 ps
Chameleon ps
Ultra 690 - 1020 < 4 < 1.9 pulsed, 1.0 - 1.5 ps
Chameleon ps
Ultra I 690 - 1040 < 4 < 1.9 pulsed, 1.0 - 1.5 ps
Chameleon ps
Ultra II 680 - 1080 < 4 < 1.9 pulsed, 1.0 - 1.5 ps
Table 2: Usable MP lasers for TCS SP5 and TCS SP5 X