Leica TCS SP II True Confocal Scanner User Manual

1 Table of Contents 1 2 3

Table of Contents ... 2 Legal notes ... 3 Usage of this manual... 4 3.1 Do a task... 4 3.2 Online help... 5 4 Confocal imaging... 5 5 Safety remarks ... 8 5.1 General Safety Directions... 8 5.2 Risk of Operation ... 8 5.3 Warnings, Cautions, and Notes... 8 5.4 Laser Class... 9 5.5 Technical Safety Measures ... 10 5.6 WARNINGS ... 10 5.7 Power Cord Set Requirements... 11 5.8 General Safety Notes ... 11 5.9 Special Safety Remarks for Users ... 12 5.10 Laser Safety... 13 5.11 Laser Hazard Classification and Laser Safety Control Measures... 14 5.12 Laser Hazards ... 14 6 Maintenance and cleaning ... 15 6.1 Selecting an installation site ... 15 6.2 Protect the microscope against dust and grease ... 15 6.3 Be careful when using aggressive chemicals... 15 6.4 Cleaning the optical system of the microscope ... 15 6.5 Cleaning the microscope surface ... 16 7 The Leica Confocal Software: An overview ... 16 7.1 Starting the software... 16 7.2 The experimental concept of the software ... 17 7.3 Basic structure of the user interface... 17 7.4 Opening records ... 19 7.5 Saving images ... 19 7.6 Organizing data by grouping experiments... 20 7.7 Compiling experiments ... 20 8 Starting the Operating System Windows NT™... 20 8.1 Using a Mouse... 21 8.2 The Windows NT interface ... 22 8.3 The Start menu ... 23 8.4 Starting a Program ... 23 8.5 The Taskbar... 24 8.6 Setting the Time and Date... 25 8.7 Getting Help... 25 8.8 Shut Down Windows NT... 26 9 Help from the Internet... 26 10 Softwarefunctions ... 27 10.1 Software functions for data acquisition... 27 10.2 Softwarefunctions for data display... 52 10.3 Software functions for measurements and analysis... 77 10.4 Softwarefunctions for documentation of data ... 80 10.5 Softwarefunctions for data handling ... 84 10.6 Softwarefunctions for customization... 88 11 Glossary ... 90

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2 Legal notes Version 1.4, May 2000, Made in Germany. © Copyright 1999/2000, 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 or its authorized agent. DISCLOSURE 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 Leica Microsystems Heidelberg GmbH reserves the right to revise this document and/or improve products described herein at any time without notice or obligation. Information 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 express 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. TRADEMARKS Throughout this manual, trademarked names may be used. Rather than put a trademark (™) symbol in 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.

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SAFETY This instrument is designed and manufactured to comply with applicable performance standards for Class 1 laser products as defined by USHHS (Class 1 laser products shall not emit hazardous laser radiation during normal customer operation), CDRH/FDA, OSHA and EN standards and regulations known to be effective at the date of manufacture. Every possible hazardous situation cannot possibly be anticipated, therefore, the user must exercise care, common sense, and observe all appropriate safety precautions applicable to Class IIIb lasers and high-voltage electrical equipment during installation, operation and maintenance. Deviation from published operating or maintenance procedures is not recommended. Operation and maintenance procedure changes are performed entirely at the user’s risk. SOFTWARE LICENSE 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.

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3 Usage of 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 book usually introduces a set of steps to accomplish a particular task with a distinctive heading.

3.1 Do 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. Menu commands are shown with an arrow between the single items, for example, File→ →Save means you should pull down the File menu and choose the Save option. Paragraphs with a diamond bullet, like this one, are optional steps. Usually there is a sequence. Do one or more of the steps to cause the designated action or actions. We recommend that you review this book again after you have used a Leica TCS SP II for a while. You will discover useful features that you breezed over the first time.

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3.2 Online help Leica TCS SP II comes with a context sensitive online help system that teaches you the different functions of the system. To retrieve the contents of the online help system: Choose Help → Contents. The online help system opens with an activated CONTENTS window. It shows a collapsible table of contents. Double-click an entry of the table of contents to show the corresponding information. To retrieve information using a keyword: Choose Help→ →Index. The online help system opens with an activated INDEX window. Enter a word you would like to look for and the online help system will show the most similar keyword defined. To perform a full-text-search: Choose Help→ →Search. The online help system opens with an activated SEARCH window. Enter a word you would like to look for, press the LIST TOPICS button and the online help system will show a ranked list of topics. On the right side of the field where you enter the word, there is a fly out menu to perform fulltext search using logical operators.

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4 Confocal imaging 4.1.1 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. An instrument of this kind represents a „fusion product“ of contributions from many fields: 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. In 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 sample 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 epiillumination imaging mode.

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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 (i.e., a photo-diode) positioned behind the confocal aperture, and 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: 1.The stage-scanning type in which the microscope stage with the specimen is moved in a scanning motion while the optics remain stationary. 2. The beam-scanning or mirror-scanning type in which the illuminated point is scanned over the fixed specimen by small, fast, galvanometer-driven mirrors as used by LEICA. The LEICA TCS SP II system makes it possible to image a single, in-focus plane – horizontal or vertical – as well as a series of planes. A single vertical section, or xz-scan, allows you to see your sample as though from the side. The advantage of having a stack of serial optical sections through the specimen in digital form is that either a composite projection image can be computed, or a volume-rendered 3-D representation of the specimen can be generated on a graphics computer.

4.1.2 Optical Resolution power The term resolution refers to the ability to distinguish fine details in a structure. An ideal microscope would have optics totally free from aberrations of any kind. In such a hypothetical instrument the resolving power would only be limited by diffraction. This limit can be expressed as the minimum distance between two points in the specimen for which they still appear as two points (Raleigh’s criterion). Beyond this limit the points will merge and cannot be resolved as two different points. This distance can be calculated from the size of the diffraction image formed by an infinitely small point in the specimen. It is equal to the radius of the first minimum in this diffraction image. This in turn is related to the numerical apertures of the objective and condenser. The numerical aperture is defined by the refractive index of the lens and the width of the cone of light that can pass through it. In analogy with the reasoning above, the axial resolution can be defined as the radius of the first minimum along the microscope Version: RK09052000 page 6

axis of the diffraction image of a point object. According to the theory for such three-dimensional diffraction images, the radius of the first axial diffraction minimum is approximately twice that of the lateral. The axial resolution is thus approximately half of the resolution within the plane of focus. The LEICA TCS SP II is a no-compromise true point scanning system with 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 smaller the pixel size, the more easily two close objects can be distinguished. The scan resolution is restricted to the maximum optical resolution power.

4.1.3 Detection 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 subvolumes (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 reflectity of the specimen. The sensitivity and noise produced by the detector.

The detector is a central component of the confocal microscope, too..

4.1.4 Image processing In 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. In 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: 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 these manipulations do not improve the quality of the collected data, they serve the purpose of improving the visibility and facilitating the qualitative interpretation of the data.

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4.1.5 Light source Lasers are favorable as light sources for confocal microscopy because they feature high brightness, small divergence of the beam, are easy to focus and are stable in intensity. The stability is important for making quantitative measurements Therefore an online reference correction is made for each pixel.

4.1.6 Integration The Leica TCS SP II 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 hard copy output. •

5 Safety remarks This section introduces you to standard safety precautions, warnings and cautions.

5.1 General Safety Directions Your safety is extremely important. Read and follow all warnings and cautions in this book before handling and operating Leica equipment. You can be seriously injured, and equipment and data can be damaged if you do not follow the safety warnings and cautions.

5.2 Risk of Operation The entire 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.

5.3 Warnings, Cautions, and Notes The warnings, cautions, and notes in this manual use the following format.

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Danger 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.

Caution A 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 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 Site of Installation This system was tested and complies with the permissible limit values for the emission of electromagnetic radiation (EMC). However, the remaining risk of effects on other devices cannot be ruled out.

Warning This system must not be mounted next to life-saving equipment

Warning This system must not be mounted next to life-saving equipment.

5.4 Laser Class In order to take into account any possible danger resulting from inadmissible usage we have assigned the Leica TCS SP2 to the laser safety class IIIb. However, improper usage can lead to conditions under which this laser class can no longer be guaranteed. For this reason we are not able to generally declare our system to belong to Class 1 This instrument is designed and manufactured to comply with applicable performance standards for Class 1 laser products as defined by USHHS (Class 1 laser products shall not emit hazardous laser radiation during normal customer operation), CDRH/FDA and OSHA standards and regulations known to be effective at the date of manufacture.

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Hazardous situation cannot possibly be anticipated. Therefore, the user must exercise care, common sense, and observe all appropriate safety precautions applicable to Class IIIb lasers and high-voltage electrical equipment during installation, operation and maintenance. Deviation from published operating or maintenance procedures is not recommended. Operation and maintenance procedure changes are performed entirely at the user’s risk. The laser light emitted from the Leica TCS SP2 used in accordance with the instructions is harmless.

5.5 Technical Safety Measures In accordance with the laser safety directives EN 60825 (European standard) and CDRH (USA) Leica Microsystems Heidelberg GmbH employs various safety measures. Warning labels have been affixed near apertures or moveable parts where exposure to laser light is possible. Furthermore a laser-interlock-switch is installed.

Note Note that it is a requirement of EN 60825-1: „Safety of Laser Products, Part 1. Equipment classification, requirements and user’s guide“, that for installations where class IIIb devices are used, a Laser Safety Officer (or Laser Protection Advisor) should be appointed. It is the laser safety officer’s responsibility to review and designate appropriate controls for the use of the equipment.

5.6 WARNINGS Warning Do not look directly into or at a reflection of the laser beam while the laser is scanning. Long term exposure to the laser beam can damage your vision. For the purposes of laser safety, a direct laser beam which has been deflected from a mirror or polished surface is considered to be as intense as the direct beam.

The user is responsible for the safe operation and maintenance of this instrument at all times. Class 1 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 lethal high-voltages and Class IIIb laser radiation of sufficient power to cause severe eye injury, burns, and property damage. Version: RK09052000 page 10

Tampering with, or deactivation of, any safety system and/or interlock terminates Class 1 laser product performance, will expose personnel to hazardous Class IIIb laser radiation, bodily injury, and void warranty coverage.

5.7 Power Cord Set Requirements The power cord set supplied with your instrument meets the requirements of the country where you purchased the instrument. If you use the instrument in another country, you must use a power cord set that meets the requirements of that country.

Danger 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.

Caution 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 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 reconnect power, or turn the instrument on

5.8 General Safety Notes In 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 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.

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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 SP2 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. Conduct safety inspection and checks on the instrument. These inspections are defined by VDE (FDA in USA) and the laser device safety regulations. The user must perform these inspections as described. Only service engineers authorized by Leica Microsystems Heidelberg GmbH are allowed to carry out repair work. Do not electrically connect the product to devices which are not mentioned in this accompanying documentation. Before connecting the product to any such devices, consult the local Leica Service Agency or Leica Microsystems Heidelberg GmbH directly.

5.9 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 SP2 in accordance with the instructions in order to ensure safety. Several important points are listed below: Do not change objectives during scanning operations How to change objectives: •

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•

Ascertain that the Scan mode is switched off. The Scan button must visibly released. Be sure that there is no laser light in the focal plane. Close the safety shutter at the tube. Turn the lens turret so that the lens to be exchanged appears outside of the optical axis and points outwards. Do not apply strong moments to the turret, as this may reduce the resolution of the turret scan permanently. Screw in the new lens and turn the lens turret again into the optical axis.

All unused positions on the objective nosepiece must be capped.

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Do not insert reflective objects into the beam path during scanning operations. Do not change specimen during scanning operations. How to exchange a specimen: •

•

Ascertain that the Scan mode is switched off. The Scan button must visibly released. Be sure that there is no laser light in the focal plane. Close the safety shutter at the tube. Exchange the specimen.

5.10 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, from laser pointers to CD players. Lasers can produce a very intense and very narrow (collimated) beam of electromagnetic radiation in the frequency 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 the body is generally at the surface. The eye and the skin are critical organs for laser radiation exposure, and 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 energy may be focused by the lens of the eye onto the retina causing severe damage. If laser radiation is of high enough energy, 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 of concern if exposure occurs.

Spectrum Region

Wavelength Range

Organ Effected

UV-C 100 nm to 280 nm

All absorbed in cornea and conjunctiva

UV-B 280 nm to 320 nm

Almost all absorbed in cornea and conjunctiva

UV-A 320 nm to 380 nm

Cataract formation

380 nm to 760 nm

Retina

IR-A 760 nm to 1400 nm

Retina, lens, skin

Infrared IR-B 1400 nm to 3000 nm

Cornea and skin

IR-C 3000 nm to 106 nm

Cornea and skin

Ultraviolet

Visible

Lasers are often identified by type, i.e. Argon-Krypton, wavelength(s), and Laser Hazard Classification. The Laser Hazard Classification is determined by the wavelength(s), maximum duration of exposure, and the average power output of the laser. Version: RK09052000 page 13

5.11 Laser Hazard Classification and Laser Safety Control Measures Note Direct exposure of the eye by a laser beam should always be avoided with any laser, no matter how low the power.

Class I Exempt lasers that cannot produce a hazard. Class II Low power visible laser which, due to the reflex response, does not normally present a hazard unless viewed directly. Class IIIa Lasers that normally do not produce a hazard if viewed momentarily with the unaided eye; may produce a hazard if viewed using collecting optics. Class IIIb Lasers that can produce an eye injury if viewed directly, including intrabeam viewing of specular reflections. Class IV Laser that can produce an eye injury or skin burns from direct, specular or diffuse reflections; may be fire and skin hazards. Class I, II, and IIIa lasers should have a yellow and black „Caution“ label, while Class IIIb, and IV lasers should have a red, black, and white „Danger“ label. Embedded systems: Class II, III or IV 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.

5.12 Laser Hazards The danger from lasers can be divided into the following major categories:

Danger

•

Eye hazards such as retinal or cornea burns.

RETINAL INJURY IS PERMANENT • • •

Skin hazards such as burns. Electrical hazards from high voltage equipment. Fire hazards.

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6 Maintenance and cleaning Please refer to the corresponding manuals for information on how to maintain the conventional LEICA Research microscope. The instructions and additional information relating to the components of the TCS SP II confocal system are summarized below.

6.1 Selecting an installation site Do not expose the system to draft. Therefore, do not install your TCS SP II system next to elevators, air conditioners and other inlets and outlets.

Warning This system must not be mounted next to life-saving equipment

6.2 Protect the microscope against dust and grease When not in use, the system should be covered with a plastic foil (part of delivery) or a piece of cotton cloth. The system should be operated in a room which is kept as dust and grease-free as possible. Dust caps should always be placed over the objective turret positions when no objective is in place.

6.3 Be careful when using aggressive chemicals You must be particularly careful if your work involves the usage of acids, lyes or other aggressive chemicals. Make sure to keep such substances away from optical or mechanical components.

6.4 Cleaning the optical system of the microscope The optical system of the microscope must be kept clean. Under no circumstances should users touch the optical components with their fingers or anything which may bear dust or grease. Remove dust by using an air puffer (not solvent based) or a fine, dry hair pencil. If this method fails, use a piece of cloth, moistened with distilled water. Persistent dirt can be removed from glass surfaces by means of pure alcohol, chloroform or naphtha. If an objective lens becomes accidentally contaminated with inappropriate immersion oil or mounting medium, contact your next Leica representative for advice about cleaning with solvents. Take this seriously, because some solvents may dissolve the glue which holds the lens in place.

Caution Do not open objectives for cleaning. Oil should be removed from oil immersion lenses after use. Once most of the oil has been removed with a clean tissue, a piece of

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lens tissue should be placed over the immersion end of the lens. A drop of recommended solvent should be applied, and the tissue gently drawn across the lens surface. This should be repeated as often as is necessary to attain total cleanliness. Use a clean piece of lens tissue each time.

6.5 Cleaning the microscope surface Use a linen or leather cloth (moistened with naphtha or alcohol) in order to clean the surfaces of the microscope housing or the scanner (varnished parts).

Caution Never use acetone, xylol or nitro thinners as they attack the varnish. All LEICA components and systems are carefully manufactured on the basis of the latest production methods. If you encounter problems in spite of our efforts, do not try to fix the devices or the accessories yourself, but contact your Leica representative.

7 The Leica Confocal Software: An overview 7.1 Starting the software 7.1.1 Requirements for starting the software The LCS software is copy protected to prevent it from being used on two computers at the same time. This protection system allows all additional application packages to be used. To enable the software, the provided dongle must be inserted into the parallel port of your computer. The dongle does not affect the functionality of the parallel port (e.g. for printing, etc.). To use the software on a second computer, the dongle must be fitted to its parallel port.

Note If you remove the dongle from the confocal system control computer, the software cannot be started, preventing operation of the confocal system. The LCS software can be started in two operating modes: hardware mode and simulation mode. In hardware mode, all hardware components are accessed and initialized by the software. For this reason, you should switch on the hardware first and then wait about 20 seconds before starting the software. In simulation mode, the software runs independently of the hardware. This mode is intended for secondary installations on another computer, for example for training or offline analysis of existing data.

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7.1.2 Starting the software Select Start|Programs|Leica Confocal Software. The initial screen of the Leica Confocal Software appears. In this window, one of three profiles can be selected. Company With this option, the Leica Confocal Software is started with the default settings. In this mode, the configuration and position of toolbars is fixed and cannot be changed. Personal In this mode, a user-specific configuration profile can be selected. The user name depends on the account under which a person logs on to the operating system. If the user does not yet have a personal configuration profile, the default settings are used. Last Exit With this option, the last used configuration profile is used. For advanced users: If you have several configuration profiles, you can load to these at startup by clicking the button with the three small black dots at the lower right edge of the profile options. Here, you can also reset your current personal configuration profile to the default settings. When you press the Start button, the Leica Confocal Software starts with the selected configuration profile.

Note If you do not press the Start button, the software will start by itself after a specified time with the selected configuration profile.

7.2 The experimental concept of the software With the Leica Confocal Software, image data or the results of image processing steps can be combined in groups. These groups are referred to as "experiments" and are stored in a specific file format (*.lei). In these experiments, original experimental image data can be stored together with image viewing data. For further details, see chapter "Organizing data by grouping experiments".

7.3 Basic structure of the user interface The visual appearance of the Graphical User Interface (GUI) depends to a large extent on the chosen configuration profile. It does, however, contain a number of standard elements. The following standard elements are common to all GUI configurations: The menu bar

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The menu bar contains the File, View, Macro, Tools, Window and Help menus. These menus contain commands and information for viewing data, making changes to settings and customizing the program for specific users. It does not contain functions for the direct control of scan functions. These are contained in the TCS menu (View→ →Menu→ →TCS Menu). The menu bar itself cannot be configured. The Viewer window (TCS_Viewer) This window displays image data, experimental conditions and user information. The image window can be configured (see chapter "Modifying the user interface and defining user-specific settings" on page 16). Beside confocal image records, the image window also shows experimental data such as system settings. An image window for a new experiment can be opened with File → New. TCS menu (TCS_Menu) The TCS menu contains the buttons for the individual device functions. It is arranged in individual working steps. The number of working steps varies depending on the installed software. The default set of working steps consists of data acquisition (Acquire), image viewing (View), surface reconstruction (3 D), the measuring functions (Quantify), the image processing and analysis functions (Process) and the documentation functions (Annotate). If the TCS menu is not available in the current configuration profile, you can activate it with View → Menu → TCS Menu. The toolbars Individual button arrangements can be set up for each individual user. A key feature of the toolbars is that they can be toggled on and off complete with all buttons. This is done with the menu option View → Menu → Container. Viewer window for documents (Experiment Overview) In this window, existing experiments and their content are listed in the form of a directory tree. The viewer window can be opened with View → Experiment Overview. Status bar The status bar appears at the lower edge of the Leica Confocal Software user interface. In it, the following information is displayed: • • •

The progress when loading image data (progress bar) The software version number The name of the machine configuration (system type)

For details about individual functions, see chapter "Software reference".

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7.4 Opening records 7.4.1 Readable file formats The following file formats can be opened and viewed with the Leica Confocal Software Experiments (*.lei) This is a Leica-specific binary file format. Files of this format contain data for complete experiments. TIFF files (*.tif) These are Leica image files in single and multiple tagged image file format (TIFF). Both image files in the previously used TCS formats and external files in RGB TIF format can be read. Annotation files (*.ano) This is also a Leica-specific binary file format. In these files, annotation sheets are stored. The elements on the annotation sheets, such as images, text and graphics, are each available as individual objects. When a file of this type is opened, both the image data and the experiment settings are loaded.

7.4.2 Automatic reuse of instrument parameters With the Leica Confocal Software, the hardware settings saved with experiments or single images can be carried over to a new experiment. This allows subsequent experiments to be carried out with the same settings. To carry over previous settings, open the viewer window for the record whose settings you want to reuse. Then click "Apply" (in the default configuration profile on the toolbar).

Note If you cannot find the "Apply" button in any of the open windows, you

can

assign

the

button

to

any

window

with

Tools→Customize. In the dialog that opens, select the "File" category in the "Commands" tab. Left-click the "Apply" button and keep the button pressed while pulling it to the window of your choice. To insert the button in its current position, release the mouse button.

7.5 Saving images Individual images and experiments can be saved in the file formats described in section "Readable file formats" above. To save images and experiments, select File → Save. When an experiment is first saved, the "Save as" dialog opens, where you must enter a file name. You can also select a file format in this dialog. Experiments can only be saved in the Leica-specific *.lei format. When you are saving experiments, you may be able to save existing individual images in *.tif or *.raw format. Version: RK09052000 page 19

Note If the experiment or image has already been saved, the previous data is overwritten each time the experiment or image is saved. To prevent this, you must save the data under a new filename with File →Save as.

7.6 Organizing data by grouping experiments The Leica Confocal Software allows single images, image series, and the results of image processing steps to be combined in a group (an experiment). The experiment overview window provides an overview of all loaded experiments. If the experiment overview window is not visible, you can open it with View → Experiment Overview. With File → New and File → New(Template) a new experiment can be created. In the same way, existing files that are being opened are managed as separate experiments.

7.7 Compiling experiments After you have created a new experiment with File→ →New or File→ →New(Template), you can assign data to it.

Note Images acquired with the continuous scan function are automatically overwritten the next time a scan is started. If you want to permanently keep a single image as part of an experiment, choose the single scan function. An experiment contains data acquired with the single or series scan function. If you perform image processing functions on a record, you can also save the results as part of the experiment. To do this, double-click the desired image or series in the experiment overview. Now carry out the image processing functions (e.g. maximum projection or topology image). Mark the area within the Viewer window that you want to add to the experiment. With the right mouse button (context menu), select Send to→ →Experiment. The Selection (raw) option creates a copy of the raw data for the selected object as a new, separate component of the experiment. The Selection (snapshot) option creates an RGB image (no 3D data, photo only) of the selected object as a new component of the experiment.

8 Starting the Operating System Windows NT™ You don’t have to start Windows NT ™– it starts automatically when you turn on your PC. You will first see a splash screen. Next you have to log on to your computer. As you can see from the instructions in the box, pressing the Ctrl, Alt and Delete keys at the same time will log you on.

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