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.

3 Copyrights
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
registered trademarks of the respective
j) manufacturers.
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
10

-* 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.
3
3
3
3
11

3 Safety instructions and their meaning
■5
In the following chapter, we explain the meaning of the safety instructions.
Safety instructions contain very important instructions and helpful information. It is very
important that you observe the safety instructions. They are for your personal protection
and are intended to protect you from incorrect operation and damage of the instrument.
The following safety instructions are used in these operating instructions:
DANGER
This kind of warning alerts you of an operating procedure, practice,
condltion, or statement in the operating instructions that must be
strictly observed and followed, as otherwise you expose yourself to
the riskof fatal injury.
WARNING! HIGH VOLTAGE
This kind of high-voltage warning alerts you of an operating
procedure, practice, condition, or statement in the operating
¡nstructions that must be strictly observed and followed, as otherwise
you expose yourself to the risk of fatal injury.
3
B
WARNING! LÁSER RADIATION
A warning of láser radiation indicates an operation, a process, a
condition or an instruction that must be observed strictly to prevent
serious eye or skin injuries to the persons using the system and
persons in the vicinity of the system.
3
13

CAUTION
This kind of safety caution alerts you to an operating procedure,
practice, condition, or statement that must be strictly observed to
prevent severe system damage or destruction, or data corruption or
loss.
This kind of note is a statement that either provides extra information
about a topic or contain special instructions for handiing the product.
14

3
4 Safety instructions
4.1 General safety instructions
This system consists of components and peripheral devices that comply with EN 61010-
1/IEC 1010-1, safety standards for electrical measurement, control and laboratory
equipment. In order to maintain this condition and to ensure safe operation, you must
follow the instructions and warnings contained in these operating instructions.
So that you can opérate 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. For further information, refer to the chapter on , page 37.
Safety equipment must not be disabled!
SETTING UP THE SUPPLY UNIT
For safe handling and operation the Supply Unit always has to be set
up in upright position. The Supply Unit must not be set up other than
upright despite a possible lack of space!
5
a
Repairs and servicing may only be performed by authorized Leica
Microsystems CMS GmbH service personnel. In particular, the supply
unit, the sean head and the light waveguides may be opened or
worked on by authorized Leica service personnel only. If you have any
further questions, please contact the Leica branch office in your
country (see Chapter 15Contact, page 111).
Unauthorized alterations to the system or noncompliant use shall void
all rights to any warranty claims! The owner/operator is fully Hable for
all consequences resulting from the use of the device if it is modified,
opened, serviced, repaired, assembled or dismantled by persons
other than authorized Leica service representatives.
The Leica TCS SPE system may be used indoors only.
15

The TCS SPE system must not be used together with life-support
systems, such as those used in intensive-care fields.
To power down the system and/or individual components and
peripherals ¡n the event of danger, actúate the power switch on the
suppiy unit immediately (see Chapter Disconnecting the power suppiy,
page 55) and disconnect the unit from the power suppiy.
Switch off the On/Off switch on the individual components and
peripheral devices and unplug the power suppiy line from the power
suppiy. Always observe all of operating instructions provided for the
individual components and peripheral devices!
Within the suppiy unit and the sean head there are installed
components that can cause fatal injury ¡f handled improperly. Opening
these components will result ¡n danger to people and the device.
Therefore, only authorized Leica service personnel may open or work
on the suppiy unit and the sean head. If you have any further
questions, please contact the Leica branch office ¡n your country (see
Chapter 15Contact, page 111).
Never look into the light guide or the láser light. The láser light can
cause permanent eye damage!
Do not connect any external equipment.
For safety reasons, only those components and peripheral devices
that are mentioned in these Operating Instructions may be connected
to the TCS SPE system.
If you have any further questions, please contact the Leica branch
office in your country (see Chapter 15Contact, page 111).
The microscope must not be subjected to temperatures above 35°C!
16

3
Electrical components and peripheral devices must be placed at least
10 cm away from the wall and from flammable substances.
9
The cable and light waveguide may not be folded, stretched, pinched
or rolled up tightly!
Be careful not to trip over the cable and light waveguide. Be
absolutely certain to familiarize yourself with the system and the
surrounding área.
i
Be absolutely certain to prevent the optics and mechanical parts from
coming into direct contact with acids, bases and other aggressive
chemicals.
To prevent loss of data, make regular backups of your data and
images on a suitable data carrier.
Also, be sure to back up your data before any service or repair work is
performed! Leica Microsystems CMS GmbH shall not be liable for any
loss of data.
If the software crashes the image data will not be lost. When the LAS
AF software is restarted without restarting the workstation (Supply
Unit) the data are automatically restored. If the software crash is
caused by a crash of the workstation (Supply Unit), the image data
will be lost. After a software crash do not switch the workstation
(supplv unitt off but restart the LAS AF software after 15 seconds.
17

4.2 Láser safety
Please observe appiicable national laser-safety instructions and laser-safety regulations
for láser class 3B / Illb. The laser-safety instructions are provided in Chapter 5Laser
safety, page 23. The operator is responsible for observing the láser safety regulations.
4.3 Electrical safety
Make sure that the supply voltage at the system remains in an
approved tolerance range (100 V- - 240 V- ±10%)!
If the tolerance valué is exceeded, we cannot guarantee that the
system will opérate. The unit may be damaged and become a hazard
to the user! If necessary, use a suitable supply unit to ensure that the
required supply voltage of the system lies within the specified
tolerance valúes. If you have any further questions, please contact the
Leica branch office in your country (see" Chapter 15Contact, page
111).
The system may be connected to a power supply with ground
protection conductor only! Do not interfere with the grounding function
by using an extensión cord without a ground wire. Any interruption of
the ground wire inside or outside of the system, or reléase of the
ground wire connection, can cause the system to become hazardous.
Intentional ground interruption is not permitted!
By connecting them to ground, peripheral devices which have their
own and/or sepárate power supplies and which are connected to the
microscope can be connected to the same ground potential. For
power supplies without ground protection conductor, contact the Leica
branch office in your country (see Chapter Contact 15, page 111)
Use only the supplied power cables or the cables supplied by your
local Leica service technician to connect the peripheral devices to the
power supply.
Only authorized Leica service personnel may replace fuses in the
Leica TCS SPE system. If you have any further questions, please
18

i
contact the Leica branch office in your country (see Chapter
15Contact, page 111).
When replacing fuses and lamps in other components and perípheral
devices, the devices and their components must be switched off and
disconnected from the power supply. Always observe all of operating
instructions provided for the individual components and peripheral
devices!
■i
The entire system and all components and peripheral devices
microscope are not protected against water ingress. Water ingress
can cause electric shock. Do not assemble the microscope and its
accessory components in the immediate vicinity of a water connection
or other áreas where the possibility of water ingress exists.
i
i
9
"9
Protect the microscope from excessive temperature fluctuations. Such
fluctuations can lead to the accumulation of condensation, which can
damage the electrical and optical components.
19

4.3.1 General specifications
Supply voltage:
100V~to240V~±10%
Frequency:
Power consumption:
Line input fuse
Powersupply on the rear
side of the supply unit:
Ambient temperature:
Relative humidity:
Overvoltage category:
Pollution degree:
50/60 Hz
max. 800 VA
2x T8A/250V"
max. 150 VA
+15°Cto+35°C
max. 80% (non-condensing)
20

' 4.4 Standards
This system and the peripheral devices supplied by Leica Microsystems CMS GmbH for
this system comply with the following standards:
■ IEC/EN 61010-1
"Safety requirements for electrical equipment for measurement, control and
$ laboratory use"
í • IEC/EN 60825-1
^ "Safety of láser products,
Part 1: Equipment classif¡catión, requirements and user's quide"
- IEC/EN 61326
"Electrical Equipment for Measurement, Control and Laboratory Use - EMC
"J Requirements"
~Í ■ IEC/EN 61000-3-2
■j "Electromagnetic compatibility" (EMC) Part 3-2: Limits - Limits for harmonio
currents
■ IEC/EN 61000-3-3
' "Electromagnetic compatibility" (EMC) Part 3-3: Limits - Limits -
"j Section 3: Limits for voltage fluctuations and flicker in low-voltage networks.
^ ■ For use in the USA
-. CDRH21 CFR 1040.10:
Láser Products U.S. Food and Drug Administration (FDA) ("Complies with FDA
9 performance standards for láser products except for deviations pursuant to láser
notice No. 50, dated 26 July, 2001").
Within the scope of the CDRH/FDA (USA), "Illb" must be written in place of
"3B" in the description, "láser class 3B".
21

4.5 Instructions for handling glycerin solution
Glycerin solution for glycerin lens is hygroscopic, meaning that it absorbs moisture from the
air and thus changes its refraction Índex, which can ¡mpair the optical imaging performance.
However, this can be compensated for to a certain extent using the correction ring. The
effect of the absorption of moisture by the glycerin solution will be stronger the larger the
surface and the smaller the volume of the quantity used.
Handling of glycerin solution
Vials of glycerin solution may not be left open, and should be closed tightiy immediately after
use. Glycerin solution may not be refrigerated, but must be stored tightiy closed at room
temperature. Changes to the ambient conditions affect the glycerin/water mixture ratio and
thus cause differences in the refraction index.
Storage life of glycerin solution
In its original closed container, Glycerin solution ¡s prepared for optimal imaging performance.
The original closed container has a storage life of one year. The storage life after the
container is opened is limited and depends largely on the type of use; refer to the previous
section. Refrigeration decreases the storage life rather than increasing it. If the glycerin
solution absorbs too much moisture from the air, you will not be able to obtain perfect
microscopic images even you readjust using the correction ring. However, this does not harm
the objective.
22
<5