Leica TCS SP User manual

True Confocal Scanner
Leica TCS SP
User Manual

Contents
Contents ............................................................................................................................................. 2
General............................................................................................................................................... 3
How to Use this Manual................................................................................................................... 3
Confocal imaging ................................................................................................................................ 5
Help via the nternet ........................................................................................................................ 7
Legal Notes ..................................................................................................................................... 8
Safety Notes...................................................................................................................................10
Starting the system ............................................................................................................................22
Starting the Operating System........................................................................................................22
Setting Up Users ............................................................................................................................26
Starting the Confocal System .........................................................................................................27
Setting the Köhler llumination ........................................................................................................34
Maintenance and Cleaning .............................................................................................................36
The Leica Confocal Software: Overview.............................................................................................38
Opening and Saving Data Sets.......................................................................................................40
Keyboard Shortcuts ........................................................................................................................41
Menu Functions..............................................................................................................................42
LCS File Formats............................................................................................................................45
Specification of the "Lei" File Format (Version beta 2.000) .............................................................47
General..............................................................................................................................................54
ntroduction to the Leica Confocal Software Help ...........................................................................55
Opening the Context-Sensitive Help ...............................................................................................55
Procedures and Exercises for Selected Processes ............................................................................63
Setting nstrument Parameters for the First mage Recording.........................................................63
Creating User-Defined nstrument Parameter Settings ( PS) ..........................................................69
Setting nstrument Parameters Using the Control Panel .................................................................75
Recording Spatial mage Series .....................................................................................................78
Recording Time mage Series ........................................................................................................87
Recording Lambda Series (Spectral mage Series) ........................................................................95
Recording Sequential mage Series..............................................................................................102
Recording mages of Regions of nterest (RO Scanning).............................................................112
Linking Single mages or mage Series.........................................................................................116
Separating Single mages or mage Series...................................................................................124
Data Recording Functions................................................................................................................132
Data Viewing Functions ...................................................................................................................165
Data Processing...............................................................................................................................212
Measurement and Analysis Functions..............................................................................................238
Data Documentation ........................................................................................................................249
Data Handling ..................................................................................................................................253
User-Specific Adjustments ...............................................................................................................256
Optional Software Packages ............................................................................................................257
Material (optional).........................................................................................................................257
Appendix..........................................................................................................................................260
Adaptation Function for Correcting Trend Curves .........................................................................260
Operating Principle of Boolean Operations...................................................................................262
Specifications...................................................................................................................................275
Dimensions of the Leica TCS SP..................................................................................................275

Leica Microsystems Heidelberg GmbH
General
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
Page 3 of 278
General
How to Use this Manual
About this book
This manual is both a tutorial and a reference manual for the Leica TCS SP. We intend this manual
for the novice and the experienced user. f you are a new user, you should start with the chapter
"Running the LCS Software," use the software for a while, and then read through the rest of the
manual. Experienced users can search through the chapters, table of contents, and index to find
special topics explained.
This book also covers the way your Leica Confocal Software and the Leica Microscope interact.
How to use this manual
We urge you to use this book, not just read it. To learn about the Leica Confocal Software, use the
book as a tutorial. We show how to accomplish a variety of tasks. Work through the instructions to
learn. The manual usually introduces a set of steps to accomplish a particular task with a distinctive
heading.
Performing a task
Following the heading that introduces a task are either sequential steps or optional steps, each with
its distinctive style:
1. Numbered paragraphs, like this one, designate step-by-step instructions. Follow them to learn how
the tools work.
2. f several menu commands must be selected in sequence, an arrow separates the individual menu
commands: for example, File →
→→
→Save indicates that you click on File and select Save in the displayed
pull-down menu.
Usually there is a sequence. Perform one or more of the steps to cause the designated action or
actions.
We recommend reviewing this manual again after you have used the Leica TCS SP for a while. You
will come across useful functions during the second reading that may have been overlooked during
the first reading.
Online Help
Leica TCS SP comes with a context-sensitive online help system that explains the different functions
of the instrument.
To retrieve the contents of the online help system:
Select Help
→
→→
→ Contents.The online help system opens with an activated CONTENTS window. t
shows a collapsible table of contents. Double-click an entry of the table of contents to view the
corresponding information.
To retrieve information using a keyword:
Select Help →
→→
→Index.The online help system opens with an activated NDEX window. Enter the word
you want to look up. The online help shows the key word that represents the closest match to the
word you entered.
To perform a full-text-search:
Select Help →
→→
→Search.The online help system opens with an activated SEARCH window. Enter the
term or definition you want to look up and click on the L ST TOP CS button. A hierarchically structured
list of topics is returned. On the right side of the field where you enter the word is a menu to perform a
full-text search using logical operators.

Leica Microsystems Heidelberg GmbH
General
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
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ocumentation Conventions
Button
The buttons provided on the user interface of the Leica Confocal Software. Buttons are marked with
icons and/or have an (often abbreviated) English label. They either trigger actions directly or open
dialog windows.
Menu
The menus are divided into the categories of File, View, Macro, Tools, Window and Help and are
displayed in the menu bar located at the upper edge of the user interface.
Option
Options refer to the selectable items that are hierarchically listed below the menus. Options either
trigger actions directly or open dialog windows.
ialog Window
Both buttons and options open dialog windows. Dialog windows are used to set various parameters
and select functions.
Tab
Tabs are found in dialog windows. Tabs thematically combine the parameters and functions that can
be configured in dialog windows. Some registers are divided into fields.
Viewer window
The Leica Confocal Software offers two types of viewer windows: The Viewer window is called up
using the New button and shows the recorded images. The Experiment Overview viewer window
displays the recorded images in a directory tree. This viewer window is called up from the View menu
and appears as a separate window at the left side of the user interface.
Legend
The Leica Confocal Software provides two legends, which display the parameters and settings of an
image recording. The Experiment legend can be placed at the right edge of the Viewer window. The
Hardware legend is called up from the View menu and appears as a separate window at the left side
of the user interface.
Context Menu
Context menus appear when you click the right mouse button while holding the mouse pointer over
certain areas of the user interface. Context menus contain various, context-sensitive commands.

Leica Microsystems Heidelberg GmbH
General
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
Page 5 of 278
Confocal imaging
What is confocal imaging?
Conceptualized in 1953, the Confocal Laser Scanning Microscopy has only in the past 10 years
become a practical technique. Today it is the technique of choice for biological research, chemical
analysis, and materials testing. The results of many years of research and development in many
different areas are combined in such an instrument: microscopy, laser technology and optics for
coherent light, video technology, electronics and computer technology.
Confocal microscopy detects structures by collecting light from a single focal plane of the sample,
excluding light that is out of focus.
n a point scanning confocal system, the microscope lenses focus the laser light on one point in the
specimen at a time (the focal point). The laser moves rapidly from point to point to produce the
scanned image. Both fluorescent and reflected light from the specimen pass back through the
objective.
The microscope and the optics of the scanner module focus the light emitted from the focal point to a
second point, called the confocal point. The pinhole aperture, located at the confocal point, allows
light from the focal point to pass through the detector. Light emitted from outside the focal point is
rejected by the aperture.
The confocal principle is illustrated schematically for the epi-illumination imaging mode.
As in conventional epifluorescent microscopes, one lens is used as both condenser and objective.
The advantage is eliminating the need for exact matching and co-orientation of two lenses. A
collimated, polarized laser beam from an aperture is reflected by a beam splitter (dichroic mirror) into
the rear of the objective lens and is focused on the specimen. The reflected light returning from the
specimen passes back through the same lens. The light beam is focused into a small pinhole (i.e., the
confocal aperture) to eliminate all the out-of-focus light, i.e., all light coming from regions of the
specimen above or below the plane of focus. The achieved optical section thickness depends on
several parameters such as the variable pinhole diameter and the wavelength. The in-focus
information of each specimen point is recorded by a light-sensitive detector (e.g., a photodiode)
positioned behind the confocal aperture. The analog output signal is digitized and fed into a computer.
The detector is a point detector and only receives light from one point in the specimen. Thus, the
microscope sees only one point of the specimen at a time as opposed to the conventional microscope
where an extended field of the specimen is visible at one moment. Therefore, to obtain an image it is
necessary either to move the illuminated point or to move the specimen. These two possibilities have
given rise to two different types of confocal microscopes:
Microscopes with movable objective stage (stage scanning): The objective stage with the specimen is
moved forward after every finished recording, while the optical system remains stationary with this
type of scanning.
Microscopes with beam or mirror technology: The illuminated point is scanned over the fixed
specimen using small, fast, galvanometer-driven mirrors as used by LE CA.
The LE CA TCS SP system allows you to image a single focal plane as well as a series of planes—
horizontal or vertical. A single vertical section or xz-scan allows for a side view of the specimen.
f a sequence of optical sections of the specimen is combined to form an image stack and then
digitally processed, it offers the advantage of using this multidimensional data set to create either a
calculated two-dimensional image (projection) or a reduced scale 3D representation of the specimen
using a suitable computer.
Optical resolution
The term resolution refers to the capability of distinguishing finest details in a structure. n a perfect
microscope, the optical system would be free of any type of aberration. n such a hypothetical
instrument, the resolution would be limited only through diffraction. One could express this as the
smallest distance between two points of a specimen at which they are still visible as two separate
points (Rayleigh criterion). Beyond this limit, the two points merge (i.e., their diffraction discs overlap
completely or partially) and can no longer be recognized as two different points. This distance can be
calculated using the size of the diffraction image of an infinitely small point of the specimen. t
corresponds to the radius of the first minimum in this diffraction image. This, in turn, is related to the
numeric apertures of the objective and the condenser. The numeric aperture is defined by the
diffraction index of the lens and the size of the luminous cone that may enter.
Analogous to the argumentation above, the axial resolution can be defined as the radius of the first

Leica Microsystems Heidelberg GmbH
General
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
Page 6 of 278
minimum along the microscope axis of the diffraction image of a point object. According to the theory
for such 3D diffraction images, the optical resolution along the z-axis is smaller than the lateral optical
resolution by a factor of 2. Thus, the optical resolution along the z-axis amounts to approximately one
half of the resolution within the focal plane.
The LE CA TCS SP microscope is a no-compromise true point-scanning system with extremely high
sensitivity and theoretical maximum x-, y- and z-resolution.
Scan resolution refers to image clarity as determined by the number of pixels and pixel size. The
larger the number of pixels and the larger the scanning format, the more easily two close objects can
be distinguished. The scan resolution is restricted to the maximum optical resolution power.
etection
Confocal imaging, or to be more precise, the measurement of the optical properties of tiny sub-
volumes of a specimen, is limited not only by the optical quality of the microscope. Other limitations
are:
- The measurement of continuous specimen only in discrete sub-volumes (because of sampling and
digitalization).
- The accuracy with which the sub-volumes are defined, determined by the scanning mechanism.
- The brightness of the light source in relation to the reflectivity of the specimen.
- The sensitivity and noise produced by the detector.
Another central component of the confocal microscope is the detector. Due to its very high signal-to-
noise ratio, LE CA Microsystems Heidelberg uses photomultipliers as detectors.
Image processing
n the first confocal microscopes, the detector was connected to an oscilloscope with long-persistence
phosphor which would display an image as it was being scanned. n the instruments of today, the
signal is digitized and recorded in a computer. This makes it possible to manipulate the image in a
multitude of ways. The following options are available:
- Contrast enhancement by thresholds, linear contrast stretching and gamma correction (curvature of
the image intensity value versus source intensity graph).
- Superimposition of images in experiments.
- Digital filtering for edge enhancement, smoothing, noise suppression, etc.
- Reconstruction of three-dimensional views from stacks of images of optical sections. This allows, for
instance, an image of an xz plane to be reconstructed from a stack of images of xy planes. Complete
3D models of the specimen can also be rendered and examined from any direction.
- Assembly of digital movies from time-sequences of microscope images.
- Quantification and Measurements.
Although this type of image manipulation does not improve the quality of the collected data, it serves
the purpose of improving the visibility and facilitating the qualitative interpretation of the data.
Light source
Lasers are extremely well suited as light sources for confocal microscopy because they emit a very
bright light and small divergence of the beam. n addition, they are easy to focus and stable in
intensity. The stability is especially important for quantitative measurements.

Leica Microsystems Heidelberg GmbH
General
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
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Integration
The Leica TCS SP was designed as an integrated system. Optical and mechanical elements work
seamlessly with computer hardware and software. The integrated Leica Confocal Software package
supports the complete imaging process, from optical sectioning, through image processing and
analysis (which is the main application), to hardcopy output.
Help via the Internet
First contact your local Leica subsidiary or the local dealer.
You can also find information on Leica Microsystems Heidelberg GmbH and confocal microscopy on
ourwebsiteat
http://www.confocal-microscopy.com.
Or send an e-mail to [e-mail].

Leica Microsystems Heidelberg GmbH
General
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
Page 8 of 278
Legal Notes
Version 2.3, March 14, 2003. Made in Germany.
© Copyright 2002/2003, Leica Microsystems Heidelberg GmbH. All rights reserved.
No part of this publication may be reproduced or transmitted in any form or by any means, electronic
or mechanical, including photocopying, recording, or storing in a retrieval system, or translating into
any language in any form without the express written permission of Leica Microsystems Heidelberg
GmbH.
ISCLOSURE
This document contains Leica Microsystems Heidelberg GmbH proprietary data and is provided solely
to its customers for their express benefit of safe, efficient operation and maintenance of the product
described herein. Use or disclosure of Leica Microsystems Heidelberg GmbH proprietary data for the
purpose of manufacture or reproduction of the item described herein, or any similar item, is prohibited,
and delivery of this document shall not constitute any license or implied authorization to do so.
REVISIONS AN CHANGES
Leica Microsystems Heidelberg GmbH reserves the right to revise this document and/or improve
products described herein at any time without notice or obligation. nformation and specifications in
this manual are subject to change without notice.
WARRANTY
Leica Microsystems Heidelberg GmbH provides this publication "as is" without warranty of any kind,
either expressed or implied, including but not limited to the implied warranties of merchantability or
fitness for a particular purpose. All reasonable precautions have been taken in the preparation of this
document, including both technical and non-technical proofing. Leica Microsystems Heidelberg GmbH
assumes no responsibility for any errors or omissions. Leica Microsystems Heidelberg GmbH shall
not be responsible for any direct, incidental or consequential damages arising from the use of any
material contained in this document.
TRA EMARKS
Throughout this manual, trademarked names may be used. Rather than including a trademark (™)
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.
SAFETY
The terms used of the laser safety class refer to the standard EN 60825-1. Within the
scope of the CD H, the laser class 4 should be replaced by IV in the text, the laser
class 3B by IIIb, the laser class 3A by IIIA, the laser class 2 by II and the laser class 1
by I.
This instrument is designed and manufactured to comply with applicable performance standards for
Class 3B laser products as defined by USHHS, CDRH/FDA, OSHA and EN standards and regulations
knowntobeeffectiveatthedateofmanufacture.
Every hazardous situation cannot be anticipated, therefore, the user must exercise care, common
sense, and observe all appropriate safety precautions applicable to Class 3B lasers and high-voltage
electrical equipment during installation, operation and maintenance.
Deviating from published operating or maintenance procedures is not recommended. Operation and
maintenance procedure changes are performed entirely at the user's risk.
SOFTWARE LICENSE

Leica Microsystems Heidelberg GmbH
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Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
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The software described in this document is furnished under a License Agreement which is included
with the product. This agreement specifies the permitted and prohibited uses of the product.

Leica Microsystems Heidelberg GmbH
For your safety
Leica TCS SP User Manual English
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Safety Notes
This section introduces the standard safety precautions, warnings and safety cautions.
General Safety irections
Your safety is extremely important. Read and observe all warnings and safety cautions listed in this
manual and attached to the instrument before operating any Leica instruments. Serious injuries can
occur, and equipment and data can be damaged if the safety warnings and safety cautions are not
followed.
Risk of Operation
The risk for the performance of the device is assumed by the operator or the owner of the product.
The owner or operator will be fully liable for all consequences which may result, if the device is
opened by persons other than authorized Leica service staff, if it is not serviced or maintained
properly or if it is used for purposes other than those described in the accompanying documentation
and the online help facility. Leica Microsystems Heidelberg 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 Heidelberg GmbH.
Warnings, Safety Cautions, and Notes
The warnings, safety cautions, and notes in this manual use the following format.
Such a warning alerts you of an operating procedure, practice, condition, or statement
that must be strictly observed to avoid death or serious injury to the persons working on
the equipment.
Such a safety caution alerts you to an operating procedure, practice, condition, or
statement that must be strictly observed to prevent equipment damage or destruction,
or corruption or loss of data.
Notes are statements that either provide extra information about a topic or contain
special instructions for handling a particular condition or set of circumstances.
Special Conditions of the Installation Site
Even though the system was tested and complies with the permissible limits of electromagnetic
radiation (EMC), a residual risk of effecting other devices cannot be ruled out.
This system must not be mounted next to medical life-saving equipment.

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Before moving the confocal system, it should be thoroughly cleaned. The same also
applies to the removal of components. This applies in particular to systems that are
located in biomedical research labs.
This is necessary to remove a possible contamination and, thereby, avoid carry-over of
dangerous substances and pathogens and its accompanying risk of persons. Pay not
only attention to surfaces, but especially to fans and cooling devices since dust can
frequently accumulate at these locations.
The use of the instrument for unintended purposes as well as unauthorized opening of
the instrument can create the risk of electrical voltage as well as laser radiation.
The terms used of the laser safety class refer to the standard EN 60825-1. Within the
scope of the CD H, the laser class 4 should be replaced by IV in the text, the laser
class 3B by IIIb, the laser class 3A by IIIA, the laser class 2 by II and the laser class 1
by I.
Laser Class
This instrument is designed and manufactured to comply with applicable performance standards for
Class 3B laser devices as defined by USHHS, CDRH/FDA, OSHA and EN 60825-1 standards and
VBG 93 regulation for prevention of accidents known to be effective at the date of manufacture.
Every hazardous situation cannot be anticipated by the manufacturer, therefore, the user must
exercise care and common sense, and observe all appropriate safety precautions applicable to Class
3B lasers and high-voltage electrical equipment during installation, operation and maintenance.
Operation and maintenance procedure changes are performed entirely at the user's risk.
The Leica TCS SP used in accordance with the instructions is harmless.
The user is responsible for compliance with current safety regulations. For safety
reasons, only trained Leica service personnel is permitted in the laser area.
Technical Safety Measures
n accordance with the laser safety directives EN 60825 (European standard) and CDRH (US-
American standard) Leica Microsystems Heidelberg GmbH employs various safety measures.
Warning labels have been affixed near all apertures or removable parts where exposure to laser light

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Part no.: 159330193 / Vers.: 31102002
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is possible.
Furthermore, several laser interlock-switches were installed.
Observe the specification of the standard EN 60825-1: "Safety of laser products, Part 1. Equipment
classification, requirements and user's guide", which requires that a laser safety officer or a laser
protection advisor must be named for installations using instruments of class 3B. t is the user's
responsibility to review and designate appropriate controls for the use of the equipment.
Never look directly into a laser beam or a reflection of the laser beam. Exposure to the
laser beam can cause irreversible damage to your vision. A reflected laser beam is just
as intensive as a direct beam.
The user is responsible for the safe operation and maintenance of this instrument at all
times. Class 3B laser product and electrical safety compliance is assured only when all
safety devices, interlocks, and laser containment systems are in serviceable condition
and operating. Disabled or damaged safety devices or systems will expose personnel to
Class 3B laser radiation of sufficient power to cause severe eye injury, burns or
property damage.
Every interference with or any deactivation of one of the safety systems and/or one of
the safety locks results in the loss of warranty claims.
Power cord set
The power cord set supplied with your instrument meets the requirements of the country where you
purchased the instrument. f you use the instrument in another country, you must use a power cord
set that meets the requirements of that country.
The following wiring is specified by the manufacturer:
The power cord of the microscope must be plugged into the supplied connector strip. f a high-
pressure mercury lamp is used, it should be connected to a separate power supply. This prevents it
from continuously being switched on and off together with the control computer and the scan
electronics and, therefore, increases the service life of the source of illumination.
This equipment is designed for connection to a grounded (earthed) outlet. The
grounding type plug is an important safety feature. To reduce the risk of electrical shock
or damage to the instrument, do not disable this feature.
To reduce the risk of fire hazard and electrical shock, do not expose the unit to rain or
humidity. Do not open the cabinet. All maintenance is to be performed by an authorized

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Leica technician.
Do not allow any liquid to enter the instrument cabinet, or come into contact with any
electrical components. The instrument must be thoroughly dry before you connect
power, or turn the instrument on.
General Safety Notes
n accordance with general safety regulations as well as laser device safety regulations comply with
the following stipulations:
Observe the operating instructions!
Use the product only according to the information given in this documentation and in the online help
facility of the Leica Confocal Software. Carefully read the complete operating instructions before you
start the system. As a preparation for your single work steps always read the relevant description of
function in the online help and in particular observe the safety regulations indicated for handling the
system. You can get an overview of the single functions in the contents file of the online help.
Use the product only for purposes described in this documentation and in the online help
facility of the Leica Confocal Software.
The system is designed for making confocal laser scan images and quantitative measurements in the
discipline of material science. Using the Leica TCS SP for any other purposes or applications is
inexpert and inadmissible handling. The user assumes the entire risk for all experiments with this
system and for the consequences resulting from such experiments. This is particularly true if the
device has been opened or modified by the user.
Observe the maintenance instructions.
Please read the chapter 'Maintenance and Cleaning' in this manual.
o not perform any adjustments on the equipment that are not described in this manual or in
any subsequent addendum to the manual.
Only service technicians authorized by Leica Microsystems Heidelberg GmbH are allowed to carry out
repair and service work.
Electrically connect only those electrical devices to the product that are mentioned in this
accompanying documentation. Never electrically connect devices that are not mentioned in
this documentation.
Before connecting any such devices to the product, consult the local Leica Service Agency or Leica
Microsystems Heidelberg GmbH directly.
o not use an S70 microscope condenser.
n the worst-case scenario, the large working distance and the low numeric aperture of the S70
condenser could result in impermissible high laser radiation loads if the safety notes are not observed
(see "Special Safety Notes for Users"). Therefore, only S1 and S23 Leica condensers should be used.
Special Safety Remarks for Users
Leica Microsystems Heidelberg GmbH has done everything possible to maximize user safety and
minimize health risks applying special safety measures in accordance with several safety directives.
The user must operate the TCS SP in accordance with the instructions in order to ensure safety.
Several important points are listed below:
o not change objectives during scanning operations
How to change objectives:
Ensure that the Scan mode is switched off. Be sure that there is no laser light in the focal plane.
Turn the objective nosepiece so that the objective lens to be exchanged appears outside of the optical
axis and points outwards. Do not apply any force to the objective nosepiece, as this may permanently
reduce the resolution of the turret scan.
Screw in the new objective lens and turn the objective nosepiece back into the optical axis.

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All unused positions on the objective nosepiece must be capped!
o not insert objects into the beam path during scanning operations!
Insert the specimen correctly into the specimen holder!
o not change specimen during scanning operations!
How to exchange a specimen:
Ensure that the Scan mode is switched off. Be sure that there is no laser light in the focal plane. Close
the safety shutter at the tube.
Laser Safety
The word 'LASER' is an acronym for Light Amplification by Stimulated Emission of Radiation. The first
laser was demonstrated in 1960 and used a ruby as the lasing medium. Lasers have been used in
many applications from surgery to bar code readers at supermarket checkouts, laser pointers, laser
printers to CD players.
Lasers can produce a very intense and very narrow (collimated) beam of electromagnetic radiation in
the wave range of 150 nm to 1 mm. This radiation is generally in the form of intense visible light.
Because laser light is not an ionizing type of radiation, interaction with matter is generally at the
surface. The eye and the skin are very sensitive organs for laser radiation exposure. The resultant
effects vary depending on the type of laser (wavelength of the radiation) and beam energy output.
Laser energy of the proper wavelength and power may be focused by the lens of the eye onto the
retina causing irreversible damage. f laser radiation energy is high enough, skin burns may also
result if extremities or other body parts are placed in the laser beam. The following table summarizes
the various regions of the electromagnetic spectrum produced by lasers and the organs that are at
risk if exposure occurs.
Spectrum Region Wavelength Range Organ Effected
UV-C 100 nm to 280 nm All absorbed in cornea and conjunctiva
Ultraviolet UV-B 280 nm to 320 nm Almost all absorbed in cornea and conjunctiva
UV-A 320 nm to 380 nm Lens (lens cloudiness)
Visible 380 nm to 760 nm Retina
R-A 760 nm to 1400 nm Retina, lens, skin
nfrared R-B 1400 nm to 3000 nm Cornea and skin
R-C3000nmto10
6nm Cornea and skin
Lasers are frequently identified based on the type of laser medium. The Laser Hazard Classification is
determined by the wavelength(s), maximum duration of exposure, and the average power output of
the laser.
Laser hazard classification
Never look directly into a laser beam regardless of the type of laser and the power
used.
Class 1
Harmless laser (usually embedded).
Class 2
Low power visible laser which, due to the reflex response, does not normally present a hazard unless
viewed directly.
Class 3A
Lasers that are generally harmless if inadvertently viewed directly, including intrabeam viewing;
however, these lasers may represent a danger if collector optics are used.
Class 3B

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Lasers that can produce an eye injury if viewed directly, including intrabeam viewing of specular
reflections.
Class 4
Laser that can produce an eye injury or skin burns from direct, specular or diffuse reflections. Danger
of burns may develop.
Embedded systems
Class 2, 3A, 3B or 4 lasers or laser systems contained in a protective housing and operated in a lower
classification mode may be classified at a lower classification. Specific control measures may be
required to maintain the lower classification.
Laser Hazards
The danger from lasers can be divided into the following major categories:
1. Eye injuries such as burns of the retina or cornea.
ETINAL INJU Y IS I EVE SIBLE
2. Skin hazards such as burns.
3 Electrical hazards from high voltage equipment.
4. Fire and explosion hazard.
Specification of laser radiation in use
Laser
type
Wavelength
[nm]
Maximum luminous power at
laser output [mW]
Maximum luminous power in
focal plane [mW]
Pulse duration
Ar-UV 351, 364 < 50 < 2.5 continuous wave
(non-pulsed)
Diode 405 < 25 < 4 continuous wave
(non-pulsed)
Diode 430 < 10 < 4 continuous wave
(non-pulsed)
HeCd 442 < 30 < 3 continuous wave
(non-pulsed)
ArKr 488, 568, 647 < 125 < 10 continuous wave
(non-pulsed)
Ar 458, 476, 488,
514
< 200 < 50 continuous wave
(non-pulsed)
HeNe 543 < 1.5 < 0.5 continuous wave
(non-pulsed)
Kr 568 < 25 < 5 continuous wave
(non-pulsed)
HeNe 594 < 3 < 1 continuous wave
(non-pulsed)
HeNe 633 < 15 < 4 continuous wave
(non-pulsed)

Leica Microsystems Heidelberg GmbH
For your safety
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
Page 16 of 278
The following laser safety labels are used
1. at all openings where light can be emitted
For laser emission in the visible spectral region
(Ar/ ArKr/ Kr/ HeNe/ HeCd/ diode)
For laser emission in the ultraviolet spectral region
(Ar-UV)
LASER RADIATION
I
S
E
M
I
T
T
E
D
F
R
O
M
T
H
I
S
A
P
E
R
T
U
R
E
AVOID EXPOSURE
VISIBLE AND INVISIBLE
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE
VISIBLE AND INVISIBLE
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE
2. on all housing parts that can be opened with tools and emit laser radiation
For laser emission in the visible spectral region
(Ar/ ArKr/ Kr/ HeNe/ HeCd/ diode)
For laser emission in the ultraviolet spectral region
(Ar-UV)
LASER RA IATION WHEN OPEN
AVOI EXPOSURE TO BEAM
DANGER
VISIBLE AN INVISIBLE
LASER RA IATION WHEN OPEN
AVOI EXPOSURE TO BEAM
DANGER
3. on the cover of the supply unit
For laser emission in the visible spectral region
(Ar/ ArKr/ Kr/ HeNe/ HeCd/ diode)
For laser emission in the ultraviolet spectral region
(Ar-UV)
AVOID DIRECT EXPOSURE TO BEAM
< 15 mW HeNe 633 nm
CLASS IIIb LASER PRODUCT
LASER RADIATION
AVOID DIRECT EXPOSURE TO BEAM
< 400 mW ArKr 457-675 nm
CLASS IIIb LASER PRODUCT
LASER RADIATION

Leica Microsystems Heidelberg GmbH
For your safety
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
Page 17 of 278
AVOID DIRECT EXPOSURE TO BEAM
< 400 mW ARGON 457-514 nm
CLASS IIIb LASER PRODUCT
LASER RADIATION
AVOID DIRECT EXPOSURE TO BEAM
< 400 mW KRYPTON 568 nm
CLASS IIIb LASER PRODUCT
LASER RADIATION
AVOID DIRECT EXPOSURE TO BEAM
< 500 mW Ar-Kr-HeNe 457-633 nm
CLASS IIIb LASER PRODUCT
LASER RADIATION
AVOID DIRECT EXPOSURE TO BEAM
< 500 mW Ar-Kr-HeNe-HeCd 442-633 nm
CLASS IIIb LASER PRODUCT
LASER RADIATION
On a UV laser (optional):
V S BLE AND NV S BLE
LASER RAD AT ON S EM TTED
FROM TH S APERTURE AVO D EXPOSURE
INVISIBLE AN VISIBLE
LASER RA IATION. AVOI
EYE OR SHIN EXPOSURE
TO IRECT OR SCATTERE
RA IATION. CLASS 4
LASER PRO UCT PER
EN60825-1 (1994)
ARGON ION LASER
2 WATTS MAX CW
TH S PRODUCT COMPL ES W TH DHHS RAD AT ON PERFORMANCE STANDARDS
21 CFR SUBCHAPTER J EXCEPT W TH RESPECT TO THOSE CHARACTER ST CS
AUTHOR ZED BY VAR ANCE NUMBER 89V-0201 EFFECT VE JULY 1998.
MOD F CAT ON OR USE AS A COMPONENT OF ANOTHER PRODUCT MAY
RESULT N T NOT COMPLY NG W TH THE ABOVE REGULAT ONS.
5100 PATR CK HENRY DR VE SANTA CLARA CA 95054 MADE N U.S.A.
Installation on the laser itself:
Confocal systems operated with a UV laser feature a special microscope optics. For
this reason, retrofitting a confocal system designed for the visible spectral range with a

Leica Microsystems Heidelberg GmbH
For your safety
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
Page 18 of 278
UV laser cannot be done easily.
Position of laser safety labels
1. Inverted microscope
Location 1 and Location 2 mark
the mounting locations of the
laser safety labels on the
inverse microscope stand.
Location 1 for systems W THOUT UV laser
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE
for systems W TH UV laser
VISIBLE AND INVISIBLE
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE
Location 2 for systems W THOUT UV laser
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE
for systems W TH UV laser
VISIBLE AND INVISIBLE
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE

Leica Microsystems Heidelberg GmbH
For your safety
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
Page 19 of 278
2. Upright microscope
Location 1 and Location 2 mark
the mounting locations of the
laser safety labels on the
upright microscope stand.
Location 1 for systems W THOUT UV laser
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE
for systems W TH UV laser
VISIBLE AND INVISIBLE
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE
Location 2 for systems W THOUT UV laser
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE
for systems W TH UV laser
VISIBLE AND INVISIBLE
LASER RADIATION
IS EMITTED
FROM THIS APERTURE
AVOID EXPOSURE

Leica Microsystems Heidelberg GmbH
For your safety
Leica TCS SP User Manual English
Part no.: 159330193 / Vers.: 31102002
Page 20 of 278
3. Scan head
Mounting locations of the
laser safety labels on the
scan head
.
for systems W THOUT
UV laser
LASER RA IATION WHEN OPEN
AVOI EXPOSURE TO BEAM
DANGER
for systems W TH UV
laser
VISIBLE AN INVISIBLE
LASER RA IATION WHEN OPEN
AVOI EXPOSURE TO BEAM
DANGER
4. Supply unit (cover)
for systems with HeNe laser
LASER RA IATION WHEN OPEN
AVOI EXPOSURE TO BEAM
DANGER
for systems with ArKr laser