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Leica Confocal Software LCS User Manual

1. User Manual Leica LCS english LCS Software Functions Deleting Regions of Interest ROIs L Function The Clear button deletes all defined ROIs at once To delete an individual ROI click the Select ROI button then the ROI in the image and press the DELETE key on the keyboard Other Requirements The definition of a region of interest in the image is required for quantification functions Therefore the Clear is deactivated until you activate a quantification function using one of the following buttons see Measuring a Profile Within a Region of Interest ROI see Calculating a Histogram LL see Measuring Roughness Within a Region of Interest ROI Documenting Data Creating an Annotation Sheet Function Use the Annotation button to open a window in which you can prepare recorded images for presentation purposes If you have opened the annotation sheet the Snap Line Rectangle and Text buttons are also enabled Use these buttons to copy the image loaded in the Viewer window into the annotation sheet to highlight specific areas of the copied image with lines and rectangles and to enter an image comment into a text field see Copying an Image A see Drawing a Line A see Drawing a Rectangle see Adding a Text Field If you hold the mouse pointer over the annotation sheet and press the right mouse button a context menu containing the following commands appears Page 122 of 164 Art N
2. SFP Icon Simulated Creating SFP Projection Images Fluorescence Process Running SFP Projection Images as 3D gt gt User Manual Leica LCS english Rotation Animation Creating a Red Green Stereo View of the SFP Projection Creating an SFP Projection Image see Principles and Types of Projections In the SFP tab in the Light Direction field you define the projection angle for the first computing step of the SFP projection This angle symbolizes the angle of incidence of the laser beam onto the specimen Enter one coordinate each for x and y Click on Rescale in the Options field to standardize the intensity values of the image to the maximum possible intensity values when creating the projection image This ensures that the calculated gray values of the projection image remain within the value range of for example 0 to 255 at 8 bits This allows for brightening dark images If you click on Invert in the Options field the projection axis is mirrored The calculation of the projection image is thus in the opposing direction This function makes sense above all in the case of inverse microscopes to match the calculation of the projection image to the inverse beam path of laser light If you click on Filter before Rendering in the Options field the image stack is filtered in a lowpass filter in all three spatial directions before the SFP projection image is calculated In this way strong noise can be removed from the recorded
3. Defining the Begin Point for a Spatial Series Function Use the Begin button to define the begin point of a spatial image series First set the exact z position and or y position using the z y position button or on the control panel using the corresponding radio button Then click Begin This saves the position value for the start point The end point is set in the same manner LL see Setting the z y Position LL see Controlling Functions via Control Panel Note You can also conveniently set the begin point and end point of a spatial series in the Series Scan Overview dialog window Open this dialog window by pressing the Series button the small button not the button that starts the Series Scan function see The Series Scan Overview Dialog Defining the Begin Point for a Wavelength Series nara i A Function For a wavelength series this function records a stack of individual images each of which are detected at a specific wavelength from a single optical level Use the Lambda Scan Begin button to define the wavelength at which recording should begin User Manual Leica LCS english Page 37 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Note The Lambda Scan Begin button is only active if you used the Mode button to select a scanning mode with the wavelength dimension see Selecting a Scan Mode Press Beam to open the Bea
4. rrrrrnnnrrnnrnranernnnennnnen 84 Taste NAO eget arses netcad oer sete cance ene eeeeters 123 TEN Even 129 Template Button rrranrrnnrernnrrnnnnrrnnennnre 129 PRP 125 Text FO atnstvatauciancmatiassinhiietateetnansniuantassantns 125 The arithmetical mean 06 110 112 Thickness of the Optical Sections 29 Page 162 of 164 Leica Microsystems Heidelberg GmbH ME 87 Tiled Button erenervoroonorrorvnnavervanenerranenverenenre 87 Time BUON sorrisos inie 33 Time Configuration Dialog Window 33 Time Series Lunn Aces daetdsnsisaceee 33 41 Topographical Image c cceeeeee 61 105 Topography rrnrrrnnrvnnrvnnrvnnrvnnrnnnrnnnnnnn 61 105 Topography Button rrrnnrrnrrrnnrennrrnnnrnnnr 105 Transparent Factor 56 64 92 102 103 Transparent Projection 56 64 92 95 96 102 103 Triple DIGKrole sic scace swecvbich cnntncs onndesionewacvetaveds 138 Triple dichroic filterS rrrarrnnnrrrrnnrrnnnrnnnre 22 Turning the Scan Field rrrnrrrnnrrrnnrnnnnrnnnre 44 Two point measurement 53 110 113 134 Undersampling ccccceeceeeeeeeeeeeeeaeeeaeeenees 30 INGO REE EE 106 Unidirectional rennwrronanrasnnrannnrnanennavsnnenre 43 Unidirectional Scan rrrrnnnnnnnnnnvnnrrnnvnnrennnn 43 Unidirectional Bidirectional Scan Button 43 UV Lens Wheel rrrnnnnnrnnnrrnnnnnrvnnnrrnnnnrennn 48 Variable
5. Viewer Options Dialog Window Display Icon Function To open the Viewer Options window select the Experiment Overview option in the View menu The Experiment Overview window appears on the left side of the user interface The top part of the viewer window displays the recorded images in a directory tree The bottom part displays the Viewer Options window This dialog window is used to perform basic settings for different software functions The left side shows the icons corresponding to the functions and the right side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons The tabs of the Display icon can be used to perform optional settings for the following functions see Starting and Ending a Film User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Page 52 of 164 Leica Microsystems Heidelberg GmbH n 1 LCS Software Functions On the Settings tab use the graphic zoom to enlarge or reduce the image displayed in the Viewer window Displaying n pixel as 1 pixel in the resulting image reduces the image Automatic The image is displayed in the original image format 1 n Note Displaying 1 pixel as n pixel in the resulting image enlarges the image The Leica Confocal Software provides three different zoom functions the graphic zoom the 3D zoom and the electronic zoom see Zooming the 3D View
6. A topographical image is the prerequisite for the generation of a height profile For this reason the Roughness Profile button is active only when a topographic image of the data record has been created in advance with the Topography button Only one detection channel can be quantified at a time LL see Creating a Topographical Image Click on the Roughness Profile button The measurement section is displayed as a white line in the image in the Viewer window You can change the length and position of the line segment by clicking the line and while clicking and holding the grab point with the left mouse button dragging it to the desired position gt A window opens automatically to display the measurement curve for every detection channel as well as the statistical values Parameter Meaning Formula Evaluation Length of the plotted section 5 5 length I P according to DIN EN ISO 4287 L xo 4 y2 z y4 Max Peak Height of the highest profile Max Pp Pp peak based on the average height according to DIN EN ISO 4287 Min Valley Depth of the greatest profile Min Pv Pn valley based on the average height according to DIN EN ISO 4287 Deviation Arithmetic average of the Pa profile ordinates within the measured section average height according to DIN EN ISO 4287 RMS Pq Root mean square value of User Manual Leica LCS english the profile ordinates within the measured section according to DIN EN ISO 4287
7. Favorites If you select Favorites in the dialog window of the online help tab you can include the current help topic in a list making them easily available for future use Opening the Context Sensitive Help Function 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 Click the Help button 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 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 under the Help menu Page 20 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions 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 displ
8. dialog Try again Then click the OK button Your new password will be in effect the next time you log on Caution Do not forget your password if you set one Without the right password you cannot access your computer anymore The Welcome dialog box is now displayed Take a moment to read the Did you know tip and then click the Close button to begin using Windows NT Using a Mouse You need a mouse to work most efficiently in Windows NT Here are the mouse actions you need to know Point means to move the mouse pointer onto the specified item by moving the mouse The tip of the mouse pointer must touch the item User Manual Leica LCS english Page 7 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Click on an item means to move the pointer onto the specified item and press and release the mouse button once Unless specified otherwise i e right click use the left mouse button Clicking usually selects an item Double click on an item means to move the pointer to the specified item and press and release the left mouse button twice quickly Double clicking usually activates an item Drag means to move the mouse pointer onto the item hold down the mouse button and move the mouse while holding down the button Unless specified otherwise i e right drag use the left mouse button The Windows NT Interface The basic interface of Windows NT is called the Deskt
9. gt A viewer window opens that displays the measurement curve for each detection channel and the statistical values Parameter Meaning Formula Pixel The total number of User Manual Leica LCS english pixels that are included in the calculation of the histogram This corresponds to the configured scan format Page 109 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Mean The arithmetical mean value Maximum Maximum value Minimum Minimum value Variance Variance Average Average deviation Deviation Standard Standard deviation Deviation Mean Average image energy Energy Root Mean Root mean square value Square RMS Skewness Skewness of the Note distribution LCS Software Functions RMS M Pixel 1 S i Ii wll ARO Skew gt Some of the parameters are specified in scientific exponential notation e g Pixel 3 28e 005 327680 corresponds to a scan format of 640 x 512 Using the maximum and minimum values of intensity the gain values and offset values can be optimally configured for the detectors Measuring a Profile Within a Region of Interest ROI Function The Profile z function measures a measurement variable within a region of interest displays it in a curve and calculates various statistical values The profile is measured from the data record selected in the Viewer window This data set can be a spatial
10. see Controlling Functions Using the Control Panel Note A detector is not enabled unless its corresponding Active checkbox is checked in the Signal or Beam Path Setting dialog windows The Electronic Zoom Function In confocal microscopy the magnification of an image is determined both by the objective and the electronic zoom The objective generates a first image whose magnification is based on the objective s magnification factor The electronic zoom provides additional magnification A zoom factor of 1 scans the maximum scan field size with a specific number of dots If the zoom factor is set to 2 the same number of dots is used to scan a scan field with half the page length of the maximum scan field 1 4 the original scan field This achieves stronger magnification and thus better image resolution too because a smaller scan field is scanned with the same frequency which results in a higher density of data In the dialog window that was opened by pressing the Zoom button you can select one of the preset zoom factors If you click on the Others button you can set a different zoom factor in two ways Use the mouse to move the slide of the scale The corresponding value is shown in the middle of the dialog window gt Double click the boldface numerical value located in the middle of the dialog window This opens a second dialog window where you can enter an exact value You can also set the zoom factor using the corres
11. Amplitude Maximum Position of maximum Arg Max I 2 t A amplitude value position Minimum Minimum value Min I Amplitude Minimum Position of minimum Arg Min I 2 t A amplitude value position Average Average deviation 1 Deviation DISP Haa Ii wel Standard Standard deviation r 5 Deviation s I JAR T Ta Li pel i Variance Variance The following four measurement values appear only when the Two Point Measurement function is selected in the Viewer Options dialog window Select the View menu the Experiment Overview option and the Charts icon LI see Viewer Options Dialog Window Charts Icon Inthe Measurement field you can switch two point measurement on or off by selecting 2 Point or Off um I s Intensity measured at measuring point 1 z 4 ti I l4 nm t A I Z I t Intensity measured at measuring point 2 Z I z I t I I I A t A dl dz Difference of values between measuring I Z4 I Za l ti point 1 and 2 I t2 IC M l2 User Manual Leica LCS english Page 111 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions d z t Height z recording time t bandwidth A Z4 Zal ti tel lL A Note between the measuring points 1 and 2 l2l Typical Applications This function can be used to determine the extreme values of the intensity within an image stack They in turn allow for
12. Selecting a unidirectional or bidirectional scan Page 161 of 164 Leica Microsystems Heidelberg GmbH Selecting an Image rrrnnnnnnnnnnnnnannnnennnnennn 90 Selecting an Objective rrrrrrrrarrrrnrevnnnennn 24 Selecting Color Look Up Tables 48 83 84 selecting Color Look Up Tables LUT 84 Selecting the Excitation Beam Splitter 22 Selecting the Excitation Wavelength 22 Selecting the Region of Interest 120 SELEC NO 1 48 90 Selection Button rrrrnrnnrrrnnnrrnvnnnrnnnnrennnnr 90 Separating Images rrrnrrnnnnrnnnnnnanrnnnnennnnennn 71 SE ENN nade nanes 88 Series Button rrrnnnrernnnernnnnvrnnnnvvnnnvnvnnnere 36 Series Image rarrnnnrnnnnnnnnnnnnnnnnnnannnanenunennnen 91 Series Scan Button rrrnrrrnnnrrnvnnrrrnnnrernnnr 41 Series Scan Overview Dialog Window 36 Setting Scan Paramete ls cccccceeee sees 35 Setting the Photomultiplier ee 25 Setting the Z y Position rrrrrernrrrrnrrvnnnnernn 33 SFP Projection Image 56 64 92 104 VA 0 EE EE 64 131 Short Pass Filter rrrnnnrrrnnnnornnnnvrvnnnennnn 138 Signal to noise Ratio rrarrrnrernrernnennnennre 138 Simulated Fluorescence Process SFP 104 Simultaneous or Sequential Image Recording EEE 22 Single MAJE ude 86 SKAGE SCAN seeders 34 Skewness of the Distribution 109 SMOG eear
13. rrrrrrnnrrnnrrnnrrnnrnnnrnnnnnnn 24 Nyquist Theorem rrrnnrnnnnrrnanrnnanennanennnnennn 30 ODJECUVE gsi covcctand cisosdedacuanasdiavandelcavesdesceunsenoce 24 Objective Button rrrarrrnnrennnrernnrrrnnnvanennnnr 24 Objective Dialog Window c ccceeceeeees 24 Objective NOSEDPIECE rrrnnrrnnnnrnnnnrnnnnnnanennnrr 24 GE EE 44 Offset Value rrerrnnnrnrnnnnrnnnnrrvnnnvnvnnnvnvnnnere 25 OJ eg EE EEE EE EE 127 Open Button sais wteenosevcieidian vieauaetedendeeerteieenies 127 Opening a File ccccscccsseeceseeceeeeeeeeeees 127 Opening the Context Sensitive Help 20 Optimal ZOOM Factors rrrnrrrnnnnrnnnnranennnrr 26 Option New WINdOW ccceeceeeeeeeeeeeees 129 Options Dialog Window rrarrrnnnrnnnnennnnnnnn 129 Re 143 Original Image rarrrnnnrrnnnnrvnnrnnnnnnnnnennnnennn 106 Orthogonal Projections 64 71 95 97 100 101 102 MON oes re a E 29 OE EE EE E 57 Overlay Button ararrarennnrernnnennnnnnnnrnnanennnre 87 Phase Button rrasarovnarannervananraunnransernaveene 44 Phase Displacement ccccecceseeeeeeeeeees 44 Phase Visualization c ccceseeeeseeeeeeeees 138 Ping pong Mode rrnnrnnnrnnnrvnnrnnnrnnnrnnnnnnnnnnn 52 Pinhole Button cccccccseeeeeseeeeeeeeeeseeeeees 29 Plane Objectives rrrarrrnnrnnnrnnnrnnnnnnnnnnn 24 138 Play Stop Button rrronrrrnnrrrnnrnrarern
14. Hardware Settings ccccceeeeeeeeeeees 45 48 POO EE 134 136 He 20 PANO EEE pemnteamsceamenteuetes 64 131 Highpass Filter rronrrrnnrrrnrernnrennnnvnnennnre 131 Histogram EE EEE 109 Image Brightness rrrrrnrrnnrrnnrrnnnrnnnn 25 71 Image Contrast rrrnrnnnnnnnnnnnnnnnnenanennner 25 77 Image Curvature Aberration 006 138 Image Raster cccccseccsseeceseeseeeeeseeseeess 30 Image Resolution rrrarrnnrrnnnrvnnrvnnrnnnrnnnnnnn 47 Image Series 33 36 37 40 41 88 89 90 91 Image Stack 40 41 92 95 96 105 Image Tool Button 62 64 77 131 Image Tool Dialog Window 62 64 71 77 131 142 Image Tool Dialog Window 3D Button 62 64 Image Tool Dialog Window Amplitude Button EE AEE NE 62 77 User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Index Image Tool Dialog Window Basic Button 62 71 Image Tool Dialog Window Materials Button EEE EA N 62 131 Image Tool Dialog Window Multi Color Button sess eee epee gs AIEE IA E AE NE 62 Immersion Objective rrrrnrarnnrrnnnrnnnrr 24 138 1 FOCUS EEE 29 Invariable Projection Axis 64 71 92 95 97 100 101 102 Isolines Image rrnnnnnnnnnnnvrnnvrnnrnnnnvnnnnnnnnnn 106 Keyboard ShortcutS rrrnnrrnanrrvanernanennnnennn 19 Lambda Scan Button rrrnrnnrennnrrrvnnrrnnnnnen 37 Lambda Steps Button rrrrrnrrn
15. O A value of gt 0 is useful for example if a reduction of one output image is to be shown at a certain position of the other output image This function is also useful for lateral image series such as those found in biomapping LL see Example for Shifting an Image The second data set is now placed next to the first data set The direction of the placement depends on whether the check box on the x y or z tab was selected Selecting this function in all the different tabs for x y and z shifts the second output image either in only one dimension or in several dimensions Here you can indicate which pixel represents the end of the merged image Definition background Absolute lengths are used to determine Page 73 of 164 Leica Microsystems Heidelberg GmbH Result Best Fit LCS Software Functions the dimensions of the merged image If the two data sets to be merged feature the same linear extension but a different number of pixels due to a different scan format it must be decided whether the number of pixels or the resulting total length is to be decisive for the merged image see Selecting a Scan Format The program uses the following procedure upon selecting Best Fit The width of a single pixel is calculated for both images um pixel If the difference of this size between the two images is smaller than a factor of 2 this procedure uses the larger image resolution i e the smaller um pixel value for the resultin
16. ararnnnnnne 134 136 Measuring Volumes rrrarrrnrrnnrennrrvnnennnnrr 113 VEN SINCE EEE 142 Merging Images rrrnnrnnnnrnnanennnnennnnennnnnnnnnn 71 Minimum value rannnnnnnnnnnnnr 77 109 110 112 Mode Button rernnnnvnnnnorvnnnvrnnnnnnnnnnnnnnnennnnne 31 Move Button rrrnnrnnnrrnnnrrnnnrennnrennvrennrrnnnre 108 MOVING corxtasacnnacatusnbetaGusnentnnesdeuncsasmasaetons 51 108 Moving an Image in the Result Image 142 Moving the 3D View rrrnnrrnnnrnnnnrnnnnennnre 108 Moving the Region of Interest 121 Moving the Region of Interest ROI 120 121 Multidimensional image data block 41 Multiple Image cccccssecceeeceeeseeeeeeeeeens 87 Multiplication reronrrrarrrrnnrrrnnrrranennnnennnnnnn 77 Multiplying Images rrrnrrrnnrrrnnnnnnnnernnnnnnnnnn 77 Neutral Filter rarnnnnnnnnnananannnnnnnnnnnnnne 22 138 New Experiment rarrnnanrnnnnrnnnnvnnnnnvnnennnne 129 New Experiment Button rrrrrrnnrrnnrnnnnnnn 129 New Window rrrnnnnnnnnnnnnnnnnnnnnnnennnnsnnnnnnnnnnn 48 NOKT EEE emecuantn 88 Next Button rernoronnervonarrosnnrannern rennarsnneere 88 Next Image of a Series rrrrrarrrnnrernnrrrnnnn 88 User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Index Number of Spatial Sections 36 40 Number of Wavelength Steps 00 41 Numerical Aperture
17. available scan modes The scan mode determines which optical levels of the specimen are to be scanned Fundamentally horizontal xy sections or vertical xz sections can be recorded To generate a three dimensional image of the specimen the optical sections are continued in the corresponding third direction in space thus recording a stack of individual images In addition to this it is possible to add the factor of time or wavelength while recording the images Function An image stack is recorded from the xy sections in the z direction An image stack is recorded from the xz sections in the y direction A line is recorded several times in succession A xy section is recorded several times in succession An xz section is recorded several times in succession An image stack is recorded several times in succession from xy sections in z direction An xy section is recorded at different wavelengths An xz section is recorded at different wavelengths All scan modes with the exception of xt are composed of at least three dimensions The device will ignore the third as well as other dimensions if you record images only from one optical plane xy or xz using the Continuous Scan function of the Single Scan function Page 31 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Note The xzy and xzt scan modes are only displayed in the list if you previously selected the selection poin
18. to open the context menu and select Send to gt Experiment 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 component of the experiment Menu Functions The following TCS SP2 functions can be carried out from the menu bar File Menu 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 window 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 defaults see the chapter on Default Settings and Templates Open Template This option opens a previously saved experiment a single data block or annotation files The way in which the viewer window is displayed can be selected from a set of templates Close User Manual Leica LCS english Page 16 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note Note Note User Manual Leica LCS english LCS Software Functions This option clos
19. 16 32x4 16 8x1 16 1 1 3 8 0 5 13 5 A B C D E Gray Values of Pixels A E in an Original 16 4 8 32 8 Image Factors of kernel size 5 1 16 4 16 6 16 4 16 1 16 User Manual Leica LCS english Page 69 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Gray value of C in the filtered image 13 5 A calculation of this kind is performed for each pixel in the original image At the edge of the image the filter mask protrudes beyond the image For these pixels the value 0 is set Inthe Kernel Size field select a value for the kernel size In the Filter Directions field set a the dimensions of the filter Select gfe the spatial axes to which the filter is to be applied sequentially One dimensional filters are applied that is the filter is calculated only via one axis at any given time Inthe Filter Type field select the Smooth filter The low pass filter described above is applied to the image 18 m 0 5 1 TO cen oe VG 35 4 45 5 4 4 Intensity profile of the image after applying the Smooth filter This filter type filters the high frequencies out of the image That is the very distinctive transitions from low to high intensity values are reduced This filter can be used to remove noise from an image gt Inthe Filter Type field select the Highpass filter After the low pass filter has been applied to the image the values of 1 lem 05 1 T
20. AA EN a EEE NE E 48 VIENNE NNN 51 Viewer Options Dialog Window 3D ICON rrrnnnennnnrnnnnnnnnnnvnnennnennnnennnnnnnnnnnanennanennanennnsnnnsennanennnsennn 51 Viewer Options Dialog Window Display ICON cccccecccseceeeeeeeeeeeeeeseeeeseeeeaeeesseeeseeeesaeeeseeeeaes 52 Viewer Options Dialog Window Charts ICON rrrnnrnnnnnnanrnranennnnernnrnrnnrnnanennnnennnnnnnernnaannnnennanennnsennn 53 Viewer Options Dialog Window Online Measure ICON cccecccceecceeeeeaeeeceeeeseeeeseeeeseeesseeesaeeees 55 Viewer Options Dialog Window Projections ICON ccccccecccseeeceeeeceeeeceeeeaeeeeeeeeseeeeseeeeeseeeseeeees 56 Viewer Options Dialog Window Overlay ICON ccccccceccceececaeeeceeceseeeeseeeeseeeseeeeseueesaeesaeesaeeeaes bl Viewer Options Dialog Window Scan Progress ICON cccccceececeeecee cece eeseeeeseeeeseeeeaeeesseeesaeeees 58 Viewer Options Dialog Window Surface View ICON 0 cccccseeccseeeeeeeeceeeeseeeeseeesaeeeseeeeseeeeseeeaes 58 Viewer Options Dialog Window Surface Measure ICON c cece ceccceeeecaeeeeeeeeseeeeeeeeeseeesaeeesaeeees 60 Viewer Options Dialog Window Surface Calculation ICON rrranrrrnnrnranrrnnnennnnrrnnnrnnnnnranernnnnennnnnn 61 Ten ee Cs OO PRE EE EEE EN EE EN iiaca Nel wt aduty 62 m ge 1001 Dialog WNAOW sus ateabr g A A seeren 62 Image Tool Dialog Window 3D Button optional rrrrnrernnnrnnnnrranennnnnrrannnnanennnnennnnnn
21. ERROR 10 VT BOOL 11 False 0 VIT VARIANT 12 VIT UNKNOWN 13 VIP UIL 1 Other constants omitted here E VARTYPE li el P Ti jol Not specified Null 2 byte signed 4 byte signed 4 byte real 8 byte real Currency Date Binary string IDispatch Scodes Boolean True l1 VARIANT FAR IUnknown FAR Unsigned char that are not valid in VARIANTs VT RESERVED int 0x8000 By reference a pointer to the data is passed Vi BIREP int 0x4000 VT ARRAY int 0x2000 A safe array of the data is passed The vt value determines the interpretation of the data of the experiment as follows Value Description User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Page 155 of 164 Leica Microsystems Heidelberg GmbH Appendix VT_EMPTY No value was entered VT Ul1 An unnamed 1 byte character was saved in bVal VT 12 A 2 byte integer value was saved in Val VT 14 A 4 byte integer value was saved in Val VT R4 An IEEE 4 byte real value was saved in fltVal VT R8 An IEEE 8 byte real value was saved in dbl Val VT CY A fixed point value is indicated It consists of 15 digits before the z point and 4 digits after the point The value was saved in cyVal Exceptions for files in LEI format A string was saved in the variable TCHAR A floating zero value was indicated Here the floating zero is not a VT_NULL NUL
22. LCS english Page 82 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note User Manual Leica LCS english vyv LCS Software Functions Procedure Select an image from the Source Image drop down list The drop down list contains all the recorded and calculated images of the experiments displayed in the Experiment Overview viewer window For multichannel images select the All Channels check box to make all changes for all channels at one time and to the same extent Change the form of the contrast transfer function by adjusting the slider Click on the Show button to obtain a preview of the calculation Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the calculation as a new data record Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment Viewing detection channels Viewing Detection Channel 1 8 Function Clicking the Channel 1 2 3 4 5 6 7 8 button displays the image data that has been recorded in detection channel 1 8 in the Viewer window You can assign any of several other color look up tables to the detection channel This setting can be changed both in the result image and while the image is being recorded To do so open the Select Look up Tables dialog window There are two ways of opening the dialog window Cli
23. Manual Leica LCS english Page 117 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions LL see Measuring a Profile Within a Region of Interest ROI see Calculating a Histogram see Measuring Roughness Within a Region of Interest ROI Defining the Region of Interest ROI as a Rectangle Function Using the Rectangle button you can define a rectangular Region of Interest ROI within the image Click the Rectangle button Click the left mouse button on the image position where you would like to place a ROI corner Then keeping the left mouse button pressed drag the mouse pointer diametrically to the opposite corner to define the second corner of the ROI Vv LL see Selecting and Moving the Region of Interest ROI LL see Moving and Rotating a Region of Interest ROI LL see Deleting Regions of Interest ROIs Other Requirements The definition of a region of interest in the image is required for quantification functions Therefore the Rectangle is deactivated until you activate a quantification function using one of the following buttons see Measuring a Profile Within a Region of Interest ROI see Calculating a Histogram see Measuring Roughness Within a Region of Interest ROI User Manual Leica LCS english Page 118 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Auto
24. Microsystems Heidelberg GmbH LCS Software Functions Setting the Detection Pinhole Function Clicking the Pinhole button opens a dialog window that you can use to set the diameter of the detection pinhole In the upper right list field in the dialog window select the units for displaying the diameter You can select from mm Airy units and digital values There are two ways of specifying a value for the diameter Use the mouse to move the slide on the scale that is displayed on the left in the dialog window The corresponding value is shown in the middle of the dialog window gt Double click the boldface numerical value located in the middle of the dialog window This opens a second dialog window where you can enter an exact value The diameter of the detection pinhole must be set to the currently used objective at all times If you click the Airy 1 button the detection pinhole is set automatically to the optimum value of 1 Airy unit depending on the objective in use In addition to the numerical aperture of the objective and the wavelength of the light the detection pinhole also determines the thickness of the optical sections Additional information The diameter of the pinhole has the optimum if it matches the diameter of the Airy disc The Airy disc refers to the inner light circle of the diffraction pattern of a point light source The diameter of the Airy disc in turn is also dependent on several optical parameters an
25. No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Creating SFP projections of an image stack optional Function The SFP button starts an SFP projection the mathematical simulation of a fluorescence process The basis for a projection is an image stack i e a series of horizontal xy sections or vertical xz sections When a projection is generated the sampling points of the individual images superimposed along the projection axis are examined throughout all optical sections In the first step the laser beam is simulated penetrating the sample and diminishing In the second step using the determined light density the simulated fluorescence for each Voxel is calculated see Principles and Types of Projections Click the SFP button A SFP projection of the current image stack is created and displayed in the Viewer window You can now change the projection axis by clicking in the image in the Viewer window and holding the left mouse button pressed gt Broken support lines are displayed that encloses the symbolically displayed image stack Turn the image stack to the desired new position by moving the mouse pointer while holding down the left mouse button Upon releasing the left mouse button the new SFP projection will be calculated and displayed The projection axis is the viewing direction of the user to the newly positioned image stack You can return to the original i
26. Result Slider This slider adjusts the brightness of the result image If the slider Brightness is exactly in the center the output image and result image have exactly the same level of brightness If the slider is moved from the center to the right the result image becomes brighter than the output image If the slider is moved from the center to the left the result image becomes darker than the output image Slider This slider adjusts the contrast of the result image If the slider is Contrast exactly in the center the output image and result image have exactly the same level of contrast If the slider is moved from the center to the right the result image has greater contrast than the output image If the slider is moved from the center to the left the result image has less contrast than the output image Click on the Show button to obtain a preview of the calculation Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the calculation as a new data record Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment Gamma Correction Setting the Contrast Transfer Function Gamma Icon Curve The contrast transfer function gamma curve can be used to influence the conversion of the output image contrast to the result image contrast in a non linear manner User Manual Leica
27. SAME 15 Data Organization by Grouping Experiments rrrrrrrnrrrrrarernnrernnnrrnnrrranrrnanennnnrnnnnennsnnnaennasennunennn 16 COMPING EXPENSE ee 16 MUST GE Le 10 NN EE NN NE 16 R VET SNOEN NN 19 LE S DOM Ware Functions reser 19 Introduction to the Leica Confocal Software Help cc ccceccceecccseceseeeeeeeeseeeseeeeseeeeseeeeeeeeesaeeeaes 19 Opening the Context Sensitive Help cccccccsecccsseeceeecceeeceucecceeccaeeceuseceueessueesseeseueessusessaeenaass 20 Documentation Conventions 9 u5h cess ciszccioceccaarneedeticestdenteneudsicens gacctieaedathapendendenaetbacaaeneecseeeedetaansuealys 21 BELE Np SC ONGIING PE EE EE EE EE EE 22 SCMIAGMNE Bean PAN s rer 22 SCIECING AN OE CE sr 24 Seinge DEE NS rer 25 TAS EIS CUO MIC Zoran eneren 26 Enlarged Recording Of a Frame ccccccccsscccseecceeeceuceceueeceeceueeseeecueecsacecaueesseeeseeeeeueessueetsneensas 28 Setting the Detection Pinhole sad eek dte 29 SCICCUNG a SCAM FO Se 30 Se Mg A SCAR MOGE NR 31 SEE MG a ocan SDC CO ee 32 Sele Z Vk INN 33 CONG UGG ATMS SEES Aden 33 ML SNE SN 34 SEN FE OMMMUOUS SAN 35 Series Scan Overview Dialog WIindow rrrnrnnnnnnnnnnnnanennnnennnnnnnnnnnnnnennnnennnnnnnnennanennunennnsnnnssnnnnennnne 36 Defining the Begin Point for a Spatial Series rrrarrrrnnrrrnrrrrarrrnnnernnnennnnnranrnranennnnennanennannnnnsennunennn 37 Defining the Begin Point for a Wavelength Series rrrrrrrrnrrrrarrrrrnnevnnnrranrnranernnn
28. 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 prt Templates for the Viewer window are saved in this format This allows for specifically saving the appearance and type of displayed information An explanation of the individual configuration options can be found in the chapter on Viewer window Ihw Calibration data and hardware settings of the confocal system are saved in this format This file may be edited only by service personnel certified by Leica Microsystems Heidelberg Warning Changes to this file may result in serious possibly irreparable damages to the confocal system Damages demonstrably resulting from changes to this file by the user will result in a loss of warranty for the complete system lut 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 Selecting Color Look Up Tables Fixed Leica specific File Formats The following file formats can be
29. U The configuration is deleted gt Optional Software packages Materials Image Tool dialog window Materials button optional Schematic Structure of the Dialog Window The numerous functions of the Image Tool dialog window are arranged on a number of levels The gray buttons 3D Basic Amplitude Materials and Multicolor on the left side of the dialog window represent the first level of the hierarchy Each of these buttons has a group of icons that appear below the selected button The icons are the next level on which functions are grouped Each icon opens different tabs and fields that are displayed on the right side in the dialog window and arrange the functions on the lowest level The following elements can be found in each tab In the Source Image drop down list at the top of the dialog window select the image data record to be processed If you click on the Use Selected Image check box the image data record currently displayed in the Viewer window is used for the calculation If you click on the Show button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the preview window in the dialog window If you click on the Apply button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the Viewer window and created as a new file in the current experiment Click on the button with the arrow symbols to show
30. an Overlay Image Additional information The stereoscopic effect of the anaglyphic image is possible since human beings use both eyes for viewing Since the eyes are approximately 6 5 cm 2 56 inch apart the brain receives two images from different perspectives Objects located at different distances are observed at different angles This angular difference is a measure of distance for the human brain The difference between the similar images generated by the two eyes is processed by the brain into one image with spatial depth impression Holding a slightly different image in front of each eye can also simulate these image differences In anaglyphic images the complete stereoscopic image information is located on one image The information of the left and right image is coded through colors For this reason glasses with a different color filter for each eye are needed in viewing these images This filter separates the image information again for each eye The red filter only passes red light while completely is absorbing the green light On the other hand the green filter absorbs the red light Each eye perceives one image both are merged in the brain into an overall spatial image Icon Orthogonal Creating projection images along the orthogonal Projection axis Creating Projection Images from Different Detection Channels Creating a Projection Image Along the Orthogonal Axis z axis with horizontal xy sections and y axis with
31. 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 Every hazardous situation cannot be anticipated therefore the user must exercise care common sense and observe all appropriate safety precautions applicable to Class 3b lasers and high voltage electrical equipment during installation operation and maintenance Deviation from published operating or maintenance procedures is not recommended Operation and maintenance procedure changes are User Manual Leica LCS english Page 5 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Legal Notes 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 User Manual Leica LCS english Page 6 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Starting up the system Starting the Windows NT Operating System You don t have to start Windows NT it starts automatically when you turn on your PC You will first see a splash screen Next you have to log on to your computer As you can see from the instructions in the box pressing the Ctrl Alt and Delete ke
32. can undo this using the Original key LL see Projection of an Image Stack with Invariable Projection Axis LL see Maximum Projection of an Image Stack with Invariable Projection Axis L see Average Projection of an Image Stack with Invariable Projection Axis LL see Transparent Projection of an Image Stack with Invariable Projection Axis On the Stereo View Tab you can create red green stereo images So called anaglyphic images Anaglyphic images create a stereoscopic sense of depth if they are viewed with corresponding 3D glasses red green stereo glasses The following settings must be made to create the two stereo images gt Inthe Stereo View Options field click on the On check box to activate the function Inthe Eye Angle field determine the angle under which the two stereo images are recorded For example if you enter the value 30 a stereo User Manual Leica LCS english Page 75 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions image is recorded at an angle of 15 each left and right from the projection axis that was set on the Rotation tab Stereo images can only be created from one detection channel If you have an image series that was recorded in several detection channels select the desired image data set in the Detection Channel field Click on the Show button to obtain a preview of the stereo images Show starts the calculat
33. displayed in the Viewer window is used for the calculation If you click on the Show button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the preview window in the dialog window If you click on the Apply button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the Viewer window and created as a new file in the current experiment Click on the button with the arrow symbols to show the preview window on the right edge of the dialog window or to hide it The projections triggered using Show are displayed temporarily in this preview window The Close button closes the dialog window User Manual Leica LCS english Page 71 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Leveling Adapting an interpolation function to icon a data set to correct trend curves e g skewed recording of a data set Procedure This procedure aims at bringing an intensity curve that features a trend curve into a horizontal position For this purpose a suitable interpolation function polynomial is first adapted to the intensity curve Next the distance of the optimal interpolation function to a horizontal intensity plateau is calculated The occurring intensity differences are added pixel by pixel to the original data set This compensates the trend curve see Adaptat
34. g lei file with PC Tiff images Size Data byte type 4 int 4 int 4 int nTC 2 wchar 4 int nTC 2 wchar 4 int nTC 2 wchar 4 int NTC 2 wchar Symbol nTC nTC nTC nTC Description Internal version number Number of images in the data collection of the experiment Length of the following string in wchar Short description of the format Length of the following string in wchar File extension of the main file Length of the following string in wchar Identifier of the single image format e g PC TIFF Length of the following string in wchar File extension for the single image format e g tif raw ID_LUTDESC memory block Description of the color look up tables LUT s User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Page 152 of 164 Leica Microsystems Heidelberg GmbH Size Data byte type 4 int 4 DWORD For the next channels until nLU 4 int 1 bool 4 int nTC wchar 4 int nTC wchar 4 int nTC wchar 4 int 4 int Appendix 1 SAFEARRAY Data Type Symbol nLU nTC nTC nTC Appendix Description Number of channels Identifier ID of the dimension whose display is being colored Internal version number bool Isinverted Length of the following string in bytes Description of the color look up table LUT Length of the following string in bytes File name of the color look up table if available Length of the following string in bytes Na
35. image series with a large number of individual images but at a low scan format you can save and open it faster in raw format than in tif Storing All Data Function Use the Save All button to save experiments lei or annotation sheets ano while giving them a specific name and file type When saving an experiment a folder is created at the file level with the name of the experiment This folder then contains the description file lei for the experiment as well as the individual image files The format of the description file is Leica specific and binary This file contains the parameter settings and the color information in the form of color look up tables for each image belonging to the experiment The image files of an experiment can be saved in tif or raw format The standard format tif also contains the experiment settings and the color information for the images In the raw format just the image data is saved Page 128 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Typical Applications The advantage of the raw format is the smaller file size but this is only significant for image recordings with relatively small amounts of data For example if you record an image series with a large number of individual images but at a low scan format you can save and open it faster in raw format than in tif Creating an Experiment a New Function Click the
36. images If you click on Calc Shadow in the Options field a shadow is calculated and displayed for the SFP projection image Move the slider in the Absorption field to vary the absorption coefficient a Page 68 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions The higher this is set the smaller the values calculated in the projection image If the coefficient is set as low many of the pixels in the projection image reach the maximum value making it increasingly difficult to distinguish between the structures Move the slider in the Threshold field to define a threshold value The intensity values below this value are not taken into account for the creation of the projection image Click on the Show button to obtain a preview of the projection images Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the projection as a new data record Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment In the Rotation and Stereo View tabs you can create a rotation animation or a stereo view of the SFP projection image LL see Running Projection Images as a 3 dimensional Rotation Animation see Creating a Red Green Stereo View Filter Linear lcon Applying low pass and high pass filters to images Filters are used
37. images according to the formula Pixel Intensity resut Pixel Intensity source image 1 X Pixel Intensity Source Image 2 Divides the intensity values of the two selected images according to the formula Pixel Intensity resut Pixel Intensity source image 1 Pixel Intensity Source Image 2 Selects for each pixel the minimum value of the two selected Images according to the formula Pixel Intensity resut MIN Pixel Intensity source image 1 Pixel Intensity Source Image 2 Selects for each pixel the maximum value of the two selected images according to the formula Pixel Intensity resut MAX Pixel Intensity source image 1 Pixel Intensity Source Image 2 Calculates for each pixel the average of the two selected images according to the formula Pixel Intensity resut 1 2 x Pixel Intensity source image 1 Pixel Intensity Source Image 2 The binary representations of the pixels of the two selected Page 78 of 164 Leica Microsystems Heidelberg GmbH OR XOR LCS Software Functions images are linked with the logical operator AND That means that pixels in the result image are given the value 1 when the value 1 is present in both of the pixels otherwise 0 is entered see Boolean Operations You achieve meaningful results using this Boolean operator when you link an image with a binary mask The pixel is displayed in the result image at the point where the pixels of the image meet the pixels of the mask with the value 1 The
38. modes are available depending on the other options you enable simultaneously Button Combination Gallery Single Channel 1 2 3 4 5 Gallery Single Overlay Channel 1 2 3 4 5 Gallery Tiled Channel 1 2 3 4 5 Gallery Tiled Overlay Channel 1 2 3 4 5 User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Display All individual images in an image series are displayed for the selected detection channel only Based on the number of individual images in an image series overlay images are generated from all selected detection channels All individual images in an image series are displayed for all selected detection channels All individual images of an image series are displayed both separately for all selected detection channels and in overlay images together Page 91 of 164 Leica Microsystems Heidelberg GmbH LCS Software Functions Projections Principles and Types of Projections Ea Seg ee 4 Per APP AD EEE Lar T BE T PHOTTTY PH HI m i 3 th eo aE g Ts Sele TT UIT TT T 2a Projection with an invariable projection axis 2b Projection with a variable projection axis Functional Principles of Projections Biological specimens are recorded with a series of images Each image is taken from a particular level in the specimen The result is a three dimensional recording that contains the entire volume of a specimen Each optical section each ind
39. of a roughness profile that show a trend into a horizontal position For this purpose a linear interpolation function polynom is fitted to the measurement curve C see Image Tool Dialog Window Basic Button LL see Adaptation Function for Correction of Trends None No adaptation function is applied to the measurement curve Automatic The measurement curve is brought into a horizontal position linear using a linear adaptation function Interactive The position of the measurement curve is corrected using the level linear adaptation function in such a way that the line segment between the two measurement points is on the horizontal plane Freeze The newly positioned measurement curve is locked so that the item of the measurement points can be changed again without interactively modifying the measurement curve The Multipoint Measurement field provides the possibility of saving the two measurement points as well as the difference in height between the measurement points Click on the Remember button to save the values The Clear button deletes them again User Manual Leica LCS english Page 60 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Viewer Options Dialog Window Surface Calculation Icon Function To open the Viewer Options window select the Experiment Overview option in the View menu The Experiment Overview window appears on the left side of the user inte
40. on the appropriate date or use the controls to change the month or year The time can also be changed by first selecting the digital display and then using the up and down arrows To change the Time Zone Select the appropriate Time Zone from the drop down list at the top of the Page 10 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions screen Notice that the option to automatically adjust the clock for daylight savings time is selected On some systems you can also drag the highlighted area on the world map and drop it on the correct location Changing the date and time information within Windows NT Workstation 4 will update the battery backup CMOS clock in your system Note Depending on the shell configuration systems connected to a network may get a time and date update from a network server every time they log on If the server s time is incorrect your workstation s time will be wrong too Please inform your network administrator Getting Help Windows NT includes a powerful help system In addition to Help menus in every window there is a standalone Help feature available from the Start menu To access it click your mouse on the Start button and click on Help There are three tabs in this box Contents Index and Find The Contents tab appears on top To move to a tab click on it Contents The Contents tab displays individual Help topics The topics are organize
41. see Electronic Zoom Click on one of the selection points on the same tab to hide or display the respective graphic element in the Viewer window Coordinates The z position of the current image is displayed Scale Grid A measurement bar with length indicators is displayed A grid is placed over the current image The length of the measurement bar and the grid width are calculated dependent on the objective the electronic zoom and the beam divergence You can use the Movie tab to set the speed for running the film sequence of an image series You can choose speeds from 6 images per minute to 25 images per second Use the mouse to drag the slide on the scale to the desired value Select the Ping Pong mode if the film sequence is to run from the first to User Manual Leica LCS english the last image and then in reverse order back to the first image If this mode is not selected the film sequence always starts with the first image again Viewer Options Dialog Window Charts Icon Function To open the Viewer Options window select the Experiment Overview option in the View menu The Experiment Overview window appears on the left side of the user interface The top part of the viewer window displays the recorded images in a directory tree The bottom part displays the Viewer Options window This dialog window is used to perform basic settings for different software functions The left side shows the icons corresponding to th
42. see Maximum Projection of an Image Stack With Variable Projection Axis L see Average Projection of an Image Stack With Variable Projection Axis LL see Transparent Projection of an Image Stack With Variable Projection Axis Setting the Projection Axis and Running Projection Images as a 3 dimensional Rotation Animation Inthe Rotation tab set the desired projection axis by entering a value for Z Y and X With these three values you rotate the image stack into a certain position and thus define the angle at which the image stack is projected The projection axis here is the viewing angle of the person viewing the rotated image stack You can verify the result in the small preview window in the tab The scheme below illustrates the rotation direction of each image level in the case of positive values for Z Y and X In the case of negative values the rotation direction is exactly the other way around gt To make settings for the rotation animation click on the Animation check box The corresponding fields are enabled Inthe Rotation field select the animation type Full turn or 360 half turn or 180 quarter turn or 90 or any other angle User Manual Leica LCS english Page 65 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH User Manual Leica LCS english LCS Software Functions In the Frames field enter the number of projection images to be created for t
43. select the desired entry If you hold the mouse pointer over any area within the Experiment legend and press the right mouse button a context menu containing the following commands opens Command Function Experiment Edit This opens the Edit Legend dialog window where you can specify a Title for the legend specify the Number of legend entries or Clear all entries Activate This displays the Experiment legend in the Viewer window Remove This deletes the current Experiment legend Add This creates a new Experiment legend in the Viewer window Experiment tab The Hardware Legend The hardware settings for image recording are registered in the Hardware legend To open the legend select the Hardware Legend option from the View menu To select the entries that you want displayed in the legend click the Edit button This opens the Edit Legend Entries dialog window The Available Entries list box shows all available entries Select the entries that you want displayed in the legend Then click Add to carry the selected entries over into the Show entries list box The Show entries list box contains the entries that are to be displayed in the legend Use the Remove button to remove entries from being displayed in the legend Use the Move up and Move down buttons to shift one or more than one entry up or down in the list You can use the Edit grid color and Edit background color buttons to modify the color of the frame and backg
44. slide bar represents the wavelength at which the recording is to end Click Lambda Scan End to save the value The begin point of a wavelength series is set in the same manner In addition you also have to set the number of wavelength steps LL see Defining the Begin Point for a Wavelength Series LL see Setting the Number of Wavelength Steps Typical Applications A wavelength series can be applied to determine the maximum emission of a fluorochrome This is useful because the Stokes shift of the emission curve of a fluorochrome is dependent on each specimen that is applied This then allows you to set the detection range precisely for a specific application User Manual Leica LCS english Page 39 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Defining the Number of Spatial Sections Function Clicking the Sections button opens a dialog window that you can use to select the number of horizontal xy sections and vertical xz sections for recording an image series If you want to specify a number of sections that is not listed click the Others option This opens the Z Y Configuration dialog window which contains the following data Parameter Description Image Dim z y The height of the entire image stack between the begin and mm end points of the image series Sections The number of configured sections Step Size mm The step size i e the distance
45. the Selection button a dialog window opens which lets you select any image of a series and display it in the Viewer window The number of individual images for the series is displayed from 1 to n as well as in an entry field the number of the currently displayed image Use the mouse to move the slide of the scale The corresponding image is displayed immediately Enter the number of the desired image into the entry field and click on Apply Starting and Ending a Film Function You can view the individual recorded images of an image series as a film sequence Use the Play Stop button to start and stop the film The film speed that is the number of single images per time unit is variable and can be set in the Viewer Options dialog window gt From the View menu select Viewer Options Click the Display icon and then on the Movie tab see Viewer Options dialog window Display icon If the Gallery button is enabled the Play Stop First and Last Next and Previous buttons are disabled and displayed in gray The Gallery button is used to show all of the individual images of an image series and does not allow film sequence mode Page 90 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Viewing the Series image Function If you click the Gallery button all of the individual images in an image series are displayed in the Viewer window The following display
46. the preview window on the right edge of the dialog window or to hide it The projections triggered using Show are displayed temporarily in this preview window The Close button closes the dialog window Filter Linear lcon Applying low pass and high pass filters to images User Manual Leica LCS english Filters are used to enhance the quality of an image As a rule this means removing undesirable pixels from the image The filter function available here a low pass filter is used to suppress static noise in an image that could be generated by the detector The principle of a filter consists of Page 131 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions taking the value of each pixel in an image and setting it off against the values of the adjacent pixels The filter kernel describes the number and weighting of the adjacent pixels that come to bear in the calculation of the new pixel The kernel used here can be described using Pascal s triangle Filter kernel size Pascal triangle Scaling 1 1 1 2 Kernel Size 3 1 2 1 1 4 1 3 3 1 1 8 Kernel Size 5 1 4 6 4 1 1 16 1 5 10 10 5 1 1 32 Kernel Size 7 1 6 15 20 15 6 1 1 64 1 7 21 35 35 21 7 1 1 128 Kernel Size 9 1 8 28 56 70 56 28 8 1 1 256 If for example kernel size 5 is selected 5 pixels of the original image are used in calculating 1 pixel in the filtered image It is always the middle pixel in the filter kernel that is the
47. the user interface The top part of the viewer window displays the recorded images in a directory tree The bottom part displays the Viewer Options window This dialog window is used to perform basic settings for different software functions The left side shows the icons corresponding to the functions and the right side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons In the tabs of the Projections icon you can make optional settings for the following functions LL see Maximum Projection of an Image Stack With Invariable Projection Angle see Average Projection of an Image Stack With Invariable Projection Angle see Transparent Projection of an Image Stack With Invariable Projection Angle see Creating an SFP Projection of an Image Stack The Projections tab contains various options for generating a projection image Select whether the intensity maximums the arithmetical average or a weighted average of the intensities are to be displayed in the projection image Vary the weight factor for the transparent projection Set a threshold value or scaling factor for the displayed intensities Type Maximum projection Average projection Transparent projection Transparent factor Drag the slide on the scale Threshold Drag the slide on the scale User Manual Leica LCS english Art No 15 9330 033 Vers 01082
48. to enhance the quality of an image As a rule this means removing undesirable pixels from the image The filter function available here a low pass filter is used to suppress static noise in an image that could be generated by the detector The principle of a filter consists of offsetting the value of each pixel in an image with the values of its neighboring pixel The filter kernel describes the number and weighting of the adjacent pixels that come to bear in the calculation of the new pixel The kernel used here can be described using Pascal s triangle Filter kernel size Pascal triangle Scaling 1 1 1 2 Kernel Size 3 1 2 1 1 4 1 3 3 1 1 8 Kernel Size 5 1 4 6 4 1 1 16 1 5 10 10 5 1 1 32 Kernel Size 7 1 6 15 20 15 6 1 1 64 1 Tf 21 35 35 21 T 1 1 128 Kernel Size 9 1 8 28 56 70 56 28 8 1 1 256 If for example kernel size 5 is selected 5 pixels of the original image are used in calculating 1 pixel in the filtered image It is always the middle pixel in the filter kernel that is the pixel to be calculated In this case the gray values of the 5 pixels corresponding to the weighting scale 1 4 6 4 1 are multiplied by the factors 1 16 4 16 6 16 4 16 1 16 and together added to the final value With respect to the calculation of the pixel C in the following schematic representation this means that the gray values of pixels A B and D E are included in the calculation of the filtered pixel C C is calculated as follows 16x1 16 4x4 16 8x6
49. used for displaying transmission recordings This is especially suited for displaying 12 bit images This is commonly used for recording specimens that have been marked with pH sensitive or ion sensitive fluorochromes and for displaying time series These are recommended for generating topographical images i e for mapping surface structures Geo Land amp Sea is especially useful for visualizing depths and graduations These are recommended for optimizing image contrast the offset and gain of the detectors Glow Over highlights in blue intensities at the upper end of the table while Glow Under represents in green intensities at the lower end of the table Glow Over and Under is a combination of these two color tables Page 85 of 164 Leica Microsystems Heidelberg GmbH LCS Software Functions Cyan These are recommended for printing images The CMY K colors Magenta are generally used for the printer system color output RGB Yellow colors such as the other color look up tables used here are applied generally for color representation on monitors Because these two color systems vary greatly the colors in the CMY K representation can differ strongly from those used in the monitor representation All color look up tables are also available with inverse color flow meaning high intensities are represented as dark and low intensities light Viewing the Single image Function Click the Single button to view only one detection ch
50. vertical xz sections Inthe Type field select one of the three projection types maximum projection average projection and transparent projection see Principles and Types of Projections The Direction field determines which single images are used for the projection Select z for stereoscopic series t for time series or I for wavelength series to create the projection from all images of a detection channel such as from image 1 to image 10 in detection channel 1 Select ch to create the projection from a certain image in all detection channels for example from image 1 in detection channel 1 and image 1 in detection channel 2 Click on Rescale in the Options field to standardize the intensity values of the image to the maximum possible intensity values when creating the projection image This ensures that the calculated gray values of the projection image remain within the value range of for example 0 to 255 at 8 bits This allows for brightening dark images If you click on Invert in the Options field the projection axis is mirrored The calculation of the projection image is thus in the opposing direction This function makes sense above all in the case of inverse microscopes to match the calculation of the projection image to the inverse beam path of laser light The Transparent Factor field is only active if the transparent projection was selected The slider is used to vary the transparent factor a The higher
51. viewing these images This filter separates the image information again for each eye The red filter only passes red light while completely is absorbing the green light On the other hand the green filter absorbs the red light Each eye perceives one image both are merged in the brain into an overall spatial image Image Separation Separating single images or image segments from a icon data set Here you perform the settings to separate a segment from an image or complete images from an image stack in one or several detection channels Select an image from the Source Image drop down list The drop down list contains all the recorded and calculated images of the experiments displayed in the Experiment Overview viewer window The dimensions x y and z and the detection channel can freely be combined in the Dimension Selection field Field Function description Dimension The detection channels of the image or the image series are Selection ch indicated here Select the detection channels from which the image segment or the individual images are to be separated Dimension The number of single images of an image series is indicated Selection z here Enter the number of images to be separated in the Start z and End z entry fields Use the R Reset button to undo the User Manual Leica LCS english Page 76 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions entries Dimension T
52. when the refraction indexes of the embedding medium and or the specimen are matched to the refraction index of the objective medium Setting the Detectors NE Function Use the Signal button to open a dialog window for adjusting the detectors so that the entire range of intensities of a color look up table is assigned and displayed in the image For this purpose a Gain value and an Offset value can be set for each detector The gain value modifies the amplification of the detected signal thus changing the brightness and contrast of the image The offset value defines a threshold value Only those signals that lie above the threshold value are detected and displayed in the image There are two methods for setting the gain and offset values Use the mouse to move the slide on the scale The corresponding value is shown below the corresponding scale gt Double click the numerical value that is displayed below the scale This opens a second dialog window where you can enter an exact value Best suited to optimization of gain value and offset values are the color look up tables Glow Over Glow Under and Glow Over and Under see Selecting Color Look Up Tables User Manual Leica LCS english Page 25 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions You can also set the gain value and offset value of the detectors using the corresponding radio buttons of the control panel
53. 001 Function From each of the examined columns of sampling points the sampling point with the highest intensity is displayed in the two dimensional projection image as the representative of all values within the column From each of the examined columns of sampling points the arithmetical average of the intensities measured in the column is displayed in the projection image From each of the examined columns of sampling points a weighted average of the intensities measured in the column is displayed in the projection image Function enabled only for transparent projection The higher transparent factor a 0 lt a lt 1 setting the stronger the intensity values flow from the lower levels in the image stack into the projection image Function enabled only for average and transparent projection Intensities that are below the specified threshold value are ignored in the calculation of the projection image Page 56 of 164 Leica Microsystems Heidelberg GmbH Scaling Rescale to maximum LCS Software Functions Function enabled only for average and transparent projection The measured intensity values are scaled to the maximum possible intensity values The SFP tab contains various options for generating an SFP projection image see Principles and Types of Projections Field Light Direction Absorption Threshold Rescale to maximum Function Here enter a coordinate for x and y for the projection
54. 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note vvv Note LCS Software Functions While the 3D view is being zoomed the resolution of the image is decreased in order to speed up processing When the left mouse button is released the image is displayed with its original resolution As an alternative to zooming a 3D view manually the Viewer Options dialog window also allows you to change the zoom scale by specifying numerical values From the View menu select Viewer Options Click the 3D icon You can then vary the zoom factor in the Navigation tab by changing the y value in the Translation field LL see Viewer Options Dialog Window 3D Icon The Leica Confocal Software provides three different zoom functions 3D zoom electronic zoom and graphical zoom L see Electronic Zoom see Viewer Options Dialog Window Display Icon Measuring and Analysis Functions Calculating a Histogram Function The Histogram function measures the frequency of a measurement variable displays it in a curve and in addition calculates various statistical values The histogram is measured from the data set selected in the Viewer window This data set can be a spatial series a time series or a wavelength series The statistical values are calculated dependent on the size of the third dimension height z for spatial series time t for time series wavelength A for wavelength series Click on the Histogram button
55. 0x objective which has a maximum Scan field size of 150 mm would result in the following raster point distance 150 gm 1024 To achieve the required 47 nm raster distance you can either raise the scan format to 2048x2048 for instance or you can decrease the scan field size using the Electronic Zoom Zoom 2 ca 146 nm The current raster distance is displayed in the Hardware legend This value is referred to as Voxel Size and recalculated as soon change the scan format the Electronic Zoom or the objective see The Electronic Zoom see Selecting an Objective Page 30 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note Mode XYZ XZY xt xyt xzt xyzt Xy XZ User Manual Leica LCS english LCS Software Functions When the sampling rate of 2 to 3 points is exceeded it is referred to as oversampling In the case of oversampling the structure is fully mapped but no additional information is gained The disadvantage of oversampling is the long scanning time and the danger of destroying bleach sensitive specimens When the sampling rate is less than 2 to 3 points it is referred to as undersampling In this case there is the danger that not all necessary information is sampled The image might illustrate structures that are not actually present in the specimen This effect is called aliasing Function Clicking the Mode button opens a dialog window containing a list of
56. 12 bits are used for the arithmetical operations As the calculation result can produce values extending beyond the interval the following three methods are available to readapt them to the interval Raw Data Options Cut at Maximum Absolute value Rescale User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Result Values less than 0 are set to 0 All values greater than 255 for 8 bit images or 4095 for 12 bit images are set to the value 255 or 4095 This concatenates all the values lying outside the interval The amounts of the result data are used This turns negative results into positive results The extreme values are determined The minimum value is assigned to the 0 The maximum value for 8 bit images is assigned to the value 255 for 12 bit images to the value 4095 Page 79 of 164 Leica Microsystems Heidelberg GmbH gt LCS Software Functions If you have selected Scaled Data the world coordinates that is the actual and non standardized values are used for the arithmetical operations The standardization of the results data into the intensity interval 0 255 and or 0 4095 is only used for the image display functions for color look up in contrast the actual result values are used for the quantification functions If you select 12 Bit Output in the Output Option field each pixel in the result image is coded with 12 bits i e the result values are saved in a value range from 0 4095
57. 4 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions The Experiment Software Concept The Leica Confocal Software allows you to join image data or edited 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 viewing data together Additional information can be found in the chapter Organizing Data by Grouping Experiments The Basic Structure of the User Interface The appearance of the Graphical User Interface henceforth referred to as GUl depends strongly on the configuration profile being used A number of elements of the GUI however are standard The GUI provides the following standard elements The menu The menu contains the submenus File View Macro Tools Window and Help The submenus contain commands and information for general display settings and user customization They do not provide any of the functions for directly controlling the scan functions These functions are located in the TCS menu View gt Menu TCS Menu The menu line cannot be customized 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
58. Averaging Method Function The Average function launches an averaging method for the image recording In this case every individual image i e every xy section or xz section is scanned several times For every sampling point the arithmetical average is calculated from the repeatedly measured intensity Page 46 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions values and represented in the result image The method used here determines a consecutive average This means every image recorded after the first image is calculated with the results of the previously displayed image and is displayed in the result image dynamic average Clicking the Average button opens a dialog window that you can use to set how often a section is to be scanned You can select from 1 to 64 scan repeats Typical Applications Recording an image using the averaging method is primarily useful for suppressing noise In fluorescence microscopy weak fluorescent specimens result in little light reaching the detector The resulting low photon count leads to noisy images In such cases you can improve the signal to noise ratio by carrying out multiple recordings and determining averages of the image Note Recording an image using the averaging method is not recommended for bleach sensitive specimens Repeated recordings and the resulting length of subjection to light can lead to the destruction of th
59. CODE VT_ERROR CY DATE VT_DATE BSTR VT_BSTR IUnknown VT_UNKNOWN IDispatch VT DISPATCH SAFEARRAY VT ARRAY unsigned char VT BYREF VT UIL short VT BYREF VT I2 long VT BYREF VT I4 float VIT BYREF VT R4 double VIT BYREF VT R8 VARIANT BOOL VT BYREF VT BOOL SCODE VT BYREF VT ERROR Art No 15 9330 033 Vers 01082001 bVal iVal lVal fltVal dblVal boolVal scode cyVal date bstrVal FAR punkVal FAR pdispVal FAR parray FAR pbVal FAR piVal FAR plVal FAR pfltVal FAR pdblVal FAR pboolVal FAR pscode Jd FE Zi Id iy Ii Fi be l Id a num of vr UT vr I2 VT I4 VT R4 VT R8 VT_CY Page 154 of 164 Leica Microsystems Heidelberg GmbH Cy Vi BYREF VT Cr DATE VT BYREF VT DATE BSTR VT BYREF VT BSTR IUnknown FAR VT BYREF VT UNKNOWN IDispatch FAR VT BYREF VT DISPATCH SAFEARRAY FAR VT ARRAY VARIANT VT BYREF VT VARIANT void ByRef y Appendix FAR pcyVal lee FAR pdate jer FAR pbstrVal I7 FAR ppunkVal es FAR ppdispval FAR pparray 17 FAR pvarVal FAR byref Generic Important Files with the LEI format never use pointers as parameters VARTYPE typedef unsigned short enum VARENUM VI EMPTY 0 VT NULL L YT I2 2 int VT I4 3 int VT_R4 4 VT_R8 5 VT_CY 6 VT DATE 7 VT_BSTR 8 VY DISPATCH 9 VT
60. Creating an Experiment 127 128 129 Creating the 3D View rrrnrnnnrnnnnennner 58 106 Data ge 10 EE 127 Deleting Regions of Interest 122 PEEL 83 Detection Channel rrrrnnnrnnnnnennnnnnnnnnrnnnnr 83 Detection Pinhole rrnnrrnnnrvnnnrnnnrrnnnrrnnnnenn 29 BEG 0 JE 22 DICRO RE EEE 138 Dichroic Filters rrrnnrennnnrernnnnnrnnnrvnnnnvnnnnre 22 Diffraction Pattern rrnnnnrnnnnnnevnnnnnnnnnrnnnnr 29 Digitalization rrrrnrrrnrrnnrrrnrrrnnevnrevarevnnenner 47 Displacement arnrnnnnnnnnrnnanrnnnnrnnnnnnnnnnnnnnnn 44 Displaying an Image r rnnrnnnnrnnnrvnnevnnrvnnenner 90 User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Index Displaying an Image From a Series 90 Displaying the Previous Image in a Series 89 Dividing Images rranrrnnnrnnnrnnnrnnnnnnnvnnnnnnnnnn IT Documentation Conventions rrrrnrnnrrvnnnnn 21 Double ICNIOIC ccseceeeeeeeeeeeeeeeeeeeeees 138 Double dichroic filters rrrnrrrrnnrrrrnnrrnnnnnen 22 Drawing a LING rrnnnrnnnnnnnrnnnrnnnnnnnnnnnnnnnnnnn 124 Drawing a Line on the Annotation Sheet 124 Drawing a Rectangle cccccceceseeeeeeeenees 124 Drawing a Rectangle on the Annotation Sheet EEE 124 Dry Objective ccccceeeceseeceeeeseeeesaees 24 138 Dynamic average rrnnrnnnrnnnrnnnrnnnrnnnrnnnnnnnnnnn 46 Edit Legend Dialo
61. In the case of scaled data the intermediate values are interpolated Click on the Show button to obtain a preview of the arithmetical operations Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the calculation operation as a new data set Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment Arithmetic With Constant Running Arithmetical Operations with Images Icon and a Constant Procedure Select an image from the Source Image drop down list The drop down list contains all the recorded and calculated images of the experiments displayed in the Experiment Overview viewer window gt Set the desired value of the constants in the Constant Value field Select the desired arithmetical image operation in the Operation field The following arithmetical functions can be run with one image and a constant Operation Result Addition Adds the constants pixel by pixel to the intensity values of the selected image according to the formula Pixel Intensity gesu Pixel Intensity scurceimage Constants Subtraction Subtracts the constants pixel by pixel from the intensity values of the selected image according to the formula Pixel Intensity gesu Pixel Intensity source image Constants Multiplication Multiplies the constants pixel by pixel by the selected image accordi
62. L pointer This value is required for a 3 state logic for example in the SQL database query language An SCODE error was indicated The error type is specified scodee A Boolean value true false was indicated VT_BOOL The value OxFFFF all bits 1 means true the value O all bits 0 means false A value corresponding to a date was indicated A date is saved as a number in double precision format All data is VT BSTR VT ERROR PE saved as differential dates in reference to January 1 1900 For example the entry 25 corresponds to 01 25 1900 User Manual Leica LCS english Page 156 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Index GE 0 e EEE 127 128 EE 127 128 129 EE sce cmse E E sarseeeooe 127 128 VZ Bliss 47 77 ZF OMAN e 53 gt 51 PPU vn 106 3D image data record rrrnnrrrnrnnrarevranennnnennn 41 3D View Button rrrnnnrnnnnrrvnnnrrnvnnrrnrnnnennnn 106 Pl 47 77 MEN 138 Absorption Coefficient rrranennnennne 92 104 PACITOMANG Lunvnapasmemsgrssdnsaesune 138 PACIWOMVEU C essenin sinen ann Ennni 138 Acousto Optical Tunable Filter 138 Adding a Text Field rrranrrrnrornnrernnrrnnnrrnnre 125 Adding a Text Field to an Annotation Sheet125 Adding Images arrnnnnennnnnnnnrnranennnnennnnennnnnn TT Adjusting phase rarennnrennnrnnrrnanevnnennnennnennnr 44 AN DEC ene 29 138 AES ser 30 138 AND 143 Angle of
63. Leica Confocal Software Ox File View Macro Tools Window Help EE gt periments an JE Hardware ES Document 3 Experiment lei User Name Institute Comment 1 Comment 2 Format 5124512 Image Dim 1 00 mmx 1 00 mmX Begin End ZN Position 0 02 um Scan Direction unidirectional Parameter Setting Sections Scan Mode Y Position Stage Gain PMT 1 Gain PMT 2 Gain PMT Trans Gain PMT 3 Gain PMT 4 Offset PMT 1 Document gs Selection ora Eicon Ready Build 0224 SIMULATOR SP 7 Leica Confocal Software LCS User Manual MICROSYSTEMS Table of Contents Table OF COMENS cies ects eden eee ident marie adden eee 2 LEA NE Jr D Starting UP NE SN PP T Starting the Windows NT Operating S SEN Harr 7 VSN ENOS T TAG VVIRGOWSINT SCI ACC PS 8 TESE Near 9 Staring a FOO Fal weve coset Sa coda G FTSE 9 TE TESE RENE 10 Sening the TIME NA Date ud 10 OTN RCO EE Se Vee 11 SMUTDOW I VVIMG OWS NI suse 11 Seting VP USES Under WINNT users 12 The Leica Confocal Software Overview ccccccccsscccseeccseeccsceceueeesueeeeueeceueeceueeseeeseessusessueesseesneeesaas 13 SEM TINS SNE Jar 13 The Experiment Software Concept rnnnrnnnnnnnnnnvanrnnnnrnnnnrnnnnnnnnnnnanrnnanrnnnnennnnennnnnnanrnnanennnnennnsnnnnne 14 The Basic Structure of the User Interface cic isosceles East 14 OPENING PAA RE OS siaa a aa cmon a a S 15
64. Look up Tables button to open a dialog window which you can use to assign color look up tables to the detection channels The color look up tables can be configured both in the result image and while the image is being recorded Click the detection channel to which you want to assign a new color look up table in the Select Channel field Select the desired color look up table in the Select LUT field Click Apply to check the results in the Viewer window It is also possible to open the Select LUT s dialog window from the Viewer window Hold the mouse pointer over any position in the Viewer window Then push the right mouse button Click on the LUT item in the context menu that appears The color bars for the active detection channels appear in the Viewer window to the right of the image window Double click one of the color bars Page 84 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Loading a second color look up table If you hold the mouse pointer over a color bar grab points appear at the top and bottom of the color bar You can use these grab points to limit the current color look up table to a specific intensity range and to load a second color look up table vvvyv Drag the top grab point down or the bottom grab point up Double click the area above or below the corresponding grab point The Select LUT s dialog window opens so that you can select a second co
65. New Experiment button to open a new Viewer window which creates a new experiment An experiment is a file that consists of one ore more individual images or image series This allows you to keep several images each recorded using different scan parameters or edited versions of images in one experiment The format of the experiment file lei is Leica specific User specific Adaptation Saving the Viewer Window as a Template Function Using the Template button you can save a user defined design of the Viewer window as a template The elements of the Viewer window that can be shown or hidden are the buttons the color bars of the color look up tables and the Experiment legend see Viewer Window Clicking the Template button opens a dialog window that you can use to enter a name for the template This configuration of the Viewer window is saved and will be loaded each time you open a file or create a new experiment Additional information In the Tools menu click on Options and select the Viewer Template tab to access the user defined templates In the Leica Templates list box you can choose from the following predefined templates Viewer Design Template Viewer Pure The view window consists only of the image window Viewer LUT The view window consists of the image window and the color bars of the color look up tables User Manual Leica LCS english Page 129 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsy
66. Page 134 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions The following four measurement values appear only when the Two Point Measurement function is selected in the Viewer Options dialog window Select the View menu the Experiment Overview option and the Charts icon LI see Viewer Options Dialog Window Charts Icon Inthe Measurement field you can switch two point measurement on or off by selecting 2 Point or Off Parameter Meaning Formula h position of the slide s Profile height at the position of the hi li left measurement point slide s left measurement point h position of the slide s Profile height at the position of the h l2 right measurement slide s right measurement point point Step Height dh The profile s height difference ha Fy between the slide s left and right measurement points dx Distance between the slide s left and IE l4 right corners alpha The angle of the left measurement point to right measurement arctan EN connection line from horizontal Saving the measurement values The software can save the values on the positions of both measurement points as well as the height difference between the measurement points gt Select the View menu Experiment Overview option Surface Measure icon see Viewer Options Dialog Window Surface Measure Icon gt To save the values click on the Remember button in the field Multipoint Mea
67. Projection Axis 64 92 96 98 100 101 103 Variance rarnnnnnnnnnnannnnnnnnannnnnnene 109 110 112 EE EE 48 Viewer Options Dialog Window 51 52 53 56 57 58 61 Viewer Options Dialog Window 3D Icon 51 Viewer Options Dialog Window Charts Icon 53 Viewer Options Dialog Window Display Icon 52 Viewer Options Dialog Window Overlay Icon57 Viewer Options Dialog Window Projections User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Index Viewer Options Dialog Window Scan Progress KO 58 Viewer Options Dialog Window Surface Calculation ICON rranrrnnernnrnnnrnnnrnnnnnnnnnnn 61 Viewer Options Dialog Window Surface View so EE 58 Viewer WIndOW ccecececeeceeececeeeeeeees 48 84 Viewing an Overlay Image 06068 57 87 Viewing Measurement Bat cc0eeceees 52 Viewing the Last Image of a Series 89 Viewing the Original Image c0008 106 Viewing the Series Image 0cceseeeeees 91 VIEWING Z Position rrrrrnnnrrrnnnrrnnrrranennnnennn 52 Ve 138 Wavelength ccccccsccseeccseeeeeeeeeesseeseeeeeeees 41 Wavelength Series rrrrrrrrnrennnennre 37 39 41 Windows NT Setting Up Users 12 Windows NT starting erorernnernnrrnnrrnrnnnnnnn 7 NTK EN 106 N RE EEE eteasnoteaeusaetateerenes 119 Working Distance rarrnnrnnnnnnanonanennner 24 138 Ru 143 ER CC
68. Rotation rarrnnnnrrnnrrrarernnnennnnennnnnn 44 Annotation aaeoa 122 123 124 125 Annotation Button rarrrnnnrrrrnnrrrnnnnrrnnnnen 122 Annotation Sheet 115 122 123 124 125 AIR eee een A eee ae eee ee 138 APT an dende 138 Aperture ene een ee ee ee 138 Apochromate ccccssecceeeeceeeceeeeceeeesaees 138 User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Index Apochromatic rrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnennnenner 138 Jo EEE EEEE 45 APP BUKOM 4 ctuwscasencpunteudewiaensaupsatabariaosand sues 45 Applying the Parameter Setting of an Experiment rranrrnnrrnnnnnnnnnnnnnnnnnnnnnnnnnnne 45 PAU ae EREE 136 ANT srren tera tel Ge nemceasie nossa Tf Arithmetic Average 060 109 134 136 Arithmetic With Constant rrrnnnrnnnnnnrnnnnr TT ASCII Data ies diccansbesezeseedncemcastapseteclaeseustasns 116 Automatically Defining the Region of Interest ed E 119 Average sive vevavwasdeedinnsacundasideeaventsbeaceucess 46 77 Average deviatiOn cccccceseeeeeeees 109 112 Average IMAGE energy ccceecceeeeeeeeeeeees 109 Average Projection56 64 92 95 96 100 101 Averaging Method rrnarrnnnrnvnnnnnnnnnnarennnnennn 46 Basic SettingS ccccccsseecseseeseseeceeeeeeeesees 51 Beam Path Setting Dialog Window 22 37 39 Begin Button rrvarrrnnnrornnrrrnnrovanrnnnnrnnnnennn 37 bidirectional ccccese
69. SFP projection View images as rotation animation Page 62 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Creating red green stereo views Applying lowpass and highpass filters to an image data record Filter Linear see Image Tool Dialog Window 3D Button Button Icon Tab Description of Function Basic Leveling Adapting an interpolation function to a data set to correct trend curves Merging Voxel Combining several images or data x y z ch sets to a new image or data set t Ar Stereo Projections Creating projection images along the Stereo View orthogonal axis Creating red green stereo views Image Cutting single images or image Separation sections from a data record see Image Tool Dialog Window Basic Button Button Icon Tab Description of Function Amplitude Arithmetic With Running Arithmetical Operations Two Images with Images and or Masks Arithmetic With Running Arithmetical Operations Constant with Images and a Constant Bit Converter Conversion of 8 bit images to 12 bit images and vice versa Brightness and Setting image brightness and Contrast contrast Gamma Setting the Contrast Transfer Correction Function Gamma Curve see Image Tool Dialog Window Amplitude Button Button Icon Tab Description of Function Materials Filter Applying low pass and high pass filters to Linear images see Image Tool Dialog Window Materials Button Button Icon Tab De
70. Save As dialog window opens It is easy to inadvertently overwrite your original data using the Save function To avoid doing so use the Save As function to save an experiment that has already been saved once or more under a new name see Saving a File As User Manual Leica LCS english Page 127 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH User Manual Leica LCS english LCS Software Functions Saving a File as Function Use the Save As button to save an experiment lei or annotation sheet ano while giving it a specific name and file type When saving an experiment a folder is created at the file level with the name of the experiment This folder then contains the description file lei for the experiment as well as the individual image files The format of the description file is Leica specific and binary This file contains the parameter settings and the color information in the form of color look up tables for each image belonging to the experiment The image files of an experiment can be saved in tif or raw format The standard format tif also contains the experiment settings and the color information for the images In the raw format just the image data is saved Typical Applications The advantage of the raw format is the smaller file size but this is only significant for image recordings with relatively small amounts of data For example if you record an
71. Scan Format Define the wavelength at which you want the wavelength series to begin see Defining the Begin Point for a Wavelength Series Define the wavelength at which you want the wavelength series to end vv v y User Manual Leica LCS english Page 42 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions see Defining the End Point for a Wavelength Series Select the desired number of recordings between the begin and end points of the wavelength series see Setting the Number of Wavelength Steps When you have finished configuring these settings press the Series Scan button Selecting a unidirectional or bi directional scan Function If you click the Unidirectional Bi directional Scan button bi directional scan mode is enabled If this button is not clicked unidirectional scan mode is automatically set In unidirectional scan mode each line is scanned from left to right No data is recorded while the laser beam is being guided to the starting point of the next line In bi directional scan mode the first line is scanned from left to right and the second from right to left In other words the return run or flyback of the laser beam is implemented for recording data too thus increasing the scan speed when using bi directional scan mode If bi directional scan mode is active you can double the scan speed using the Speed button Unidirectional Bi directiona
72. Sharp Factor field to vary the effect of the Sharp filter If this factor is set to 0 the filter is not active The value specified in the Kernel Size field as Cut off Wavelength is the size of the filter measured in pixels This value is calculated on the basis of the scan parameters enlargement of the objective wavelength of the excitation light scan format electronic zoom factor used for each image The value specified in the Filter Directions field as Cut off Wavelength is the size of the filter measured in nanometers For example with a value of 160 nm all wavelengths lesser than or equal to 160 nm are filtered Click on the Show button to obtain a preview of the filtered images Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the filtered images as a new data record Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment User Manual Leica LCS english Page 133 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Function The Roughness Profile function measures height distance and angular values of a surface along a specific line and displays these values in a curve The height profile is measured from the data record selected in the Viewer window This data record should be a spatial series
73. TRL J Opens the Objective dialog window for defining and selecting applied microscope objective lenses CTRL N Opens a new experiment CTRL O Starts the Open dialog window for opening an existing file CTRL P Opens the Printer Selection dialog window CTRL S Saves the active experiment LCS Software Functions Introduction to the Leica Confocal Software Help Three different help levels are available for the Leica TCS SP2 Quick Help When you let the mouse pointer hover over a Leica Confocal Software button a brief explanation of the function of this button is displayed This so called Help Banner automatically disappears when the mouse pointer is moved Context sensitive Help If you want to use the context sensitive help function click on the Help button The entire user interface is then frozen and a question mark appears beside the mouse pointer Then instead of triggering the button function clicking a button opens an explanation of the button s function If the Help button is not present on the user interface Select the Customize option from the Tools menu Here you will find all buttons arranged by categories vy The Help button can be found in the File category Click on it using the left mouse button and drag it to the desired window Contents of the Online Help Select the Contents option from the Help menu to view the online help table of contents which allows you to select any function in order to
74. The crystal generates an internal ultrasonic wave field the wavelength of which can be configured to any value Radiated light is diffracted vertically to the ultrasonic wave field like through a grid Aperture is the sine of the opening angle under which light enters into the front lens of a microscope objective its symbol is NA The aperture influences both the light intensity and the resolution capacity of an objective optical system Since various media can be present between specimen and objective lens such as the embedding medium for the specimen the numerical aperture NA n sin a is usually applied as the unit of measurement for the light intensity and the resolution capacity Describes a correction class for objectives The chromatic aberration for three wavelengths is corrected for objectives of this type usually 450nm 550nm and 650nm and the sine condition for at least two colors is met The image curvature aberration is not corrected The distance from the front lens of an objective to the focal point For a variable working distance the gap between the front lens of the objective and the cover slip or uncovered sample is specified Usually objectives with large working distances have low numerical apertures while high aperture objectives have small working distances If a high aperture objective with a large working distance is desired the diameter of the objective lens has to be made correspondingly large These however ar
75. U Ol EE 40 gt N S Le IONS Renee eee ae eee EEE 31 p FAR OL 0 OE EE 40 EST 0 g enire ine anean 31 POL 33 Z Configuration Dialog Window 0 40 ZF 33 zly Position ssndirsnsiesaddsendadievexedidavendaadrenesnias 37 38 z y Position Button rrronrrnnnrrnarennnrevnnnnnnnnnn 33 E OON EE 26 108 LOOM BU Ouse 108 Page 163 of 164 Leica Microsystems Heidelberg GmbH Index Zoom In Button rrarrrnnrrnrrvnnrrnrnnnrnnrnnnnnnn 28 Zooming the 3D View rrrrarennnrrnnnrnvnnrnnnre 108 User Manual Leica LCS english Page 164 of 164 Art No 15 9330 033 Vers 01082001
76. WORD ID FILTERSET 30 const DWORD ID TIMEINFO 40 const DWORD ID SCANNERSET 50 const DWORD ID EXPERIMENT 60 const DWORD ID LUTDESC 70 A memory space of 32 bits is defined for the DWORD data type 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 and is structured as follows Data symbol Description type int Internal version number int NSE Number of image series int nim Length of a file name in wchar int nExt Length of the file extension of the image file in bytes wchar File extension of image file 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 4 int nFiles Number of single images of the series 4 int Width of a single image 4 int Length of a single image 4 int Bits data points image resolution 4 int Data points pixels display resolution For the next n User Manual Leica LCS english Page 147 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH image files nlm wchar 2 ID_DIMDESCR memory block This block provides a description of an image in an n dimensional space For the next n dimensions User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Size byte A 4 nTC NTC Data type int Symbo
77. a Binary of two images representation of pixel 1 b Binary representation of pixel 2 c Binary representation of result pixel Page 143 of 164 Leica Microsystems Heidelberg GmbH LCS File Formats Appendix Formats of User specific and Device dependent The LCS software provides the following file formats for user specific data Instrument data Instrument Parameter Settings Macros Templates for operator console User profiles Directory Leica IPS Company mac pbo CompanyProfile pro Directory User IPS User mac pbo PersonalProfile pro LastExitProfile pro LastRecent Ist Templates prt prt Calibration data Machine Ihw Color Look Up Tables lut User specific profile as it was Ist defined the last time in use In detail the different file formats contain 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 You can use factory defined standard sets of instrument parameters that cannot be changed but that allow for a very quick configuration of the hardware In addition every user can save instrument parameter sets in a separate user directory This allows you to save the instrument parameters typical for your planned use
78. access the Control Panel and Printing folders Also accessible is the Task Properties window Being able to access the core system configuration utilities in this way is particularly useful when an application is already in the foreground and you want to make a quick change The Find command features an easy way to locate all system resources Within the Find category you can perform searches for three distinct types of search as described below The Taskbar The taskbar positioned at the bottom of the screen provides a constant view of which applications are running on the system and an easy way of switching between them As you add to the number of concurrently running applications the taskbar automatically re sizes its iconized view of the applications to ensure that they can always be seen To switch from one running program to another simply click on the second program as displayed in the taskbar The taskbar also provides constant additional information such as the system time and volume control if a sound card is installed All of these functions can be adjusted by the user Setting the Time and Date The current Date Time and Time Zone information can be set from the Date Time icon within the Control Panel This setting is important since Windows NT stamps the date and time on all of your files as you create and modify them The two options can be selected by clicking on the appropriate tab To change the Date and Time Click
79. ail MS Mail and Microsoft Exchange If there is already an MS Mail post office on the same network that the system is connected to the Windows NT Workstation 4 mail client can connect directly to it The Inbox lets you access your messages The Recycle Bin icon represents the holding place for deleted items As long as files are in the Recycle Bin they can easily be recovered if they have been accidentally deleted Windows NT Workstation 4 will preserve files until the system runs out of free disk space When this happens Windows NT Workstation 4 will prune the contents of the Recycle Bin on a first in first out basis Caution Files that are overwritten due to an application using a duplicate filename will not be saved to the Recycle Bin Double clicking the Bin displays its contents The empty window confirms that there are no items in the Recycle Bin The Start Menu A single click of the left mouse button on the Start button opens the start menu and presents the seven major option categories for starting work on the system A single click of the right mouse button opens a small but powerful control menu containing the options Open Explore and Find Their functionality is described below Starting a Program The Start menu contains the various categories where your applications and work are stored You can move further into the various subcategories by positioning the mouse over the category that you are interested in to automati
80. al Projection icon If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be stored as a new file in the current experiment LL see Image Tool Dialog Window 3D Button LL see Creating an Experiment User Manual Leica LCS english Page 102 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note User Manual Leica LCS english LCS Software Functions Transparent Projection of an Image Stack with Variable Projection Axis optional Function You can activate a transparent projection using the 3D Trans The projection axis is freely variable The basis for a projection is an image stack i e a series of horizontal xy sections or vertical xz sections When a projection is generated the sampling points of the individual images superimposed along the projection axis are examined throughout all optical sections From each of these sampling point columns a weighted average is calculated from all intensity values which is then displayed in the two dimensional projection image as a representative of the entire column see Principles and Types of Projections The user can vary the weighting of the sampling points to calculate the averages by setting the corresponding factors transparent factor in the Viewer Options dialog window Furthermore you can indicate a
81. anennnnennn 90 FAI 117 Page 160 of 164 Leica Microsystems Heidelberg GmbH Position of Maximum Value 0cccee 110 Position of Minimum Value c008 110 Previous BUON uasemassassalemmen deia 89 Previous Image of a Series cceeeee eee 89 Printer Selection Dialog Window 126 PANIN EEE AR 115 126 Printing Quantification Data 115 Processing Large Amounts of Data 46 Profile z Button rrrrnnrrnnrnnrernnrrnnnnrennnn 110 Profile Button rrrnnnrrnnnnrrvnnnnvrnnnrrnnnnrennnn 112 Pole PE KA 134 136 Profile Valley rrrarrrnanenanenanennnennnnrr 134 136 Progress Display ccccsccceseesseeeeeeeeeeeees 58 Projection ararnnnnnnnnnnnnnn 71 92 95 96 104 Projection Button rrnnrrarrnnrnannnrnannner 95 96 Projection of an Image Stack 92 95 96 104 Quantification 53 112 115 116 134 136 RK Du 128 Recording a Line Using the Averaging Method 45 Recording an Image Using the Averaging sd goro ERE 46 REGLENE Lise dessere 118 124 Rectangle Button rrrararnnrrrnnrrrnnrrvnnennnre 124 REAUCING snap adaaend 51 84 108 Refraction Index rrrrnnrrrrnnrvrrnrrvvnnrrvvnnrennnn 138 Region of Interest as a Polygon 117 Region of Interest as a Rectangle 118 Region of Interest as an Ellipse 116 Resolution ra rernar
82. angle of the SFP projection This angle symbolizes the angle of incidence of the laser beam onto the specimen Click on the Apply button to create the projection with the new angle You use the slider to vary the absorption coefficient a The higher this is set the smaller the values calculated in the projection image If the coefficient is set as low many of the pixels in the projection image reach the maximum value making it increasingly difficult to distinguish between the structures Use the slider to define a threshold value The intensity values below this value are not taken into account for the creation of the projection image The intensity values of the image are scaled to the maximum possible intensity values This ensures that the calculated gray values of the projection image remain within the value range of for example 0 to 255 at 8 bits This allows for brightening dark images Viewer Options Dialog Window Overlay Icon Structure of the Experiment Overview window To open the Viewer Options window select the Experiment Overview option in the View menu The Experiment Overview window appears on the left side of the user interface The top part of the viewer window displays the recorded images in a directory tree The bottom part displays the Viewer Options window This dialog window is used to perform basic settings for different software functions The left side shows the icons corresponding to the functions and the right
83. annel or several detection channels in only one image in the Viewer window The following display modes are available depending on the other options you enable simultaneously Button Combination Display Single Channel 1 2 Only the selected detection channel is displayed 3 4 5 for an image series the first image of the selected detection channel is displayed Single Gallery All individual images in an image series are Channel 1 2 3 4 5 displayed for the selected detection channel only Single Overlay A single overlay image is generated from all Channel 1 2 3 4 5 selected detection channels for an image series the first image of each selected detection channel is used Single Gallery Based on the number of individual images in an Overlay Channel 1 2 image series overlay images are generated from 3 4 5 all selected detection channels Note The Single and Tiled buttons cannot be enabled simultaneously because they carry out opposing functions User Manual Leica LCS english Page 86 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Viewing a Multiple image Function If you click the Tiled button the detection channels are displayed separately in the Viewer window The following display modes are available depending on the other options you enable simultaneously Button Combination Display Tiled Channel 1 2 3 All selected detection channels a
84. areas defined by the mask are cut out of the image The binary representations of the pixels of the two selected images are linked with the logical operator OR That means that pixels in the result image are given the value 1 when the value 1 is present in at least one of the two pixels otherwise 0 is entered see Boolean Operations You achieve meaningful results using this Boolean operator 1 When you link two binary masks The result image is a combination of the two masks 2 When you link an image with a binary mask The result image is an image in which the mask appears 3 When you link two images In contrast to the overlay image pure intensity values are overlaid here An RGB color addition does NOT take place here see Viewing an Overlay Image The binary representations of the pixels of the two selected images are linked with the logical operator XOR That means that pixels in the result image are given the value 1 when the two pixels have different values otherwise 0 is entered see Boolean Operations You achieve meaningful results using this Boolean operator when you create two binary masks of images and link them The pixel is displayed in the result image at the point where the two images differ Inthe Arithmetic Type field select whether raw data or scale data is to be used for the arithmetical operations If you have selected Raw Data the values standardized to the interval 0 255 for 8 bits or 0 4095 for
85. ata 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 proofing Leica Microsystems Heidelberg GmbH assumes no responsibility for any errors or omissions Leica Microsystems Heidelberg GmbH shall not be responsible for any direct incidental or consequential damages arising from the use of any material contained in this document TRADEMARKS Throughout this manual trademarked names may be used Rather than including a trademark symbol at every occurrence of a trademarked name we state that we are using the names only in an editorial fashion and to the benefit of the trademark owner with no intention of infringement SAFETY This instrument is designed
86. ate Due to the significantly longer life of triplet states phosphorescence these excited molecules can react with triplet oxide and be lost for further fluorescence excitation The factor by which the light velocity in an optical medium is less than in a vacuum Dichroic filters are interference filters at an angle of incidence of light of 45 The transmissivity or reflectivity of dichroites depends on a specific wavelength of light For an RSP 510 filter reflection short pass for example the excitation light below 510 nm is reflected and the excitation light above this value is transmitted The transmission values are generally between 80 and 90 and the reflection values between 90 and 95 Double dichroic filters are interference filters at an angle of incidence of light of 45 The transmissivity or reflectivity of double dichroites depends on two specific wavelengths of light For a doubledichroite DD 488 568 for example the excitation light at 488 nm and 568 nm is reflected and the excitation light above these values is transmitted The transmission values are generally at 80 and the reflection values are between 90 and 95 A digital filter consists of a computing rule used to modify image data Filters are always applied to remove disturbing image components A phase true filter ensures that quantifiable image values do not change through filtering and remain a requirement for standardized measuring methods e g characteriz
87. ation of surfaces in accordance with ISO A light optical contrast process for displaying fluorescent structures Auto fluorescent samples have a so called primary fluorescence They do not need to be enriched with additional fluorescent substances Secondary fluorescent substances on the other hand have to be treated with appropriate dyes or Stains called fluorochromes Specific dyeing methods additionally allow the precise localization of the stained structure elements of an object Fluorescence microscopy allows both potential morphological examinations and the ability to carry out dynamic examinations on a molecular level Describes a correction class for objectives Fluorite objectives are semi apochromatic meaning their degree of correction lies between the achromatic and apochromatic A frame corresponds to the acquisition of a single optical section For example if a single optical section is acquired 4 times to average the data and to eliminate noise then 4 frames are created for this optical section A microscopic objective developed with the requirements for applying immersion media The use of incorrect or no immersion medium with an immersion objective can lead to resolution loss and impairment of the correction While the optical design of conventional microscopes allows the uniform detection of focused and unfocussed image components the confocal principle suppresses the structures found outside of the focal plane of the m
88. aximum intensity value or the arithmetical average or the weighted average of all intensity values is calculated and displayed in a two dimensional projection image representing the entire column LI see Principles and Types of Projections User Manual Leica LCS english Page 95 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Click the Fix O Proj Button A maximum average or transparent projection of the current image stack is created and displayed in the Viewer window You can return to the original image anytime by clicking on the Original button The Viewer Options dialog window allows you to indicate a threshold and to scale the intensity value range for the image see Viewer Options Dialog Window Projections Icon Note You can activate a projection using either the Fix O Proj Button or the Image Tool dialog window If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be Stored as a new file in the current experiment LL see Image Tool Dialog Window 3D Button LL see Creating an Experiment Projecting an Image Stack with a Variable Projection Axis optional Function The 3D Proj Button starts the project type last used or selected in the Image Tools dialog window Projections and Animations icon Projec
89. ayed in the menu bar located at the upper edge of the user interface Option Options refer to the selectable items that are hierarchically listed below the menus Options either trigger actions directly or open dialog windows Dialog Window Both buttons and options open dialog windows Dialog windows are used to set various parameters and select functions 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 Legend The Leica Confocal Software provides two legends which display the parameters and settings of an image recording The Experiment legend can be placed at the right edge of the Viewer window The Hardware legend is called up from the View menu and appears as a separate window at the left side of the user interface Context Menu Context menus appear when you click the right mouse button while holding the mouse pointer over certain areas of the user interface Context menus contain various context sensitive comman
90. ber of Elements 512 Reset Shift 0 000 Size 51329 8 um um Image 2 Number of Elements 512 Reset Separation Shift f200 00 Size 200 00 3 um f Append Bm Shift x Result Element Number g 2 IV Best Fit Amplitude Materials Multi Color Leica Intern Apply lt lt Example of merging images with different numbers of channels a Fekk PE Se ee a First output image setting Start 2 b Second output image setting Start 1 c Merged image User Manual Leica LCS english Page 142 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Appendix How Boolean Operations Work AND Write a 1 if there is a 1 in pixel 1 AND in pixel 2 otherwise write 0 OR Write a 1 if there is a 1 in pixel 1 OR in pixel 2 otherwise write 0 XOR Write a 1 if the values in pixel 1 and in pixel 2 differ otherwise write 0 User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 AND represents the intersection of two images a Binary representation of pixel 1 b Binary representation of pixel 2 c Binary representation of result pixel OR represents the union of sets of two images a Binary representation of pixel 1 b Binary representation of pixel 2 c Binary representation of result pixel C oftfofoft 1jo o XOR represents the union of sets without the intersection
91. between two sections User Manual Leica LCS english In this dialog window you can enter any value for the number of sections and the section width The height of the image stack cannot be changed as this parameter is determined by the start and end point of the image series As the section width always has to be a multiple of the minimum section width of the z setting drive certain combinations of values are only possible if either the height of the image stack or the number of sections is adapted Depending on which of the two Calculate buttons you click one of the two parameters is not changed If you want to calculate the number of sections with the priority of leaving the height of the image stack unchanged where possible Enter the desired step size in the Step Size field Then click the Calculate button next to the Step Size field If you want to calculate the number of sections with the priority of leaving the number of sections unchanged where possible Enter the desired step size in the Step Size field Then click the Calculate button next to the Sections field If you want to calculate the step width with the priority of leaving the height of the image stack unchanged where possible Enter the number of desired sections whole numbers only in the sections field Then click the Calculate button next to the Sections field lf you want to calculate the step width with the priority of leaving the number of sections unc
92. calculated by the quantification functions as ASCII standard data This enables you to read and edit data in Excel The calculated values for the following quantification functions which are also displaying an evaluation graph can be exported see Measuring a Profile Along a Line Segment see Measuring a Profile Within a Region of Interest ROI see Measuring Surfaces and Volumes LL see Measuring Roughness Along a Line Segment This function is also available in the context menu within the quantification function window gt Move the mouse pointer into the view window opened by a quantification function Click the right mouse button and select the Export menu item Defining the Region of Interest ROI as an Ellipse Function Using the Ellipse button you can define an elliptic Region of Interest ROI within the image User Manual Leica LCS english Page 116 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Click the Ellipse button Click the left mouse button on the image position where you would like to place a ROI corner Then keeping the left mouse button pressed drag the mouse pointer diametrically to the opposite corner to define the second corner of the ROI Vv LL see Selecting and Moving the Region of Interest ROI LL see Moving and Rotating a Region of Interest ROI LL see Deleting Regions of Interest ROIs Other Requi
93. cally open the next subcategory You do not have to click the mouse The Programs command displays the Programs menu This menu lists all of the applications installed and available to you Some options are marked by an arrow This arrow indicates that a submenu follows Drag the mouse cursor over the Accessories command to see its submenu The Accessories submenu lists the set of Windows NT built in programs TIP If you drag an object either from the Desktop or Windows Explorer and drop it directly onto the Start button a link to that object will automatically appear in the Start menu There are many ways to start a program The following method is the simplest one 1 Click on the Start button 2 Click on Programs 3 Click on the group that contains the program you want to start for instance LCS 4 Click on the program you want to start for instance Leica LCS Another way to start a program is to open a document that you created in that program The program automatically opens when the document opens Double click on a document file in My Computer or Windows Explorer to open it Or click on the Start button and select a recently used document from the Documents menu You can also start a program by double clicking on its shortcut icon on the desktop Shortcut icons are links to other files When you use a shortcut Windows simply follows the link back to the original file Whenever you use a document or program frequently you migh
94. can parameters listed above and check the results directly in the image User Manual Leica LCS english Page 35 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note Note User Manual Leica LCS english LCS Software Functions With bleach sensitive specimens it is advisable to restrict the use of the Continuous Scan function to short time intervals Subjecting the specimen to the continuous light of the laser can destroy the specimen photochemically optical bleaching thus making it unusable Series Scan Overview Dialog Window Function This dialog window lets you define the begin and end points of an image series and trace the recording of the individual sections The three dimensional scan area is represented graphically as a cube Within this cube a yellow square represents the current z position or y position a green one represents the begin point and a red one the end point The corresponding position values are displayed to the right of the cube Set the begin and end point as follows Use the mouse pointer to drag the yellow square to the level in which the image series is to begin Or click the z y Position button to open a dialog window where you can enter the values of the z position or y position Click in the white box for the begin point The corresponding position value appears and is saved Use the mouse pointer to drag the yellow square to the level in which the image ser
95. ccsseeseetecserecsersaneesaess 43 bi directional rrrrnnnnnnnnnrnnnnrennnnnnnnnnrnnnnnen 44 Bidirectional scan rernnrannarvannnrannnrannevnnneene 43 Bilder addieren cccccssseecseeeeceseeeneeeens Tf Bilder CIVIGICreN cccceseeeseseeceeeteneeeeens Tf Bilder multiplizieren rrrrnnrrnnrnnnrrnnrvnnnnnnnnnn IT Bilder subtrahieren rrnnnrnnnnrernnnnrnnnnnnnnnnen 77 BIEN 35 138 Boolean Operations rrrrarrnnnnernnnevnnnnnnnnnr 143 Burst mode rersernonurrounnvnnrornnernnvennenvanserenne 46 Calculating eoii a 109 Page 157 of 164 Leica Microsystems Heidelberg GmbH Center Of mass rrernnnrrnnnnrvnnnnrnvnnnvnvnnnennnn 105 Center of mass of intensities 105 CANIM GS sorserien E ante 83 OE 122 Color MIXING sosser sinai 87 Configuring a Time Series rrrrararrarernnrennn 33 Confocalty EE 138 CONTINUOUS SCAN rrnnnnnnnnnnrnvnnnrnvnnnrnnnnnennnne 35 Contrast Transfer Function rrnrrnnnnnrrnnnr 77 Control Panel ccccsececsseeeseseeeseeeeesees 130 CONVETSION eisnbensiscsrdeacdeateataleehennctelesweudsataneiene Tf Coordinate AXES ccccccccsseeessseeeseseeeneaees 51 COD VAG van 123 Copying an Image into the Annotation Sheet Fe os EE A EE EEE AE E E es 123 Copying Quantification Graphs 115 Correcting Trend Curves 006 71 141 Creating a Topographical Image 61 105
96. ces the way in which the microscope is controlled for example the motorized objective nosepiece Settings Use this option to select the hardware settings that you want to be applied when opening a previously saved data record License This option allows you to install subsequently purchased licenses A subsequent license usually is installed if a software package offered as an option is purchased The Window Menu 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 topographical and overlay views The Help Menu Contents Opens the table of contents of the online help Search Starts the search function for the online help Index Opens the index of the online help Page 18 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Keyboard Shortcuts Special key combinations have been defined to speed up access to frequently used software functions Keyboard Function Shortcuts F1 Opens the online help ALT F8 Opens the Macros dialog window for running editing and deleting macros ALT F11 Starts the VBA development environment optional optional CTRL L Opens the Legend Info dialog window for entering user specific settings that can be applied when documenting saving and viewing the documentation of image recordings C
97. ck the Select Look up Tables button Hold the mouse pointer over any position in the Viewer window Click the right mouse button Click on the LUT item in the context menu that appears The color bars for the active detection channels appear in the Viewer window Double click the corresponding color bar see Selecting Color Look Up Tables LUT This method of assigning color look up tables only influences the currently displayed image As soon as you start a new scan the color look up tables configured in the Beam Path Setting dialog window are applied again see Setting the Beam Path Page 83 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH n 1 LCS Software Functions Zooming Image s in the Viewer Window Function Clicking the Display button opens a drop down list that you can use to set the graphical zoom This enlarges or reduces the image displayed in the Viewer window Displaying n pixel as 1 pixel in the resulting image reduces the image Automatic The image is displayed in the original image format 1 n Note Vv User Manual Leica LCS english Displaying 1 pixel as n pixel in the resulting image enlarges the image The Leica Confocal Software provides three different zoom functions the graphic zoom the 3D zoom and the electronic zoom LL see Zooming the 3D View LL see Electronic Zoom Selecting Color Maps LUT Function Use the
98. cros only if the complete integrated VBA development environment IDE is installed Record a New Macro This starts the automatic macro recorder For more information refer to the documentation provided with the optional macro development environment Pause Recording This pauses the macro recorder Stop Recording This stops the macro recorder Visual Basic Editor optional This option launches the software for developing macros and programs Page 17 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note User Manual Leica LCS english LCS Software Functions based on Visual Basic for Applications VBA Here you can edit and modify macros and even develop entire programs The Tools Menu 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 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 It is important to apply the correct data for the objectives as some of the calculated sizes such as the scan field dimensions depend on them Microscope Use this option to select the Leica microscope you are using This setting influen
99. ction axis is the viewing direction of the user to the newly positioned image stack Use the Zoom 3D View button to enlarge or reduce the projection LL see Zooming the 3D View You can return to the original image anytime by clicking on the Original button In the Viewer Options dialog window you can also adjust a threshold and scale the intensity value range present in the image LL see Viewer Options Dialog Window Projections Icon You can activate an average projection with the 3D Avg button or in the Image Tool dialog window 3D button Projections and Animations icon If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be stored as a new file in the current experiment see Image Tool Dialog Window 3D Button see Creating an Experiment Page 100 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Average Value Projection of an Image Stack with Invariable Projection Axis Function Using the button Fix Avg button starts an average projection For this function the projection axis is always the orthogonal axis Z axis for horizontal xy sections and y axis for vertical xz sections The basis for a projection is an image stack i e a series of horizontal xy sections or vertical xz sections When a projection is generated the sa
100. d Analysis FUNCIONS uses ende 109 Calculating a NN 109 Measuring a Profile Within a Region of Interest ROl rrrnrnnrrnnnnnennnrrvvnnrrnnnnnrennnrrennnnrennnrennnne 110 Measuring a Profile Along a Line Segment rarrnnanrnnnnannnnnnnnnnvanennannnnnnnnnanennnnennnnennnnnnnnennanennnnene 112 Measuring Surfaces and Volumes cccccccsscccssecceeeceuceceuceceueecseeceucecaucesueesueeeeueessueesseesseeesaas 113 Copying Quantification Graphs to the Annotation Sheet rrrarerarerarerarerarenanennnnnnanennnnnnnennnennner 115 Printing QuantitiGation GAMST 115 Exporting Quantification Data agere 116 Defining the Region of Interest ROI as an Ellipse 0 0 cc ceeccceececeeeeeee esse eeseeeeseeesseeeseeeees 116 Defining the Region of Interest ROI as a POlygon rrrannrnanrvnnnevnnnnrnnnnranennnnnrnnnnnranennnnennnnennnnne 117 Defining the Region of Interest ROI as a Rectangle rrrarrnnnnnrranrnnanennnnrnnnnrrnnrrnanennnnennnnnnnnnnn 118 Automatically Defining the Region of Interest ROI rrrararranannnnennnnernnnernnnennnnrrnnnrranennnnennnnennnnnn 119 selecting and Moving the Region of Interest ROI rrrrrnnnrrnanernnrernnnrnnnrrrannnnnnnennnnnnnnennanennnnennn 120 Moving and Rotating the Region of Interest ROl rrrrarrnnnnrrnanrrranennanernnrennnnnnnnennnnennnnennnnennnenn 121 Deleting Regions of Interest ROIS rrrrrrrnnnrnnnnnnanrnnanernnnrnnnnnnnnrnnanrnnnnnnnnnnnnnnnnnnsnnanennn
101. d can be described for the Leica Confocal System as follows ae To calculate the diameter of the Airy disc you need the excitation wavelength in the case of several wavelengths a mean value should be used the numerical aperture NA and the magnifying factor M of the objective The factor of 3 6 refers to the magnification of other optical components belonging to the Leica Confocal microscope If the pinhole is set to the Airy disc light from outside the focal plane is suppressed and the signal to noise ratio is high These conditions allow the recording of optical sections of minimum thickness The wider the diameter of the pinhole the more light reaches the detector The image becomes brighter but blurring from structures outside the focal plane will also appear in the image making it increasingly unfocused Increasing the diameter of the pinhole above 1 Airy unit is recommended only for detecting very weak signals Generally when recording material scientific specimens enough light is reflected into the detectors so that the detection pinhole can be closed completely i e set to the minimum diameter User Manual Leica LCS english Page 29 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH aa User Manual Leica LCS english LCS Software Functions Selecting a Scan Format Function Clicking the Format button opens a dialog window containing a selection list of scan
102. d function is shown in the field If you have assigned a function to all the radio buttons you can save this configuration as a template Click on the left of the three buttons located on the right side of the status bar A dialog window is opened in which you can give the configuration a name and save it Click on the middle button to select and load a configuration In addition to these user defined configurations you can also access other configurations predefined at the factory Click on the right of the three buttons located on the right edge of the status bar This opens a dialog window listing all the configurations for the control panel The configurations set as default indicated by an L Leica can only be loaded but not modified The user defined configurations are created in the drop down list under User and marked with a U User Click one of the configurations and then press the right mouse button to open a context menu which contains the following commands Page 130 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Command Function Set as default The configuration is loaded as the default configuration template when the software is started Remove default The setting of this configuration as default setting is setting undone Load Loads the configuration Rename only for Use this to change the name of the configuration U Delete only for
103. d into categories and are represented by small book icons Double click on any book to open it Sub books and documents appear Double click on sub books and documents to open them Index The Index tab displays an index of all available topics Type the word you want to look up The Index list scrolls to that part of the alphabetical listing When you see the topic on the list that you want to read double click on it Find Rather than searching for information by category the Find tab offers a full text search Enter the word s or phrase to be searched for under Help in the text entry box The text entry box is linked to a list of words in your Help files and any words or phrases that match will be displayed You can specify more than one word by separating words with a space If you wish to change a search option select Options The first time you click on this tab Windows tells you it needs to create a list Click Next and then Finish to allow this The main Find tab appears next Type the word s you want to find in the top text box Then click a word in the middle box to narrow the search Finally review the list of help topics at the bottom and double click the one you want to read When you re done reading about a document click Help Topics to return to the main Help screen or click Back to return to the previous Help topic Click the window s Close button to exit Help Shut Down Windows NT Always use the Shut Down command bef
104. d scan format specified by the objective that can be used to scan the specimen without loss of data Scan Zoom Scan Scan Resolution Objective Format Factor Field Frequency Scan sS Size nm Frequency mm Magnificatio Numerical Resolution n Aperture at 1 488 nm 20 0 6 325 1024 x 1 750 732 0 4 1024 2 375 366 0 8 4 187 183 1 8 6 125 122 2 7 512x512 1 750 1465 0 2 2 375 732 0 4 4 187 365 0 9 8 94 183 1 8 10 75 146 2 2 200x200 1 750 3750 0 1 2 375 1875 0 2 4 187 935 0 3 8 94 470 0 7 16 47 235 1 4 32 23 115 2 8 40 1 25 156 1024 x 1 375 366 0 4 1024 2 187 183 0 8 4 94 92 1 7 6 62 60 2 6 512x512 1 375 732 0 2 2 187 365 0 4 4 94 183 0 8 8 47 92 1 7 10 37 72 2 2 200x200 1 375 1875 0 1 2 187 935 0 2 4 94 470 0 3 8 47 235 0 7 16 23 115 1 3 32 12 60 2 6 100 1 4 139 1024 x 1 150 146 0 9 1024 2 75 73 1 9 3 50 49 2 8 512x512 1 150 293 0 5 2 75 146 0 9 4 37 72 1 9 6 25 49 2 8 200x200 1 150 750 0 2 2 75 375 0 4 4 37 185 0 7 8 18 90 1 5 12 12 60 2 3 User Manual Leica LCS english Page 27 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Note The Leica Confocal Software provides three different zoom functions the electronic zoom the 3D zoom and the graphic zoom LL see Zooming the 3D View LL see Viewer Options Dialog Window Display Icon Enlarged Recording of a Frame Function The Zoom In function enables an enlarged recording of a freely selectabl
105. data records but also the experiment data and system settings You can open a new experiment image window by selecting File gt New The TCS menu TCS_Menu The TCS menu contains buttons for the individual device functions It is subdivided into individual work steps The number of work steps available depends on the installed software The standard set of work steps consists of data recording Acquire image viewing View surface reconstruction 3 D measurement functions Quantify image processing and analysis functions Process and documentation functions Annotate If the TCS menu is not displayed using the current configuration profile you can activate it by selecting View gt Menu gt TCS Menu The toolbars 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 To do so select View gt Menu Container The document viewer window Experiment Overview The document viewer window displays the recorded experiments and their contents in a directory tree Open this viewer window by selecting View gt Experiment Overview The status bar The status bar is found at the bottom edge of the Leica Confocal Software user interface It displays e the progress while loading image data progress bar e the version number of the software e the name of the system configuration system type For details about th
106. depth if they are viewed with corresponding 3D glasses red green stereo glasses The following settings must be made to create the two stereo images In the Stereo View Options field click on the On check box to activate the function In the Eye Angle field determine the angle under which the two stereo images are recorded For example if you enter the value 30 a stereo image is recorded at an angle of 15 each left and right from the projection axis that was set on the Rotation tab Stereo images can only be created from one detection channel If you have an image series that was recorded in several detection channels select the desired image data set in the Detection Channel field Click on the Show button to obtain a preview of the stereo images Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the stereo images as a new data set Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment To display both stereo images in one image in the Viewer window the buttons Channel 1 Channel 2 Single and Overlay must be pressed LL see Viewing Detection Channel 1 LL see Viewing Detection Channel 2 Page 66 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions LL see Viewing a Single Image LL see Viewing
107. dialog window 3D button Orthogonal Projection icon The projections with variable projection axes are located in the Image Tool dialog window 3D button Projections and Animations icon The SFP projection is located in the Image Tool dialog window 3D button SFP icon Maximum Projection During maximum projection we assume that the maximum intensity values are the relevant information for the reconstruction of a structure For this reason the maximum value is found in each column of sampling points and is displayed in the two dimensional projection image as representative of the entire column lp Max Vn Whereby Ip is the pixel in the projection image and Vn is the investigated Voxel Average Projection During average projection every intensity value with equal weighting flows into the projection image For this reason during average projection the arithmetic average of all intensity values is calculated in each column of sampling points This value is displayed in the two dimensional projection image as representative of the entire column User Manual Leica LCS english Page 93 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions H 1 20 n Ip T whereby IP is the pixel in the projection image Vn is the investigated Voxel and N is the total number of Voxel Transparent Projection During transparent projection all intensity values should be included
108. ds See This symbol is a reference to another topic in the online help User Manual Leica LCS english Page 21 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH vv VY Vv vvv User Manual Leica LCS english LCS Software Functions Data Recording Setting the Beam Path Function Open the Beam Path Setting dialog window using the Beam button and set up the beam path and the detectors with the corresponding color look up tables used to record the image Selecting the excitation wavelength Above the spectrum in the dialog window you will find boxes to set the excitation wavelength using an AOTF Click the check box next to the laser name to activate the laser Set the output for the laser line by moving the slide on the corresponding scale to the desired value or Double click the percentage value for the laser output This opens a second dialog window where you can enter an exact value The active laser line is shown as a line in the spectrum Loading and Saving Parameter Settings Also above the spectrum is a list box for loading and saving parameter settings The manufacturer has predefined parameter settings for the most important fluorescent dyes They are labeled with the letter L for Leica and can be loaded only not modified You can also save the settings made for a specific image recording as user defined parameter configuration so that you can load them any time with a s
109. e functions and the right side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons Page 53 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions The tab of the Charts icon can be used to perform optional settings for the following functions see Measuring a Profile Along a Line Segment see Measuring a Profile Within a Region of Interest ROI see Measuring Surfaces and Volumes see Measuring Roughness Along a Line Segment The options in Charts tab can only be selected if one of these buttons was used earlier to activate a quantification function Activating the two point measurement gt Clink on 2 Point in the Measurement field to activate the two point measurement Each of the buttons listed above opens a window that displays a measuring curve and statistical values If the two point measurement is selected the window shows a measurement slider 2 with two measurement points 3 below the measurement curve 1 The position of the two measurement points in the profile curve is symbolized by small black squares 4 This allows for very precise settings of the range to be evaluated within the measurement In addition the statistics shown below the measurement curve is expanded by the following measurement values position of measurement points distance of measurement poi
110. e View menu The Experiment Overview window appears on the left side of the user interface The top part of the viewer window displays the recorded images in a directory tree The bottom part displays the Viewer Options window This dialog window is used to perform basic settings for different software functions The left side shows the icons corresponding to the functions and the right side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons In the tab of the Scan Progress icon you can make optional settings for the following functions LL see Selecting a Scan Mode User Manual Leica LCS english The currently set scan mode is shown in the Scan Progress tab The status of an image recording can also be checked on the basis of the progress display Viewer Options Dialog Window Surface View Icon Function To open the Viewer Options window select the Experiment Overview option in the View menu The Experiment Overview window appears on the left side of the user interface The top part of the viewer window displays the recorded images in a directory tree The bottom part displays the Viewer Options Page 58 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions window This dialog window is used to perform basic settings for different software functions The left side shows the
111. e assigned real z positions of the individual sampling points first and then are color coded see Creating a Topographical Image User Manual Leica LCS english Maximum Projection of an Image Stack with Invariable Projection Axis Function The 3D Max Button starts a maximum projection The projection axis is freely variable The basis for a projection is an image stack i e a series of horizontal xy sections or vertical xz sections When a projection is generated the sampling points of the individual images superimposed along the projection axis are examined throughout all optical sections From each of these columns of sampling points the maximum intensity value is displayed in the two dimensional projection image as the representative of all intensity values within the column see Principles and Types of Projections Page 98 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Click on the 3D Max Button A maximum projection of the current image stack is created and displayed in the Viewer window You can now change the projection axis by clicking in the image in the Viewer window and holding the left mouse button pressed Broken support lines are displayed that encloses the symbolically displayed image stack Turn the image stack to the desired new position by moving the mouse pointer while holding down the left mouse button If you release the left mou
112. e software downsampling In such cases the time tag must be re calculated accordingly User Manual Leica LCS english Page 151 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Appendix A time tag can be defined at any time during an n dimensional data acquisition process scan The coordinate of the time tag 0 0 2 7 indicates that the time tag was set at the start of a data record 0 0 c on the second channel 0 0 2 for the 7th optical section 0 0 2 7 ID SCANNERSET memory block This memory block contains device parameters that are set at the time of the image acquisition Further details about the SAFEARRAY structure can be found in the description of the ID FILTERSET memory block Size Data type byte 24 SAFEARRAY Symbol sa Description Microsoft type see Appendix for all elements that contain the structure sa rgsabound 0 cElements 128 wchar 64 wchar 64 wchar 20 VARIANT 4 DWORD 4 DWORD 4 DWORD 4 DWORD Identifier for the contents of this entry Name of a shorthand notation e g a TIFF description tag String contains the contents of the string Contains the data type and the data value except for the string data type which is described in the field above Separate memory space for data values Not in use Identifier ID of the memory block for internal use only Test value ID_EXPERIMENT memory block Description of the used save format e
113. e specimen Image Recording With a Digital Resolution of 8 Bits or 12 Bits LE HH Bit Function This function reads in the analog intensity values measured by the detector by means of an A D converter either as 8 bit signal or 12 bit signal Additional information While digitizing the analog intensity signal with a digital resolution of 8 bit 256 different intensity values can be displayed Taking the natural statistical variations of the typical intensity value range of fluorescence specimen into account a digitization with 8 bit is completely sufficient in most cases for an image recording without loss of information For specimens with a higher intensity dynamics e g for specimen with very intensity weak and intensity strong areas an image recording with 12 bit digitization is recommended In a 12 bit digitization 4096 different intensity values can be resolved Typical Applications For image recording with very high intensity dynamics mostly material specimens with high reflectivity of the surface and occurrences of very dark areas at the same time User Manual Leica LCS english Page 47 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Setting the UV Lens Wheel LVlens Function For each objective this function reduces the focus offset between visible and UV excitation light to a value below the optical resolving capacity In order to achieve thi
114. e square frame of a specimen Click the Zoom In button At the same time the Rectangle button is automatically activated LL see Defining the Region of Interest ROI as a Rectangle gt Specify the frame to be enlarged by drawing a rectangular evaluation area region of interest or ROI into the image using the Rectangle button see Selecting and Moving the Region of Interest ROI Use the Select button to move the frame on the image To start the image recording press the Continuous Scan Series Scan or Single Scan button Note The software automatically adapts the freely selected rectangular frame to the actual square form that can be implemented by the scanner The dashed line shows the actual frame Additional information The enlarging recording of a frame will only provide additional detailed information as long as the distance of the grid points remains larger than half the optical resolution On the other hand the resolution depends on the objective used the adjusted scanning format and the light wavelength used Empty magnification Typical Applications All applications that deal with the optical resolution of very small structures and that can do without an overview image While recording such specimens care should be taken that an objective with a corresponding high optical resolution is used for image recording User Manual Leica LCS english Page 28 of 164 Art No 15 9330 033 Vers 01082001 Leica
115. e usually low correction optic systems because maintaining extreme process accuracy through a large lens diameter can only be achieved with great effort The curved surface to which a microscopic image is to be clearly and distinctly mapped is described as image curvature aberration It is conditional on the convex shape of the lens and makes itself apparent as an error due to the short focal distances of microscope objectives Here the object image is not in focus both in the center and at the periphery at the same time Objectives that are corrected for image curvature aberration are called plane objectives plane flat image field The destruction of fluorochromes so called fluorochromes by intense lighting In fluorescence microscopy fluorochromes are excited with laser light to a high state of energy the Singlet state When the excited molecules return to their normal energy state a fluorescent signal is emitted If the intensity of the Page 138 of 164 Refraction index Dichroic Double dichroite Filter digital phase true Fluorescence microscopy Fluorite objectives Frame Immersion objective Confocality Reflection short pass filter Reflection long pass filter Empty User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Glossary excitation is too high however the color molecules can change via intercrossing from a Singlet state to a triplet st
116. ected scan mode You can record a line xt a horizontal section xyt a vertical section xzt or a stack of horizontal sections xyzt interrupted by a particular time interval many times in a row Note The Time button is not enabled unless you have selected a scan mode with time dimension using the Mode button see Selecting a Scan Mode User Manual Leica LCS english Page 33 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Use the following parameters in the dialog window to configure a time series using the xt scan mode AT The recording time for a line cannot be modified by the user Lines Number of line recordings Lines per Number of lines per stored page page Pages Number of stored pages calculated automatically Complete Total recording time i e the product of AT and number of Time recordings Using the Calculate button you can calculate how many stored pages are needed for a particular number of lines per page Use the following parameters in the dialog window to configure a time series using the xyt or xzt scan mode AT Recording time for a xy section or xz section plus pause interval Frames Number of recordings of the xy section or xz section Complete Total recording time i e the product of AT and number of Time recordings Use the following parameters in the dialog window to configure a time series using the xyzt scan mode AT Recording time f
117. ed in the two dimensional projection image as the representative of all intensity values within the column see Principles and Types of Projections Click the Fix Max Button A maximum projection of the current image stack is created and displayed in the Viewer window You can return to the original image anytime by clicking on the Original button User Manual Leica LCS english Page 97 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions In the Viewer Options dialog window you can also adjust a threshold and scale the intensity value range present in the image see Viewer Options Dialog Window Projections Icon Note LQ Note A maximum projection can be activated either by using the Fix Max Button or in the Image Tool dialog window via 3D button Orthogonal Projection icon If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be stored as a new file in the current experiment see Image Tool Dialog Window 3D Button see Creating an Experiment Note the difference between a maximum projection and a topographical image which is generated from intensity maximums In a maximum projection the intensity maximums are assigned color values directly In a topographical image based on intensity maximums the intensity maximums ar
118. ed until after the set number of averaging steps has completed The method used here determines a consecutive average This means that every line recorded after the first line is averaged with the previously displayed line and is displayed in the result image dynamic average Clicking the Line Average button opens a dialog window that you can use to set how often a line is to be scanned You can select from 1 to 8 scan repeats Using a parallel function it is possible to record complete images using the averaging method see Recording an Image Using the Averaging Method Typical Applications This function is used predominantly for recording live specimens Recording an image using the averaging method is not recommended for bleach sensitive specimens Repeated recordings and the resulting length of subjection to light can lead to the destruction of the specimen Applying the Parameter Setting of an Experiment Je Function Click the Apply button to use the hardware settings of a previous experiment for recording a new experiment This lets you configure the settings for new image recordings with a single click with the scan parameters that have been optimally configured for a previous application Page 45 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions gt Activate the image data block that is configured with the settings you want to apply to the new experime
119. educed from the flare angle of the light cone recorded by the objective and the refraction index of the medium between objective and specimen NA n sin a Objectives with greater magnification generally have larger numerical apertures but smaller entrance pupils and therefore can record light only from a relatively small scan field Objectives with larger apertures permit higher resolutions but allow less free working distance The following table illustrates this relationship Page 24 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Objective Resolution Resolution Resolution Resolution Scan xy Air z Water z Oil 2 Field Size xy HC PL 1301 19410 25879 29559 3000 FLUOTAR 5x 0 15 HC PL 651 4768 6407 7335 1500 FLUOTAR 10x 0 30 N PLAN 20x 488 2630 3566 4093 750 0 40 N PLAN 50x 260 649 948 1108 300 0 75 PL APO 100x 139 319 209 236 150 1 40 Values in nm at Values in wavelength A 488 mm nm Typical Applications Material scientists commonly use dry objectives to study surface structures Immersion Objectives are the best choice for producing images of layered structures in which material layers with varying refraction indexes come into contact with each other When working with biological specimens the choice between an oil immersion objective and water immersion objective depends on the specimen itself and its embedding medium You will achieve the best resolution
120. ee Boolean Operations XOR The binary representations of the pixels of the constants and the image are linked with the logical operator XOR That means that pixels in the result image are given the value 1 when the two pixels have different values otherwise 0 is entered see Boolean Operations In the Arithmetic Type field select whether raw data or scale data is to be used for the arithmetical operations If you have selected Raw Data the values standardized to the interval 0 255 for 8 bits or 0 4095 for 12 bits are used for the arithmetical operations As the calculation result can produce values extending beyond the interval the following three methods are available to readapt them to the interval Raw Data Result Options Cut at Values less than 0 are set to 0 All values greater than 255 for 8 Maximum bit images or 4095 for 12 bit images are set to the value 255 or 4095 This concatenates all the values lying outside the interval Absolute The amounts of the result data are used This turns negative value results into positive results Rescale The extreme values are determined The minimum value is assigned to the 0 The maximum value for 8 bit images is assigned to the value 255 for 12 bit images to the value 4095 If you have selected Scaled Data the world coordinates that is the actual and non standardized values are used for the arithmetical operations The standardization of the results data into the in
121. el as well as the statistical values Parameter Meaning Formula Length The length of the 5 5 measured section L 4ix3 317 ly2 y4 Mean Arithmetic mean of 1 Amplitude measurement variable H i 7 Fe gt Pixel Pixel Maximum Maximum value of Max I Amplitude measurement variable Minimum Minimum value of Min I Amplitude measurement variable User Manual Leica LCS english Page 112 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Average Mean standard deviation 1 Deviation of the values from the DISP 1 Te IL elr i average value of the measurement variable Standard Standard deviation of r Deviation the values of the s l VAR iT i u t i measurement variable hl 1 Variance Variance of the 1 2 measurement variable VAR I TRE ya i elt The following four measurement values appear only when the Two Point Measurement function is selected in the Viewer Options dialog window Select the View menu the Experiment Overview option and the Charts icon LI see Viewer Options Dialog Window Charts Icon gt Inthe Measurement field you can switch two point measurement on or off by selecting 2 Point or Off Parameter Meaning Formula um I s Value measured at measuring point 1 z 21 I ti I l nm t A um I s Value measured at measuring point 2 z _1I Z I t I l nm t A dl dz Difference of values between
122. el n Vn intensity of the Voxel n This calculation is completed in one step for the entire image stack for all Voxel in the direction of the incoming beam The result is a table of illumination strengths In the second step the established illumination strengths are used to calculate the emitted fluorescence 2 The recursive formula is used to calculate the intensity of the pixel in the projection image as representative of the Voxel in the observer s viewing direction Int Fln i or 7 Ttip max In addition a is the absorption coefficient of the fluorescent light in the sample and Vin is the illumination strength of the nth Voxel calculated in the first step Projecting an Image Stack with an Invariable Projection Axis Function Using the button Fix O Proj start the projection type that was used last or which was selected in the Viewer Options dialog window Projections icon Projections tab For this function the projection axis is always the orthogonal axis Z axis for horizontal xy sections and y axis for vertical xz sections The basis for a projection is an image stack i e a series of horizontal xy sections or vertical xz sections When a projection is generated the sampling points of the individual images amp 8212 superimposed along the projection axis amp 8212 are examined throughout all optical sections From each of these columns comprised of sampling points depending on the projection type the m
123. emory block This memory block contains the time taggers for image series that were acquired with a time dependent scan mode Size Data Symbol Description byte type 4 int nDims Indicates the number of User Manual Leica LCS english Page 150 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Appendix dimensions that the description of the time tagger includes 4 int Indicates which dimension was provided with a time tagger For the next n dimensions 4 DWORD Identifier ID of the dimension see the description of the D DIMDESGCR memory block 4 DWORD Size of the dimension number of elements e g 512 pixel 4 DWORD Distance between the individual dimension entries 4 int NTS Number of time taggers For the next ntime taggers NTS 64 wchar Time tagger as string string data type 4 int nTM Number of time tags per time tagger For the next m time tags nTM 4 int nC Number of dimensions of the time tagger description loop nC 4 int Coordinate in this dimension 64 wchar Time tag as string wchar data type Example Image with the following dimensions and dimension sizes X y ch Z 512 512 3 10 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 th
124. enauadde 92 Projecting an Image Stack with an Invariable Projection AXIS ccccceccceeeceeeeeeeeeeeeeeeeeeeeeeeeees 95 Projecting an Image Stack with a Variable Projection Axis Optional rrrrrrrnnrrrnnrernnrrvnnrrrnnnennn 96 Maximum Projection of an Image Stack with Invariable Projection AXIS cccceseeseeeeeeeeeeees 97 Maximum Projection of an Image Stack with Invariable Projection AXIS ccccseceseeeeeeeeeeees 98 Average Projection of an Image Stack With Variable Projection Axis optional 0006 100 Average Value Projection of an Image Stack with Invariable Projection Axis ccceeeeeees 101 Transparent Projection of an Image Stack with Invariable Projection AXIS ccccseceseeeeeeees 102 Transparent Projection of an Image Stack with Variable Projection Axis optional 103 Creating SFP projections of an image stack Optional ccccccsecceeceeeeeeeseeeeeeeeseeeeseeeeaeeeeaes 104 Creating a Topographical image ccccsscccssccceseeceeeccececeuceceueecaeeecueessucecausesueessueessueesseesseeesags 105 Viewing the Original Image cccccsscccsscccseeccsceccseeceecceeceacessueesaueesueeceueecsueecueeseeeesaeseneaeenaaes 106 Creating oD VIEWS aS 106 creatino the 30 VIEW caprine a a a a a a 106 ROAN SD NOW eosina a a a te aces tg traten haces aecatet ese ea Sens 107 M TE PI 108 ZOE He D VOW ee 108 Measuring an
125. ennanennnnen 122 LOC US MEIN Pye 122 Creating an Annotation SNe cccccccsecccesceceeeccesecceeceuceceueesaeeesueeceucecsusesueeeueeseueessusesseneneas 122 Copying an Image into the Annotation Sheet rrrrnrrnnnnrnnnrrnanernnnrnnnnrvnnnnnrnnennnnennannnanennasennnnennn 123 Drawing a Line on the Annotation Sheet rrrnnrrrnnrrnnnrrranernannrvnnnrranennnnennnnennnnnnanennnnennanennnnennnene 124 Drawing a Rectangle on the Annotation Sheet rrrranrrranrannnnernnnrvrnnrnnnnennnnennnnnnnnennanennnnennnnennnene 124 Adding a Text Field to an Annotation Sheet rrrannrrnnnrennnrennnnrrnnrnvanennnnrnnnnnnnnnnrnnennanennnnennnsennnne 125 PIN Sak tea cet ie taste th a eet il teat ee ees ta i ne ee baat or ee ar ed oa al eae eect 126 BELE g 210 1190 PER NERE tamer er nym nner tne tn etter seer Teen ee yer er ence 127 Opening as NC are 127 SAN a FNC eben aa see tae Ca sata A Carat der ae cuca tener tarts 127 Saving a FEHR 128 SONO FA OA eain a a a A OEA R 128 Creating ani EXPennNeNt save 129 USEFSPETIE AAAPA ION ee 129 Saving the Viewer Window as a Template rrrrnnnnnnnrnnnnernnnnrnnnnnanennnnernnnnvnnnnnnnennnnennnnennnnnnnnennnne 129 Controlling Functions Using the Control Panel rrrannnnanennnnrnnnnnnnnnrnanrnvanennnnennannnnnnnnnnennanennnnennn 130 Optional SOMWalre package aS 131 ME de EE EEE EEE EE EE A EE ucnelasies 131 Image Tool dialog window Materials button optional arrrranrrrrarer
126. ennarvonanrasnnrannernarennarsnneere 30 Resolution POWer rnrrnnnrrnnnrrnnnnrnnnrennenennnnn 24 User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Index ROl 28 116 117 118 119 120 121 122 Root mean square value 109 134 136 KOO EE 121 Rotate Button sunmiimnsiernmsknenksienbinnen 107 ROAU EE 107 Rotating the 3D View rrrarrrnanrennnnevnnnnnnnnn 107 Rotation Animation ccccceceseeeeeeeeneeeeees 64 BN ENE 127 DOVE EN 128 Save All Button rrrrrnrnnrrrnnnrrnnnnnrnnnnrrnnnnr 128 NES 128 Save AS Button rnrvrrnnnrrnnnnvrvnnnvnvnnnvnnnnre 128 Save BUMON Lurnssaamenmkdenaakeandnnindedne 127 Se SEN 127 128 SAVING a File rrrnnnrnnnnrrnnnnnnnnnnnnnnnnnnennnnennn 127 SAVING a File aS rrrrnrrrnnnnrvnnrrnnrrnrnnennnnennn 128 Saving Measurement Points 000 134 Scaling of the Measurement Curve 53 Scan Field Lunnan nske skveeberengkeneke 44 Scan Field Rotation Button rrrrnnrrrnnnnnrn 44 Scan 9 EEE 30 Scan MOS Luuasummaumanmienamid 31 58 Scan Speed errannrannrvnnnennnnnnnnvnnvrnuvrnuvennveen 32 Sections Button rrnrarnnnrrvvnnrrnvnnrrrnnnrennnnnen 40 Se TE EE ENE 120 Select LUT Dialog Window 0008 84 Selecting a Scan Format ararnnennnrnnnrnnnnnnn 30 Selecting a Scan Mode cccsccceeee seen eee 31 Selecting a Scan Speed rrrrararnnrevnnnrnnnr 32
127. ennnrennnnnnnrnnnnsennn 37 Defining the End Point for a Spatial Series rrrnrrrnnrrrnanrrnanennnnnnnnnrnnanennnnnnnnrnnanrnnanennnnnnnnnnnnnennnne 38 Defining the End Point for a Wavelength Series rrrrrarrrrarrnnnnrrranernanennnnrnnnrnnanennnnennnnennssnnnsennnne 39 Defining the Number of Spatial Sections rrrnnrrnnnrrnanrrnnnrrnnnnvnnrnnanrnnanennnnnnnnnnnanrnnanrnnnnnnnnnnnnnsnnnne 40 Setting the Number of Wavelength Steps arrrranrrnanrrnnnrnnnnvnnnnnrnnrrnanrnnnnennnnnnnnnnnanennanennnnnnnnnnnnnne 41 Slanga SOMES SCAN are 41 Selecting a unidirectional or bi directional SCAN cccccecceseeeeeeeeceeeeceeeeseeeeseeeseeeeseeeeseeeeseeesaeeees 43 AdUSUIG DESS 44 TIRING the Scan FE verserer 44 Recording an Image of a Line Using the Averaging Method rrrnnrnnnnnnnanennnnrnnnnnrnnnnnnnenvnnennnnennn 45 Applying the Parameter Setting of an Experiment rrrrnnrrnnnnrranrrnnrernnnrvnnnrnnnnennnnennnnnnnnennanennnnennn 45 Recording an Image Using Burst Mode ccccccccsecceecceeeeeeeeeeeeeseeseeecaeeceeecseesaeesseeseeesseeseeesas 46 Recording an image using the Averaging Method rrarrnarennnennnrvnnennnnrnnnnnnnennnennnrnnnennnennernnnennnen 46 Image Recording With a Digital Resolution of 8 Bits or 12 Bits rrrrnrrrnnrernnrrrarrrvnnrnrnnrnranennnnennn 47 SEEMING THe UV LENS VEL aa 48 BEE SA EN gt ER ES ER a acta cake de EE cate GRE NR Weeder 48 TE EVE NTN vvs E E
128. es 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 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 Recent files Opens one of the recently opened files Print Opens a dialog for printing the content of the currently active window Exit Terminates the program The View Menu Menus Use this option to switch TCS menu and the toolbar on and off 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 Experiment 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 Viewer Options Use this item to configure various options for displaying data records in the viewer window such as the display angle and calculation methods for displaying image data etc The Macro Menu 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 ma
129. ese functions refer to the chapter on Software Reference User Manual Leica LCS english Page 14 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Opening Data Records 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 Tiff files tif These are Leica image files in single and multi TIFF format Both image files in the previously used TCS formats and external files in RGB TIF format can be read Annotation ano The format of this type of file is also Leica specific and binary It is used for saving annotation 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 Automatically Applying the Study Parameters of an Experiment 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 window for the data record that contains the settings you want to apply There click the Apply button which i
130. f the following string in wchar NTC wchar Physical start position with unit 2 of length e g 10 um 4 int NTC Length of the following string in bytes Name of the image series Length of the following string in bytes Description of the image series nTC wchar 4 int nTC nTC wchar Identification numbers ID for image dimensions The following identification numbers are possible ID decimal 0 120 121 122 116 6815843 6357100 7602290 7798904 7798905 7798906 4259957 4325493 4391029 6357095 6422631 6488167 6553703 7864398 7929934 7995470 7602254 7077966 7471182 5767246 5832782 5898318 Meaning undefined X y Z t for time dimension channel number wavelength Range rotation x wide for motorized xy stage y wide for motorized xy stage z wide for z stage user1 not specified user2 not specified user3 not specified gray scale e g of histogram gray scale e g of histogram gray scale2 e g of histogram gray scale3 e g of histogram logical x not physical but logical position value logical y not physical but logical position value logical z not physical but logical position value logical t not physical but logical position value logical wavelength value not physical but logical value logical rotation value not physical but logical value logical x wide value not physical but logical value logical y wide value not physical but logical value l
131. for each zoom value and enters the corresponding values Due to the temperature dependency of the mechanics and electronics there may be a slight deviation from these default values during operation Use this function to readjust these values Turning the Scan Field Function Use the Scan Field Rotation button to rotate the scan field This neither rotates the specimen nor the direction of the scanner but rather the intermediate microscopic image The angle of rotation is infinitely variable between 10 and 100 degrees The original position 0 degrees is based on a predefined reference position in the scanning head When the unit is switched on it automatically relocates to this reference position Typical Applications This function is used to align textures on surfaces or long stretched out biological structures relative to the viewer User Manual Leica LCS english Page 44 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note User Manual Leica LCS english LCS Software Functions Recording an Image of a Line Using the Averaging Method Function The Line Average function launches an averaging method for the image recording In this case each single line is scanned several times For every sampling point the arithmetical average is calculated from the repeatedly measured intensity values in one line and represented in the result image The next line of the specimen will not be scann
132. formats Selecting the scan format selects the image raster that will be used for recording the images An image raster is the amount of points scanning the specimen in the three directions in space Besides the numerical aperture of the objective and the excitation wavelength the scan format together with the electronic zoom determines in large part the spatial resolution of the recorded data Additional information When selecting the scan format observe the influences between the image raster and the resolution of the generated image According to the Nyquist theorem or sampling theorem a structure can only be scanned without information loss if the smallest optically resolvable distance is scanned with about 2 to 3 raster points This optically resolvable distance called lateral resolution depends on the numerical aperture of the objective and the wavelength of the applied excitation light D Laterale Aufl sung A The following example should illustrate this relationship You have selected for example the PL APO 100x objective unit with a numerical aperture of NA 1 4 and a wavelength of 488 nm Based on these factors the smallest optically resolvable distance is determined as follows 1 4 460 1 4 Based on this rough formula the distance of the raster points required avoiding information loss while recording is 140 nm ca 140 nm ca 4 nm If however you select a scan format of 1024x1024 a PL APO 10
133. from being scanned The system automatically switches to zoom factor 2 or zoom factor 4 User Manual Leica LCS english Page 32 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Setting the Z Y Position Va Pos Function Use the z y Position button to define the horizontal level z position or vertical level y position of the specimen in which the images are to be recorded To record an image series using the Series Scan function define the start and end points of the image series using the z y position button and the Begin and End buttons LI see the Series Scan Overview Dialog Clicking the z y Position button opens a dialog window that provides you with two ways of specifying a position value Use the mouse to move the slide on the scale that is displayed on the left in the dialog window The corresponding position value is shown in the middle of the dialog window gt Double click the boldface position value located in the middle of the dialog window This opens a second dialog window where you can enter an exact value The z y position can also be set using the corresponding knob on the console see Controlling Functions via Control Console Configuring a Time Series Function Click the Time button to open the Time Configuration dialog window for setting up a time series recording The settings that can be changed in this dialog window depend on the sel
134. function can also be used to check the image section by zooming in on the scan field Starting a Continuous Scan Function Use the Continuous Scan button to permanently record images from a single optical level in the specimen This does not generate image series since the image being generated always replaces the previously generated image Press the button a second time to stop the continuous scan Additional information The unit applies the last used scan parameters automatically You can modify some of the parameters while the image is being recorded Others have to be configured before you start the recording Before Recording While Recording Selecting an Objective Adjusting the electronic zoom Selecting a Scan Mode Setting the Detector Selecting a unidirectional or bi directional scan Adjusting phase Before Recording While Recording Selecting an Objective Setting the Beam Path selecting a Scan Format Setting the Detection Pinhole selecting a Scan Mode Adjusting the electronic zoom Selecting a Scan Speed Setting the Detectors Selecting a unidirectional or bi directional scan Adjusting phase Setting the z y Position The buttons of the functions that cannot be changed during continuous scanning are disabled and appear gray Typical Applications The continuous scan function is best used to optimize the image quality for the first scan of a specimen While the specimen is being scanned continuously you can modify the s
135. g GmbH LCS Software Functions Note If the Gallery button is enabled the Next and Previous First and Last and Play Stop buttons are disabled and displayed in gray The Gallery button is used to show all of the individual images of an image series and does not allow film sequence mode Displaying the Previous Image in a Series Function You can view the individual images of an image series as a film sequence Click Previous to jump to the previous image in the series Note If the Gallery button is enabled the Next and Previous First and Last and Play Stop buttons are disabled and displayed in gray The Gallery button is used to show all of the individual images of an image series and does not allow film sequence mode Viewing the Last Image of a Series Function You can view the individual images of an image series as a film sequence Click on the Last button to jump to the last image in the series Note If the Gallery button is enabled the First and Last Next and Previous and Play Stop buttons are disabled and displayed in gray The Gallery button is used to show all of the individual images of an image series and does not allow film sequence mode User Manual Leica LCS english Page 89 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note User Manual Leica LCS english LCS Software Functions Displaying an Image From a Series iL Function When you click on
136. g Window rrrarrrnnrennnnnnn 48 Electronic ZOOM rrannornnrornarvnnarrennnransernnnerne 26 NOS EE N 116 Empty Magnification ccccccseceeeeeeeeeees 138 ENO PO EN EE EE ENa 36 End Point for a Spatial Series 36 38 End Point for a Wavelength Series 39 Enlarged ReECOmrdingS cccscccseeeeseeeeseeeeaes 28 ENN 45 129 Experiment anlegen ranrnnnrnnnrnnnnvnnnnnnnnnnrr 129 Experiment Legend rranarnnnrnnnnnvanrvnnnennnnennn 48 Experiment Overview Viewer Window 129 EKOT EEE EE 116 Exporting Quantification Data 116 PIS TYG einir 127 Film Sequence rrnanrnnnnnnnnnnnnnnnnnne 88 89 90 FE PENE NE 88 ER EE 88 First Image of a Series rrrrrrnrrrrarrrrarernnnennn 88 Page 158 of 164 Leica Microsystems Heidelberg GmbH Fluorescence MICFOSCOPY cccceeeeeeees 138 Fluorite Objectives rrrarararerarernnennre 24 138 MOCALIPIAING sopia Eians 29 Focal shift compensation ccccceeeeeeee 48 Format Button rrnnrennrrvnnrrrnnrrnnnrrnnnrennnnn 30 FE EE NN 28 GAV AG Larsen dl 25 HUN saw saeecaseusceederscce ence uae aie ceeeeneee nase 91 Gallery Button rrarrnnnrrnnnrrnnnnnranrnnanennnnennn 91 Gamma Curve rrrnnnrrnnnnrvnnnnrvnnnrrnnnnrrnnnnnennnnr Tf GOSSEN Lae eee 138 Graphical ZOOM rareranornreranennnevnnennnennee 52 84 Hardware Legend cccccceceeeeeeeeeseeeeeeeees 48
137. g image If the deviation of the image resolution between the two output images is larger than a factor of 2 the smaller image resolution i e the larger um pixel value is used for calculating the resulting image The number of pixels of the resulting image Element Number divided by the total length results in the resolution of the resulting image Definition background Absolute lengths are used to determine the dimensions of the merged image If the two data sets to be merged feature the same linear extension but a different number of pixels due to a different scan format it must be decided whether the number of pixels or the resulting total length is to be decisive for the merged image LL see Selecting a Scan Format Tab ch Definition of the combination of images with different numbers of detection channels Field Image 1 Start Image 2 Start Check box Append Description of Function If two images or data sets with different numbers of detection channels are to be merged this option can be used to define the channel number in the merged image at which the channels of the output image 1 should be inserted see Example for merging images with different channel numbers If two images or data sets with different numbers of detection channels are to be merged this option can be used to define the channel number in the merged image at which the channels of the output image 2 should be inserted see Example for mergin
138. g images with different channel numbers This function distributes the two output images to different channels of the merged image Stereo icon Creating projection images along the orthogonal axis Creating red green stereo views Creating projection images along the orthogonal axis z axis for horizontal xy sections and y axis for vertical xz section gt Inthe Type field select one of the three projection types maximum projection average projection and transparent projection see Principles and Types of Projections The Direction field determines which single images are used for the projection Select z for stereoscopic series t for time series or I for wavelength series to create the projection from all images of a detection channel such User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Page 74 of 164 Leica Microsystems Heidelberg GmbH LCS Software Functions as from image 1 to image 10 in detection channel 1 Select ch to create the projection from a certain image in all detection channels for example from image 1 in detection channel 1 and image 1 in detection channel 2 Click on Rescale in the Options field to standardize the intensity values of the image to the maximum possible intensity values when creating the projection image This ensures that the calculated gray values of the projection image remain within the value range of for example 0 to 255 at 8 bits This allows for brighte
139. ginal button to undo a projection image or topographical image The Viewer window then displays the recorded raw data of the image again Creating 3D Views Creating the 3D View Function Use the 3D View button to display a two dimensional data record three dimensionally When viewing an image series the data record of the series that is currently being viewed in the Viewer window is always the one applied You can display a single xy section or xz section from the raw data or a result image such as a topographical image or projection image in the 3D view Depending on the dimension represented or calculated in the output image either intensity values or height values are mapped to the z axis of the 3D view when the 3D view is generated The spatial representation of intensity in a 3D view leads easily to the assumption that the topography of the specimen is being represented Note however that you can only display the real surface structure of a specimen if you have generated a topographical image first see Creating a Topographical Image The 3D view can be generated in three different modes surface wireframe or isolines Set the display mode in the Viewer Options dialog window From the View menu select Viewer Options Click the Surface View icon and then click the Visualization tab see Viewer Options Dialog Window Surface View Icon Page 106 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems He
140. glish Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Glossary Glossary An optical image distortion conditional on the varying refraction of light rays of different wavelengths on a lens Thus light rays of shorter wavelengths have longer focal distances than light rays of longer wavelengths An optical image distortion conditional on the varying distance of paraxial light rays of the same wavelength from the optic axis Light rays that travel through outer lens zones have shorter focal distances than rays that travel through the lens center optic axis Describes a correction class for objectives The chromatic aberration for two wavelengths is corrected for objectives of this type Usually an objective of this type is corrected to a wavelength below 500nm and above 600nm Furthermore the sine condition for one wavelength is met The image curvature aberration is not corrected The Airy disc refers to the inner light circle surrounded by alternating dark and light diffraction rings of the diffraction pattern of a point light source The diffraction discs of two adjacent object points overlap some or completely thus limiting the spatial resolution capacity An image distortion caused by a sampling frequency that is too low in relation to the signal frequency The acousto optical tunable filter is an optic transparent crystal that can be used to infinitely vary the intensity and wavelength of radiated light
141. h a larger scale than that of the empty magnification do not provide any additional information about the object but instead only diminish the focus and the contrast Neutral filters are semi reflective glass plates They are used to distribute the light path independent of wavelength The incident light is partially reflected and partially transmitted Neutral filters are usually placed at a 45 angle in the path of the beam The ratings of a neutral filter are based on its reflectivity to transmissivity ratio A neutral filter RT 30 70 for example reflects 30 of the excitation light and transmits 70 The principle of phase visualization as used by Leica is an optimized alternative method to ratiometric displaying The main area of application lies in measuring ion concentrations in physiology In contrast with ratiometric procedures phase visualization obtains more information on the specimen In addition this method allows for adapting the display of physiological data to the dynamics of the human eye Detailed information on the principle of phase visualization can be obtained directly from Leica Microsystems Heidelberg GmbH An acronym based on the words picture and element A pixel represents the smallest indivisible image element in a two dimensional system In this documentation both the sampling points of the specimen as well as the image points are referred to as pixels Describes a correction class for objectives The image cur
142. hanged where possible Enter the number of desired sections whole numbers only in the sections field Then click the Calculate button next to the Step Size field If you click the Reset button the last shown values are displayed Page 40 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions LL see Starting a Series Scan LL see Series Scan Overview dialog window Setting the Number of Wavelength Steps Function For a wavelength series this function records a stack of individual images each of which are detected at a specific wavelength from a single optical level The images are recorded within a wavelength range which is limited by the begin and end points Use the Lambda Steps button to define the number of recordings that are to take place within this range Note The Lambda Steps button is only active if you used the Mode button to select a scanning mode with the wavelength dimension LL see Selecting a Scan Mode LL see Defining the Begin Point for a Wavelength Series LL see Defining the End Point for a Wavelength Series Typical Applications A wavelength series can be applied to determine the maximum emission of a fluorochrome This is useful because the Stokes shift of the emission curve of a fluorochrome is dependent on each specimen that is applied This then allows you to set the detection range precisely for a specific app
143. he animation The number of projection images and the animation type specify the intervals at which the projection images are created With a half turn angle of 180 and a number of images of for example 10 an image is created in steps of 20 not 18 as there are only 9 steps for 10 recorded images Note There is another calculation in the case of the full turn angle of 360 To prevent the first and last projection image from being recorded under the same angle 360 0 the function calculates one recorded image more but without creating the corresponding projection image In the case of 10 images for example the interval between the projection images is 36 not 40 as 11 recorded images i e 10 steps are used in the calculation However only 10 projection images are created In the Rotation Axis field vary the inclination of the rotation axis Check the changes in the preview window In the Depth Factor field enter a value to scale the z axis of the projection images Click on the Apply button to start the calculation of the projection to display the result image in the Viewer window and to create it as a new file in the current experiment To start the rotation animation click on the Movie button see Starting and Ending a Film Creating Red Green Stereo Views On the Stereo View Tab you can create red green stereo images so called anaglyphic images Anaglyphic images create a stereoscopic sense of
144. he image data record to be processed If you click on the Use Selected Image check box the image data record currently displayed in the Viewer window is used for the calculation If you click on the Show button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the preview window in the dialog window If you click on the Apply button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the Viewer window and created as a new file in the current experiment Click on the button with the arrow symbols to show the preview window on the right edge of the dialog window or to hide it The projections triggered using Show are displayed temporarily in this preview window The Close button closes the dialog window see Creating an Experiment Overview of the Function Groups Created in the Image Tool Dialog Window Button Icon Tab Description of Function 3D User Manual Leica LCS english Projections amp Projections Creating projection images Animations Rotation along a variable projection Stereo axis View Running Projection Images as 3D Rotation Animation Creating red green stereo views Orthogonal Creating projection images Projections along the orthogonal axis Creating Projection Images from Different Detection Channels SFP Simulated SFP Creating SFP Projection Fluorescence Rotation Images Process Stereo Running
145. he mouse pointer over any position in the 3D view Press and hold the left mouse button Move the mouse pointer in the direction in which you want to move the 3D view vvv Note While the 3D view is being moved the resolution of the image is decreased in order to speed up processing When the left mouse button is released the image is displayed with its original resolution As an alternative to moving a 3D view manually the Viewer Options dialog window also provides the option of specifying the coordinate values for the position of the image From the View menu select Viewer Options Click the 3D icon and then click the Navigation tab Modify the coordinate values for the x axis and z axis in the Translation field vvv see Viewer Options Dialog Window 3D Icon Zooming the 3D View Function Use the Zoom button to magnify and shrink a 3D view steplessly while maintaining the aspect ratio This function only adjusts the scale of i e zooms the generated image It cannot be used to improve its resolution To zoom in Click anywhere inside the image window of the Viewer window on the 3D and while pressing the left mouse button drag the mouse view pointer toward the lower edge To zoom out Click anywhere inside the image window of the Viewer window of the 3D and while pressing the left mouse button drag the mouse view pointer toward the upper edge User Manual Leica LCS english Page 108 of 164 Art No 15 9330
146. he number of pixels in y that is dependent upon the scan Selection y format is indicated here Enter the number of pixels to be separated in the Start y and End y entry fields Use the R Reset button to undo the entries Dimension The number of pixels in x that is dependent upon the scan Selection x format is indicated here Enter the number of pixels to be separated in the Start x and End x entry fields Use the R Reset button to undo the entries Step z y x Here you can enter an interval within a dimension For gt User Manual Leica LCS english example if you enter 2 in the Step z entry field only every 2nd image is separated If you want to separate all images of a detection channel from an image series click in the ch entry field on the channel to be separated without changing the values in the entry fields of the other dimensions If you want to separate complete single images from an image series enter the corresponding numbers in the Start z and End z entry fields without changing the values in the entry fields of the other dimensions If you want to separate a segment in all images of an image series define the size of the segment in the Start x End x and Start y End y entry fields without changing the values in the entry fields for z Schematic Structure of the Dialog Window The numerous functions of the Image Tool dialog window are arranged on a number of levels The gray buttons 3D Basic Amplitude Mater
147. his mode is intended for secondary installations on another computer for example for training or offline analysis of existing data Starting the Software Select Start Programs Leica Confocal Software The initial screen of the Leica Confocal Software appears This window allows you to select from three profiles 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 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 Last Exit This option loads the configuration profile that was last active For advanced users If 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 configuration profile Note After a delay the software starts automatically and loads the currently selected configuration profile User Manual Leica LCS english Page 13 of 16
148. ials and Multicolor on the left side of the dialog window represent the first level of the hierarchy Each of these buttons has a group of icons that appear below the selected button The icons are the next level on which functions are grouped Each icon opens different tabs and fields that are displayed on the right side in the dialog window and arrange the functions on the lowest level The following elements can be found in each tab In the Source Image drop down list at the top of the dialog window select the image data record to be processed If you click on the Use Selected Image check box the image data record currently displayed in the Viewer window is used for the calculation If you click on the Show button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the preview window in the dialog window If you click on the Apply button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the Viewer window and created as a new file in the current experiment Click on the button with the arrow symbols to show the preview window on the right edge of the dialog window or to hide it The projections triggered using Show are displayed temporarily in this preview window Page 77 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions The Close button closes the dialog
149. icons corresponding to the functions and the right side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons In the tab of the Surface View icon you can make optional settings for the following functions see Creating the 3D View In the Visualization tab you can select whether the 3D view is to be generated as Surface Wireframe or Isolines and specify the perspective in which the 3D view is displayed Render Function Mode Surface The blank spaces between the pixels are filled in with surface Wireframe All pixels are linked with lines while the blank spaces remain free Isolines Pixels that correspond to values of the same intensity are enclosed in a curve Projection Type Function Perspective The 3D view is displayed in central perspective Parallel The 3D view is displayed in parallel perspective The Stretch height factor field offers the ability to vary the scaling factor in the z direction thus stretching or shrinking the height of the 3D view Use the Downsample rate field to reduce the density of data of the 3D view to increase the speed of image processing At a pixel density of 1 1 all measured intensity values are displayed in the image At a pixel density of 1 2 only every second measured intensity value is used in the surface image Use the Isoline interval field to define a distance for separating the i
150. icroscope objective Screens are implemented in optically conjugated locations to achieve this They function as point light source excitation screen and point detector detection screen The diameter of the detection screen along with the wavelength and numerical aperture of the objective being used determines the axial extension of an optical section optical resolution Reflection short pass filters are interference filters that transmit short wave light while reflecting long wave light An optical reflection short pass filter is characterized by the reading of the wavelength edge at which the filter changes from transmission to reflection 50 threshold Reflection long pass filters are interference filters that reflect short wave light but are transparent for long wave light An optical reflection long pass filter is characterized by the reading of the wavelength edge at which the filter changes from reflection to transmission 50 threshold A magnification without additional gain of information Empty magnification is Page 139 of 164 magnification Neutral filter Phase visualization Pixel Plane objectives ROI Signal to noise ratio Stokes shift Triple dichroic Dry objective Voxel User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Glossary used as soon as distances are displayed that are smaller than the optical resolution Magnifications wit
151. idelberg GmbH Note vvv Note vvv User Manual Leica LCS english LCS Software Functions A separate 3D view is created for each detection channel Therefore it is not possible to generate a single 3D view from one overlay image Overlay button Rotating the 3D View Function Use the Rotate button to rotate a 3D view in all three spatial directions Note that the 3D view is rotated around a fixed point which is located in the center of the image Hold the mouse pointer over any position in the 3D view Press and hold the left mouse button Move the mouse pointer in the direction in which you want to rotate the 3D view While the 3D view is being turned the resolution of the image IS decreased in order to speed up processing When the left mouse button is released the image is displayed with its original resolution As an alternative to rotating a 3D view manually the Viewer Options dialog window also provides the option of specifying the angles of rotation for the three spatial axes From the View menu select Viewer Options Click the 3D icon and then click the Navigation tab You can then specify the angles of rotation for all three axes in the Rotation field see Viewer Options Dialog Window 3D Icon Page 107 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Moving the 3D View Function Use the Move button to shift a 3D view Hold t
152. ies is to end Or click the z y position button to open a dialog window where you can enter the values of the z position or y position Click in the white box for the end point The corresponding position value appears and is saved The entire height of the image stack between the begin and end points is calculated and displayed total Now click the Series Scan button The dialog window stays open and you can follow the process of the image series being recorded The start point and end point can also be defined using the control panel and the separate keys Begin and End LL see Defining the Start Point for a Spatial Series LL see Defining the End Point for a Spatial Series LL see Setting the z y Position Additional information The following additional parameters which have to be set before recording an image series are specified in the Series Scan Overview dialog window Page 36 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Scan mode top line to the right of the graphic image gt Scan format red digits at the upper edge of the cube gt Number of optical sections division between begin and end points The red number on the vertical edge of the cube represents maximum travel of the z actuator that cannot be changed LL see Selecting a Scan Mode LL see Selecting a Scan Format LL see Defining the Number of Spatial Sections
153. in the projection image as well In contrast to average projection the intensity values from the various individual images are weighted differently The intensity values from the lower images in the image stack are weighted less than the intensity values from the upper images For this reason during transparent projection the weighted mean of all intensity values is calculated in each column of sampling points This value is displayed in the two dimensional projection image as representative of the entire column In Vo lp Tyee lg i a Tnxal gt l vg Ip Th xe Slat Yn Whereby In is the pixel in the projection image Tn 1 Vn Vmax is the standardization a is the variable transparent factor In 1 is the value of the previously calculated Voxels and Vn is the currently investigated Voxel Calculation of the projection image begins with the lowest and ends with the highest individual image in the image stack Weighting of the intensity values is based on two factors The first factor Tn is calculated from the relationship of the respective intensity value to the maximum possible intensity standardization This factor ensures that an image s gray value range from 0 to 255 at 8 bit for example will not be exceeded The second user adjustable transparent factor a 0 lt a lt 1 determines the weighting of the previously measured intensity value The higher the transparent factor is adjusted the more the intensity
154. ingle click Click the Save button This opens a dialog window for entering a name for the parameter configuration The parameter configuration is stored in the list box under User and is labeled with the letter U for User Furthermore you can individually select the parameters that you want saved when using the Save command Select the Settings option from the Tools menu In the Settings dialog window click the Instrument Parameters tab Select individual parameters by clicking the corresponding checkboxes The Select all and Deselect all buttons allow you to select all or no parameters quickly Click one of the user defined or preset parameter configurations and then press the right mouse button to open a context menu which contains the following commands Page 22 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Command Function Set as default The standard setting for the parameters is loaded when setting you Start the software Remove default This removes the default setting property from the setting selected configuration Load Loads the parameter configuration Rename only for Use this to change the name of the parameter U configuration Delete only for U Deletes the parameter configuration Selecting the excitation beam splitter The symbol green tilted line for the excitation beam splitter is located to the left of the spectrum Click on the s
155. intensity was measured is selected When determining the center of mass a mean value is calculated from all superimposed sampling points the center of mass of the area that is limited by the curve of the measured intensity values The intensity maximum or center of mass is then assigned the real z position of the corresponding sampling point and then color coded Based on this assignment a topographical image maps the real surface structure of the specimen Using the default settings higher structures appear light and deeper structures dark Use the Viewer Options dialog window to set the selection criterion for the topographical image gt From the View menu select Viewer Options Click on the Surface Calculation icon to view the Topography tab see Viewer Options dialog window Surface Calculation icon By applying a second step you can display the topographical image as a three dimensional graphical image using the 3D View function see Creating the 3D View Typical Applications Displaying the image data in a topographical image is especially informative in material scientific research For the application of quantification functions the topography function is indispensable User Manual Leica LCS english Page 105 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note User Manual Leica LCS english LCS Software Functions Viewing the Original image Function Click the Ori
156. ion Axis Function Using the button Fix Trans start the transparent projection For this function the projection axis is always the orthogonal axis Z axis for horizontal xy sections and y axis for vertical xz sections The basis for a projection is an image stack i e a series of horizontal xy sections or vertical xz sections When a projection is generated the sampling points of the individual images superimposed along the projection axis are examined throughout all optical sections From each of these sampling point columns a weighted average is calculated from all intensity values which is then displayed in the two dimensional projection image as a representative of the entire column see Principles and Types of Projections The user can vary the weighting of the sampling points to calculate the averages by setting the corresponding factors transparent factor in the Viewer Options dialog window Furthermore you can indicate a threshold and scale the intensity value range for the image LL see Viewer Options Dialog Window Projections Icon Click the Fix Trans button A transparent projection of the current image stack is created and displayed in the Viewer window You can return to the original image anytime by clicking on the Original button Note You can activate a transparent projection using the Fix Trans button Alternately it can be activated using the Image Tool dialog window 3D button Orthogon
157. ion Function for Correcting Trend Curves The target function to be optimized in determining the coefficients of the adaptation function is fl 2 de min Procedure for calculating the adaptation function gt Select an image from the Source Image drop down list The drop down list contains all the recorded and calculated images of the experiments displayed in the Experiment Overview viewer window gt Inthe Fitting Interpolation Function field select the character of the adaptation function Fitting Interpolation Formula Function Linear f x a bx cy Bilinear f x a bx cx dxy Quadratic f x a bx cx dxy fx Cubic f x a bx cx dxy fx gxy hx ly Select the step size in the Sample Step Width field The step size sets the number of support positions for the polynomial The smaller the step size the more time consuming and precise the calculation of the adaptation function Ifthe data set should show noise it is recommended to hide the low intensity noise signals in the Threshold field by setting a threshold All intensity values whose values are below the threshold are not used in the calculation This prevents noise signals from being erroneously used as support positions for the adaptation function Click on the Show button to obtain a preview of the arithmetical operations Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Clic
158. ion and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the stereo images as a new data set Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment To display both stereo images in one image in the Viewer window the buttons Channel 1 Channel 2 Single and Overlay must be pressed L see Viewing Detection Channel 1 LL see Viewing Detection Channel 2 LL see Viewing a Single Image pa A see Viewing an Overlay Image Additional information The stereoscopic effect of the anaglyphic image is possible since human beings use both eyes for viewing Since the eyes are approximately 6 5 cm 2 56 inch apart the brain receives two images from different perspectives Objects located at different distances are observed at different angles This angular difference is a measure of distance for the human brain The difference between the similar images generated by the two eyes is processed by the brain into one image with spatial depth impression Holding a slightly different image in front of each eye can also simulate these image differences In anaglyphic images the complete stereoscopic image information is located on one image The information of the left and right image is coded through colors For this reason glasses with a different color filter for each eye are needed in
159. ion of Interest ROI see Deleting Regions of Interest ROIs gt A window opens automatically to display the statistical values Parameter Meaning Pixel Area Avg Height Pa Number of pixels within the selected region of interest Area Average height of the profile ordinates Arithmetic average of the profile ordinates within the measured section average height according to DIN EN ISO 4287 RMS Pq Root mean square value of the profile ordinates within the measured section according to DIN EN ISO 4287 Min Valley Minimal height of the profile ordinates User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Formula A A da A i iA H Z xy dA i A Fa ale pda i A Py fiz i v JA i with 2 6 v Z Z Min Pv Page 136 of 164 Leica Microsystems Heidelberg GmbH LCS Software Functions Pn based on the average height Max Peak Maximal height of the profile ordinates Max Pp Pp based on the average height Note This quantification function is only available off line only available for previously recorded data records User Manual Leica LCS english Page 137 of 164 Art No 15 9330 033 Vers 01082001 Aberration chromatic Aberration spherical Achromatic Airy disc Aliasing AOTF Acousto Optical Tunable Filter Aperture numerical Apochromatic Working distance Image curvature aberration Bleaching optical User Manual Leica LCS en
160. ions 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 Experiment Overview window provides a general overview of the open experiment To open the Experiment Overview window select View gt Experiment Overview Create a new experiment by selecting File gt New or File gt New Template Files that were previously saved are handled as separate experiments when opened Compiling Experiments After you have defined a new experiment by selecting File gt New or File gt New Template you can start filling it with data Note Images that have been recorded in continuous scan mode are overwritten automatically 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 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 functions 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
161. ish Page 99 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note User Manual Leica LCS english LCS Software Functions Average Projection of an Image Stack With Variable Projection Axis optional Function Use the button 3D Avg to start an average projection The projection axis is freely variable The basis for a projection is an image stack i e a series of horizontal xy sections or vertical xz sections When a projection is generated the sampling points of the individual images superimposed along the projection axis are examined throughout all optical sections From each of these columns of sampling points the arithmetic average is calculated from all intensity values and displayed in the two dimensional projection image as the representative of the complete column see Principles and Types of Projections Click on the 3D Avg button to create an average projection of the current stack of images The projection is displayed in the Viewer window You can now change the projection axis by clicking in the image in the Viewer window and holding the left mouse button pressed Broken support lines are displayed that encloses the symbolically displayed image stack Turn the image stack to the desired new position by moving the mouse pointer while holding down the left mouse button After you release the left mouse button the computer calculates and displays the new average projection The proje
162. it is set the more the intensity values from the lower levels in the image stack affect the projection image see Principles and Types of Projections gt Move the slider in the Threshold field to define a threshold value The intensity values below this value are not taken into account for the creation User Manual Leica LCS english Page 67 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note LCS Software Functions of the projection image Click on the Show button to obtain a preview of the projection images Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the projection as a new data record Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment The three projection types can also be enabled directly using the corresponding keys Fix O Proj Fix Max Fix Avg Fix Trans If you activate the function using the button the projection image will just be a temporary display on the monitor You can undo this using the Original key LL see Projection of an Image Stack With Invariable Projection Axis LL see Maximum Projection of an Image Stack With Invariable Projection Axis see Average Projection of an Image Stack With Invariable Projection Axis see Transparent Projection of an Image Stack With Invariable Projection Axis
163. ividual image depicts only the information from a single level in the specimen Therefore structures that extend throughout the entire specimen will be partially represented in several images Projections are necessary to view these structures in their entirety Using projection algorithms it becomes possible to select the relevant information from many images and to depict this information in a single two dimensional image This method can be used to reconstruct connected structures Accordingly a series of images is the foundation of any projection The series may consist of horizontal xy sections or vertical xz sections In order to best understand the functionality of a projection it is practical to think of an image series as a stack of individual images In turn each individual image consists of individual laser sampling points Furthermore the individual sampling points or Voxels 1 the intensity values measured in each individual image lie on top of each other During creation of a projection the sampling points of the individual images which lie on top of each other along the projection axis 2a 2b are scanned through all of the optical sections Then either the intensity value that fulfills the selection criteria or another calculated value is depicted in a two dimensional image 4a 4b as a representative of all intensity values User Manual Leica LCS english Page 92 of 164 Art No 15 9330 033 Vers 01082001 Leica Micr
164. jection the actual surface is always at least as large in general even larger Ratio A A This value represents the relation of surface to base area The larger it is the more the two dimensional surface is embedded in the three dimensional space The following four measurement values appear only when the Two Point Measurement function is selected in the Viewer Options dialog window Select the View menu the Experiment Overview option and the Charts icon LI see Viewer Options Dialog Window Charts Icon Inthe Measurement field you can switch two point measurement on or off by selecting 2 Point or Off V Z lt upper This value represents the volume located below the upper position value position value of the measurement slider right corner point of the slider V z lt lower This value represents the volume located below the lower position value position value of the measurement slider right corner point of the slider dV Volume between the position values of the slider dz Distance between the position values of the slider Note This quantification function is only available off line only available for previously recorded data records User Manual Leica LCS english Page 114 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Copying Quantification Graphs to the Annotation Sheet QSnap Function The Snap Quantification Data function copies the data calcu
165. k on the Apply button to create the calculation operation as a new data set Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment Merging Combining several images or data sets to a new image or data icon set User Manual Leica LCS english Voxel tab Adaptation of different image resolutions and additional settings for calculating the resulting image Page 72 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Field Amplitude Image 1 2 Min Amplitude Image 1 2 Max Result 8 bit 12 bit Maximum Value Average Value LCS Software Functions Description of Function The minimum intensity value is shown here For raw data this value is 0 For scaled data the actual minimum value is shown here The minimum is adjustable so that differently scaled data sets can be adapted to each other If a data set with a small interval of intensity values is combined with a data set featuring a large intensity value range the first one is shown darker than the second one Adapting the minimum values of both data sets scales them in such a way that equal parts of the intensity values of both data sets are included in the merged image The maximum intensity value is shown here For raw data this value is 255 with 8 bit data or 4095 with 12 bit data For scaled data the actual maximum value is shown here The
166. l int DWORD DWORD int nTC wchar int nTC wchar int nTC wchar int int nDims DWORD DWORD DWORD int nTC wchar Appendix Name of the next image Description Internal version number of the VOXEL description Voxel type e g RGB or GRAY see the following VOXEL TYPES table Byte size of a pixel Typical 1 or 2 or 3 or 6 for RGB 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 Maximum value of measured variable intensity value or length value of a distance for a voxel Length of the following string in wchar Minimum value of measured variable intensity value or length value of a distance for a voxel Length of the following string in wchar Designation of measured variable for intensity z for length value Internal version number Dimension of the image e g x y ch z 4 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 unit of length e g 10 um Page 148 of 164 Leica Microsystems Heidelberg GmbH Appendix 4 int NTC Length o
167. l 200 Currently not available Image lines per second 400 800 Image lines per second 800 1600 Image lines per second 1000 2000 Image lines per second see Selecting a Scan Speed Note In order to accurately align the pixels of the forward sweep and flyback the phase between the forward sweep and the flyback can be adjusted Use the Phase button or the appropriate knob on the control panel see Setting the Phase see Controlling Functions using the Control Panel If the speed levels 800 or 1000 are set technical limitations prevent the maximum scan field from being scanned The system automatically switches to zoom factor 2 or zoom factor 4 see Electronic Zoom User Manual Leica LCS english Page 43 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Adjusting phase we Function During bi directional image recording there may be a phase displacement between the scanning beams moving forward and backwards Click on the Phase button to open a dialog window that allows you to correct the displacement Use the mouse pointer to move the slider on the scale until the pixel displacement disappears from the image It is also possible to adjust the phase with the respective knob on the control panel see Controlling Functions using the Control Panel Additional information Before delivery of the instrument the factory performs a phase adjustment
168. lated by the quantification functions to the annotation sheet The calculated values for the following quantification functions which are also displaying an evaluation graph can be copied to an annotation sheet see Creating an Annotation Sheet see Measuring a Profile Along a Line Segment see Measuring a Profile Within a Region of Interest ROI LL see Measuring Surfaces and Volumes LL see Measuring Roughness Along a Line Segment Printing Quantification Graphs Function The Print Quantification Data function sends the data calculated by the quantification functions to the standard printer The calculated values for the following quantification functions which are also displaying an evaluation graph can be printed see Measuring a Profile Along a Line Segment LL see Measuring a Profile Within a Region of Interest ROI LL see Measuring Surfaces and Volumes see Measuring Roughness Along a Line Segment User Manual Leica LCS english Page 115 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions This function is also available in the context menu within the quantification function window gt Move the mouse pointer into the view window opened by a quantification function Click the right mouse button and select the Print menu item Exporting Quantification Data QExp Function Using he Export Quantification Data function you can export the data
169. lication Starting a Series Scan Function Use the Series Scan button to create an image series This creates a multidimensional image data block of the specimen The available dimensions for recording an image series are the three directions in space x y Z as well as the dimensions of time t and wavelength A This allows you to record a three dimensional spatial image stack consisting of xy or xz sections with the additional factor of time or wavelength User Manual Leica LCS english Page 41 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Note Before recording an image series configure all required scan parameters using the Continuous Scan function to ensure optimal image quality see Starting a Continuous Scan Before recording a spatial image series configure the following settings Select the scan mode using the Mode button LL see Selecting a Scan Mode Select the scan format using the Size button isee Selecting a Scan Format gt Set the desired z position or y position using the z y position button or the corresponding knob on the console see Setting the z y Position see Controlling Functions Using the Console Set and store the begin point for an image series using the Begin button or in the Series Scan Overview dialog window see Defining the Begin Point for a Spatial Series gt Set the desired z position or y position using the z y po
170. lor look up table The upper and lower intensity ranges are represented in the colors of the second color look up table Note This method of assigning color look up tables only influences the currently displayed image As soon as you Start a new scan the color look up tables configured in the Beam Path Setting dialog window are applied again see Setting the Beam Path Typical Applications Generally the selection of a suitable color look up table for a specific use depends on the user s own judgment and needs Experience shows however that certain color look up tables are particularly useful for specific uses Color Map Green Red Blue Gray P Color 1 P Color 2 3 4 5 6 Geo Land Geo Sea Geo Land amp Sea R amp B Glow Glow Over Glow Under Glow Over amp Under User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Application This is commonly used for recording specimens that have been marked with FITC Cy2 DTAF or other similar fluorochromes which emit within the green spectral range This is commonly used for recording specimens that have been marked with TRITC Texas Red Cy3 Rodamine or other similar fluorochromes which emit within the red spectral range This is commonly used for recording specimens that have been marked with UV fluorochromes such as DAPI or Hoechst or other similar fluorochromes which emit within the blue spectral range This is commonly
171. m Path Setting dialog window gt Activate a detector by clicking its corresponding check box A cast shadow now links the activated detector to the corresponding slide bar on the scale of the spectrum gt Double click this slide bar to open the Range Properties dialog window where you can specify the end point of the series Drag the slide bar on the scale of the spectrum to the desired start position The left edge of the slide bar represents the wavelength at which the recording is to begin Click Lambda Scan Begin to save the value The end point of a wavelength series is set in the same manner In addition you also have to set the number of wavelength steps LL see Defining the End Point for a Wavelength Series LL see Setting the Number of Wavelength Steps Typical Applications A wavelength series can be applied to determine the maximum emission of a fluorochrome This is useful because the Stokes shift of the emission curve of a fluorochrome is dependent on each specimen that is applied This then allows you to set the detection range precisely for a specific application Defining the End Point for a Spatial Series End Function Use the End button to define the end point of a spatial image series First set the exact z position or y position with the z y Position button or with the corresponding rotary knob on the control panel Then click End This saves the position value for the end point The begin point is set in the
172. mage anytime by clicking on the Original button Note This SFP projection can be activated using the SFP button and the Image Tool dialog window 3D button SFP icon If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be stored as a new file in the current experiment LL see Image Tool Dialog Window 3D Button LL see Creating an Experiment User Manual Leica LCS english Page 104 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Creating a Topographical image JIEN Topa Function Use the Topography function to select specific intensity data from an image stack i e a series of xy or xz sections and transfer them into a two dimensional topographical image This function examines the sampling points voxels that are superimposed along the z axis throughout all optical sections From each of these columns of sampling points only the intensity value that is met by the selection criterion is displayed in the two dimensional topographical image as the representative of all values within the column You can make your selection based on either the maximum intensity or the center of mass of the measured intensities If you want to represent the intensity maximums in the topographical image only the sampling point at which the maximum
173. matically Defining the Region of Interest ROI Function You can use the Wizard button to have the computer define any structure in the image as a Region of Interest ROI automatically in the image Depending on which of the buttons you have clicked an elliptical rectangular or polygonal region of interest is placed around the structure Click on the Ellipse Rectangle or Polygon buttons then on the Wizard button gt Double click with the left mouse button on the structure in the image that you want to mark as region of interest gt The structure is automatically marked as an elliptical rectangular or polygonal region of interest pa LL see Defining the Region of Interest ROI as Ellipse LL see Defining the Region of Interest ROI as a Polygon LL see Defining the Region of Interest ROI as a Rectangle pa LL see Deleting Regions of Interest ROIs Other Requirements The definition of a region of interest in the image is required for quantification functions The Wizard button is only enabled when you have previously enabled a quantification function using one of the following buttons see Measuring a Profile Within a Region of Interest ROI see Calculating a Histogram see Measuring Roughness Within a Region of Interest ROI User Manual Leica LCS english Page 119 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg G mbH LCS Software Fu
174. maximum is adjustable so that differently scaled data sets can be adapted to each other If a data set with a small interval of intensity values is combined with a data set featuring a large intensity value range the first one is shown darker than the second one Adapting the maximum values of both data sets scales them in such a way that equal parts of the intensity values of both data sets are included in the merged image This setting defines the resolution of the merged image An 8 bit resolution can display 256 different intensity levels A 12 bit resolution can display 4096 different intensity levels This option takes the brightest pixel each from the two images to be merged to calculate the corresponding pixel of the merged image This option calculates the arithmetic mean from the two images to be merged to calculate the corresponding pixel of the merged image Note If two strongly different images are calculated to a merged image by using the average method the black image background causes a reduction of the image intensity Tab x y z t la Definition of the stereoscopic arrangement of the output images in the resulting image before combining Field Image 1 2 Shift Append check box Result Element number User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Description of Function The position in x y z t A of the output image within the merged image is set here This value is typically
175. me username is an open parameter which must be replaced by the currently defined user name Note Factory installed hard disks feature 2 partitions C and D The user directory should be set up on partition D User Manual Leica LCS english Page 12 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions The Leica Confocal Software Overview Starting the Software 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 Note 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 T
176. me of the color look up table Field for internal use Dimension of the color look up table typedef struct FARSTRUCT tagSAFEARRAY unsigned short cDims dimensions in this array unsigned short fFeatures unsigned long cbElements of the array size of unsigned long cLocks void HUGEP pvData data SAFEARRAYBOUND rgsabound 1 dimension SAFEARRAY Count of DON T CARE Size of an element Does not include pointed to data 17 DON T CARE Pointer to the One bound for each Important The Leica specific file format LEI uses only one dimensional data fields arrays SAFEARRAYBOUND Structure Represents the bounds of one dimension of the array The lower bound User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Page 153 of 164 Leica Microsystems Heidelberg GmbH User Manual Leica LCS english of the dimension is represented by 1Lbound and cElements Appendix represents the number of elements in the dimension The structure is defined as follows typedef struct tagSAFEARRAYBOUND unsigned long cElements elements long LLb und SAFEARRAYBOUND VARIANT and VARIANTARG typedef struct FARSTRUCT tagVARIANT VARIANT typedef struct tagVARIANT VARTYPE vt DON T CARE unsigned short wReservedl unsigned short wReserved2 unsigned short wReserved3 union unsigned char short long float double VARIANT BOOL VT_BOOL S
177. measuring _ I Z I Z2 I t1 point 1 and 2 I to I l I l2 d z t A Height z recording time t bandwidth 21 Zol ti tol li Note User Manual Leica LCS english between the measuring points 1 and hl 2 This quantification function is only available off line only available for previously recorded data records Measuring Surfaces and Volumes Function The Materials function calculates surfaces and volumes of the three dimensional spatial data set Click on the Materials button to automatically open a window in which a measurement curve and the size of the accumulated volume are displayed depending on the z position Accumulated volume means that for each z position the value of the volume of the data set lying below it is calculated A requirement for calculating the surfaces and volumes is a typographical Page 113 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions image Thus the Materials button is only active if the Topography button was used before to create a topographical image of the data set Only one detection channel can be quantified at a time LL see Creating a Topographical Image Parameter Meaning scanned This value represents the horizontal projection of the surface of Area A a xy section Surface This value represents the actual surface of a xy section In Area A relation to the horizontal pro
178. mpling points of the individual images superimposed along the projection axis are examined throughout all optical sections From each of these columns of sampling points the arithmetic average is calculated from all intensity values and displayed in the two dimensional projection image as the representative of the complete column see Principles and Types of Projections Click the Fix Avg button An average projection of the current image stack is created and displayed in the Viewer window You can return to the original image anytime by clicking on the Original button In the Viewer Options dialog window you can also adjust a threshold and scale the intensity value range present in the image see Viewer Options Dialog Window Projections Icon Note An average projection can be activated either by using the Fix Avg button or in the Image Tool dialog window via 3D button Orthogonal Projection icon If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be stored as a new file in the current experiment LL see Image Tool Dialog Window 3D Button LL see Creating an Experiment User Manual Leica LCS english Page 101 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Transparent Projection of an Image Stack with Invariable Project
179. n the factory default configuration profile is located in the toolbar Note 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 gt Customize In the dialog window that appears select the Commands tab under the File category With the left mouse button click the Apply button and drag it while holding the mouse button pressed to the desired window Release the left mouse button to insert the button at the current location 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 Experiments can only be saved in the Leica specific lei format When you are saving experiments you may be able to save existing individual images in tif or raw format Note If you are saving an experiment or image that has already been saved before the old data will be overwritten If you do not want to do this select File gt Save as fo save the new or changed data to a new file with anew name User Manual Leica LCS english Page 15 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Funct
180. nctions Selecting and Moving the Region of Interest ROI M vvvyv Function Using the Select ROI button you can select and move a Region of Interest ROI within the image Click first the Select ROI button and then the ROI you want to move ove the mouse pointer into the ROI As soon as the mouse pointer changes to a crossed double arrow press and hold the left mouse button Keeping the left mouse button pressed move the ROI You can change the size of the region of interest you have drawn Hold the mouse pointer over one of the ROI s edges until a double arrow appears Press the left mouse button to shift the shape of the ROI in one di gt if rection you place the mouse pointer exactly over a corner of the ROI you can change its shape in two directions at once AR AR User Manual Leica LCS english see Moving and Rotating a Region of Interest ROI see Deleting a Region of Interest ROI Other Requirements The definition of a region of interest in the image is required for quantification functions Therefore the Select ROI is deactivated until you activate a quantification function using one of the following buttons LL see Measuring a Profile Within a Region of Interest ROI see Calculating a Histogram see Measuring Roughness Within a Region of Interest ROI Page 120 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functio
181. ndividual isolines in mm This allows you to limit the number of isolines in the 3D view Use the Isoline detail level field to enter a limiting value specifying that only isolines of a particular length are to be displayed in the 3D view This limits the displayed isolines to those which correspond to an intensity value that has a specific frequency of occurrence User Manual Leica LCS english Page 59 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Viewer Options Dialog Window Surface Measure Icon Function To open the Viewer Options window select the Experiment Overview option in the View menu The Experiment Overview window appears on the left side of the user interface The top part of the viewer window displays the recorded images in a directory tree The bottom part displays the Viewer Options window This dialog window is used to perform basic settings for different software functions The left side shows the icons corresponding to the functions and the right side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons In the tab of the Surface Measure icon you can make optional settings for the following functions see Measuring Roughness Along a Line Segment In the Leveling field there are options for an adaptation function to bring measurement curves
182. ndow and created as a new file in the current experiment Click on the button with the arrow symbols to show the preview window on the right edge of the dialog window or to hide it The projections triggered using Show are displayed temporarily in this preview window The Close button closes the dialog window Projections and Creating Projection Images Along a Definable Animations Icon Projection Axis User Manual Leica LCS english Running Projection Images as 3D Rotation Animation Creating Red Green Stereo Views Creating Projection Images Along a Definable Projection Axis In the Projections tab select one of the three projection types maximum projection average projection and transparent projection see Principles and Types of Projections Click on Rescale in the Options field to standardize the intensity values of the image to the maximum possible intensity values when creating the projection image This ensures that the calculated gray values of the projection image remain within the value range of for example 0 to 255 at 8 bits This allows for brightening dark images If you click on Invert in the Options field the projection axis is mirrored The calculation of the projection image is thus in the opposing direction This function makes sense above all in the case of inverse microscopes to match the calculation of the projection image to the inverse beam path of laser light The Transparent Factor field is only active if the
183. ndow or an annotation sheet can be printed When you initiate the Print command the image currently loaded in the Viewer window or the currently open annotation sheet is printed using the default printer and default page layout You can change these default settings in the Printer Selection dialog window to suit your needs Use the following buttons provided in the dialog window to do so Button Function Printer setup This opens the dialog window for selecting a printer and changing the printer settings Print This prints the image or the annotation sheet Background Use this to open a dialog window for selecting a background color color for the page Center on page This centers the image on the page Fit to page This option changes the size of the image to fit the size of the printable area Aspect ratio The height to width ratio remains the same when the size of the image changes Use the Image field to change the height Size Y and width Size X of the image and to define a top margin Offset Y and a left margin Offset X The image size and margins cannot be modified if the Fit to page option is enabled If you have enabled Center on Page you can change the size but not the margins Check the results of your settings in the Print Preview field Use the Page field to specify the height and width of the printable area which is not identical to the paper size The two bottom lines of the dialog window display the currently in
184. ng a Rectangle on the Annotation Sheet Rect Function Use the Rectangle button to draw a rectangle on the annotation sheet On the annotation sheet click the position where you want the corner of the rectangle to be Keeping the left mouse button depressed drag the mouse pointer across the page to define the size of the rectangle If you mark the rectangle and then press the right mouse button a context menu containing the following commands appears Command Function Style Color User Manual Leica LCS english This opens a dialog window where you can configure among other options the style and thickness of the line This opens a dialog window for selecting a color for the rectangle Page 124 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note Note LCS Software Functions Bring to This brings the rectangle to the foreground front sent to This sends the rectangle to the background back Delete This deletes the rectangle You can change the size of the rectangle by dragging one of the image grab points of the rectangle To move the rectangle without changing its size click in the middle of the rectangle and move it while pressing and holding the left mouse button The Rectangle button can be applied only if the annotation sheet has been opened using the Annotation button first Adding a Text Field to an Annotation Sheet Function Use the Text button to inse
185. ng to the formula Pixel Intensity peau Pixel Intensity suremas X Constants Division Divides the selected image by the constants according to the Min formula Pixel Intensity gesu Pixel Intensity source image Constants Selects for each pixel the minimum value of the selected image and constants according to the formula Pixel Intensity zeu MIN Pixel Intensity sarema Constants Max Selects for each pixel the maximum value of the selected image and constants according to the formula Pixel Intensity gear MAX Pixel Intensity scrceimace Constants Average Calculates for each pixel the average of the selected image and constants according to the formula Pixel Intensity gear 1 2 x Pixel Intensity scrceimace Constants AND The binary representations of the pixels of the constants and User Manual Leica LCS english the image are linked with the logical operator AND That means that pixels in the result image are given the value 1 when the value 1 is present in both of the pixels otherwise 0 is entered see Boolean Operations Page 80 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH OR LCS Software Functions The binary representations of the pixels of the constants and the image are linked with the logical operator OR That means that pixels in the result image are given the value 1 when the value 1 is present in at least one of the two pixels otherwise 0 is entered s
186. ning dark images If you click on Invert in the Options field the projection axis is mirrored The calculation of the projection image is thus in the opposing direction This function makes sense above all in the case of inverse microscopes to match the calculation of the projection image to the inverse beam path of laser light The Transparent Factor field is only active if the transparent projection was selected The slider is used to vary the transparent factor a The higher it is set the more the intensity values from the lower levels in the image stack affect the projection image LI see Principles and Types of Projections gt Move the slider in the Threshold field to define a threshold value The intensity values below this value are not taken into account for the creation of the projection image Click on the Show button to obtain a preview of the projection images Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the projection as a new data record Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment Note The three projection types can also be enabled directly using the corresponding keys Fix O Proj Fix Max Fix Avg Fix Trans If you activate the function using the button the projection image will just be a temporary display on the monitor You
187. nnenrnnennnnennn 64 Image Tool Dialog Window Basic Button ranrrnnrrnnrrnnrrnnnrnnnrrnnrrnnrrnnnrnnnrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnne 71 Image Tool Dialog Window Amplitude ButtOn cc cccecccceeeeeeeneeeee eens esse esse eeseeeseeeseeeseeeseenes IT VIEWING detection Nanne lS aereas a a A sene 83 VIEWING Detection Channel 14 83 Zooming Image s in the Viewer Window ccccecccseeceeeeeeeeeeseeeeseeeeseeeeseeeseeeeseueeseeeeseesaeeesseeees 84 Selecting Cobr MAPS LUT ernea a a E 84 Viewing the Single Mag E HSS a 86 VIEWING Mutipl Image Lasse a a 87 VIEWING all Overlay NIM AGC See 87 VIEWING Mage SONOS Lee 88 Viewing the First Image of a Series ccccccccssccceseeceseeceeecceeceuceceuceseueeseessueeceaeessueesseeesseesaeeeess 88 Display the Next Image in a Series cece cecccceecccseeceececeuceceueecaeeeceeceucecsueessueeseessueeseueessueesseeeseas 88 Displaying the Previous Image in a Series rrrarrnnnrrnnnrnvnnrrranennanennnnennnnnnanrnnannennnsnnnssnnanennnsennnnennn 89 Viewing the Last Image of a Series rrnnrrnnnnrnnnnnnnnrrnanernanennnnennnnnnnnrnnanrnnnnennnnennnnnnnnrnnanennnnennnnse 89 Displaying an Image From a SETPICS ccccccccescccsecceececeucecaueecueeccseecececeusesaueesaeeseueessueessueessneesaes 90 Staningand Endin Pave 90 Viewing the Series NIE sr 91 PrO 2164 18 ER ER EE NR NE EE 92 Principles and Types of Proeclons suaraeasvadsrdgakkemaaenaebksl
188. nnnernnrrrnnnnranrnnnnenrnnnnrnnennnne 131 Measuring Roughness Along a Line Segment rrannnnannnnanannanennnnnnnnnnnanennnnnennnnnnnnennnnennuennnnennnene 134 Measuring Roughness Within a Region of Interest ROl rrrrrrrrnnnrnanrrrannennnnrnnnernarennnnennnnnnnnnnn 136 GOSA Sister esata ated date deci ate aco ethene Ube eae duatel a cate tame eae te euatetteaetetee 138 APPEAR 141 LES FE FOTEN 144 Formats of User specific and Device dependent Data rrrnnnnnnnnnnannnnnnennnnennnnnnnnrnnnnennnnennnnennnene 144 Fixed Leica specific File FOMals aves diesel Gis vc cradiedi avandia ocedicaicees tied duievellats oisielleddvwnellatactoeeted sne 145 Leica Microsystems Heidelberg GmbH Legal Notes Legal Notes Version 2 0 August 01 2001 Made in Germany Copyright 2000 2001 Leica Microsystems Heidelberg GmbH All rights reserved No part of this publication may be reproduced or transmitted in any form or by any means electronic or mechanical including photocopying recording or storing in a retrieval system or translating into any language in any form without the express written permission of Leica Microsystems Heidelberg GmbH DISCLOSURE This document contains Leica Microsystems Heidelberg GmbH proprietary data and is provided solely to its customers for their express benefit of safe efficient operation and maintenance of the product described herein Use or disclosure of Leica Microsystems Heidelberg GmbH proprietary d
189. notation 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 Specification of the Lei file format version beta 2 000 When a file is saved in the LCS software a subdirectory is first created under the current directory The following files are saved in this subdirectory 1 A description file its structure is described below This file is used for interpreting the data of an experiment through the LCS software 2 The individual image files the following convention is used in assigning the name of the image files NAME channel number dimensionindex Thus Chloroplast C1 Z1 means that Chloroplast is the name of the data record assigned by the user that the image corresponds to the first optical section of a three dimensional data stack Z1 and that the data was acquired in the first detection channel C1 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 Meaning Possible values Byte arrangement 0x49494949 Intel LSB 0x4D4D4D4D Motorola MSB Identification for Leica 0x016033F0 Lei format Version number current x20000000 Address of directory table any possible addre
190. nrrnnrrnnrnnnnnnn 41 Last Button EEE 89 Last Image of a Series rrrrrnnnrrnnrnnnrnnnrnnnnnnn 89 LCS file formats ccccccssccceeeeceseeeneees 144 Legal notes r arrrnnrnnnnnnnnonnnnnnnnnnnnnnnnnnnnnnnvnnn D LETE Ol re 48 Leica Confocal Software LCS rrrrnnrrnnnnrr 13 Length of the measured section 112 LEVEN Hk 61 141 EE 124 line averaging rrrnnnnrnnnnrnanrnnanrnnnnnnnnnnnnnennnne 45 LINE BULON NE 124 Linear Filters rrrnnnnnnnnnnnnnnnannnnnnnnuner 64 131 Loading and Saving Parameter Settings 22 Long Pass Filter rrrarnnnrnnnrrvnrrnnrrnnrennnrnnnr 138 Look Up Table rrrarorarerarerarerarerarennnennre 48 84 Look up Table Button rrrarernnrrnnrrnnrnnnnnnn 84 Lowpass Filter rrrrrrrrnnrrnnrrnnrnnrnnnnnnn 64 131 OE tenon aecena denen 48 84 Magnification cccccceeeeees 26 51 84 108 Materials Button rrrnnrnnrrnnnrrennnnrrvnnrrnnnnr 113 Page 159 of 164 Leica Microsystems Heidelberg GmbH Maximum Intensity rronrrnnrrnnrrnnrrnnrnnnnrnn 105 Maximum Projection 56 64 92 95 96 97 98 105 Maximum value 06 77 109 110 112 Measurement Slider 2 0 0 eeeeeeeeeeeeeeeeees 53 Measuring ranrnnnrrnnrvnnrrnnrvnnrnnnnvnnnnnnnnnnnsnn 112 Measuring a Profile Along a Line Segment112 134 Measuring a Surface rrnnnrrnnnrnnnnnrnanernnrr 113 Measuring Roughness
191. ns Moving and Rotating the Region of Interest ROI Function Using the Rotate ROI button you can move and rotate a Region of Interest ROI within the image Click first the Rotate ROI button and then the region of interest you want to rotate Place the mouse pointer exactly on the corner of the selected region of interest As soon as the mouse pointer changes to a double arrow press and hold the left mouse button Keeping the left mouse button pressed rotate the region of interest The region of interest will be rotated around its center point vv wv yy You can change the size of the region of interest you have drawn Hold the mouse pointer over one of the ROI s edges until a double arrow appears Press the left mouse button to shift the shape of the ROI in one direction LL see Moving and Rotating a Region of Interest ROI LL see Deleting Regions of Interest ROIs Other Requirements The definition of a region of interest in the image is required for quantification functions Therefore the Rotate ROI is deactivated until you activate a quantification function using one of the following buttons see Measuring a Profile Within a Region of Interest ROI see Calculating a Histogram see Measuring Roughness Within a Region of Interest ROI User Manual Leica LCS english Page 121 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH a AI AI AI
192. nt Click the Apply button Mode Recording an Image Using Burst Mode Function If the workstation must process large amounts of data during the recording of images it may lead to a delay of the scanning process This is because the recorded image is first displayed on the monitor before the scanner can scan additional images The Burst function allows for decoupling the scan process of the laser and the refreshing of image data on the screen For this purpose the transmission of image data from the program memory to the monitor is delayed but the scanning process is not By clicking on the Burst button you can select operating modes with different delay times Description No Burst Image data are recorded and simultaneously and continuously displayed on the monitor Frame Burst Image data are not displayed on the monitor until a single image has been recorded Complete Image data are not displayed on the monitor until an image Burst series has been recorded Automatic The software automatically sets the optimum operating mode Note User Manual Leica LCS english for a specific application The following parameters can increase the amount of data to be processed to the extent that it will delay the display on the monitor Scanning format scanning speed bi directional scan number of active detection channels calculation of an overlay image and size of the Viewer window Recording an image using the
193. nts To move the image without changing its size mark the image and move it while pressing the left mouse button The Snap button can be applied only if an annotation sheet has been opened using the Annotation button first and the Viewer window has been clicked Page 123 of 164 Leica Microsystems Heidelberg GmbH LCS Software Functions Drawing a Line on the Annotation Sheet Function Use the Line button to draw a line on the annotation sheet On the annotation sheet click the position where you want the line to begin Keeping the left mouse button depressed drag the mouse pointer across the page to the position where you want the line to end If you mark the line and then press the right mouse button a context menu containing the following commands appears Command Function Style This opens a dialog window where you can configure the style thickness and length of the line Color This opens a dialog window for selecting a color for the line Bring to This brings the line to the foreground front Send to This sends the line to the background back Delete This deletes the line Note You can change the length of the line by dragging one of its grab points To move the line without changing its size click the center grab point and move the line while pressing and holding the left mouse button The Line button can be applied only if the annotation sheet has been opened using the Annotation button first Drawi
194. nts measurement value at the measurement points difference of the measured variable between the measurement points 12 22 pm Changing the position and length of the measurement slider gt To change the position move the mouse over the slider click and hold the left mouse button and drag it to the desired position gt To change the length of the slider grasp it at one end with the mouse and enlarge or reduce it while pressing the left mouse button Changing the scaling of the measurement curve By default the scaling of measurement curves is automatically adapted by the software to the intensity value range that is present in an image recording However the scaling can also be changed manually User Manual Leica LCS english Page 54 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Deselect the Auto check box to deactivate automatic scaling You can now redefine the upper and lower limit value of the value range separately for each measurement curve each detection channel Check the results of the changed scaling directly in the window Vv Viewer Options Dialog Window Online Measure Icon Function To open the Viewer Options window select the Experiment Overview option in the View menu The Experiment Overview window appears on the left side of the user interface The top part of the viewer window displays the recorded images in a directory tree The bo
195. o 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note LCS Software Functions Command Function Line Use this to insert a line of default size onto the annotation sheet Rectangle Use this to insert a rectangle of default size onto the annotation Text Zoom Grid sheet Use this to insert a text field with a default font onto the annotation sheet Use this to select from four magnifications for viewing the annotation sheet This displays a grid on the annotation sheet which cannot be printed The Line Rectangle and Text commands can be activated both from the context menu and by clicking their corresponding buttons Saved annotation sheets are given the ano file extension Copying an Image into the Annotation Sheet Function If you click the Snap button the image loaded in the Viewer window is copied onto the annotation sheet If you mark the copied image and then press the right mouse button a context menu containing the following commands appears Command Function Original size This displays the image in its original size Fit to page This enlarges the image to the size of the annotation sheet Bring to front This brings the image to the foreground Send to back This sends the image to the background Delete This deletes the image User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 You can change the size of the image by dragging one of the image grab poi
196. o s s t Dg 3 5 4 45 5 the original image are subtracted ii ii from the values of the low pass filtered image The result corresponds to an image that was processed with a high pass filter Intensity Profile of the Image After Application of the Highpass Filter This filter type exclusively represents the clearly defined transitions from low to high intensity values in the image gt Inthe Filter Type field select the Sharp filter After the low pass filter has been applied to the image the values of the high pass filtered image are genomes a added to the values of the original image Intensity Profile of the Image After Application of the Sharp Filter This filter type filters the low frequencies out of the image That is the very distinctive transitions from low to high intensity values are intensified This filter is also used to amplify the noise in an image User Manual Leica LCS english Page 70 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Use the slider in the Sharp Factor field to vary the effect of the Sharp filter If this factor is set to 0 the filter is not active The value specified in the Kernel Size field as Cut off Wavelength is the size of the filter measured in pixels This value is calculated on the basis of the scan parameters enlargement of the objective wavelength of the excitation light scan format electronic
197. of objectives and the symbolic representation of the slots on the objective nosepiece Find in the list the objective that you are using and select it with the mouse Click and hold the left mouse button and drag the objective onto the symbol representing the slot in which the objective is installed The assignment is saved in the software and the objective appears in the selection list which you can open by pressing the Objective button Repeat this procedure for every objective that you have installed in the objective revolver You can use the Add Remove and Edit buttons in this dialog window to add new objectives remove objectives or edit existing objective labels The microscope models DM RXA DM RXE DM IRBE and DM IRBE2 use a software program to control the objective nosepiece The program automatically rotates the selected objective into the beam path whenever you select an objective by using the button or Objective dialog window All other microscope models require not only software adjustment of the objective but also manual rotation of the objective into the beam path Additional information When selecting the correct objective for a specific application the objective s correction class achromatic apochromatic fluorite objectives and plane objectives and especially the magnification factor and the numerical aperture are of great significance The numerical aperture determines the resolution capacity of an objective and is d
198. ogical z wide value not physical but logical value 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 Presently only three entries of the SAFEARRAY structure are used 1 sa cDims should 1 Presently only one dimensional data fields arrays are used 2 sa cbElements 3 sa rgsabound 0 cElements Size of elements in byte indicates the number of elements contained in the structure User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Page 149 of 164 Leica Microsystems Heidelberg GmbH Appendix size Datatype Symbol Description byte 24 SAFEARRAY sa Microsoft type see Appendix 1 for all elements that contain the structure sa rgsabound 0 cElements 128 wchar Identifier for the contents of the entry 64 wchar Name of a short description e g a TIFF description tag 64 wchar String contains the contents of the string 16 VARIANT Contains the data type and the data value except for the string data type which is described in the field above 4 DWORD Separate memory space for data values 4 DWORD Not in use 4 DWORD Identifier ID of the memory block for internal use only 4 DWORD Test value ID TIMEINFO m
199. ontrol Panel icon in the My Computer window to view and change any system component The Control Panel contains numerous icons that allow you to control your system The particular icons that you see on your PC may be slightly different from those illustrated due to that fact that you may have different hardware installed and may or may not be connected to a network or modem You may also have different Windows NT Workstation 4 options installed Double clicking the Network Neighborhood icon displays the Network Neighborhood dialog box which gives you information about who and what is connected to your workstation It provides an easy mechanism for browsing any network systems and resources that you may be able to connect to in a way that is independent of the actual type of network vendor Traditionally if a system needed to be simultaneously connected to different types of network the way in which each could be connected and viewed would be vendor specific Windows NT Workstation 4 is capable of displaying a common view of your entire network even though it may actually comprise resources from Windows NT Novell NetWare Banyan Vines or others The Inbox icon is used if Microsoft Exchange is active on your system Windows NT Workstation 4 has built in electronic mail services based on User Manual Leica LCS english Page 8 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Microsoft M
200. op It provides a background for the items it contains The initial icons on the desktop allow the user to view and interact with the system in a logical way The Windows NT screen contains many special elements and controls Here is a brief summary The background on which all the pictures and boxes rest is the desktop The taskbar shows the windows and programs that are open You can switch between open windows and programs by clicking the name on the taskbar The Start button opens a menu system from which you can start programs Click on the Start button then click on your selection from each menu that appears Some icons appear on your desktop You can activate one by double clicking on it We now take a brief tour of the items you see on the screen A standard desktop item is the My Computer icon Double clicking this icon opens the My Computer window The My Computer window gives you easy access to the major components like hard and floppy disk drives of your computer system or workstation For example by double clicking the Hard disk C icon you can see the contents of your PC s hard drive This allows the user to view local resources as objects The Windows NT Workstation 4 control panel and print control support are also accessible from My Computer If you installed one of the additional applications such as Dial Up Networking at installation time it will also appear in the My Computer window You can use the C
201. opened and viewed in the Leica Confocal Software 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 image uses 2 bytes whereby the first byte contains the higher valued bits in accordance with the INTEL format little endian Each optical section is saved for each channel in a separate file with the file extension raw using the following naming convention name of experiment_name of data record_channel number_z dimension_number of optical sections raw 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 format or RAW format Tiff files tif These are Leica image files in single and multi TIFF format Both image files in the previously used TCS formats and external files in RGB TIF User Manual Leica LCS english Page 145 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note Appendix format can be read Annotation ano The format of this type of file is also Leica specific and binary It is used for saving an
202. or a stack of xy sections plus pause interval Stacks Number of recordings of the image stack Complete Total recording time i e the product of AT and number of Time recordings Each parameter can be calculated in relation to the other parameters When entering the values note how the dialog window operates The entry field you click on will be activated Click on the parameter to be calculated The corresponding entry field is grayed out gt Now enter the values for the other parameter Click on Apply to calculate the parameter By clicking on Reset the last saved values are displayed When you have finished configuring these settings press the Series scan button Starting a Single Scan Function Use the Single Scan button to record a single image from a single optical level in the specimen Before recording an image using the single scan function configure all User Manual Leica LCS english Page 34 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions required scan parameters using the Continuous Scan function to ensure optimal image quality see Starting a Continuous Scan Typical Applications The single scan function is designed for recording bleach sensitive specimens Use this function in the case of bleach sensitive specimens not only for image recording but also as an alternative to the continuous scan function for setting the scan parameters The single scan
203. ore you turn off your PC The Shut Down option allows the user to close the Windows NT Workstation operating system and ensure all running processes can halt cleanly and are given the chance to flush any data out to the disk that may be in cache memory Several options are available when shutting the system down User Manual Leica LCS english Page 11 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Caution Powering down your computer without prior shutting it down may result in severe data loss Setting Up Users Under WinNT 1 Logging on as administrator Log on as administrator by using the ID Administrator and the password Admin 2 Opening the User Manager Select Start gt Programs Administrative Tools User Manager 3 Defining a new user Enter at least the following information in the opened dialog window 1 User name 2 Password must be re entered in the following line for confirmation purposes Select the following two check boxes 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 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 userna
204. osystems Heidelberg GmbH LCS Software Functions within the column This is repeated for each column of sampling points 3a 3b Projections with invariable or variable projection axes There are two main groups of projection types available in the Leica Confocal Software These are distinguished by the type of projection axis There are projections with invariable axes as well as projections with variable axes The first type is orthogonal projections The projection axis is always the optical axis that runs vertical to the stack of images the z axis with horizontal xy cuts and the y axis with vertical xz cuts In contrast the projection axis for the second type may be freely selected With the exception of the SFP projection the same three projection types are available in both of the two main groups The Projection Types Projection Type Invariable Projection Variable Projection Axis Axis Maximum Projection Average Projection Transparent T Projection Trans SFP Projection j lt o ad gt i SFP Activate the projection type either with the corresponding button or in the Image Tool dialog window If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be stored as a new file in the current experiment The projections with invariable projection axes are located in the Image Tool
205. ow 3D Icon Function To open the Viewer Options window select the Experiment Overview option in the View menu The Experiment Overview window appears on the left side of the user interface The top part of the viewer window displays the recorded images in a directory tree The bottom part displays the Viewer Options window This dialog window is used to perform basic settings for different software functions The left side shows the icons corresponding to the functions and the right side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons The tabs of the 3D icon can be used to perform optional settings for the following functions LL see Rotating the 3D View LL see Moving the 3D View LL see Zooming the 3D View The Navigation tab displays the numerical values of the actions that are carried out using the Rotate Move and Zoom buttons and the mouse pointer Use the Rotation field to tilt a 3D view in all three spatial directions by modifying the angular degrees of the three axes The 3D view is rotated around a fixed point which is located in the center of the image To best understand the rotation function modify the angle of one axis at a time while leaving the other two axes set to 0 User Manual Leica LCS english Page 51 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Ro
206. pixel to be calculated In this case the gray values of the 5 pixels corresponding to the weighting scale 1 4 6 4 1 are multiplied by the factors 1 16 4 16 6 16 4 16 1 16 and together added to the final value With respect to the calculation of the pixel C in the following schematic representation this means that the gray values of pixels A B and D E are included in the calculation of the filtered pixel C C is calculated as follows 16x1 16 4x4 16 8x6 16 32x4 16 8x1 16 1 1 3 8 0 5 13 5 A B C D E Gray values of pixels A E in the original 16 4 8 32 8 image Factors of kernel size 5 1 16 4 16 6 16 4 16 1 16 Gray value of C in the filtered image 13 5 A calculation of this kind is performed for each pixel in the original image At the edge of the image the filter mask protrudes beyond the image For these pixels the value 0 is set Inthe Kernel Size field select a value for the kernel size m In the Filter Directions field set the 0 5 1 ER 22 ae 9 35 4 45 5 dimensions of the filter Select the jeep spatial axes to which the filter is to Intensity profile of an unfiltered be applied sequentially One image dimensional filters are applied that is the filter is calculated only via one axis at any given time gt Inthe Filter Type field select the Smooth filter The low pass filter described above is applied to the image is m fs i This filter type filters the high Intensity profile of
207. pographical image Topography Function Processing Invert height The column of sampling points is examined in reversed direction This reverses the height information A negative of the topographical image is displayed Level The horizontal position of a recording is corrected using a linear interpolation function polynom see Image Tool Dialog Window Basic Button Threshold Function Drag the slider on the Intensities that are below the specified threshold scale or enter a numerical value are ignored in the calculation of the value topographical image User Manual Leica LCS english Page 61 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Image Tool Image Tool Dialog Window Schematic Structure of the Dialog Window The numerous functions of the Image Tool dialog window are arranged on a number of levels The gray buttons 3D Basic Amplitude Materials and Multicolor on the left side of the dialog window represent the first level of the hierarchy Each of these buttons has a group of icons that appear below the selected button The icons are the next level on which functions are grouped Each icon opens different tabs and fields that are displayed on the right side in the dialog window and arrange the functions on the lowest level The following elements can be found in each tab In the Source Image drop down list at the top of the dialog window select t
208. ponding rotary knob on the control panel LL see Controlling Functions via Control Panel Additional information While zoom factors from 1 to 32 can be set the useful magnification of the electronic zoom cannot be increased infinitely The limit is achieved using the smallest optically resolvable distance which is determined by the resolution capacity of the objective According to the Nyquist theorem this smallest optically resolvable distance can only be mapped without information loss if it is scanned with about 2 to 3 raster points If the scan frequency is exceeded at a relatively high zoom factor and the specified scan format further magnification is no longer useful because no more optical details can be resolved zero order magnification see Selecting a Scan Format User Manual Leica LCS english Page 26 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions see Selecting an Objective Note With bleach sensitive specimens it is advisable to restrict the use of the electronic zoom to short time intervals Since you are recording a segment of the specimen with a relatively higher scan frequency in the case of high zoom factors the specimen is exposed to a stronger impact of light This can lead to photomechanical destruction of the specimen optical bleaching Optimal Zoom Factors The following table shows which zoom factors marked red depending on the scan field size an
209. re displayed 4 5 separately in the case of image series the first image in each is applied Tiled Gallery All individual images in an image series are Channel 1 2 3 4 5 displayed for all selected detection channels Tiled Overlay All selected detection channels are displayed both Channel 1 2 3 4 5 separately and in an overlay image together In the case of image series the first image in each is applied Tiled Gallery All individual images of an image series are Overlay Channel 1 2 displayed both separately for all selected detection 3 4 5 channels and in overlay images together Note The Tiled and Single buttons cannot be enabled simultaneously because they carry out opposing functions Viewing an Overlay Image Function Clicking the Overlay button displays all selected detection channels together in an overlay image in the Viewer window The following display modes are available depending on the other options you enable simultaneously Button Combination Display Single Overlay A single overlay image is generated from all Channel 1 2 3 4 5 selected detection channels for an image series the first image of each selected detection channel is used Gallery Single Based on the number of individual images in an Overlay Channel 1 2 image series overlay images are generated from 3 4 5 all selected detection channels Tiled Overlay All selected detection channels are shown User Man
210. rements The definition of a region of interest in the image is required for quantification functions Therefore the Ellipse button is deactivated until you activate a quantification function using one of the following buttons see Measuring a Profile Within a Region of Interest ROI see Calculating a Histogram see Measuring Roughness Within a Region of Interest ROI Defining the Region of Interest ROI as a Polygon a Poly Function Using the Polygon button you can define any polygonal Region of Interest ROI within the image Click the Polygon button Click the left mouse button on the image position where you would like to place a ROI corner gt In order to define the other polygon corners release the mouse button move the cursor to the next polygon corner and click the left mouse button again Repeat this step for each desired polygon corner gt If you want to draw the polygon by hand keep the left mouse button pressed and draw the ROI into the image Close the polygon with a double click Vv LL see Selecting and Moving the Region of Interest ROI LL see Moving and Rotating a Region of Interest ROI LL see Deleting Regions of Interest ROIs Other Requirements The definition of a region of interest in the image is required for quantification functions Therefore the Polygon button is deactivated until you activate a quantification function using one of the following buttons User
211. rface The top part of the viewer window displays the recorded images in a directory tree The bottom part displays the Viewer Options window This dialog window is used to perform basic settings for different software functions The left side shows the icons corresponding to the functions and the right side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons In the tab of the Surface Calculation icon you can make optional settings for the following functions see Creating a Topographical Image The Topography tab contains various options for generating a topographical image Select whether the intensity maximums or centers of mass of the intensities are to be interpreted as height in the topographical image Specify the type of visualization for height in the topographical image and set a threshold value for the displayed intensities Surface Function Reconstruction Search maximum From each of the examined columns of sampling points intensity the sampling point with the highest intensity is displayed in the two dimensional topographical image as the representative of all values within the column Calculate center of From each of the examined columns of sampling points mass of intensities the center of mass of the surface which is restricted by the curve of the measured intensity values is displayed in the two dimensional to
212. ri es kieo iane 64 131 SN cae PEES E P E I E 123 Nap PUNO EE 123 User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Index Software Reference Menu Functions 16 Spatial Image Series rrrarrrnnrrnarenanennnrr 40 41 Spatial Representation rrrrnnrrnnnrnnannennn 106 Spectral Scan r rnnnnnnnnnnnnnnnnnnnnnnnnnnennner 37 41 Spectral Series ccccccseeceeeeseeeseeeeeees 37 41 Speed Button rrarrrnnrnnnrnnnnevnnevnnrnnnnnnnnnnnnnnn 32 Speichern adecuswinscrandemwupiudadian db tween 127 Stack Processing of Data rrrrrrrnnnnnnanennnrr 46 Standard deviation ararnunnnnnnr 109 110 112 SL PO 36 Start Point for a Spatial Series 36 37 Start Point for a Wavelength Series 37 Starting a Series SCAN rrarernnennnennrnnnrnnnnnnn 41 Starting and Ending a Film eronnornnnrrnnnrnnre 90 Statistical Calculations rrrrrnnrrnrnnrrrnnnrr 109 SEP Widtsoe 40 Stereo VICW cecececeecececeeeecececeseeaeeeeeeess 64 71 Stokes Displacement cccceeeeeeeeeeees 138 Subtracting IMAGES rrrnnnrnnnnnnnnnnvnnnnnanennnne 77 SUD FACTION sc icciasdcamctensehsaasenccahccessiescanwatonsdensijuiens 77 Suppressing NOISE cccceecceeeeeeeseeeeeeeees 46 Surface Structure rrrnnrnrrnnnrvrnnnnenvnnnvnnnnre 105 TASC FP Luren 88 Taste Image Tool rronrnnnnnnrnnnnnnrnnnnnnnnn 77 131 Taste Look up Table
213. round of the legend The Hardware legend is aligned automatically along the right edge of the user interface You can change however the size and position of the legend as desired To change the width of the legend use the mouse pointer to drag the edge to the position you desire gt To change the position of the legend double click on the double lined edge of the legend or click the 5 icon once This releases the legend from the user interface turning it into a window Now you can drag the legend to any position by pressing and holding the left mouse button User Manual Leica LCS english Page 50 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Note If the legend has been enlarged to cover the entire width of the user interface you first have to shrink the height of it in order to drag it back to the edge of the user interface Option New Window You can open a second Viewer window containing the same image as the currently open Viewer window by clicking the New Window option in the Window menu This command does not create a new experiment but rather opens a second view of the current image data This feature is useful for viewing different representations of the same image data at the same time Sequential numbers are added behind the file extension to distinguish between the resulting copies of the current Viewer window Viewer Options Viewer Options Dialog Wind
214. rt a text field onto the annotation sheet On the annotation sheet click the position where you want the text field to be placed Keeping the left mouse button depressed drag the mouse pointer across the page to define the size of the text field If you mark the text field and then press the right mouse button a context menu containing the following commands appears Command Function Font This opens a dialog window used to select from various fonts and other formatting options Transparent This makes the background visible through the text field Bring to This brings the text to the foreground front Send to This sends the text to the background back Delete This deletes the text User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 You can change the size of the text field by dragging one of the image grab points of the text field To move the text field without changing its size click in the middle of the text field and move it while pressing and holding the left mouse button The Text button can be applied only if the annotation sheet has been opened using the Annotation button first Page 125 of 164 Leica Microsystems Heidelberg GmbH LCS Software Functions Printing Function You can trigger the print command either by clicking the Print button or from the Printer Selection dialog window To open this dialog window select the Print option in the File menu The image loaded in the Viewer wi
215. rted image Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the converted image as a new data record Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment Brightness amp Contrast Icon Setting image brightness and contrast Procedure Select an image from the Source Image drop down list The drop down list contains all the recorded and calculated images of the experiments displayed in the Experiment Overview viewer window For multichannel images select the All Channels check box to make all changes for all channels at one time and to the same extent For images that do not use the entire dynamic range select the Linear Model check box If the check box has been clicked there is a linear relationship between the intensity in the output image and the result image Whether an image uses the entire dynamic range available can be seen by creating a histogram of the image If the histogram does not cover the entire intensity range see Min and Max the dynamic range is NOT being used in full LL see Calculating a Histogram If you have images in which you wish to highlight the medium intensities more strongly select the non linear model Linear Model check box NOT enabled Inthe Options field use the sliders to change the brightness and or image contrast Options
216. s a suitable correction lens is brought into the beam path for each objective suitable for use with UV excitation light To use this corrective feature proceed as follows Click the Objective button standard procedure in Acquire step see Selecting an Objective Select a UV capable objective from the list see indication on list Note User Manual Leica LCS english In this case the corrective lens for the selected objective is automatically moved into the beam path The possible occurrence of a focus offset between visible light and UV light has physical reasons and is not a malfunction of the device Other Requirements In order to achieve an automatic correction of the physically based color length fault between visible and UV light it is not sufficient to manually rotate the objective into the beam path Data Viewing The Viewer Window ee In the default setting the Viewer window consists of three important areas The image window 4 in which the recorded images are displayed is located in the middle Button pads 1 and 2 can be arranged to the left and below the image window and the Experiment legend 3 to the right of it It is possible to select other default settings of the Viewer window and to save user defined configurations as a template Page 48 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions see Saving Viewer Window Viewer a
217. s a Template Button pads 1 and 2 By default the button pad to the left of the image window contains the buttons for image viewing functions and the button pad below the image window contains the buttons used for scrolling through the individual images of an image series Both button pads can be moved within the Viewer window or removed from it as separate windows To do so click on the double lined edge of the button pad and drag it while keeping the mouse button depressed to the desired position Image window 4 If you place the mouse pointer at any position within the image window and then press the right mouse button a context menu containing the following commands opens Command Function Send to Experiment Selection This copies the raw data of a raw selected area in the image window as a new image in the current experiment The new image can be processed Selection This creates a new image in the snapshot current experiment from the snapshot of a selected area in the image window The new image can no longer be processed All This creates a new image in the snapshot current experiment from the snapshot of the entire current image window The new image can no longer be processed Printer Selection This prints the area selected in the image window All This prints the entire current image window Left This shows or hides the button pad to the left of the image buttons window Bottom This shows or hides the b
218. same manner see Setting the z y Position see Controlling Functions via Control Panel Note You can also define the begin and end points for a spatial series in the Series Scan Overview dialog window Open this dialog window by pressing the Series button the small button not the button that starts the Series Scan function User Manual Leica LCS english Page 38 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions LL see The Series Scan Overview Dialog Defining the End Point for a Wavelength Series Function For a wavelength series this function records a stack of individual images each of which are detected at a specific wavelength from a single optical level Use the Lambda Scan End button to define the wavelength at which recording should end Note The Lambda Scan End button is only active if you used the Mode button to select a scanning mode with the wavelength dimension see Selecting a Scan Mode gt Press Beam to open the Beam Path Setting dialog window gt Activate a detector by clicking its corresponding check box A cast shadow now links the activated detector to the corresponding slide bar on the scale of the spectrum gt Double click this slide bar to open the Range Properties dialog window where you can specify the end point of the series Drag the slide bar on the scale of the spectrum to the desired end position The right edge of the
219. scription of Function Multicolor Cross Removing cross talk between detection Talk channels User Manual Leica LCS english Page 63 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH gt LCS Software Functions Schematic Structure of the Dialog Window The numerous functions of the Image Tool dialog window are arranged on a number of levels The gray buttons 3D Basic Amplitude Materials and Multicolor on the left side of the dialog window represent the first level of the hierarchy Each of these buttons has a group of icons that appear below the selected button The icons are the next level on which functions are grouped Each icon opens different tabs and fields that are displayed on the right side in the dialog window and arrange the functions on the lowest level The following elements can be found in each tab In the Source Image drop down list at the top of the dialog window select the image data record to be processed If you click on the Use Selected Image check box the image data record currently displayed in the Viewer window is used for the calculation If you click on the Show button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the preview window in the dialog window If you click on the Apply button at the bottom of the dialog window the selected calculation is activated and the result image is displayed in the Viewer wi
220. se button a new maximum projection is calculated and displayed The projection axis is the viewing direction of the user to the newly positioned image stack Use the Zoom 3D View button to enlarge or reduce the projection see Zooming the 3D View You can return to the original image anytime by clicking on the Original button In the Viewer Options dialog window you can also adjust a threshold and scale the intensity value range present in the image see Viewer Options Dialog Window Projections Icon Note A maximum projection can be activated either with the 3D Max Button or in the Image Tool dialog window with the 3D button and the Projections and Animations icon If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be stored as a new file in the current experiment LL see Image Tool Dialog Window 3D Button LL see Creating an Experiment Note Note the difference between a maximum projection and a topographical image which is generated from intensity maximums In a maximum projection the intensity maximums are assigned color values directly In a topographical image based on intensity maximums the intensity maximums are assigned real z positions of the individual sampling points first and then are color coded LL see Creating a Topographical Image User Manual Leica LCS engl
221. series a time series or a wavelength series The statistical values are calculated dependent on the size of the third dimension height z for spatial series time t for time series wavelength for wavelength series Click on the Profile z button and draw a region of interest ROI in the image The buttons used to define a region of interest are activated User Manual Leica LCS english together with the Profile z button Art No 15 9330 033 Vers 01082001 1 see Defining the Region of Interest ROI as Ellipse LL see Defining the Region of Interest ROI as a Polygon Page 110 of 164 Leica Microsystems Heidelberg GmbH LCS Software Functions LL see Defining the Region of Interest ROI as a Rectangle LL see Automatically Defining the Region of Interest ROI LL see Selecting and Shifting the Region of Interest ROI pa LL see Deleting the Region of Interest ROI gt A window opens automatically to display the measurement curve for every detection channel as well as the statistical values Parameter Meaning Formula pixel ROI Number of pixels within the selected region of interest Area Area of the region of Fi interest AS ae A 0 Length Height of image 5 5 stack spatial series L 4ix3 x4 ly2 y1 Recording time time series Bandwidth wavelength series Mean The arithmetical 1 Amplitude mean H 7 No Pixel Pixel Maximum Maximum value Max I
222. side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons The tab of the Overlay icon can be used to perform optional settings for the following functions see Viewing an Overlay Image On the Overlay tab you can select among three types of creating an overlay image from raw data and the corresponding color look up tables User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Page 57 of 164 Leica Microsystems Heidelberg GmbH LCS Software Functions Color Merging Function True Color add The color values of the pixels in the original images RGBs are averaged and displayed in the overlay image True Color add The color values of the pixels in the original images RGBs with dynamic are averaged scaled to the maximum possible color adjustment values and displayed in the overlay image Fast bit wise OR The color values of the pixels in the original images RGB are mixed using a fast bit by bit calculation process and displayed in the overlay image see Boolean Operations Coloring Function Only Red The color look up tables Red Green and Blue are always used Green Blue to create the overlay image independent of the current color look up tables of the original images Viewer Options Dialog Window Scan Progress Icon Function To open the Viewer Options window select the Experiment Overview option in th
223. sition button or the corresponding knob on the console LL see Setting the z y Position LL see Controlling Functions Using the Console Set and store the end point for an image series using the End button or in the Series Scan Overview dialog window see Defining the End Point for a Spatial Series Select the number of spatial sections using the Sections button LL see Defining the Number of Spatial Sections When you have finished configuring these settings press the Series Scan button You can track the recording process of the image stack in the Series scan Overview dialog window Before recording a time series configure the following settings Select a scan mode with time dimension using the Mode button 1 see Selecting a Scan Mode Select the scan format using the Size button LL see Selecting a Scan Format gt Set the desired z position or y position using the z y position button or the corresponding knob on the console LL see Setting the z y Position LL see Controlling Functions Using the Console gt Set the number of recordings the pause interval between the recordings and the complete editing time see Configuring a Time Series Before recording a wavelength series configure the following settings Select a scan mode with wavelength dimension using the Mode button LI see Selecting a Scan Mode Select the scan format using the Size button see Selecting a
224. so that they can be reproduced and made available for other experiments mac Macros are saved in this format Macros contain VBA Visual Basic for Application program code Leica provides the individual program objects for controlling the confocal system in an object model Additional information about the development of macros for controlling the confocal system can be found in the chapter on 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 pbo Templates for the assignment of the control panel are saved in this format These templates define which scan parameter is controlled by which rotary knob This format also includes factory presets that have proven useful for most standard applications Self defined templates are saved in the user specific directory pro Specific profiles of the user interface are saved in this format This includes information on the type of key and its location in a specific toolbar In addition new toolbars can be defined or existing defined toolbars can be hidden or displayed The position of the toolbars User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Page 144 of 164 Leica Microsystems Heidelberg GmbH Appendix whether embedded or freely moveable is also stored in a profile
225. ss within the complete A length of the file Directory table A Address Meaning Entry data type Address of Number of entries in this table DWORD A A A 4 Index of the first entry represents a logical memory DWORD block e g acquisition parameter hardware settings or image parameter dimensions A 8 Address A 1 of the 1st entry DWORD A 12 Index of the first entry represents a logical memory DWORD User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 block e g acquisition parameter hardware settings or image parameter dimensions Page 146 of 164 Leica Microsystems Heidelberg GmbH A 14 Appendix Address A2 of the 2nd entry DWORD 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 optical sections etc Each entry in the directory table corresponds to a logical memory block Block table Xn Address Meaning Data type A1 or A2 or Check digit DWORD A3 A A 4 bytes Description of the contents of the DWORD block table currently not used A 8 bytes Version of entry DWORD A 12 bytes Size of entry DWORD A 16 bytes Begin of entry The data type depends Size byte 4 KAR nExt 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 D
226. stalled printer and configured paper size Note Close the Printer Selection dialog window by clicking OK to save the new settings for the active Viewer window This allows you to store different printer settings and page layouts for different images However these settings are not permanent and are lost when you exit the Leica Confocal Software User Manual Leica LCS english Page 126 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Data Handling Opening a File Function With the Leica Confocal Software you can open various file types using the Open button File Type Description Experiment A Leica specific binary data format When the experiment is lei loaded both the image data and the experiment settings are loaded Annotation A Leica specific binary data format The elements on the sheet ano annotation sheet such as images texts and graphic images are each available as individual objects Tiff files tif These are Leica image files in single and multi Tiff format External files in RGB Tiff format can be loaded as well LL see Creating an Experiment L Note see Creating an Annotation Sheet Saving a File Function Click the Save button to save the data of the current experiment lei or the current annotation sheet ano If you use the Save button to save an experiment or annotation sheet for the first time the
227. stems Heidelberg GmbH LCS Software Functions Viewer The view window consists of the buttons the image window Standard the color bars of the color look up table and the Experiment legend User Manual Leica LCS english In the Personal Templates List box the user defined templates are entered Using the Add Active Viewer and Remove Template buttons you can save a new template or delete an existing one In the Tools menu click on Options to indicate in the Workspace tab how many view windows if any Viewer should open when the Leica Confocal Software is started Controlling Functions Using the Control Panel Function At frequent points during an image recording various parameters have to be reset For this reason the control panel can be used to control functions quickly and directly You can freely allocate the radio buttons of the control panel to a selection of functions In the View menu select the Status Bars option and then Control Panel Status Bar The status bar for the control panel is displayed at the bottom of the user interface of the Leica Confocal Software Viewed from left to right the status bar consists of the icon for the control panel the seven fields that represent the radio buttons of the control panel and three buttons Click on one of the fields arranged in the same way as the radio buttons of the control panel A list is opened where you can select the desired function The name of the allocate
228. surement The Clear button deletes them again Note This quantification function is only available off line only available for previously recorded data records User Manual Leica LCS english Page 135 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Measuring Roughness Within a Region of Interest ROI Function The Roughness Area function measures height distance and angular values of a surface within a region of interest The profile is measured from the data record selected in the Viewer window This data record should be a spatial series A topographical image is the prerequisite for the generation of a height profile For this reason the Roughness Area button is active only when a topographic image of the data record has been created in advance with the Topography button see Creating a Topographical Image Click on the button Roughness Area and draw a region of interest ROI into the image The buttons with which you define a ROI are activated together with the Roughness Area button AI AI Ty AI hy AI hy AI hy Yr Yam om A see Defining the Region of Interest ROI as an Ellipse see Defining the Region of Interest ROI as a Polygon see Defining the Region of Interest ROI as a Rectangle see Automatically Defining the Region of Interest ROI see Selecting and Moving the Reg
229. t consider creating a shortcut for it on the desktop To do so just use the right mouse button to drag an object out of Windows Explorer or My User Manual Leica LCS english Page 9 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Caution Caution User Manual Leica LCS english LCS Software Functions Computer After releasing the mouse button a menu appears Select the option Create Shortcut s Here Some programs automatically create a shortcut during their installation procedure Windows NT Workstation 4 does not actively track a link between an original and a shortcut For instance if you create a shortcut of a program and subsequently move rather than copy the original to a different folder then the shortcut may no longer function The Startup folder is special in one respect any programs located in this folder will start automatically when Windows NT Workstation 4 is started The Documents menu shows the names of the 15 files you created most recently You can open any of these files and its related application at the same time by clicking the file s name in this menu Document files that are opened within an application typically by selecting the File Open command within the application will not be displayed here Only documents opened directly from the Desktop will be displayed here The Settings menu offers three commands for changing your system s settings You can directly
230. t Galvo using the Z Scan button Likewise the scan modes for a xy and xzA wavelength will only be displayed if only one detector is activated in the Beam Path Setting dialog window see Setting the Beam Path Selecting a Scan Speed Speed Function Clicking the Speed button opens a dialog window that you can use to select from four scan speeds Speed 200 Image lines per second 400 Image lines per second 800 Image lines per second 1000 Image lines per second The data recording speed is further increased in combination with the bi directional scan see Unidirectional or Bi directional Scan Additional information The higher the set scan speed the shorter the dwell time of the laser point Furthermore the scan format i e the number of sampling points in a row needs to be taken into account The higher the scan format at a constant speed is the shorter the dwell time of the laser point over a sampling point The longer the light point of the laser beam dwells over the individual sampling points of the specimen the more light is detected by the detector So using a lower scan speed results in a better signal to noise ratio The disadvantage of a lower scan speed is that the relatively long impact of the light on the specimen can bleach the specimen photochemically making it unusable This is especially important with fluorescence applications Note If the speed levels 800 or 1000 are set technical limitations prevent the maximum scan field
231. t above these values is transmitted The transmission values are generally at 80 and the reflection values are between 90 and 95 A microscopic objective used without immersion media Between the objective lens and the specimen is air An acronym based on the words volume and pixel A Voxel represents the smallest indivisible volume element in a three dimensional system In this documentation both the volume elements of the specimen as well as the 3D image points are referred to as voxels Page 140 of 164 Leica Microsystems Heidelberg GmbH Appendix Appendix Adaptation Function For Correcting Trend Curves see Image Tool Dialog Window Basic Button 1 Selecting the step width that is the distance of the support positions 2 Selecting the character of the interpolation function to be adapted 20 ap 60 30 100 120 142 3 Calculating the interpolation function f x a bx cy 4 Adding the intensity differences to the leveling of the trend curve um 20 40 60 80 100 1420 140 160 after leveling User Manual Leica LCS english Page 141 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Appendix Example of moving an image in the result image m Source Image 1 froughness249 tit Selection OD v M Use Selected Image Leveling Source Image 2 froughness249 tit Projection_00 v I Use Selected Image Voxel amp E Jch m Image 1 Num
232. tation X from 0 to 45 Y from 0 to 45 Z from 0 to 45 LCS Software Functions Function The 3D view is rotated 45 around the fixed point in the direction of the negative z axis The 3D view is rotated 45 around the fixed point in the direction of the negative y axis The 3D view is rotated 45 around the fixed point in the direction of the negative y axis Use the Translation field to shift a 3D view to the right or left up or down and enlarge or shrink it by modifying the coordinate values Users of all regional versions should be sure to use the dot as the decimal placeholder Translation Function X Y Z Positive values shift the 3D view to the right negative to the left Positive values enlarge the 3D view negative shrink it Positive values shift the 3D view up negative down Use the Predefined field to change the viewing perspective of the image to Top view or Side view by clicking these buttons respectively In the Display tab click on one of the listed commands to show or hide the corresponding graphic element of the 3D view in the Viewer window Graphic Elements Function Show LUT Show scale Show the selected color look up table on the z axis Show the scale Show bounding box Show the bounding box which limits the measurement volume Show axes Show the coordinate axes Show data during Show the image data while the 3D view is being rotated 3D motion shifted enlarged or shrunken
233. tensity interval 0 255 and or 0 4095 is only used for the image display functions for color look up in contrast the actual result values are used for the quantification functions If you select 12 Bit Output in the Output Option field each pixel in the result image is coded with 12 bits i e the result values are saved in a value range from 0 4095 In the case of scaled data the intermediate values are interpolated Click on the Show button to obtain a preview of the arithmetical operations Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the calculation operation as a new data set Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment Bit Converter Conversion of 8 Bit Image Resolution to 12 Bit Image Icon Resolution and Vice Versa gt gt User Manual Leica LCS english Procedure Select an image from the Source Image drop down list The drop down list contains all the recorded and calculated images of the experiments displayed in the Experiment Overview viewer window In the New Resolution field select the image resolution into which you want to convert the selected image Page 81 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Click on the Show button to obtain a preview of the conve
234. the image after frequencies out of the image That is applying the Smooth filter the very distinctive transitions from low to high intensity values are reduced User Manual Leica LCS english Page 132 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions This filter can be used to remove noise from an image gt Inthe Filter Type field select the Highpass filter After the low pass filter has been applied to the image the values of em 0 5 1 EO sn s d OG 35 4 45 5 the original image are subtracted i aj from the values of the low pass Intensity Profile of the Image After filtered image The result Application of the Highpass Filter corresponds to an image that was processed with a high pass filter This filter type displays only the very distinctive transitions from low to high intensity values in the image gt Inthe Filter Type field select the Sharp filter After the low pass filter has been applied to the image the values of 0 5 1 TS oa se SA 35 4 45 5 the high pass filtered image are T 4 added to the values of the original image Application of the Sharp Filter Intensity Profile of the Image After This filter type filters the low frequencies out of the image That is the very distinctive transitions from low to high intensity values are intensified This filter is also used to amplify the noise in an image Use the slider in the
235. the optimized setting of the detectors In addition this function can be used to determine the emission maximum of a wavelength series By adapting the width and the position of the screens in front of the detectors more emission light from the specimen can be recorded than with conventional optical filters In time series this function can be used to determine the time of the highest fluorescence activity This quantification function is only available off line only available for previously recorded data records Measuring a Profile Along a Line Segment Function The Profile function measures a measurement variable along a line segment displays it in a curve and calculates various statistical values The profile is measured from the data record selected in the Viewer window This data set can be a spatial series a time series or a wavelength series The statistical values are calculated dependent on the size of the third dimension height z for spatial series time t for time series wavelength for wavelength series Click on the Profile button The measurement section is displayed as a white line in the image in the Viewer window You can change the length and position of the line segment by clicking the line and while clicking and holding the grab point with the left mouse button dragging it to the desired position gt A window opens automatically to display the measurement curve for every detection chann
236. threshold and scale the intensity value range for the image see Viewer Options Dialog Window Projections Icon Click the 3D Trans button A transparent projection of the current image stack is created and displayed in the Viewer window You can now change the projection axis by clicking in the image in the Viewer window and holding the left mouse button pressed Broken support lines are displayed that encloses the symbolically displayed image stack Turn the image stack to the desired new position by moving the mouse pointer while holding down the left mouse button Upon releasing the left mouse button the new transparent projection will be calculated and displayed The projection axis is the viewing direction of the user to the newly positioned image stack Use the Zoom 3D View button to enlarge or reduce the projection LL see Zooming the 3D View You can return to the original image anytime by clicking on the Original button A transparent projection can be activated using either the 3D Trans button or alternately the Image Tool dialog window 3D button Projections and Animations icon If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be stored as a new file in the current experiment LL see Image Tool Dialog Window 3D Button LL see Creating an Experiment Page 103 of 164 Art
237. tions tab The projection axis is freely variable The basis for a projection is an image stack i e a series of horizontal xy sections or vertical xz sections When a projection is generated the sampling points of the individual images superimposed along the projection axis are examined throughout all optical sections From each of these columns comprised of sampling points depending on the projection type the maximum intensity value or the arithmetical average or the weighted average of all intensity values is calculated and displayed in a two dimensional projection image representing the entire column LL see Principles and Types of Projections Click the 3D Proj Button A maximum average or transparent projection of the current image stack is created and displayed in the Viewer window You can now change the projection axis by clicking in the image in the Viewer window and holding the left mouse button pressed gt Broken support lines are displayed that encloses the symbolically User Manual Leica LCS english Page 96 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions displayed image stack Turn the image stack to the desired new position by moving the mouse pointer while holding down the left mouse button Upon releasing the left mouse button the new projection will be calculated and displayed The projection axis is the viewing direction of the user to the ne
238. transparent projection was selected You use the slider to vary the transparent factor a The higher it Page 64 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions is set the stronger the intensity values flow from the lower levels in the image stack into the projection image In general this includes more information in the projection image see Principles and Types of Projections gt Move the slider in the Threshold field to define a threshold value The intensity values below this value are not taken into account for the creation of the projection image gt To set the projection axis click on the Rotation tab see below for description Click on the Show button to obtain a preview of the projection images Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the projection as a new data record Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment Note The three projection types can also be enabled directly using the corresponding keys 3D Proj 3D Max 3D Avg 3D Trans If you activate the function using the button the projection image will just be a temporary display on the monitor You can undo this using the Original key LL see Projection of an Image Stack With Variable Projection Axis LL
239. ttings as follows gt Activate each of the desired detectors by clicking on its corresponding Active check box below the color look up table icon A cast shadow now links the activated detector to the corresponding slide bar on the scale of the spectrum gt Specify the wavelength range for the detection by dragging the two ends of the slider to the desired positions on the scale or gt Double click the slide bar to open an additional dialog window in which you can enter a precise value for the start point and the end point of the wavelength range For fluorescence applications list fields with common fluorescence dyes have been set up for each detector Select the desired fluorescence dye to display its emission curve in the spectrum Select a color look up table by clicking on the corresponding icon see Selecting Color Look Up Tables User Manual Leica LCS english Page 23 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note User Manual Leica LCS english LCS Software Functions Selecting an Objective Function Click the Objective button to open a list of objectives and make an appropriate selection This list contains only the objectives that were previously assigned to one of the maximum of seven threaded ports on the objective nosepiece You can assign objectives as follows Select the Objectives option from the Tools menu A dialog window opens containing an extensive list
240. ttom part displays the Viewer Options window This dialog window is used to perform basic settings for different software functions The left side shows the icons corresponding to the functions and the right side shows the related tabs When you open the dialog it contains the icons of the functions that you are currently using Click Show all to view all icons In the tab of the Online Measure icon you can make optional settings for the following functions The slider on this function can be used to set the number of optical sections for which the measured values of an online measurement are displayed graphically The developing trend in an experiment can be seen from the online measurement graph Note The trend data only exists in temporary form and it cannot be saved The temporarily saved data can however be exported via the context sensitive menu right mouse button or copied into an annotation sheet LL see Exporting Quantification Data LL see Copying Quantification Data to the Annotation Sheet Typical Applications Quantitative evaluation of data records User Manual Leica LCS english Page 55 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Viewer Options Dialog Window Projections Icon Function To open the Viewer Options window select the Experiment Overview option in the View menu The Experiment Overview window appears on the left side of
241. ual Leica LCS english Page 87 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH Note User Manual Leica LCS english LCS Software Functions Channel 1 2 3 4 5 separately as well as together in an overlay image In an image series the first image is used Tiled Gallery All individual images of an image series are Overlay Channel 1 2 displayed both separately for all selected detection 3 4 5 channels and in overlay images together Use the Viewer Options dialog window to select from three different methods of color mixing for generating an overlay image From the View menu select Viewer Options Click on the icon Overlay see Viewer Options dialog window icon Overlay Viewing Image Series Viewing the First Image of a Series Function You can view the individual images of an image series as a film sequence Click First to jump to the first image in the series If the Gallery button is enabled the First and Last Next and Previous and Play Stop buttons are disabled and displayed in gray The Gallery button is used to show all of the individual images of an image series and does not allow film sequence mode Display the Next Image in a Series Function You can view the individual images of an image series as a film sequence Click Next to jump to the next image in the series Page 88 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelber
242. utton pad located below the image buttons window LUT This shows or hides the color look up tables of the current image Legend This shows or hides the Experiment legend Full screen This enlarges the Viewer window to full screen Viewer This opens the Viewer Options dialog window Options User Manual Leica LCS english The color look up tables 5 are displayed as color bars to the right of the image window If you hold the mouse pointer over a color bar grab points appear at the top and bottom of the color bar You can use these grab points to limit the current color look up table to a specific intensity range and to load a second color look up table This allows you to increase the contrast of the image graphically Page 49 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions Drag the top grab point down or the bottom grab point up Double click the area above or below the corresponding grab point The Select LUT s dialog window opens so that you can select a second color look up table The upper and lower intensity ranges are represented in the colors of the second color look up table vvvyv see Selecting Color Look Up Tables LUT The Experiment 3 Legend Various image parameters of an image recording are registered in the Experiment legend To select the parameters that you want displayed click any area of the legend This opens a list of all available entries Then
243. values from the lower levels in the image stack will be included in the projection image SFP Projection Simulated Fluorescence Process mathematical simulation of a fluorescence process This projection type uses arithmetic operations to simulate in a specimen the process of absorbing laser light and emitting fluorescent light This procedure is common in fluorescence microscopy It consists of two arithmetic steps 2 1 A 3 The first step simulates the laser beam 1 which penetrates the sample User Manual Leica LCS english Page 94 of 164 Art No 15 9330 033 Vers 01082001 Leica Microsystems Heidelberg GmbH LCS Software Functions and is thereby weakened We assume a lateral light source striking the Voxel of the image stack The weakening of the incoming beam while traversing the image stack is represented by the following expression vn In fl oe n Inf VE Whereby In 1 is the remaining intensity of the incoming light after absorption in the nth Voxel In is the intensity of the incoming light in the nth Voxel a is the absorption coefficient of the laser light in the sample Vn is the intensity in the Voxel and Vmax is the maximum Voxel intensity The quotient Vn Vmax corresponds to the standardization For each Voxel the illumination strength Vin In x Vn is calculated using the calculated intensities In This information is temporarily saved in a table In intensity of the incoming light in Vox
244. vature aberration is corrected for objectives of this type Correcting this error requires lenses with stronger concave surfaces and thicker middles Three types of plane objectives plane achromate plane apochromate and plane fluorite are based on the type of additional correction for chromatic aberration Abbreviation for Region of Interest ROI encloses an area for which a measurement analysis is to be performed On top of that an ROI can also designate the area of a specimen to be scanned ROI scan The ratio of signals detected in the specimen to the unwanted signals that are caused randomly by various optic and electronic components which are also recorded by the detector The Stokes shift is a central term in fluorescence microscopy If fluorescent molecules are excited with light of a specific wavelength they radiate light of another larger wavelength This difference between excitation light and fluorescent light is referred to as Stokes shift Without Stokes shift separating the high intensity excitation light from the low intensity fluorescence signals in a fluorescence microscope would not be possible Triple dichroic filters are interference filters at an angle of incidence of light of 45 The transmissivity or reflectivity of triple dichroites depends on three specific wavelengths of light For a tripledichroite TD 488 568 647 for example the excitation light at 488 nm 568 nm and 633 nm is reflected and the excitation ligh
245. view information on it User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Page 19 of 164 Leica Microsystems Heidelberg GmbH LCS Software Functions Keyword Search Index Select the Index option from the Help menu to view an index of key words Select a key word View the corresponding content pages by double clicking the key word or selecting it and then clicking the Display button Full text Boolean Search Select the Search option from the Help menu to launch the full text search engine Enter your search word in the input field Click 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 Examples Result Pinhole AND This phrase finds help topics containing both the word Sections pinhole and the word sections Pinhole OR This phrase finds help topics containing either the word Sections pinhole or the word sections or both Pinhole This phrase finds help topics containing the word pinhole and NEAR the word sections if they are located within a specific search sections radius This method also looks for words that are similar in spelling to the words specified in the phrase Pinhole NOT This phrase finds help topics containing the word pinhole and Sections not containing the word sections vv vv User Manual Leica LCS english
246. window Arithmetic With Two Running Arithmetical Operations with Images Images Icon and or Masks Procedure Select two images from the Source Image 1 and Source Image 2 drop down lists The two drop down lists contain all the recorded and calculated images of the experiments displayed in the Experiment Overview viewer window Select the desired arithmetical operation in the Operation field Note A requirement for running arithmetical operations is equal dimensions or a 1 n relationship of the two data records Equal dimensions means that two images must have the same format in x and y and that two image stacks must have the same number of single images z The 1 n relationship exists when for example 1 single image is offset against each nth single image of an image stack The following arithmetical functions can be run with two images and or masks Operation Addition Subtraction Multiplication Division Min Max Average AND User Manual Leica LCS english Art No 15 9330 033 Vers 01082001 Result Adds the intensity values of the two selected images according to the formula Pixel Intensity resut Pixel Intensity source image 1 Pixel Intensity Source Image 2 Subtracts the intensity values of the two selected images according to the formula Pixel Intensity resut Pixel Intensity source image 1 Pixel Intensity Source Image 2 Multiplies the intensity values of the two selected
247. wly positioned image stack Use the Zoom 3D View button to enlarge or reduce the projection see Zooming the 3D View You can return to the original image anytime by clicking on the Original button The Viewer Options dialog window allows you to indicate a threshold and to scale the intensity value range for the image see Viewer Options Dialog Window Projections Icon Note You can activate a projection by either using the 3D Proj Button or the Image Tool dialog window If you activate the function using the button the projection image will just be a temporary display on the monitor When activated through the dialog window the projection image will be Stored as a new file in the current experiment LL see Image Tool Dialog Window 3D Button LL see Creating an Experiment Maximum Projection of an Image Stack with Invariable Projection Axis Function Using the button Fix Max Button starts a maximum projection For this function the projection axis is always the orthogonal axis Z axis for horizontal xy sections and y axis for vertical xz sections The basis for a projection is an image stack i e a series of horizontal xy sections or vertical xz sections When a projection is generated the sampling points of the individual images superimposed along the projection axis are examined throughout all optical sections From each of these columns of sampling points the maximum intensity value is display
248. ymbol and select the desired beam splitter gt Neutral filters such as the RT 30 70 filter are used in reflection applications to lead the excitation light to the sample and the reflected light to the detection pinhole Dichroic filters such as the reflection short pass filter RSP 510 are used in fluorescence applications to split and detect fluorescent light of a particular wavelength range from the excitation light Double dichroic filters are used for image recording where the preparation is marked with two fluorescent dyes and two excitation wavelengths The DD 488 568 double dichroic for instance is used to split excitation light with a 488 nm and 568 nm wavelength from the detection light Depending on the system configuration you can also select DD 488 543 and DD 458 514 type double dichroites gt Triple dichroic filters are used for image recording if the preparation is marked with three fluorescent dyes and three excitation wavelengths The TD 488 568 633 triple dichroic for instance is used to split excitation light with a 488nm 568nm and 633nm wavelength from the detection light Depending on the system configuration you can also select TD 488 568 647 and TD 488 543 633 type triple dichroites Setting detectors and color look up tables LUT Below the spectrum in the dialog window the boxes for the four detectors PMT 1 to 4 as well as for the PMT Trans transmitted light detector are shown Adjust the se
249. ys at the same time will log you on After pressing the Ctrl Alt and Delete keys the Login information dialog box appears 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 Windows NT separate files are created for managing user specific profiles of the LCS software For information about setting up users under Windows NT please refer to the chapter Setting Up Users under WinNT in this manual After logging on with your user ID you may change your password by pressing the keys Ctrl 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 Note If you miss type the confirmation password you will see a warning
250. zoom factor used for each image The value specified in the Filter Directions field as Cut off Wavelength is the size of the filter measured in nanometers For example with a value of 160 nm all wavelengths lesser than or equal to 160 nm are filtered gt Click on the Show button to obtain a preview of the filtered images Show starts the calculation and displays the resulting images in the Image Tool dialog window Or Click on the Apply button to create the filtered images as a new data record Apply starts the calculation displays the resulting images in the Viewer window and saves them as a new file in the current experiment Schematic Structure of the Dialog Window The numerous functions of the Image Tool dialog window are arranged on a number of levels The gray buttons 3D Basic Amplitude Materials and Multicolor on the left side of the dialog window represent the first level of the hierarchy Each of these buttons has a group of icons that appear below the selected button The icons are the next level on which functions are grouped Each icon opens different tabs and fields that are displayed on the right side in the dialog window and arrange the functions on the lowest level The following elements can be found in each tab Inthe Source Image drop down list at the top of the dialog window select the image data record to be processed If you click on the Use Selected Image check box the image data record currently

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