Leica TCS SP5 X User manual

Leica TCS SP5
User Manual

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. Notes about this manual..................................................................................... 7
1.1 Copyright ............................................................................................................ 7
1.2 Patents ............................................................................................................... 8
2. General ................................................................................................................. 9
2.1 About these operating instructions ..................................................................... 9
2.2 Intended use....................................................................................................... 9
3. TCS SP5 specifications .................................................................................... 11
3.1 System overview .............................................................................................. 11
3.2 Dimensions....................................................................................................... 12
3.2.1 System with inverted stand:............................................................................ 12
3.2.2 System with upright stand............................................................................... 12
3.3 Requirements regards ambient conditions ....................................................... 13
3.4 Permissible ambient conditions ........................................................................ 13
3.5 Electrical connections....................................................................................... 14
3.6 Waste heat/required cooling performance........................................................ 14
3.7 Required safety measures for VIS and UV systems......................................... 14
3.8 Required laser safety measures for MP systems ............................................. 14
4. Safety instructions and their meaning ............................................................ 15
5. Safety instructions ............................................................................................ 17
5.1 Which standards does this product meet?........................................................ 17
5.2 Which laser class does the product have? ....................................................... 18
5.3 Laser class for VIS and UV systems ................................................................ 18
5.4 Laser class for MP systems.............................................................................. 19
5.5 What should the user of the laser scanning microscope observe? .................. 19
5.6 Safety Notes for the User ................................................................................. 20
5.7 Safety Notes for Operation ............................................................................... 21
5.8 Specific Safety Notes ....................................................................................... 23
5.8.1 Operational reliability ...................................................................................... 23
5.8.2 De-energizing the system ............................................................................... 24
5.8.3 Maximum current load of the power outlet strip at the supply unit.................. 24
5.9 Laser safety devices......................................................................................... 25
5.9.1 Eye protection for VIS or UV systems ............................................................ 25
5.9.2 Detachable-key switch.................................................................................... 25
5.9.3 Emissions warning indicators ......................................................................... 26
5.9.4 Remote interlock connector on the supply unit............................................... 27
5.9.5 Remote interlock connectors on additional external lasers............................. 28
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5.9.6 Interlock connection on scanner ..................................................................... 28
5.9.7 Function and position of safety switches ........................................................ 29
5.9.8 Transmitted-light lamp housing for inverted stands ........................................ 30
5.9.9 Transmitted-light lamp housing for upright stands.......................................... 31
5.9.10 Mirror housing on upright stand ...................................................................... 32
5.9.11 Special laser safety equipment....................................................................... 34
5.9.11.1 Safety beam guide and beam collector...................................................... 34
5.9.11.2 Eye protection for MP systems .................................................................. 35
5.9.11.3 Shielding in MP systems (IR lasers) .......................................................... 35
5.10 Overview of usable VIS/UV lasers.................................................................... 36
5.11 Overview of usable MP lasers (IR lasers)......................................................... 37
5.12 Safety label on TCS SP5 system ..................................................................... 37
5.12.1 Inverted stand DMI 6000 CS .......................................................................... 38
5.12.2 Upright stand DM 5000/6000 CS.................................................................... 40
5.12.3 Scan head....................................................................................................... 42
5.12.4 Supply unit ...................................................................................................... 43
5.12.5 MP beam coupling unit ................................................................................... 44
5.12.6 Cover (for replacement flange) ....................................................................... 45
5.12.7 Mirror Housing ................................................................................................ 46
5.13 Installation and storage site requirements ........................................................ 47
5.14 Changing the installation site............................................................................ 47
5.15 Scanner cooling................................................................................................ 48
6. Care and Maintenance ...................................................................................... 49
6.1 Cleaning the optical system of the microscope ................................................ 49
6.2 Cleaning the microscope surface ..................................................................... 50
7. Startup of the system........................................................................................ 51
7.1 Starting and logging on..................................................................................... 51
7.2 Setting Up Users .............................................................................................. 52
7.3 Starting up ........................................................................................................ 53
7.4 Starting the LAS AF.......................................................................................... 55
8. Introduction to LAS AF ..................................................................................... 57
8.1 General............................................................................................................. 57
8.2 Online help ....................................................................................................... 57
8.2.1 Structure of the online help............................................................................. 57
8.2.2 Calling Online Help ......................................................................................... 58
8.2.3 Full-text search with logically connected search terms................................... 59
8.3 Structure of the user interface .......................................................................... 60
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8.3.1 General structure of the user interface ........................................................... 60
8.4 Key combinations ............................................................................................. 61
9. Introduction to Confocal Work......................................................................... 63
9.1 Preparation ....................................................................................................... 63
9.1.1 The objective .................................................................................................. 63
9.1.2 Conventional microscopy................................................................................ 64
9.1.3 Why scan? ...................................................................................................... 67
9.1.4 How is an optical section created? ................................................................. 68
9.2 Acquiring optical sections ................................................................................. 70
9.2.1 Data acquisition .............................................................................................. 70
9.2.2 Illumination...................................................................................................... 72
9.2.3 Beam splitting ................................................................................................. 73
9.2.4 Emission bands .............................................................................................. 74
9.2.5 The pinhole and its effects.............................................................................. 76
9.2.6 Image detail and raster settings...................................................................... 77
9.2.7 Signal and noise ............................................................................................. 81
9.2.8 Profile cuts ...................................................................................................... 83
9.3 Multiparameter fluorescence ............................................................................ 84
9.3.1 Illumination...................................................................................................... 84
9.3.2 Beam splitting ................................................................................................. 86
9.3.3 Emission bands .............................................................................................. 86
9.3.4 Crosstalk......................................................................................................... 86
9.3.5 Sequential capture.......................................................................................... 87
9.3.6 Unmixing......................................................................................................... 87
9.4 3D Series.......................................................................................................... 88
9.4.1 z-stack ............................................................................................................ 88
9.4.2 Layer thicknesses ........................................................................................... 88
9.4.3 Distances ........................................................................................................ 89
9.4.4 Data volumes.................................................................................................. 89
9.4.5 Depictions ....................................................................................................... 90
9.4.5.1 Gallery........................................................................................................ 90
9.4.5.2 Movie.......................................................................................................... 90
9.4.5.3 Orthogonal projections ............................................................................... 91
9.4.5.4 Rotated projections .................................................................................... 92
9.5 Time series ....................................................................................................... 92
9.5.1 scan speed ..................................................................................................... 93
9.5.2 Points.............................................................................................................. 93
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9.5.3 Lines ............................................................................................................... 93
9.5.4 Planes............................................................................................................. 93
9.5.5 Spaces (time-space)....................................................................................... 93
9.5.6 FRAP measurements ..................................................................................... 94
9.6 Spectral series.................................................................................................. 94
9.6.1 Data acquisition and utilization ....................................................................... 94
9.6.2 About spectral resolution ................................................................................ 94
9.7 Combinatorial analysis ..................................................................................... 95
10. Switching off the system .................................................................................. 97
11. Disposal ............................................................................................................. 99
12. Contact ............................................................................................................. 101
13. Declaration of conformity............................................................................... 103
14. Glossary ........................................................................................................... 105
15. Safety data sheets........................................................................................... 111
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1. Notes about this manual
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 of any kind may be assumed for the accuracy of this
manual's contents. If you have 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 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.
Manufactured in the Federal Republic of Germany.
© Copyright Leica Microsystems CMS GmbH.
All rights reserved.
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1.2 Patents
The product is protected under the following 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.
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2. General
2.1 About these operating instructions
The main focus of these operating instructions is directed to the safety instructions that must
be observed while working with the laser scanning microscope.
In addition, these operating instructions provide a rough overview of the operating principle of
laser scanning microscopes. It presents you with the first steps for starting and startup of the
system and provides a description of the software Leica Application Suite
Advanced Fluorescence (LAS AF).
The Leica TCS SP5 is supplied with the latest version of the licensed Leica Application Suite
Advanced Fluorescence. To maintain information on the most current level, the description of
software functions was intentionally omitted from these operating instructions. Instead, you
are referred to the online help of the Leica Application Suite Advanced Fluorescence in which
you can obtain the most up-to-date explanations and instructions about the corresponding
software functions.
Please read the chapter "Introduction to the help of the Leica Application Suite Advanced
Fluorescence" in these operating instructions to familiarize yourself first with setup and
operation. Please refer to the online help for further information on particular functions.
2.2 Intended use
The system was designed for confocal recording (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 according to the medical products law are excluded from
proper intended use.
The manufacturer assumes no liability for any improper intended use and for use outside the
specifications of Leica Microsystems CMS GmbH as well as any risks resulting from it. In
such cases, the declaration of conformity becomes invalid.
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3. TCS SP5 specifications
3.1 System overview
Figure 1: System components (overview)
1 TCS SP5 Scanner
2 Main switch board
3 TCS workstation
4 Supply unit
5 Control panel
6 Microscope stand
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3.2 Dimensions
3.2.1 System with inverted stand:
1400 mm
600 mm 730 mm
1200 mm
2540 mm
Figure 2: Dimensions of the TCS SP5 with inverted stand
3.2.2 System with upright stand
600 mm
730 mm
2400 mm
1490 mm
1200 mm
Figure 3: Dimensions of the TCS SP5 with upright stand
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3.3 Requirements regards ambient conditions
Do not expose the system to draft.
Ensure that the system is not installed next to air conditioners or ventilation systems. For this
reason, the installation site should be carefully planned.
Ensure that the environment is as dust-free as possible.
Please also read the notes on protection against dust in the chapters on maintenance and
cleaning.
Installing the system in darkened rooms is also advisable.
For installation, maintenance and transport, the TCS SP5 system requires doors with inside
spans of 1.00 m.
With regard to the load-bearing capacity of the floor, please note that the system will apply a
static load of 200 kg/m².
3.4 Permissible ambient conditions
Permissible temperature range for operation:
+18 to +25 ºC
Temperature range for optimum optical
behavior: +22°C to ±1°C
Permissible relative humidity: 20 - 80 % (non-condensing)
Permissible vibration speeds:
Frequency range [5 Hz–30 Hz]:
Frequency range [larger 30 Hz]:
< 30 μm/s (RMS)
< 60 μm/s (RMS)
Pollution degree: Class 2
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3.5 Electrical connections
Supply voltage: 100 - 240 V AC ± 10 %
Frequency: 50/60 Hz
Power consumption: 3200 VA
Overvoltage category: II
The building installation must feature three separate power connections with the following
fuse protection:
•3 x 100 V - 120 V mains at 20 A
or
•3 x 200 V - 240 V mains at 12 - 16A
For the specifications of external lasers such as UV and MP lasers, please refer to the
manufacturer's documentation.
3.6 Waste heat/required cooling performance
The TCS SP5 system features a maximum power consumption of 3.2 kW (VIS system) or
6.2 kW (MP system), respectively.
For the specifications of external lasers such as UV and MP lasers, please refer to the
manufacturer's documentation.
3.7 Required safety measures for VIS and UV systems
Please observe valid country-specific regulations pertaining to laser safety measures for
laser class 3B/IIIb. The operator is responsible for observing the laser safety regulations.
3.8 Required laser safety measures for MP systems
Please observe valid country-specific regulations pertaining to laser safety measures for
laser class 4/IV. The operator is responsible for observing the laser safety regulations.
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4. Safety instructions and their meaning
DANGER
This kind of warning alerts you of an operating procedure, practice, condition, or
statement in the operating instructions that must be strictly observed and followed,
as otherwise you expose yourself to the risk of fatal injury.
WARNING! LASER RADIATION
A laser warning points out an operation, a process, a condition or an instruction
that must be observed strictly to prevent serious eye injuries to the persons using
the system.
WARNING! HIGH VOLTAGE
A high-voltage warning points out an operation, a process, a condition or an
instruction that must be observed strictly to prevent possible injury or death of the
persons using the system.
WARNING! HARMFUL SUBSTANCES
A harmful substances warning points out a substance that can be harmful to your
health.
CAUTION
A safety note points out an operation, a process, a condition or an instruction that
must be observed strictly to prevent severe damage to the system or loss of data.
Notes either contain additional information on a specific topic or special
instructions on the handling of the product.
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5. Safety instructions
The instrument is Class 3B or 4 laser equipment (depending on the laser used) in
accordance with IEC/EN 60825-1.
This laser equipment may be operated only by persons who have been instructed
in the use of the system and the potential hazards of laser radiation.
As it is not possible to anticipate every potential hazard, please take care and apply common
sense to the installation, operation and maintenance of this product. Observe all safety
precautions relevant to Class 3B/IIIb lasers and Class 4/IV (MP systems).
Do not deviate from the operating and maintenance instructions provided herein.
The failure to observe these instructions shall be exclusively at the user's own risk and may
void the warranty.
5.1 Which standards does this product meet?
This device was 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 device. Operating this device on a public low-voltage
grid may result in radio interference. The operator must take suitable
measures should this occur.
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 for voltage fluctuations and flicker in low-voltage
networks.
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For use in the USA:
CDRH 21 CFR 1040.10:
Laser Products U.S. 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 scope of the CDRH/FDA (USA), the designation Laser Class 3B in the text must be
replaced by IIIb and Class 4 by IV.
5.2 Which laser class does the product have?
Laser
type Wavelength range Configuration Laser class
VIS 400 - 700nm,
(visible laser radiation)
Laser combinations as of
chapter 5.10
(except laser wavelengths
350 - 400nm)
3B/IIIb
UV
350 - 700nm,
(visible and invisible
laser radiation)
Combination of lasers
from Chapter 5.10
(VIS and UV lasers)
3B/IIIb
MP
350 - 1050nm,
(visible and invisible
laser radiation)
Combination of lasers from
chapter 5.10 (VIS/UV lasers)
and chapter 5.11 (IR lasers)
4/IV
5.3 Laser class for VIS and UV systems
According to IEC/EN 60825-1, this laser scanning microscope is a laser device of Class 3B.
Never expose eyes or skin to direct radiation! The laser light can cause permanent
eye damage!
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5.4 Laser class for MP systems
According to IEC/EN 60825-1, this laser scanning microscope is a laser device of Class 4.
Never expose eyes or skin to direct or indirect radiation! Laser light can cause
permanent eye damage and skin injuries!
5.5 What should the user of the laser scanning microscope observe?
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 liable 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.
This laser equipment may be operated only by persons who have been instructed
in the use of the system and the potential hazards of laser radiation.
The user is obligated to perform and monitor suitable safety measures according
to IEC / EN 60825-1 and the corresponding national regulations.
To assure classification as a 3B or 4 laser product according to IEC/EN60825-1
and electrical safety compliance, all safety devices, interlocks, and safety systems
of the laser device must be in operational condition.
Deactivating or damaging these safety devices or any intervention in any of these
safety devices may lead to serious eye injuries, physical injuries or property
damages. In these cases, Leica Microsystems CMS GmbH does not assume any
liability.
According to IEC/EN 60825-1: "Safety of laser products, Part 1: Equipment
classification, requirements and user's guide," the user is required to designate a
Laser Safety Officer or a Laser Protection Advisor.
Repairs and servicing may only be performed by authorized Leica Microsystems
CMS GmbH service personnel.
The user is fully liable for all consequences resulting from the use of the system if
it is opened, improperly serviced or repaired by other persons than authorized
Leica service representatives.
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If repairs or service measures are performed that require opening parts of the
housing, only trained Leica service technicians may occupy the room in which the
laser scanning microscope is located.
Leica Microsystems CMS GmbH will not be liable for damages resulting from non
observance of the above information. The above information does not, in any way, implicitly
or explicitly, modify the warranty and liability clauses contained in the general terms and
conditions of Leica Microsystems CMS GmbH.
5.6 Safety Notes for the User
Read and observe the safety notes in the Operating Instructions and the safety labels
located on the system. Failure to observe the safety notes may lead to serious injuries and to
significant damages to the system and loss of data.
This laser equipment may be operated only by persons who have been instructed
in the use of the system and the potential hazards of laser radiation.
Before performing operating steps for the first time with the system, read the
corresponding description of the function in the online help first.
You can get an overview of the single functions in the contents file of the online
help.
Do not connect any external equipment.
Connect only those electrical devices to the product that are listed in the Operating
Instructions. Otherwise, please contact your local Leica service agency or Leica
Microsystems CMS GmbH.
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