Leica TCS SPE User manual

MICROSYSTEMS

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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3
The instructions contained in the following
3 documentation reflect state-of-the-art technology and
^ knowledge standards. We have compiled the texts
and illustrations as accurately as possible.
^ Nevertheless, no liability of any kind may be assumed
for the accuracy of this manual's contents. If you have
•5 any comments on these operating instructions or on
^ any of our other documentation we would be pleased
to receive them. The information in this manual is
^ subject to modification at any time and without
notification.
^ 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
9 copyright laws. All rights reserved. Reproduction,
adaptation or translation of these programs is
prohibited without prior written permission from Leica
i) Microsystems CMS GmbH.
■■9 The term "Windows" may be used in the following text
- without further identificaron. It is a registered
trademark of the Microsoft Corporation. Otherwise, no
*) inference with regard to the free usability of product
ñames may be drawn from the use of those ñames.
3 All other brand ñames and product ñames in this
document are brands, service marks, trademarks or
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9 © Leica Microsystems CMS GmbH
3

The product ¡s protected under the following US
patents:
5,903,688; 6,137,627; 6,211,988; 6,222,961;
6,285,019; 6,418,153; 6,423,960; 6,444,971;
6,466,381; 6,614,526; 6,657,187; 6,678,443;
6,754,003; 6,801,359; 6,809,815; 6,850,358;
6,954,306
Further patents are pending.

Contents
~ 1 Important information about the operating instructions 9
1.1 Intended use 10
2 The Leica TCS SPE system 11
$
| 3 Safety instructions and their meaning 13
4 Safety instructions 15
) 4.1 General safety ¡nstructions 15
4.2 Láser safety 18
'" 4.3 Electrical safety 18
3 4.3.1 General specifications 20
4.4 Standards 21
* 4.5 Instructions for handling glycerin solution 22
5 Láser safety 23
* 5.1 Láser and láser class 24
^ 5.2 Safety labels on the Leica TCS SPE system 25
5.2.1 Microscope DMI4000 26
9 5.2.2 Microscope DM2500 28
-^ 5.2.3 Microscope DM5500 30
5.2.4 Sean head 32
^ 5.2.5 Supply unit 33
m 5.2.6 Mirror housing 35
3 6 Safety devices on the microscope 37
^ 6.1 Transmitted light lamp housing on the inverted microscope 37
6.2 Transmitted light lamp housing on the upright microscope 38
3 6.3 Mirror housing on the upright microscope 40
^ 6-4 Safety beam guide and beam collector 42
6.5 Láser keyswitch and láser emission display 44
5 6.6 Safety switches 46
a 6.7 Interlock 47
6.8 Remote interlock 48
^ 7 Work safety 49
7.1.1 Specimen área 51
3 7.1.2 Changing the specimen 52
7.1.3 Changing theobjective 53
7.1.4 Changing filter cube/beam splitter 54

7.2 Disconnecting the power supply
55
8 System overview
58
57
8.1 System components and peripheral devices
8.1.1 Microscope
8.1.2 Supply unit with workstation integrated cft
8.1.2.1 Dongle "'".! 59
8.1.3 Scanhead on
60
9 Installation location
9.1 Ambient conditions 62
9.1.1 Air-conditioning of the installation location 62
9.1.2 Power supply
9.2
63
Installation of the system 63
10 Starting up
10.1 Switching on the system 65
10.2 Switching off the system 67
11 LAS 6g
11.1 Calling up the online help 6g
11.2 Full-text search with logically connected search terms 70
11.3 General structure of the user interface 71
12 Introduction to confocal work 73
12.1 Preparation 73
12.1.1 Theobjective 74 «
12.1.2 Conventional microscopy 74
12.1.3 Why sean? ".".".'"I" 77
12.1.4 How is an optical section created? 78 •
12.2 Acquiring optical sections 80 f
12.2.1 Data acquisition 80 v
12.2.2 Illumination '"' Q2 «
12.2.3 Beam splitting 83
12.2.4 Emission bands 83
12.2.5 The pinhole and its effeets 85 ?
12.2.6 Image detail and ráster settings 86
12.2.7 Signal and noise go
12.2.8 Profilecuts "'.".'.".".'"".".'.' 92 5
12.3 M ultipara meter fluorescence 92
12.3.1 Illumination \ZZZZZ'".'.'.". 93 *
12.3.2 Emission bands 95 («
12.3.3 Sequential capture g5
12.4 3D series """.'.".'.".'"'".'.'." 96
í

ít
i y 12.4.1 z-stack 96
. ^ 12.4.2 About section thickness 96
I ~~ 12.4.3 ...and spacing 96
'3 12.4.4 Data volumes 97
^ 12.4.5 Depictions 97
12.4.5.1 Gallery 98
? 12.4.5.2 Movie 98
- 12.4.5.3 Orthogonal projections 98
12.5 Time series 100
d 12.5.1 Sean speed 100
3 12.5.2 Unes 100
12.5.3 Planes 100
? 12.5.4 Spaces (time-space) 100
^ 12.6 Spectral series 100
12.6.1 Data acquisition and utilization 101
S 12.7 Combinatorial analysis 101
13 Care and maintenance 103
5
13.1 Filter 104
13.1.1 Changing the filter 104
13.2 Moisture 105
5 13.3 Dust cover 105
13.4 Clinging dirt 106
13.5 Cleaning the specimen stage 106
sj 13.6 Cleaning glass surfaces 106
13.7 Cleaning the objective 107
13.8 Removing immersion oil 107
j» 13.9 Handling acids and bases 107
13.10 Cleaning the supply unit 108
13.11 Cleaning the sean head 108
% 13.12 Cleaning the monitor 108
13.13 Cleaning the keyboard 108
a 14 Disposal 109
15 Contact
16 índex 113

1 Important information about the operating
^ instructions
- These operating instructions are an essential component of the Leica TCS SPE system
and must be read carefully before the microscope ¡s put into operation or used. The
* emphasis is on the safety instructions, which must be strictly observed during all work with
^ the Leica TCS SPE system. Keep these instructions in a convenient, safe place.
^ Please read and observe the instructions provided for the individual components and
^ peripheral devices and keep them in a safe place.
sf These operating ¡nstructions present the first steps for activating the system and describe
^ the basic functions of the Leica Application Suite Advanced Fluorescence (LAS AF)
software.
a
Detailed descriptions of the software functions have not been included in these
instructions. Instead, please refer to the online help of the LAS AF, which presents the
$ most up-to-date explanations and instructions of the corresponding software functions.
Please read Chapter LAS AF to familiarize yourself with the structure and use of the
online help.
5

1.1 Intended use
The system was designed for confocal recording (láser scanning images) of fluorescence-
marked living and fixed specimens as well as for quantitative measurements in the área of
life science.
This system is intended solely for laboratory use. The system is not intended for use for in
vitro diagnostics, or as defined by the Germán medical devices law
(Medizinproduktgesetz).
The user of this product is responsible for proper and safe operation and safe
maintenance of the system as well as for following all applicable safety regulations. The
user is fully Hable for all consequences resulting from the use of the system for any other
purposes than those listed in the Operating Instructions or the online help.
The manufacturer assumes no responsibility or liability for any use outside of the intended
use or use outside of the specifications from Leica Microsystems CMS GmbH or any risks
resulting from such use. In such cases, the EU Declaration of Conformity shall be invalid.
Due to potential hazards, only personnel who are familiar with its operation and who nave
appropriate training should be assigned responsibilities with the Leica TCS SPE system. «
Such training can be obtained from the manufacturer, from a láser protection officer or
from a recognized outside organizaron, and should cover at least the following points:
í
■ Familiarization with the operating processes of the system (under consideration of
all safety instructions provided in these operating instructions)
■ Correct use of láser protection for preventing hazards, use of warning signs etc.
■ Necessary personal protection
■ Biológica I effect of the láser on eyes and skin /;
(5
a
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-* 2 The Leica TCS SPE system
The Leica TCS SPE is a true confocal microscope system for entry-level áreas.
^ This extremely compact system is particularly easy to use while offering all of the
^ advantages of a confocal microscope.
^ Ultrathin z-sections with one-of-a-kind, pinpoint sharp image quality are recorded in
^ conjunction with the integrated, high-precision Galvo-z stage and composed into three-
dimensional visualizations with the new LAS AF software.
-^ The system achieves máximum signal output with the optimized tuning of the special,
highly dynamic photomultiplier, which comes with new control technology, motorized
^ variable pinhole and efficient prisms. The spectral detector ensures the perfect adjustment
to the emission spectrum of today's dyes and the dyes of the future.
^ The TCS SPE can be equipped with up to four lasers. Long-lasting solid-state lasers with
emission wavelengths of 488 nm, 532 nm and 635 nm are included with the standard
system. An optional versión with an additional 405 nm láser is also available. Compared
ó) to conventional gas lasers, solid state lasers provide a significantly longer service life and
excellent stability.
^ Using the standard AOTF, it is possible to optimally adjust the láser intensity infinitely
between 0 and 100 % to prevent irradiation and unnecessary bleaching.
$ Based on the high-level integration of the supply unit, the TCS SPE can be used in any
laboratory; extensive cooling or special power termináis are not necessary.
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