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1. Dipl BE 40 BNA oil PL APO 125 85 Lens Formatl Scan Gon Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 2 Note Note Page 4 Selecting the objective only influences the image legend on the right hand side of the image window and some internal parameters like the optimum pinhole size The computer control program cannot turn the lens turret Therefore always ensure that the objective in use matches the lens de scription If the lens description and position do not correspond to your microscope setup you will have to change the PowerScan preferences Please refer to the chapter PowerScan Software Reference if you want to do this The laser system has now been running long enough to deliver a very stable intensity of laser light Adjust the laser power using the rotary button on the laser box The laser power reading at the right side of the image windows image legend is only in relative units and does not dis play an absolute value For FITC turn the rotary button to a position be tween 10 and 12 o clock If the laser is too intense this may result in a poor quality image This is because fluorochromes saturate at sufficiently high power densities of excitation Any increase in laser power beyond that point does not in crease fluorescence but only reduces image quality and increases the rate of bleaching Now press the Scan button in the TCS_Tools window t
2. This step is only available if you have installed the optional Power3D soft ware In this step you can create true multi dimensional representations of your data Movies showing location changes can be generated 3D mode Opens a new image window on top of the normal image as in Pseudo 3D mode This new window contains additional picture processing tools Rendering processes work with a huge quantity of data and build graph ical representations applying several algorithms More free RAM stor age and a bigger Windows NT pagefile will help speed up this process Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 1 Page 9 Note All movement processes should be carried out in low resolution mode This saves time and requires little virtual storage Resolution can be defined in the preferences window Settings Preferences in the menu at the top of your screen Switch back to the desired higher resolution to render the final image If you want to change several parameters one after the other either in the x image window or in the preferences dialog you should suppress imme of Render diate rendering by pressing the Don t render button in the tools section You must release Don t render again in order to see any changes Physiology optional TCS_Tools MAD AO AED A AE AUR Ab ad Gadd red Selec Ne Note This step is only available if you have installed the optional PowerPhys s
3. Fine focusing 8 Use the automatic turret in order to turn the next lens into the beam path Automatic exchange of objectives Upper button change in clockwise direction Lower button change in anticlockwise direction 9 Repeat steps 7 through 8 until all the magnification factors of all the lenses have been defined 10 Repeatedly press the CHANGE key until the EXIT message flashes on the display 11 Then press the LEARN key All changes are saved now Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for inverted LEICA DM IRBE Microscopes Page 9 2 Settings for incident light observations For incident light applications please proceed as follows 1 Switch the incident light lamp on unless it is already on Allow at least 10 minutes time for the lamp to reach a steady state Brightness control 3 If you wish to study a fluorescent specimen and if a mercury dis charge lamp is available you should now select the appropriate fluo rescence filter cube Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for inverted LEICA DM IRBE Microscopes Page 10 Select a filter The revolver positions are factory equipped with the following beam splitters Position 2 13 or L4 for FITC fluorescence Position 3 N2 1 for Rhodamine fluorescence Position 4 Scan position Select
4. Depending on the configuration of the microscopes options and the application you must switch on the following components O the integrated power supply unit for halogen lamp and motor focus e the external power supply units for the Hg or Xe lamp for fluores cence microscopy When you switch on the mercury high pressure lamp Hg lamp it must be on for at least one hour before you can switch it off again Allow the lamp to cool down for at least 15 to 20 minutes before switching it on again Otherwise the Hg lamp may implode which will result in mercury contamination Leica TCS Manual Rev 99011 3 Using the confocal system 3 2 System procedures Starting up the procedure Note Page 3 2 Switch on the laser The TCS NT system can be equipped with a number of different lasers Depending on the type of laser installed you must observe different in structions for switching on the system Key switch high voltage supply for laser tubo Intensity of laser light Minin Euuter Laser box The instructions below relate to the OMNICHROME lasers that are in stalled in the standard version of the system The OMNICHROME laser is switched on in three steps 1 Set the toggle switch Main to ON in order to switch on the la ser cooling unit 2 Turn the key switch to the right position ON in order to switch on the high voltage supply for the laser tube W Adjust the desired lase
5. Ext Foc e Page 15 Set the scale factor for the averaging techniques in extended focus image creation not available as far as version 1 6 x If set to 0 the scale factor is set internally equal to the number of sections The Threshold level sets the gray value below which all information is suppressed This function is helpful to reduce background noise Sets the representative gray value of each optical section to the maximum gray value Sets the representative gray value of each optical section to the gray value of the center of mass be low the distribution curve Invert image colors Defines the difference in viewing direction be tween the red and green image for stereo repre sentations not available as far as version 1 6 x Sets frame rate frames per second for online movies presentation of data stack Defines which ratio is used when you press the ratio button TCS_Image window Close dialog window Display the original scanned data Display an extended focus image average gray scale value Display a maximum projection image maximum gray scale value Proj Topo ATA Leica TCS Manual Generate a red green stereo image overlay Generate a topological presentation that translates the height of a struc ture within a series of optical sections into a color code Rev 99011 4 Reference 4 2 PowerScan software reference View mode Page 16 Co Contra st Change contrast and or
6. Leica TCS Confocal Systems User Manual Version 1 0 January 1999 Made in Germany Copyright 1998 99 Leica Microsystems Heidelberg GmbH All rights reserved Edited and written by EDV Service Dr Kehrel Heidelberg Germany No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechani cal 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 cus tomers 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 repro duction of the item described herein or any similar item 1s 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 de scribed 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 ex
7. Press Next to display the next image of a movie Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 6 Alay Select Note Note Sawe Page 9 If you want to select a specific image from your movie press Select This opens a new dialog box Drag the slider to select a frame or enter 1ts number into the input field Click OK to confirm your entry Set current frame a E Display frame fiO of 10 Press Play to display your movie The Play button changes to a Stop button Click on the same button again to stop the movie You can set the movie speed in the Preferences dialog See the PowerScan software reference for details After the last image has been displayed the movie loops back to the first image The PowerScan software can only play back movies calculated with the Pseudo 3D software It is not possible to load movie files from disk To save your movie to disk press the Save button Define a file name and confirm by clicking OK The video compression window appears Video Compression Compressor Microsoft ideo 1 ee Cancel Compression Quality 75 al oen About W Key Frame Ever 15 frames M Data Rate 300 KB sec Accept the default values by clicking OK For more information on the video compression option see the Leica TCS online help Your movie is saved to disk To display it double click on the file name in the NT
8. parabolic hd Treshald Surface fit Select between two surface fit procedures flat optimal plane or parabolic optimal ellipsoid Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Quantify mode Line Rect Alip se Poly ari Gear Profile a a bi n YO e UN Surface Page 18 Threshold Define a threshold value 0 255 for the fre quency intensity that has to be exceeded for calculation of roughness parameters Close Close dialog Quantify mode tools Draw straight lines as region of interest ROI Draw a rectangle or square as ROI Draw an ellipse or circle as ROI Draw polygons as ROI Delete all drawn objects Create an intensity profile Create an intensity histogram Determine statistical data on the geometry of a marked area such as sur face and various roughness parameters Produce a xz intensity profile of a series Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Pseudo 3D mode Page 19 Pseudo 3D mode Menu File Open File Save as File Save Selected File Default Legend File gt Exit Settings Preferences Settings Timelapse Help Help Help Help on Help Help gt About Leica TCS PC Leica TCS Manual Open images from a storage device Save entire data set to a new file in TIF or TCS EXP format Save frames of a selected pane to a new file Load a d
9. Click on the Start button then click on your selection from each menu that appears O Some icons appear on your desktop You can activate one by double clicking on it We now take a brief tour of the items you see on the screen A standard desktop item is the My Computer icon Double clicking this icon opens the My Computer window The My Computer window gives you easy access to the major components like hard and floppy disk drives of your computer system or workstation For example by double clicking the Hard disk C icon you can see the contents of your PC s hard drive This allows the user to view local resources as objects Access to the Windows NT Workstation 4 control panel and print support con trol are also accessible from My Computer If at installation time you installed one of the additional local applications such as Dial Up Networking this will also appear within My Computer Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software The Windows NT interface Page 5 My Computer File Edt View Help Control Panel Printers Dial Up Networking E objectis You can use the Control Panel icon in the My Computer window to view and change any system component The Control Panel contains numerous icons that allow you to control your system The particular icons that you see on your PC may be slightly different from those illustrated due to that fact that you may have di
10. Slice Don t Render Page 21 2 5 D viewer settings 2 5D is used instead of Pseudo 3D Scale Z axis Scale the z axis to achieve a reasonable aspect ra tio between xy plane and z axis Default mode Set default viewing mode when entering Pseudo 3D mode Contour lines Number of contour lines 10 500 used for con tour mode 3D viewer settings Refer to 3D mode preferences Close Close dialog Pseudo 3D mode tools Calculate a flat projection of the current image Show the intensity of the image as a 3D grid Render a pseudo 3D image with the intensity of the individual pixels converted to height values Calculate a contour map with the intensity of individual pixels converted to height values Simultaneously visualize xz and yz sections from any region of your three dimensional data stack Visualize arbitrary sections from any region of your three dimensional data stack Suppress immediate rendering Click again to restart Display the first image of the movie Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Pseudo 3D mode O on T cn La re lv am a H iy rm azt E End fii Steps Record Page 22 Display the previous image of the movie Select a specific image out of the movie Press the Play button to display the movie Click on the same button now labeled stop again to stop the movie Display the next image o
11. When you install a TCS NT sys tem for the first time you will always receive instructions for operating the unit from a Leica technician The following information is not in tended to replace this initial training If you do not have a working knowledge of the Windows NT operating system please read the chapter Running the Leica TCS Software first You should then continue with the Overview of Leica TCS Software This tutorial is not intended to introduce all the functions available with this software since these are covered comprehensively in other chapters The following instructions cover only confocal data acquisition and pro cessing When setting up the microscopes for conventional operation please refer to the manual that accompanies them or read the chapter Setting up the Microscopes Note Your safety is extremely important Please read the information in this book before handling and operating the equipment and pay strict attention to all warnings it contains Failure to heed these safety warnings can lead to seri Ous injury as well as damage to equipment and data Please read the Safety Notes before working through this tutorial This tutorial is subdivided into sections each with exercises You should work through the sections in the sequence suggested They are designed to cover topics you will normally need to understand Once you have worked through this tutorial you should be confident using the Leic
12. e High spatial resolution only photons from a very small sub volume should be detected at any moment e High temporal resolution the detector should respond linearly whether the frequency of incident photon events is high or low e Low noise In the absence of incoming photons the PMT still pro duces a small signal There can also be some random variation in the output signal between identical photons Cooling the detector can re duce the noise A PMT with its internal amplification produces fewer random noise compared against a CCD Image processing In the first confocal microscopes the detector was connected to an oscil loscope with long persistence phosphor which would display an image as it was being scanned In the instruments of today the signal is digi tized and recorded in a computer This makes it possible to manipulate the image in a multitude of ways e Contrast enhancement by thresholds linear contrast stretching and gamma correction curvature of the image intensity value versus source intensity graph e Superimposition of images in experiments with multiple fluorophores or for background subtraction O Digital filtering for edge enhancement smoothing noise suppression etc O 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 Leica TCS Manual Rev 99011
13. tor focus After you have released the upper key the CAL message is dis played This message indicates that the calibration mode is active Press the lower one of the two keys and keep it depressed Turn the fine focus wheel in order to adjust the magnification of the lens currently in use The magnification factors of all available Leica lenses are stored These values can be displayed and saved by turning the fine focus wheel Release the lower button and turn the next lens into the beam path Repeat steps 6 through 8 until all the lenses have been defined 10 Switch the microscope off All changes are saved now 2 Settings for incident light observations For incident light applications please proceed as follows l Switch the incident light lamp on unless it is already on Allow at least 10 minutes time for the lamp to reach a steady state Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for upright LEICA DM RE Microscopes Page 3 2 Switch the transmitted light lamp off if necessary WT GN El a UN a es ay A AAN r K 3 If you wish to study a fluorescent specimen and if a mercury dis charge lamp is available you should now select the appropriate fluo rescence filter cube 4 Switch the light stop device off if necessary in order to clear the in cident light beam path Select a
14. Europe Daplight Time To change the Date and Time O Click on the appropriate date or use the controls to change the month or year The time can also be changed by first selecting the digital display and then using the up and down arrows To change the Time Zone O Select the appropriate Time Zone from the drop down list at the top of the screen Notice that the option to automatically adjust the clock for daylight Date Time Properties Ei Date Time Time one M Automatically adjust clock for daylight saving changes Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software Getting Help Note Page 11 savings time is selected On some systems you can also drag the highlighted area on the world map and drop it on the correct location Changing the date and time information within Windows NT Workstation 4 will update the battery backup CMOS clock in your system Depending on the shell configuration systems connected to a network may get a time and date update from a network server every time they log on If the servers time Is incorrect your workstations time will be wrong too Please inform your network manager Getting Help Help Topics Windows HT Help Contents Index Find Click a topic and then click Display Or click another tab such as Index 2 IF You ve Used Windows before Y Introducing Windows MT da How To g Tips and Tricks g Troubleshoot
15. Explorer or open the AVI file with the Windows Media Player Movies use a large amount of disk space even when they are compressed Please ensure that you have enough free disk space several megabytes before you try to save a movie Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 7 Page 1 Tutorial 7 This tutorial covers the preparation of documentation in the annotation workbook It allows you to compose a document consisting of images and data The snap function displayed in all TSC_Image windows transfers the image to the Scanner sheet of the TCS_Annotation work book while the data is transferred to the Quantification sheet of the same workbook Data is only produced in the quantification step The quantification sheet is a normal Excel sheet 3 TCS Annotation Miel Era 4 X Position um Channel 1 Channel 2 Channel 3 0 9 00 8 00 9 00 0 35 9 00 7 00 9 00 0 71 9 00 7 00 9 00 EN 1 06 9 00 7 00 9 00 9 1 41 5 00 7 00 9 00 10 1 77 8 00 7 00 9 00 EEN 2 12 8 00 7 00 8 00 2 47 8 00 7 00 8 00 2 83 9 00 7 00 9 00 EEJ 3 18 9 00 7 00 8 00 15 3 53 8 00 7 00 8 00 16 3 89 9 00 7 00 8 00 4 24 8 00 7 00 9 00 18 4 59 8 00 6 00 8 00 19 4 95 8 00 6 00 8 00 53 8 00 6 00 8 00 5 65 8 00 7 00 9 00 4 4 iS Scanner h Quantification 4 FP To save it to a disk file choose File gt Save as in the menu The file type is Excel Workbook xls and both sheets
16. IS PERMANENT 2 Skin hazards such as burns 3 Electrical hazards from high voltage equipment 4 Fire hazards Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Technical Specification of the System Page 11 Technical Specification of the System dimensions of the desk 190cm x 90cm x 142 cm without chair scanner electronic box integrated in desk dimensions of the laser 75 cm x 27 cm x 60 cm box number of laser boxes 2 for 3 laser system 1 laser box placed under desk HeNe laser within scanner electronic box weight of basic system max 320 kg without UV system max 428 kg with complete UV system supply voltage 230V main voltage fluctuations 10 main frequency 50 60 Hz main frequency fluctuations 10 main fuse 10 A microscope workstation 16 A ArKr or Kr Laser 16 A Ar Laser 32 A UV Laser heat load max 5 kW without UV system 10 kW with complete UV system heat load average 3 kW standby mode 2 kW room temperature 10 C to 28 C separate cooling only UV laser coolant air Room requirements height of room minimum 1 8m opening minimum lmx15m magnetic protection shield only in proximity of high magnetic fields room darkening recommended mains supply 2 x or 3x for 3 laser system 230 V 16 A separate supply environment avoid close pro
17. Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Pseudo 3D and 3D view windows Page 4 Pseudo 3D and 3D view windows Translate image to new position i Enlarge or reduce the current image zoom in zoom out oom 3D rotate the image Rotate Perpendicular view all angles set to 0 Oblique view X 45 Y 15 Z 10 i eke Oblique Set rotation angles p cc CE Set light vector Light Display a coarse preview image Previa li Print currently displayed image Prt Copy currently displayed image and data to a separate annotation sap worksheet Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Tools window Page 5 Tools window Main Return to the main control screen Acquisition Acquire scan new images or series View Load and process images and series Quantity Evaluate your images statistically Pseudo 3D Calculate pseudo 3D representations intensity maps taal a Calculate real 3D representations optional Physiology Make offline physiological analysis of confocal data sets optional CUE UU EYE Annotate Prepare documentation inside the annotation workbook Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Acquisition mode Acquisition mode Menu File Open File Save as File Save Selected File Default Legend File gt Exit Setti
18. Maximum alri 20 60 3 00 97 00 y al El 22 30 0 00 ro 00 by snai m 51 71 5 06 5 00 28 00 If you select more than one object the system displays an error mes sage Microsoft Excel To print out the intensity profile press the Print button in the TCS_Im age window The Snap function copies the contents of the profile window into the annotation workbook You can use this annotation sheet to create your Siap own documentation The data in the graphs is copied automatically into a sheet labeled Quantification Images are copied into a sheet labeled Scanner Note It is not possible to save and reload objects Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 5 Page 6 To produce an xz intensity profile of a series along the edge of a marked area press the Stack button stack f The system also calculates the ratio of channel 1 to channel 2 Stack Profile 1 Channel 1 Channel 2 Channel 3 Ratio c2 c1 2 Ko 2 3 2 Z Position um To create an intensity histogram for a marked area of an image press Histo Histo l a ee l o o A A histogram of the intensity distribution provides statistics on the distri bution of gray values in your image within a marked area Other param eters are shown in the image legend al Histogram 5 Channel 1 Channel 1 Area urn E Mean Std dev Minimum Maxim
19. Setting the K hler illumination 4 4 Setting the parfocality A AE AAA ee N L a 5 Setup instructions for inverted LEICA DM IRBE Microscopes 6 1 Mounting and defining the lenses 6 2 Settings for incident light observations 9 3 Setting the K hler illumination sas 11 4 Setting the parfocality A a G a l oa 1 2 System procedures 1 General VERE ET AENA A ee ee ee 1 Starting Up the procedure toto toto totor 2 Shutdown procedure i ee ee ke Oe Se eo ee se ee a 5 Leica TCS Manual Rev 99011 Page 2 System Care 1 2 002 eee ees 1 Safety Notes 2 eae 1 General Safety Directions ttt tt tt tes 1 Risk of Operation 1 Warnings Cautions and Notes sss ttt ttt 1 Laser Class A E Ow he Oe CO 2 Technical Safety Measures sss tt ttt tt te 2 WARNINGS i ae a eh eee oe kn se oe ee ee eS eh ee ce Se Be 4 Power Cord Set Requirements o ooroo orooro 5 Cooling Fan Obstruction o eee eee bee ee 5 Installation of the Laser Box totor 5 General Safety Notes ssc ttt ttt tes 6 Special Safety Remarks for Users o o ttre 7 Laser Safety sc otoo ee 9 Laser Hazards E ce oe ae eS O lO a oe ee OF 1 0 Technical Specification ofthe System roso 11 Specification of Laser Radiation o o
20. Several important points are listed below Do not remove the protective shield only for inverted microscopes Do not change objectives during scanning operations How to change objectives 1 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 2 Turn the lens turret so that the lens to be exchanged appears outside of the optical axis and points outwards 3 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 Do not insert reflective objects into the beam path during scanning operations Do not change filter cubes and beam splitters during scanning operations How to change filter cubes 1 Ascertain that the Scan mode is switched off The Scan button must visibly released Be sure that there 1s no laser light in the focal plane 2 Pull the fluorescence module and insert or exchange filter cube as de scribed in the microscope s documentation 3 After having changed the filter cubes insert the fluorescence module completely and attach again the cover in front of the fluorescence fil ter block to close the opening Do not change specimen during scanning operations How to exchange a specimen 1 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 Leic
21. able to access the core system configuration utilities in this way is partic ularly useful when an application is already in the foreground and you want to make a quick change Ff B04 bmp 29 E05 bmp FF BO6 bmp 27 B07 bmp B11 bmp desktop tif 9 Imagel bmp 49 Leica bmp Aunbrrs zdr Ea start tit TCS Documentation lt cz start sla Bo tos_start sla 2 test2Bd avi The Find command features an easy way to locate all system resources Within the Find category you can perform searches for three distinct types of search which are described as follows Find The Microsoft Network On The Microsoft Network If you have pre configured Windows NT Workstation 4 to connect to the Microsoft Net work MSN via a modem link selecting this option will automatically connect to the MSN and perform a network wide search for whatever item you specify as the search criterion Find All Files Files or Folders Here you may perform a highly powerful file name and actual file content search By using the Find program it is possible to specify a text string that will occur within a file even if the file 1s a binary file You may also search for a particular type of file such as searching for all wave sound files ending in an extension of WAV Find Computer Computer If connected to a network or workgroup the Find computer option is very useful for locating a particular computer or
22. confocal system 3 5 Tutorial Tutorial 5 Page 4 If you acquired or loaded a series of images you can switch back and forth between View and Quantify mode to select a different image This way an object can be continued to another section so that lengths and in tensity profiles can be measured in a z series To create an intensity profile for a marked area of an image click the bas Profile button Profile This function calculates the gray values along the edge of a drawn ob ject within your image The horizontal axis of the intensity profile corre sponds to the distance from the starting point upper left corner the vertical axis indicates the gray values Bi Profile 11 5 x Channel 1 Channel 1 Length prm i dev Minimum Maximum 91 32 E 4 00 167 00 You can obtain statistical values such as Sum Mean Deviation Maxi mum and Minimum from the legend CAUTION The length is only correct if the right lens is selected during acquisition Note Intensity profiles are only available for the edge of a geometrical object one dimensional Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 5 Page 5 If you mark your region of interest on an overlay image each channel is analyzed separately hi Profile 12 Channel 1 Channel 2 Channel 3 417 Position pm Length prn std der Minimum
23. detector The signal noise ratio be comes better signal stronger than background However low speeds may have disadvantages as well The fluorescence stain may become photochemically destructed bleaching effects due to the intensive laser beam In addition the scanning time increases at low scanning speeds This fact is particularly important when scanning living organisms Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 3 Speed Page 6 The scanning speed is specified in image lines per second Therefore the time the beam remains on a spot depends on the beam speed and the dimensions of the area to be scanned To change the scan speed click on the Speed button in the TCS_Tools window This opens a dialog window displaying the 6 available speed categories Each class of velocity slow medium fast is present twice Speeds marked with the number 2 use a bi directional scan This means that in contrast to usual confocal scanning systems the TCS NT system also uses the flyback of the laser beam for data acquisition The advantages of this are lower acquisition time and reduced bleaching of the sample The Leica TCS has six different scanning speeds Slow 220 lines per second Slow2 440 lines per second bi directional Medium 450 lines per second Medium2 900 lines per second bi directional Fast 950 lines per second Fast2 1900 lines per second bi direction
24. diaphragm or the condenser until the greater part of the field of view is illuminated Now close the field diaphragm again Focussing Focus the border of the light spot by adjusting the height of the con denser Centering Turn the centering screws of the field diaphragm or the condenser in order to center the light spot in the field of view The centering is easier if you slightly open the field diaphragm in order to enlarge the light spot Close the field diaphragm after centering the spot Opening the field diaphragm Open the field diaphragm until the border of the light spot just disap pears at the border of the field of view Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for inverted LEICA DM IRBE Microscopes Page 12 7 Closing the aperture diaphragm Close the aperture diaphragm until you have set the desired contrast 8 If you change the objective It may become necessary to readjust the Kohler illumination after you have changed the objective 4 Setting the parfocality Due to the different working distances of the different lenses their focus lies at different absolute specimen slide heights Setting the parfocality means that the focus position of every single lens is stored After a changing of the lens the focus plane is then approached automatically with the inverse stand 1 Place a specimen on the specimen slide and if necessary perfor
25. hand mouse button on the Start button will invoke the start menu and present you with the seven major categorized options for start ing work on the system A single click of the right hand mouse button will invoke a small but powerful control menu containing the options Open Explore and Find Open Find Open All Users Explore All Users Their functionality is described as follows Item Function Open The contents of the Start menu can be viewed Folders and icons can be deleted copied and moved using drag and drop techniques Explore Will invoke the NT Explorer The Explorer can display a hierarchical representation of the entire system and permit further Desktop manipulation and configuration Find Will invoke the Windows file searching utility from which you may search for any file either by its name or by its actual content Starting a Program The Start menu contains the various categories where your applications and work are stored You can move further into the various subcategories by posi tioning the mouse over the category that you are interested in to automatically open the next subcategory You do not have to click the mouse The Programs command displays the Programs menu This menu lists all of the applications installed and available to you An arrow meaning that there is a submenu follows some items Drag the mouse cursor over the Accessories command to see its submenu The Accessories submen
26. have difficulties in file handling This type of file save only stores the picture in the TCS_Image win dow Some textual information from the legends panel is written as cap tion info into the file but only very few software programs such as Adobe PhotoShop for example are equipped to display this data To save full documentation including scan parameters and legends you need to use the annotation facilities of the Leica TCS control program We will practice this in a later tutorial The PowerScan software uses a special TIF format to save multi channel pictures Multipage TIFF Every channel is stored as a separate image in one common uncompressed file PowerScan can reimport this data when such a file is opened but most image process ing software can only display the first page Use the Save selected com mand to save only one active channel to a regular single page TIF file Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 2 Note Page 7 If more than one channel is selected PowerScan writes a Multipage TIFF even with Save selected At the end of this tutorial we will manipulate our scanned image using different look up tables The analog intensity signal from the detector PMT is converted into digital values There are a maximum of 256 distinct levels A color look up table CLUT is used to convert the digital values of the input image into the gray scale or color values r
27. if Microsoft Exchange is active on your system Win dows NT Workstation 4 has in built electronic mail services based on Microsoft Mail MS Mail and Microsoft Exchange If there is already an MS Mail post office on the same network that the system is connected to the Windows NT Workstation 4 mail client can connect directly into it The Inbox lets you access your messages The Recycle Bin icon represents the holding place for deleted items As long as files are in the Recycle Bin they can easily be recovered if they have been acci dentally deleted Windows NT Workstation 4 will preserve files until the sys tem runs out of free disk space When this happens Windows NT Workstation 4 will prune the contents of the Recycle Bin on a first in first out basis File Edit iew Help Name Original Location Date Deleted Type _ O objectis Files that are overwritten due to an application using a duplicate filename will not be saved to the recycle bin Double clicking the Bin displays its contents The empty window con firms that there are no items in the Recycle Bin Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software The Start menu Page 7 The Start menu Program d a0 di Documents d Settings d Eind Help 1OA Run Windows NT Workstation Shut Down A single click of the left
28. light within it The Brightness control is a quick way of ad justing a picture that is too dark after it has been scanned Contrast var ies the difference in shading between areas Move the Brightness slider to the right to increase brightness and to the left to decrease brightness At 200 the image is solid black Move the Contrast slider to the right to increase contrast and to the left to decrease it All changes will be visible immediately in the TCS_Image window Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 4 Gamma Page 4 Contrast and Brightness Correction for channel 1 mM 4 Contrast fi 00 4 Brightness lo To confirm the changes click OK Press Cancel to undo them The Gamma function enables you to modify the contrast using loga rithms This allows you to display dark areas with increased brightness without significantly changing the bright areas When you click Gamma the system displays a dialog box in which you can adjust the gamma curve When converting the intensity signal into gray scale values the non linear logarithmic contrast method expands the lower signal range and compresses the higher range This results in an increased contrast range The curve below shows the assignment of original gray scale values in to converted gray scale values out Logarithmic contrast expansion 300 250 150 o Q o o mm o o o D oS o o t fu co o
29. most recent already averaged image and the current image are averaged again To define the maximum number of averaging processes click on the Accum button and select one of the pre defined values between 1 and 32 powers of 2 Default is 1 The recommended value for fluorescence samples is 4 Accumulation of frames improves image quality at the expense of scan ning duration and possible sample degradation Press the Series button in the TCS_Tools window to start scanning the series of optical sections The sections are collected and the motor advances the image plane by the specified z interval after each image You will be able to view each image on the screen as the series 1s collected The Leica TCS system au tomatically stops scanning when the predefined number of sections is reached Now you can Save your pictures to a storage device like your hard disk Click on the File menu and select Save as Set the save type to ScannerFile tif Choose an appropriate name and directory It is a good idea to relate the name of the z series file to the sample type but prefix it with Z so that you know that it is a z series Scan speed can influence the intensity and quality of your image signal The scanning speed determines how long the laser beam remains on a specific spot of the object The lower the scanning speed the longer the beam remains on a specific spot At a lower scanning speed more pho tons from the object can reach the
30. on change password The Change Password dialog box displays Change Password User Mame Administrator Domain ADMIN Old Password New Password Confirm Hew Password Type your current password in the Old Password field passwords are case sen sitive so be sure you use the right case then press the Tab key Pressing the Tab key moves the cursor to the next field Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software Using a Mouse Page 3 Type your new password then press the Tab key again Retyping the new pass word confirms that you aren t making a typing error This 1s important since the characters you type appear as asterisks on the screen Note If you miss type the confirmation password you will see a warning dialog Try again Then click the OK button Your new password will be in effect the next time you log on Do not forget your password if you set one Without the right password you can t access your computer anymore Help Contents Nest Tip Show this Welcome Screen next time you start Windows NT The Welcome dialog box is now displayed Take a moment to read the Did you know tip and then click the Close button to begin using Windows NT Using a Mouse You need a mouse to work most efficiently in Windows NT Here are the mouse actions you need to know O Point means to move the mouse pointer onto the specified item by moving the mouse Th
31. or program frequently you might consider cre ating a shortcut for it on the desktop To do so just use the right mouse button to drag an object out of Windows Explorer or My Computer On the menu that appears select Create Shortcut s Here Some programs automatically create a shortcut during their installation procedure Windows NT Workstation 4 does not actively track a link between an original and a shortcut For instance if you create a shortcut of a program and subse quently move rather than copy the original to a different folder then the short cut may no longer function The Startup folder is special in one respect any programs held within it will start automatically when you start Windows NT Workstation 4 The Documents menu shows the names of the 15 files you created most re cently You can open any of these files and its related application at the same time by clicking the file s name in this menu Document files that are opened within an application typically by selecting the File Open command within the application will not be displayed here Only doc uments opened directly from the Desktop will be displayed here The Settings menu offers three commands for changing your system s settings You can directly access the Control Panel and Printing folders Also accessible is the Task Properties window Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software The Taskbar Page 9 Being
32. point in the globe on which you have clicked Moving the mouse turns the globe and the image inside it Release the mouse button when you reach the desired position To turn the axes by defining certain angles press Angle The Set rotation angles dialog opens Set rotation angles 2 El f gt fn OK Ta fio z al j Foo peel Either use the sliders or type a number between 180 and 180 in the text boxes Click on the Set button to assign the new values without closing the dialog window or press OK to assign the values and close the dialog box PowerScan offers two predefined sets of rotation angles Press the Oblique button to switch to X 45 Y 15 Z 10 After testing several viewpoints you can always come back to this view with a single click of the mouse The other predefined view is the perpendicular view To obtain this view press Perp This view is equivalent to setting all angles to 0 Only the xy plane is displayed The image below shows a contour plot see below in perpen dicular view Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 6 Page 4 As with all view windows there is a Print and a Snap button e h Snap Print Two buttons still remain The Preview button displays a coarse preview image that uses only a quarter of the pixel information to gain more Previa speed The effect is more dramatic with high resolutio
33. screen Microsoft Copyright E 1985 1996 Microsoft Corporation Workstations with Microsoft Internet Explorer This product is protected by US and international copyright laws as described in the About Box 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 Begin Logon y Fress Chl Alt Delete to log on on Once you press the Ctrl Alt and Delete keys the Logon Information dialog box is displayed Typing your password identifies you as a valid user for this computer Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software Startup Windows NT Page 2 Logon Information Erte a user name and password that i wald foe this EJ al system 2 3 User name TCS_User Password ma Domain Lab J Get Help Shut Down The default User name for the Leica TCS system is TCS_User No default password A password should be assigned to keep unauthorized per sonal out of your computer After being logged in you can change your pass word by pressing Ctrl Alt and Delete keys at the same time Windows NT Security Logon Information Tou are logged on as 4DMIN Administrator Logon ate Use the Task Manager to close an application that is not responding Lock Workstation Logoff Shut Down l Task Manager Cancel Then click
34. sleeve which is to be installed between condenser and transmission light detector Service engineers authorized by Leica Microsystems Heidel Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Technical Safety Measures Page 3 berg GmbH Service Letter 10 can only carry this out In scanning mode the entire range is between objective and transmitted light detec tor is laser safety range of the class IIb 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 instal lations where class Illb devices are used a Laser Safety Officer or Laser Protection Advisor should be appointed It is the laser safety officer s re sponsibility to review and designate appropriate controls for the use of the equipment Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes WARNINGS Page 4 WARNINGS 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 de flected 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 in strument at all times Class 1 laser product and electrical safety compliance is assured only when all sa
35. the Leica TCS for any other purposes or appli cations is inexpert and inadmissible handling The user assumes the en tire 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 Follow the maintenance instructions Please refer to the chapter Maintenance and Mounting of the online help facility of the TCS software Conduct safety inspection and checks on the instrument These inspections are defined by VDE FDA in USA and the laser de vice safety regulations The user must perform these inspections as de scribed 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 di rectly Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Special Safety Remarks for Users Page 7 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 in accordance with the instructions in or der to ensure safety
36. the image currently displayed To translate the image to a new position inside the view window press the Pan button Position the mouse cursor anywhere on the image Press the left mouse button keep it depressed and move the mouse pointer to reposition the image Release the mouse button when you reach the desired position During translation your image is replaced by a three dimensional bounding box to indicate its actual position This bounding box is al ways shown even if Show bounding box is deselected in the prefer ences To enlarge blow up or reduce the current image press Zoom Position the mouse cursor anywhere on the image Press the left mouse button keep it depressed and move the mouse pointer to resize the im age Move to the top or to the right to enlarge the image Move to the bottom or to the left to reduce image size Release the mouse button when you reach the desired position As with the pan function the sys tem displays a bounding box that represents the outer limits of your im age during zoom Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 6 O Rotate Page 3 To rotate the image press Rotate Position the mouse cursor anywhere on the image Press the left mouse button keep it depressed and move the mouse pointer to rotate the im age Think of your image as enclosed in a globe and the mouse pointer as a stick positioned at the
37. to draw a circle Ellipses can only have vertical or horizontal axes To select image data within an arbitrary line multiple lines click on the Polygon button Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 5 Note Note ear Page 3 This tool is used for drawing polygons The points of the polygon are placed by making consecutive clicks on the drawing surface As you add points the polygon will continue to grow until it is finished with a dou ble click This tool does not close the shape of the polygon Starting and end points do not meet The polygon tool enables you to draw lines freehand 1 Position the cursor where you want to start painting 2 Press and hold the left mouse 3 Drag the mouse to paint a freehand line 4 To end a paint stroke release the mouse button Each time you release the mouse button you end the paint stroke and set a polygon point You can add straight lines by making consecutive clicks on the drawing surface This allows you to combine freehand and straight lines A double click always ends the drawing mode It is not possible to edit a single point of a polygon curve Resizing always affects the whole polygon Sample of different objects To delete all objects at once press the Clear button To delete single objects mark them with the pick tool and press the De lete key on the keyboard Leica TCS Manual Rev 99011 3 Using the
38. user you should start with the chapter Running the Leica TCS software use the software for a while and then read through the rest of the book 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 TCS software and the Leica microscope interact How to use this book We urge you to use this book not just read it To learn about the soft ware use the book as a tutorial We show how to accomplish a variety of tasks Work through the instructions to learn The book usually intro duces a set of steps to accomplish a particular task with a distinctive heading 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 instruc tions Follow them to learn how the tools work 2 Menu commands are shown with a bar 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 Leica TCS for a while You will discover useful features that you breezed over the first time Online tutorial Leica TCS c
39. 011 3 Using the confocal system 3 5 Tutorial Tutorial 5 Page 1 Tutorial 5 Line In this tutorial the measurement on images like intensity distribution or histograms will be described Before you can start you must first mark a region of interest in your picture The Leica TCS software enables you to mark geometrical areas of your images for quantitative analysis The functions include e Selecting image data along a line e Selecting image data within a rectangle e Selecting image data within an ellipse e Selecting image data within a polygon Before you begin obtain an image or a z series or load a file from a disk If you are using a series select a picture in View mode Display one channel or overlay in single mode in the TCS_Image window This is done automatically when you switch to Quantify mode because tiled mode is disabled during quantification Select a certain geometrical shape as your region of interest ROI To select image data along a line click on the line button This tool is used for drawing straight lines Lines are drawn by click dragging Clicking and releasing the mouse button sets the two endpoints 1 Position the cursor at the point where you want the line to start 2 Depress the left mouse button 3 Drag the cursor to where you want the line to end PowerScan draws a dotted line between the starting point and the cursor 4 Release the mouse button PowerScan draws a line between the two
40. 2 Leica TCS 2 1 Confocal Microscope Systems What is confocal imaging Page 6 models of the specimen can also be rendered and examined from any direction e Assembly of digital movies from time sequences of microscope im ages Although these manipulations do not improve the quality of the col lected data they serve the purpose of improving the visibility and facili tating the qualitative interpretation of the data Light sources Lasers are favorable as light sources for confocal microscopy because they have high brightness small divergence of the beam are easy to fo cus and are stable in intensity The stability is important for making quantitative fluorescence measurements For fluorescence microscopy it is of course necessary to have a light source that can excite the fluorophore with which the sample has been stained More generally one would like to have a single light source capable of exciting all fluorophores of interest This calls for the use of multi mode lasers which emit light at several wavelengths or laser lines For excitation in the UV range a separate UV laser is normally needed The argon ion laser has become a standard light source because of its general applicability and reliability It can excite most common dyes that are excited by visible light Moreover many new dyes are designed spe cifically for the argon ion laser An alternative laser choice is an ar gon krypton laser The argon krypton laser is
41. 2048 option Detection Confocal imaging or to be more precise the measurement of the optical or fluorescent properties of tiny sub volumes of a specimen is limited not only by the optical quality of the microscope Other limitations are e The continuous specimen is measured only in discrete sub volumes because of sampling and digitalization e The accuracy with which the sub volumes are defined determined by the scanning mechanism Leica TCS Manual Rev 99011 2 Leica TCS 2 1 Confocal Microscope Systems What is confocal imaging Page 5 e The brightness of the light source in relation to fluorescence satura tion and photodamage of the specimen e The sensitivity of and noise produced by of the detector The detector is another central component of the confocal microscope LEICA uses a photo multiplier tube PMT A PMT reacts to incoming photons by accelerating a cascade of electrons through a stack of dynode plates The theoretical limit of the detection lies in the Poisson statistics of the flux of incoming photons For making quantitative anal ysis of fluorescence it is desirable that the detector responds linearly 1 e the output signal is a linear function of the incident light intensity A good detector would have the following properties e High photon efficiency as many as possible of the photons from the specimen must be detected The sensitivity should be high in the whole wavelength interval of interest
42. Administration FDA publishes and enforces legal requirements on lasers in the US In Canada the Radiation Protec tion Bureau RPB regulates lasers The laser safety standard that applies outside of the US and Canada is IEC 825 1 Safety of Laser Products Part 1 Equipment Classification Requirements and User s Guide The product must satisfy the safety requirements of IEC 825 1 under the environmental conditions specified in IEC 1010 1 or another relevant general product safety standard e g IEC 950 for information technol ogy IEC 204 for industrial equipment or IEC 601 2 22 for medical la Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Laser Safety Standards Page 14 sers Such conditions include temperature and relative humidity shock and vibration and electromagnetic susceptibility European versions identical to the IEC 825 1 standards have been pub lished as EN 60825 1 Most documents covering the safety of laser users are based on the ANSI Z136 series of standards particularly ANSI Z136 1 Standard for the Safe Use of Lasers Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Page 1 Tutorial This tutorial provides an introduction to operating the Leica TCS system and describes the various basic ways of manipulating it to obtain high quality images It is assumed here that the system has already been in stalled and aligned and is ready to run
43. N t co 00 CO ON t g q N N 0N hJ m o o Oo Gray scale value out q o D Gray scale value in Move the Gamma slider to the right to increase the gamma factor and to the left to decrease 1t flat curve Gamma Correction for channel 1 x al J gt foo El i i Cancel To further improve the appearance of your image you can smooth low pass filter or sharpen the contours high pass filter All filters are fixed Press the Filter button to display the filter dialog Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 4 hlo city Page 5 Filter Channel 1 Hone C Sharpening Cancel C Smoothing Cancel C Sharpening Smoothing C Smoothing Sharpening None returns your image to its original state Sharpening builds a 3x3 matrix of each pixel and its neighbors and multiplies this pixel matrix by a special mathematical high pass filter Smoothing builds a 3x3 matrix of each pixel and its neighbors and mul tiplies this pixel matrix by a special mathematical low pass filter The other two options apply sharpening and smoothing in succession in different sequences The Contrast Gamma or Filter functions do not modify your original data If you want the changes to become permanent press Modify This action cannot be reversed Please take note of the following before us ing this function 1 Have you save
44. On Gontrast Gamma Filte i Topa E F Meri Las apes E This step allows you to view and edit images that you have just acquired or that have been retrieved from a storage device There are tools for ba sic adjustments like contrast and gamma correction or for sharpening fil ters as well as more sophisticated tools that calculate extended projections of image series Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 1 Page 8 Quantify TCS_Tools TCS_Tools This step allows you to evaluate your images statistically It displays the tools required for selecting an area and for statistical analysis Pseudo 3D lalalala ESEI gelen e TCS_Tools MAD AIDA ADA MED Piyotony In this step you can calculate pseudo three dimensional representations of your images with the z axis corresponding to an intensity code Vari ous tools enable you to calculate and render different projections of your data or to create movies from image series You can save movies in stan dard windows AVI format for use out with the PowerScan software Pseudo 3D mode opens a new image window on top of the normal one to display an optical section of a series presented as a pseudo three dimensional image This new window contains additional picture processing tools 3 D optional Ao E eA a ay
45. _ es Cancel idth gt Custom Length gt Style Color y ATEO 7 p Weight To delete objects select them with a single mouse click and press De lete To keep images and corresponding text and arrows together you can group them The objects must be selected in order to be grouped To se lect more than one object press the Shift key and click any object you want to pick After selecting the objects press Group A group behaves like a single object To ungroup several objects select the group to want to separate by a single mouse click and press Ungroup Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 7 Page 4 Note You can use this feature to separate image and legend because the snap function ties them together as two grouped objects 3 TCS Annotation Al E Format 512 x 512 x 32 ImageSize 141 2 x141 2x17 5 Scalebar 100 um LaserPower 1237 DateTime W297 11 25 AM Method FITC TRITCICYS 400y n 4 gt dif Scanner Quantification I lt You can transfer data on calculations or documentation from the quanti fication sheet of the TCS Annotation workbook to a second instance of Export Excel that opens automatically when you press Export Save your data in Excel to retain it Images from the scanner sheet of the TCS_Annotation workbook are transferred to an external graphics program Note l Only selecte
46. a TCS confocal microscope and familiar with the general functions of the Leica TCS software Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 1 Page 1 Tutorial 1 This first tutorial lets you practice switching your Leica TCS machine on and off and gives you an overview of the Leica PowerScan software Before collecting an image you should familiarize yourself with the way in which the software and display are controlled We shall then stroll through all the steps possible with the software without actually collecting any data Switching on the Machine 1 Start your Leica TCS machine by following the procedures described in steps 1 through 3 of the chapter System Procedures 2 Log on to the operating and control computer and start the Leica TCS PowerScan software as described in steps 4 through 6 If you look at the startup screen carefully you will see that Microsoft Excel starts first The Leica PowerScan software runs on top of Excel using some of its functions but hiding most of the complexity of the pro gram After a few seconds the main control screen appears on your monitor X LEICA TCS NT TCS_Tools HR File Settings Help 181x TCS NT Version 1 6 582 Copyright 1995 1996 1997 1998 Leica Lasertechnik GmbH Heidelberg Features installed PowerScan Power3D PowerPhys To begin select one of the buttons Leica Load Images View Images Exit Program Acqui
47. a TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Special Safety Remarks for Users Page 8 2 In case of an inverted microscope tilt the light arm 3 Exchange the specimen 4 In case of an inverted microscope put the light arm into upright posi tion Do not disconnect fiber when the system is in operation Exchange the scan head only after having switched off the system How to exchange the scan head refer to the online help facility Look for the Topic Maintenance If a TCS UV system is ordered without a remote control unit for the UV Laser the Laser Power Supply has to be located in the same room as the Leica TCS If your are using the tube to observe the specimen switch off the scan mode The Scan button must visibly be released Be sure that there is no laser light in the focal plane Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Laser Safety Page 9 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 The laser produces a very intense and very narrow collimated beam of electromagnetic radiation in the frequency range of 200 nm to mm This radiation is generally in the form of intense vi
48. adius of the first minimum in this diffraction image This in turn is related to the numerical apertures of the objective and condenser The numerical aper ture 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 axis of the dif fraction image of a point object According to the theory for such three dimensional diffraction images the radius of the first axial diffrac tion minimum is approximately twice that of the lateral The axial reso lution is thus approximately half of the resolution within the plane of focus The LEICA TCS is a no compromise true point scanning system with high sensitivity and theoretical maximum x y and z resolution In re flection mode the LEICA TCS achieves an x y resolution of 0 18 um FWHM and a corresponding z resolution of better than 0 35 um FWHM at A 488 nm N A 1 32 glass air interface ideal environ mental conditions These figures are guaranteed for both upright and in verted configurations 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 more pixels the larger the scanned area that can be covered at a given pixel size The LEICA TCS uses arrays up to 1024 x 1024 pixels 2048 x
49. al The default is medium speed The scan speed is independent of the im age size in x direction The time it takes to collect a frame depends on the number of lines i e the y distance and the speed in lines per sec ond This means that the dwell time per pixel is less with a larger image Medium speed is generally used for small and medium images Choose a Slow scan speed with large images for improved image quality The Leica TCS offers five different acquisition formats If you click on the Format button you can specify the number of pixels for scanning number of sampling points in x and y direction Default is 512x512 pixels Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 3 Note oor Page 7 1 0xb4 obxcob 512x512 1Ue24e1024 Settings for acquisition and viewing formats are independent of each other If both formats match every pixel is reproduced 1 1 If the view ing format differs from the acquisition format the PowerScan software scales the image to fit the viewing area which leads to loss of data All images are saved with their original acquisition size regardless of the ac tual viewing size While changing the viewing format does not provide you with more data zoom can make more information visible Used in conjunction with the Pan arrows the function allows you to manipulate the area of interest more finely The Leica TCS confocal laser scanning microsco
50. are reference Contour 25 lines Contour 75 lines The Ortho slicer function enables you to display xz and yz sections from any region of your three dimensional data stack simultaneously Ortho selection of xy position Selectian of z position xiy slice 019 viz slice be xiz slice 333 size urm 05 3x65 3x1 3 5 Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 6 Slice l Page 7 Click with the left mouse button on the large image and move the mouse pointer to define the xy position for slicing You can define the z posi tion within the two small outer images Values for the slices currently selected are displayed in the bottom right hand corner The Slice function extends the function of the ortho slicer for arbitrary slicing Both the bounding box of your image and the slicing plane can be paned and rotated independently To the left of your image you will see a small representation of the bounding box white box and the slicing plane red plane You can control their movement with your mouse and two pairs of radio buttons The pair on the left specifies the action rotate or translate pan and the pair on the right specifies the object bounding box or slicing plane Choose any of the four possible combinations and click on the control image with the left mouse button keep the mouse button depressed and drag to rotate or pan the selected object Releas
51. are saved in one file You can open this file with every version of Excel version 7 or later for processing and use all the Excel functions and tools such as statistics charts and drawing tools to refine your data and images Archive the files to document your work The scanner sheet of the workbook is an exact copy of your image If you snap a scanned image the system uses the original image size of the scanned area which is independent of the actual viewing size in the TCS_Image window If you copy more than one picture to the annota tion workbook you can drag the individual pictures in the scanner sheet to rearrange them The PowerScan software offers tools from the TCS Tools window to add text boxes and arrows to your image Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 7 Page 2 There are two buttons for black and white text boxes and two for black and white arrows Select those appropriate for your image background Apart from the color button pairs are the same To add text to an object select the object image with a single mouse click Press one of the Text buttons A text box will pop up automatically in the top left hand corner of the object Overwrite insert text here with your own text Box text is a tool to edit text with determined margins The box can be assigned line and fill attributes A line of simple text will begin a new line of text when it reaches the e
52. 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 your self but contact your Leica representative Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes General Safety Directions Page 1 Safety Notes This section introduces you to standard safety precautions warnings and cautions 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 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 1s used for purposes other than those described in the accompanying docu mentation 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 im plicitly or explicitly modify the warranty and l
53. asses back through the same lens The light beam is fo cused into a small pinhole 1 e the confocal aperture to eliminate all the out of focus light 1 e all light coming from regions of the specimen above or below the plane of focus The achieved optical section thick ness depends on several parameters such as the variable pinhole diame ter The in focus information of each specimen point is recorded by a light sensitive detector 1 e a photo multiplier positioned behind the confocal aperture and the analog output signal is digitized and fed into a computer Up to 4 confocal detectors offer the simultaneous acquisition of multiple signal data with subsequent combined display Since the beam affects all the detectors simultaneously the reconstructed images show perfect reg istration with each other This allows simultaneous imaging of two or more different fluorescent stains 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 e The stage scanning type in which the microscope stage with the spec imen is moved in a scanning motion whi
54. ault format of text or a text box double click on the selection frame of the text The Format Object window appears Select the formats you want to assign to the text or the text box Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 7 Group hop Page 3 Format Object Pattems Font Alignment Protection Properties Font Font Style Size Arial F egular i 0 Pp Algerian El a _ Cancel Bold Y Arial Narrow Bold Italic El Underline Coo A a Not Fo a Preview Strikethrough Superscript AaBbCcY ydr Subscript This is a TrueType font The same font will be used on both pour printer and your screen This dialog allows you to assign fills and pattern to the text box You can create colored borders or round the corners The text can have any font and size available in your system If you want red or yellow text here is the place to select it The third tabstrip allows you to change the horizontal and vertical alignment and the orientation of the box text To add an arrow press one of the corresponding Arrow buttons A default arrow appears in the upper left corner of the object You can move and size an arrow just like any other object Shift drag an arrow to copy it Double click on an arrow to change its format Format Object HEI poe Biens Protection Properties Line Arrowhead Automatic Al Style
55. better suited for certain fluorochromes that are excited by green light such as Texas Red and Cyanin Blue In addition this laser has an orange line that is very good for exciting Cy 5 The argon krypton laser however is less stable than the argon ion laser and has a shorter lifetime Laser Wavelengths nm Argon ion 458 476 488 514 Argon krypton 488 568 647 Upright versus inverted models Light collecting capability of both types of microscopes is virtually the same If you work primarily with living samples or need access to the sample for micromanipulators microinjectors and the like the flexibil ity provided by the inverted configuration will probably best suit your needs The compact LEICA TCS confocal module can be exchanged between upright and inverted microscopes by the user in less than 5 minutes without the need for realignment Leica TCS Manual Rev 99011 2 Leica TCS 2 1 Confocal Microscope Systems What is confocal imaging Page 7 Photodestruction Photosensitivity limits the amount of time a specimen can be exposed to the scanning process Many fluorescent dyes are sensitive to the laser light used to excite them The accumulated stimulus of light causes the dyes to break down This phenomenon is called bleaching or photodestruction This imposes a limitation on the precision of the quan titative fluorescence measurements that can be made The photodestruction rate varies amon
56. box has to be installed so that the laser emission indicator red light visibly points to the front Do not obstruct the air ventilation slots minimum distance 15 cm Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes General Safety Notes Page 6 General Safety Notes In accordance with general safety regulations as well as laser device safety regulations comply with the following stipulations Operation only after instruction Only authorized trained personnel is allowed to operate the Leica TCS Observe the operating instructions Use the product only according to the information given in this docu mentation and in the online help facility of the TCS software Carefully read the operating instructions before you start the system As a prepara tion for your single work steps always read the relevant chapters in the online help and in particular observe the safety regulations indicated for handling the system You can get an overview of the single chapters in the contents file of the online help In addition you can take advantage of a tutorial program to learn step by step about the functions and char acteristics of the Leica TCS Use the product only for purposes described in this documentation and in the online help facility of the TCS software The system is designed for making confocal laser scan images and quantitative measurements in the disciplines of biology medicine and material sciences Using
57. brightness for the active image Modify contrast in a logarithmic way Gamma 2 Smooth low pass filter or sharpen high pass filter the current image T T Make changes permanent destroying original data Modi not available for some versions Display the first image in a series E Display the previous image in a series O Select a specific image from a series on T i La re Press the Start button to displays the images as a movie Click on the same button now labeled stop again to stop the movie av a Display the next image in a series a H iv Display the last image in a series ol a Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Quantify mode Page 17 Quantify mode Menu File Open Open images from a storage device File Save as Save entire data set to a new file in TIF or TCS EXP format File Save Selected Save frames of a selected pane to a new file File Default Legend Load a default legend not available as far as version 1 6 x File gt Exit Terminate the PowerScan program Settings Preferences Open the quantify mode preferences Settings Timelapse Not available in view mode Help Help Open the TCS NT online help Help Help on Help Open chapter How to use the Online Help Help About Leica Copyright info TCS PC Quantify mode preferences Preferences surface quantification surface fit
58. cal systems using upright and inverted microscopes Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 3 Page 3 Before you can assign new positions you have to press the Begin End buttons again to reset the position markers 8 Click on Scan again to stop continuous scanning for maximum sam ple protection You can now define a number of optical sections in the vertical z plane between the begin and end positions You can accept the system s auto defaults or specify your own num ber The auto function calculates the number of sections on the basis of certain plausibility assumptions To specify your own value click on the Sect button You can choose from among some predefined values If you select a user defined number of optical sections the resulting z stack size referred to as image size in the dialog box If you select a certain number of sections for acquisition 200 used for demonstation only and press the Calculate button the system responds with one of two reactions User detined Sections ImageSize Um 10 661 Sections 200 atepsize Wm 0 081 OK Cancel 1 If there is only one way to find the best combination of Image size and step size the system calculates this set of parameters automati cally Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 3 Page 4 Due to hardware limitations which occur w
59. can software switches automatically to View mode as soon as you load images 2 Press the Gallery button in the TCS_ Image window 3 Select Single mode to display all optical slices of a single channel in the TCS_ Image window 4 Select Tiled mode to display all optical slices of all channels in the TCS_ Image window The gallery function enables you to image all available optical sections on One screen Try switching channels on and off Overlay mode mixes the active channels When you are in single image view the overlay picture re places the detection channel image It uses the fourth quadrant in tiled mode Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 4 icy T A iv fat a A aj y a H T Select Page 2 Return to single mode If you have loaded a series of images you can navigate through your pictures using the buttons which are like those on a VCR Press First to display the first image in a series Press Last to display the last image in a series Press Prev to display the previous image in a series Press Next to display the next image in a series If you want to select a specific image from a series press Select This opens a new dialog box Drag the slider to select a frame or enter its number in the input field Click OK to confirm your entry Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tuto
60. ct Options The first time you click on this tab Windows tells you it needs to create a list Click Next and Finish to allow this Then you see the main Find tab Type the word s you want to find in the top text box Then click a word in the middle box to narrow the search Finally review the list of help topics at the bottom and double click the one you want to read Shut Down Windows Are You sure you want to Shut down the computer C Restart the computer Close all programs and log on as a different user ez Ho Help When you re done reading about a document click Help Topics to return to the main Help screen or click Back to return to the previous Help topic Click the window s Close button to exit Help Shut down the computer Flushes all unwritten data from memory to disk and closes the system Restart the computer Flushes all data and totally restarts the sys tem Close all programs and log Flushes all data and re runs the network on as a different user connection routine to allow the user to re connect to a network server Shut Down Windows NT Always use the Shut Down command before you turn off your PC The Shut Down option allows the user to close the Windows NT Workstation operating system and ensure all running processes can halt cleanly and are given the chance to flush any data that may be in cache memory out to the disk Several options are available
61. ctive lens with a small numerical aperture limits your ability to per form confocal sectioning ability When using a lens with a high numeri cal aperture the size of the detection pinhole determines the limit of the confocal sectioning ability The Leica TCS uses a detection pinhole of variable size To increase the number of photons available for detection you can open the detec tion pinhole This increases the signal to noise ratio of the image at the expense of optical sectioning performance Due to diffraction a point of light in the focal plane is imaged as a bright disc surrounded by increas ingly dimmer rings The bright center is called Airy disc The diameter of the Airy disc depends on several optical parameters such as wave length the numerical aperture of the objective the magnification pro vided by the objective and additional magnification factors of the system You can achieve the best possible performance by setting the size of the detection pinhole to the size of the Airy disc You can change the detection pinhole size using a panel box potentiom eter if assigned or using the mouse If you are in Acquisition mode you will see the Pinhole tool inside the TCS_Tools window Click on the button and keep the mouse button depressed Moving the mouse changes the pinhole size Move it upwards or to the left to increase the value and downwards or to the right to decrease it Release the mouse button to accept the value as
62. ctly by means of the motor focus device 1 Place a specimen on the specimen slide and if necessary perform the adjusting steps described in section 2 Settings for incident light observations 2 Turn the lens with the highest magnification factor into the beam path 3 Focus a distinct point of your specimen 4 Switch the microscope off 5 Switch your microscope on again while pressing the upper of the two keys to the right of the display on the microscope base motor focus 6 Now briefly press the upper of the two keys to the right of the dis play 7 Switch the microscope off again and then switch it on again while keeping the upper key depressed 8 You have now defined the focus plane of the lens with the highest magnification factor as the upper stop position of the specimen slide You can now press the motor focus key in order to easily find the fo cus plane again 9 Release the upper key change lens and focus again 10 Briefly press the lower key 11 Change the lens and press the lower key again 12 Repeat step 10 until you have defined all the lenses Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for inverted LEICA DM IRBE Microscopes Page 6 Setup instructions for inverted LEICA DM IRBE Microscopes The settings depend on the optional components with which the DM stand may be fitted This description refers to a fully equipped sy
63. d objects are exported You can define the external program in the preferences You can use programs from MS Paint to Adobe PhotoShop to process your image E tmp2 bmp Paint File Edit View Image Options Help Fou 1 Foret 512 x52 x32 InregeSize 14 2x 12x 17 5 77 To open the image in an external program a temporary file is created To keep the image you should save it under a new name in the graphics pro gram Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software Startup Windows NT Page 1 Running the Leica TCS software The Leica TCS software of your system runs under the Microsoft Windows NT operation system An operating system controls the way in which you as a user deal with and operate the computer For that it is necessary to have at least a good working knowledge of Windows NT Windows NT and the Leica TCS software are both installed and therefore ready for use when you take over the system from a Leica technician Therefore this short tutorial does not cover program installations or other administrative tasks Windows NT operates using an intuitive and usable Graphical User Interface GUI that makes your computer easy to use by providing menus and pictures to select Before you can take advantage of it however you must learn some Windows NT basics Startup 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
64. d the original data 2 Are you sure you want to replace the original data You can also use mathematical operations to further process a series of images This offers the possibility to represent 3D data on screen The information of the entire record and not only one single optical section is used with it Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 4 Page 6 To display an extended focus image press the ExtFoc button An extended focus image is an image in which the intensity values of a pixel of overlaid sections are added and then divided by a scale factor You can select the scale factor in the Preferences dialog box see PowerScan software reference If you set the scale factor to 0 the system assigns it the same value as to the number of optical sections to produce the arithmetic mean Ext Foc Crianded focus Image result Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 4 Page 7 To display a maximum projection of the sum of the acquired optical slices press Proj The system determines the maximum of all intensity values in the view direction in the different optical sections of a record This is displayed in the projection This method is particularly useful for detecting struc tures Direction of view Intensity Liz LZ 2 _2 UY MY MEE ARE AY AD Y JO Oy A H ZT Y Wh Wh He W Intensi
65. dge of the box or when you enter a re turn If you type more text into a box than will fit with the current text and box size the overflow characters are not displayed on the screen To scale the box select it and click drag the handles of the selection frame You must open text to edit it Place the mouse cursor on the text element and click when the mouse cursor changes to a text cursor This places the write cursor l in the text element The write cursor can be moved around in the text with the arrow keys Home positions the cursor at the beginning of the line End positions it at the end of the line When the write cursor is in the text you can add element characters behind the write cursor and delete characters using the Del or Backspace key You can close text elements by doing one of the following e Click outside of the text element e Open another text element O Activate another tool You can move objects like text boxes and arrows from one position on the screen to another by placing the mouse cursor over the frame of the element and click dragging it to the new position When the mouse but ton is depressed the bounding box follows the movements of the mouse When you release the mouse button the object is redrawn in this position It remains selected until you click on another position in the workspace To scale an object select it and click drag one of the eight handles on its selection frame To change the def
66. e field of view Then turn the centering screws of the field diaphragm or the condenser until the greater part of the field of view is illuminated Now close the field diaphragm again Aperture diaphragm Field diaphragm Focussing Focus the border of the light spot by adjusting the height of the con denser Centering Turn the centering screws of the field diaphragm or the condenser in order to center the light spot in the field of view The centering is easier if you slightly open the field diaphragm in order to enlarge the light spot Close the field diaphragm after centering the spot Opening the field diaphragm Open the field diaphragm until the border of the light spot just disap pears at the border of the field of view Closing the aperture diaphragm Close the aperture diaphragm until you have set the desired contrast If you change the objective It may become necessary to readjust the Kohler illumination after you have changed the objective Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for upright LEICA DM RE Microscopes Page 5 4 Setting the parfocality Due to the different working distances of the different lenses their focus lies at different absolute specimen slide heights Setting the parfocality means that the focus position of every single lens is stored After a changing of the lens the focus plane can be approached dire
67. e the mouse button when you are finished The image on the right reflects the settings you have made immediately Movement control C rotate C bounding box translate plane Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 6 Pegin 0 jiii Record icy T A iv fat a A aj y a H Page 8 The PowerScan software allows you to produce movies Windows AVI files of your rotated or paned pseudo 3D images To create a movie please proceed as follows 1 Select a certain orientation and or size of the current representation using the view tools in the Pseudo 3D view window 2 Press Begin in the TCS_Tools window to define the start position for the movie 3 Change the orientation of the current representation using the view tools in the Pseudo 3D view window 4 Press End in the TCS_Tools window to define the end position for the movie 5 Define the number of animation frames using the Steps button Use the default value of 10 slices or define your own Set number of animation frames I 4 Il L Ro Oe Record fi frames Cancel 6 Press the Record button to start the calculation of the movie You can navigate through your movie with buttons like those on a VCR Press First to display the first image of the movie Press Last to display the last image of the movie Press Prev to display the previous image of a movie
68. e tip of the mouse pointer must touch the item O Click on an item means to move the pointer onto the specified item and press and release the mouse button once Unless specified otherwise i e right click use the left mouse button Clicking usually selects an item O Double click on an item means to move the pointer to the specified item and press and release the left mouse button twice quickly Double clicking usually activates an item O Drag means to move the mouse pointer onto the item hold down the mouse button and move the mouse while holding down the button Unless speci fied i e right drag use the left mouse button Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software The Windows NT interface Page 4 The Windows NT interface The basic interface of Windows NT is called the Desktop which provides a background for the items it contains A Start EF Paint Shop Pro A LEICA TCS NT TCS_Tools 7 51 PM The initial icons on the Desktop allow the user to view and interact with the system in a logical way The Windows NT screen contains many special elements and controls Here s a brief summary O The background on which all the pictures and boxes rest is the desktop O The Taskbar shows the windows and programs that are open You can switch between open windows and programs by clicking the name on the Taskbar O The Start button opens a menu system from which you can start programs
69. ed any pa rameters your sample is bleaching Reduce the laser power until the im age stops darkening in continuous scanning mode Try to compensate with a higher PMT gain to brighten the image or examine another posi tion of your sample Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 2 Ph Contra st Sic ar Page 6 An alternative to using the panel box is to use the mouse to modify the sensitivity of the selected detector channel Press the Contrast button keep the left mouse button depressed and move the mouse up and down to change the sensitivity of the detector You will see that the brightness of the selected image changes TCS_Image TCS SP UV175021 User Name LLT Demo City State Comment Sections 2 Accu 1 Format 512 x 512 x 2 ImageSize 250 0 x 250 0 x 0 0 Zoom 1 00 PMT1 100 PMT2 100 PMT3 100 PMTTrans 100 Offset1 0 Offset 0 Offset3 0 OffsetTrans 0 Pinhole 2 24 Speed medium Scalebar 100 pm LaserPower 0 DateTime 11 21 98 5 38 PM Method FITC Lut Ch1 green Ex Splitter RSP500 BFWheell BP530 30 As soon as you have attained a satisfying result stop continuous scan ning by clicking on the Scan button again Now you can save this picture to a storage device like your hard disk Click on the File menu and select Save as Set the save type to ScannerFile tif Choose an appropriate name and directory Refer to the chapter Running the Leica TCS software if you
70. ee om ee Se Om 6 Acquisition mode preferences toto ttt tt tte 6 Acquisition mode time lapse ttt tt tt te 9 Acquisition mode tools E A AA ee ee ee ee a a 1 2 View mode A ee ee a ee a ke a o ee ee 1 4 Menu ao E A A a a u a mm A a 1 4 View mode preferences ss ttt ttt ttt tes 14 View mode tools E i et AA AAA sea 1 5 Quantify mode E O A oe ce ce a A 1 T Menu a al a o A A ee E A iy a 1 7 Quantify mode preferences ttt tte 17 Quantify mode tools x a th th ee ne 1 8 Pseudo 3D mode ae ee A ee ee ee AE ee ee 19 Menu SE A A A A Oe Oh 1 9 Pseudo 3D mode preferences totor 20 Pseudo 3D mode tools Be om a e A A E A a a 21 3D mode optional a ce ie eo e a ee we ee a 23 Menu O te ma ee ee a a 23 3D mode preferences Qe ay le Ge O a a a a a a 23 3D mode tools ce se G oe ee a we Se eS ee Se eo Oe a o 24 Physiology mode A E ee a ee ee a ee i 26 Menu ee ee a e E A ee ee ee o a 26 Physiology mode preferences ss ttt ttt 26 Physiology mode tools A me ee tee Si 26 Annotation mode E ee ee ACA AO 28 Menu eom ee AE ee ee me ee AAA 23 Annotation mode preferences sss ttt ttt 28 AnnotationmodetoolS s s s a s s soa 29 Leica TCS Manual Rev 99011 1 About this book How to use this book Page 1 About this book This book is both a tutorial and a reference manual for the Leica TCS We intend this book for the novice and the experienced user If you are a new
71. efault legend not available as far as version 1 6 x Terminate the PowerScan program Open the Pseudo 3D mode preferences Not available in Pseudo 3D mode Open the TCS NT online help Open chapter How to use the Online Help Copyright info Rev 99011 4 Reference 4 2 PowerScan software reference Pseudo 3D mode Note Page 20 Pseudo 3D mode preferences Preterences Common settings Show rotation angles wW Show bounding box Resolution Minimum Gamma _ gt 10 Movie Speed E 5 D viewer settings Scale Y axis 1 00 times Default mode Flat Contour lines eb ka ID viewer settings Default Mex Proj z mode O Mirror Z SFP Emission gt 52 SFPLightabs f gt 33 Transparency Pseudo 3D and 3D share the same preferences dialog Common setting Show rotation Display rotation angles x y and z axis in the up angles per right corner Show bounding box Always display a bounding box around the image Resolution Adjust the resolution of the image to be calculated in five steps Gamma Modify contrast in a logarithmic way 1 0 Back to preset gamma value 1 0 Movie Speed Adjust movie speed between 1 and 30 frames per second This setting 1s used for storing AVI files Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Pseudo 3D mode Flat b hule sh Surface Contour Pek Ortho
72. em is aligned the epifluorescence image is parfocal with the confocal fluorescence image When you want to stop studying the specimen activate the light stop de vice to protect the specimen from bleaching Push the lower lever to full Push lever up to 1 position Stop Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 2 Page 3 If you are satisfied with your image switch the microscope to confocal operating mode Turn the filter revolving wheel into scan position 1 Pull the lever on the left of the binocular tube right for inverted This closes the binocular light path for laser safety 2 Turn the thumb wheel on the lens tube to scan position if you have not already done so The computer monitor should display the main control screen of the PowerScan software 1 Click Acquire Images 2 A small dialog box appears where you can select a predefined filter setting FilterSettings 3 Choose FITC for now Click on FITC in the list on the left then click OK The control program displays the image and tools windows Acquisition mode is preselected Select your current objective with the Lens button in the TCS_Tools window Clicking on this button opens a dialog window containing a list box Click on the objective to select it The dialog window closes automati cally To close the dialog without selecting a different objective click on the Lens button again
73. ens will hit the specimen or the speci men slide during the automatic approaching of the focus plane thereby damaging the system When using oil immersion lenses note that these lenses should be wetted with oil before the parfocality adjusting step in order to avoid misalignment of the focus plane Leica TCS Manual Rev 99011 3 Using the confocal system 3 2 System procedures General Page 1 System procedures General The LEICA TCS system consists of several components that have sepa rate power supply units The actual number of components depends on the individual system configuration The components belong to one of the following categories the LEICA DM R and LEICA DM IR conventional microscopes the illumination units such as Hg Xe lamp or halogen lamp the laser s Ar laser Ar Kr laser UV laser HeNe laser the electronics the monitor s for system operation and image display the operating and Host computer PC Leica DM R Upright Leica DM IR Inverted The electrical components are connected to one phase of mains via a common distribution block Due to the considerable power consumption of the laser tube we recom mend that you connect the system to a second phase of the local voltage supply Leica TCS Manual Rev 99011 3 Using the confocal system 3 2 System procedures Starting up the procedure Page 2 Starting up the procedure 1 Switch on the LEICA DM R LEICA DM IR microscopes
74. equired for the output image The output image is usually written to a separate area of the frame store so that the original data remains unmodified It is safe to try different CLUTs on your image because you can always return to the original one A color look up table CLUT can be color or grayscale It is simply a way of mapping the various intensities in the image to new intensities The colors can overlap either to produce emulations of different stains or for purely aesthetic reasons The use of non continuous CLUTs can provide an instant visual indication of intensity information or sudden changes in intensities CLUTs can even map bands of intensities to bands of color This can be used to highlight minor changes in intensity as a sudden change of color Click the CLut button on the left side of the TCS_Image window to dis play the active look up table as a color bar on the right side of the im age If your image uses more than one channel every channel can use its own CLUT WN TCS_Image Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 2 Page 8 To select a new look up table click on the corresponding color bar This opens a dialog box listing up to 15 CLUTs LutSelection Channel 1 E Cancel glow glowdark glowoy er glowoyun poli rh rot bit ro M blue gray Choose a CLUT by clicking on the color strip The representation of your image changes immediately If
75. erical Aperture NA is not large enough to pro duce the thinnest optical sections it does have the practical advantage of a long working distance This reduces the risk of accidentally bringing the lens into contact with the coverslip when manipulating the position of the stage You can use more powerful lenses with greater numerical apertures once you have familiarized yourself with the working distance First you should have a look at your specimen using conventional epifluorescence illumination Refer to the microscope handbook and Setting up the Microscopes in this manual for exact instructions Do not switch on the fluorescence lamp mercury discharge lamp of your microscope while your computer is booting Electric disturbances caused by the lamp can affect the PC during startup The following are the basic settings required 1 Turn the filter thumb wheel to position 2 for FITC fluorescence 13 filter Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 2 Page 2 2 Push the lever on the right inverted or left upright of the binocu lar tube to open the binocular light path 3 Turn the thumb wheel on the lens tube to scan position no filter in the rear receptacle or Pos 1x for UV light Find an interesting area of your sample for imaging using the micro scope binocular eyepieces and conventional epifluorescence illumina tion Focus carefully Since the Leica TCS syst
76. ers coupled simultaneously with new merge module pat pend Up to 5 detectors 4 reflected light fluorescence 1 transmitted light for simultaneous acquisition Patented fiber coupling of UV laser All filters and beam splitters motorized High speed high resolution z stage for live vertical sectioning and 40 nm re positioning accuracy PC workstation with operating system Windows NT for stability and seamless network integration Single or dual monitors with Cinemascope desktop 2D measurements and Pseudo 3D surface reconstruction software standard Powerful 3D module based on AVS Express with 24 bit three channel rendering option Application Software for Physiology option Quantify Leica TCS Manual Rev 99011 2 Leica TCS 2 1 Confocal Microscope Systems What is confocal imaging Page 2 What is confocal imaging Although conceptualized in 1953 the Confocal Laser Scanning Micro scope has only in the past 10 years become a practical instrument To day it is the instrument 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 video technology electronics and computer technology laser technology and optics for coherent light 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 con
77. f the movie Display the last image of the movie Save a movie to disk AVI file Define the start position for a movie Define the end position for a movie Define the number of animation frames default 10 Calculate the movie frames automatically switches Don t Render off Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference 3D mode optional 3D mode optional Menu File Open File Save as File Save Selected File Default Legend File gt Exit Settings Preferences Settings Timelapse Help Help Help Help on Help Help gt About Leica TCS PC 3D mode preferences Page 23 Open images from a storage device Save entire data set to a new file in TIF or TCS EXP format Save frames of a selected pane to a new file Load a default legend not available as far as version 1 6 x Terminate the PowerScan program Open the 3D mode preferences Not available in 3D mode Open the TCS NT online help Open chapter How to use the Online Help Copyright info 3D and Pseudo 3D share the same preferences dialog Preferences Common settings Show rotation angles Cl Show bounding box Resolution Minimum w oe 6 aS Movie Speed al TE 2 5 D viewer settings Scale asis 1 00 r times Default mode Flat Contour lines 25 30D viewer settings Default Shading ti tst i sY mode Cl Miroz SFF Emissio
78. ference 4 2 PowerScan software reference 3D mode optional iv a l of ie Hi 0 a m T to z mF 0 O on T cn La re a H iy fat a a Pegin Haii End fii Steps Record Page 25 Set a threshold level for each channel to display Suppress immediate rendering Click again to restart Display the first image of the movie Display the previous image of the movie Select a specific image from of the movie Press the Play button to display the movie Click on the same button now labeled stop again to stop the movie Display the next image of the movie Display the last image of the movie Save a movie to disk AVI file Define the start position for a movie Define the end position for a movie Define the number of animation frames default 10 Calculate the movie frames automatically switches Don t Render off Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Physiology mode Page 26 Physiology mode Menu File Open Open images from a storage device File Save as Save entire data set to a new file in TIF or TCS EXP format File Save Selected Save frames of a selected pane to a new file File Default Legend Load a default legend not available as far as version 1 6 x File gt Exit Terminate the PowerScan program Settings Preferences Not available in physiology mode Sett
79. fety devices interlocks and laser containment systems are in service able condition and operating Disabled or damaged safety devices or sys tems will expose personnel to lethal high voltages and Class Illb laser radiation of sufficient power to cause severe eye injury burns and property damage Tampering with or deactivation of any safety system and or interlock termi nates Class 1 laser product performance will expose personnel to hazard ous Class lllb laser radiation bodily injury and void warranty coverage DANGER INVISIBLE LASER RADIATION The Leica TCS with UV system uses a Class Illb Argon lon Laser working in the UV A wave length range The output beam is by definition a safety and fire hazard Precautions must be taken during unprotected laser operation and mainte nance to prevent accidental exposure to direct or reflected radiation from the laser beam Wear ANSI OSHA approved UV laser protective eyewear any time there is an opportunity for unprotected laser exposure If you use S70 condensers it is possible in the worst case no specimen objective with low numerical aperture that the laser beam freely propa gates over the working distance of 70 mm This distance is large enough that the user may unintentionally insert reflective objects in the beam path For reasons of safety we therefore advice you not to use S70 condensers in confocal applications DANGER HIGH VOLTAGE Both the laser box and system electronic
80. fferent hardware installed and may or may not be connected to a net work or modem You may also have different Windows NT Workstation 4 op tions installed J Control Panel File Edit View Help E E 2 Y Accessibility Add Remove Console Date Time Devices Dial Up Display Options Programs Monitor ay es a a D a Fonts Internet Keyboard Modems Mouse Multimedia Network 7 usos PC Card Ports Printers Regional SCSI Adapters Server Services PCMCIA Settings Sound System Tape Devices Telephony 26 object s Double clicking the Network Neighborhood icon displays the Network Neighborhood dialog box which gives you information about who and what is connected to your workstation It provides an easy mechanism for browsing any network systems and resources that you may be able to connect to in a way that is independent of the actual type of network vendor Traditionally if a system needed to be simultaneously connected to different types of network the way in which each could be connected and viewed would be vendor specific Windows NT Workstation 4 is capable of displaying a common view of your entire net Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software The Windows NT interface Page 6 work even though it may actually comprise resources from Windows NT Novell NetWare Banyan Vines or others Network Neighborhood File Edit View Help 2 object s The Inbox icon is used
81. filter An The revolver positions are factory equipped with the following beam splitters Position 2 13 or L4 for FITC fluorescence Position 3 N2 1 for Rhodamine fluorescence Position 4 Scan position Select the desired filter by turning the revolver wheel 5 You can now study your specimen in the conventional fluorescence mode Now you can focus an interesting structure of your specimen fine focus wheel if you have not already done this 6 When you do not wish to study the specimen any longer please acti vate the light stop device in order to protect the specimen against bleaching Note You should work as quickly as possible in order to avoid bleaching of the specimen or to reduce at least bleaching effects Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for upright LEICA DM RE Microscopes Page 4 3 Setting the Kohler illumination l Focussing Focus an area of the object neglect the quality of the illumination for the time being Opening the aperture diaphragm Fully open the aperture diaphragm It will be closed at a later point in time for adjusting the desired contrast Closing the field diaphragm The field of view darkens in most areas You will see an unfocused light spot If the spot disappears the system is out of center In this case open the field diaphragm until you can just see the light spot at the border of th
82. focal system the microscope lenses focus the la ser light on one point in the specimen at a time the focal point The la ser 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 fluores cent light emitted from the focal point to a second point called the con focal 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 Detector Pinholes SERA Confocal Beamsplitter Objective ems Object NOT in Focal Plane Object in Focal Plane V E AE Object NOT in Focal Plane wee ASA SIS AAA Leica TCS Manual Rev 99011 2 Leica TCS 2 1 Confocal Microscope Systems What is confocal imaging Page 3 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 or emitted longer wavelength fluorescent light returning from the specimen p
83. g different dyes but is constant for any one dye The Leica TCS features a short emission light path and a minimum number of optical elements between sample and detector This design most efficiently collects the precious fluorescent light and avoids excessive excitation light allowing you to acquire high quality images with the least possible photodestruction Integration The Leica TCS was designed as an integrated system Optical and me chanical elements work seamlessly with computer hardware and soft ware Filters apertures controls and laser controls are available at your finger tips The integrated software package supports the complete im aging process from optical sectioning through image processing and analysis to hard copy output Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for upright LEICA DM RE Microscopes Page 1 Setting up the Microscopes Before recording confocal images with your TCS NT system you should first study the specimen by means of a conventional microscope and adjust the interesting specimen structures in the observation field Specimen adjusting procedures and the necessary microscope settings are described in this chapter References to the different recording tech niques in the confocal mode are given at the end of this chapter Which specific recording method you choose will depend on the specimen to be studied For more detailed inf
84. g the selection by moving the mouse 4 When the selection reaches the desired position release the mouse button To size the zooming area move the mouse to any corner of the square until the mouse pointer changes to a double headed arrow Press the left mouse button keep it pressed and move the mouse pointer to resize the square This is the same as the action for windows Note The maximum zoom factor with this function is 32 In this tutorial you have learned how to perform a z series and zoom and pan into your image In the next tutorial you will learn how to per form basic image processing Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 4 Page 1 Tutorial 4 Single Tiled Querlay The View mode in the PowerScan software contains some powerful im age processing features that allow you to work on single images or en tire image series In view mode you can process both images recently acquired and images retrieved from a storage device The TCS_Image window contains the same tools as the acquisition mode which will now be familiar to you We will be using some of these tools during this tutorial As a first step we will display a gallery of all acquired optical slices 1 Acquire or load a data set from a disk To open a file choose Files gt Open in the menu Single images or image series in Multipage TIF files are loaded into the RAM of your Leica TCS system The PowerS
85. ge height of a measuring surface Rt Rh Rd These figures represent the difference between the maximum and mini mum of a profile span Ra This figure represents the average peak to trough height of a measuring surface Rq This figure represents the scatter of amplitude values around the zero line Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 5 Page 8 Stock This figure represents the amplitude symmetry factor This measurement of the symmetry of the amplitude distribution can yield both positive and negative values e Negative value most of the peaks project into the material e Positive value most of the peaks project over the material Area This value is the area of the marked region of interest and the plan pro jection of the real surface of the specimen Surface This function provides the total surface of the specimen within the re gion of interest Ratio This value indicates the degree to which the surface is fractured The to tal surface is divided by the area of the region of interest The result compares the surface value with the value of the area of interest and de termines the fracturing degree of the surface Volume This figure indicates the volume of the object within the region of inter est Note l For further details please see the online help in the PowerScan software Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tu
86. h your view press the Pseudo 3D arrow button to switch to pseudo 3D mode PowerScan Opens a new window called Pseudo 3D View to display its output Default mode for pseudo 3D is flat mode You can change this in the preferences dialog see PowerScan software reference for details To calculate a flat projection of the current image press Flat In contrast to the normal viewing mode you can define a different view point within flat mode zoom rotate etc Pseudo 3D requires a large amount of CPU power Generating a new view requires some time Press the Don t Render button to suppress immediate rendering if you plan to change several viewing parameters in quick succes sion Click on the button again to restart the PowerScan rendering engine Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 6 Note oom Page 2 PowerScan offers five distinct resolutions of the image to be calculated Al ways use minimum if you try different types of presentation since the res olution setting has a very substantial influence on CPU time Increase the resolution in the preferences dialog step by step to achieve the final pic ture Higher resolutions do not always result in a better image Since the preferences dialog is a non modal window you can leave it open while ad justing other view parameters The Pseudo 3D View window has its own set of tools in the panel on the left They all process
87. he group that contains the program you want to start for instance Leica TCS NT 4 Click on the program you want to start for instance TCS NT Due to initialization of the hardware the start up process might take a while Upon program start some adjustments of the microscope are per formed Imaging cannot occur during this period Leica TCS Manual Rev 99011 3 Using the confocal system 3 2 System procedures Shutdown procedure Page 5 Shutdown procedure Please check the following before switching off the system Have you saved all images contained in the frame store If you do not save the image information to the hard disk or an optical disk you will lose this data as RAM is a volatile memory Save the image information single images or image series from the file menu of the Leica TCS software Save as or Save selected When do you want to use the system again Frequent switching on and off considerably reduces the service life of the laser This also applies to the microscope lamps Therefore if the interval between two sessions with the system is less than six hours you should not power down the system Simply reduce the laser power to minimum by turning the rotary button laser box counter clockwise to the left stop This is of course only a recommendation you may power down the system as you want You must note however that the service life of the laser is reduced by frequent on and off cycles t
88. his also applies to opera tion with too much laser power The steps required to power down the system are described below Please always proceed in the correct sequence Switching off the system 1 Switch off the laser This is done in two phases Phase 1 Reduce the laser power by turning the rotary button all the way to the left stop e Switch off the laser by turning the key switch to the left until you reach the left stop e The cooling fan is still active e The fan should remain active for approximately five minutes after powering down the laser in order to entirely cool down the unit Phase 2 e Switch off the power supply unit which also switches off the fan If your system features a laser other than the OMNICHROME laser please refer to the manufacturer s instructions Leica TCS Manual Rev 99011 3 Using the confocal system 3 2 System procedures Shutdown procedure Page 6 2 While the fan cools down the laser you can perform the next steps in order to power down the system e Check whether you saved your images If not select File gt Save as After saving your images to disk terminate the PowerScan software To end the session select File Exit e Close the Windows NT Workstation 4 operating system Always use the Shut Down command from the start menu before you turn off your PC See Chapter Running the Leica TCS software about log off procedures e Switch off the electronics box N
89. iability clauses contained in the general terms and conditions of Leica Microsystems Heidelberg GmbH Warnings Cautions and Notes The warnings cautions and notes in this manual use the following for mat Warning 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 state ment that must be strictly observed to prevent equipment damage or de struction or corruption or loss of data Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Laser Class Page 2 Notes Note Notes are statements that either provide extra information about a topic or contain special instructions for handling a particular condition or set of cir cumstances Laser Class This instrument is designed and manufactured to comply with applica ble 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 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 In order to take into acc
90. in the current sequence Del Delete the current interval Save Save interval data of this sequence to disk itv Load Load interval data of this sequence from disk itv Basic Back to basic interval mode Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Acquisition mode Page 12 Note a If you have defined more than one interval for this sequence you will loose all but the first if you switch back to basic mode TCS Timelapse All intervals but the first have to be removed before switching to basic mode Do pou really want to switch to basic mode 7 Acquisition mode tools Open the filter settings dialog Filters ae Select objective as defined in preferences T i Select scanning mode You can choose xy and xz sections and xyt or xzt time series Scanning size in pixels Format lode p Start continuous scanning press again to stop scanning Scanning speed Modify brightness and contrast of the selected detector channel by mov Contrast ing the mouse O Modify pinhole size by moving the mouse Fink al Electric zoom factor oor Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Acquisition mode Page 13 Move pan scan field for zoom factor gt 1 17 HN 4 Starting position for a z series Art Tn Begin Ending position for a z series Number of optical sections in z axis Number of accumulations f
91. information or parts from it user definable is shown in the legend panel on the right side of the images or copied to the annotation workbook on Snap Panelbox Display the panel box dialog to assign functions to potentiometers Preferences Panelbox Configuration RARE A com Default To change the panel box configuration click on the arrow to the right of the drop down list and choose one of the available parameters Auto parameters are active for the currently selected 1m age channel Fiore PMT auto PHT 1 PRT PRT 4 PTT Offset auto Offset 1 Offset 2 Offset 3 Offset 4 Offset T Pinhole Phaze Fos r Fos Y Pos Z oom Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Acquisition mode Reset Default Close Y Selection Bleach Y Selection Z Wide Simple Time lapse delay UV Lenswheel Warnings Micro Objectives Leica TCS Manual Page 8 Undo changes go back to default configuration Set actual configuration as new default Assign values and close dialog Make moveable y bar visible in xy mode to spec ify y position for a subsequent xz scan Open the wizard to calibrate the spot bleaching feature Activate the Z Wide drive of your electronically driven LEICA microscope stand e drive instead of the z fine focusing stage Select your LEICA microscope type within the list box Define waiting intervals between individual scan n
92. ing g windows NT Command Glossary Print Cancel Windows NT includes a powerful help system In addition to Help menus in ev ery window there is a standalone Help feature available from the Start menu To access it click your mouse on the Start button and click on Help There are three tabs in this box Contents Index and Find The Contents tab appears on top first To move to a tab click on it O Contents The Contents tab displays individual Help topics The topics are organized into categories and are represented by small book icons Double click on any book to open it Sub books and documents appear Double click on sub books and documents to open them Index The Index tab displays an index of all available topics Type the word you want to look up The Index list scrolls to that part of the alphabetical listing When you see the topic on the list that you want to read double click on it Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software Shut Down Windows NT Page 12 O Find The Find tab provides a text entry box for you to type the specific word s or phrase you want to search for under Help rather than searching for informa tion by category The text entry box is linked to a list of words in your Help files and any words or phrases that match will be shown You can specify more than one word by separating words with a space If you wish to change a search option sele
93. ing processes to obtain time series of individual xy sections Toggle lens wheel warning If on pops up a warn ing message when an UV lens is used that does not match to one of the three available pinhole op tics Allocate objectives lenses and turret positions The identification of the objective supplies you with additional information about every single ob jective e g lateral and axial solution working distance Micro Objectives Slot M S500 0TH dry M PLAN sxi iM dry PLAM 100 348 dry PL FLICT AF 160 54 imm PL FLUOT AR 200 44 dry A PLAN 200 454 imm MW PLAN 200 55 dry PL FLUOT AR Ly 250 758 ol PL FLUOT AR DY dd oil PL FL 1 0 5 Uy adh oil PL FLUOTAR q i TEMA dry PL APO 400725 R4 oil PL APO 1 25 0 75 Ly 631 215 w PL APO Uy 631325 oil PL APO sixt A w PL APO j A A sb Pim Rev 99011 4 Reference 4 2 PowerScan software reference Acquisition mode Page 9 Acquisition mode time lapse Time lapse offers a widely flexible tool to define your own scan se quences for the series scan mode You can define up to seven different sequences for optical series and one additional series that is triggered by an external signal on the remote user port TCS Timelapse 1 7 Click a button to immediately start a defined se quence Note If you press a button the new series starts immediately terminating other running sequences InPort This series is triggered by an electrical signal o
94. ings Timelapse Not available in physiology mode Help Help Open the TCS NT online help Help Help on Help Open chapter How to use the Online Help Help About Leica Copyright info TCS PC Physiology mode preferences None Physiology mode tools Draw straight lines as region of interest ROD Line Draw a rectangle or square as ROI Rect Draw an ellipse or circle as ROI Hlip ze Draw polygons as ROI AEN Poly ari Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Physiology mode Page 27 S IN Observe changes in xt images for multiple regions of interest ROI Delete all drawn objects Start analysis for all regions of interest Display the first image in a series Fi le Display the previous image in a series Fre Select a specific image from a series mm T cn La re Press the Start button to display the images as a movie Click on the same button now labeled stop again to stop the movie av ae Display the next image in a series a x iv Display the last image in a series rm ast Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Annotation mode Page 28 Annotation mode Menu File Open Open Excel workbook xIs from a storage device File Save as Save workbook to a new file in XLS format File Save Selected Not available in annotation mode File Default Legend Load a default lege
95. ith every confocal system the minimum step size for the Leica TCS system is 40 5 nm This is far better than the xz resolution of all objectives currently used All step sizes must be multiples of 40 5 nm The system calculates the corre sponding step size as a multiple of 40 5 nm under the restriction that the image size which is the current height of the complete stack of optical sections should be matched as exactly as possible and the number of sections should not be smaller than the value you defined Alternatively you can stipulate the step size and the system will calcu lated the number of sections 2 If there are several possible combinations for a given set of param eters the system opens a stack parameter correction window If you originally defined the number of sections the system will offer you two alternatives Either you vary the number of sections in order to minimize the differ ence between real and calculated image size or you vary the image size so that 1t matches the product of the number of sections and the step size If you originally defined the step size the system will offer you two al ternatives a Either you vary the step size in order to minimize the difference be tween real and calculated image size or b you vary the image size so that it matches the product of the number of sections and the step size Stack parameters correction One or more values have to be adjusted to match the current ha
96. ive pane has a red dotted border e Single click on another pane this is now the active pane and it now has the red dotted border Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 1 Note CH1 Chenay m Single Page 4 The functions Contrast Brightness Gamma Filter and Save Selected re late to the image in the active quadrant only Save As Snap and Print refer on the entire image In this section the In this quadrant the 1st detection channel 2nd detection channel is displayed is displayed The active detection channel is marked with a red doted line In this quadrant the In this quadrant either 3rd detection channel an overlay image or an is displayed optional 4th channel is displayed The first detection channel is displayed in the first top left quadrant The second detection channel appears in the second quadrant top right and the third in the third quadrant bottom left The fourth quadrant bottom right represents a superimposition picture overlay or an op tional fourth detection channel Individual detection channels can be switched on and off by pressing or releasing the appropriate channel button You can switch the overlay image up to 3 channels superimposed in red green and blue RGB on and off using the relevant Overlay but ton If you are in single image view the overlay picture represents all detec tion channels Only activated detec
97. l Measures Note Direct exposure of the eye by a laser beam should always be avoided with any laser no matter how low the power Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Laser Hazards Page 10 Class e Exempt lasers that cannot produce a hazard Class Il e Low power visible laser which due to the reflex response does not normally present a hazard unless viewed directly Class Illa O Lasers that normally do not produce a hazard if viewed momentarily with the unaided eye may produce a hazard if viewed using collect ing optics Class Ilib O Lasers that can produce an eye injury if viewed directly including intrabeam viewing of specular reflections Class IV e Laser that can produce an eye injury or skin burns from direct specu lar or diffuse reflections may be fire and skin hazards Class I IL and IIIa lasers should have a yellow and black Caution la bel while Class IIb 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 hous ing and operated in a lower classification mode may be classified at a lower classification Specific control measures may be required to main tain the lower classification Laser Hazards The danger from lasers can be divided into the following major catego ries 1 Eye hazards such as retinal or cornea burns RETINAL INJURY
98. l dialog box in which you can select a predefined filter setting Fiter ating 3 Choose FITC Since we are not going to perform a scan at present this selection is not important Click on FITC in the list on the left then click OK A filter setting allows you to select the wavelength of the excitation FITCYTRIT C400 bros FITC TRITC CY5 100 E and emission light This adjustment can be stored under a specific name for later use 4 The screen that pops up now is divided into two windows the TCS_Image window and the TCS_Tools window All images are displayed in the TCS Image Window The TCS Tools window contains tools You can size and drag both windows in the same way as any regular Windows NT window The picture in the TCS_Im age window is fixed to one out of three possible sizes It will normally be most practical to align the Image window above the Tools window If your system is equipped with a dual monitor display you can dedicate an entire screen to the Image window Leica TCS Manual Rev 99011 3 Using the confocal system oon 3 5 Tutorial Tutorial 1 312 1024 Tiled Page 3 TCS Image Window TCS_Image C users TCS_User Images fibro inf TCS NT 165141 User Name LLT Demo City State Stephan s Test Comment Sections 36 Accu 0 Format 512 x 512 x 36 ImageSize 20 5 x 20 5 x 57 6 Zoom 5 26 PMT1 453 PMT2 100 PMT3 100 PMTTrans 100 Offset O Offset 0 Offset3 0 Off
99. le the optics remain station ary e The beam scanning or mirror scanning type in which the illuminated point is scanned over the fixed specimen by small fast galvanome ter driven mirrors as used by LEICA The LEICA TCS 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 z 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 Leica TCS Manual Rev 99011 2 Leica TCS 2 1 Confocal Microscope Systems What is confocal imaging Page 4 Resolution The term resolution refers to the ability to distinguish fine details in a structure An ideal microscope would have optics totally free from aber rations 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 r
100. m the adjusting steps described in section 2 Settings for incident light observations 2 Turn the lens with the highest magnification factor into the beam path 3 Press the LEARN and subsequently the CHANGE key below the display The PARF message should flash on the display now Hielal ODER PARA OB lea LVR 4 Press the LEARN key again The ADJUST amp LEARN message should flash on the display now 5 Now use the motor focus or the fine focus wheel in order to focus a distinct point of your specimen Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for inverted LEICA DM IRBE Microscopes Page 13 6 Press the LEARN key again The display should now show the ADJUSTED message 7 Use the automatic turret in order to change the lens The display should now show the ADJUST amp LEARN message 8 Press the LEARN key again The display should now show the ADJUSTED message 9 Repeat steps 7 and 8 until all the lenses are defined in terms of their parfocality 10 Repeatedly press the CHANGE key until the EXIT message flashes on the display 1 1 Press the LEARN key in order to exit the calibration mode Note If there are any unused lens positions in your lens turret define the lower stop position as the focus plane This way you avoid that an existing l
101. ment in resolution just empty magnification Please see the zoom ta ble in the online help system for the optimum zoom values Exceeding these values will make the image bigger but with no further improvement in reso lution You zoom in toward the center of the image If the zoom factor is greater than 1 0 you can pan the image within the maximum scan field by clicking on the arrows to the right of the zoom button Press the appropriate arrow to move the scanned area in the required di rection Clicking on the square at the center of the Pan arrows centers the zoomed area If you are using a slow scan speed you will have to wait a second before seeing the results Newer versions of the PowerScan software contain a faster and more vi sual method of zooming and panning in a single action the ZoomIn function To use it you must acquire an image in xy mode and then stop scanning click the Scan button again Next click the ZoomIn button in the TCS_ Tools window A square selection appears at the top left hand corner of the image It defines the area of interest square where the system zooms in during the next scan Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 3 Page 9 Drag the square to the area of the picture you want to enlarge 1 Place the mouse cursor over the border of the selection It changes to a four headed arrow 2 Press the left mouse button and keep it depressed 3 Dra
102. n SFF Light abs Transparency CEET a oe Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference 3D mode optional Ext Foc MaxP ro Mo Light shading MA UA Solid SFP Page 24 Common settings See Pseudo 3D mode 2 5D viewer settings Not applicable to 3D 3D viewer settings Default mode Select the default type of 3D presentation Mirror Z Mirror the z axis to compensate for a different definition of TOP and BOTTOM between upright and inverted microscopes SFP Emission Define the strength of the simulated emission of every voxel for SFP simulated fluorescence pro cess SFP Light abs Define the strength of the simulated absorption of every voxel for SFP Transparency Define the amount of transparency for every voxel Close Close dialog 3D mode tools Averages the gray scale values of a 3D image and displays the result as an extended focus image Calculates a flat projection of a 3D record The pixel intensities repre sent the maximum gray scale value along the z axis of the 3D record Direct compositing raytracing rendering method Render Phong shading proportional to intensity Render an interpolated surface presentation Raytracing rendering mode that simulates the absorption of excitation light and emission of fluorescence light by the specimen simulated flu orescence process Show contour with equal brightness Leica TCS Manual Rev 99011 4 Re
103. n the remote user port pin 2 Load Load all sequence information from a disk file sq Save Save all sequence information to a disk file sq Close Close dialog window without saving Define Define one type of interval for a series of optical sections Interval Definition Button 1 Set Output Signal krones i each time interval 13 run Number of Frames to scan fo Accumulated frames i Frame duration lo seconds Delay after interval lo seconds Repeat Interval i timez General Settings M Set marker 1st run Continue with Advanced gt enones Finish Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Acquisition mode Note Type Set Output Signal Number of Frames to scan Page 10 Definition is for a time lapse series or for spot bleaching Trigger external device remote user port pin 3 4 7 or 8 or none Sets the number of optical sections acquired be tween top and bottom position within your speci men Top and bottom position must be set with the Begin and End buttons within TCS_ Tools window if you intend to do xyzt series Accumulated Frames Frame duration Delay after interval Repeat interval Set marker Continue with Number of scans to be accumulated and averaged per optical section Minimum
104. n images The Light vector button activates a light vector Position the mouse cursor anywhere on the image Press the left mouse Light button and keep 1t depressed until the light vector becomes visible as a white bar Pseudo 3D View 20 lo d y E a x ry A i m Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 6 Page 5 The direction of the arrow corresponds to the direction of illumination The light vector can be rotated in the same way as images The use of a light vector makes your image appear even more realistic To end this setting release the mouse button All work was done in flat mode up to now To present the data as a mesh press Mesh Mesh ne l l E aii A mesh indicates the intensity of the image on a 3D grid The resolution see preferences determines the number of gridlines Mesh low resolution Mesh medium resolution The Surface function renders a pseudo 3D image in which the intensity of the individual pixels is converted to height values Surface Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 6 Page 6 The Contour function creates a pseudo 3D image where the intensity of the individual pixels is converted to height values Pixels of the same in Contour tensity are connected by contour lines like those on a map You can set the number of contour lines in the preferences dialog see PowerScan softw
105. nd not available as far as version 1 6 x File gt Exit Terminate the PowerScan program Settings Preferences Open the annotation mode preferences Settings Timelapse Not available in annotation mode Help Help Open the TCS NT online help Help Help on Help Open chapter How to use the Online Help Help About Leica Copyright info TCS PC Annotation mode preferences Please enter the complete path pointing to the application you wish to use for data export IC Win324pp Photashp photoshp exe Browse _ Cancel Export sends image data to this external graphics program Browse lets you browse your hard disk for the required executable Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Annotation mode Page 29 Annotation mode tools Add black text to your images 5 Add white text to your images um a ut Add white arrows to your images Add black arrows to your images Group objects Separate grouped objects Lig rou po Delete selected object 2 e NE Delete li Print annotation worksheet Pret Export quantification data to an Excel worksheet or image data to an ex i Export ternal graphics program Leica TCS Manual Rev 99011
106. ngs Preferences Settings Timelapse Help Help Help Help on Help Help gt About Leica TCS PC Page 6 Open images from a storage device Save entire data set to a new file in TIF or TCS EXP format Save frames of a selected pane to a new file Load a default legend not available as far as version 1 6 x Terminate the PowerScan program Open the acquisition mode preferences Open the time lapse dialog Open the TCS NT online help Open chapter How to use the Online Help Copyright info Acquisition mode preferences Preferences Legend Info Panelbox T Secleckion Bleach i T Zelection wide Simple Timelapse Dela UY Lenswheel Warnings O active Calibration E ackive OM IRBE 5 seconds O active Micro Objectives Slot O e o h Magnification Optical Resolution at 488 nm theoretical values HA immersion Indez Tape WorkDist CoverGlass uy micron micron nm zz nm 0 75 dry 1 PL APO 0 51 0 1 mo 5027 643 53 125 oil 1 518 PL APO 1 25 0 75 El 0 17 TES 156 16 334 40 Close Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Acquisition mode Page 7 Legend info Display the legend info dialog Institute Institute of Cellology Ine Address 1 Golgi Way 25 Address 2 Scan City UBA sss i sSSSCS Comment 1 FITC stained elephant Comment 2 Comment 3 Comment 4 Enter the information into the edit boxes All
107. o 12 Serial Number and Year of Manufacture Identification Label 2 se eee ee ooo 12 Laser Safety Labels st tt ttt tes 12 Laser Safety Standards o o sooo roo noron 13 Tutorial eos owe eh eee ew wee oe 1 Tutorial 1 i th ew me eo ee 1 Tutorial 2 oe ee Nw e et me oO we ow ew ee oe se oe ee Me Oe a 1 Tutorial 9 E O a ae we E ee ee a o e a 1 Tutorial 4 E a a we 8 ee ee ee Oe es ee oe oe ee Oe Oe we OU E 1 Tutorial 5 ir cn ee we ee eS kee Oe eo ewe Oe ee we 1 Tutorial 6 Eo an a eae a a a o eae a a 1 Tutorial 7 i k ea AE EA A Se ee ke ee EEE me te 1 Running the Leica TCS software 1 Startup Windows NT ie ee ee oe oe oS eS ae oe ee Oe ee Se Oe 1 Using a Mouse A A A A E 3 The Windows NT interface sss ttt totor 4 The Start menu tt tt tt ee 7 The Taskbar ee ee A Se ae OO eh ae Oe 9 Setting the Time and Date sss sett tts 10 Getting Help ee e ee Oe oe E A A a OR Oe 1 1 Shut Down Windows NT ttt tt ts 12 Leica TCS Manual Rev 99011 Page 3 PowerScan software reference 1 Starting the program ee ee nunun onun ouu un ouuu 1 Main control screen o osooso wowo aono ee 2 Menu a e re ee RA A AE a AA A A E 2 TCS_Image window tools str ttt motors 3 Pseudo 3D and 3D view windows sss str sss 4 Tools window ke ek ce ee ek he ee o a ee ee he G 5 Acauisition mode chine ee ke Ss ee ee ee on se ee ae oe Se ee 6 Menu ote oe oe ee Oe he Oh ee Se ee ee
108. o achieve an image of reasonable brightness Adjust the laser power as necessary 4 Use the motor focus device to move the specimen slide z position potentiometer of the panelbox As the sample is lowered upright microscope the image will change and then become black At this point all regions of the sample are out of focus As the sample is raised again the focal plane moves deeper into the sample Optical sections are produced from the top to the bottom of the sample With inverted microscopes the direction is reversed Due to absorption of light in the upper sample regions and aberrations image quality and intensity become worse The maximum depth at which im ages can be produced from inside the sample will vary from sample to sample and with the objective lens in use 5 Go back to the brightest part of the sample and adjust gain and offset again to achieve an optimal image Now you have to define the start and stop positions for series scanning mode 6 Move to the start position and click on Begin in the TSC_Tools win dow to record this position as the starting level 7 Next move to the end position and click on End to record this posi tion as the stopping level The z distance is displayed with positive values If you press Begin in the lowest focus position and End in the highest focus position the dis tance is displayed as negative values The definition of these values al lows for consistent operations with confo
109. o channels usually only vary within a small interval you can select a window with a certain width All values in this window are spread over the 256 values of a look up ta ble You can define the starting point of your window using the offset slider Off while the range of the ratio is defined using the range slider Rn Press CLut to display a color bar to the right of the image pane Usually all intensity values measured by the detectors are evenly divided into 256 gray values and translated to a certain color by means of a color look up table You can select a different look up table by clicking on the color bar Each individual detection channel can be converted to a com plete glow scale LutS election Channel 1 o Cancel rh rod bit blue gray q a oo glow glowdark qlowoy er glowoy un poli We will learn more about color look up tables in a later tutorial Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 1 Page 6 The Snap function copies the contents of your image window into the annotation workbook You can use this annotation sheet to create your own documentation You will find this workbook by clicking on Anno tate in the TCS_ Tools window An annotation sheet is in reality a page of an Excel spreadsheet It can be saved as a separate file for additional processing You can use all the Excel word processing and drawing functions to polish up yo
110. o start the con tinuous scan The beginning of the scanning process is indicated by the faint sound of the shutter opening An image should appear shortly in the TCS_Image window You can adjust some parameters in the Leica TCS system using 7 poten tiometers in the panel box normally located in front of your monitor s You can configure the individual digital potentiometers as you wish choosing from a large number of parameters To learn how to change settings please see the chapter PowerScan Software Reference A standard configuration is as follows from left to right Photomultiplier 1 PMT1 gain detector voltage PMT2 gain PMT3 gain Phase Pinhole Zoom Position Z Y Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 2 Page 5 Preferences Panelbox Configuration amp O gt Reset Default The gain is the voltage across the PMT Increasing the gain produces a brighter image but also increases image noise If the gain is set too high the image will be saturated This means that too many pixels are at peak brightness The gain is set correctly if only a few pixels reach peak brightness The offset is a DC offset on the PMT voltage It is used to adjust the level below which no signal is seen Only values above the offset are spread over the entire color range and displayed When this is set correctly all parts of the image not representing de tected pho
111. oft ware The Physiology software package is designed for offline analysis of data sets The analysis software calculates the mean intensity across the de fined regions of interest ROI Most tools allow you to define a certain number of ROIs similar to the Quantify step You can define your own formulas Annotate TCS_Tools nn J royo y Paete30 20 Poy Y Ase Ada RRE El Text Teti Arro Arrow Groupi Ungrpf Delete Print por This step allows you to compose a document consisting of images and data sets It uses a regular Excel workbook There are two sheets Scan ner and Quantification A apro Quantification Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 1 Page 10 Only data produced in the quantification step is copied into the quantifi cation sheet of the annotation workbook You can use the Snap function in image windows to copy images to the Scanner sheet A selection of tools allow you to add text and arrows to your image and to group ungroup or delete objects Annotation workbooks can be exported You can export data on intensity distributions and histograms as well as ex porting pictures to your own external image processing software defined in the Preferences menu such as Adobe PhotoShop not available from Leica Now that we have seen all the available steps and tools it is time to shut down the machine again Switching off the machine The components sho
112. omes with an online tutorial that teaches you how to use the software and hardware It s an online workbook because you can practi cally enforce the steps that make up the tutorial to learn how to use the Leica TCS software To start the tutorial choose Help Help in the TCS NT control program Under the appearing menu bar you find a Leica TCS Manual Rev 99011 1 About this book Online help Page 2 switch button with the writing Tutorial on which you must click to open the online tutorial Online help There are many online help pages available throughout the Leica TCS software To start the online help choose Help Help from the control programs main menu Most windows offer a context sensitive help Press the F1 key to get assistance with a specific task Leica provides several other ways you can find answers to your ques tions Use the online help and online documentation 1 Choose the Help gt Contents switch button 2 You will see a page that contains links to various types of online help and online documentation For example you can click on the link to the Leica TCS system handling Search for help by looking up a word 1 Choose the Help Index switch button You will see a page with a Find tab that lets you search the help file 2 Type a word or words in the first field You will see a list of sections that contain those words You can double click on a link to move to a section or p
113. or averaging Start a series of optical sections Interactive zoom and pan Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference View mode View mode Menu File Open File Save as File Save Selected File Default Legend File gt Exit Settings Preferences Settings Timelapse Help Help Help Help on Help Help gt About Leica TCS PC View mode preferences Page 14 Open images from a storage device Save entire data set to a new file in TIF or TCS EXP format Save frames of a selected pane to a new file Load a default legend not available as far as version 1 6 x Terminate the PowerScan program Open the view mode preferences Not available in view mode Open the TCS NT online help Open chapter How to use the Online Help Copyright info Preferences ExtendedFocus scale by Topological Image Threshold 0 pa times 1 a Maximum ll C Center of Mass Invert image Y Stereo Angle degree Movie Max framerate Frames s Ratio Image a 1 channel 2 channel C 2 channel 1 channel Close Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference View mode Extended Focus Scale by Topological Image Threshold Maximum Center of Mass Inverted image Stereo Angle Movie Ratio Image Close View mode tools On gi rial
114. ormation regarding operation and use of your mi croscope please see the user documentation for the LEICA DM R and LEICA DM IR microscopes Setup instructions for upright LEICA DM RE Microscopes The settings depend on the optional components with which the DM stand may be fitted This description refers to a fully equipped vertical system E version so that your system does not necessarily possess all the components mentioned in this description It is assumed that the lighting modules are fitted and that the lamps are adjusted 1 Mounting and defining the objectives The LEICA DM R microscope is fitted with a non motorized lens turret The following explanations refer to this type of microscope 1 Sort your lenses We would recommend sorting the lenses according to their magnifi cation factor Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for upright LEICA DM RE Microscopes Page 2 8 J Move the specimen slide to its lowest position Press and hold down both keys to the right of the display on the microscope base motor focus while you switch on the microscope This makes it easier for you to screw in the lenses Screw the previously sorted lenses into the appropriate holes Switch your microscope off Switch your microscope on again while pressing only the upper of the two keys to the right of the display on the microscope base mo
115. ote Never switch off the PC without a shutdown 3 After you have deactivated the system software you may switch off the hardware components at any order 4 Switch off the laser cooling system by way of the laser power supply unit Leica TCS Manual Rev 99011 3 Using the confocal system 3 3 System Care Page 1 System Care Please refer to the corresponding manuals for information on how to maintain the LEICA DM R or LEICA DM IR conventional micro scopes The instructions and additional information relating to the components of the TCS confocal system are summarized below Selecting an installation site Do not expose the system to draft Therefore do not install your TCS system next to elevators air conditioners and other inlets and outlets Protect the microscope against dust and grease When not in use the system should be covered with a plastic foil 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 Be careful when using aggressive chemicals You must be particularly careful if your work involves the usage of ac ids lyes or other aggressive chemicals Make sure to keep such sub stances away from optical or mechanical components Cleaning the optical system of the microscope The optical system of the microscope must be kept clean Under no ci
116. ount any possible danger resulting from inadmissible usage we have assigned the Leica TCS to the laser safety class IIIb Every potential hazardous situation cannot possibly be anticipated Therefore the user must exercise care common sense and observe all appropriate safety precautions applicable to Class IIb lasers and high voltage electrical equipment during installation operation and maintenance Deviation from published operating or maintenance proce dures is not recommended Operation and maintenance procedure changes are performed entirely at the user s risk The laser light emitted from the Leica TCS used in accordance with the instructions is harmless Technical Safety Measures In accordance with the laser safety directives EN 60825 European stan dard and CDRH USA Leica Microsystems Heidelberg GmbH em ploys various safety measures Warning labels have been affixed near apertures or moveable parts where exposure to laser light is possible Furthermore laser interlock switches are installed Beneath the con denser of the inverted research microscope LEICA DM IR a special ra diation protection shield is mounted to suppress scattered laser light Use only the condensers S1 and S23 These condensers have low nu merical aperture This results in low divergence and therefore in higher intensities of scattered light Fibers are not replaceable without special tools For this purpose inverted microscopes need a laser protection
117. ouse pointer to resize the object When the outline reaches the desired size release the mouse button You can use the horizontal handles to change the height of the object and the vertical handles to change its width Corner handles can be used to change height and width simultaneously To select image data within a rectangle click on the Rect button In drawing mode the mouse cursor changes to a cross when it is placed in the workspace To draw a rectangle by defining two opposing corners 1 Point the cursor at a position where you want a corner of the rectan gle to appear 2 Press the left mouse button and keep it depressed 3 Drag the cursor from one corner to the opposite corner to create the rectangle As you move the cursor an outline of the shape appears and the bounding box follows the movements of the mouse as long as the mouse button remains depressed 4 When the outline is the size you want release the mouse button The system draws the rectangle You can also use the rectangle tool to draw a square It is not possible to draw rotated rectangles To select image data in an ellipse click on the Ellipse button Ellipses are defined by placing two opposing corners To draw an el lipse click where you want to set the imaginary corner of the ellipse Keep the mouse button depressed while you drag the mouse to draw the ellipse Release the mouse button to complete the ellipse You can also use the ellipse tool
118. pe generates an image in two stages In stage 1 the objective generates a magnified representa tion of the object the so called intermediate image The size of the intermediate image depends on the magnification factor of the objective The scanning process is responsible for stage 2 of the magnification With maximum deflection of the scanning laser beam the object is imaged with a zoom factor of 1 If the deflection of the la ser beam gets smaller but the number of scanned spots and the image size on screen remain unchanged the imaged area is magnified Zoom factor 2 means that a square area with half the side length of the maximum scanning field is scanned This results in an image that dis plays only 4 of the entire scanning field area In contrast with the optical enlargement through the objective phase 1 the enlargement process in phase 2 is named electronic zoom Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 3 m 00m Note i Note oon Page 8 To zoom into your image click Zoom and highlight one of the options KJ E hal I ke Il ee Cs L If you select the other option you can specify your own zoom factor Press OK to confirm your entry and return to acquisition mode oom oom factor ok OF Cancel E Each lens has a maximum useful zoom beyond which there is no more im prove
119. permanent The Leica TCS PowerScan software contains a very handy shortcut pressing the F9 function key sets the diameter of the detection pinhole to 1 Airy Unit This can only be done provided the objective in use was se lected previously You can change the lens settings with the Lens button This tutorial begins where the previous tutorial left off If you just com pleted Tutorial 2 skip the next 5 steps and start scanning You can use the sample of tutorial 2 further Starting the machine and finding an interesting region of your sample in conventional epifluorescence illumination is performed in the same way as in tutorial 2 The next 5 steps are also the same 1 Go to confocal operation mode Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 3 ES Filters a Lens Scan Page 2 2 Switch to Acquisition mode in your PowerScan software 3 Open the filter settings dialog by clicking on the Filters button 4 Select the predefined FITC method within the settings window 5 Match the objective in use and the lens description To select the ob jective click the lens button in the TCS_Tools window Before you start with your first z series you should examine the optical sectioning property of your sample with continuous scan as you did in tutorial 2 1 Press F9 to set the detection pinhole to 1 Airy unit 2 Click on the Scan button to start scanning 3 Adjust gain and offset t
120. points Only simple lines connecting two points can be drawn with the line tool To draw longer straight lines use the polygon tool see below PowerScan switches back to Pick mode see below after the line is drawn To draw another line click on the line button again Pick mode is active as long as no other tool is selected The pick cursor is mainly used to select objects It can also move resize and copy ob jects Click on an object for example a line to select it A selected ob ject shows handles small rectangles To select multiple objects lasso them by dragging the pick cursor from a blank area across the edges of the objects or encase them within the selection area Another way of se lecting a set of objects is to click on the objects one after another with the Shift key depressed Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 5 Rect Note Alip se Note Poly ari Page 2 When the cursor is positioned on a selected object or a group of selected objects it changes to a four headed arrow You can drag an object or a group of selected objects anywhere in your image To duplicate a selec tion keep the Ctrl key depressed while dragging To resize a single object position the pick cursor on one of the boundary handles It is not possible to resize a group of objects The cursor changes to a double headed arrow Press the left mouse button keep it depressed and move the m
121. press 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 assume no responsibility for any errors or omissions Leica Microsystems Heidelberg GmbH shall not be responsible for any direct incidental or consequen tial 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 oc currence of a trademarked name we state that we are using the names only in an editorial fashion and to the bene fit of the trademark owner with no intention of infringement 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 cus tomer operation CDRH FDA OSHA and EN standards and regulations known to be effective at the date of man ufacture 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 IIb lasers and high voltage electrical equipment during installation operation and maintenance Deviation from
122. 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 prod uct This Agreement specifies the permitted and prohibited uses of the product Rev 99012 Page 1 Contents About this book 1 How to use this book ere OE ee ee eC eK Se Se we He ce 1 Doatask o so ttt u a u u 1 Online tutorial ee ee ee ee ee ee ee ee ee ee ee ee ee ee ee 1 Online help TES E A A CR 2 Use the online help and online documentation 2 Search for help by looking upaword 2 Get contact information for help with technical questions 2 Find help on the Internet tt ttt ts 2 Leica TCS euros ee ee 1 What is confocal imaging ts ttt ttt tt 2 Resolution a EA i o A E AR 4 Detection osos s s so so ee 4 Image processing O a r L er a 5 Light SOUIC S soso xo x n u u n es 6 Upright versus inverted models gt o soosoo ooo 6 Photodestruction s s a hee ee ee ee 7 Integration ld A E JE a A 7 Setting up the Microscopes 1 Setup instructions for upright LEICA DM RE Microscopes 1 1 Mounting and defining the objectives 1 2 Settings for incident light observations gt 2 3
123. r cumstances 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 dis tilled 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 inappropri ate immersion oil or mounting medium contact your next Leica repre sentative for advice about cleaning with solvents Take this seriously because some solvents may dissolve the glue which holds the lens in place Do not unscrew the 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 lens tissue should be placed over the immersion end of the lens A drop of recom mended solvent should be applied and the tissue gently drawn across Leica TCS Manual Rev 99011 3 Using the confocal system 3 3 System Care Page 2 the lens surface This should be repeated as often as is necessary to at tain total cleanliness Use a clean piece of lens tissue each time 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 var nished parts Never use acetone xylol or nitro thinners
124. r power with the rotary button If you should interrupt your measurements for less the 3 4 hours you should not switch off the laser Each switching of the laser within one hour costs approx 3 4 hours lifetime If you should interrupt your measurements for more than 30 minutes mini mize the laser power In standby mode you can reach a 3 4 times higher lifetime compared with operation in maximum power mode In critical experiments you should wait at least 30 minutes after each change of the laser power After 30 minutes you have a very stable intensity of the laser light Leica TCS Manual Rev 99011 3 Using the confocal system 3 2 System procedures Starting up the procedure Page 4 3 Switch on the system electronics box key switch optional HeMe laser main switch Switches are located in a small box on top of the laser box just beneath the tabletop 4 Switch on the monitor s 5 Switch on the operating and control computer PC PONG When you switch on the PC the system automatically loads the operat ON OFF ing system Windows NT 4 Workstation Default User Name TCS_User no default password See Chapter Running the Leica TCS software about Windows NT ba sics and log on procedures 6 Start LEICA TCS PowerScan software Start the program by double clicking on its shortcut icon on the desktop or start it from the programs menu 1 Click the Start button 2 Click Programs 3 Click on t
125. rdware resolution 16 134 10 702 ImageSize Lim 200 133 Sections 0 081 0 081 StepSize um Change StepSize Cancel Example You used the Begin and End buttons to define an image size height of current stack of optical sections of 10 661 um In the user defined sec tions dialog you specified 200 sections Because there are two solutions the system prompts you with the corrections window Your first choice is to leave the number of 200 sections as it is and cor rect the height from 10 661 um to 16 134 um light blue attribute in first column Your second choice is to reduce the number of optical sections Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 3 Note Series Page 5 to 133 light blue attribute in second column in order to match the height of the stack as closely as possible 10 661 10 702 Change Image Size is the default choice When deciding on the number of sections to be collected please note that a single 512x512 image 1 channel requires approximately 260KB of disk space For large collections you need a high capacity mass storage device typically an optical disk since your hard disk will eventually become full Use the hard disk for work space only and archive images to an inter changeable mass storage device You can improve the signal noise ratio by averaging the signal collected over several frames with a floating averaging function The
126. re Images This screen displays the version number of your software release and which feature modules are installed Some of them are optional and are not available on all machines The bottom of the screen contains four buttons These represent the three main working modes of the program together with an exit button Acquire Images This function makes all tools available in data acquisition mode This is the main mode for obtaining confocal images Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 1 Page 2 Load Images This function allows you to open images you stored earlier Single images or series of images are loaded from a storage device such as hard disk into the memory of your Leica TCS system The control program automatically switches to view mode View Images This function switches to view mode In this mode you can view loaded single images and image series and edit them with the image processing tools available in the Leica TCS system Exit Program Clicking this button terminates the Leica TCS control program Be fore switching off your PC you should always terminate all programs running and close the Windows NT Workstation operating system us ing the Shut Down command in the start menu Before collecting an image please familiarize yourself with the way in which the software and display are controlled 1 Click Acquire Images 2 The system displays a smal
127. ress the Display button The word you searched for will be highlighted For more help on Help please refer to chapter Running the Leica TCS software because Leica uses the standard Windows NT help system in the entire software Get contact information for help with technical questions 1 Within the online help choose the Help gt Contents switch button 2 Open the Generals book and double click on the Leica world wide topic 3 Select your region and country to get the address just as phone and fax numbers of your local Leica representative Find help on the Internet You can find information on Leica Microsystems Heidelberg GmbH and confocal microscopy on our web site http www Ilt de Send email to support llt de Leica TCS Manual Rev 99011 2 Leica TCS 2 1 Confocal Microscope Systems Page 1 Leica TCS The Confocal Microscope System for highest demands in multi user facilities The Leica TCS is a universal Laser Scanning Confocal Microscope System for the bio medical and materials research environment Ultimate optical design and stability with extreme sensitivity and su perb image quality Perfect registration of UV and visible fluores cence images from 350 900 nm in all four dimensions 3 spatial 1 time dimension Highly ergonomic user interface Integrated Multimedia Tutorial Programmable interactive control panel with 7 dials for quick set up of instrument parameters Up to 5 Las
128. rial 4 Note Original Contra st Page 3 Framel 05 et 4 gt Frame to set 1 ali Se Cancel A particularly spectacular mode is the Movie mode Press the Start but ton to display the images as a movie The Start button changes to a Stop button Click on the same button again to stop the movie You can specify the speed of the movie in the Preferences dialog Please see the PowerScan software reference for details After the last image the movie loops back to the first one Movie mode is disabled if you display a Gallery There are a number of situations in which you will want to modify the original data The PowerScan system provides a whole range of tools for such purposes e Look up tables see tutorial 2 e Contrast methods linear and logarithmic e Contour filters softening sharpening The Original mode is the usual display mode for presenting recorded or loaded images in the TCS_Image window If you press Original after processing and manipulating image data the system restores the original data as recorded The Modify function see below overwrites the original data irretrievably You can change the contrast and brightness of the active image channel currently displayed in the TCS_Image window When you click Con trast the system displays a dialog box in which you can adjust the con trast and brightness The Brightness Contrast command brightens or darkens an image and the range of
129. s control unit contain electrical circuits operating at lethal voltage and current levels All maintenance is to be performed by an authorized Leica techni cian Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Power Cord Set Requirements Page 5 Power Cord Set Requirements The power cord set supplied with your instrument meets the require ments 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 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 per formed 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 be fore you reconnect power or turn the instrument on Cooling Fan Obstruction The UV Laser is equipped with a separate cooling system The cooling fan shall remain unobstructed at all times Do not operate the instrument if the cooling fan is blocked or obstructed in any manner Installation of the Laser Box The laser
130. setTrans 0 Pinhole 1 86 Speed medium Scalebar 100 pm LaserPower 1237 DateTime 19 12 1995 Objective 100 0 1 4 OIL This window visualizes your data display window The window is di vided into three main sections The large middle section is reserved for image display the smaller section on the left contains image display tools and the right hand section contains some user and image specific information As soon as you start the PowerScan software it displays a single 512x512 pixel window pane To the left there are three buttons that allow you to change the image size You can choose between the following options 256x256 312x312 1024x1024 pixels This tool only influences the on screen representation of the image This size is independent from of the pixel size of the scanned area which you can define using the Format button described later on Please note that there are no scroll bars below and to the right of the im age when the image pane does not fit in the image window In this case please adjust the window size as necessary e Click with the left mouse button on the edge of the window or any of its corners Keep it depressed and drag the mouse changing the size and shape of the outline When you are satisfied with the new size and shape release the button to drop the border in its new place Clicking on the Tiled button will divide the display area into four panes referred to as quad mode The act
131. shared resource on the network Once located you can double click the name of the computer or shared resource and perform any network action such as Ex ploration or mapping a network drive LEICA TOS NT E Document WordPad GJ Calculator The Taskbar The Taskbar positioned at the bottom of the screen provides a constant view of which applications are running on the system and an easy way to switch between them As you add to the number of concurrently running appli cations the Taskbar automatically re sizes its iconized view of the applications to ensure that they can always be seen To switch from one running program to another simply click on the second program as displayed in the Taskbar The Taskbar also provides constant additional information such as the system time and volume control if you have a sound card fitted all of which can be further tailored by the user Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software Setting the Time and Date Page 10 Setting the Time and Date The current Date Time and Time Zone information can be set from the Date Time icon within the Control Panel This setting 1s important since Win dows NT stamps the date and time on all of your files as you create and modify them The two options can be selected by clicking on the appropriate tab Date Time Properties El Ei Date amp Time Time Zore 1 09 50PM 4 Current time zone WW
132. sible 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 la ser radiation exposure and the resultant effects vary depending on the type of laser wavelength of the radiation and beam energy output La ser 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 sum marizes the various regions of the electromagnetic spectrum produced by lasers and the organs of concern if exposure occurs Spectrum Wavelength Range Organ Effected Region UV C 100 nm to 280 nm All absorbed in Cornea and Conjunctiva Al 11 tunctiva Ultraviolet TE Da 2 a most a porosa in Cornea and Conjunctiva Cataract formation UV A 320 nm to 380 nm All absorbed in lens Cataract Formation Visible 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 10 nm Cornea and Skin Lasers are often identified by type 1 e Argon Krypton wavelength s and Laser Hazard Classification The Laser Hazard Classification is de termined by the wavelength s maximum duration of exposure and the average power output of the laser Laser Hazard Classification and Laser Safety Contro
133. stem E version so that your system does not necessarily possess all the components mentioned in this description It is assumed that the lighting modules are fitted and that the lamps are adjusted 1 Mounting and defining the lenses The LEICA DM IRBE microscope is fitted with a motorized lens turret The following explanations refer to this type of microscope 1 Sort your lenses We would recommend sorting the lenses according to their magnifi cation factor 2 Move the lens turret to its lowest position Press and hold down both keys on the right side of the microscope base motor focus while you switch on the microscope This makes it easier for you to screw in the lenses Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for inverted LEICA DM IRBE Microscopes Page 7 3 Screw the lens with the highest magnification factor into the lateral hole Screw down the lens Hella EU UU fr PARFLO BM lc EHR 5 Press the CHANGE key twice so that the OBJ message flashes on the display 6 Press the LEARN key 7 Turn the fine focus wheel until the magnification reading corre sponds to the magnification factor of your lens Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for inverted LEICA DM IRBE Microscopes Page 8
134. the desired filter by turning the revolver wheel 4 Switch the light stop device off if necessary in order to clear the in cident light beam path 5 You can now study your specimen in the conventional fluorescence mode 6 Now you can focus an interesting structure of your specimen fine focus wheel if you have not already done this 7 When you do not wish to study the specimen any longer please acti vate the light stop device in order to protect the specimen against bleaching Note You should work as quickly as possible in order to avoid bleaching of the specimen or to reduce at least bleaching effects Leica TCS Manual Rev 99011 3 Using the confocal system 3 1 Setting up the Microscopes Setup instructions for inverted LEICA DM IRBE Microscopes Page 11 3 Setting the Kohler illumination 1 Focussing Focus an area of the object neglect the quality of the illumination for the time being Field diaphragm Aperture diaphragm Opening the aperture diaphragm Fully open the aperture diaphragm It will be closed at a later point in time for adjusting the desired contrast Closing the field diaphragm The field of view darkens in most areas You will see an unfocused light spot If the spot disappears the system is out of center In this case open the field diaphragm until you can just see the light spot at the border of the field of view Then turn the centering screws of the field
135. time per frame If the actual scanning time is lower than this setting the PowerScan software inserts a waiting period before it pro gresses to the next section Additional waiting time at the end of the interval Number of repetitions for this interval at least 1 Set a interval starting marker within the physiol ogy graph Start this sequence at the end of the current se quence CAUTION If you loop back you can create infinite loops Finish Leica TCS Manual Stop creating this interval OK Rev 99011 4 Reference 4 2 PowerScan software reference Acquisition mode Page 11 Advanced Define advanced intervals Sequence Editor Button 1 Interval 245 Set Output Signal each time interval i run Number of Frames Accumulated frames Frame duration fo seconds Delay after interval lo seconds Repeat Interval fi times First Frew Mest Last Hew Del General Settings W Set marker 1st run Save lt lt Basi continue with Load Bao rones y Advanced mode lets you define sequences of intervals with different characteristics Additional topics Interval x y Values of interval x out of y First Jump to the first interval within the defined sequence Prev Jump to the previous interval within the defined sequence Next Jump to the next interval within the defined sequence Last Jump to the last interval within the defined sequence New Create a new interval with
136. tion channels with their channel button depressed contribute to the overlay image This makes 1t very easy for you to cre ate different views of your scanned data The original data remains un modified throughout the entire process To return to single image view press Single Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 1 Page 5 If you have scanned a series of pictures of the same object for example individual z sections you can display an overview of these using the Gallery function You can combine Gallery mode with Single and Tiled mode to display all the individual images from one or more detection channels at the same time Gallery and Overlay mode can be merged Using these display modes to create interesting images will not alter your Original data So there is no danger of damaging anything If your system is equipped with the optional physiology software pack age for on and offline analysis of data sets it will also display a Ratio button This can be used to generate an online ratio picture during a scan This is done by dividing the intensity values of two channels You can define which channel is to be divided by which in the preferences dialog see PowerScan software reference Because changes of intensity values using a ratio image are very small and often hardly visible the Leica TCS software uses a special magnify ing procedure Since the ratio values of tw
137. tons are displayed as black All parts of the image represent ing detected photons are displayed as a real intensity A good image uses the full range of intensities between black and peak brightness Adjust the gain and offset of PMT1 until the image is bright enough and shows a good contrast The image will appear green since a green color look up table CLUT see below is used as standard for FITC fluores cence If necessary increase or decrease the laser excitation laser power after adjusting the gain Do not saturate the image If you see large areas of the brightest color within the active CLUT you should re duce the laser power To assist you in finding the best settings you can temporarily switch to a special CLUT called Glowovun short for glow over and under with strong colors at the extremes of its spectrum so that you can easily rec ognize over and undershot regions All the settings mentioned above are interdependent This means that you may can change additional parameters after resetting certain values For example if you set the brightness by means of the gain and then re duce the laser power less light will reach the detector In order to re cover the previous brightness you will now have to increase the detector voltage slightly and correct the offset As you gain experience you will be better able to use the right combination to obtain an optimum image If the image becomes darker even though you have not chang
138. torial 6 Page 1 Tutorial 6 Note Flat Note x Don t Render The representation in pseudo 3D mode is based on the actual represen tation in view mode This form of display presents an optical section of a series or a single image as a pseudo 3D image The xy plane of this view corresponds to the real appearance of a selected optical section The z axis does not represent any real three dimensional height informa tion but rather corresponds to an intensity code The brighter a point of a given optical section the higher its position in this view Color coding is used as an additional visual effect Because pseudo 3D does not evaluate any real three dimensional struc tures it can be applied to single images If you use a z series the pseudo 3D image does not create a 3D representation of any real object within your sample but rather a representation of intensities from a single section as seen in view mode If you need real 3D information you have to use the Power3D software that is available as an optional PowerScan component Before starting acquire an image or a z series or load a file from disk If you are using a series select a picture in View mode Display one chan nel or overlay in single mode in the TCS_Image window If you are us ing tiled mode only the image in the active quadrant is processed Use any projection original extended or intensity focus or topographic mode except stereo If you are satisfied wit
139. turn to the operating system Menu File gt Open File Save as File Save Selected File gt Default Legend File Exit Settings Preferences Settings gt Timelapse Help Help Help gt Help on Help Leica TCS Manual Open images from a storage device same as Load Images button Not available in main control screen Not available in main control screen Load a default legend not available as far as version 1 6 x Terminate the PowerScan program same as Exit Program button Not available in main control screen Not available in main control screen Open the TCS NT online help Open chapter How to use the Online Help Rev 99011 3 Using the confocal system 3 1 Tutorials TCS_Image window tools Page 3 Help gt About Leica Copyright info TCS PC TCS Image window tools i Display a single channel Single Switch channel 1 2 or 3 on and off Chr hn Cnn Divide display area into four panes Select active quadrant with mouse click nooo Display all available optical sections on one screen Gallery for every active channel Display an overlay image multi channel in RGB Generate an online ratio picture Derda Select image display size independent of scanning size 1024 Select color look up tables for selected pane Lut i Print currently displayed image Copy currently displayed image and data to a separate annotation sah worksheet
140. ty Maximum Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 4 Page 8 To display a stereo image of acquired optical slices press Stereo Stereo generates a red green stereo image overlay from a single image in the view window If you view it with special red green glasses you can see the 3D effects The PowerScan software calculates two different images by rotating the viewing direction by a certain angle The higher the pixel shift between both pictures the greater the depth Sterea Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 4 Page 9 To display a topological image height as color press Topo AE Topo The topological presentation translates the height of a structure into a color code in a series of optical sections Viewing direction O um Intensity EL ELE DD Maximum intensity Center of mass MF Z A a LA Y LA 7 LA Z LA a Lm 200 um Topological image The z position of the maximum intensity center of mass is represented by a color coded pixel The height is represented by the intensity 1 e the higher the brighter This completes our range of image processing options You should have learned how to alter the contrast brightness and color balance of an im age glance through a series of images and generate new and exciting views from sequential scans Leica TCS Manual Rev 99
141. u lists the set of Win dows NT built in programs TIP If you drag an object either from the Desktop or from the Windows Ex plorer and drop it directly onto the Start button a link to that object will auto matically appear in the Start menu There are many ways to start a program The simplest is here 1 Click the Start button 2 Click Programs 3 Click on the group that contains the program you want to start for instance Leica TCS 4 Click on the program you want to start for instance TCS NT Leica TCS Manual Rev 99011 4 Reference 4 1 Running the Leica TCS software The Start menu TESMT Move Here Copy Here Create Shortcut s Here Cancel Windows NT Workstation Page 8 JF Accessories FA Startup pee Command Prompt 2 Microsoft Excel yy Windows AT Explorer ic Administrative Tools Corman Leica TCS NT a Paint Shop Pro Startup Another way to start a program is to open a document that you created in that program The program automatically opens when the document opens Dou ble click on a document file in My Computer or Windows Explorer to open it or click the Start button and select a recently used document from the Docu ments menu You can also start a program by double clicking on its shortcut icon on the desktop Shortcut icons are links to other files When you use a shortcut Win dows simply follows the link back to the original file Whenever you use a document
142. uld be shut down in the following order Laser PowerScan software Windows NT operating system all hardware com ponents including cooling fans 1 Stop your laser by following the procedures as described in the Sys tem Procedures chapter step 1 phases 1 and 2 2 Stop the Leica TCS PowerScan software and the Windows operating system as described in step 2 3 Switch off all hardware components as described in step 3 The next tutorial will help you perform image acquisition for the first time and carry out basic image optimizations Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 2 Page 1 Tutorial 2 In this tutorial we will obtain our first confocal image of an FITC stained object You need a sample that is suitably prepared for imaging 1 e suitably sustained with Fluorescein isothiocyanate FITC and mounted in a non fluorescing medium As this tutorial can require some time you should use a durable object that will not degrade too quickly If you do not have such a preparation at hand continue with tutorial 4 and use an image provided by Leica for the next steps Start the machine as you did in tutorial 1 Log on to Windows NT and start the PowerScan software This will initialize the z position of the fine focusing stage You can now start preparing for the conventional microscopy We sug gest to a low powered objective lens 10x while working through this tutorial While the Num
143. um 17 50 14050 61 09 40 46 400 172 00 Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 5 Note Topo Surface Note Note Page 7 Intensity histograms are created for the whole area of a geometrical object two dimensional To measure surface parameters go back to View mode and press the Topo button for one channel or overlay within a single image Switch to Quantify mode again and select a region of interest geomet rical object Press Surface This determines additional statistical data on the geometry of a marked area such as surface and various roughness parameters Roughness parameters are calculated for rectangular areas only Surface parameters are calculated for the whole area selected two dimensional Volume parameters are calculated for the whole set of optical sections within the marked area three dimensional 3 Surface 2 al Fe 32 18 pm Mean value Rh 30 67 um Maximum peak Rd 34 27 pm Minimum valley Rt 64 93 pm Maximum peak to trough height Ra 9 01 pm Average peak to trough height Rg 6 09 pm Square average peak to trough height Sk 0 12 Bias of the amplitude distribution Area 1403 71 pm 2D area of region of interest Surface 10483 64 pr 3D area of object surface bounding rect Ratio 7 47 Ratio of area and surface Volume 42351 30 pm Object volume inside region of interest 3 Print Snap Mean This figure represents the avera
144. ur docu mentation If you copy a graph to the annotation workbook in the quantification step the data is copied automatically from the graphs to a sheet labeled Quanti fication Images are copied into a sheet labeled Scanner The Print function allows you to print out the image currently selected from your default printer When you press the print button the system displays a standard print preview Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 1 Page 7 TCS _ Tools Window The TCS Tools window is subdivided into two fields an upper field dark gray in color containing arrow shaped buttons for individual imag ing and image processing steps and a light gray area field containing relevant individual tools See the PowerScan software reference for explanations of every single function For most applications you can go through the steps from left to right in the direction of the arrows If you have selected a certain step the tools in the lower field are organized accordingly This approach helps you to obtain the desired results more easily TCS_Tools Luli Formal E series Main This button returns you to the main control screen described above Acquisition This step allows you to record new images It displays the tools required for data acquisition The step is represented in the above picture View TCS_Tools
145. when shutting the system down Powering down your computer without prior shutting it down may result in se vere data loss Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Starting the program Page 1 PowerScan software reference This chapter provides a short reference to the PowerScan menus and commands The basic working of the PowerScan user interface is de scribed in full detail within the tutorial chapters Starting the program a Double click on the TCS NT program icon on your desktop or b Click on 1 Start taskbar 2 Programs 3 Leica TCS NT group 4 TCS NT icon Leica TCS Manual Rev 99011 4 Reference 4 2 PowerScan software reference Main control screen Main control screen Page 2 The Leica PowerScan software starts with the main control screen X LEICA TCS NT TCS_Tools E File Settings Help Acquire Images TCS NT Version 1 6 582 Copyright 1995 1996 1997 1998 Leica Lasertechnik GmbH Heidelberg Features installed PowerScan Power3D PowerPhys To begin select one of the buttons Leica Load Images View Images Exit Program The four buttons at the bottom of the screen switch to the three main work modes of the program or exit the program e Acquire Images switch to data acquisition mode e Load Images open images from a storage device e View Images view and process acquired or loaded images e Exit Program and re
146. ximity to air conditioning equipment protect from dust Heat load ArKr laser cooling air flow rate 240 320 m h heat load lt 3 kW Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Specification of Laser Radiation Page 12 Specification of Laser Radiation Laser type Wavelength nm Maximum performance Maximum performance at the laser outlet mW in the focal plane mW ArKr 488 568 647 lt 125 lt 10 Ar 458 476 488 514 lt 100 lt 10 Ar UV 351 364 lt 50 lt 3 Kr 568 lt 25 lt 5 HeNe 633 lt 15 lt 5 Serial Number and Year of Manufacture Identification Label The serial number as well as the year of manufacture of the system are indicated on the identification label Identification labels are applied to the following positions 1 workstation rear 2 microscope if supplied by Leica Microsystems Heidelberg GmbH rear 3 electronic box on the front right 4 laser box rear 5 scan head rear Laser Safety Labels The following adhesive laser safety labels are attached to the Leica TCS Leica TCS Manual Rev 99011 3 Using the confocal system 3 4 Safety Notes Laser Safety Standards Page 13 The user should note the positions of these labels and from time to time check that they are present and securely attached to the equipment Laser Safety Standards The CDRH Center for Devices and Radiological Health agency within the U S Food and Drug
147. you are satisfied with the result you can close the dialog by clicking OK Some CLUTs like the poli table have remarkable visual effects To assist you in finding the best settings for laser power gain and offset there is a special CLUT called Glowovun short for glow over and un der The extremes of its spectrum contain strong colors so that you can easily recognize over and undershot regions in your image Overshot blue Undershot green Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 2 Page 9 In normal circumstances large green and blue areas should not be visi ble This ensures that the full range of intensities is displayed Use the Glowovun CLUT during scanning and change to a different CLUT to evaluate and process your scanner data When you have completed tutorial 2 you can close your system as you did before or continue with tutorial 3 to collect image series Leica TCS Manual Rev 99011 3 Using the confocal system 3 5 Tutorial Tutorial 3 Page 1 Tutorial 3 The majority of standard applications are covered by horizontal mode xy mode This mode corresponds to conventional microscopy since you always look vertically onto the xy plane By moving the z stage ver tically you can record complete image series for optical sectioning In this tutorial you will learn how to perform a z series The thickness of an optical section will depends on several factors An obje

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