Home

TCS SP2 Users Manual

1. Page 2 7 1 Which safety labels are used 0 0 ccc ees Page 28 7 7 1 On the upright microscope stand 000 cee eee Page 28 7 2 On the inverted microscope stand 0 0 cece eee Page 33 hess OM INE SCAM NEOAG inn e4y5 ot taari ia nedat Shi Rey Gehan aS Page 34 7 7 4 On the supply unit 0 0 ees Page 36 7 7 5 On an external 405 nm diode laser optional Page 3 7 8 Overview of usable lasers 0 0 00 ccc ee ee ees Page 38 7 9 Which requirements are placed upon the installation storage site for safety reasons 2 1 0 eee eens Page 40 7 10 What must be observed when moving the installation site of a Leica laser scanning microscope 000 eae Page 41 3 Confocal IMAGING ccsnc2ccce tee cew dandiee a tie ee tose Page 42 8 1 What is confocal imaging 0 0 cee eee Page 42 S 2 OptGal FeSOWMON 22 nicnrch eieioraredexoiededdparayesenoaeddas Page 43 Os DeC ION i pareen wil ete een e aia eee eee etek eet toes Page 43 8 4 Image processing cece eee eee ees Page 44 O20 LON SOURCE 3 ccuesn tap EEA dum aietars E we eters ache E cle we ater Page 45 OONA O enaren wile te een e eae Sethe estan wet the Page 45 9 Starting up the confocal system 00000e Page 46 10 Starting the Operating System 0000ee Page 57 1021 SEMMI UD USCIS i 444 6 oi naka wed ode eee oe aN da ew eae Page 58 Leica Microsystems Heidelbe
2. a T rue Confocal Scanner Leica TCS SP2 MP User M anual Seica MICROSYSTEMS Table of Contents 1s General orereta enin EVEA seeecenewas esate Page 1 1 1 About these operating instructions 0 0 0 ccc eee Page 1 2 Legal NOLES a062s sheen 6 ee een ae ad te dae ean Page 2 3 Intended use purpose 24 xta0ek ena aan we ta eae aero ee Page 4 4 Explanation of Document Symbols 5 Page 5 2 DIMENSIONS 224 26 o5 selec ews e een ee iwaksredesakssasgionnes Page 7 5 1 Leica TCS SP2 MP System direct coupling 0 Page 5 2 Leica TCS SP2 MP System fiber coupling 0 005 Page 9 5 3 Technical Specifications and connection requirements TOF LCS OPZ MP eiecviwevee Legere dae be tow kts eet oe ees Page 10 6 Description of the multiphoton IR laser sys tem Page 11 6 1 Specifications of the multiphoton IR laser system Page 11 6 2 Details of the laser safety housing for the beam coupling Page 11 6 3 Installation of the multiphoton IR laser system Page 12 6 4 Laser system warranty 0 00 eee ees Page 12 6 5 Service calls and product modifications 0 0000 Page 12 7 General Safety Notes 0c c ee eee Page 13 7 1 Safety Notes for the User 0 0 ccc ees Page 13 7 2 Which standards does this product meet 00 08 Page 13 7 3 What should the user of the
3. Figure 3 Connecting the transmitted light lamp housing During the time when no lamp housing is connected to the microscope stand the lasers must not be switched on since laser radiation could otherwise exit from the opening in the microscope stand Leica Microsystems Heidelberg GmbH Page 19 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 5 4 Eye protection System with inverted microscope stand It is not necessary to wear eye protection If the device is used as prescribed and the sa fety notes are observed the limit of the laser radiation is maintained so that eyes are not endangered System with upright microscope stand The laser beam can be deflected or scattered by the specimen or objects moved into the specimen area For this reason a danger to the eyes cannot be ruled out in any case Using protective eyewear specification 680 990 DIR L5 990 1064 DIR L4 is required Corresponding eye protection is included in the delivered system The supplied eye protection only provides safe protection against the infrared la sers supplied by Leica Microsystems Heidelberg GmbH Page 20 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 6 What laser safety devices does the laser scanning microscope have 7 6 1 Shielding The light of all employed VIS lasers wavelength ran
4. 50 const DWORD ID_EXPERIMENT 60 const DWORD ID LUTDESC 70 A memory space of 32 bits is defined for the DWORD data type Page 82 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format e ID_SERIES memory block This data block contains information about the size of the complete data series combined in an experiment It always occurs only once per experiment The data block is structured as follows Size byte Data Sym Description type bol dint Internal version number Number of image series 4 Jint nim Length of a file name in wchar int nExt Length of the file extension of the image file in bytes snExt wcehar File extension of image file e ID_IMAGES memory block This block contains all file names of all image files that are part of a series The data block is structured as follows Fr i el Number of single images of the series les O int Wein ofa single image Oa int Length of a single image 4 int Bis data points image resolution int __ Data points pixels display resolution For fe next n image files nm 2 wehar Name of the next image e ID _DIMDESCR memory block This block provides a description of an image in an n dimensional space Size Data Sym Description lever ype bon R 4 int Internal version number of the VOXEL des D a e eee en 4 int Voxel typ
5. Changing the Scanner Page 65 Cleaning Page 98 Climatic requirements Page 10 Confocal imaging Page 42 Confocal observation Page 50 Context sensitive Help Page 93 Conventional observation Page 48 Cooling power Page 10 D Declaration of Conformity Page 103 Description file Page 81 Detection Page 43 Dimensionen Page 7 Dimensions Page 10 Direct coupling Page 7 Disclosure Page 2 Document Symbols Page 5 E E Mail Page 97 Electrical connection requirements Page 10 F Fiber coupling Page 9 File Formats Page 78 Full text search Page 94 H Heat emission Page 10 Help Page 93 Help topics Page 94 Image processing Page 44 Installation site Page 98 Internet Page 97 K Keyboard Shortcuts Page 73 Keyword Page 94 K hler illumination Page 59 L Laser Class Page 10 Laser safety housing Page 11 Leica Confocal Software Page 68 Leica subsidiary Page 97 Light source Page 45 Maintenance Page 98 Menu Page 69 Menu Functions Page 74 Microscope surface Page 99 Multiphoton system Page 11 O Operating System Page 57 Optical resolution Page 43 Q Quick Help Page 93 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 RS English Art No 159330113 Vers 20112003 Index R T Readable File Formats Page 71 TCS Menu Page 69 Toolbars Page 70 S Trademarks Page 2 Saving Images Page 72 Trigger control panel Page 100 Service c
6. Emissions warning indicators Figure 7 Emission warning indicators on the control panel Figure 8 Emission warning indicator on the optional external 405 nm laser Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 The readiness of the laser is signaled by an emission warning indicator The emission warning indicators are located above the detachable key switches and are yellow when lit As soon as the emission warning indicator of the laser is lit it is possible from a functional standpoint that laser radiation is present in the specimen area The optional 405 nm laser features the emis sion warning indicator 1 above the detacha ble key switch For lasers whose readiness is not indicated as described above please refer to the supplied manual of the laser manufacturer for the location of the emission warning indicator Page 23 Safety Notes 7 6 4 Remote interlock connector on the supply unit of the TCS system The remote interlock connection is located on the rear side of the supply unit 15 VDC supply voltage The remote interlock connector is plugged into this connection The remote interlock connector features a shorting bridge that jumpers Pin 1 and Pin 5 as shown in the following illustration Remote interlocks that are connected to the room the door or other locally fixed safety interlocks can be connected to the remote
7. Maintenance and Cleaning Do not open objectives for cleaning Oil should be removed from oil immersion lenses after use First remove the immersion oil using a clean cloth Once most of the oil has been remo ved with a clean tissue a piece of lens tissue should be placed over the immersion end of the lens A drop of recommended solvent should be applied and the tissue gently drawn across the lens surface This should be repeated as often as necessary to attain total cleanliness Use a clean piece of lens tissue each time 18 3 Cleaning the microscope surface Use a linen or leather cloth moistened with naphtha or alcohol to clean the surfaces of the microscope housing or the scanner varnished parts Never use acetone xylene or nitro thinners as they attack the var nish All LEICA components and systems are carefully manufactured using the latest produc tion methods If you encounter problems in spite of our efforts do not try to fix the devices or the accessories yourself but contact your Leica representative Before moving the confocal system it should be thoroughly cleaned This applies in particular to systems that are located in biomedical research labs This is necessary to remove any existing contamination and to prevent any carry over and endangering of others Pay not only attention to surfaces but especially to fans and coo ling devices since dust can frequently accumulate at these locations Leica
8. on Leica Microsystems Heidelberg GmbH Page 5 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting the Operating System 10 1 Setting Up Users 1 Logging on as Administrator To log on as administrator use the ID Administrator and the password Admin J 2 Open the User Manager Select Start Programs Administrative Tools User Manager 3 Defining a new user Enter the following information in the dialog window being displayed e Username e Password must be re entered in the next line for confirmation purposes 4 From the check boxes displayed select the following two a User must change password at next logon this allows the new user to define his or her own password at logon b Password never expires this allows a defined password to be valid either until it is changed in the User Manager or the user is deleted 5 Select the Profiles option in the bottom section of the dialog In the Local path field enter the following path for storing user specific files d users username uSername is an Open parameter which must be replaced by the currently defined user name Factory installed ha rd disks are set up with two partitions C and D The user directory should be set up on partition D Page 58 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Setting th
9. tes define which scan parameter is controlled by which rotary knob This format also inclu Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 The Leica Confocal Software des factory presets that have proven useful for most standard applications Self defined templates are saved in the user specific directory e pro Specific profiles of the user interface are saved in this format This includes which button is located in which position on which toolbar In addition new toolbars can be defined or existing toolbars can be displayed or hidden The position of the toolbars whether em bedded or freely movable is also stored in a profile This allows every user to save an individual user interface For example system administrators can use this tool to create a separate user interface for each knowledge level of the user beginner advanced expert ist Besides the individual user specific profiles the user directory also contains the profile of the last session as well as a list of the last four data records applied by the user e prt Templates for the Viewer window are saved in this format This allows for specifically sa ving the appearance and type of displayed information An explanation of the individual configuration options can be found in the chapter on Viewer window e jhw Calibration data and hardware settings of the confocal
10. Documentation Conventions 0 cc ees Page 95 16 62 Performing a task So 6 02 32 tte eee oul ee oa i eS ae ee a he Page 96 17 Help Via the Internet 0260i0deeiewdes teristiw Senden de Page 97 18 Maintenance and Cleaning 00e eee eee eee Page 98 18 1 Selecting an installation site 2 0 0 0 eee Page 98 18 2 Cleaning the optical system of the microscope Page 98 18 3 Cleaning the microscope surface 0 00 naen eee eens Page 99 19 Optional trigger control panel Page 100 19 1 DESCniption Of FUNCION 2201 02 se24eeds sinwe nE EEEE E Page 100 192 MSas er d wae Leak he EE obese Page 102 TOs Se ONC AMOMN enset arta dada octa godt hare aoe aegis go rare ce eae Page 102 20 Declaration of Conformity 00 cece eee Page 103 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art No 159330073 Vers 20112003 General 1 General 1 1 About these operating instructions These operating instructions direct their main area of concentration to the safety notes which must be strictly observed while working with the laser scanning microscope In addition these operating instructions provide the user with a rough overview of the ope rating principle of laser scanning microscopes present the first steps for activating and starting up the system and provide a description of the Leica Confocal Software The Leica
11. It corresponds to the radius of the first minimum in this diffraction image This in turn is related to the numeric apertures of the objective and the condenser The numeric aperture is defined by the diffraction in dex of the lens and the size of the luminous cone that may enter Analogous to the argumentation above the axial resolution can be defined as the radius of the first minimum along the microscope axis of the diffraction image of a point object According to the theory for such 3D diffraction images the optical resolution along the Z axis is smaller than the lateral optical resolution by a factor of 2 Thus the optical reso lution along the z axis amounts to approximately one half of the resolution within the focal plane The LEICA TCS SP2 MP microscope is a no compromise true point scanning system with extremely high sensitivity and theoretical maximum x y and z resolution Scan resolution refers to image clarity as determined by the number of pixels and pixel size The larger the number of pixels and the larger the scanning format the more easily two close objects can be distinguished The scan resolution is restricted to the maximum optical resolution power 8 3 Detection Confocal imaging or to be more precise the measurement of the optical properties of tiny sub volumes of a specimen is limited not only by the optical quality of the microscope Other limitations are The measurement of continuous specimen only in d
12. Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Due to the live voltage of 230 V replacing the shorting plug by an exter nal interrupt circuit e g door interlock switch may only be performed Page 25 Safety Notes 7 6 6 Safety beam guiding and beam shield at inverted micro scope stands The safety beam guiding and beam shield are used with inverted microscopes to protect against laser radiation and are located between condenser and transmitted light detector Figure 11 1 Safety beam guiding 2 Beam shield Page 26 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 6 7 Function and position of safety switches When the safety switches are released the light path of the laser beam is interrupted Figure 12 Position of the safety switch on the upright am microscope stand left and on the inverted microscope stand right Position Activated by Type of micro Activated if Function scope Switching lever Upright DMR An integrated Prevents stray at the beam DML FS beam splitter light if the user splitter in the prism is moved switches from tube of the mi into the beam confocal obser croscope path of the mi _ vation to eye croscope piece observa tion tion Transmitted Inverted DMIR The illuminator Prevents laser light illuminator arm is tilted light while wo
13. Software appears This window allows you to se lect from three profiles e Company This option starts the Leica Confocal Software using the default factory settings This means that the configuration and the position of the Toolbars is preset These settings cannot be changed e Personal This option lets you use a user defined configuration profile The user name is dependent upon the account used to log on to the operating system If the custom profile does not yet exist at the first initialization the default factory settings are applied to the personal profile e Last Exit This option loads the configuration profile that was last active Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 The Leica Confocal Software For advanced users lf you have more than one configuration profile you can load them at startup by clicking the button with the three black dots which is located at the right lower edge of the profile options You may also use this option to reset your custom configuration profile to the standard factory profile Clicking the Start button starts the Leica Confocal Software using the corresponding confi guration profile After a delay the software starts automatically and loads the currently selected configura tion profile 14 1 3 The Experiment Software Concept The Leica Confocal Software allows you to join image data or edited
14. allows you to install subsequently purchased licenses A subsequent license usually is installed if a software package offered as an option is purchased 14 4 5 The Window Menu The following functions can be carried out from the menu bar e New Window This option opens a new Viewer window within the same experiment For example you can use this function to view the same data record using different views such as topogra phical and overlay views 14 4 6 The Help Menu The following functions can be carried out from the menu bar e Contents Opens the table of contents of the online help e Search Starts the search function for the online help e index Opens the index of the online help e Tutorials Opens a tutorial in which the user is guided through selected processes step by step e Set Language Allows for selecting the language of the online help Leica Microsystems Heidelberg GmbH Page 77 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 78 The Leica Confocal Software 14 5 LCS File Formats 14 5 1 Formats of user specific and device dependent data The LCS software provides the following file formats for user specific data Directory Leica Directory User Instrument data Instrument Parameter IPS IPS Settings User profiles CompanyProfile pro PersonalProfile pro LastExitProfile pro LastRecent st Calibration data Machine hw
15. images into groups Each group is called an experiment and is stored in a special data format lei This makes it possible to store both the original experimental image data and the image vie wing data together Additional information can be found in the chapter Organizing Data by Grouping Experi ments 14 1 4 The Basic Structure of the User Interface The appearance of the Graphical User Interface henceforth referred to as GUI de pends strongly on the configuration profile being used A number of elements of the GUI however are standard The GUI provides the following standard elements e The Menu The menu contains the submenus File View Macro Tools Window and Help The sub menus contain commands and information for general display settings and user customi zation They do not provide any of the functions for directly controlling the scan functions These functions are located in the TCS menu View Menu TCS Menu The menu line cannot be customized e The Viewer Window TCS_Viewer The Viewer window displays the image data experimental conditions and user data The image window can be configured see the chapter on Modifying the User Interface and Defining User Specific Settings The image window shows not only the confocal image data records but also the experiment data and system settings You can open a new ex periment image window by selecting File New e TCS Menu TCS Menu The TCS menu
16. images of xy planes Complete 3D models of the specimen can also be rendered and ex amined from any direction Assembly of digital movies from time sequences of microscope images Quantification and Measurements Although this type of image manipulation does not improve the quality of the collected data it serves the purpose of improving the visibility and facilitating the qualitative inter pretation of the data Page 44 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Confocal Imaging 8 5 Light source Lasers are extremely well suited as light sources for confocal microscopy because they emit a very bright light and small divergence of the beam In addition they are easy to fo cus and stable in intensity The stability is especially important for quantitative measure ments 8 6 Integration The Leica TCS SP2 MP was designed as an integrated system Optical and mechanical elements work seamlessly with computer hardware and software The integrated Leica Confocal Software package supports the complete imaging process from optical sectio ning through image processing and analysis which is the main application to hardcopy output Leica Microsystems Heidelberg GmbH Page 45 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 46 Starting up the confocal system 9 Starting up the confocal system
17. interlock connection The laser beam path is interrupted if the contact is open The overall length of the cable between the two connecting pins of the remote interlock connector should not exceed 10 m Trigger out Shutter UW 5 O TETE Figure 9 Wiring of the remote interlock connector on the rear side of the supply unit Page 24 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 6 5 Remote interlock connector for 405 nm laser optional Figure 10 Wiring of the remote interlock connector on the rear side of the laser power supply A 230 VAC If the laser scanning microscope is equipped with the optional 405 nm laser non pulsed the Interlock remote interlock connection is located on the rear side of the laser power supply The remote interlock connector contains a shorting bridge Remote interlocks that are connected to the room the door or other locally fixed safety interlocks can be connected to the remote interlock connection The laser beam path is interrupted if the con tact is open The supply voltage of the remote interlock circuit of the 405 nm laser is For this reason the remote interlock circuit of the 405 nm laser must never be connected to other remote interlock circuits but instead must be securely separated from them A by a qualified electrician Leica Microsystems Heidelberg GmbH
18. objective It may become necessary to readjust the K hler illumination after you have changed the objective Leica Microsystems Heidelberg GmbH Page 61 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Switching off the laser microscope 12 Switching off the laser scanning micro scope 1 Save the image data From the menu select File Save as to save the data record 2 Close the LCS Software From the menu bar select File Exit to exit the LCS software 3 On the control panel turn off the swit ches for the scanner and the laser 4 Shut down the computer From the toolbar select Start Shutdown to shut down the computer Figure 31 Shutting down the computer 5 Switch off the microscope and any fluorescent lamps that are switched on Leica Microsystems Heidelberg GmbH Page 63 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Changing the Scanner 13 Changing the Scanner to another Leica Microscope Stand 1 Switch off the system completely 2 Remove the cable connector for the la ser safety switch from the microscope tube See step 1 in the following illustra tion LUTAS j 3 Remove the RS 232 cable from the plug on the rear side of the microscope See step 3 in the following illustration Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 1593300
19. of the objective lens and is focused on the specimen The reflected light returning from the specimen passes back through the same lens The light beam is focused into a small pinhole i e the confocal aperture to eliminate all the out of focus light i e all light coming from regions of the specimen above or below the plane of focus The achieved optical section thickness depends on several parameters such as the variable pinhole diameter and the wavelength The in focus information of each specimen point is recorded by a light sensitive detector e g a photodiode positio ned behind the confocal aperture The analog output signal is digitized and fed into a computer The detector is a point detector and only receives light from one point in the specimen Thus the microscope sees only one point of the specimen at a time as opposed to the conventional microscope where an extended field of the specimen is visible at one mo ment Therefore to obtain an image it is necessary either to move the illuminated point or to move the specimen These two possibilities have given rise to two different types of confocal microscopes Microscopes with movable objective stage stage scanning The objective stage with the specimen is moved forward after every finished recording while the optical system re mains stationary with this type of scanning Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 15933007
20. plates e Close This option closes the current experiment It does not save the experiment automatically before closing it You are not asked whether you want to save the experiment before it is closed e Close all This option closes all open experiments It does not save the experiment automatically before closing it You are not asked whether you want to save the experiment before it is closed Save Saves the current experiment Save as Saves the current experiment under a different name Save all Saves all experiments and the annotations sheets If one or several experiments or annotation sheets were not saved previously using the Save command it is necessary to enter file name and savings location for the corres ponding experiment using a dialog window Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 The Leica Confocal Software e Recent files Opens one of the recently opened files e Print Opens a dialog for printing the content of the currently active window e Exit Terminates the program 14 4 2 The View Menu The following functions can be carried out from the menu bar e Menus Use this option to switch TCS menu and the toolbar on and off e Status bar This option switches the display of the status bar on and off at the bottom edge of the user interface of the LCS software e Experi
21. series left Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 52007 017 Zust 02 DANGER E f VORSICHT LASER RADIATION CLASS 38 LASERSTRAHLUNG KLASSE 3B W H WENN ABDECKUNG GE FFNET HEN OPE AVOID EXPOSURE TO BEAM NICHT DEM STRAHL AUSSETZEN DANGER VORSICHT VISIBLE AND INVISIBLE SICHTBARE UND UNSICHTBARE LASER RADIATION CLASS 3B LASERSTRAHLUNG KLASSE 3B WENN ABDECKUNG GEOFFNET WHEN OPEN j AVOID EXPOSURE TO BEAH NICHT DEM STRAHL AUSSETZEN DANGER VORSICHT VISIBLE AND INVISIBLE SICHTBARE UND UNSICHTBARE LASER RADIATION CLASS 4 LASERSTRAHLUNG KLASSE 4 WHEN OFEN WENN ABDECKUNG GEOFFNET BESTRAHLUNG VOM AVOID EYE OR SKIN EXPOSURE TO AUGE ODER HAUT DURCH DIREKTE DIRECT OR SCATTERED RADIATION ODER STREUSTRAHLUMG VERMEIDEN 52008 005 Zust 02 mark conte AVOID EXPOSURE AUSTRITT VON BESTRAHLUNG SRO THES IPER TURE LASERSTRAHLUNG VERHELDEW VESTRSER RADIATION f AVOID EXPOSURE STCHTBARER UMD BESTRAHLUNG UNS TCHTBARER VERHEIDEN IS EMNITED FROH THIS APERTURE LASERSTRAHLUNG 52006 0806 Zust 02 LARTE ere AVOID EXPOSURE AUSTRITT VON BESTRAHLUNG O ATEN NE LASERSTRAHLUNG VERMEIDEN VISIBLE AHD INVISIBLE AUSTRITT VON A AVOID EXPOSURE SICHTBARER UND BESTRAHLUMG UMSICHTBARER LASER RADIATION I5 EXRITED VERMEIDEN FROH THIS APERTURE LASERSTRAHLUNG Figure 14 Safety label on the stand of the DM Rxxx product series right Leica Mic
22. system are saved in this format This file may be edited only by service personnel certified by Leica Microsystems Heidel berg Changes to this file may result in serious possibly irreparable damages to the confocal system Damage demonstrably resulting from changes to this file by the user will result in a loss of warranty for the complete system Color look up tables are saved in this format They are used to assign the intensity values measured by the detectors to each color This allows to bring out not only the true color display but also certain intensity areas More details can be found in the chapter on Se lecting Color Look Up Tables Leica Microsystems Heidelberg GmbH Page 79 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 80 The Leica Confocal Software 14 5 2 Fixed Leica specific file formats The following file formats can be opened and viewed in the Leica Confocal Software e Raw data raw The RAW format saves data as a linear two dimensional array in the INTEL format The column index of the array corresponds to the fastest scan dimension in general the x axis while the line index corresponds to the slower scan dimension in general the y axis In an 8 bit image each measured variable is saved as a single byte A 12 bit re cording uses 2 bytes whereby the first byte contains the more significant bits according to the INTEL format little endian Each optica
23. unit of length e g 10 um Length of the following string in wchar Physical start position with unit of length e g 10 um Page 84 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format e Identification numbers ID for image dimensions The following identification numbers are possible ID deci Meaning mal 0 jundefined EO X Z eee OCC me tfortime dimension SS SCS 7798905 4259957 Fe es oe 6357095 6422631 6488167 6553703 D a i 7864398 7929934 7995470 7077966 7471182 5832782 5898318 e ID_FILTERSET memory block This memory block describes the hardware settings such as pinhole diameter or selected filters The memory block starts with a SAFEARRAY header that describes the number and size of the entries A complete description of the SAFEARRAY structure can be found in Appendix 1 4325493 user2 not specified 4391029 users not specified Presently only three entries of the SAFEARRAY structure are used 1 sa cDims should be 1 Presently only one dimensional data fields arrays are used Leica Microsystems Heidelberg GmbH Page 85 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format 2 sa cbElements Size of elements in byte 3 sa rgsabound 0 cElements indicates the number of el
24. 008 006 ust 02 VIS FROM THIS APERTURE VISIBLE AHD IMVISIBL IMP IS ENNITED FROM THIS APERTURE z 52007 017 Zust 02 a LASER RADIATION CLASS 36 WHEN OPEN AVOID EXPOSURE TO BEAN VISIBLE AND INVISIBLE LASER RADIATION CLASS 3B WHEN OPEN AVOID EXPOSURE TO BEAN DANGER VISIBLE AND INVISIBLE LASER RADIATION CLASS 4 WHEN OPEN AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION AUSTRITT WOH BESTRAHLUNG LASERSTRAHLUNG VERHETDEM AUSTRITT VON SICHTBARER UND BESTRAHLUNG UNS ICHTBARER VERMEIDEN LASERSTRAHLUNG VORSICHT LASERSTRAHLUNG KLASSE 3B WENN ABDECKUNG GEOFFNET NICHT DEM STRAHL AUSSETZEN VORSICHT SICHTBARE UND UNSICHTBARE LASERSTRAHLUNG KLASSE 3B WENN ABDECKUNG GE FFNET NICHT DEM STRAHL AUSSETZEN VORSICHT SICHTBARE UND UNSICHTBARE LASERSTRAHLUNG KLASSE 4 WENN ABDECKUNG GEOFFNET BESTRAHLUNG VON AUGE ODER HAUT DURCH DIREKTE ODER STREUSTRAHLUNG VERMEIDEN Figure 18 Safety label on the stand of the DM IRxxx product series Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 33 Safety Notes 7 7 3 On the scan head Zusatzlich bei MP Systemen Additional in case of MP systems Figure 19 Safety label on the scan head left Page 34 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 iT f i A Figure 20 Safet
25. 2 tp seat exchanger various laser combinations corresponding to the infra possible red laser system used ae e eer ee Electrical connec 230 V AC 10 A 50 230 VAC 6A tion requirements or 110 V AC 15 A 50 60 Hz 110 V AC 10 A 50 60 Hz Dimensions see individual configuration see individual configuration LxWxH mm dependent installation dia dependent installation dia grams grams Weight kg Mira Verdi 145 Mira Verdi 11 Tsunami Millenium 120 Tsunami Millenium 40 Mai Tai 95 Mai Tai 40 Optical bench maximum 280 Heat emission sy stem Cooling power heat exchanger Environmental cli optionally with pressurized air matic requirements cushioning for vibration ab sorption a a 6 4 E Page 10 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Description of the multiphoton IR laser 6 Description of the multiphoton IR laser sys tem The Leica TCS SP2 MP multiphoton system consists of a Ti Sa Laser Tsunami Mai Tai or MIRA type The Ti Sa Laser itself is optically pumped by a frequency doubled solid state laser Millenia or Verdi type The pump laser itself is in turn optically pumped by a diode laser that is located in the solid state laser s power pack The radiation generated by the diode laser is coupled to the solid state laser The radiation generated by the solid state laser is led to the Ti Sa laser th
26. 3 Vers 20112003 Confocal Imaging Microscopes with beam or mirror technology The illuminated point is scanned over the fixed specimen using small fast galvanometer driven mirrors as used by LEICA The LEICA TCS SP2 MP system allows you to image a single focal plane as well as a se ries of planes horizontal or vertical A single vertical section or xz scan allows for a side view of the specimen If a sequence of optical sections of the specimen is combined to form an image stack and then digitally processed it offers the advantage of using this multidimensional data set to create either a calculated two dimensional image projection or a reduced scale 3D re presentation of the specimen using a suitable computer 8 2 Optical resolution The term resolution refers to the capability of distinguishing finest details in a structure In a perfect microscope the optical system would be free of any type of aberration In such a hypothetical instrument the resolution would be limited only through diffraction One could express this as the smallest distance between two points of a specimen at which they are still visible as two separate points Rayleigh criterion Beyond this limit the two points merge i e their diffraction discs overlap completely or partially and can no longer be re cognized as two different points This distance can be calculated using the size of the dif fraction image of an infinitely small point of the specimen
27. 7 PC TIFF Length of the following string in wchar 4 in niC 2 wchar File extension for the single image format e g tif raw e ID _LUTDESC memory block Description of the color look up tables LUTs Size byte Data Sym Description type bol J4 int nLU Number of channels 4 R Identifier ID of the dimension whose display D is being colored For the next channels until nLU int LS Internal version number ei bool IsInverted int Length of the following string in bytes nt J O aN wchar Description of the color look up table LUT Length of the following string in bytes wchar m File name of the color look up table if avai i a a lable Length of the following string in bytes wehar Name of the color look up table int __ Field for internal use int lt Dimension of the color look up table ee eS a Lee le Le o o e Ao o me je G do Leica Microsystems Heidelberg GmbH Page 89 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format e SAFEARRAY Data Type typedef struct FARSTRUCT tagSAFEARRAY unsigned short cDims Count of dimensions in this array unsigned short fFeatures DON T CARE unsigned long cbElements Size of an element of the array Does not include size of pointed to data unsigned long cLocks DON T CARE void HUGEP pvData Pointer to the data SAFE
28. 73 Vers 20112003 Page 65 Changing the Scanner 4 Loosen the Allen hexagon screw bet ween the scanner and tube See step 2 in the following illustration To do so use a 3mm metric Allen screw j ff il For microscopes equipped with separate electronic control units LEICA CTR MIC electronics box the RS 232 cable should not be removed from the micro scope stand but instead from the electronic control unit 5 Carefully pull the scan head back and off The scan head weighs approx 13 kg Handle the scan head very carefully in order to prevent damage during the changing process 6 Set the scanning head onto the new mi croscope stand as shown in the follo wing figure 7 Rotate the scan head 90 degrees and place it on to the tube optics in the direc tion of the arrow 1 Page 66 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Changing the Scanner 8 Fasten the scan head with the Allen screw See 2 in the illustration above Winn eamm 9 Insert the RS 232 plug into the rear side of the microscope See 3 in the above illu stration For microscopes equipped with separate electronic control units LEICA CTR MIC electronics box the RS 232 cable should not be removed from the micro scope stand but instead from the electronic control unit Leica Microsystems Heidelberg GmbH Page 67 Operating Instru
29. AR ppunkVal IDispatch FAR SAFEARRAY FAR FAR pparray VARIANT FAR pvarVal void FAR byref e VARTYPE typedef unsigned short VARTYPE enum VARENUM Specification of the Lei file format VT_DISPATCH VT_ARRAY VT_BYREEF VT_UI1 VT_BYREF VT_l2 VT_BYREF VT_14 VT_BYREF VT_R4 VT_BYREF VT_R8 VT_BYREF VT_BOOL VT_BYREF VT_ERROR VT_BYREEF VT_CY VT_BYREEF VT_DATE VT_BYREF VT_BSTR VT_BYREF VT_UNKNOWN FAR ppdispVal VT_BYREF VT_DISPATCH VT_ARRAY VT_BYREF VT_VARIANT Generic ByRef i Files with the LEI format never use pointers as parameters VT_EMPTY 0 Not specified VT NULL 1 Null VT_ 2 an 2 byte signed int VT_14 o 4 byte signed int VT_R4 4 4 byte real VT_R8 5 8 byte real VT_CY 6 Currency VT_ DATE 7 Date VT_BSTR 8 Binary string VT_ DISPATCH 9 Dispatch VT ERROR 10 Scodes VT _BOOL 11 VT VARIANT 12 VT_UNKNOWNE 13 VT Ul Si Boolean True 1 False 0 VARIANT FAR Unknown FAR Unsigned char Other constants that are not valid in VARIANTs omitted here 3 VT RESERVED int 0x8000 By reference a pointer to the data is passed Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 91 Specification of the Lei file format VT
30. ARRAYBOUND rgsabound 1 One bound for each dimension SAFEARRAY The Leica specific file format LEI uses only one dimensional data fields ar rays e SAFEARRAYBOUND Structure Represents the bounds of one dimension of the array The lower bound of the dimension is represented by Lbound and cElements represents the number of elements in the di mension The structure is defined as follows typedef struct tagSAFEARRAYBOUND unsigned long cElements _ num of elements long ILbound DON T CARE SAFEARRAYBOUND e VARIANT and VARIANTARG typedef struct FARSTRUCT tagVARIANT VARIANT typedef struct tagVARIANT VARTYPE vt unsigned short wReservedt unsigned short wReserved2 unsigned short wReserveds union unsigned char bVal VT_UH short iVal NT_le long Val 1 VT_ 14 float fltVal NT_R4 double dblVal VT_R8 VARIANT _BOOL boolVal VT _BOOL SCODE scode VT ERROR CY cyVal I NT_CY DATE date 1 VT_DATE BSTR bstrVal VT_BSTR Unknown FAR punkVal VT_UNKNOWN Page 90 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 IDispatch FAR pdispVal SAFEARRAY FAR parray unsigned char FAR pbVal short FAR piVal long FAR plVal float FAR pfltVal double FAR pdblVal VARIANT BOOL FAR pboolVal SCODE FAR pscode CY FAR pcyVal DATE FAR pdate BSTR FAR pbstrVal Unknown FAR F
31. Check whether the hardware dongle is plugged into the parallel port of the computer The LCS software can NOT be started without the provided dongle Switch on the microscope s and the light sources All manual Leica microscope stands DM LM DM IRB DM R DM LFSE can be switched on and off using the switch located at the side of the stand All automatic Leica microscope stands DM LFSA DM RXA DM RXA2 DM RE DM IRBE can be switched on and off using the switch located at the side of the stand The Leica microscope stands DM LFSA DM IRE2 and DM RXA2 can be switched on and off using the separate electronics unit LEICA CTR MIC Electronics box All fluorescent lamps are equipped with individual power supplies For this re ason they can only be switched on and off at the power supply 3 Activate the switches on the control pa nel for the workstation PC confocal scanner Scanner and lasers Lasers Log on to the computer Use your personal user ID if one has been set up This ensures that the user specific settings are saved and maintained for this user only If the system administrator has not yet assigned a personal user ID log on as TCS_User A password is not requi red Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting up the confocal system 5 Start the LCS program To do so click on the program icon on the desktop of th
32. DA USA the designation Laser Class 4 in the text must be replaced by IV Page 16 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 5 2 Safety notes for operating the laser scanning microscope A During scanning the laser radiation is freely accessible after exiting the objective in the specimen area of the laser scanning microscope This circumstance demands special attention and caution If the laser radia tion comes in contact with the eyes it may cause serious eye injuries For this reason prudent handling is absolutely necessary as soon as one or several laser emission warning indicators are lit If used as prescribed and observing the safety notes during the opera tion of a laser scanning microscope there are no dangers to the user Never look directly into a laser beam or a reflection of the laser beam Avoid all contact with the laser beam Otherwise your eyesight may be permanently damaged A reflected laser beam is just as dangerous as a direct beam Never deactivate the laser protection devices Read the chapter What are the safety devices of the laser scanning microscope to fa miliarize yourself with the safety devices of the laser scanning microscope Note that objects such as micromanipulators in the specimen area may cause laser light to exit the safe beam path during the scanning in an uncontrolled man ner
33. Microsystems Heidelberg GmbH Page 99 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 100 Optional trigger control panel 19 Optional trigger control panel 19 1 Description of Function The optional trigger control panel is used for controlling incoming and outgoing trigger si gnals In addition trigger signals can be ac tuated manually to control recording sequen ces The control panel can be ordered from the appropriate Leica branch office using part number 156108330 Figure 33 Front side of the trigger control panel Signal lamp indicates when a trigger signal was manually actuated on trigger channel 1 or an external trigger signal arrives via T1 con nection rear side of the trigger control panel Signal lamp indicates when a trigger signal was manually actuated on trigger channel 2 or an external trigger signal arrives via T2 con nection rear side of the trigger control panel Signal lamp indicates when a trigger signal was manually actuated on trigger channel 3 or an external trigger signal arrives via T3 con nection rear side of the trigger control panel Signal lamp indicates when a trigger signal was manually actuated on trigger channel 4 or an external trigger signal arrives via T4 con nection rear side of the trigger control panel signal lamp illuminates if an outgoing trigger signal is emitted via trigger channel 4 standard TTL signal Signal lamp ind
34. TCS SP2 MP is supplied with the latest version of the licensed Leica Confocal Software To always keep the information up to date the description of software functions was intentionally omitted from these operating instructions Instead reference is made to the online help of the Leica Confocal Software which presents the most up to date ex planations and instructions to the corresponding software functions First read the chapter Introduction to the Leica Confocal Software help in these opera ting instructions to familiarize yourself with its design and operation Additional information about particular functions can subsequently be viewed directly on the screen in electronic form Leica Microsystems Heidelberg GmbH Page 1 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 2 Legal Notes 2 Legal Notes Made in Germany Copyright 2001 2004 Leica Microsystems Heidelberg GmbH All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying recording or storing in a retrieval sy stem or translating into any language in any form without the express written permission of Leica Microsystems Heidelberg GmbH DISCLOSURE This document contains Leica Microsystems Heidelberg GmbH proprietary data and is provided solely to its customers for their express benefit of safe efficient operation and
35. _BYREF VT_ARRAY int Ox4000 int Ox2000 A safe array of the data is passed The vt value determines the interpretation of the data of the experiment as follows VT_EMP No value was entered TY VT 2 A 2 byte integer value was saved in Va VT 4 A 4 yte integer value was saved in Va VT_CY A fixed point value is indicated It consists of 15 digits before the point and 4 digits after the point The value was saved in cyVal VT_BST Exceptions for files in LEI format R A string was saved in the variable TCHAR VT_NUL A floating zero value was indicated Here the floating zero is not L a NULL pointer This value is required for a 3 state logic for ex ample in the SQL database query language VT _ER An SCODE error was indicated The error type is specified sco VT_BOO A Boolean value true false was indicated L The value OxFFFF all bits 1 means true the value O all bits 0 means false VT_DAT A value corresponding to a date was indicated E A date is saved as a number in double precision format All data is saved as differential dates in reference to January 1 1900 For example the entry 25 corresponds to 01 25 1900 Page 92 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Introduction to the Help 16 Introduction to the Leica Confocal Software Help The system comes with a context sensitive onli
36. age 81 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format Directory table A Address Entry data ee tno Address Number of entries in this table DWORD AeA neS 0 Index of the first entry represents a logical memory DWORD block e g acquisition parameter hardware set tings or image parameter dimensions Address A1 of the 1st entry Index of the first entry represents a logical memory DWORD block e g acquisition parameter hardware set tings or image parameter dimensions Address A2 of the 2nd entry DWORD e e ee The length of the directory table depends upon the number of images and image data to be saved in an experiment acquisition parameters number of channels number of opti cal sections etc Each entry in the directory table corresponds to a logical memory block Block table Xn Address Meaning Datatype A1 or A2 or Check digit DWORD A 4 Bytes Description of the contents of the DWORD block table currently not used A 8 Byte Version of entry DWORD A 12 Byte size of entry DWORD DWORD A 16 Byte Begin of entry The data type depends upon the entry type The following logical memory blocks are available const DWORD ID_SERIES 10 const DWORD ID IMAGES 15 const DWORD ID DIMDESCR 20 const DWORD ID FILTERSET 30 const DWORD ID_ TIMEINFO 40 const DWORD ID SCANNERSET
37. all Page 12 Setting the Kohler illumination Page 60 V a a oe Viewer Window Page 69 Software License Page 3 Starting the Software Page 68 Starting up Page 46 W Status bar Page 70 Warranty Page 2 Switching off Page 63 Weight Page 10 Leica Microsystems Heidelberg GmbH Page 105 Operating Instructions Leica TCS SP2 RS English Art No 159330113 Vers 20112003
38. at have been recorded in continuous scan mode are overwritten auto matically when the next scan is started If you want to keep a single recording as a permanent part of an experiment you should use the single scan function The Selection raw option stores a copy of the raw data of the selected object as a new separate component of the experiment The Selection snapshot option stores an RGB image no 3D data just a pure snapshot of the selected object as a new separate com ponent of the experiment Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 The Leica Confocal Software 14 3 Keyboard Shortcuts In order to accelerate recurring software functions special key combinations have been defined ALT F8 Opens the Macros dialog window for laun ching editing and deleting macros ALT F9 Opens a dialog window containing predefi ned macros that can be started from this dialog window ALT F10 Starts the online help for the LCS macro language ALT F11 optional Launches the VBA developmental environ ment optional CTRL This option allows you to install subse quently purchased licenses CTRL J Opens the Objective dialog window in or der to define and select the microscope objectives CTRL L Opens the Legend Info dialog window in order to enter user specific data that can be stored and displayed when documen ting image recordi
39. at the same time will log you on After pressing the Ctrl Alt and Delete keys the Logon information dialog box appears 2 Typing your password identifies you as a valid user for this computer The default user name for the Leica TCS SP2 system is TCS_ User A standard password was not set It is recommended setting up a separate user ID for each user system administrator This will create individual directories that can be viewed only by the respective user Since the LCS software is based on the user administration of the operating system separate files are created for managing user specific profiles of the LCS software For information about setting up users please refer to the chapter Setting Up Users in this manual 3 After logging on with your user ID you may change your password by pressing the keys Cirl Alt and Delete at the same time Then click on Change Password The Change Password dialog box displays Type your current password in the Old Password field passwords are case sensitive so be sure you use the right case Then press the Tab key Pressing the Tab key moves the cursor to the next field Type your new password then press the Tab key again Confirm your new password by re entering it This will eliminate any typing errors This is especially important since the characters you type appear as asterisks on the screen 8 Then click the OK button Your new password will be in effect the next time you log
40. aved be fore the old data will be overwritten If you do not want to do this select File Save as to save the new or changed data to a new file with a new name 14 2 4 Data Organization by Grouping Experiments The concept of the Leica Confocal Software allows you to group individual images image series and the results of edited images into single groups or experiments The Experi ment Overview window provides a general overview of the open experiment To open the Experiment Overview window select View Experiment Overview Create a new experi ment by selecting File New or File New Template Files that were previously saved are handled as separate experiments when opened 14 2 5 Compiling Experiments After you have defined a new experiment by selecting File New or File New Template you can start filling it with data An experiment contains data acquired with the single scan or series scan function If you Carry out image processing functions on a data record you can save the results as additional components of the experiment To do this double click the desired image or series in the experiment overview window Carry out the desired image processing func tions Such as maximum projection or topological image or others Mark the region within the viewer window that you want to keep as part of the experiment Click the right mouse button to open the context menu and select Send to Experiment Images th
41. by means of reflection or scattering and endanger the environment Do not change a specimen during scanning Procedure Upright microscope Inverted microscope Switch off the lasers Switch off the lasers Tit the transmitted light arm back Exchange the specimen Insert the speci Exchange the specimen Insert the speci men correctly into the specimen holder men correctly into the specimen holder Tilt the transmitted light arm back into the working position Leica Microsystems Heidelberg GmbH Page 17 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 18 Safety Notes Do not change any objectives during scanning Procedure Switch off the lasers Rotate the objective turret so that the objective to be changed is swiveled out of the beam path and points outward Exchange the objective All unoccupied positions in the objective turret must be closed using the supplied caps Close all unused positions of the objective turret with a cap Do not change any filter cubes or beam splitters during scanning Procedure Upright microscope Inverted microscope Switch off the lasers Switch off the lasers Remove the cover of the fluorescence mo Pull out the fluorescence module dule see Microscope Stand operating in structions Remove the filter cube beam splitter Remove the filter cube beam splitter Insert the desired filter cube beam splitter Inser
42. chnique and a special type of sample preparation For example the FITC TRITC designation refers to the settings for a two channel recording simultaneous for the two fluorescent dyes FITC and TRITC It is always possible to define and store your own methods in addition to the factory set methods For details refer to the chapter on Software User Con figurations Page 52 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting up the confocal system 10 Selecting the microscope objective C PLAN 10x 0 22 m Press the Obj button in the Acquire Empty operating step Empty m Select the objective lens to be used in PL FLUOTAR L 63x 0 70 CORR amp the opened dialog window Empty 7 Empty Empty E 11 Selecting the scan mode With automatic microscope stands the selected microscope objective is automa tically moved into the beam path Other stands require a manual operation m Press the Mode button to select the scan mode xyzt KYA XZA The scan mode is used to select the type of data stack to be recorded The follo wing options are available m spatial scan mode xyz xzy m time scan mode xt xyt xzt xyzt m spectral scan mode xyA XZA Leica Microsystems Heidelberg GmbH Page 53 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting up the confoca
43. contains buttons for the individual device functions It is subdivided into individual work steps The number of work steps available depends on the installed soft Leica Microsystems Heidelberg GmbH Page 69 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 The Leica Confocal Software ware The standard set of operating steps consists of data recording Acquire image vie wing View surface reconstruction 3 D measurement functions Quantify image pro cessing and analysis functions Process and applications Applications If the TCS menu is not displayed using the current configuration profile you can activate it by selec ting View Menu TCS Menu e The Toolbars Toolbar This area allows you to insert and customize individual function buttons The advantage of the container is that it can be switched on or off with its entire contents of buttons For this purpose proceed as follows View Menu Container e The Document Viewer Window Experiment Overview The Document Overview window displays the recorded experiments and their contents in a directory tree Open this viewer window by selecting View Experiment Overview e The Status bar The Status bar is found at the bottom edge of the Leica Confocal Software user interface It displays the progress while loading image data progress bar the version number of the software the name of the system configuration system
44. ctions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 68 The Leica Confocal Software 14 The Leica Confocal Software 14 1 Starting the Software 14 1 1 Requirements for starting the software The LCS software is copy protected to prevent it from being used on two computers at the same time This protection system allows all additional application packages to be used The protection system consists of a dongle that must be inserted into the parallel port of the workstation The functionality of the parallel port e g for printing etc is not affected To use the software on a second computer the dongle must be attached to its parallel port If you remove the dongle from the workstation of the confocal system the software cannot be started thereby preventing operation of the confocal system The LCS software can be started in two operating modes hardware mode and simulation mode In hardware mode all hardware components are accessed and initialized by the software For this reason you should switch on the hardware first and then wait about 20 seconds before starting the software In simulation mode the software runs independently of the hardware This mode is inten ded for secondary installations on another computer for example for training or offline analysis of existing data 14 1 2 Starting the software Select Start Programs Leica Confocal Software The initial screen of the Leica Confocal
45. devices to the product that are listed in the Operating In structions Otherwise please contact your local Leica service agency or Leica Microsy stems Heidelberg GmbH 7 2 Which standards does this product meet This device was tested and meets the requirements of the following standards e IEC EN 61010 1 Safety requirements for electrical equipment for measurement con trol and laboratory use e IEC EN 61326 Electrical equipment for measurement control and laboratory use EMC requirements Class A e IEC EN 60825 1 Safety of laser products Part 1 Equipment classification require ments and user s guide e CDRH 21 CFR 1040 10 Laser Products U S Food and Drug Administration FDA Complies with FDA performance stan dards for laser products except for deviations pursuant to laser notice No 50 dated 26 July 2001 Leica Microsystems Heidelberg GmbH Page 13 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 3 What should the user of the laser scanning micro scope observe The user of this product is responsible for proper and safe operation and safe maintenance of the device as well as for following all applicable safety regulations The user is fully liable for all consequences resulting from the use of the device for any other purposes than those listed in the Operating Instructions or the online help The user is required to perfo
46. e e g RGB or GRAY see the follo fe over wing VOXEL TYPES table D iiiad Byte size of a pixel Typical 1 or 2 or 3 or 6 for RGB hae eas el Resolution of scan data 8 12 or 16 bit Length of the following string in wchar wchar wide character character format that uses a 2 byte coding of characters unlike the ASCII format 1 byte Leica Microsystems Heidelberg GmbH Page 83 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format nTC wcehar 2 Maximum value of measured variable inten sity value or length value of a distance for a 4 int nTC Length of the following string in wchar nTC wchar Minimum value of measured variable inten 2 sity value or length value of a distance for a 4 jit nTC Length of the following string in wchar nTC wchar Designation of measured variable I for in 2 tensity z for length value a ae ee 4 it Internal version number 4 Jint nDims Dimension of the image e g x_y_ch_z 4 For the next n dimensions 4 DWORD Identification number ID of the dimension see the following list Identification numbers ID for image dimensions Size of dimension e g 512 pixel Distance between sub dimensions e g byte distance between the image series of two consecutive channel numbers Length of the following string in wchar Physical length with
47. e Kohler Illumination 11 What is the Kohler illumination In a microscopic image only a certain area of a specimen can be displayed image field K hler illumination allows for illuminating only this particular area The technical back ground for the illumination of the image field is described as follows If the illuminated area is smaller than the image field the luminous cone detected by the objective lens as well as the numeric aperture becomes smaller Since the optic resolution is directly dependent upon the numeric aperture a lower illumination also reduces the op tic resolution which is not desired in most cases If the illuminated area is larger than the image field it leads to increased scattered light This in turn leads to a reduction of the image contrast possibly resulting in the situation where optically dissolved structures of the microscopic image can no longer be observed Kohler illumination represents a compromise between maximum contrast and maximum resolution The most efficient microscope objectives frequently reach their optimum optic performance only with exactly adjusted K hler illumination Leica Microsystems Heidelberg GmbH Page 59 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Setting the Kohler Illumination 11 1 Setting the Kohler illumination 1 Focussing Focus an area of the object Neglect the quality of the illumination for the time being 2 Open
48. e computer 6 Next select a software profile A sepa rate profile can be created for each user containing the structure of the graphic user interface as well as the definition of user specific settings of the confocal sy stem MEE TOLT me Copyright 1997 2003 by Leica Microsystems Heidelberg GmbH Hello please choose your profile Personal C Company C Basic Company Start Cancel Options Do not perform the next step until the initialization of the hardware components is complete 7 Affix a specimen and adjust the specimen using the conventional microscopy mode First focus on a position of your specimen to be observed Next set the Kohler illu mination see the chapter on Kohler Illumination Leica Microsystems Heidelberg GmbH Page 47 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting up the confocal system Adjusting to conventional observation for upright microscopes of series DM Rxxx DM LFxx Turn the fluorescence filter dial to the appropriate filter cube Figure Pos 1 1 DAPI optional 2 TRITC 3 FITC 4 empty position scan Set the control bar to VIS Figure Pos 2 Ay 2 Vis TVIPHOTO O TCs Page 48 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting up the confocal system Adjusting to conventional observation for inverted
49. e for biological re search chemical analysis and materials testing The results of many years of research and development in many different areas are combined in such an instrument micro scopy laser technology and optics for coherent light video technology electronics and computer technology Confocal microscopy detects structures by collecting light from a single focal plane of the sample excluding light that is out of focus In a point scanning confocal system the microscope lenses focus the laser light on one point in the specimen at a time the focal point The laser moves rapidly from point to point to produce the scanned image Both fluorescent and reflected light from the speci men pass back through the objective The microscope and the optics of the scanner module focus the light emitted from the fo cal point to a second point called the confocal point The pinhole aperture located at the confocal point allows light from the focal point to pass through the detector Light emitted from outside the focal point is rejected by the aperture The confocal principle is illustrated schematically for the epi illumination imaging mode As in conventional epifluorescent microscopes one lens is used as both condenser and objective The advantage is eliminating the need for exact matching and co orientation of two lenses A collimated polarized laser beam from an aperture is reflected by a beam splitter dichroic mirror into the rear
50. ements contained in the structure Size Data type Symbol Description 24 SAFEARRAY Microsoft type see Appendix 1 M e aq elements that contain the structure Sa rgsabound 0 cElements 128 wehar Identifier for the contents of the entry 64 wchar Name of a short description e g a ee er ywener String contains the contents of the VARIANT Contains the data type and data value except for the string data value which is described in the field above lee ee lues lew Notinuse 4 DWORD ate Nov orhe memon blockt ID of the memory block for internal use ae J4 pwo SCSTestvalueSOS S Page 86 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format e ID_TIMEINFO memory block This memory block contains the time taggers for image series that were acquired with a time dependent scan mode size Data Sym Description ites ype bot P int nDims Indicates the number of dimensions that the SINS desonpion of he tme tagger includes gt Indicates which dimension was provided with a imetagger OSOVO a du the next n dimensions DWOR Identifier ID of the dimension see the descrip gt tion of oh D_ DIMDESCR memory block Size of the dimension number of elements a e g 512 pixel im T E Distance between the individual dimension en tries 4 jit nTS_ Number
51. erforming a task Following the heading that introduces a task are either sequential steps or optional steps each with its distinctive style Usually there is a sequence Perform one or more of the steps to cause the designated action or actions Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 96 Help via the Internet 17 Help via the Internet First contact your local Leica subsidiary or the local dealer You can also find information on Leica Microsystems Heidelberg GmbH and confocal mi croscopy on our web site at http www confocal microscopy com Or send an e Mail to CLSM Support leica microsystems com Leica Microsystems Heidelberg GmbH Page 97 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 98 Maintenance and Cleaning 18 Maintenance and Cleaning Please refer to the corresponding manuals for information on how to maintain the Leica research microscope The instructions and additional information relating to the components of the confocal sy stem are summarized below 18 1 Selecting an installation site Do not expose the system to draft Ensure that the system is not installed next to elevators air conditioners or other inlets and outlets For this reason the installation site should be carefully planned Protect the microscope from dust and grease When not in use the s
52. escription le ee SAFEAR Microsoft type see Appendix Oa aa a OaS for all elements that contain the structure Sa rgsabound 0 cElements J128 wchar identifier for the contents of this entry 64 wchar Name of a shorthand notation e g a TIFF description tag 64 wehar String contains the contents of the string Si RRNTT _ oe the data type and data value except for the string data value which is described in the field above 4 DWORD __ Separate memory space for data values I woro a eg ay space Tor data values in use nono e ties TIOy af ire memon bokir ID of the memory block for internal use only pwo estve o Page 88 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format e ID _EXPERIMENT memory block Description of the used save format e g lei file with PC Tiff images Size Data sym Description byte type bol a 4 Internal version number 4 int Number of images in the data collection of the experiment 4 Length of the following string in wchar nTC 2 wchar Short description of the format 4 jint nTC Length of the following string in wchar o nTC 2 wcehar _ File extension of the main file 4 4 it nTC Length of the following string in wchar pee anal Identifier of the single image format e g
53. ge 400 700 nm visible spectrum and UV lasers wavelength range lt 400 nm invisible is fed through an optical waveguide and therefore completely shielded until it leaves the microscope objective and reaches the specimen For systems with infrared laser wavelength gt 700 nm the beam is passed through a sa fety beam guiding and if necessary also passed through an optical waveguide This shields the laser beam and makes it inacces sible to the user until it leaves the microscope objective and reaches the specimen Figure 4 1 Safety beam guiding 2 IR laser Leica Microsystems Heidelberg GmbH Page 21 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 6 2 Detachable key switch The detachable key switches for protection against unauthorized use of the laser devices are located on the control panel Figure 5 Detachable key switch for internal laser In the case of an optional external 405 nm laser the detachable key switch for the laser is located on its power pack Figure 6 Detachable key switch of the optional external 405 nm laser For lasers that are not connected as described above please refer to the supplied manual of the laser manufacturer for the position of the detachable key switches Page 22 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 6 3
54. icates the line signal This lamp illuminates during the time in which data is recorded during the line scan Signal lamp indicates the frame signal This lamp illuminates during the time in which a single frame is recorded The logical AND operation between the line signal L and the frame signal F Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Optional trigger control panel Push button activating this push button creates a trigger signal on trigger channel 1 This signal can be recorded and processed by the confocal system Push button activating this push button creates a trigger signal on trigger channel 2 This signal can be recorded and processed by the confocal system Push button activating this push button creates a trigger signal on trigger channel 3 This signal can be recorded and processed by the confocal system Push button activating this push button creates a trigger signal on trigger channel 4 This signal can be recorded and processed by the confocal system Figure 34 Rear side of the trigger control panel Trigger Connection socket for connecting the trigger out socket on the out rear side of the supply unit 9V DC Voltage supply of the trigger control panel 100 mA T2 i Connection for i incoming trigger signals V 100 MS 2 2 kOhm Out4 Connection which is used to emit a Aa signal generated by a confocal sys
55. indow for the data record that contains the settings you want to apply There click the Apply button which in the factory default configuration profile is located in the toolbar If you do not find the Apply button in one of the open windows you can load it into any of the windows by selecting Tools Customize In the dialog window that appears select the Commands tab under the file category File With the left mouse button click the Apply button and drag it while holding the mouse but ton pressed to the desired window Release the left mouse button to insert the button at the current location Leica Microsystems Heidelberg GmbH Page 71 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 2 The Leica Confocal Software 14 2 3 Saving Images As described above under Readable File Formats individual images and experiments can be saved using the same data formats Select File Save to save your images and experiments The first time an experiment is saved the Save as dialog opens automatically allowing you to specify a file name In addition to defining a suitable file name you can also select a file format here Experi ments can only be saved in the Leica specific lei Format When you are saving experi ments you may be able to save existing individual images in tif or raw format If you are saving an experiment or image that has already been s
56. ing the aperture diaphragm Fully open the aperture diaphragm It will be closed at a later point in time until the desired contrast is adjusted Figure 30 DM LFS LSFA Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 60 Setting the Kohler Illumination 3 Closing the field diaphragm The image field darkens in most areas You will see an unfocused light spot If the spot disappears upon closing the field diaphragm the field diaphragm must be centered In this case open the field diaphragm until you can just see the light spot at the border of the image field If no light spot is visible the condenser could be set to the wrong height You should therefore adjust the height of the condenser until the field diaphragm is visible 4 Focussing Focus the border of the light spot by adjusting the height of the condenser 5 Centering Turn the centering screws of the condenser until the light spot is centered in the middle of the image field The centering is easier if you slightly open the field diaphragm to enlarge the light spot 6 Opening the field diaphragm Open the field diaphragm until the light spot just disappears at the border of the image field 7 Closing the aperture diaphragm Close the aperture diaphragm until you have set the desired image contrast open to ap proximately 70 of the maximum diameter 8 If you change the
57. iscrete sub volumes because of sampling and digitalization Leica Microsystems Heidelberg GmbH Page 43 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Confocal Imaging The accuracy with which the sub volumes are defined determined by the scanning me chanism The brightness of the light source in relation to the reflectivity of the specimen The sensitivity and noise produced by the detector Another central component of the confocal microscope is the detector Due to its very high signal to noise ratio LEICA Microsystems Heidelberg uses photomultipliers as detectors 8 4 Image processing In the first confocal microscopes the detector was connected to an oscilloscope with long persistence phosphor which would display an image as it was being scanned In the in struments of today the signal is digitized and recorded in a computer This makes it possi ble to manipulate the image in a multitude of ways The following options are available Contrast enhancement by thresholds linear contrast stretching and gamma correction curvature of the image intensity value versus source intensity graph Superimposition of images in experiments Digital filtering for edge enhancement smoothing noise suppression etc Reconstruction of three dimensional views from stacks of images of optical sections This allows for instance an image of an xz plane to be reconstructed from a stack of
58. isk of fire hazard and electrical shock do not expose the AN unit to rain or humidity Do not open the cabinet Do not allow any liquid to enter the instrument cabinet or come into contact with any electrical components The instrument must be tho roughly dry before connecting it to the power supply or turning it on Page 40 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 10 What must be observed when moving the installa tion site of a Leica laser scanning microscope Before moving the laser scanning microscope it should be thoroughly AN cleaned The same also applies to the removal of components This ap plies in particular to systems that are located in biomedical research labs This is necessary to remove a possible contamination and thereby avoid carry over of dangerous substances and pathogens and its accompanying risk of persons Pay not only attention to surfaces but especially to fans and cooling devices since dust can frequently accumulate at these locations Leica Microsystems Heidelberg GmbH Page 41 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 42 Confocal Imaging 8 Confocal Imaging 8 1 What is confocal imaging Conceptualized in 1953 the Confocal Laser Scanning Microscopy has only in the past 10 years become a practical technique Today it is the technique of choic
59. l section is saved for each channel in a sepa rate file with the file extension raw using the following naming convention name of expe riment_name of data record channel number_z dimension_number of optical section raw e Experiments lei The format of this type of file is Leica specific and binary This format is provided for the data of entire experiments A file description is saved in this file format referencing a series of image files in TIF for mat or RAW format e Tiff files tif These are Leica image files in single and multiple TIF format Both image files in the pre viously used TCS formats and external files in RGB TIF format can be read e Annotation ano The format of this type of file is also Leica specific and binary It is used for saving annota tion sheets The elements on the annotation sheet such as images texts and graphic images are each available as individual objects Information on whether the stored measured variable is an intensity value or a height value is saved in the corresponding LEI file Displaying a 12 bit number in the INTEL format Most significant byte according to INTEL format Se ee eee Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format 15 Specification of the Lei file format version beta 2 000 When a file is saved in the LCS software a
60. l system 12 Selecting the scan format m Press the Format button to select the scan format 32 32 64 64 128 128 256 256 512 32 512 64 512 128 512 256 512 512 640 512 1024 128 1024 256 1024 512 1024 1024 13 Optimizing the acquisition parameters in continuous scan The scan format determines the number of grid points used by the scanner to scan the scan area m Press the Continuous button to start the continuous scan As soon as the continuous scan runs you can use the control panel and its defined acquisition parameters to optimize image quality The optimization parameters include m the exact z position within the specimen m the amplification factor of the selected detector the diameter of the detection pinhole m the zoom factor Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 54 Starting up the confocal system 14 Acquisition of a three dimensional spatial data set 3D series Define the upper limit of the data set to be acquired Use the corresponding knob for adjusting the z position of the control panel i The default setting of the z position is position 7 of the control panel feseeses Gain PMT 1 Reedy Figure 28 Standard layout of the control panel bottom part of the image m Press the Begin button to define the starting position of the 3D
61. laser scanning microscope observe Page 14 7 4 Operational reliability 2 0 0 0 00 eee Page 15 7 4 1 De energizing the system 0 0 ccs Page 15 7 4 2 Maximum current load of the power outlet strip atthe SUPPIY UNIT 24 2235 t6 Gada eae gidetes ne Gab Sek ce tke seca ee Page 15 TO Laser SAICLY 3 sete t ceaa e ia aiedctrars Rude aang hace S ENE E slaves Page 16 7 5 1 Which laser class does this product have Page 16 7 5 2 Safety notes for operating the laser scanning microscope Page 17 7 5 3 Transmitted light lamp housing at upright stands Page 19 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP Englisch Art No 159330073 Vers 20112003 Table of Contents 7 5 4 Eye protection 0 0 cc eens Page 20 7 6 What laser safety devices does the laser scanning MIGPOSCODE AVG ind aioe cedeeeee bei eeeeBheeeeiaheueeensnds Page 21 10 Mc NC Oae tsi crccap s wave Bebe Gb als eke hayes E EE Mek E a eee Page 21 7 6 2 Detachable key switch 0 00 eee ees Page 22 7 6 3 Emissions warning indicators 000 cece es Page 23 7 6 4 Remote interlock connector on the supply unit of the TCS systemPage 24 7 6 5 Remote interlock connector for 405 nm laser optional Page 25 7 6 6 Safety beam guiding and beam shield at inverted microscope standS 0 eens Page 26 7 6 7 Function and position of safety switches
62. lt 25 Continuous lt 30 wave 0 5 4 8 1 405 405 430 442 442 lt lt lt lt lt wave non pulsed 458 476 488 lt 200 496 514 non pulsed 458 476 488 lt 500 lt 125 Continuous 496 514 wave non pulsed 543 Continuous wave non pulsed lt Continuous wave non pulsed re wave non pulsed 594 lt lt Continuous wave non pulsed Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes HeNe 633 lt 15 lt 4 Continuous wave non pulsed TiSa 700 1000 lt 2000 lt 500 pulsed 1 3 ps mean power Leica Microsystems Heidelberg GmbH Page 39 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 9 Which requirements are placed upon the installation storage site for safety reasons The limits concerning the emission of electromagnetic radiation EMC are met by this de vice to EN 61326 A residual risk of affecting other devices cannot be ruled out This device was designed for use in a lab and may not be set up in an AN area with medical devices serving as life support systems This equipment is designed for connection to a grounded earthed out AN let 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 r
63. maintenance of the product described herein Use or disclosure of Leica Microsystems Heidelberg GmbH proprietary data for the purpose of manufacture or reproduction of the item described herein or any similar item is prohibited and delivery of this document shall not constitute any license or implied authorization to do so REVISIONS AND CHANGES Leica Microsystems Heidelberg GmbH reserves the right to revise this document and or improve products described herein at any time without notice or obligation 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 expressed or implied including but not limited to the implied warranties of merchantability or fitness for a particular purpose All reasonable precautions have been taken in the preparation of this document including both technical and non technical proo fing Leica Microsystems Heidelberg GmbH assumes no responsibility for any errors or omissions Leica Microsystems Heidelberg GmbH shall not be responsible for any direct incidental or consequential damages arising from the use of any material contained in this document TRADEMARKS Throughout this manual trademarked names may be used Rather than including a trade mark symbol at every occurrence of a trademarked name we state that we are using the names only in an editorial fashi
64. ment Overview This option opens a window displaying the individual experiments and their components data records graphs etc in the form of a tree Double click an experiment component within the Experiment Overview window to bring the corresponding viewer window to the foreground e Hardware Legend This option toggles the display of the hardware legend on and off 14 4 3 The Macro Menu The following functions can be carried out from the menu bar e Macros Depending on the software installed either use this option to edit macros directly or just list and run them You can edit and modify macros only if the complete integrated VBA development envi ronment IDE is installed e Run Macros Opens a dialog window containing predefined macros that can be started from this dialog window Leica Microsystems Heidelberg GmbH Page 75 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 6 The Leica Confocal Software Macro Interface Help Starts the online help for the LCS macro language e Record a New Macro This starts the automatic macro recorder For more information refer to the documentation provided with the optional macro deve lopment environment e Pause Recording This pauses the macro recorder e Stop Recording This stops the macro recorder e Visual Basic Editor optional This option launches the software for developing macros and pr
65. microscopes of series DM IRxx Turn the fluorescence filter dial to the appropriate filter cube figure pos 1 1 DAPI optional 2 TRITC 3 FITC 4 empty position scan Pull the control bar out completely removes additional filters from the beam path figure pos 2 Set the tube lens selector to the scan position VIS stands figure pos 3 Insert the control bar for the beam splitter side exit completely off position fi gure pos 4 Insert the control bar for the beam splitter front exit completely off position fi gure pos 5 Figure 24 DM_IRxx microscopes Leica Microsystems Heidelberg GmbH Page 49 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting up the confocal system 8 Changing to confocal operation Switching to confocal observation for upright microscopes of the DM Rxxx DM LFxx series Turn the fluorescence filter dial to the Scan position Figure Pos 1 Pull out the control bar completely posi tion TCS figure pos 2 TwiPHOTO O T Tcs Turn the fluorescence filter dial to the position 4 figure pos 1 m Pull out the control bar completely posi tion TCS figure pos 2 Figure 26 DM LFSx microscope line Page 50 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting up the confocal
66. n 2 252 tera esos OES ERE E EED AER Page 74 1442 The View Men 2060855 ee oe oe ae ee ae eke E A Page 75 14 4 3 The Macro Menu 352 stung Saudi wie a Gay chelator ia wee GR alate Page 75 14 4 4 The Tools Menu 2 ooceciceo nner ete Sones eee ane oud sone Page 6 14 4 5 The Window Menu 0 0 ccc eens Page 14 4 6 The Help MCNU kee ide hoa eh ERK OLS ETRE PAHS OS ELS Page 145 CS RIG FOmMal wie kta e ets ee eid ETETEN te acd heh Page 8 14 5 1 Formats of user specific and device dependent data Page 78 14 5 2 Fixed Leica specific file formats 0 0 00 eee Page 80 15 Specification of the Lei file format version beta 2 000 Page 81 15 1 Structure of description file 0 0 0c eee Page 81 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP Englisch Art No 159330073 Vers 20112003 Table of Contents 16 Introduction to the Leica Confocal Software Help Page 93 1l QUCK HEID sec sssz ax a 5 deb sans dns ok vig se aloe ob Abe de added di Geshadhee we edd abcde vindech 4 Page 93 16 2 Context sensitive Help 0 0 0 ccc ee ees Page 93 16 3 Retrieving help topics using the table of contents Page 94 16 4 Retrieving help topics using a keyword 0000 00 eee Page 94 16 5 Retrieving help topics using the full text search Page 94 16 6 FaAvVOINCS oc 20 ak caked cake Rae Re Oe RO a eee Page 95 16 7
67. n mm The maximally equipped configuration is shown Standard configurations and options can be found on the individually applicable price lists Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 8 Specifications 5 2 Leica TCS SP2 MP System fiber coupling 330 a a8 0 o 2 oO O T oo Fo Power supply g E D Millenia laser Pc 9 z Q s me C 2 eS lt 230 V 230 V Chiller for Power supply Tsunami laser Millenia laser i to 2 230 V Microscope _ 230 V an rele VIS laser ug i Chiller for 320 x 300 Tsunami Laser h Millenia pump laser Laser ventilation P Tube go WAT i rO rp Instrumentation Shelf wll 1 0 Tsunami laser Anti vibration table Newpart 900 Safety Box Safety device beam routing 1155 1500 max 600 7 685 1310 length overall min 4300 Dimensions in mm The maximally equipped configuration is shown Standard configurations and options can be found on the individually applicable price lists Leica Microsystems Heidelberg GmbH Page 9 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specifications 5 3 Technical Specifications and connection require ments for TCS SP2 MP The Leica TCS SP2 MP is classified as Over voltage Class II Pollution Degree
68. ne help system that explains the different functions of the system Three different help levels are available 16 1 Quick Help When you let the mouse pointer hover over a Leica Confocal Software button a brief ex planation of the function of this button is displayed This so called Help Banner automati cally disappears when the mouse pointer is moved 16 2 Context sensitive Help Click Help to open the context sensitive online help function which provides you with short explanations for the various buttons and functions of the Leica Confocal Software 1 Click on the Help button 2 A question mark appears next to the mouse pointer This temporarily disables the functions of all buttons Use the mouse pointer to click the button that you want an explanation of 4 Online help opens directly to the description for the corresponding button Online help also provides you with an index of key words and a search function so that you can search for specific topics and buttons Furthermore you can print the individual descriptions You can also open the online help by selecting Contents Search or Index option un der the Help menu If the Help button is not present on the user interface 1 Select the Customize option from the Tools menu Here you will find all buttons arran ged by categories 2 The Help button can be found in the File category 3 Click on it using the left mouse button and drag it to the desired wind
69. nglish Art no 159330073 Vers 20112003 Intended use 3 Intended use purpose The system was designed for confocal recording laser scanning images of fluorescence labelled living and fixed specimens as well as for quantitative measurements in the area of life science This device was designed for use in a lab Use for in vitro diag nostics for me dicinal purposes is not included in the intended use The manufacturer assumes no responsibility or liability for any use outside of the intended use or use outside of the specifications from Leica Microsystems Heidelberg GmbH or any risks resulting from such use In such cases the certificate of conformity is invalid Page 4 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Explanation of Document Symbols 4 Explanation of Document Symbols Such a warning alerts you of an operating procedure practice condi tion or statement that must be strictly observed to avoid death or se rious injury to the persons working on the equipment Such a safety caution alerts you to an operating procedure practice condition or statement that must be strictly observed to prevent equipment damage or destruction or corruption or loss of data Notes are statements that either provide extra information about a topic or contain special instructions for handling a particular condition or set of circumstances Leica Microsys
70. ngs CTRL M Use this option to select the Leica micro scope you are using CTRL N Opens a new experiment CTRL O Starts the Open dialog window to open an existing file CTRL P Opens the Printer Selection dialog window CTRL S Saves the active experiment CTRL B Opens the Beam Path Setting window to set the parameters required for the image recording Leica Microsystems Heidelberg GmbH Page 73 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 4 The Leica Confocal Software 14 4 Menu Functions 14 4 1 File Menu The following functions can be carried out from the menu bar e New This option opens a new experiment in a new window The display properties of the new window are determined by the active defaults see the chapter on Default Settings and Templates New Template This option opens a new experiment in a new window The way in which the viewer win dow is displayed can be selected from a set of templates Open This option opens a previously saved experiment or a single data block image data or annotation The display properties of the new window are determined by the active de faults see the chapter on Default Settings and Templates Open Template This option opens a previously saved experiment a single data block or documentation files The way in which the viewer window is displayed can be selected from a set of tem
71. ngth generation is to be modified damage through incorrect use or import duties for replacement parts 6 5 Service calls and product modifications Service calls and product modifications within the warranty period are transacted by the laser manufacturer s local branch offices Direct product return shipments to the laser ma nufacturer should not be made without prior approval of the local branch Serial numbers may not be altered or removed The laser manufacturer s products may not be modified Exceptions are those alterations described in the multiphoton IR laser system s operating manual Page 12 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 General Safety Notes 7 1 Safety Notes for the User Read and observe the safety notes in the Operating Instructions and the safety labels located on the instrument Failure to observe the safety notes may lead to serious injuries and to significant dama ges to the instrument and the data Observe the instructions for operating the instrument located in the Operating Instructions Before performing certain operating steps using the instrument always read the corresponding description of the function in the online help first You can get an overview of the single functions in the contents file of the online help Do not connect any external equipment Connect only those electrical
72. of time taggers Be the next n time taggers nTS a Time tagger as string string data type Number of time tags per time tagger Pen the aim m time tags nTM Number of dimensions of the time tagger des kha ee In e in this di T Froo wchar Time tag as string a data pe Example Image with the following dimensions and dimension sizes 512 512 For example the description of a time tag is 512 512 3 10 This description matches the description of the image The time tagger description 1024 1024 3 10 for the above sample image means that the original image featured an xy dimension of 1024 1024 and was reduced by the software downsampling In such cases the time tag must be recal culated accordingly Leica Microsystems Heidelberg GmbH Page 87 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specification of the Lei file format A time tag can be defined at any time during an n dimensional data acquisition process scan The coordinate of the time tag 0 O 2 7 indicates that the time tag was set at the start of a data record O_O c_ onthe second channel O_O 2 for the 7th optical sec tion 0 0 2 7 e ID _SCANNERSET memory block This memory block contains device parameters that are set at the time of the image acqui sition Further details about the SAFEARRAY structure can be found in the description of the ID_FILTERSET memory block Size Data type Symbol D
73. ograms based on Visual Basic for Applications VBA Here you can edit and modify macros and even develop en tire programs 14 4 4 The Tools Menu The following functions can be carried out from the menu bar Legend Info This option opens the dialog window for entering user specific data This data e g name of institute name of specimen etc is saved with each data record and can be displayed next to the acquired images in the legend e Objective This option opens the dialog window for defining applied objectives The objectives you use can be made known to the software by copying them using drag and drop from the list provided in this dialog window The correct information of the objective is important since some calculated values depend on it e g scan field dimensions automatic adaptation of the detection pinhole e Microscope Use this option to select the Leica microscope you are using This setting influences the way in which the microscope is controlled for example the motorized objective nose piece e Stains This option allows for manually entering values for dye characteristics Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 The Leica Confocal Software e Settings Use this option to select the hardware settings that you want to be applied when opening a previously saved data record e License This option
74. on and to the benefit of the trademark owner with no intention of infringement SAFETY The terms used of the laser safety class refer to the standard EN 60825 1 Within the scope of the CDRH the laser class 4 should be replaced by IV in the text the laser class 3B by Illb the laser class 3A by IIIA the laser class 2 by Il and the laser class 1 by I This instrument is designed and manufactured to comply with applicable performance standards for Class 3B laser products as defined by USHHS CDRH FDA OSHA and EN standards and regulations known to be effective at the date of manufacture Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Legal Notes Every hazardous situation cannot be anticipated therefore the user must exercise care common sense and observe all appropriate safety precautions applicable to Class 3B la sers and high voltage electrical equipment during installation operation and maintenance Deviating from published operating or maintenance procedures is not recommended Operation and maintenance procedure changes are performed entirely at the user s risk SOFTWARE LICENSE The software described in this document is furnished under a License Agreement which is included with the product This agreement specifies the permitted and prohibited uses of the product Leica Microsystems Heidelberg GmbH Page 3 Operating Instructions Leica TCS SP2 MP E
75. ons directly or open dialog windows Dialog Window Both buttons and options open dialog windows Dialog windows are used to set various parameters and select functions e Register Registers are found in dialog windows Registers thematically combine the parameters and functions that can be configured in dialog windows Some registers are divided into fields Viewer window The Leica Confocal Software contains two types of viewer window The Viewer window is called up by pressing the New button and displays the recorded images The Experiment Overview viewer window displays the recorded images in a directory tree This viewer window is called up from the View menu and appears as a separate window at the left side of the user interface e Legend The Leica Confocal Software provides two legends which display the parameters and set tings of an image recording The Experiment legend can be placed at the right edge of the Viewer window The Hardware legend is called up from the View menu and appears as a separate window at the left side of the user interface Context Menu Context menus appear when you click the right mouse button while holding the mouse pointer over certain areas of the user interface Context menus contain various context sensitive commands Leica Microsystems Heidelberg GmbH Page 95 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Introduction to the Help 16 8 P
76. ow Leica Microsystems Heidelberg GmbH Page 93 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 94 Introduction to the Help 16 3 Retrieving help topics using the table of contents Select Help Contents The online help system opens with an activated CONTENTS win dow It shows a collapsible table of contents Double click an entry of the table of contents to view the corresponding information 16 4 Retrieving help topics using a keyword Select Help Index The online help system opens with an activated INDEX window Enter the word you want to look up The online help shows the key word that represents the closest match to the word you entered Select a key word View the corresponding content pages by double clicking the key word or selecting it and then clicking the Display button 16 5 Retrieving help topics using the full text search Select Help Search The online help system opens with an activated SEARCH window Enter the term or definition you want to look up and click on the LIST TOPICS button A hierarchically structured list of topics is returned Click on the triangle to the right of the input field to view the available logical operators Select the desired operator Enter the second search word you would like to associate with the first search word behind the operator Pinhole AND Sections This phrase finds help topics containing both the word pinhole and the
77. r arm e g for working king on the spe on the speci cimen men Switching lever Inverted DMIR An integrated Prevents stray at the beam beam splitter light if the user splitter in the prism is moved switches from tube of the mi into the beam confocal obser croscope path of the mi __ vation to eye croscope piece observa Leica Microsystems Heidelberg GmbH Page 27 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 28 Safety Notes 7 7 Which safety labels are used The corresponding safety labels are selected dependent on the laser configuration VIS UV MP and attached in the following locations 7 7 1 On the upright microscope stand 92007 017 Zust 02 DANGER LASER RADIATION CLASS 3B WHEN OPEN AVOID EXPOSURE TO BEAM DANGER VISIBLE AND INVISIBLE LASER RADIATION CLASS 3B WHEN OPEN AVOID EXPOSURE TO BEAM DANGER VISIBLE AND INVISIBLE LASER RADIATION CLASS 4 AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION VORSICHT LASERSTRAHLUNG KLASSE 3B WENN ABDECKUNG GEOFFNET NICHT DEM STRAHL AUSSETZEN VORSICHT SICHTBARE UND UNSICHTBARE LASERSTRAHLUNG KLASSE 3B WENN ABDECKUNG GEOFFNET NICHT DEM STRAHL AUSSETZEN VORSICHT SICHTBARE UND UNSICHTBARE LASERSTRAHLUNG KLASSE 4 WENN ABDECKUNG GEOFFNET BESTRAHLUNG VON AUGE ODER HAUT DURCH DIREKTE ODER STREUSTRAHLUNG VERMEIDEN Figure 13 Safety label on the stand of the DM Rxxx product
78. r damages resulting from no nobservance of the above information The above information does not in any way impli citly or explicitly modify the warranty and liability clauses contained in the general terms and conditions of Leica Microsystems Heidelberg GmbH Page 14 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 4 Operational reliability 7 4 1 De energizing the system The main circuit breaker is located on the right side of the supply unit It is used to de energize the complete system using a single switch The main circuit breaker functions as a switch and as an overcurrent fuse This device is intended for service tasks or for emergencies It is not to be used as the regular on off switch for the system Figure 1 Supply unit with main circuit breaker 7 4 2 Maximum current load of the power outlet strip at the sup ply unit The total power consumption of all loads con nected to the power outlet strip must not ex ceed 8A Figure 2 Power outlet strip rear side of supply unit Leica Microsystems Heidelberg GmbH Page 15 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 5 Laser safety 7 5 1 Which laser class does this product have According to IEC EN 60825 1 this laser scanning microscope is a laser device of Class 4 For the scope of the CDRH F
79. re 15 Safety label on the stand of the DM LFSxxx product series Page 30 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 52007 017 Zust 02 DANGER VORSICHT LASER RADIATION CLASS 3B LASERSTRAHLUNG KLASSE 3B WHEN OPEN WENN ABDECKUNG GEOFFNET AVOID EXPOSURE TO BEAM NICHT DEM STRAHL AUSSETZEN DANGER VORSICHT VISIBLE AND INVISIBLE SICHTBARE UND UNSICHTBARE LASER RADIATION CLASS 3B LASERSTRAHLUNG KLASSE 3B WHEN OPEN WENN ABDECKUNG GEOFFNET AVOID EXPOSURE TO BEAM NICHT DEM STRAHL AUSSETZEN DANGER VORSICHT VISIBLE AND INVISIBLE SICHTBARE UND UNSICHTBARE LASER RADIATION CLASS 4 LASERSTRAHLUNG KLASSE 4 WHEN OPEN WENN ABDECKUNG GEOFFNET BESTRAHLUNG AVOID EYE OR SKIN EXPOSURE TO VON AUGE ODER HAUT DURCH DIREKTE DIRECT OR SCATTERED RADIATION ODER STREUSTRAHLUNG VERMEIDEN Figure 16 Safety label on the stand of the DM 6000 product series front side Leica Microsystems Heidelberg GmbH Page 31 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes nna res Wee a j 3 Vie rosy A S Al O Leh AA ee AA Figure 17 Safety label on the stand of the DM 6000 product series rear side Page 32 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 7 2 On the inverted microscope stand 52
80. rg GmbH Operating Instructions Leica TCS SP2 MP English Art No 159330073 Vers 20112003 Table of Contents 11 What is the Kohler illumination 00005 Page 59 11 1 Setting the Kohler illumination 0 0 0 0 0c cee eee Page 60 12 Switching off the laser scanning microscope Page 63 13 Changing the Scanner to another Leica Microscope Stand 0c eee eee Page 65 14 The Leica Confocal Software 000 cee eeee Page 68 14 1 Starting the Software 0 ee ees Page 68 14 1 1 Requirements for starting the software Page 68 14 1 2 Starting the software 0 0 0 ccc eens Page 68 14 1 3 The Experiment Software Concept 00 eee Page 69 14 1 4 The Basic Structure of the User Interface Page 69 14 2 Opening and saving data sets 0 ccc ees Page 71 14 2 1 Readable File Formats 0 0 0 ccc eens Page 71 14 2 2 Automatically Applying the Study Parameters olan EXDEIMEN sisi amp nn arta Sys eta 5s Sate tee are Raa eae Reese ee Page 71 14 2 32 SAVING IMAGES rasire Kak eee ANSE S L ORS a ER eS Page 2 14 2 4 Data Organization by Grouping Experiments Page 72 14 2 5 Compiling Experiments 0 0 00 eee Page 2 14 3 Keyboard Shortcuts 0 ccc eens Page 73 144 Menu FUNCUONS s 24 2 occcab lt deeetetoaees ooendwrueted ease nada Page 4 14A Fe MENU seene
81. rm and monitor suitable safety measures accor ding to IEC 60825 1 and the corresponding national regulations Users must have received instructions concerning the risk potential associated with the operation of laser devices To assure class 4 laser product and electrical safety compliance all safety de vices interlocks and safety systems of the laser device must be in operational condition Deactivating or damaging these safety devices or any intervention in any of these safety devices may lead to serious eye injuries physical injuries or property damages In these cases Leica Microsystems Heidelberg GmbH does not assume any liability According to IEC EN 60825 1 Safety of laser products Part 1 Equipment classifica tion requirements and user s guide the user is required to designate a laser safety offi cer or a laser protection advisor Repairs and service measures may only be performed by service technicians authorized by Leica Microsystems Heidelberg GmbH The user is fully liable for all consequences resulting from the use of the device if it is ope ned improperly serviced or repaired by other persons than authorized Leica customer service representatives f repairs or service measures are performed that require opening parts of the housing only trained Leica service technicians may occupy the room in which the laser scanning microscope is located Leica Microsystems Heidelberg GmbH will not be liable fo
82. rosystems Heidelberg GmbH Page 29 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 52007 017 Zust 02 DANGER LASER RADIATION CLASS 3B WHEN OPEN AVOID EXPOSURE TO BEAM DANGER VISIBLE AND INVISIBLE LASER RADIATION CLASS 36 WHEN OPEN AVOID EXPOSURE TO BEAN DANGER VISIBLE AND INVISIBLE LASER RADIATION CLASS 4 WHEN OPEN AVOID EYE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION 52008 005 Zust 02 LASER RADIATION TS ENHITED AVOID EXPOSURE FROWN THIS APERTURE WISIBLE AWD INVISIBLE LASER RADIATION AVOID EXPOSURE IS EMMITED FROM THIS APERTURE 52008 006 Zust 02 LASER RADIATION 15 EMNITED AVOID EXPOSURE FROH THIS APERTURE VISIELE AND INVISIBLE l r LASER RADIATION AVOID EXPOSURE TS EMMITED FROM THIS APERTURE VORSICHT LASERSTRAHLUNG KLASSE 3B WENN ABDECKUNG GEOFFNET NICHT DEM STRAHL AUSSET ZEN VORSICHT SICHTBARE UND UNSICHTBARE LASERSTRAHLUNG KLASSE 3B WENN ABDECKUNG GEOFFNET NICHT DEH STRAHL AUSSETZEN VORSICHT SICHTBARE UND UNSICHTBARE LASERSTRAHLUNG KLASSE 4 WENN ABDECKUNG GE FFNET BESTRAHLUNG VON AUGE ODER HAUT DURCH DIREKTE ODER STREUSTRAHLUNG VERMEIDEN AUSTRITT VON BESTRAHLUNG LASERSTRAHLUNG VERMEIDEN AUSTRITT VON SICHTBARER UMD BESTRAHLUNG UNSICHTEARER VERHEIDEN LASERSTRAHLUMG AUSTRITT VOM BESTRAHLUAG LASERSTRABLUAG VERHET DER AUSTRITT VON SICHTBARER UND BESTRAHLUNG UNSTCHTBARER VERHEIDEN LASERSTRAHLUNG Figu
83. rough a safety beam conductor With the exception of the laser power pack the entire laser system is located on a vibration proofed optical stage 6 1 Specifications of the multiphoton IR laser system Please refer to the corresponding manuals provided for specifications of the multiphoton laser system 6 2 Details of the laser safety housing for the beam cou pling The following diagram shows the inner struc ture of the laser safety housing for the beam coupling between infrared laser system and microscope IR LASER Figure 1 1 Auto correlator optional 2 Spectrometer 3 Laser safety enclosure 4 Laser sa fety shutter 5 Optical fiber Leica Microsystems Heidelberg GmbH Page 11 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Description of the multiphoton IR laser system 6 3 Installation of the multiphoton IR laser system Installation of the multiphoton IR laser system is performed directly by the manufacturer or one of its official branches or agencies 6 4 Laser system warranty The warranty period for the multiphoton IR laser system is 1 year or 3 000 hours whiche ver comes first but a maximum of 15 months after delivery The warranty period begins on the day of installation The warranty includes replacement parts and travel costs Not included maintenance work adjustment work or exchange of consumable items such as fuses or mirrors if the desired light wavele
84. sd User specific profile as it was defined the last time in use In detail the different file formats contain e IPS Instrument parameters are saved in this format This includes hardware based settings such as intensity of the individual laser lines number of channels used simultaneously type of main beam splitter used position and bandwidth of individual spectrophotometer diaphragms and sensitivity of detectors In addition to the default unchangeable instru ment parameter sets that allow a quick adjustment of the hardware any user can save custom instrument parameter sets in his personal user directory This allows you to save the instrument parameters typical for your planned use so that they can be reproduced and made available for other experiments e mac Macros are saved in this format Macros contain VBA V isual B asic for A pplication pro gram code Leica provides the individual program objects for controlling the confocal sy stem in an object model Additional information about the development of macros for con trolling the confocal system can be found in the manual The LCS Macro Language The software includes factory predefined macros that can be used without modifications or modified according to your particular needs The self designed or modified macros are stored in the respective user defined directory e pbo Templates for the assignment of the control panel are saved in this format These templa
85. series Define the lower limit of the data set to be acquired Use the corresponding knob for adjusting the z position of the control panel Press the End button to define the end position of the 3D series m Stop the continuous scan by pressing the Cont button again m Press the Sect button to define the number of optical sections m Press the Aver button to define the number of sampling times frame average i This method averages the recordings of individual optical sections Leica Microsystems Heidelberg GmbH Page 55 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting up the confocal system Next press the Series button to record the 3D series Continuous 15 Saving image data From the menu select File gt Save as to save the data record For additional details on saving data records and on saving formats see the chapter entitled Opening and Saving Data Records Page 56 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting the Operating System 10 Starting the Operating System You do not have to start the operating system it starts automatically when you turn on your PC You will first see a splash screen 1 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
86. subdirectory is first created under the current directory The following files are saved in this subdirectory 1 A description file whose structure is described below This file is used for interpreting the data of an experiment through the LCS software 2 The individual image files naming image files is based on the following naming conven tion NAME_channel number_dimensionIndex Thus Chloroplast C1_Z1 means that Chlo B My Computer i I pha ii En roplast is the name of the data record assi 3 C E Chioroplast C1 Z2 gned by the user that the image corresponds m mages r t ti n n eon E Chioroplast C221 to the first optical section of a three dimensio AA Chinroplasi inet nal data stack Z1 and that the data was C Greenplant eee eel acquired in the first detection channel C1 Contents of C _Images Chloroplast Figure 32 Example for saving an experiment in Lei format 15 1 Structure of description file The description file can be read only by the LCS software It consists of a collection of various tables and contains the following structure Header table Byte Meaning Possible values Byte arrangement 0x49494949 Intel LSB lil S Identification for Leica 0x016033F0 Version number current x20000000 T 12 Address of directory table any possible address within the complete 15 A length of the file Leica Microsystems Heidelberg GmbH P
87. system Adjusting to confocal observation for inverted microscopes of series DM IRxx Turn the fluorescence filter dial to the Scan position figure pos 1 Insert the control bar completely position Stop figure pos 2 Turn the tube lens selector to the Scan position figure pos 3 Pull out the control bar for the beam splitter side exit completely on position fi gure pos 4 Insert the control bar for the beam splitter front exit completely off position fi gure pos 5 qui Figure 27 DM_IRxx microscopes Leica Microsystems Heidelberg GmbH Page 51 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Starting up the confocal system 9 Selecting a specific set of acquisition parameters application m Press the Beam button in the Ac quire operating step m Select a method in the open Beam Broce E Path Setting dialog window top right 0 0 25 0 0 0 0 0 L Ciy window L REFLECTION L Transmissior L TRITC L TRITCwide ye oe a a FE el L YFP 458 476 488 501 514 520 568 647 U user nm nm nm nm nm nm nm nm AOBS Specimen Eo SEE oo PMT 1 PMT 2 PMT 3 PMT 4 PMT Trans None GFP sNone gt None o a a O a Active M Active E Active E Active Acti A method is a set of hardware settings IPS Instrument Parameter Setting spe cifically identifying a certain acquisition te
88. t the desired filter cube beam splitter Reattach the cover to the front of the fluo Reinsert the fluorescence module rescence module Never disconnect an optical waveguide Never remove the scan head from the microscope stand during operation Before removing the scan head the system must be completely switched off m Do not use an S70 microscope condenser The large working distance and the low numeric aperture of the S70 microscope conden sers could result in a threat from laser radiation Therefore only S1 and S23 Leica microscope condensers should be used Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 9 3 Transmitted light lamp housing at upright stands x Upright stand without transmitted light lamp housing If there is no trasmitted light lamp housing connected to the upright stand it is necessary to close the opening with the enclosed cover in order to prevent the emission of laser radiation Upright stand with transmitted light lamp housing If there is a transmitted light lamp housing connected to the upright stand and you want to replace it proceed as follows Switch off the lasers Disconnect the lamp housing from the power supply Remove the lamp housing Perform the intended tasks at the lamp housing After finishing the tasks screw the lamp housing back onto the microscope stand
89. tem standard TTL Line signal This signal is on logical 1 during the time in which data is recorded during a line scan Frame signal This signal is on logical 1 during the time in which data is recorded during a single image frame The logical AND operation between the line signal L and the frame signal F Leica Microsystems Heidelberg GmbH Page 101 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Optional trigger control panel 19 2 Installation The trigger control panel is connected to the confocal system as follows 1 The supplied ribbon cable is connected to the Trigger out connection socket at the rear side of the operator panel and to the connection of the same name at the rear side of the supply unit 2 The trigger control panel must be con nected to the supplied power supply and the power pack must be connected to 230 V AC Figure 35 Connection of the trigger control panel 19 3 Application External trigger signals connection sockets T1 T4 can be transmitted to the confocal system via trigger control panel using a BNC cable Outgoing trigger signals can be transmitted to external devices via Out4 connection sok ket The TM1 TM4 push button can be used for testing purposes Page 102 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Declara
90. tems Heidelberg GmbH Page 5 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specifications 5 Dimensions 5 1 Leica TCS SP2 MP System direct coupling Sg 58 a ra pe Le Power supply D 2 ar Millenia laser o S mi oO 5 te co ao Fs 5 LO LO qm gm 110 ee 230V ae VIS laser A3 0 a 230 V 230 V ae pi at Power supply Chiller for sal Millenia laser Tsunami laser one O As p Microscope and a00 r Accessories meee be Chiller for Safety device 320 beam routing N Tsunami Laser Safely Box g Tsunami laser ze iN E 9 Anti vibration table Newport Millenia pump laser 1500 length overall min 3700 Dimensions in mm Leica Microsystems Heidelberg GmbH Page 7 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Specifications lt b ao od oe O O V Q Of gs c at Ox Power supply 5 D D Millenia laser gt Q 5 oc m Oo uy i gt Y r 110 A 230V os i VIS laser AZO gat 1 230 V 230 V coe i At Power supply Chiller for ip y Millenia laser Mai Tai laser 230 V 4 ri Y Microscope and a00 ah Accessories Ok Chiller for Mai Tai laser i Mai Tai single box a R D Anti vibration table Newport _ E 300 1200 length overall min 3400 Dimensions i
91. tion of Conformity 20 Declaration of Conformity Manufacturer Leica Microsystems Heidelberg GmbH Address Am Friedensplatz 3 Germany 68765 Mannheim Product TCS SP2 MP Confocal Laser Scanning Microscope We declare that the product described herein complies with the following European Directives 89 336 EEC Directive on Electromagnetic compatibility 73 23 EEC Directive on Low voltage equipment The product conforms to the standards EN 61326 1 1997 EN 61326 A1 1998 EMC requirements for Class A electrical equipment for measurement control and laboratory use EN 61000 3 2 2000 Electromagnetic compatibility EMC Part 3 2 Limits Limits for harmonie current emissions EN 61000 3 3 1995 Electromagnetic compatibility EMC Part 3 Limits Section 3 Limitation of voltage fluctuations and flicker in low voltage supply systems for equipment with rated current lt 16A EN 61010 1 2007 Safety requirements for electrical equipment for measurement contro and laboratory use Part 1 General requirements EN 60825 1 1994 A11 1996 A2 2001 Safety of laser products Part 1 Equipment classification requirements and user s guide Manager Research amp Development Mannheim Germany LC 3 January 01 2004 Dr Rafael Storz Leica Microsystems Heidelberg GmbH Page 103 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 104 Index B Beam coupling Page 11 C
92. type For details about these functions refer to the chapter on The LCS Software Functions Page 70 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 The Leica Confocal Software 14 2 Opening and saving data sets 14 2 1 Readable File Formats The following file formats can be opened and viewed in the Leica Confocal Software Experiment lei The format of this type of file is Leica specific and binary This format is provided for the data of entire experiments e TIFF files tif These are Leica image files in single and multiple TIF format Both image files in the pre viously used TCS formats and external files in RGB TIF format can be read e Annotation ano The format of this type of file is also Leica specific and binary It is used for saving annota tion sheets The elements on the annotation sheet such as images texts and graphic images are each available as individual objects When these files are loaded both the image data and the experiment settings are loaded 14 2 2 Automatically Applying the Study Parameters of an Experi ment With the Leica Confocal Software hardware settings that have been saved for previous experiments or single images can be applied to new experiments This allows you to carry out different experiments using constant settings To carry over settings from a previous experiment open the viewer w
93. word sec tions Pinhole OR Sections This phrase finds help topics containing either the word pinhole or the word sec tions or both Pinhole NEAR Sections This phrase finds help topics containing the word pinhole and the word sections if they are located within a specific search radius This method also looks for words that are similar in spelling to the words specified in the phrase Pinhole NOT Sections This phrase finds help topics containing the word pinhole and not containing the word sections Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Introduction to the Help 16 6 Favorites If you select Favorites in the dialog window of the online help tab you can include the cur rent help topic in a list making them easily available for future use 16 7 Documentation Conventions Button The buttons provided on the user interface of the Leica Confocal Software Buttons are marked with icons and or have an often abbreviated English label They either trigger actions directly or open dialog windows Menu The menus are divided into the categories of File View Macro Tools Window and Help and are displayed in the menu bar located at the upper edge of the user interface e Option Options refer to the selectable items that are hierarchically listed below the menus Op tions either trigger acti
94. y label on the scan head right Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes Page 35 Safety Notes 7 7 4 On the supply unit 52005 002 52023 001 Figure 21 Safety label on the supply unit Page 36 Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Safety Notes 7 7 5 On an external 405 nm diode laser optional 52007 017 Zust 02 405 nm 25 mW Figure 22 Safety label on an external 405 nm laser Leica Microsystems Heidelberg GmbH Page 37 Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003 Page 38 Safety Notes 7 8 Overview of usable lasers The laser scanning microscope features a combination of the lasers listed below Laser type J gt UV Diode Diode Solid state HeCd DPSS442 Ar external HeNe DPSS561 A a x x A HeNe Wavelength Maximum lumi Maximum lumi Pulse duration nm nous power at la nous power in fo ser output mW cal plane mW 351 364 lt 60 Continuous wave non pulsed Continuous wave non pulsed lt 0 3 pulsed 60 ps mean power Continuous wave non pulsed Continuous wave non pulsed 25 5 a i a 12 Continuous wave non pulsed a a 1 5 12 40 A lt 4 F T gt O mean power A A lt 3
95. ystem should be covered with a plastic foil part of delivery or a piece of cotton cloth The system should be operated in a room which is kept as dust and grease free as possible Dust caps should always be placed over the objective nosepiece positions when no objec tive is in place Exercise care in the use of abrasive chemicals You must be particularly careful if your work involves the usage of acids lyes or other ag gressive chemicals Make sure to keep such substances away from optical or mechanical components 18 2 Cleaning the optical system of the microscope The optical system of the microscope must be kept clean Under no circumstances should users touch the optical components with their fingers or anything which may bear dust or grease Remove dust by using an air puffer not solvent based or a fine dry hair pencil If this method fails use a piece of cloth moistened with distilled water Persistent dirt can be removed from glass surfaces by means of pure alcohol chloroform or naphtha If an objective lens is accidentally contaminated by unsuitable immersion oil or by the spe cimen please contact your local Leica representative for advice on the use of certain sol vents for cleaning purposes Take this seriously because some solvents may dissolve the glue which holds the lens in place Leica Microsystems Heidelberg GmbH Operating Instructions Leica TCS SP2 MP English Art no 159330073 Vers 20112003

TCS SP2 Users Manual

Contents

Download Pdf Manuals

Related Search

Related Contents