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Leica Application Suite

1. LAS User Manual 4 176 Power Mosaic Scan Direction Scan Direction The stage Scan Direction may be either side to side or front to back The options are Allow the software to determine the best direction Automatic Select side to side Horizontal or Select front to back Vertical Generally Automatic is the best option to ensure the most efficient scan especially when Turboscan is being used 1 Click on the arrows to the right of the Direction header to reveal the options drop down menu 2 Click to select the required direction Although both specimens illustrated are the same a vertical scan on Figure 4 would be best because there are fewer direction changes 4 177 2007 Leica Microsystems Switzerland Ltd Power Mosaic Advanced Options Advanced The Advanced options allow individual image tiles to be saved temporarily Buffered to a nominated folder and if they are saved set the size of the thumbnails in that folder It is important that there is sufficient disk space to accommodate all of the images otherwise the scan will stop The recommended approach is to have a partitioned section of the disk D PMTemp for example in which to buffer the images Make sure that computer current user has privileges extended to the partition Thumbnail size is also important in that they are initially stored in RAM for fast access If the thumbnails are t
2. s ssasesesesussnunnunusunnsennununununnunununnnunnnnanununenennnnnn 4 187 Predictive Focus Continued s asasesesesseneennunununnnernunununennnnununununnnnanununenennnnnn 4 188 Au t f cUS O a a e A N 4 189 Greate Pattern Grids oisein raora paara a a Rar raa aad Aaa 4 190 Create Pattern Grid Continued a asesessesesununusunnnernununununnununnnnnunnnnununnnnnnnnnnnn 4 191 Create Pattern Grid Continued ooo cccccsssssssescsssessevsessessessessesessevnsaesaes 4 192 Save Configuration cccssssesssesssseeesseeesseeestseeseseeasseeasseeaeseseesteanseeeestsenateess 4 193 GIS 4 194 E CHE 4 195 Z Stacks Continued oo cccscseescesssssescsseesesseesessesseesevseuseesevausevaesaesansaasanses 4 196 Z Stacks Continued oo csssseecessesescsseesssseesessesseusesseuseesevaussevausaesneaesanses 4 197 Browse Save amp Display wo cc ccccccsssssessessessessessessevsevessessesaessesaesaesansaesaes 4 198 CN UCNE 4 199 SAVEIMOSAIC gpass iania 4 200 Save Mosaic Continued asasasenesuenseunrnununnnunnununununnnnununununnnnnnunununnnnnnnnnnnnnana 4 201 Retrieving the Mosaic csscsssscsssscssssesessesseeaeseceeseeatseeeeseeseatsenseerestsenatsees 4 202 Optional Applications een 5 3 Reticule agciccucianatabacciihisdiivasccdciecbissadevsdscsacueasseeevecusuus 5 4 Sep eisereen ebessi 5 5 AGUER CT CR 5 6 Reticules on Live IMages uu cceescscssesscsscessessesesseesesssssesseseesaesaeseeseusees
3. Deleting a Configuration Quality merges sz 4 When a configuration is selected the Delete button becomes active Click it to delete the selected configuration Testing the Movie length With all of the settings made or an existing configuration selected check the movie length by 5 Clicking the Test button After a short delay the Approximate maximum length 6 and Frames per second count 7 will appear Aim for a frame count of at least 15 frames second fps and preferable closer to 26 fps Movie name M S abre If the frame count is too low adjust x the exposure time on the Camera tab or increasing the light level at Quality Reen 2 5 the microscope or by selecting a Live Capture binning option that Include timestamp results in smaller image sizes After making changes click the Test button to review the movie length and frame count 4 43 2007 Leica Microsystems Switzerland Ltd Movie Start Recording and Progress To start recording the movie 1 Click on the Acquire Movie button The Recording Progress panel shows Leica GE the Current Clip progress 2 with 3 Time elapsed as hh mm ss 4 Computed Time to completion as hh mm ss and 5 Real time Completion expected at There is also a progress bar 6 Li which provides a graphical Time elapsed representation of time elapsed for the current clip Time to completion 7 To stop the current clip and skip to Comple
4. Total Area mm fi Scan Haight imm irs 4 181 2007 Leica Microsystems Switzerland Ltd Power Mosaic Random Scan Pattern Random and Random without overlap patterns The options are Random the number of tiles some of which may overlap Random without Overlap the number of tiles none of which will overlap Random pattern creates a specified number of tiles randomly inside a specified boundary Some of the tiles may overlap Click on the Target Width and Target Height text boxes and enter values for the scan area boundary The scan area does not have to cover the entire image Click on the Total Fields tiles text box and enter a value for the number of tiles Click Apply to apply the settings Repeat from Step 2 the change the values Click on the Close button to save and exit Random Pattern without Overlap This option is the same as Random except that none of the tiles will overlap Ban area enabled for e implement the change Wan aroa enabled ar implement the change p Target Width m o Total Area mmr i Fee IS Ar Apply LAS User Manual 4 182 Power Mosaic Annular Scan Pattern Annular Options are Full Coverage and Inside only This option automatically populates an area defined between two concentric circles with a calculated number of tiles 1 Click on the Diameter text box and enter a value for
5. Call the Online Help DM Operation overview LAS User Manual 3 40 DM Microscope Operation lllumination Control The current settings for the active light axis are displayed in the control Depending on which light axis is activated TL or IL light settings can be modified Use left mouse button the mouse wheel or the cursor buttons to change the values for light intensity Int field diaphragm Fld and aperture diaphragm AP respectively For precise adjustment of the light intensity the Fine Mode is automatically selected FIM Fluorescence Intensity Manager in FLUO mode the intensity of the excitation light can be reduced in 5 steps to protect the sample from bleaching For COMBI mode FLUO DIC or FLUO Phase use the tabs to switch between the control panel of TL and IL axis Mirrorhouse To change the fluorescence illumination with the Mirrorhouse move the mouse into the IIluminination Window of LAS Acquire Press Right Mouse Key Select Mirrorhouse Select Light Path NB Modifications in the light settings are stored and automatically recalled when the microscope in turned on Please use the right mouse button to get a context menu for additional functions Call the Online Help DM Operation overview 3 41 2007 Leica Microsystems Switzerland Ltd DM Microscope Operation Objective Nosepiece C All learned in objectives are displayed in the control window The current object
6. Sharpening enhances the edges of indistinct features on the image making is clearer and crisper Flip Horizontal and Flip Vertical re orientate the image top to bottom or side to side Acquire Camera Processing Shading Shading is the name given to variations in the background light level across an image The examples show transmitted light through a microscope on the left the light source and the optics conspire to create a bright spot in the centre of the image which gradually becomes less and less bright toward the edges Even illumination on live images can be achieved in software by applying a blank area value to the entire image area The effect is shown in the right image The light source and any of the optical elements will produce different shading levels so each microscope element combination should have its own shading setting LAS User Manual 3 102 Camera Processing Sharpening Use the Sharpening tool to improve the clarity and crispness of indistinct detail on a image Sharpening is a software function in which the boundaries between tonal values are enhanced The level of enhancement is selectable between Low and Robust very high but too much sharpening can make the image appear grainy and speckled It is a fast SC process so the best approach is to start with the Low setting and work toward Robust step by step 1 Click on the arrows to the right of t
7. in this case Windows Media Player See Preferences Play Movie Using Movie File Type Frames per second 00 00 02 demm Creating AVI File LAS User Manual 4 34 SS yo The Movie module must be installed and enabled before it is available See Registration for details of installation The Movie module captures images or frames in real time as a single continuous file directly to the computer hard drive Frames are captured as quickly as the camera and the computer will allow broadly governed by the resolution and exposure time Aim for low resolution without compromising viewing quality with fast exposure to achieve a frame rate of 20 to 25 frames per second This will provide smooth playback Movies may be split into a number of chunks or clips all stored as separate files Between the clips capture is suspended so time and disk space are not wasted on periods of specimen inactivity 4 35 2007 Leica Microsystems Switzerland Ltd Movie Select or Create a Folder To select or create a folder in which to store the Movie 1 Click on Options on the header bar 2 From the drop down menu select Preferences 3 On the Save Images panel check that the path in the In this folder window is correct or change it 4 Click on the Browse button and 5 Navigate to the required folder and Click it to select Alternatively 6 Click on the Mak
8. 3 Click on the Define sequence button With this option the selected sequence setup panel appears then go to Defining Clip Sequence See Defining Clip Sequence GB Close shutter when not acquiring 2007 Leica Microsystems Switzerland Ltd Movie Define Clip Sequence On the Sequence panel 1 Set the number of clips for the sequence by clicking the up down arrows to the right of the Number of clips text box Number of clips b E 1 Recording time 2 Set the recording time for the clip s by clicking on the appropriate field in the Recording time window and typing in a number or using the up down arrows to the right of the window The fields are hours minutes seconds hh mm ss If more than one clip is required each will record for the same time 3 If there is more than one clip set the interval between them by clicking on the appropriate field in the Interval window and typing in a number or using the up down arrows to the right of the window The fields are hours minutes seconds hh mm ss Each clip will be separated by the same interval WW Close shutter when not sequiring 4 Click on the Add Sequence icon to load the sequence to the program window 5 Almost any number of sequences may be added to a program In the example there are 3 sequences with a total of 5 clips 6 Remove a sequence by clicking on it and then on the Delete icon 7 Change the order b
9. 3 27 2007 Leica Microsystems Switzerland Ltd Preferences Save Images Image Name All images saved automatically are given a name followed by a sequential number You create the name which remains the same for all the images but the software increments and appends the number In the example Gamma_Section has been typed as the image name in the With this name entry box As images are saved the names and numbers progress like this Gamma_Section 001 Gamma_Section 002 Gamma_Section 003 Gamma_Section 010 and so on Each is unique due to the sequential number but belongs to a group because of the common image name The image name may be changed at any time To set a new image name 1 Double click in the text entry box beneath the With this name caption Any existing text will be highlighted 2 Type a new name Choose a name which is appropriate to the task or the images you are going to capture 3 Click anywhere outside the text entry box window to save the name Pressing the Enter key or the OK button will save the name but will also close the Preferences dialog LAS User Manual 3 28 Preferences Save Images Zero Padding The sequential numbers appended to the image name may be padded with leading zeros so that regardless of actual value it always has the same number of digits and the images are listed in the correct numerical order In the example Zero
10. 4 Check the camera and adjust the exposure On a clear field of view debris in the optical path will be seen as fixed dark regions on the live image when the specimen is moved If cleaning is necessary a qualified technician should perform it e Check that there is no visible debris in the optical path e For a colour camera Saturation 1 75 Gamma 0 6 Gain 1 Make fine tuning adjustments around these values to achieve the required image e For a mono camera Saturation 1 75 Gamma 1 0 Gain 1 e Ensure that the black and white levels on the histogram are reset See Input Options Continued on the next page LAS User Manual 4 156 Operating Steps Continued Adjusting the camera for Turboscan Turboscan the fastest scanning method can be used if e the camera is a Leica DFC FX type using progressive scan mode e there is sufficient light to give an exposure of less than 200 us and e the specimen is flat enough not to use focus adjustment or is suitable for predictive focus e Select an exposure mode with Progressive scan e Set Exposure to approximately 100 us e On the Histogram display click to enable Show Under Over Exposure e Adjust the lamp voltage until red flecks appear on the image white highlights Note On a DM microscope fine lamp voltage control is achieved by pressing both stand lamp buttons together Expect a high lamp voltage and very bright image e If there is insufficien
11. 4 134 Class names were created on the Setup tab and now they may be selected and applied to types of objects identified on the image When a Class name is selected it is displayed on the results against each of the tools used It can also be displayed on the image against the relevant measurement if Class is enabled on the Properties panel To apply a Class name to a measurement or range of measurements 1 Click on the arrows to the right of the Select Class header bar to reveal the panel 2 Scroll to the Class name required and click on it Class names may be colour coded and that colour will be reflected in the tools used For example the Root Leader Class name has been selected in the illustration Its colour code is red and therefore any tool outline used subsequently will also appear coloured red The Class name may be changed at any time but only subsequent tools will reflect the change Interactive Measurements Measure Select Class Select Class 4 135 2007 Leica Microsystems Switzerland Ltd The Properties panel provides simple and quick methods for changing tool attributes such as line text and background colours font styles and line thickness Each tool displays a label with essential numeric information such as distance or angle but it is also possible to display the Class name the main parameter as text such as Distance and a comment 1 Reveal the Proper
12. 6 If a sub folder is required click on the Make New Folder button and type a new sub folder name 7 8 Click OK 9 In the With This Name text box type a name which will be automatically applied to the MultiFocus Image when it is created Whilst in Preferences 10 Check that the Zero Padding value is correct 11 that the Bitdepth is correct and 12 the Saved Image Format is correct Click OK to save the Preference settings Acquire MultiFocus F3 hardware Solup Firmware Vodats Full Screen Play Movie Files Using Meainstream_2006 Abrams_Diagnostic E Dalglas Fellch_Bio Harford_Medical E Hayward_Analytical O Project_Alpha Project_Beta D Project_Deka Project_Epsilon QO n eg x 4 51 2007 Leica Microsystems Switzerland Ltd MultiFocus Options 1 Click on the Acquire Workflow to select it 2 Click on the Z tab to display the MultiFocus control panels 3 If necessary click on the arrow to the right of the Options header bar to reveal the Options panel Create MultiFocus after stack acquire 4 Selecting this option clicking the checkbox will automatically create a MultiFocus Image when the last Z slice is acquired Left un checked the MultiFocus Image Dein can be created manually on the Croats Mut tocus after stack acquire Viewer z Save sub images Save Sub images
13. Alternatively e Mark new objective in the selection list with left mouse button Open dialog with right mouse button and use select 3 19 2007 Leica Microsystems Switzerland Ltd DM Microscope Configuration Fine Tuning Node e All available objectives are mechanically adjusted to the parfocalising distance of 45 mm e Despite the extremely high precision it makes sense to correct the parfocality electronically e The sequence to set the parfocality values is designed as guided wizard and leads the user through all necessary steps NB The focus value of the dry objective with the highest magnification factor is stored as focus plane and can be recalled in the module DMOperation The parfocality compensation of this focus plane is performed for each objective automatically after setting the parfocality values Learn in parfocality Press Start to run the learn sequence e Find focus the objective with the lowest magnification factor is selected automatically This focus point is not stored but it facilitates finding the specimen on the slider This step of the sequence can be skipped if not necessary Adjust for each objective the z focus with hand wheel or SmartMove and press O K to store the value The next objective is inserted automatically When switching from dry objectives displayed in blue to immersion objectives displayed in yellow the user is asked to apply the appr
14. Fine adjustment 7 Use the mouse wheel if fitted to move the zoom in small steps Continued CO 20 25 32 0 EZ ojo LAS User Manual Stereomicroscope Operation Zoom Magnification 1 If the Objective Changer feature is fitted and is enabled on the Hardware Setup panel High and Low magnification levels are available 2 Click to enable High magnification or 3 Click the Low magnification option 3 gt CED DUAA D 4 The Zoom Drive Scale changes to reflect the magnification level selected Please select the comeraframegrabber you want to use as image source See Installation and Licensing for selecting hardware and the Hardware Setup panel Continued 3 57 2007 Leica Microsystems Switzerland Ltd Stereomicroscope Operation Motorised Focus There are three options for driving the Motorised Focus Focus Control 1 Fine focus FF and motor focus MF buttons are available with MF designated microscopes and Z6APO A and Z16APO A macroscopes 2 Click on fine F or FF to move the focus in small increments 3 Click on coarse C or MF to move the focus in large increments 4 Click hold and drag the Focus Control up or down Release the mouse button when the desired focus is reached Generally start with coarse C or MF selected to get close to optimum focus and then select fine F or FF to tune the focus position Type a posit
15. The zept m het chohged What wasit yas Dhe t ds 6 On the Confirm panel click Apply to keep the new orientation or Discard to start again 7 If Apply is clicked the Save Changes prompt appear Click Replace to save the cropped image and replace the original Save As to save the cropped image to a new name or Discard to restore the image and start again Before proceeding to colour adjustment it is good practice to make a copy of the re orientated image as a backup 3 137 2007 Leica Microsystems Switzerland Ltd Enhance Colour Two panels provide a range of powerful tools to adjust and modify the image colours Brightness Contrast Gamma BCG and Hue Saturation Intensity HSI To reveal the panels click on the arrow to the right of the bar While the panels use complex mathematics to manipulate the image ultimately colour is simply a matter of perception what our eyes and our brains see and if what we see suits our purpose then the colour is correct Before adjusting the image colour consider how it is going to be presented For archiving or electronic transmission perhaps very little adjustment will be necessary for projection in a Powerpoint presentation for example Saturation may need to be increased to maintain colour vibrancy on the screen for paper printing Gamma and Intensity may need increasing to keep colours pure and clean The Leica Application Suite a
16. LAS User Manual 3 24 The Acquire Workflow The Acquire Workflow gives access to the control of the connected microscope and camera while displaying a live image on the screen This enables the easy composition of images that are acquired stored in a defined folder and added to a Gallery With Leica Application Suite all microscope and camera controls can be set to individual requirements from objective magnification contrast method exposure gain and gamma For more advanced acquisition options additional modules can be added such as SG MultiFocus Montage Image Overlay MultiStep SG MultiTime Timelapse and SG MultiTime Movie Recording For these options an additional tab will appear on the Acquire Workflow operation of the module is described in the appropriate section 3 25 2007 Leica Microsystems Switzerland Ltd Preferences 7 Preferences settings allow you to configure the Leica Application Suite to best harmonise with your way of working To reach the Preferences dialog 1 Click Options on the top toolbar The Options Menu appears 2 Click Preferences The Preferences dialog appears Fast track to the Preferences dialog by pressing CTRL O from any screen Depending on the installed and enabled Modules there will be one two or three tabs along the top edge of the dialog The Image Output tab is always present Movie Settings and Save and Recall only appear
17. Leica Application Suite Montage provides advanced versatile features for producing excellent extended depth of focus images Uses the renowned technology of Auto Montage from Syncroscopy Digital images from a Z Stack spread over the focus range of the specimen are acquired using the same facilities as in LAS MultiFocus Montage has finely tuned algorithms producing a single sharp image of exceptional quality Essentially it is a more advanced version of LAS Multifocus LAS MultiFocus provides only one Montage method and is effective only on narrow range of specimen types There are no facilities for adapting to different situations and no means of enhancing result image or alternative means of visualising the result image LAS Montage has many additional capabilities to extend the imaging conditions that can be used improve the quality of the resulting image create a corresponding depthmap evaluate the quality of the result and add visualisation capabilities in order to examine the surface appearance in more detail Simple profile measurements can also be made LAS Montage is required for the use of the LAS 3D visualisation module The Acquisition of the Z stack that LAS Montage is applied to is identical to the method used by LAS Multifocus and so to find out about it Go there LAS User Manual 4 100 Montage Alignment R i i This panel controls the functions relating to the generation of a Montage
18. SMS can control Motorised focus Motorised zoom X and Y stage positioning Filter wheel Zoom iris aperture Zoom objective changer Internal light source TL External light source IL and CCIC and Fluorescence shutters Five memory locations allow settings to be saved and recalled precisely replicating the microscope setup Before using SMS please check that the motorised focus cable is fitted that Stereomicroscope Operation SMS Int roduct The following motorised coded microscope devices are currently supported by SMS Schott KL 2500LCD 31 250 200 amp 31 250 201 Photonic CLS 150XD and CLS 150LS External light sources 30 111 480 amp 30 110 481 Motorised focus 300mm and 500mm 10 446 176 amp 10 447 041 MZ 16A microscope 10 447 103 MZ 16FA microscope 10 447 063 Z6 APOA microscope 10 446 368 TL RCI Internal light source 10 446 352 UMC Universal Manual Control 10 447 080 Foot switch 10 447 398 X Y Motorised stage 10 447 305 The following functions are automatically detected and can be controlled from SMS Internal and external light source intensity On and Off CCIC shutter Open and Close Zoom magnification Objective changer Motorised focus position Fine focus position Filter wheel position Fluorescence shutter Open and Close Zoom iris aperture the security clamp is properly fitted and The following manual microscopes and devices have reduced i
19. To continue to use class definition for this image Click No axa emm Define Classes K Add Classes to measurements Node Leader Clear All 4 129 2007 Leica Microsystems Switzerland Ltd There is a wide range of powerful tools in Interactive Measurement which will make your images relevant and informative The measurement derived from each tool may be displayed on the image and also in the results tables alongside the tool graphic Apart from their basic measurement most tools have additional features or parameters which will be listed in the results For example the Distance tool will draw a line and measure the distance between two points the ends of the line Its additional parameter is Line Angle from the Horizontal shown on the results as either above the horizontal or for below the horizontal Additional parameters do not appear on the image but can be shown in the measurement list on the Measure tab and in a table on the Results tab 1 To reveal the Specify Measurement tools click on the arrows to the right of the header bar 2 To reveal a tool s additional parameters click on the arrow to the left of the tool name 3 To show an additional parameter on the results check the parameter box 4 Revert to the default parameters by clicking the Default button Interactive Measurements Measurement Tools Specity Measurements Select Parameters
20. the image should be sharp The Score bar will extend across to the right 10 The Threshold value represents a percent of the highest focus score in the image For Auto Focus to succeed the calculated value must be at or above the Threshold Below the Threshold and there probably is not sufficient detail to guarantee accuracy This is used to ensure that autofocus is not performed on a blank field where an alternate focal plane such as the top of a cover slip may be found 11 Set the Threshold by clicking in the text box typing a new value and clicking the Set Threshold button 12 Click Apply The Threshold line will be drawn to reflect the new value After changing Threshold repeat Auto Focus to ensure that the value is not so high that it prevents focussing 13 Click Close 2636510 00 score BM trends 5 OED LAS User Manual 4 170 Power Mosaic Calibration Calibration Input options and lighting levels must be set and the specimen in place The Calibration process is automatic It will determine the pixel micron value for the objective being used As objective magnification increases so that the actual field of view on the specimen decreases Calibration correlates the field dimensions microns to the number of camera elements pixels required to capture it It must be carried out at the start of a session and whenever the optics are changed Calibration a
21. 2 If the Input Options panel is concealed click on the arrow to the right of the header to reveal it The panel provides options for 3 Using a pre saved Input Options Configuration or creating a new one Captured Colour Depth Captured Format 4 Selecting the Image Type colour or greyscale 5 Setting the Colour Depth 6 Determine the Captured Format i e how the image will be shown and saved by Power Mosaic and 7 Select the Live Format i e how the live image will be displayed on the Camera tab Selecting the Camera If Turboscan is going to be used only designated high speed progressive scan cameras with a trigger facility can be selected OFC_320 Bets 3 OFC_320_Delta_1 DFC_320 Delta_2 Multi_1 Choosing a Configuration If an Input Options configuration has been previously saved it can be recalled from the Configuration menu Save Current Delete Current 8 Click on the arrows to the right of the Configuration header 9 From the drop down menu click to select the saved configuration Each will have a unique name All of the saved settings will be loaded and the remainder of the Input Options may be skipped A newly created configuration can also be saved using the Configuration menu and selecting the Save Current option See Acquire Camera Input Options for detailed procedures Continued on the next page LAS User Manual 4 166 Power Mosaic
22. 2007 Leica Microsystems Switzerland Ltd Power Mosaic Predictive Focus Continued Predictive Focus Continued For multiple focus points 1 The illustration shows a range of pre determined points that have been added manually to comprehensively cover the specimen 2 Click on the Autofocus on all Points button or for a single point click on the Autofocus button The program will cycle through each of the set points focussing automatically As each is completed the point turns green and the value appears in the Z column of the dialog 3 Check failed points circled for clarity by 4 selecting the Move Stage To button and then select the focus point Manually check the point for detail Either delete the point or adjust the Focus Threshold Setting Reset the points and repeat the process LAS User Manual 4 188 Power Mosaic Autofocus Setup Autofocus Skipping To speed up the scan especially on specimens that are more or less uniform automatic focussing can be skipped on some tiles The number of tiles to skip is set up with Autofocus It is also possible to resume focussing on a tile immediately following a focus failure rather than skipping tiles to maintain focus integrity Autofocus failure generally occurs when there is insufficient detail in the specimen 1 Click to enable the Autofocus check box 2 Click on the Setup button and the Setup Autofocus dialog ap
23. 5 If the MultiFocus Image is created a automatically See 4 above it may E Nica images balora combining not be necessary to save all of the Z slices or sub images Check this a Perform manual focus option to discard the layers Save sub images must be selected if Create MultiFocus is unchecked otherwise the prompt 6 appears If Save sub images is checked the MultiFocus Image cannot be re created again the entire process Ether Save Sub Images or Create Multtocus Aher Acquite must be checked in ceder te acquire will have to be repeated Zeie 0 ms Wait before acquire Enter the Stack Name 7 Click in the Stack Name text box and type an appropriate name for the Z stack The letter Z is automatically prefixed to denote a MultiFocus group and a sequential number is appended every time a new stack is acquired Continued LAS User Manual 4 52 MultiFocus Options continued Align Images before combining 8 Optical variations predominantly in stereo microscopes can cause an apparent shift in the point of focus from layer to layer The shift will be corrected in software if the Align Images option is checked so that all X Y points of focus are in exact alignment Perform manual focus 9 Check this option if the microscope does not have a motorised focus and each image is to be focussed manually Wait before acquire 10 Measured in milli seconds this option inserts a delay between
24. Click on the Set End arrow which will become red The virtual stack will display a graphical representation of the distance between the Start and End positions 4 The Z Range window displays the difference between the Start and End microscope readings See Set Start position Manual Microscopes 4 63 2007 Leica Microsystems Switzerland Ltd MultiFocus Set Z steps Manual Microscopes With the Start and End points set the distance between the two is calculated and displayed in the Z Range and Z Step windows There are two manual methods of setting the number of layers or steps 1 Click in the Steps text box and type the number of steps required The distance between each is calculated and shown in the Z Step text box or 2 Click on the Z Step text box and type the distance required between each step The range value is divided by the entered figure and the resulting number of steps displayed in the Steps text box Optimise step size 3 Enable the Optimise step size button to have the steps calculated automatically using the microscope aperture On stereo microscopes check that the actual and displayed iris settings correspond 4 With the Stack Definition complete click the Options Save button to save the settings See Acquire Z stack images Manual Microscopes EI Creste Multtfocus after stack acquire a e er GE EE gt SE LAS User Manual 4 64 MultiFocus Set Z
25. Green and Blue are the average colour values of the sum of the target points Each value is shown separately on the result together with the maximum and minimum value for each colour LAS User Manual 4 132 Measurement Tools Cross and Parallel Lines 1 Cross This tool measures both the major and minor axes of an object simultaneously by drawing a Cross over it The Cross may be drawn at any angle Click and hold on an edge of the area to be measured and drag to the opposite side Major and minor axes dimensions are displayed Drag the Cross at an angle if necessary To adjust axis lengths click on the Picker tool and then on the Cross to reveal handles for re sizing and rotating Click hold and drag in the centre to re position Parameter Angle to the Horizontal reports the skew angle of the major axis 2 Parallel Lines Draws lines at any angle and displays the distances between them Click on the image Rotate the line about its central axis to the required position Click and drag a line to the first distance point and continue to do this across the area to be measured Double click on the last line and the distances between each of the lines is displayed together with the total line count Parameter Angle to the Horizontal displayed with a for angles smaller that 180 degrees and for angles greater 4 133 2007 Leica Microsystems Switzerland Ltd The Measure tab pro
26. Mosaic to Capture Folder 4 The mosaic can also be saved to the Windows Clipboard for loading into another application Click on the Copy Mosaic to Clipboard button Large image files are not shown in the Gallery because they take too long to display Generally large files are saved to be used in third party applications Go to Retrieving the Mosaic 4 201 2007 Leica Microsystems Switzerland Ltd Power Mosaic Retrieving the Mosaic Retrieving a Mosaic When a mosaic is retrieved and re displayed a new full size mosaic is created This could be saved again as a mosaic either as a selected region or at a different size To retrieve a mosaic 1 Click on the arrows to the right of E the Select Workspace header i Bee Loading mosale Am 2 From the drop down list click to 52 complete time remaining 00 00 01 select a Workspace 3 A progress panel Loading Mosaic esses Dest Cancal lo what images appears and the tiles are displayed as a mosaic on the viewer 4 Detail of the mosaic can be displayed by clicking the Details button and 5 the Workspace Details panel appears specifying the number of tiles and image area To delete a Workspace 6 Click on the Delete button to delete the selected Workspace Steps 1 and 2 above Deleted Workspaces cannot be retrieved Total elds 8 Total Area sq mm 27253 LAS User Manual 4 202 Chapter 5 Optional Applicatio
27. Multifocus Image 2 A Colour Relief image offers a view of the montage image color coded GN Depth Map with depth information from the depth map The color relief image i Confidence Map is the same size as the montage image used to generate it but Anaglyph always comprises 3 RGB planes 3 The 3D Model is a 3D surface view SE of the image based on the depth map information where you can view the 3D surface from different angles Colour Relief 3D Model 4 109 2007 Leica Microsystems Switzerland Ltd Montage 2D Options The 3D model can be viewed in 3 different ways 1 Solid Colour The model is displayed as a surface relief with a single colour The colour can be set by clicking on the Set button Surface Cdour IS 2 Multifocus Image The model is displayed as a surface relief Ambient light We overlayed with the multifocus image Directionallight gi iiiiiiijtisiittii istitiii i 3 Scale The model is displayed as a Rendering Speed surface relief overlayed with the colour relief image showing the Z axis scale KE depth map contours Display Mode Solid colour Select the display mode from the drop down box The brightness of the image can be adjusted using the Illumination slider Set Rendering to Speed or Accuracy to change the response time of the 3D Model display in Colour Scale mode LAS User Manual 4 110 Montage Process Images Image stacks compri
28. Power Mosaic button 6 To re position the scanned image within the grid click on the Move Scan Pattern button click and hold on the scanned image and drag it to reposition 7 The Magnifier may help in the repositioning Click on the tool and use the mouse buttons to enlarge left button or reduce right button the view Continued on the next page 4 191 2007 Leica Microsystems Switzerland Ltd Power Mosaic Create Pattern Grid Continued Create the Pattern Grid Continued Having re positioned the image within the pattern grid it is possible that there are too many tiles Excess tiles may be removed as columns or rows only by 1 Clicking on the Details button and on the dialog change the fields tiles to remove any excess 2 Click the Apply button and 3 click the Close button 4 Select the Move Scan Pattern button and re position the grid to make sure the fit over the specimen is acceptable EI 5 If an alternative Pattern Type is ol required click on the arrows to the right of the Pattern Type header and from the drop down menu click to select the required pattern It may be necessary to re adjust the pattern grid to specimen fit using the Move Scan Pattern button Go to Save Configuration Britian 5 Scan Pattern Direction Rectangular Scan x Automatic Focus Met Pees if an area enabled for editing Is modified Press Apply to implement th
29. and go directly to the Sequence panel to set up the time lapse At date and time start 2 Click on the At date and time button 3 Click on the arrow to the right of the date window and the calendar appears 4 Use the left right arrows to scroll Sat Sun through the months and years 12 3 5 6 7 5 Click on the day date on which the 8 3 10 11 12 13 14 sequence will start 22 23 24 2 2 27 2 6 The time of day is divided into three fields hours minutes seconds Double click on a field to select and then use the up down arrows to the right of the window to set the required value Click and hold on an arrow for a fast scroll The hours field 24 hour clock rolls over at a count of 24 and the Manal minutes and seconds at 60 kiami Continued ot Aen 3 fiacoce Go to the Sequence panel O Ae Ger Bos ey AEN en f Sequence step 00 0025 i 4 25 2007 Leica Microsystems Switzerland Ltd Time Lapse Set up Options continued After delay start The delay can be from 1 second to 23 hours 59 minutes 59 seconds 7 Click on the After delay button 6 The time is divided into three fields hours minutes seconds Double click on a field to select and then use the up down arrows to the right of the window to set the required value Click and hold on an arrow for a fast scroll The hours field 24 hour clock rolls over at a count of 24 and the minutes and
30. clicking the appropriate Memory button 6 Continued Filter Name Empty Fluorescence Shutter Closed Visual Magnification 10 ix Tris Diaphragm 0 MAIN OBJECTIVE 0 63x Main Focus 215 929 mm LAS User Manual 3 52 Stereomicroscope Operation The SMS Controls Revealing the SMS controls The Stereo and Macroscope controls in the Leica Application Suite are revealed by 1 Click on the Acquire Workflow 2 Click on the Mic1 tab Depending upon the microscope and the functions available the controls may be displayed on additional tabs named n sequence Mic2 or Mic3 The components of the Stereo and Macroscopes must be setup in the Setup Workflow before the Acquire Mic tabs can be used Continued 3 53 2007 Leica Microsystems Switzerland Ltd The Shutter button on the fluorescence panel opens and closes the shutter Close the shutter when the microscope is not in use to protect delicate specimens 1 Click on the Shutter button This is a toggle action the Shutter opening and closing on successive clicks The Shutter status Opened or Closed is shown on the Fluorescence panel header bar and when it is open a red dot appears on the button 2 If an Empty filter wheel position is selected the Shutter control is not available 3 and remains closed Continued Green Shutter q Shutter LAS User Manual 3 54 Stereomicros
31. inspection 3 109 2007 Leica Microsystems Switzerland Ltd Browse Opening Browse The Browse workflow tab is the entry point to locating and displaying previously saved images 1 Click on the Browse tab The Image panel appears There are three sub panels on the Image panel 1 File Information provides detailed data about the image currently displayed 2 Directory Browser allows navigation to folders and files and 3 Device Settings lists the microscope and camera settings when the image was stored This is only available if the Save and Recall module is installed To select a folder and display the images 4 Click on the expand arrow to the right of the Directory Browser bar The directory tree is revealed If an image has been previously displayed it will re appear automatically together with complete path leading to its folder 5 Navigate to the folder required by clicking on the expand arrows Harford_Medical Helvist Hydro Cy Packard Compton Proca LAS User Manual 3 110 Browse Directory Browser To display the images in a selected folder 1 Double click the folder that contains the images required 2 Thumbnails small representations of the images in the folder appear in the lower window called the Gallery 3 The saved image appears in the Viewer together with 4 the Image Control icons on the side bar 3 111 2007
32. positions The Z Range window 5 will show the numerical difference between the Start and the End positions Z Step 6 represents the distance between each of the Z steps and Steps 7 is the number of images that will be captured See Set Start position Manual microscopes 4 61 2007 Leica Microsystems Switzerland Ltd MultiFocus Set Start Position Manual Microscopes Generally the Start position is closest to the stage because the specimen can be set against a textured background which will also be in focus This gives the software a benchmark for the background so that the out of focus background of subsequent steps will be ignored 1 Focus on the start region of the specimen making a note of the microscope scale reading 2 Click on the Set Start arrow which will turn black and type in the scale reading 3 Click on the Set Start arrow which will become red again The virtual stack displays a graphical representation of depth 4 The Z Range value reflects the entered Start position See Set End position Manual Microscopes __ Steps ZStep 5 _ mer DS aes LAS User Manual 4 62 MultiFocus Set End Position Manual Microscopes 1 Focus on the region of the specimen that represents the End position Make a note of the microscope scale reading 2 Click on the Set End arrow which will turn black and type the scale reading in the window 3
33. reference for the software In subsequent slices the background will be out of focus and so ignored 1 Click on the Set Start arrow It will turn black indicating it is active and will automatically track the microscope movements 2 Adjust the microscope either with its manual controls or by using the Application Suite interface until the specimen at the Start position is in focus 3 The microscope settings are reflected in the Start window and the Start arrow moves down the virtual stack 4 5 Click on the Start arrow which will become red and locked at the focussed position Set Start Adjust Z Stack Er i Focus Drive 531 mm ao Sal Z Range ZStep pm GEES Steps 1000 Te e ror LAS User Manual 4 56 MultiFocus Define Z Stack End Automatic Microscopes See Define Z Stack Start 1 Click on the Set End arrow It will turn black to indicate it is active and tracking the microscope movements Adjust Z Stack Set End d 2 Focus on the part of the specimen that represents the last Z step using either the microscopes manual controls or the Application Suite interface The End arrow will move along the virtual stack in response to the microscope 3 Click on the End arrow which will become red again and locked in the End Z step position 4 57 2007 Leica Microsystems Switzerland Ltd MultiFocus Define Z Stack End contin
34. seconds at counts of 60 er Sequence 1 ster 207075 Go to the Sequence panel LAS User Manual 4 26 Time Lapse Sequencing 1 On the Sequence panel click the up down arrows to the right of Number of images acquired window to set the number of images in the sequence 2 The Interval Between Images time lapse window is divided into three fields hours minutes seconds Double click on a field to select and either type a value or use the up down arrows to the right of the window to set the required value Click and hold on an arrow for a fast scroll The hours field 24 hour clock rolls over at a count of 24 and the minutes and seconds at counts of 60 Intervals may extend from 1 second to 23 hours 59 minutes 59 seconds interval between images 3 The number of images is multiplied by the interval and the overall sequence duration is shown in the Total time window In the example 6 images are required with an interval of 10 seconds Including an interval for capture and data writing for the 6th image in case further images are added to the sequence this equates to 60 seconds or 1 minute 00 01 00 However if no further images are added the final interval will be ignored 4 Click on the Add Sequence icon and the image count and interval appear in the Sequence window Options 5 6 When using Fluorescence techniques enable Close shutter when not acquiring to avoid damage to the
35. 22 Time Lapse Image Exposure 1 Click on the Acquire Workflow tab Both exposure and capture format depend to a great extent on the time lapse duration between individual images For example a short time lapse of say 1 second will demand a short exposure time to allow the image to be captured processed and written to disk for the control files to be created and written and for the imaging elements in the camera to be reset ready for the next image If these functions together take ww We Select Camera longer than the time lapse images will be lost Configuration For a short time lapse Make sure the exposure time is image Type also short as a guide 150mS should be a maximum Adjust the Captured Colour Depth Ee necessary to achieve a sho exposure Zell Consider using VGA or 4x4 Binning fet Color Binning 520 x 384 for the Captured Format avoid using High Quality HQ and set the Progressive VGA 696 x 516 Captured Bitdepth to 8 bits keeping 22 Color Binning 1044 x 772 disk files small and processing 22 HG Col Binning 1044 x 772 times low 3 or Binning 696 x 514 Turn off Store and Recall in 3G HO Col Binning 696 x 514 Preferences sw 4x4 Color Binning 520 x 334 See Store and Recall 4x4 HO Col Binning 520 x 334 Progressive Red 1044 x 775 Long time lapse y Whilst the exposure time may not be as critical still consider the lower resolution capture formats Using high resolution for
36. Acquire is found by selecting the Mode Change button to the left of the Workflow bar On Selection of the MultiStep Mode Icon a new tab will appear in both the Acquire and Browse Workflow Items labelled S 4 69 2007 Leica Microsystems Switzerland Ltd MultiStep Acquire Images On the Acquire Workflow panel an S tab will be visible if the MultiStep option is active Selection of this shows four Control Panels e Scan Position e Options e Define MultiStep Sequence LAS User Manual 4 70 MultiStep Scan Position Control This shows the position of the current field of view The user can mark points on the specimen by driving the stage manually The live window will be updated to reflect the true position on the stage The Navigation or Soft Joystick can also be used to navigate to new positions A position is located using the microscope e g the corner of an area of interest to mark the initial field in the scan pattern The Create button is then pressed to mark this co ordinate on the graphic See right Cross hair in red the Create button then changes to the Expand shown right When a second coordinate is added Again by driving the stage to the desired position and then selecting the Expand button a graphic representation of the scan area is shown Grey grid show right The Clear button allows the user to remove the scan pattern and start the process again The Shrink button
37. After calibration Scale Bars and Distance Lines will display the chosen measurement unit To calibrate an image 1 Click on the Calib rate tab and on the 1 1 icon on the Side Bar This will ensure the image is shown at its original size 2 On the Calibration dialog click on the Manual button A Calibration Line will be drawn automatically on the image and its length in pixels displayed against it 3 Click hold and drag the Calibration Line so that its left hand end butts the start point of the known distance 4 On the right hand end of the line click and hold on the handle and drag it to the other end point of the known distance 5 Type the known distance in the Length box On the example the known distance across the bud is three millimetres 6 Click on the arrows to the right of the Units bar and from the drop down menu select the known units on the example millimetres 7 Click on the Apply button The ratio between pixels and the known distance will be calculated and applied to other images in the Gallery When a new Gallery is created or opened the Used Stored Values button Image Calibration Method Use Stored Values BO Manual Draw Line Image Calibration Mathod Centimeters k is normally selected This means that a previous calibration applies to this image 3 133 2007 Leica Microsystems Switzerland Ltd This group of controls allows images fu
38. BA Ges when not acquiring If the Auto checkbox is selected this value will equal the dimensions of one field of view If the Auto checkbox is unselected the step size is user defined The Pattern Size gives the x and y dimensions of the rectangular pattern The Fields boxes show the number of whole fields of views in the pattern Changing the Step Size will change the Pattern Size as will changing the number of fields of view Continued LAS User Manual 4 72 MultiStep Define Sequence continued Autofocus This option allows the user to turn on Predictive Focus Pressing the associated button that appears when Predictive Focus is selected displays the Predictive Focus Setup dialog The Predictive Focus Setup focus dialog allows the user to set a minimum of three predictive focus points Selecting Add creates a new predictive focus point based on the current stage position Shutter Allows the User to define whether the shutter is closed during stage moves and only opened prior to an acquisition by selecting Close when not acquiring This is particularly useful if not essential when undertaking fluorescence specimens 4 73 2007 Leica Microsystems Switzerland Ltd MultiStep Options The Configuration section allows the user to store and recall previously saved configurations To Save a new configuration based on the current settings the user just needs to type a name into the configurati
39. DRY or IMMersion and reasonable light settings These reasonable light settings include light intensity and diaphragms settings The current objectives and their position on the nosepiece are displayed in the status list The objectives can be removed or exchanged NB After learning in a new objective it is recommended to re adjust the parfocality values of all objectives This can be performed either in Fine Tuning or DMOperation module Learn in a new objective from the selection list e Mark position in the status list with the left mouse button e Mark new cube in the selection list with left mouse button and exchange or add objective with a double click of the left mouse button Alternatively e Mark new objective in the selection list with left mouse button Open dialog with right mouse key and use select Continued LAS User Manual 3 18 DM Microscope Configuration Nosepiece Objectives contin Continued Learn in a new objective which is not registered in the selection list e Although the objective selection list is updated frequently it may happen that one objective is not implemented In this case please use one of the predefined objectives which comes as close as possible to your objective e Mark position in the status list with the left mouse button e Mark new cube in the selection list with left mouse button and exchange or add objective with a double click of the left mouse button
40. Delete Del key on the keyboard 5 Click on the Undo Changes icon The last item created will be deleted and the previous item created will be highlighted 6 Click on the Trash Can icon The last item created will be deleted and the previous item created will be highlighted Merge text graphics with the image When annotating is complete and checked all of the text and graphics may be merged with the image to make one unified bitmap To avoid coloured annotations on a monochrome image being merged as greyscale convert the mono image to colour before annotating 7 Click on the Merge button This process cannot be reversed Neither text nor graphics will be editable There are differences in pixel structure between image display and image storage so after Merge the annotations may appear slightly different from the original image Name Image Description image Date H ScaleBar a Image Rectangle Ki Ellipse Line e Rectangle E Rectangle E image Rectangle E tipse LAS User Manual 4 20 Frage The Leica Application Suite MultiTime optional module is a highly effective solution for the automatic acquisition of images over time The time span and acquisition may range from many images per second or just a few delayed by minutes After acquisition they may be visualised enhanced and documented Other LAS modules can perform analysis and measurement of the images There are
41. File name LASMeasuremerts dot ov Fie of type Template Pied Gol LAS User Manual 4 150 Output Results Output to new Excel spreadsheet There are two methods of publishing your results to Microsoft Excel Create a new spreadsheet or Append the results to an existing spreadsheet Excel is not included in the Leica Application Suite It must be purchased and installed separately preferably to the default drive 1 When a new spreadsheet is created Excel is opened automatically and the results loaded to it All of the usual facilities of font change colour fill and column width and height are available within Excel Save and print your results in the normal way 4 151 2007 Leica Microsystems Switzerland Ltd Output Results Output to ASCII Text file Publish to ASCII coded text file 1 To save the results in an ASCII coded text txt file the navigation dialog appears 2 Navigate to the required folder or 3 create a new one and type a filename 4 Click the arrow to the right of the Save as Type window and from the file type list select Text Files txt 5 Click Save The file may be imported into a wide range of graphics and word processing applications For fast simple reporting open the file in Windows Wordpad The data is presented in columns and basic text formatting is available Save ix E Haid Ansyicad A ciis BUET eet Deka ED Project _Epsi
42. Input Options Continued Selecting the Input Options Continued J See vamera a Image Type Colour cameras may be used in either colour Configuration or monochrome Greyscale mode Last Used Di eg Type 1 Click on the arrows to the right of the Image Type header and from the drop Ps down menu Image Types i 2 select either Colour or Greyscale Captured Colour Depth Captured Colour Depth 3 Click on the arrows to the right of the Captured Colour Depth header and select 8 bit Avoid the 16 bit option if it is memi caoi Depth available It will greatly increase stored image size slow the scan and may not be Captured Format compatible with other image processing software Captured Format Power Mosaic has been designed to capture high quality images so avoid using the low resolution options The example dialogs on this page are from a camera that is not suitable for Turboscan For Turboscan select one of the Progressive capture formats from a omg Leica DFC FX camera Full Frame 2083 x 1550 4 Click on the arrows to the right of the Full Frame 2088 x 1550 Captured Format header and from the drop down menu 1 Foli Frame ye VGA 696 x 516 5 choose the highest possible resolution 122 Color Binning 1014 x72 The binning options will save disk space 22 HO Col Binning 1044 x 772 but are unlikely to improve scan speed 36 Color Binning 696 x 514 Use the navigation a
43. Leica Microsystems Switzerland Ltd Montage Colour Relief The Montage Colour Relief operation generates the color relief image which offers a view of the montage image color coded with depth information from the depth map The color relief image is the same size as the montage image used to generate it but always comprises 3 RGB planes Experiment with the controls to find a representation which works well with your particular images Images of material surfaces consisting mainly of highlight detail tend to look good with mode set to Light Features and Saturation slightly left of center Images of biological samples with dark fibers tend to look good with mode set to Invert You may find that a pair of red green or red cyan spectacles as used for anaglyphs help to interpret color as depth The Colour relief image will be saved as one of the result image files LAS User Manual 4 116 Montage Colour Relief Method 1 Select Method from dropdown box Experiment with this control to find which mode works best with your particular images e Light Features applies the color effect mostly to the lighter parts of the montage image Dark Features applies the color effect mostly to the darker parts of the montage image Invert applies the color effect to the darker parts of the montage image and darkens the background 2 Click on Apply Confidence to use the confidence image to suppress unfocused parts
44. Linking Shading Shading Shading Linking associates a specific microscope setup with a specific shading level Shading is the name given to variations in the background light level across an image In the example the left image represents transmitted light through a microscope The light source and the optics conspire to create a bright spot in the centre of the image which gradually becomes less and less bright toward the edges Even illumination can be achieved by electronically applying a blank area value to the entire image area The effect is shown in the right image The light source together with any of the optical elements will produce different shading levels so each microscope element combination should have its own shading setting Shading Linking when enabled checks the microscope Objective or Zoom level for stereo microscopes Mag changer SG Camera and port and Contrast method and automatically tries to find and apply a matching Shading Link If a match is found the status icon on the Linking header bar will be GREEN Without a match it will be RED and no shading settings applied Shading Links are not supplied as presets they have to be created by the user 1 Turn Shading Linking on or off by clicking the Shading Link button LAS User Manual 3 90 Camera Linking Shading Snapshot To create an immediate Shading Link if one does not already exist t
45. Padding has been set to 2 which results in 001 002 003 up to 009 and then 010 011 up to 099 and finally 100 101 up to 999 Set Zero Padding by 1 Click the up and down arrows to the right of the Zero Padding window to increase or decrease the number of leading zeros 3 29 2007 Leica Microsystems Switzerland Ltd Preferences Save Images Bitdepth The Bitdepth is a digital value which determines the colour range and precision of the saved image A Bitdepth value of 8 bits provides a spectrum of 256 separate colours whereas 16 bits yields 65536 colours The Bitdepth setting has a considerable effect on the disc file size so generally 8 bit should be chosen unless more colour subtlety and variation are needed The 16 bit option is not always compatible with some third party software To set the Bitdepth 1 Click on the up down arrows at the right end of the Bitdepth window A drop down list of the available options appears 2 Click on the required option and the value appears in the window The Bitdepth setting overrides the camera setting LAS User Manual 3 30 Preferences Save Images Image Format Three options are provided for the format in which images are stored Tiff is the preferred method for storing and exchanging images and text It retains the colour detail and clarity File sizes are similar to bitmaps Jpeg produces the smallest files by compress
46. Transparent C Sio 4 11 2007 Leica Microsystems Switzerland Ltd Extended Annotations Create Shapes To draw rectangles squares ellipses and circles 1 Select the Rectangle to draw a rectangle or square or Ellipse to draw an ellipsis or circle 2 For a rectangle or ellipse click and hold on the image and drag the cursor downwards until the required shape is achieved 3 To draw a square or circle click on the image HOLD DOWN the Shift key on the keyboard and drag the cursor downwards to the size or diameter required 4 The outline colour of the drawn shape will appear in the Line window The outline colour can be changed by clicking on the Line box The Select Colour dialog will appear Choose a colour and click OK 5 The shape outline may be hidden or revealed by clicking the Outline button 6 To change the outline width click on the Up Down arrows next to the Width window to increase decrease the width 7 Fill shapes with solid colour or leave them open by clicking the Transparent button This is a toggle which alternately fills or clears the shape 8 Filled shapes may appear solid or translucent by clicking the Properties Opaque button The Translucent feature allows the background to show through the shape as though it were made from stained glass 9 Change the fill colour by selecting the Select tool and clicking on the Properties Fill window which sh
47. When the Manual Sequence Prompt appears turn the turret to the filter position specified on the prompt and 8 Click Continue The image will be acquired and appear as a thumbnail in the Working Gallery The prompting process repeats for all of the selected filters 9 To end a capture sequence prematurely click on the Finish button Default Sequer PE_Overtay Manual acquire D o Always create new overlay tolder Default Sequence Name PE_Overiay d O a r Manual acquire Auto create overlay Delete channels after overlay creation Always create new overlay folder About to acquire image 1 of 4 Please select filter position 3 L5 FLUO and click Continue Click Finish if you wish to terminate the sequence now Continue 4 93 2007 Leica Microsystems Switzerland Ltd Image Overlay Creating and Viewing Overlays When the Channel images have been captured 1 The Browse Workflow opens with 2 the Image Overlay or a preview selected Thumbnails of the captured channel images together with the composite overlay if auto create were selected will be displayed in the Gallery The Viewer shows the last image captured or the Overlay There are three panels on the Image Overlay tab The General panel on which 3 The capture folder Image Groups and therefore the required images can be selected The current folder and its images are the default 4 Overlay Method deter
48. Whole Image No Effect LAS User Manual 4 114 Montage Stereo Pair The Montage Stereo Pair operation generates the stereo pair image which when viewed correctly offers a 3 dimensional color view of the montage image with depth information synthesized from the depth map The stereo pair image comprises two similar images in color or greyscale depending on the source image each the same size and pixel depth as the montage image used to generate it The stereo pair image will be saved as one of the result image files 1 Select Source from dropdown box E Montage Stereo Pair o The stereo pair may be generated from a monochrome representation Source of the montage image from the R original color montage image if the source images were color or from the color relief image if that has been generated Layout Left Right Sanaration ee 2 Select Layout For normal use eech select Left Right However some people find it easier to view a stereo pair with the images flipped Right Left 3 Set the amount of Separation the maximum amount in pixels by which left and right eye images are displaced to provide the stereo effect Lower values are recommended in most cases Hint f your mouse has a wheel for scrolling click on the slider and use the wheel to adjust the parameter value 4 Click Create Stereo Pair to see the finished Stereo Pair image in the Image viewer 4 115 2007
49. a loss of initialisation values reem Stage Speed 40 10 00mm sec 1 Click on the Initialise and Set Focus Speed 50 10 00mm sec Speed button 2 On the Automation Settings dialog set the Drive Speed slider to a high by 8 S Stage Initialise Are you sure value start at 90 3 Click the Initialise Stage button O GID 4 Click Yes on the Confirm message ee Sh E Een ee If the stage stalls reduce the speed Drive Speed and re initialise e Stage Speed 40 10 00mmisec italise Stage 5 After initialisation completes meets Focus Speed 50 10 00mm sec successfully reduce the speed by 10 and click on the Apply button LAS User Manual 4 168 Power Mosaic Focus Initialisation Focus Initialise The speed of the focus drive mechanism can be adjusted below its maximum to allow for possible overrun and to minimise shaking Optics Settings o lObiective I 3HCXPL Cd Objective FLUOTAR DRY Additionally the limits between which the focus mechanism can operate are set to prevent the specimen colliding with the objective Upper and allow sufficient clearance for the specimen to be accessed Lower Select Live Image and focus Initiafising the stage or focus will automatically move to the limits in order to determine the range of travel Make sure that there is nothing that will obstruct free movement 1 Click on the Initialise and Set Speed button Adjust th
50. allows annotations to be displayed with or without a background 7 When Transparent is checked the image shows through the text 8 If Transparent is unchecked annotations sit on a coloured opaque background 9 To change the background colour uncheck Transparent Click on the Background window The Select Colour dialog appears 10 Select a new colour either from the swatches or from the colour wheel and slider Click OK The functions available on the Properties panel change depending upon the feature selected AvortGeede Md BT BarkGothic Md BT Any Bick Saal Nanow Mant irre AvartGade Bk ST Effects I Sukoon F Undeaine Tole mr d Custom CLLK CELL 4 5 2007 Leica Microsystems Switzerland Ltd Extended Annotations Text and Captions New annotations or longer captions may be added to the image using the text drawing and alignment tools in Extended Annbtation To create a new caption or annotation 1 Click on the Text icon 2 Position the cursor a black cross on the image click and drag to draw a text box A white box with a flashing cursor appears 3 Click in the box and type the caption 4 Press the keyboard Enter key to end the text 5 Typed text will automatically wrap to the next line DO NOT use keyboard Enter to start a new line Instead add extra spaces at the end of a typed line to take the cursor to the next line Keying Enter wil
51. always compatible with some third party software and is only available for HQ Capture Formats mla 22 Color Binning 144 x 772 E High Sensitivity Binning To set the Bitdepth 1 Click on the up down arrows at the right end of the Bitdepth window 2 From the drop down menu click on the required Bitdepth The Bitdepth setting made in Preferences overrides this setting Configure Camera The Configure Camera feature acts as a reset for the camera re establishing a lost connection and re loading the current configuration To reset the camera 3 Click on the Configure Camera button LAS User Manual 3 80 High Sensitivity Binning If binning is used for Live Format but not for the Captured Format when the image is retrieved it will appear darker than expected To maintain binned live image brightness on the captured image 1 Check the High Sensitivity Binning check box Configuration Save With the camera setup complete the configuration may be saved and used on another occasion to perfectly replicate current values 2 Click on the arrows to the right of the Configuration header bar 3 From the drop down menu select Save Current 4 When the Save Current Configuration panel appears type in a unique name for the settings Make the name appropriate to the camera model the Serial Number is not important here and the subject to aid retrieval later 5 Click OK Configurat
52. and right eye images are displaced to provide the stereo effect Lower values are recommended in most cases Hint f your mouse has a wheel for scrolling click on the slider and use the wheel to adjust the parameter value 4 Check Preview to see the effect of your settings in the Preview window 5 Click Create Anaglyph to see the finished Anaglyph image in the Image viewer 4 113 2007 Leica Microsystems Switzerland Ltd Montage Enhance Confidence Filter The Montage and depth map images may be cleaned up prior to publication by adjusting the image based on the Background Confidence confidence map 0 Hard Edges gt Enhance Multifocus Image Setting Confidence filter Adjusting the Background Confidence value will define a new background mask layer which can be applied to both Background No Effect the Multifocus and Depth Map images The Background layer is defined as the Whole image CEEE bottom image of the z stack Background Confidence can be set to a Enhance Depth Map Image value between 0 and 100 Any pixel with a Confidence value in the Berne i Ale range 0 Background Confidence will be regarded as a background pixel The boundary between the background Preview SSIs mask layer and the rest of the image may be smoothed depending on the value of the smoothing factor Select from Hard edges Soft Edges Min Soft Edges Med Soft Edges Max
53. available enabled options Items can be selected marked by the left mouse button Additional dialog windows are opened with the right mouse button Microscope tree e All physical parts of the microscope are represented by a node Each node can consist of various sub nodes These nodes can not be changed actively but may be modified automatically after selecting new items e Selecting one of the nodes with the left mouse button opens automatically the specific sight Each sight displays the appropriate status list the selection list and an image of the device NB The display of the microscope tree with all nodes and sub nodes may vary due to different configurations of the microscopes 3 7 2007 Leica Microsystems Switzerland Ltd DM Microscope Configuration Condenser Turret The condenser turret node allows adding and removing IC prisms light rings dark stops The learned in items are automatically considered for each objective and each corresponding contrast method respectively Remove one item from the condenser turret e Mark item which has to be deleted in the status list with left mouse button e Use right mouse button and select delete prism Learn in a new item from the selection list e Mark position in the status list with the left mouse button e Mark new item in the selection list with a double click of the left mouse button Alternatively e Mark new cube in the selection list with left
54. available for DM6000 stands It is helpful to adjust features of devices to personal preferences and needs as well as to adapt the microscope to specific specimen if necessary It allows to set parfocality for all objectives change the operation mode to Combi adjust the step size of Z focus for each objective and to set and delete thresholds LAS User Manual 3 22 DM Microscope Configuration Tube __ The current tube which comes with the stand is already learned in In addition alternative tubes eyepieces and devices for the documentation ports can be defined and learned in This is pivotal for auto calibration purposes Tube e All current available tubes for the DM series of microscopes are displayed and selectable The display of the sub nodes Visual and Docu are adapted to the selected tube type manual or motorized Visual port This sub node allows the user to define the current eyepieces in use e The proper definition of the eyepiece magnification is a prerequisite of correct calculation of the total magnification e The total magnification is displayed on the Leica Screen NB The symbol S which is displayed on the LeicaScreen refers to the total magnification with respect to the current objective and eyepieces Learn in a new eyepiece from the selection list e Mark position in the status list with the left mouse button e Mark new cube in the selection list with left mouse button and e
55. be used NB Memory requirements can only be shown accurately if the image is to be saved as in BMP or TIF formats JPGs will show the maximum size for the image Disk space requirements are additional because field images need disk Space too Even if system is set up to acquire 16 bit images mosaic images will be acquired in 8 bits Selecting the Acquire MultiStep button will start the Scan Pattern acquisition process as defined above A Progress bar will show how the process is proceeding Pause Will halt the acquisition which can then be restarted Stop Will halt the acquisition where it is resulting in either a partial mosaic being created and or a partial set of images depending upon your settings Configuration Save Create Mosaic image Save sub images S Mosaic2 Show During Acquisition Q Mosaic E Single images Reduction Factor Final image vumm mm 5 Acquiring image 3 9 4 75 2007 Leica Microsystems Switzerland Ltd MultiStep Browse MultiStep Images Under the MultiStep tab labelled S there are two control panel e View Image Set e MultiStep View Image Set This allows the user to select a previously saved set of multi step acquisition images The images may then be stepped through like a slide show by using the video play controls The rate at which images can be displayed is altered by entering a value into the Delay box The video co
56. button to select the images Create Multifocus image 3 Click the Play button It will change to red with the pause symbol 4 The current image and total images are displayed in the Progress Window and howing stack image 4 4 817 t 5 An indicator moves across the aa a gt a i pi Progress Bar Each image is Delay between og x displayed in turn in the Viewer images Click the Play button again to freeze the current image PF te ve ep 6 Move to individual images in the set by clicking and holding the Progress Bar indicator and dragging it left or right 7 Skip an image backward or forward by clicking the Skip buttons 8 Choose the first or last images by clicking the Beginning or End buttons 9 Select and display the MultiFocus Image by clicking the MultiFocus Image button LAS User Manual 4 68 This Chapter contains the following topics Introduction Select Acquistion Mode Acquire MultiStep images Browse MultiStep images amp O zi No Seqeence image Ovoriay Introduction eee This Module allows for the creation of obectes 5 oi Scan Patterns using Motorised X Y Stage control through the Leica Application Suite interface The subsequent images may then be joined together to create one large image or Mosaic On installation of the MultiStep module and the subsequent starting of the LAS the MultiStep Module can be z
57. by e mail or fax do not remove any programs from the computer or install any additional software until after the Licence Key has been successfully installed Doing so will invalidate the Site Code Go to Installing the Licence Key Registration Form Please anter the following information Fields marked with a are mandatory Once completed send the information to Leica Microsystems e mail LASConiCentGieica microsystems com Fax 41 071 726 33 83 Name GH Company Ir O O Addressiine TI y O Address Dee 2 Ir O Zip Post code OT om gp Telephone A Fax _ t e mail Si Site Code d 48DC 5F15 0687 BD 1234ABCDS5678EFGHE t Licence Number Cancel Namibia Nau Nepal Netherlands Netherlands Antilles New Caledonia New Zealand Nicaragua 2 11 2007 Leica Microsystems Switzerland Ltd Registration Installing the Licence Key Installing the License Key 1 From the Licensing tool bar select Options 2 Click on Registration The Enter Licence Key dialog appears 3 If the Licence Key has been sent from Leica Configuration Centre by e mail highlight the Key and copy it CTRL C Click in the Licence Key text box 3 and paste the Key CTRL V Alternatively carefully type the Licence Key 4 Click OK The purchased modules will be licensed and available without restriction Problems that do occur during licensing are usually because o
58. com www microscopy imaging com LAS User Manual At Leica Microsystems Cambridge Ltd we are continually striving to improve the standards of our manuals and would welcome customer feedback If you would like to comment on any aspect of this manual or our manuals in general please send email to LMIS manuals leica microsystems com Alternatively send a fax to 44 0 1223 412526 or write to Manual Feedback Marketing Department Leica Microsystems Cambridge Ltd PO Box 80 515 Coldhams Lane CB1 3YZ United Kingdom Please quote the title and date of the manual These can be found on the previous page Table of Contents Leica Application Suite Introduction eene 1 3 COMMON Core aiiccccccsseesssneecceeeeseseessneeeeeeessnsesssnneesaeeesenees 1 4 Optional Modules icsecssccssseeeeeeeeeeeeeeeeeeeeeeeneeeeeeeeeeeeeeeeeneees 1 5 Manual and Help Organisation 00 0 cccccsssssesssssseeeeeeseeeeeees 1 6 Combined User Manual and Help 1 7 Other Important Documents cceeesssceeeeeeessseesssneeeeeeeeeesees 1 8 Release Notes uv cvccccsccsccscssssssessessessessessessessessesessaesaeseecaeseesaeseuseesesseesesaess 1 9 Release Notes Continued oo ccc ccscsccssesssssessessessessevesseesessessesesseseesassesees 1 10 Installing Configuring and Licensing Een 2 3 Installing EAS ee 2 4 The Framework and Evaluation AEN 2 5 Module and Application Evaluation cscsssessssesessssesseesseeestsseatereassee
59. drag images forward or back through the Kb Depth Map sequence This display also shows the position in the sequence while i A Confidence Map the sequence is played Anaglyph Stereo Pair Colour Relief 3D Model 4 105 2007 Leica Microsystems Switzerland Ltd Montage Multifocus Image 1 Click here to display the current Multifocus Image This image is based on the settings from the Process Workflow There are z multiiocus 11 Se several methods for creating this z im age No stack images Fixed The Fixed algorithm selects the single source image plane cgon which is in best focus at each pixel location Delay rsch 00 B Sec Blended The blended algorithm s takes into account the effects of X X adjacent in focus planes at any one pixel location and attempts to predict the point of maximum focus Stack images more accurately Weighted The weighted algorithm Multifocus Image takes into account multiple in focus planes at any one pixel location Depth Map Compound Weighted Exponentially Weighted The Confidence Map exponentially weighted and compound weighted algorithms Anaglyph take into account multiple in focus planes at any one pixel location but biased more strongly towards the best focus Max Projection The image is based on the brightest point at each pixel location Min Projection The image is based on the darkest point of each pixel location Stereo Pa
60. dt View Favortes Toots Help View system information Add or remove programs E Change a setting SD Eject this disk Other Places 9D P P Search D Folders E Name Type amp Files Stored on This Computer E Shred Documents Pie Folder D leia s Documents File Folder Hard Disk Drives Sa Local Disk Cs Local Disk a wdata 0 Local Disk Se Extension E Local Disk My Network Places 3 From the popup menu select My documents Devices with Removable Storage AutoPlay and the CD should start to Saved E D Documents Haus Floppy A 3 Inch Floppy C l See SR See Release Notes continued En techn Details AutoPlay ED Search Open Explore Sharing and Security Scan with AVG manaren y e a e ag fer 1 9 2007 Leica Microsystems Switzerland Ltd Other Documents Release Notes continued From the opening screen of the Leica Application Suite CD 1 Click on the Installation Information button 2 On the About LAS screen click on the Release notes button Microsoft Word or Write will be launched with the notes displayed 3 Click on the Back button to return to the opening screen and Exit The Feedback and Hotline button will display the latest e mail and fax addresses for Leica Microsystems 4 If the program CD is not available the same Release Notes file may be accessed from the hard drive Launch Microsoft Word and open C Program Files Leica Microsystems Leica A
61. greyscale 9 Gain should be as low as possible Start with a value of 1 See Acquire Camera for more detailed information Continued on the next page LAS User Manual 4 164 Power Mosaic Imaging Setup Continued Imaging setup Continued The image must be properly focussed On the Histogram 1 Set the Black level to 0 and 2 the White level to 255 by clicking and dragging the sliders 3 Enable Under Over Exposure by clicking the check box Run Auto Exposure 4 Click on the Auto Exposure icon and then click again to turn Auto Exposure off Manual Exposure setting for Turboscan 5 Adjust the Exposure slider to give a reading of about 100us Gradually increase the light intensity see previous page until intermittent red flecking indicating slight over exposure appears Set the white balance 6 Click and drag a Region of Interest around a white area 7 Select White Balance from the menu Fine tuning If necessary fine tune the image with the Saturation and Gain controls 4 165 2007 Leica Microsystems Switzerland Ltd Power Mosaic Input Options Selecting the Input Options Note that the Power Mosaic live image is defined by the Captured Format This is done to ensure that images are acquired at the fastest rate 1 Click on the Camera tab Select Camera DFC 320 004123603 H Configuration 3 i Last Used image Type Colour gt
62. i The light path is set to 50 50 Please use the right mouse button to get a context menu for additional functions Call the Online Help DM Operation overview 3 45 2007 Leica Microsystems Switzerland Ltd DM Microscope Operation Memory Control Only available for DM6000 Allows to store the current combination of objective and contrasting method e g 20x D Using the right mouse key settings the current combination can be stored available combinations recalled or deleted from the memory Store Current objective and contrast method are stored Recall the selected combination and the last used light settings for this combination are recalled Please press the right mouse button to get the context menu for each button memory position Clear the data on the selected memory position Save the current state on the selected memory position DM Operation overview LAS User Manual 3 46 DM Microscope Operation Autofocus Control This is an optional module and needs to be licensed before use The digital Autofocus is valid for the following cameras DFC280 DFC350 DFC480 For other cameras please see the document Systems Requirements rtf By calculation the digital Autofocus finds a reasonable focus plane and adjusts the z focus level automatically For proper functionality please adjust the parfocality values for each objective Depending on the samples two appropriate modes can be selected Se
63. if they are enabled Input Output Settings tab determines how and where captured images are stored any actions that should follow image capture and the application to use when showing movies Movie Settings tab allows you to allocate disc space to movies and to the maximum file size for them Store and Recall tab determines whether or not the microscope and camera setting are stored with a captured image Automated microscopes may be automatically adjusted to the stored setting with Recall Click on a tab to bring it to the front and reveal its controls The active tab is indicated by a red line along the tab top edge LAS User Manual 3 26 Preferences Save Images To Folder Captured images may be saved automatically to a Capture Folder of your choice 1 While Auto Save is enabled all images will be saved to the Capture Folder The Capture Folder may be changed at any time to correspond with your working patterns as follows 2 Click the Browse button A Windows Navigation pane opens 3 Navigate to the required Capture Folder and click to Select 4 Click OK 5 The new Capture Folder appears the In this folder window All selected Capture Folders are stored and may be easily re selected from a History List 6 Click on the arrows to the right of the In this folder window and from the drop down list 7 Click on the folder in which to save images
64. image by using the full potential of this function knob This line or superimposed strip can be moved across the image or positioned to any location in the image with the Pos key The LED Magnification calibration indicates two conditions of the comparison bridge LED green the comparison bridge has identical magnifications left and right taking all optics and objectives into account The specified accuracy is less than 1 per mille LED red the comparison bridge can have different magnifications of the right and left imaging paths by as much as 5 3 69 2007 Leica Microsystems Switzerland Ltd FS Operation Illumination Intensity lllumination Intensity of the cold light sources This window allows for the control of two independent cold light sources and displays the illumination intensity in degree Kelvin The allocation of the light sources L amp R depends on the connectors on the rear of the FSC LAS User Manual 3 70 FS Operation Magnification Changer Magnification Changer The Mag Changer window allows for the direct control of the 1 5x additional magnification factor to be introduced at any time with any objective magnification It acts on both the eyepieces and the photo port After clicking on the desired factor 1x or 1 5x the current status is indicated in red It will further be correlated into the total magnification with the auto calibration function 3 71 200
65. in red This is a display function only The turret of the FSC is coded but not motorized LAS User Manual 3 68 FS Operation Comparison Bridge Control Comparison Bridge Control This module enables the direct control of the bridge modes as well as the position and width of the dividing line It displays the current status for the a m functions and indicates whether the bridge is in the calibrated LED green or Zoom mode LED red Four direct control keys are available that switch the comparison bridge into one of the following modes L full left image right side not used R full right image left side not used LR Split center image The dividing line will be positioned to its default position and default width previously teached in in DM control Later adjustments of either line position and or line width result in the switch off of the LED in the LR key This mode is then called split image and no longer split center MIX Superimposed image of both the full left and full right image mode The rotary button Pos controls the dividing line position that is used to introduce more or less of the left and right half image In its minimum and maximum settings a full left or full right image and all intermediate positions can be achieved The rotary button Size controls the width of the dividing line and can be set from a very thin almost invisible line to a full superimposed
66. initialisation sets the travel range of the auto focussing mechanism and also the threshold value for focussing success Select the split screen display option 1 Click on the Auto Focus tab 2 The current stage position on the Z Axis is shown by a vertical green line ee ON 3 A blue horizontal line represents the Focus Curve 4 Using the on screen Joystick SmartMove Se or stage controls navigates in the live image panel to a part of the specimen that Score Mil mee contains some detail avoiding repetitive or mais SES symmetrical patterns It may be helpful in L proving the Auto Focus effectiveness to slightly de focus the specimen 5 The Range value in microns determines the travel limits of the focussing mechanism It reflects the thickness of the specimen Click in the Range text box and enter a value the mechanism will drive upward from the current stage Z position by 50 of the value and downward by an identical distance Between these limits Auto Focus will achieve the best focus possible 6 Click the Apply button ALWAYS click Apply when changing values 7 Click on the Auto Focus button 8 If there is sufficient detail and contrast in the specimen area chosen the curve will appear centred in the new stage Z Axis position Note that if the image is in focus and the curve is not visible it may be drawn of scale IN this case click the Set Threshold button to reset the scale and 9
67. label 6 Unit will display the measurement units mm pixels and u for example Enable the label options by clicking on them The check mark alongside indicates that an option is enabled Not all options are available for all tools because they may not be applicable Measurement Labels Set Default Properties o MB Auto Size E tex Transparent TT Background 3 wan HB Line Marker Size Label measurement with _ Parameter V Value Comment 7 Node_Leader Distance 64 ege to Dudburst 4 139 2007 Leica Microsystems Switzerland Ltd Set Default This function applies all of the current properties to any subsequent measurements The Class name may be changed but its colour coding will be ignored 7 Use the current Property settings as a default by clicking the Set Default button Apply to All To make changes to all measurement labels lines fills and font styles quickly and easily set the Property values required and 8 Click the Apply to All button All measurements will then have identical properties _ Parameter V Value Comment LAS User Manual 4 140 Interactive Measurements Measure Using M The illustration shows an image that has had a range of measurements applied to it Font and line colours have been changed to distinguish between the measurement tools and the Classes The Area tool has been filled and
68. left to right the result to the Wi ZA i image of a swing of 300 to 300 with the middle image representing the original captured value O on the brightness scale A maximum negative value will produce a black image and a maximum positive value a white image See Process Enhance Contrast amp Gamma 3 139 2007 Leica Microsystems Switzerland Ltd Enhance Contrast and Gamma 1 Contrast increases or decreases the colour values individually both with respect to each other and also white levels It is a proportional adjustment The three illustrations represent a swing of 1000 in both directions with the original image in the centre The perception of contrast depends upon the ambient light levels The three small squares opposite are identical in both illustrations and yet those surrounded by black are perceived as having a lower contrast they look closer to each other in terms of colour than those surrounded by white If your image is to be projected in dim lighting conditions consider increasing the contrast to compensate 2 Gamma is a value applied to colour levels to compensate for different ways in which the image is viewed Liquid crystal displays LCDs have a specific Gamma setting cathode ray tube CRT monitors will have another and printers yet another Changes in Gamma are applied automatically so when an image is printed for example the printer software will make adjustments b
69. microscope The scanning area is permanently stored and can be recalled after switching off the microscope Please use the right mouse button to get a context menu for additional functions Advanced Settings define a new stage region Call the Online Help DM Operation overview LAS User Manual 3 48 DM Microscope Operation Multi user Package This is an optional module and needs to be licensed before use Each user can store their own profile A profile consists of the complete hardware set up of the microscope in combination with individual user settings such as the light intensity for one specific combination of objective contrast method Depending on the user level user management different users have different rights Only the administrator logged in as service engineer is allowed to create profiles which can be published and hence used by every other user Standard users only have access to their own profiles Users who are logged in as guest can not store their own profile To save the current status of the microscope press save profile The profile is than stored for the current user Show profile creates a html file with the current set up of the microscope If no default profile is assigned the microscope always starts with the last used profile before switching off If necessary select your own profile The adjustment of the microscope may take several minutes NB it is highly recommended to store a
70. mouse button e Open dialog with right mouse key and use select LAS User Manual 3 8 DM Microscope Configuration Function Keys The stands of the Leica DM microscope series and the SmartMove have variable function keys At date of delivery the individual function keys are programmed with useful and reasonable functions They allow a fast and convenient control of the stand and SmartMove For advanced users it may be helpful to assign the function keys according to special needs or preferences NB It is highly recommended especially for DM4000 and DM4500 not to delete the function key for Change contrast and if implemented FLUO The current meanings for each variable function key are displayed in the status list Replace a function of a variable function key Mark position in the status list with the left mouse button Mark new item in the selection list with a double click of the left mouse button Alternatively Mark new function in the selection list with left mouse button Open dialog with right mouse key and use select 3 9 2007 Leica Microsystems Switzerland Ltd DM Microscope Configuration Microscope Data Status list The status list displays the current items These items can be removed or exchanged by new items from the selection list The system recognizes the new item and adapts microscope settings automatically if the changes are confirmed when leaving the
71. name for the image and click Save Places ie The image is now available in both Fio pme image_Crop_ z the current folder and the new Saveashpe PEGI po D Cancel folder 3 117 2007 Leica Microsystems Switzerland Ltd Browse Fast Navigation Three icons at the top of the Image panel provide quick and easy ways to Set the Capture Folder Go directly to the Capture Folder and Go to the last folder used To set the Capture Folder into which all captured images will be automatically saved 1 In the Directory Browser navigate to the new Capture Folder 2 Click on the Set Capture Folder icon This folder will now override the Capture Folder nominated in Preferences To return to the Capture Folder 3 Click on the Go to Capture Folder icon The Directory Browser will be updated to point to the Capture Folder To return to the last folder used 4 Click on the Last Folder icon The Directory Browser will be updated to point to the last folder used LAS User Manual 3 118 Browse Thumbnail Re size Sometimes not all of the images ina folder can be displayed as a thumbnail simply because there are too many Use the scroll bar to the right of the Gallery to view hidden thumbnails or re size them to fit the Gallery space To re size the thumbnails 1 Click anywhere in the Gallery 2 Hold down the Control key on the keyboard 3 Press either the or key on the
72. numeric keypad to increase or decrease the thumbnail size Alternatively spin the mouse wheel to increase or decrease the thumbnail size There are preset maximum and minimum sizes for the thumbnails Select All Images 4 Right click on any Gallery thumbnail to reveal the Gallery Control menu 5 Click on Select All Images to select all of the thumbnails Unselect Images 6 Right click on any Gallery thumbnail to reveal the Gallery Control menu 7 Click on Unselect All Images to de select all of the selected thumbnails SA In ji 2 0005 z 0006 20007 Unselect AN Images Delete Selected Images Hide Selected Images Open Current Image With Copy Selected Images To Import From File Send To Leica IM Rename Currentimage Refresh Thumbnail 2007 Leica Microsystems Switzerland Ltd Browse Delete Hide Images Delete Selected Images 1 If more than the current image needs to be deleted select multiple gt images by holding down the Control ul key on the keyboard and clicking the thumbnails of the images to delete z 007i 2008 20019 2 0020 Unselect All Images Uelete Selected image 3 Hide Selected Images 2 Right click on any of the selected thumbnails to reveal the Gallery Control menu 3 Click Delete Selected Images A i Aainstream_Projects Mainst Delete Images confirmation panel S appears bk Open Current imag
73. or anticlockwise to the desired position and release the mouse button Or 2 Click on the outline of the control and it will rotate to the selected position The CT light colour k value and the CCIC opening are displayed on the panel header 3 Click on the Shutter to open it as indicated by the red dot or 4 Click again to close it and the red dot disappears LAS User Manual 3 62 The Motorised Stage is represented on the control panel as a rectangle with rulers along the top and left side Actual X and Y co ordinates are displayed in two text boxes and the stage initial traverse speed is selected using one of the three buttons Fast Slow and Auto for precise positioning within the field of view The traverse speed changes automatically as the stage nears the required position To move the stage to target 1 Select the desired speed by clicking on the appropriate button Choose Fast for longer distances 2 Double click within the rectangle approximately on the X Y position required The Target Marker will move to the selected position and the stage will start to track toward it As the stage approaches the Target the traverse speed will drop to Slow and within the Target Auto precise will be selected Click in either the X or Y text box and then use the Mouse Wheel 5 to fine tune the position in 2um increments To move the stage interactively 3 Click and hold within
74. profile of the original delivery status DM Operation overview 3 49 2007 Leica Microsystems Switzerland Ltd DM Microscope Operation ExMan and IFW Motorized Excitation Manager ExMan e Allows the balancing of different fluorochrome intensities e Suitable for the following Leica dual and triple filter cube systems G R BFP GFP CFP YFP B G R C Y R For access to the balancing slider select activate the appropriate dual or triple cube in Acquire and use right mouse key to open the Excitation Manager control window NB This function is only available if the microscope is equipped with the appropriate fluo axis Internal Fast Filterwheel IFW For fast excitation in red blue or green e Suitable for the following Leica dual and triple filter cube systems G R BFP GFP CFP YFP B G R C Y R For access select activate the appropriate dual or triple cube in Acquire The filter wheel positions are then directly accessible NB This function is only available if the microscope is equipped with the appropriate fluo axis DM Operation overview LAS User Manual 3 50 Stereo and Macroscope Systems SMS is available on Leica Application Suite if an automated micro or macroscope is connected to the computer SMS provides control of the micro or macroscope from the computer and if a digital camera is also fitted to the microscope will be especially helpful in optimising specimen images
75. stack tiles can be processed in several ways and eventually into a single composite image that represents the best focus across the entire specimen regardless of thickness Z Stacks can occupy very large amounts of disc space especially if all of the tiles are saved so ensure that a liberal amount of space is available The best arrangement for image storage is to have a partitioned area on the hard drive Drive D for example separate from other programs and data or a completely independent drive 1 Click on the arrows to the right of the Z Stacks panel to reveal it 2 Click the Enable Z Stack check box to enable scanning 3 To select the required processing method click on the arrows to the right of the Method header and from the drop down menu 4 Select the method Z Stack will save all of the captured stack tiles for further individual viewing Best Focus chooses the tile with the sharpest image from each stack and discards the rest Z Stack Best Focus combines both options retaining all of the stack tiles but also selecting the best from each stack which it places at the lowest level Extended Focus examines all of the tiles from a stack choosing the best pixel from each at a given location These are then combined into a single tile and the rest discarded Maximum and Minimum Projections emulate Extended Focus to create composite images based upon the darkest and lightest pixels in each pixel column
76. the Background window which shows the current background colour and when the Properties v Select Colour dialog 2 appears Auto Size E click on either a swatch or the ES colour wheel Bd Transparent OK Background 3 Click OK to apply the new colour 6 sE width Ge Line To alter existing Label Backgrounds click on the Picker tool click on the label to be changed and then click on the Background window Marker Size Comment Label measurement with V Number Class 4 Make the label transparent by clicking on the Transparent button The text will then appear directly on the image The Transparent facility may be applied to filled shapes ellipses rectangles and area so that the image beneath shows through To change Line colour and thickness for all tools including those with an outline 5 Click on the Line window which Cit ie shows the current colour and when EE a the Select Colour dialog 2 EEH appears click on either a swatch or ERWWSIT IR the colour wheel Click OK 3 to Seeaee0 TI apply the new colour L le a To alter existing lines or outlines Custom click on the Picker tool click on the WWEHT UH item to be changed and then click BEB HAs on the Line window Current C To increase or decrease Line fed pss BR te b B thickness Green he RH Saturation fs B 6 Click on the Up Down arrows to the Blue he BR Brightness Ess Add to custom right of the Width window For large zB
77. the current capture folder may be TVeeGonOptons OI played by oo 1 Clicking on the arrows to the right of the View Image Set window and from the drop down list 10 Click to select the project required and then proceed from step 2 above 4 33 2007 Leica Microsystems Switzerland Ltd Time Lapse Convert to Movie A sequence of images captured as a Multi Time project may be converted into a movie file which can then be played on most media software To convert a project into a movie 1 Click on the arrow to the right of the Save Time Lapse Image Set as Movie header to reveal the panel 2 Click on the arrows to the right of the Movie File Type text box and from the list select the movie file format The avi Audio Video Interleave format is the default and the most popular for video exchange 3 Set the number of frames per second The smoothest least jerky movies will have a larger number of frames per second The range is 1 to 25 Double click on the Frames text box and type a value 4 Click on the Save as Movie File button and the Creating xxx File progress panel appears 5 xxx refers to the file format 6 Movie files are stored in the same folder as the Multi Time project and can be recognised by the file extension in this case avi and they also appear in the Gallery as thumbnails 7 The resulting movie file can be shown most popular video software packages
78. the filter sequence 10 To save the channel dialog sequence click the Create New Sequence button See Capturing the Channels LAS User Manual 4 82 Image Overlay Channel Dialog The Dialog is a list of the filters available on the microscope with details of 1 The filter position on the turret Initially this will also represent the image capture sequence in automatic mode but may be changed See Changing sequence order 2 The filter name FLUO 3 The contrast method Right click on the entry to reveal a menu with FLUO 324 5 ms options based upon the filter type FLUO 503 7 ms Edit Description to change the description JE 293 Refresh to update the filter information Reset Pseudo Colour reverts to the filter s default value This is only available on monochrome Ke e E cameras _ EditDescription Extended Filter reveals an ee me Refresh additional menu to determine how Ce the filter works with the internal aA fo Intensity Manager filter wheel TLEF 295 1 Internal Filter Whee Excitation Manager 4 Image exposure time Initially this value will be the same for all filters but may be changed individually to achieve the best results 5 The Gain value Again these will be set to the same common value but may be altered for each filter 6 To the left of each filter entry is a check box When this is enabled ticked the filter will be actively used in the sequence a
79. the image is moved by moving the camera or panning or if the magnification is changed the reticule remains fixed and unchanged Scalable Reticule Scalable reticules have an absolute size associated with them They can be moved and rotated by the user however they cannot be resized by the user If the image is moved or zoomed the centre of a scalable reticule will remain over the feature in the image where the reticule is positioned by the user and the reticule will re size to be correctly scaled to the image Scalable reticules are defined in physical units e g mm Example A rectangular reticule placed over a feature will remain centred over the feature and scaled to the image when the image zoomed in or out The LAS Reticule application is started from the LAS Application Launcher or directly from the Desktop icon LAS User Manual 5 4 Reticule Setup Select the Setup workflow Click on Reticule tab to show the Reticule setup panels There are two collapsible panels e Reticule Settings e Available Reticules Reticule Settings This panel defines the way reticules look on he display and what happens when an image using a reticule is saved Colour Select one of e Default The displayed reticule has the colour defined when it was created Complimentary The displayed reticule takes on a colour complimentary to the underlying colour in the image e User Defined colour The displayed reticule has a colour d
80. the outer larger circle in millimetres The circle does not have to cover the entire image Click on the Inner Diameter circle text box and type a value for the smaller circle shown coloured on the illustrations for clarity It must be smaller than the outer diameter otherwise the settings will be ignored Click on the arrows to the right of Coverage text box and from the drop down menu click to select either Full Coverage or Inner only Inner only places the tile within the outer circle avoiding inner circle Full Coverage ensures the entire outer circle is covered without including the area of the smaller circle Click Apply to apply the settings Repeat from Step 1 to change the values Click on the Close button to save and exit lf an area enabled for editing is modified Press Apply to implement the change Diameter ram 2 O_O 4 183 2007 Leica Microsystems Switzerland Ltd Power Mosaic Focus Method Focus Method Three automatic focus methods are available Predictive Focus which uses a set of known focus points to interpolate predict any unknown points Predictive Focus is fast with good results but should be used only when the specimen is either flat or has a uniform slope across focus points Use objectives up to 10x or for very flat specimens up to 20x Autofocus uses contrast differences in groups of adjacent pixels to es
81. the stage rectangle The 4 Way Arrow appears 4 Drag in the required direction The Arrow changes to reflect the direction The stage will follow the mouse position until the button is released Stage traverse speed automatically slows as the mouse position is approached 5 Click in either the X or Y text box and then use the Mouse Wheel to fine tune the position in 2im increments Continued X Y Stage 67 mm 44mm x 67 184mm 43 615 mm 0 4 120 XY Stage 67 mm7 4 mm O fst Siow aro ral 67 184 mm y 43 615 mm 3 63 2007 Leica Microsystems Switzerland Ltd Stereomicroscope Operation X Y Stage control continued To move the stage to entered co ordinates To be used when actual X Y co ordinate values are known Both co ordinates are entered in the same way 1 Click on the X or Y text box and type a new value 2 For positions measured in millimetres type mm after the value otherwise the units will default to um 3 Press the Enter key on the keyboard The stage will go to the entered positions 4 If necessary fine tune the position in 2um increments with the Mouse Wheel after clicking in either the X or Y text boxes 5 Optional accessories such as the Joystick SmartMove or UMC controls may be used together with the SMS controls LAS User Manual 3 64 Stereomicroscope Operation Wiring Diagrams 1 K d P
82. users with privileges of Administrator can alter settings and calibration In situations in which a large number of people have access to the same microscope this can be a valuable security asset LAS User Manual 3 34 Preferences Movie Disk usage Because movies can be disk space hungry the Movie Settings tab provides two ways of limiting movie size Maximum Movie Size limits file size in terms of free disk space whereas Limit Movie Size prevents files exceeding a physical size measured in Megabytes MBytes If Limit Movie Size is enabled both features will run together to control movie size 1 Click on the Movie Settings tab to reveal the Disk Usage panel To limit the size of movies in terms of disk space 2 Click on the up down arrows to the right of the Maximum Movie Size window to increase decrease the percentage of disk space that can be allocated to movies Note that at least 1 Gigabyte GByte of disk space is required simply to run the movie application To limit movie files to a specific size 3 Click on the Limit Movie Size checkbox to enable size limiting The checkbox will become red with a white tick 4 Click on the up down arrows to the right of the Limit Movie Size window to increase decrease the maximum file size Each click is a iMByte step E Ge movie size in MBytes Mowe _ NOTE At least 1GB of free space is required to use the Movie m
83. which at this point are still being stored in a temporary file and thumbnails of the tiles that are in volatile RAM and then saving the tiles as a composite image or mosaic 1 N Click on the PM Power Mosaic tab Click on the Save Workspace As button A Workspace is a detailed description of the display and image settings saved as a file with the extension sws within the Capture Folder Additionally a folder to contain the individual tiles is created within the Capture Folder A thumbnail image of each tile is also stored in the folder The Capture Folder opens in a Windows Navigation dialog The Tile folders and sws files may be seen in the illustration Type a unique name for the Workspace and click on the Save button A Saving mosaic images progress message appears and the tiles are saved in the format and resolution selected on Camera Input Options en Save as type Workroace wt d Geet S 4 199 2007 Leica Microsystems Switzerland Ltd Power Mosaic Save Mosaic Save the Mosaic The composite image or mosaic file is saved directly in the Capture Folder with several associated control files As mosaic images can be very large in terms of disk space occupied and can take up several gigabytes of space there is a facility to reduce the size of the saved mosaic This is called the reduction factor Define by drawing rectan
84. which is then placed over the coloured rectangle so the colour shows as a background to the text Start by drawing a rectangle or square 1 Click on the Rectangle tool on the tools panel and draw a rectangle or square large enough to contain the caption 2 Select an outline colour and width and a background colour as Ge wy ES longert before a Ez an Uncheck Outline to remove the outline if preferred 3 Select the Select tool Click hold and drag the rectangle to the required position on the image Next create the text 4 Click on the Text tool on the tools panel and then click and drag to draw a text box next to and slightly smaller than the rectangle 7 ve GENEE K A at CH 5 Type in the text If necessary Al 4 select a font font style size and colour 6 Check the Transparent checkbox to ensure the text background is transparent f rectangle or Now combine rectangle and text A 3 square with or 7 Click on the Select tool then click and hold on the typed text Drag the text onto the rectangle 8 Adjust its height and width if necessary using the corner handles The text should sit just inside the rectangle Select text Align Left Right or Centre if necessary LAS User Manual 4 14 Once calibration has been carried out it is possible to make real distance measurements directly on the image To measure between two points on the image 1 Click on the Extended tab and th
85. 0 2005 z_0014 jpg 4 Change the circle outline colour by clicking on the Line window and on the Colour Selector dialog that appears selecting either a swatch or a colour from the wheel and slider Click OK To draw the leader line 5 Select either Line or Tools Arrow 6 Click and hold on the main image area of interest and drag up toward the circle When the circle and D line arrow meet release the mouse 3 Change the line arrow thickness by clicking on the up down arrows to the right of the Width window 4 Change the line arrow colour by clicking on the Line window and on the Colour Selector dialog that appears selecting either a swatch or a colour from the wheel and slider Click OK 200 jem L Cara mou ir 2007 Leica Microsystems Switzerland Ltd Extended Annotations Elements Deleting and Merging The Elements panel is a list of all of the text and graphics on the image To display the Elements list 1 Click on the arrows to the right of the Elements bar The list appears 2 To hide or reveal all of the text and graphics elements check or un check the Hide button The items are not deleted only temporarily hidden 3 To clear all of the text and graphics elements click on the Clear button Use with caution Clearing the elements is permanent Delete text or graphics items 4 Click on the Select tool Click on the text or graphic item to be deleted Press the
86. 1 Two methods Z Stack and Z Stack Best Focus have a slider 2 associated with them which allows each stack step to be viewed individually a powerful image analysis tool The slider scale represents the number of steps or multiples of steps Click hold and drag the slider to display the step results in turn sesa ECH Number otstes E B BR Set Step Site To Depth of Focus 4 197 2007 Leica Microsystems Switzerland Ltd Power Mosaic Browse Save amp Display Save and Display After a scan or scans the tiles are saved in a temporary file on the hard drive as thumbnails in RAM Both may be saved to a nominated folder i e the Capture Folder on the hard drive Select the Capture Folder 1 Click on the Browse Workflow tab 2 Click on the Image tab and 3 click on the arrows to the right of the Directory Browser panel to reveal it 4 Navigate to the folder in which the tiles and mosaic are to be stored and click to select it 5 Create a new folder or rename the existing one by right clicking on the selected folder and from the pop up menu choose an option 6 Click the Set Capture Folder icon Continued on the next page Create New Folder Rename Get images from sub directories Delete LAS User Manual 4 198 Power Mosaic Save Tiles Save Scanned Tiles There are two parts to saving the scanned images i et saving the collection of individual tiles
87. 1 Enable Profile with Edit Line disabled and to draw a straight line Enable with the mouse on the image Each new line will replace any previous one and the Profile panel will show the graph of the surface section The line colour can be changed by clicking on the coloured rectangle Line Mode aad BB Profile 2 Enable Edit Line to interact with an existing line or move a reference point The line can be moved as a body or the end points can be Reference Point moved individually Any Reference point can also be repositioned The profile graph changes accordingly Placing a reference point on the image sets a zero level for Z values and the profile display will change to show heights both above and below the reference level 4 121 2007 Leica Microsystems Switzerland Ltd Montage Measure Tools Profile Panel Controls Profile Panel Controls The Profile Panel has a context menu selected on a right mouse button click The chart can be copied to the clipboard saved to a named file and printed Selecting Show Point Values allows a read out of the graphs coordinates when the cursor is placed near the graph The chart can be zoomed and panned To zoom a portion of the graph select that part with a rectangle move the mouse pointer to the required top left point press and hold the mouse button while moving the mouse pointer to the desired bottom right point Context menu controls provide an Un Zoom fun
88. 1281 Gain 1 000000 Sensitivity Binning Enabled 0 Grabbed Pixel Depth 8 Colour Enabled 1 Auto Brightness Enabled 0 Brightness Level 500 000000 contents Microscope Stand DM3000 Nosepiece 10x 0 25 11506084 Ports 1 Magnification Changer 1 Il Turret D Lamp 83 Switchable Condenser Top 1 5 13 2007 Leica Microsystems Switzerland Ltd Image Organiser Image Data The Image Data panel shows data associated with the currently selected images This can be e An image selected from the Gallery or e An image selected from the Search panel search results A list of data labels and their associated values is shown With the Audio bar buttons on the bottom left side bellow Image Data the audio comments can be recorded and saved together with the image With the Document bar the external document can be attached and is saved together with this image buttons on the bottom middle side Show attach delete an external document By means of buttons on the bottom right side you can move in the Image Gallery to the right or to the left side to next or to previous image LAS User Manual 5 14 Leica Microsystems Switzerland Ltd Telephone 44 1223 411411 Stereo and Macroscope Systems FAX 44 1223 412526 CH 9435 Heerbrugg Hotline 44 1223 401824 Switzerland di support leica microsystems com www microscopy imaging com
89. 2 distinct components to MultiTime that are operated independently SG MultiTime Time lapse Images are acquired with a delay starting at 1 sec SG MultiTime Movie Images are acquired in a compressed image stream directly to the hard drive as fast as possible equivalent to making a video recording If images are to be collected over a long time the user must ensure that the specimen and microscope are unaffected by changes in temperature focus position and the electrical supply Achieving these conditions is not the remit of the MultiTime module 4 21 2007 Leica Microsystems Switzerland Ltd Time lapse is an imaging technique that acquires images with a predefined delay between each image acquired The images are stored to the hard drive at defined intervals and can be recalled individually in a continuous loop or as an AVI movie file This delay time is the distinguishing feature between normal imaging and time lapse imaging Time lapse imaging is best suited for continuous image acquisition over long periods or where there is no need for image data at full frame rates during the operation Time lapse imaging typically has a significant time delay between each image processed The camera can acquire images at its full frame rate but only one image is processed per period This is because it is not efficient to process every single image if only certain images are of interest LAS User Manual 4
90. 24 Fileinformation smsen eege 3 125 RE CN 3 126 The Process Workflow ccccssccseseseseesseeseesseeseesnseeenes 3 127 Annotate eegene 3 128 Fontand Text Colours sett e ok A aides eas Gh 3 129 Information and Scale Bar 3 130 lee HEI CS 3 131 Hide Autosize and Merge A 3 132 Calibrate Enhance Crop Crop Continued eege A weas Abily sale ee Aas eed ee 3 136 Orientation ede eae A eee ee ee 3 137 Ee De 3 138 Elei CHE 3 139 EIER UE 3 140 Contrast and Gamma continue 3 141 NEIE e EE 3 142 une 3 143 The Analysis Workflow 00ssssssseessesesssesesessssssseeseeeeess 3 144 Optional Modules eessen 4 3 Extended Annotation cccccccsssssesssssseessssesesesseeeeesssseeeesesaes 4 4 Font Colour and Background seen 4 5 Text and Captions EEN 4 6 Change Text properties 4 7 Change fextcoloun giiia aiiai asata Eed gege 4 8 Lines Arrowsand Distances see 4 9 Annotations Scale Bar Eege 4 11 Create Shapes sasssscsssesscssacsccsesncansscscetesesssnacnacscassarsecaesneccbecransaensnaecaesesneitine 4 12 HE EE 4 13 Boxed Captions sasscstisiesscscscesdccvecsevecseatersseateccsescastic Eegen 4 14 Distance Lime sssscecssevassissectsensssacceccaccastacapsecatsnasesesveassnccosnecautsncedceveaseseesaas 4 15 Edit aDistance LINE aaccccscsesscssscssscccccassecscesescsssnerscstossaesecassnereseerasternerec ncsestertine 4 16 A CR 4 17 E Oe LC 4 18 Insert Leader Dne casscszssecassissectecexecacscc
91. 4 Camera Region of Interest Region of Interest Rol A Region of Interest is created by drawing a rectangle on an image The area within the rectangle the Region of Interest is then the target of several special functions the image outside the Region being ignored Four special functions may be applied to a Region of Interest Crop to Region of Interest When the image is acquired only the part within the Rol is saved the rest is discarded Spot Exposure Automatic exposure is applied to the entire image but using only the values contained within the Rol Find Focus Defines a moveable Region of Interest in which to focus White Balance displays all of the neutral tones as shades of black and white but only within the Region of Interest Each rectangle drawn around a Region of Interest is colour coded to denote its function ber s S WhiteBalance Spot Exposure ti C Find Focus sd gg Region of Interest gt 3 105 2007 Leica Microsystems Switzerland Ltd Camera Region of Interest Crop Crop This feature will save only the image within the Region of Interest rectangle The rest of the image is discarded 1 Click hold and drag diagonally to create a Region of Interest When the mouse button is released a menu appears 2 gt d A 2 Select Region of Interest The Ze EZ nl WhiteBalance H d A Bei Kr a rectangle colour is green
92. 7 Leica Microsystems Switzerland Ltd FS Operation Tube and Photo Port Tube amp Photo port This is a display only function The beam splitter has a fixed factor of 50 50 LAS User Manual 3 72 SE hE SS The Camera panel provides convenient control over the functions of a Leica DFC digital camera ranging from colour balance to histogram black and white levels Images can be acquired in a variety of sizes colour depths and file formats to provide even more flexibility Setting the sharpening option and shading reference acquires images of the highest quality so that further processing requirements are minimal Leica Application Suite also allows a focusing region to be defined on a live image so that areas of significance can be easily identified and focused more rapidly All parameters and configurations can be saved and recalled at a later date All Leica DFC cameras are controlled from the same interface although the features available do depend on the camera type 3 73 2007 Leica Microsystems Switzerland Ltd Camera Image Controls To access the camera selection and configuration 1 Click on the Acquire work flow tab t s File Options Help The microscope and camera panels appear 2 Click on the Camera tab There are eight panels on the Camera tab together with the primary Image Controls and the Acquire Image button Hide or reveal a panel by clicking on the arrow to the ri
93. By collecting digital images at these Z positions they can be combined by an image processing algorithm into one single sharp composite image that the effectively extends the depth of focus of the image Specimens that can benefit from LAS Multifocus are predominately those that are imaged by light reflecting from the surface such as geological and fossil specimens plant and marine biology histology and materials such as paper electronic components metallurgy surface coatings and fractures LAS Multifocus provides fully integrated software controlling microscopes with motorised focus cameras with high resolution and colour fidelity that are combined with a computer that comfortably handles digital images The performance and relative economy of modern computing A R C N F F makes the use of sophisticated imaging algorithms practicable for acquiring and processing Z stacks of multiple images The principle is simple An image that varies in focus is taken at steps across the thickness of the specimen These slices are then mathematically combined to form a MultiFocus Image all of the slices blended into a single uniformly sharp image Almost any number of slices together called a Z Stack can be captured the higher the number generally the better the MultiFocus Image and each may be saved and examined The MultiFocus application is suitable for both manual and automatic microscopes LAS User Ma
94. C Serial Bus CHEN Footswitch 2 ee connect to free CTL2 port on last footswitch UMC Continued 3 65 2007 Leica Microsystems Switzerland Ltd Stereomicroscope Operation Wiring Diagrams 2 Motorfocus Power Connaction PC Serial Bus 80 240V ep E Serial Bus with Motorfocus OR USB port with TL ACI PC USB DCI Controlbox Connector from Cross Stage connect with free CTL2 port on UMC LAS User Manual 3 66 FS Operation Introduction This module serves as remote control and status display of all motorized functions of the FS C and FS CB If a camera is attached to the microscope it can be controlled simultaneously The module can consist of the following control plug in windows depends on the connected hardware FS C or FS Comparison Bridge only Objective Magnifications Comparison Bridge Control Illumination Intensity Cold light sources Magnification Changer Tube and Photo Port zc X Y Stage Focus Drive 3 67 2007 Leica Microsystems Switzerland Ltd FS Operation Objective Magnifications Objective Magnifications This window shows the status of the objectives available and teached in for both the left and right hand revolver turret of the FSC Switching over between left and right takes place by clicking the corresponding button L amp R The currently used objective magnification is indicated
95. Configuration module Deleting one item from the status list e Mark position in the status list with the left mouse button e Press delete item Continued LAS User Manual 3 10 DM Microscope Configuration Microscope Data continued Add a new item to the status list e Mark position in the status list with the left mouse button e Mark new item in the selection list with left mouse button and exchange the items with a double click of the left mouse button Alternatively e Mark new item in the selection list with left mouse button e Open dialog with right mouse button and use select Continued 3 11 2007 Leica Microsystems Switzerland Ltd 8 DM Microscope Configuration Microscope Data continued Selection list All available and appropriate items for one node are registered in the selection list In addition in most cases the customer can define and create new items NB The items within each list can be individually arranged E g all items can be displayed according to increasing value of the order number For this purpose click in the highlighted header of the desired column Learn in a new item from the selection list e Mark position in the status list with the left mouse button e Mark new item in the selection list with left mouse button and exchange the items with a double click of the left mouse button Alternatively e Mark new item in the selection list with left
96. Date and Time from Core annotation to the image with Process Annotate These will be carried forward into Extended Annotation and appear on the re selected image 1 Click on the Extended tab to display the alignment text and drawing tools 2 To reposition annotations click on the Select tool In the example Image Name Date and Time have been moved close to Description and the Scale Bar has been moved down 3 Click on the Select tool and then on the annotation to be moved The Select tool will be in the correct position when the cursor changes to a small target sight Each annotation sits in a text box and when it is correctly selected small square handles appear at the corners of the box Click hold and drag the box to the required position 4 Fine tune positioning with the keyboard arrow keys File Options Help LAS User Manual 4 4 To change the Font Font Style or Font Size 1 Click on the annotation to be changed with the Select tool 2 Click on the Properties Font button The Font dialog will appear 3 Change the Font properties and click OK The change is immediately reflected in the annotation To change the Font colour 4 Click on the annotation to be changed with the Select tool 5 Click on Text window The Select Colour dialog appears 6 Select a new colour either from the swatches or from the colour wheel and slider Click OK The Transparent checkbox
97. EENENERENEKERENEEEEENEEEEEEEEEEEEEENEENAENENeN Fine Tuning Montage Montage Alignment we ICT ENEE Montage Optimize Preview scssssssssessssessssesrsseaeseearseeeeseceeatsenstenestsetatsees Browse Montage Images Stackimages eaea Te aae aa A A a a Laa Aai Multifocus Image EE Depth Map nase Confidence Map Anagly ph ouuw ccc ccc ccsesssscescsssessessessevsevsessevsesaessesausaesaneaesaes Stereo Pair Colour Relief 3D Model cssssscscsesssssssssscecsessseseessscetacesessenes 3D Options Process Images Source Images Create Anaglyph Image Ss Enhance Confidence Filter Stereo Pit eege EE 4 115 VTT EE 4 116 Colour Relief Method eege ENEE eege EE 4 117 Edit Mode Continued euer e EE dE EEN 4 118 Measure Tools Position css0scscecscscessccetencceccorancesesssnesecenecnecorseanentenseneasests 4 119 Measure Tools Reference Lime Mode ENEE 4 120 Measure Tools Profile Edit Line seen 4 121 Measure Tools Profile Panel Controls ENEE 4 122 Interactive MeaSurements ccccsscceeeessseeessseceesssseeeneeees 4 123 CITT WEE 4 124 Configuration Option sasccscws veces cada ERAN 4 125 Create a new Copnflguratlon 4 126 Select and Delete a Results le 4 127 Createia Class eise csc aeieeevleeees sie abstinence veces Gnade 4 128 Define Delete and Clear Classes AA 4 129 Measurement Tool sssini arainn e aeaa ee 4 130 Measure ein eege ere 4 134 Select Class L
98. Focus Show Image Set When all of the MultiFocus images are captured the Browse Workflow opens with the Show MultiFocus Image Set panel displayed Show Mutstocus Image Set The current set of images is not automatically selected so 1 Click on the arrows to the right of the Image Set widow 2 From the drop down list choose the set required by clicking its file name The Gallery will be populated with the set thumbnails and the first image will be displayed in the Viewer Creating a MultiFocus Image 3 If Create MultiFocus after stack acquire was not checked in Options only the Z step images will be present in the Gallery To create the MultiFocus Image click the Create MultiFocus Image button The software creates the image a thumbnail will appear in the Gallery and the full sized image in the Viewer 4 67 2007 Leica Microsystems Switzerland Ltd MultiFocus Show Image Set continued The panel provides the controls necessary to view the entire set as a slide show a single layer or the MultiFocus Image With the required image set Z Lichent_3 selected See Show Image Set 1 Set the delay between images in seconds by Either clicking on the Delay Delay between Between text box and typing a s images is Cl number or i y Using the up down arrows to the right of the text box ER eh Stock images 9 ra Multifocus image 2 Click on the Stack Images
99. Horiz 002mmsvg the list Check the Show Reticule box Show Reticule to apply it to the image SES Saving the image will associate the selected reticule with this image Only one reticule may be associated with an image If the Merge reticule into the image option is selected in Setup then you will EE be asked to confirm that you want to SORES e EN continue with this action Show Reticule If it is scalable the reticule may be moved over the image e The image being saved has a reticule overlay associated with t but the system is currently configured ta merge reticulas into the image If you continue the reticule overlay will be lost Do you want to continue and merge the reticule into the image Click and hold down the mouse over the reticule within the boundary defined by the markers Drag the reticule pattern to the desired position To rotate the reticule click and drag on the rotation handle LAS User Manual 5 8 Image Organiser we z The Image Organizer is specifically for use with the Leica DM1000 Leica DM2000 Leica DM2500 Leica DM3000 and Leica DM4000 B microscopes for clinical applications If the Image Organizer application is loaded the Browse capability is enhanced Click on the Browse Workflow tab to open the Image Browse panel that contains the Image Organizer browse controls There are four principle panels displayed e Category Selection e Search e Device Settings e
100. Image Data 5 9 2007 Leica Microsystems Switzerland Ltd Image Organiser Category Selection The desired Category of images can be selected and displayed by means of the pull down menu The images captured in Acquire Workflow by clicking on the button Acquire Image will be stored in the selected category By clicking on the button Show details the check box Activate category autosave is displayed Patient Data By activation of this check box the desired selected category will be loaded A Hide details and displayed automatically on start up A The captured images will be saved in LA Activate category autosave this category SCH Description Image C Documents and l Path Settings Administrator My Documents My l ImageOrganiser UserTemplate Created 10 12 2005 Modified 10 12 2005 No of Images 2 LAS User Manual 5 10 Image Organiser Template Management With the button Advanced the new categories can be created and the corresponding data fields modified you can delete or add new data fields By clicking on the button Create category a new category will be added You can also Clear data or Delete categories please note that only the new created categories can be deleted With button Save amp Close you can return to the previous mode Opareten On this page you can manage al your image categories YOu Can create and delete catagones or cinar at ire in on atready eating Categ
101. Leica Application Suite LAS V2 7 LAS User Manual Seica MICROSYSTEMS LAS User Manual All reasonable steps have been taken to ensure that this publication is correct and complete but should any user be in doubt about any detail clarification may be sought from Leica Microsystems Switzerland Ltd or their accredited representative The information in this document is subject to change without notice and should not be construed as a commitment by Leica Microsystems Switzerland Ltd Leica Microsystems Switzerland Ltd accepts no responsibility for any errors that may appear in this document 2007 Leica Microsystems Switzerland Ltd No part of this publication may be reproduced transmitted transcribed stored in any retrieval system or translated into any human or computer language by any means or in any form without the prior written permission of Leica Microsystems Switzerland Ltd Due to a policy of continuous development we reserve the right to change specifications without notice Microsoft and MS DOS are registered trademarks and Windows the Windows logo the Windows Vista logo the Windows 2000 logo and the Windows XP logo are trademarks of Microsoft Corporation Date May 2007 Software Version LAS V2 7 Leica Microsystems Switzerland Ltd Telephone 44 1223 411411 Stereo and Macroscope Systems FAX 44 1223 412526 CH 9435 Heerbrugg Hotline 44 1223 401824 Switzerland di support leica microsystems
102. Leica Microsystems Switzerland Ltd Browse Select an Image Select another image to display in the Viewer 1 Click on the thumbnail in the Gallery A brown border appears around the thumbnail to indicate the selection Image Control Same Size 2 Click on the Same Size icon or 3 Right click on the Viewer to reveal the Image Control menu and then 4 Click Zoom and Original Size Both options display the image at its actual size that is one image pixel occupies one Viewer pixel If there are more pixels in the image than there are in the Viewer this may result in some of the image not being displayed Use the Pan Window option below to view the parts that are not being displayed Paste Show Pan Window In Out See Pan Window LAS User Manual 3 112 Browse Pan Window E i To view parts of the image which are not visible in the viewer 1 Click on the Pan Window icon on the side bar or Right click on the Viewer image Click on the Show Pan Window caption The Pan Window appears To re position the Pan window click and hold the Title Bar and drag the window to the required position The Pan Window shows the entire image at a small scale with a red outlined Viewpoint 5 representing the part of the image displayed in the Viewer Click inside and drag the Viewpoint to display hidden parts of the image in the Viewer To close the P
103. Line Length End Distance YD vB Area Too 1 gt Circle Too LAS User Manual 4 130 Measurement Tools Distance Line and Area 1 Distance Measures distance between two points by drawing a line between them Click and hold on the start point and drag to the end point Constrain to vertical horizontal or any multiple of 45 degrees by pressing and holding the Shift key whilst dragging The distance line may be moved extended or reduced by using the Select Arrow tool clicking holding and dragging on a line end point Parameter Line Angle from the Horizontal shown as either above the horizontal or below the horizontal Line Draws an irregular shape around an object and measures the line length Click hold and drag around the object to be measured Parameter End Distance which is the distance between the open ends of the shape Area Draws an irregular shape around an object and measures the area within Click hold and drag around the object to be measured Double click to finish and join the ends of the outline Parameter Perimeter is the length of the containing line Parameter Mean Intensity represents the brightness of the enclosed area compared with the overall image brightness or intensity Parameter Mean Red Green and Blue are the average colour values of the area within the outline Each value is shown separately in the result together with the maximum and minimum value
104. Manual 5 6 Reticule Reticules on Live Images Reticules on Live Images e Click on the Camera tab to open the Camera control panel A reticule selection panel is added to the Camera tab in the Acquire workflow The required reticule is selected from the list Check the Show Reticule box to apply it to the live image Choose a reticule size that will fit within the image boundary to ensure that it is visible If a scalable reticule is larger than image size then it will not be movable an may be truncated If it is scalable the reticule may be moved over the image e Click and hold down the mouse over the reticule within the boundary defined by the markers e Drag the reticule pattern to the desired position e Once moved the new position is saved and cannot be reset e To rotate the reticule click and drag on the rotation handle handle Acquire 5 7 2007 Leica Microsystems Switzerland Ltd Reticule Reticules on Captured Images Reticules on captured images Click on the Reticule tab in the Browse Workflow The reticule selection panel is displayed Select an image from the gallery If it is associated with a reticule the reticule will be displayed on the image The Select Reticule panel will show the reticule name in the definition file Current image box and will indicate Current Image Definition file in the list box e The required reticule is selected from CANALA AScale
105. Microsystems Switzerland Ltd Camera Calibration Settings Retrieve Calibration Links made previously may be retrieved and applied to the current image by 1 Click on the arrows to the right of the Configuration window 2 From the drop down list of existing Calibration Links click to select The settings are applied immediately and if a Scale Bar is displayed its value will be updated Acquire Camera Calibration Settings Delete 3 Click on the Delete button to delete pe e the current setting The Default Calibration Settings Si Calibration values are then applied Acquire Camera Calibration Settings Confirm Calibration on Acquire 4 If the Confirm Calibration On Acquire check box is enabled at acquisition the Confirm message 5 appears Confirm or change the Objectives and Mag Changer settings 6 Click OK LAS User Manual 3 100 Camera Scale Bar Scale Bar The Scale Bar panel controls the attributes of the Scale Bar and whether or not it is displayed over the live image To expand the Scale Bar panel 1 Click on the small arrow to the right of the Scale Bar header bar To display a Scale Bar on the image 2 Click to enable the Scale Bar Show button This is a toggle to turn on and off the Scale Bar 3 The default display position for the Scale Bar is top left but may be re positioned by clicking and holding the bar and dragging it to the new position To change t
106. Montage Multifocus o Multifocus image Click on Create Multifocus Image to generate an image Align source images first using the parameters set in the panel Mian Mono Alignment If the source images are not exactly Sample Size registered with each other having been captured with a stereo microscope or from scanned photographs this should be corrected with the Align Source Images operation before proceeding Optimize with the Montage operation Method Blended Patch Size JH 1 Click on Align source images first SES WW Preview 2 Select Align Method from the drop e down box Orthogonal Shift will correct for X and Y shifts between the images planes Sreate Multifocus Image 3 Select Sample Size from drop down box This should normally be set to Maximum for best results 4 101 2007 Leica Microsystems Switzerland Ltd Montage Method Montage Method The optimum Montage method will depend upon the sample type Some experimentation may be necessary Select the method from the drop down box e Fixed The Fixed algorithm selects the single source image plane which is in best focus at each pixel location Blended The blended algorithm takes into account the effects of two or more in focus planes at any one pixel location The resulting depth map values may be fractional which shows slopes within the sample more accurately Weighted The weighted algorithm takes into account multi
107. OK button 8 The new name appears in the Configuration window and the settings may be used again for future Overlays See Selecting an existing Configuration WW Manual acquire BB Auto create overlay Ei Always create new overlay folder Default Sequence Name 4 89 2007 Leica Microsystems Switzerland Ltd Image Overlay Auto Create Overlay For speed and efficiency select 1 Auto create overlay by clicking the check box and 2 Click on the Always create new overlay folder check box 3 Click on the Acquire Overlay button These options will create a new folder within the capture folder with the Default Sequence Name as part of its name The images for each of the filter channels 4 will be saved inside the folder and they will be automatically combined to produce the Overlay 5 Adjusting Channels before creating 1 Uncheck the Auto create overlay check box for a preview before committing Each of the images may then be examined and adjusted for colour balance and brightness before creating the overlay LAS User Manual 4 90 1 Click on the Auto create overlay check box to enable it 2 Disable Always create new overlay folder by clicking the check box until the tick symbol disappears 3 Click on the Acquire Overlay button The Channel image location dialog appears 4 There are 3 options on the dialog Create a new sequence folder 5 will create
108. VS La a Spot Exposure j WD AP Find Focus 3 To move the Region of Interest click and hold on the green border not a handle and drag it toa new position 4 To resize the Region click hold and drag on any one of the eight handles 5 Click on the Acquire Image button When the image is acquired only the area within the Region is saved 6 In this example as soon as the image is acquired the Browse Workflow opens and the cropped image is displayed in the Viewer Browse opened because in Preferences it is the function selected to automatically open when an image is acquired This selection may be changed by going to Preferences 7 Click the arrows to the right of the Region of Interest header to reveal the panel 8 Click the Crop to Rol button to hide the green crop rectangle To reveal it again click the Crop to Rol button It will retain its size and position LAS User Manual 3 106 Camera Region of Interest Spot Exposure Spot Exposure This feature will automatically adjust the exposure using only the area with the Region of Interest rectangle as a reference The new exposure values are then applied to the entire live image The Rol mask may be moved around the image to compare different exposure results 1 Click on the image hold and drag KN n diagonally to create a rectangle ni mri ain vii This is the Region of Interest e 2 On the menu that appears when w
109. a Reset Black White points fk Reset Gamma Value Histogram Options Auto White Auto Black Ay bamma AU Reset Black White points Reset Gamma Value Histogram Options 3 97 2007 Leica Microsystems Switzerland Ltd Camera Calibration Settings I g The process of calibration compensates for any manufacturing variations in the objectives mag lenses and camera element array and mount Automatic microscope settings are available to the software which calculates a default suitable for less demanding applications but for greater precision calibrate for every objective and Mag setting as they are used Changes in camera or camera mount will require new calibrations The same applies to manual microscope Each calibration setting is given a unique name and can be recalled and applied to an image at any time As a reminder to check calibration there is a Confirm Calibration facility that prompts for settings every time an image is captured 1 If the Calibrations Settings panel is not showing click on the arrows to right of the Calibration Setting header bar Calibration Setings Ol Actual jength of line shown on image 35 024 Millimeters s em D gt Confirm Calibration On Acquire LAS User Manual 3 98 Camera Calibration Settings Calibrate Calibration requires a Calibration Slide fitted to the stage in place of a specimen Make sure the Calibra
110. a changes swipe the window to highlight it and type in a new value 4 137 2007 Leica Microsystems Switzerland Ltd The Count tool target marker has several size options To change the marker size 1 Click on the arrows to the right of the Marker Size header bar and from the drop down menu choose the required marker size For densely packed small objects on the image choose the smallest marker and disable text Auto Size Change the Class name for existing measurements by 2 Clicking on the Select tool and then on the object that requires a change of Class name 3 Scroll to select the new Class name la in the Select Class window 4 Click on the Apply Class button L eafks BI LAS User Manual Interactive Measurements Measure There are six label options available for display with measurements 1 Number is a sequential number for the measurement For a new image or after all measurements are cleared with the Clear button it starts at 1 and increments for every measurement 2 Class if checked will display the Class name on the image and also in the results table 3 Parameter displays the main function name on the image display For example using the Circle tool will display the word Area as well as the numeric value 4 Value displays the numeric result of the measurement 5 Comment will include the words typed into the Comment window on the tool
111. a new folder within the capture folder and save the images to it the equivalent of checking the Always create new overlay folder check box Empty the current sequence folder 6 will delete all of the images inside the current folder and replace them with the new acquisition Add to the current sequence folder 7 is a powerful option that allows any number of channel images and subsequent overlays to be stored in the same folder Each acquisition is given its own sequential number to differentiate between the sets 10 8 The entire acquisition sequence may be stopped by clicking the Cancel button Select the required option by clicking the button next to it and clicking on the OK button 9 11 When multiple images are stored in the same folder the Working Gallery fills with all of the images Hayward_PE3 BB Manual acquire Auto create overlay BBE Delete channels after overlay creation E Always create new overlay folder Empty the current sequence solder Add to the current sequence folder Press Cancel to stop this acquisition sequence Default Sequence Nome pu a 2_Epsilon_Aa2jpg gun 5_Epsilon_BGR RU A Epsilon_N212jp9 HU Sai 4 91 2007 Leica Microsystems Switzerland Ltd Image Overlay Auto Overlay With Delete Channels Enabling the Delete channels after overlay creation option removes the individual Channel images after the Overla
112. age 1 The monochrome Channel image with the Adjust Appearance panel as it looks when capture is complete Had this been a colour image the Apply False Colour check box would not be available Select another image by clicking on its thumbnail in the Gallery It will be displayed in the Viewer 2 To apply colour click on the Apply False Colour check box to enable it A basic colour may be applied to the image based upon the type of filter used in its capture 3 Move the Channel Colour slider to the desired colour 4 The applied colour is immediately displayed on the Viewer Continued Adjust Appearance Channel Colour 527 nm Apply False Colour 4 97 2007 Leica Microsystems Switzerland Ltd Refine the image by adjusting the sliders below the Histogram and also The Gamma value The Mixing Strength slider controls the predominance the Channel image will have in the overall result The range is from 0 to 200 The higher the value then the greater the presence of the image in the Overlay Test the result by clicking the Preview check box All of the Channels are combined in a temporary overlay 10 If the result is acceptable click on the Apply button to create an Overlay A new thumbnail will appear in the Gallery In this way any number of Overlays can be created to illustrate various aspects of the image 11 To reset the Channel images
113. ain to reveal them Show Default Values only 6 The Show default values ON shows the default parameters for all of the measured objects OFF shows only the selected parameters for a single object See Measurement Actions Continued Interactive Measurements Measure Measurement Actions Measurement pia s Node Runner Prior to budbu DRA 6 Node Runner Prior to budbu SST LAS User Manual Interactive Measurements Measure Merge Measurements and Image 7 Measurement information is stored in a separate file associated with the image and each time the image is displayed the tools and their values are re drawn over it Sometimes it will be necessary to export the image and measurements to another application which will not be able to re draw the details so the Merge button combines image and tools in a single bitmap After Merging measurements cannot be altered or modified Clear Measurements 8 Use Clear with caution because it removes all of the measurements from both the image and measurement panel They cannot be retrieved Group Measurements Individual measurements may be grouped together and summed Only the Area and Circle tools have this facility 9 Select Area or Circle 10 Click on the Group check box Draw the shapes and the areas will be summed and displayed as a single item on the Measurements panel Subtract Measurement
114. allows the user to reduce the size of the scan area This is achieved by placing the red cross hair at the position the Scan Pattern is to shrink to The Zoom In and Zoom Out buttons allow the user to enlarge the Scan Position display This display can then be navigated using the scroll bars that will appear or by holding the mouse button down over the scan position graphic and panning the position by moving the mouse Releasing the mouse stops the panning Double clicking on the stage graphic causes the stage to move to that position Units Millimetres E cD D D f X 51 02 mm Y 19 63 mm Z 5 39 mm oroz ees Je 4 71 2007 Leica Microsystems Switzerland Ltd MultiStep Define Sequence This is a data textual representation of the scan pattern It allows fine tuning of the scan pattern Scan Origin This is the location of the first field of view in the pattern and is the central point of that field Go To Drives the stage to the Scan Origin Set Takes the current stage position and sets it as the Scan Origin and the St scan pattern is shifted appropriately Pp ep Size attern Sie 5762 besi Fars E E Scan Definition Step Size This is the distance between one field and the next both horizontally and vertically If Auto is selected the component images will be adjoining De selecting Auto allows the user to acquire images with overlaps or images with spaces T SE them d
115. am Auto Black White amp Gamma Auto White and Black Because under and over exposed areas of the image do not usually contribute to its quality those levels may be ignored without detriment Auto White and Auto Black automatically crops either or both under and over exposed levels 1 Right click on the Histogram window 2 From the drop down menu select the Auto White or Auto Black option The selection becomes checked and active Select again to uncheck and disable the option Beneath the Histogram window the slider corresponding to the white or black option moves to reflect the ignored light levels 3 To reset the black and white values right click on the Histogram window and from the drop down left click on the Reset Black White Points option Auto Gamma The Auto Gamma option sets the gamma level based upon the active light levels Under and over exposed levels are not included if they have been cropped either manually or automatically Whilst Auto Gamma is enabled the Gamma control on the Exposure Adjust panel is disabled 1 Right click on the Histogram window 4 From the drop down menu select the Auto Gamma option The selection becomes checked and active Select again to uncheck and disable the option 5 To reset the gamma value right click on the Histogram window and from the drop down left click on the Reset Gamma option E Show Under Over Exposure Auto Gamm
116. amination affecting the Shading a very small amount of de focus may be used 3 Click the Next button 4 The Wizard will create the Shading Link for the current objective When complete it will automatically select the next objective change the objective manually for non auto microscopes and return to Step 1 5 When a Shading Link for all of the required objectives has been created click the Close button Linked Shading Wizard r Q Q 8 H 4 A m Stat AN 20x DW Il 2 Sal Apoy 1004 Frish 11506145 1S6 11506507 1tI506504 11505083 mmen 11506197 4 om Move the stage to an aves with a blank specimen regon or remove the H H t 3 120x a E LS Apply m5506 11506507 11506504 11505083 immessi 6 a 100 Eech 11505197 4 gt 8 e o A Stat a Low DM Il 2 Sal Apoy 11505145 11506506 11505507 11506504 11505083 immen 6 a 1004 Frish 11506197 Createg shading 3 93 2007 Leica Microsystems Switzerland Ltd Camera Linking Delete Links 1 Both Exposure and Shading Linking may be enabled together In this case the status icons on the Linking header bar will appear combined 2 Exposure and Shading links may be deleted by clicking on the appropriate Delete button Use with care This will delete ALL of the Shading Link files LAS User Manual 3 94 Camera Histogram l Select format To select the display format 1 Right click on the histogram display win
117. amp slightly 3 so that the camera may be rotated Rotate it by a very small amount and repeat the calibration until the rotation angle is within limits Tighten the clamp Once set correctly and providing the camera and stage do not change rotation should seldom require adjustment The camera rotedon in exter cxcentive itis recommecded tut the comers be resigned LAS User Manual 4 172 Power Mosaic Shading Set the Shading Reference The Shading Reference electronically corrects any light level fall off which appear towards the edges of the image which is often inherent in optical systems The required exposure light intensity objective and mag level must all be properly set with the specimen in place If there is a change in objective the Shading Reference must be carried out again Select Live Screen view 1 Navigate to a portion of the image that is free of detail with a clear background Objective 2 50 07 N PLAN DRY 2 If necessary click on the Optics Setting header to reveal the panel If a Shading Reference has not been carried out the check box will appear greyed out 3 Click the Set Shading button and 4 on the dialog click the Set button EE specimen and ensure that there Le minimal detail shown Check that the exposure is set correctly Now click Set 5 When complete the Set Shading box 7 will be checked and shading will be enabled If required
118. an Window click on the icon again 3 113 2007 Leica Microsystems Switzerland Ltd Browse Fit to Screen and Zoom ki The image may be scaled to fit the Viewer area by 1 Click on the Fit to Screen to display all of the image or 2 Right click on the Viewer and 3 Select Zoom and Fit Increase or decrease the image size by 4 Click on the Zoom In or Zoom Out icons or 5 Right click on the Viewer and CI o V Fit Original Size 6 Select Zoom and 7 In or Out or 8 Click on the Viewer hold down the Ctrl key on the keyboard and spin the mouse wheel to re size the image Show Pan Window d Fit Original Size LAS User Manual 3 114 Browse Open Image An image may be imported from another folder by 1 Click on the Open icon on the side bar or Right click on the Viewer to reveal the Image Control menu Click on Open A Windows navigation pane opens Navigate to the required folder and then the image Click Open The selected image appears in the Viewer with A thumbnail in the Gallery The imported image is not copied to the original folder Save an Image 7 Click the Save icon on the side bar or on the Image Control menu to save the displayed image It is saved under its existing name and overwrites the image previously saved Save Save As Print Copy Paste Zoom gt Show Pan Windo
119. ance Automatic Exposure Fine Tune 1 If necessary reveal the Exposure Adjust panel by clicking on the arrows right of the header bar 2 Click on the Image Controls Automatic Exposure icon 3 Adjust the Brightness Saturation and Gamma controls if necessary to achieve your optimum image Automatic White Balance All of the neutral tones white through grey to black are adjusted to remove any colour content to maintain a clean well defined image 4 Click on the Image Controls Automatic White Balance icon White Balance is applied to the entire image 5 If the image is too dark or too light Automatic White balance may fail and an error message displayed It may be possible to lighten or darken the image with the Exposure Adjust controls or change the lighting conditions at the microscope 6 To undo Automatic White Balance right click on the White Balance icon and 7 Left click the Reset label Reset White Balance LAS User Manual 3 84 Camera Exposure Adjust Manual Exposure Description Manual Exposure Description The image controls change when Exposure Adjust is in manual mode Brightness is replaced by Exposure Saturation and Gamma remain but another control Gain is added 1 Exposure controls the time that the camera sensing elements are exposed to the specimen It is sometimes called the scanning rate At the start of the exposure time period all
120. and details of specific measurement tools and options to use 1 Click on the arrows to the right of the Configuration drop down window 2 Click to select a Configuration from the list 4 125 2007 Leica Microsystems Switzerland Ltd Interactive Measurements Setup Create a new configuration 1 Click in the Settings Configuration text box and type a new Configuration name 2 Click the Save button The new name will be used for the file containing the Classes and measurement tools and options for the current image It will also appear in the drop down and can be applied to subsequent images Delete Configuration 3 The selected Configuration may be deleted by clicking the Delete button The deleted Configuration may not be retrieved Create a New Results File The Results File is the name given to the file in which the measurement data and analysis will be saved The default name is the same as the image name To create a new file name 4 Click on the Settings Results File text box and type a new file name 5 Click on the Save button The new file is created and the name added to the drop down list This method allows multiple sets of measurements to be made on a single image and recalled individually LAS User Manual 4 126 Interactive Measurements Setup Select and Delete a Results file It is possible to use an existing Results File name to store new results The original d
121. appears when the optional Analysis modules are installed In this version of the Leica Application Suite the Interactive Measurements may appear here and you can view its capabilities by clicking this link See Interactive Measurements LAS User Manual 3 144 Chapter 4 Optional Modules Optional Modules The underlying capabilities of the core functions can be enhanced with a range of advanced modules and applications Each Leica Application Suite module provides the flexibility to tailor a system solution to fulfil individual needs with upgrade options available for future requirements In this section SG Extended Annotoation SG Manual Measurements SG MultiTime Time Lapse SG MultiTime Movie MultiFocus MultiStep Image Overlay and J Montage These modules are available for a 60 day evaluation period after which a licence for their continued use has to be purchased 4 3 2007 Leica Microsystems Switzerland Ltd Extended Annotation The Extended Annotation module will only appear if it has been installed It is accessed by an additional tab on the Process Workflow Using Extended Annotations new annotations and more detailed captions may be added and repositioned on the image using a variety of fonts colours and background effects including Transparent NOTE For clarity and convenience you may choose to add and save the default annotations Image Name Description
122. arch starts from pre selected focus suitable for sample with approximately the same thickness Adjustment of parfocality is a prerequisite for this mode Search starts from the current z position suitable for samples with variable thickness Within each mode the span of search can be selected Near Search of focus position considers only the near proximity Global Range of search is extended NB For correct operation please install the newest camera driver It is highly recommended to adjust the exposure time of the camera to values lt 10 msec to ensure fast response time Advanced Settings define start mode for AF search P Call the Online Help DM Operation overview 3 47 2007 Leica Microsystems Switzerland Ltd DM Microscope Operation Stage Control The speed of the stage can be modified using the control buttons fast or precise The speed mode fast refers to the value set within the module Fine Tuning Current coordinates and a graphical position marker is displayed in the control window Up to 5 positions can be stored and recalled Use the pull down menu to select the position number position 1 5 and load position with the left mouse key Store current position with the control button Store and recall the position with the control button GO TO The stage region scanning area can be defined in the advanced settings NB The positions are not stored permanently and are lost after switching off the
123. arrabide iad aia aaa aa aa adaa 4 135 Cha ge Text attributes ino iriad are aia aT A EE TAE 4 136 Background Line colour and dickness 4 137 Count marker and Class name 4 138 Measurement Labels usais luca ioia aaa eia Edadi 4 139 Measurement Labels Continue 4 140 Using Measurement Tools AA 4 141 Measurement ACtOnS i s 2 co ncaa intial Soi 4 142 Measurement Actions Continue 4 143 Results 0 ere eebe 4 144 Select Classes sangane aeee e aa hia ee a ea 4 145 Results Table murman e e EEN eg 4 146 Results SummMaAry sabaosas ida ee laa 4 147 Results Summary Continue 4 148 Output Results eekleg et cain shee dees 4 149 Power Mosaic amp Power Mosaic Plus Power Mosaic Features aaaaanaana Power Mosaic Features Continued ccscsssssssssseseessssssessssesersrseseesseessaeaes 4 155 Operating Steps iecsssssssessssessssesesscseseeeesteeeseseeaeseeseseeaeseeeeseeataseenesteenatsess Operating Steps Continued 3 Operating Steps Continued n nnsnnanononsaenennnsnrnrnranennnsnsnnnrnnnnnennsurnrennnnnnnenna Starting User Interface User Interface Tools User Interface Views ccsssssssesccsssssssssssssesssssessssessaracsessersseesaracenseensesatanaes On screen Joystick 2 Imaging EI HR Imaging Setup Continured cssessesessssessssesesseesssesesteseeseetseeseerestseeateees 4 165 Input Options na ws 4 166 Input Options Continued oe esssessssesesseesseeaeseeeeseeaestenese
124. aseeees 2 6 Hardware Configuration ccccsssscssssesesessessesessseeesssseeeseeaes 2 7 Hardware Configuration MicroSCope u u cccscccessscsecsssesssesessesssesseesessensenees 2 8 Hardware Configuration Cameras csssssssesssseesssseseesesteeestsstateneatseeaseeees 2 9 Registering Modules and Applications KEREN 2 10 Obtaining a Licence Key icc ccccscscessessessessessessessesseseusseveseessesaesaeseesaesense 2 11 Installing the Licence Key ooo ccc csccscscsseessssessessessessessesseesessessessesseseeseusesees 2 12 Registration Intormaton REENEN 2 13 Using the Core Features een 3 3 Starting Leica Application Suite 3 4 The Setup Workflow ccccccssssssesessseeesesseeeesssensensssaneeeeasseeess 3 5 DM Microscope Configuration ccsssssssssesessssssssssssescscstsssesssssssseseseseseeeseees 3 6 Configuration Overview cc cccccececeeeeeeeseeeeeceeeaeeeeseeeeaeseaeseeesaeesaeseaeseeeeneeeaeees 3 7 Condenser Turret 3 8 Function Keys Microscope Data Microscope Data continue Microscope Data continued Microscope Data continue IG T ret aarti aed IL Turret IL Turret eontinue eneee ereree erreneren Microscope aasifecesiensi eddie ete ee NosepieCe aassssssissrsrsersssssssrrsns Nosepiece continue Parfocality Control Objective Tune Control FiIn sTUNING sAAdtentai ege Tube OedEEeE eege d Tube continued eeeeceeseeeceeeeeeeeeeeeeeeeteeeeeeeeeee
125. ata is retained and the new data is added so nothing is lost Files are saved automatically if the Workflow is changed or another image selected To select an existing file name 1 Click on the arrows to the right of the Results File text box 2 From the drop down click to select the file name to use Delete Results File 3 Click to select the Results File name to be deleted 4 Click the Delete button Deleted files cannot be retrieved 4 127 2007 Leica Microsystems Switzerland Ltd Interactive Measurements Setup Create a Class A Class is an association of measurements often made with a range of tools In this example a tiny plantlet is being measured and one of its Classes is Node Main Bud There are two measurements in this Class Bud diameter and Bud circumference The Class name associates the pertinent measurements and results Create a Class by 1 Click on the Define Classes Add button The text box is high lighted 2 Type in a new Class name Press the keyboard Enter key 3 The Colour dialog appears Each Class may be displayed in a different colour to distinguish between them 4 Select a colour from the swatches or colour wheel and click OK 5 The new Class name appears in the drop down list Red be B pe bp B Green k B Saturation bss B Blue k B Brightness bss B ni i L Clear All LAS User Manual 4 128 Interactive Measurements Setu
126. ated to increase or decrease j brightness 2 The light source may be turned on or off by clicking the Power button A red dot indicates that the source is on 3 Click hold and drag the red handle on the rotary control clockwise to increase brightness or anti clockwise to decrease it The Kale light output value is displayed on y External Illumination 2600k LA the header bar Power 4 4 Alternatively click on the outer rim of the Brightness Control which will rotate to the selected position Continued 3 61 2007 Leica Microsystems Switzerland Ltd Stereomicroscope Operation Internal Illumination co There are three controls for the Internal Transmitted Light Base CT Colour Temperature which D controls the lamp average voltage Keane KE and therefore its brightness This also affects the light colour CCIC Constant Colour Intensity Control which acts like a window blind reducing the amount of light reaching the specimen without affecting the colour Shutter is a toggle action button which stops light reaching the specimen altogether The CT 1 and CCIC 2 controls work in combination so for some procedures it will be necessary to adjust the light colour using the CT control and then adjust the brightness with the CCI control Both controls are adjusted in the same way 1 Click and hold on the red dot on the periphery of the control Drag either clockwise
127. be yellow E KN RW ei ba MI og p 4 The level bar moves left to right to l o ee y g indicate the focus precision The ei E further the bar is to the right the w EI ml ea warembo better the focus i SN x a na ns 5 To move the Region of Interest and check another point on the image click and hold on the yellow border not a handle and drag the mask to a new position 6 To resize the Region click hold and drag on any one of the eight handles 7 In this example the Region of Interest has been moved and resized The new Region of Interest has better focus precision as indicated by the level bar and the bar indicator will remain in this position until a better level is found 8 Turn off Find Focus by clicking the Off button 9 Click the Find Focus button to reveal the Region of Interest at its last size and position LAS User Manual 3 108 Browse i The Browse Workflow provides access to all associated information for each stored image such as the time of acquisition the bit depth and the calibration The Directory Browser means that images can be located quickly and easily through a simple navigation system whereby folders can be effortlessly located The integrated Gallery stores each image as a thumbnail to speed up the process of locating and retrieving image files Furthermore images can be copied and pasted to other applications or zoomed and panned for a more detailed
128. bnail Reduction Factor will help prevent filling the RAM too quickly The tiles are stored initially in a temporary file on the hard drive The individual images are much larger and are used to paint the mosaic at greater resolution to preserve detail Clearing previous scans removes the thumbnails from RAM and the tiles from the temporary file If the previous scan is left intact new thumbnails and tiles will be added to the existing with sequential image numbers 1 Click on the Clear Tiles button to clear the previous scan This is only the previous scan and does not remove any prior to that 2 The Clear Mosaic message appears Click Yes to continue 3 Click on the Acquire Power Mosaic button 4 If Turboscan is selected and the exposure speed is too high a dialog appears giving the option to use Standard scan instead or to cancel Clear Mosaic Coure PowerMosaic 4 Turboscan Information itis recommended a Camera Exposure lt 200 uSecs is used for Turboscan Perform a Standard Scaa emp EOD aD LAS User Manual 4 194 Power Mosaic Z Stacks Z Stacks If Power Mosaic Plus is installed and enabled the Z Stack option is available For every tile position on the XY plane Power Mosaic Plus creates a stack of tiles in the third dimension or Z plane The number of tiles in the stack and the distance between them can be set to be reflected in the specimen Once captured the individual
129. bry scoddentad or ntertiona systery Debugger users can debug processes on thes machine both iocahy Group for the Help ond Support Center E Siriten Tooti Evert Viewer F Parod Folders Local Users snd Grouse T Users J Gop D Performance Logs and terts Device Manager Sg bech ator fj Rosge B cos Removable Ror ape Debugger Users Orch Ondragmreeter HalpServicesGroup Cech Man s t a Servees and Arr sen Bckregir hoes Backup Operators eg Sudo develioers can author web ates on thes congue ser Hardware Stup Firmware Update Registration Registration Info Leica DM Leica DM Leica DN 2 7 2007 Leica Microsystems Switzerland Ltd Hardware Configuration Microscopes Hardware selection Microscope continued The options and accessories available depend upon the microscope chosen Automatic and motorised models will certainly have a computer connection option in the example an RS232 COM port but USB is sometimes an option so the list should be checked against the microscope manual 1 Click on the arrows to the right of the Connection window 2 Use the up down arrows to scroll through the list 3 Select an available connection by Clicking it Selecting Accessories In this example a motorised stage is an option although may not be fitted 4 Click on the arrows to the right of the Accessory Stage Controller window and from the list choose 5 None if th
130. cactntgeapsccaeseasceeanesssncsosnecons sects asiana 4 19 Elements Deleting and Merging seen 4 20 MultiTiM gonsyrn eanna aaa aa 4 21 TiMesLapse ee ee 4 22 Image Exposure EE 4 23 e CT SE 4 24 Setup Options pre 4 25 Set up Options continued anini nidan aneis 4 26 Ge WEE 4 27 Testing EE 4 28 Ee ui e VBE 4 29 Projectnames waviness 4 30 Capture Display Options A 4 31 Capture and Display continue 4 32 Replay eebe eu retin eee ie as 4 33 ConverttoMovie casman enap vida iil E 4 34 Mee Eege ees 4 35 Select or Create a Fold hi tivacsccascse ect et ce dd veer Eed 4 36 IO 4 37 Setup continued dee eee eege ee aes 4 38 Sel ct Reconding dE 4 39 Define Clip le 4 40 Name and Compressi0titucc caucisnwed anne ane Aae ES 4 41 Time Stamp and Saving Configuration cccccecceeceseeeeeseeceeeeeeeseeeeeeereeaeeaes 4 42 Existing Configuration and Testing 4 43 Start Recording and Progress 4 44 SAS ue CM 4 45 Playand Frame Capture ege EIERE 4 46 Play Ee Be 4 47 MultiFocus aac casenessseessececssneessesenssetenssneeesanenssaeenssseensas 4 48 Emable MultiFOCus eege 4 49 Select Image Format ccccccs cccscccecsssazcascetscesstseseeseestossosssbcactstascoceatiatoesanonecetineat 4 50 Select or Create Capture Folder ooo csssscssesssssssssssssessssesssssnsstsnsesnsensaeens 4 51 Options Options continued Configuration Define Z Stack Automatic Microscopes ccccsssssssescessessesseeseseseeseeeeeees Define Z Stack Start Automat
131. caled to fit in the Image box appears 21 10 2005 _O014 jpg 200 um 4 17 2007 Leica Microsystems Switzerland Ltd Extended Annotations Insert Image Outline Shapes and lines may be drawn over the images On this example a circle on the inserted image will indicate where the split in the paint flake is and a line from the circle will point to the enlarged split on the main image To achieve this first of all a white outline is drawn around the new image by 1 Select Rectangle and click on the top left hand corner of the small image Hold and drag until the rectangle fits the image 2 Enable Outline 3 Enable Transparent 4 To change the outline thickness click the arrows to the right of the Width window up arrow to increase the width and down arrow to decrease it 5 To change the outline colour click on the Line window and when the 211072008 Colour Selector dialog appears either select a swatch or use the z_0014 jpg colour wheel and slider Click OK 200 jem LAS User Manual 4 18 Extended Annotations Insert Leader Line To draw the circle and leader line from the point of interest on the small image 1 Select Circle 2 Click and hold on the small image press and hold the Shift key on the keyboard and at the same time drag the mouse to create a circle 3 Change the circle outline thickness by clicking on the up down arrows to the right of the Width window 21 1
132. ce designed for ultimate operator convenience and hence increased productivity The Z digital microphotography environment is automated through the computerised features of the Leica microscopes LAS is used for acquiring storing annotating and displaying high quality images with a thumbnail gallery Moreover LAS is highly extensible and additional modules can be licensed to include specific application starting from routine applications to cutting edge research procedures LAS is based on personal computers using Windows operating system and provides a cost effective and uniform environment compatible across the Leica range of microscopes and Digital FireWire cameras Leica Application Suite comprises Common Core functions Optional Modules Applications 2007 Leica Microsystems Switzerland Ltd Introduction Common Core The Common Core provides the basic software for configuration and control of the selected microscope as well as for the acquisition analysis and processing of high quality digital images The Core components include the following features e Microscope and digital camera configuration and control in a fully integrated manner e Auto and manual exposure adjustments to allow fully optimised imaging conditions Image calibration based on data read from Leica microscopes and cameras A scale bar displayed on the live image to indicate image size e Acquisi
133. cope Operation Filter Wheel control 1 Place the mouse cursor over a Filter button to reveal its specification 2 Click the button and the Filter Wheel will rotate to select the filter To change a filter 3 Manually turn the Filter Wheel to the required position Each filter BLUE has an identifying tag on its rim Excitation Filter 470 40 nm Carefully slide out the filter and Barrier Filter 515 00 nm insert the new one After filters have been changed 4 Click on the Refresh button All of the Filter button labels will clear the Filter Wheel will turn and the fitted filters identified 5 The button labels will be displayed with the correct filter data Continued 3 55 2007 Leica Microsystems Switzerland Ltd There are five control options for the Motorised Zoom Drag and drop 1 Click and hold the Scale Indicator and drag it to the required zoom position The shadow indicator 2 remains in the starting position until the mouse button is released Click on the Scale 3 Click on the Scale Bar at the desired zoom position The Scale Indicator will move to the selected position Type a value 4 Click in the Zoom Drive text box and type the required zoom position Values larger or smaller than the zoom limits will be ignored Preset positions 5 Click on the arrows to the right of the Zoom Drive text box and from the drop down list click to select a preset position 6
134. crosystems Switzerland Ltd Interactive Measurements Results Results Table To determine which parameters to display on the Results Table 1 Click the Details checkbox to hide or reveal the Results Table Click on the right arrow to reveal the Results Table details All of the parameters for all of the tools are available and may be turned on or off as appropriate by Checking or clearing the checkbox alongside the parameter to display or hide it The Results Table headings refer to the following parameters Image Name Image name under which the image is stored on disk Measure ment Sequential measurement number Class Class name if selected Line Length Distance and Line tools Width Circle Distance x y co ordinate Line Area Angle and Cross tools Height Circle Distance x y co ordinate Line Area Angle and Cross tools Diameter Circle tool Radius Circle tool Area Circle and Area tools Perimeter Area tool Angle Distance Angle and Cross tools End Distance Line tool between line ends Major Axis Cross tool Minor Axis Cross tool Count Count tool Parallel Distance Parallel lines Colour data is listed under Intensity Brightness Mean Maximum and Minimum Red Blue and Green Mean Maximum and Minimum for enclosed tools Circle Line and Area arses Area Perimeter Ilm 0O Mean Intensity E Max intens
135. ction To Pan a zoomed chart hold down the left shift key while moving the mouse LAS User Manual 4 122 Interactive Measurement The Analysis Workflow Interactive Measurement Module IMM has a wide range of tools for measurement and reporting It is an optional module and must be installed and licensed Options Help Calibrated measurements are made directly on an image and are based upon vector lines drawn by the user Point to point angle and area measurements are possible together with counting of related parameters The module has three tabs Setup allows you to give the measuring process an name or Configuration which refers to the file that contains details of the types and measurements made Once saved a Configuration may be applied to any stored image Measure allows actual measurements to be made and determines how lines values and captions are displayed Results displays sorts and if necessary prints the results Images must be selected before starting Analysis 1 Click on the Browse Workflow allow the Directory Browser to load and navigate to the required folder Double click to open it and display the Gallery Re size the Gallery thumbnails if necessary See Browse Thumbnail Re size 2 Click on the Analysis Workflow tab The Gallery and thumbnails of the selected folder will appear with the last image that was worked on displayed in the Viewer Select another image if
136. d cube e Set name and parameters for the new cube in the dialog window The newly defined cube is displayed instantly in the selection list and can be selected Rename an existing cube and changing parameters e Go to selection list e Open dialog window with right mouse button e Go to copy cube name e Define new name and if necessary additional parameters The newly defined cube is displayed instantly in the selection list and can be selected NB Only cubes with 1 in the column of dazzle protection can be copied LAS User Manual 3 16 DM Microscope Configuration Microscope This node displays the features and identification characteristics of the connected stand according to your order Among others the following items are listed 1 3 Serial number of the stand 1 4 Date of production 1 5 Stand name 1 6 All possible and allowed contrast method for transmitted light axis TL and incident light axis IL These items cannot be changed actively by the customer 3 17 2007 Leica Microsystems Switzerland Ltd DM Microscope Configuration Nosepiece Objecti The nosepiece node allows to learn in new objectives to exchange and remove objectives If a new objective is learned in the system automatically recalls from a predefined data base all settings which correspond to this new item This includes all possible contrast methods with correct light rings IC prisms etc the operation mode
137. d with the image Automated microscopes will adopt the settings Ask prompts if the settings should be retrieved or not Never switches off settings recall Image Mode 2 When a stored image is selected two options are available Live After Recall will switch LAS to the Acquire View and display the live image from the microscope Still After Recall will remain in the Browse View and display the selected image in the Viewer Undo Recall 3 If Undo Recall is checked the current microscope and camera settings are saved before any recalled settings are applied to the microscope and camera Reverting to the current settings is then possible Save and close 4 Click OK to save the preferences and close the dialog 3 37 2007 Leica Microsystems Switzerland Ltd DM Control provides remote control for all motorized functions of the Leica DM microscope series If a camera is attached to the microscope the camera can be controlled at the same time DM Control consists of the following control control windows depending on the connected microscope DM Microscope Operation Contrast Methods Control Fluorescence Control Illumination Control Objective Nosepiece Control Magnification Changer Control Focusdrive Control Stage Control Motorized Tube Control Autofocus Control optional module LAS User Manual 3 38 DM Microscope Operation Contrast Methods Control All available contrast met
138. dow 2 From the drop down menu left click on Histogram Options 3 From the display options click on the option required Combined shows all of the colours and the average Red Blue or Green show that colour level only Average displays the average of all of the red green and blue values Auto White Auto Black Auto Gamma Reset Black White points Reset Gamma Value d V Combined Red Only Green Only Blue Only Average Only 3 95 2007 Leica Microsystems Switzerland Ltd Camera Histogram Under amp Over Exposed Under amp Over Exposed The one click Show Under Over Exposure is a fast guide to displaying those areas of the image that are not exposed properly and are probably not adding to the image quality 1 Click on the Show Under Over Exposed button to check it On the Histogram widow two coloured panels will appear at the extreme ends of the display The blue panel indicates under exposure at the black end and the red panel over exposure at the white end The graphics are also applied to the live image so 2 this is the image before Under Over Exposed was turned on and 3 this is the same image with Under Over Exposed turned on pale blue shading showing the under exposed parts and red shading the over exposed areas Generally over exposed areas on an image should be avoided unless for a specific reason LAS User Manual 3 96 Camera Histogr
139. dy to el connect to your computer d The steps that you need to follow can be summarised as Check that you computer meets the LAG specification Install LAS software Check the Release Notes for late breaking updates Perform the Hardware Configuration Licence the purchased optional modules Enable Demo mode for the evaluation Setup Hardware essentials Complete your Preferences 2 3 2007 Leica Microsystems Switzerland Ltd Installing LAS Please read the document System Requirements rtf that is on the Leica Application Suite disc for details of compatible hardware and software before installation Go there now LAS installation is described in the document LAS Install Guide You should read this document before installing your copy Go there now Installation is described for Windows XP and Windows Vista separately as there are important differences in the procedure used The Vista operating system imposes some changes because of Microsoft s enhanced security features Extra Windows confirmation dialogs occur during the installation and you must specifically run LAS in Administrator Mode in order to modify its licence You must have full Windows Administrator privileges to install this software In a corporate network environment it may be necessary to obtain assistance from the customer s IT department The LAS disc also contains some Exampl
140. e 1 1 icon on the Side Bar to ensure the image is being viewed at its original size 2 Click on the Distance Line icon on the tool box 3 On the image click on the left hand start point of the distance to be measured 4 Hold down the mouse button and drag to the right hand end of the distance to be measured Release the button Lines may be drawn at any angle 5 The distance is displayed in the selected units immediately To change the text font style or size 6 Click on the Select tool and then click to select the Distance Line Caption 5 7 Click on the Font button The font dialog appears 8 Select the Font Style and Size Click OK Extended Annotations Distance Line Auto Size EA Transparent E E ive 4 15 2007 Leica Microsystems Switzerland Ltd Increase the Distance Line thickness 1 Click on the Up Down arrows on the Width box to increase decrease the Distance Line thickness Change the Distance Line Ends 2 Click on the arrows to the right of the Scale Bar Ends bar From the drop down select the End Stroke style to appear on the Distance Line Change the Distance Line colour by 3 Click on the Line colour window The Select Colour dialog appears 4 Click on a colour swatch or select a colour from the wheel and slider Click OK The text colour may be changed by 5 With the Select tool selected click on the Distance L
141. e Additional scans can be added to the initial scan to include all parts of a specimen Scan Patterns Rectangle Circular Annular Cross and x Random User defined Overlap of tiles allows joins to be merged smoothly Correction for camera rotation is performed Save load scan patterns Microscope Automation An Oasis XY stage and Z focus control drive board is used Software joystick for stage and focus movement Compatible with Leica Microsystems LAS configured microscopes controlling focus turret condenser and lamp control as available Stage and focus speed defined per objective linked to LAS Leica DFC Camera Support Exposure saturation gain and gamma control from LAS controls Triggered acquisition from progressive scan and FX cameras for fastest scans Automatic and manual white balance Colour or monochrome acquisition 8 or 16 bit Pseudo colour from mono cameras Frame rate shading correction Shading correction on live display Continued on the next page LAS User Manual 4 154 Power Mosaic Features Continued Export Import Save and load of mosaic scan pattern and Power Mosaic environment as a workspace Select workspace for recall from LAS Load last used workspace on start up Images exported to tif bmp or jpg Export mosaic as full resolution bitmap Export mosaic as reduced resolution bitmap Export user selected region of interest Export image to current LAS capture folder Specimen Map Switch be
142. e Demo button See Enable and disable modules LAS User Manual 2 6 Hardware Configuration Access to Hardware Configuration will depend upon the Windows User level Administrator level is required for installation If MS Windows standard Users are required to update firmware or setup hardware they must be assigned to the LAS Administrators User Group under Windows 1 From the Control Panel select Administrative Tools Computer Management 2 Double click on LAS Administrator and add the user to the list Hardware selection Microscope 3 Select Options on the Licensing tool bar 4 From the drop down menu select Hardware Setup by clicking it 5 Click on the arrows to the right of the Microscope window The microscope selection list appears 6 Use the up down arrows to scroll through the list 7 Click on the required microscope The list will close and the microscope selection will appear in the window sE omputer Managenwemi Desorption 5 Acdrarints os have complete and urreitreted socess to the cong Bacup Ope stors con override sonaty restratons for the sole p Guests have the seme access as members of the Users group bet Members in this group can heve some administr alive privleges tes Power Users possess most ghet ativo powers with some restr Remote Oesktop Users Members thes group are gated the rigtt to logon remotely Supports Ale replication in a doman Users ore preverted from
143. e Images These are useful for trialling the optional modules and can be installed after LAS ea pplication Suite D Installation information Feedback and Hotline Details install Leica Application Suite Install Example Images Instell DFC TWAIN Drivers install E240 Drivers LAS User Manual 2 4 The Framework and Evaluation Launching the Framework Running the Framework The Framework is the underlying software upon which Leica Application Suite is built Normally the Framework is invisible to users because it is hidden by the Application Suite However there are several setup utilities which require the Framework alone to be running The utilities are e Selecting Demo Demonstration mode to evaluate optional modules Leica Application Suite File e Enabling and disabling modules through the Registration Info option Firrware Update Regisiretion Registration Info e Selecting microscopes and cameras and Licensing optional modules These utilities are not accessed though the LAS interface instead shut down LAS if it is running Please ensure you have Administrator rights on the computer before performing configuration and licensing With the desktop displayed 1 Click and hold down the Shift key on the keyboard 2 For Windows XP users Right click on the LAS Icon For Vista users Right click on the LAS Icon and select Run as Administrato
144. e New Folder button to name and create a new folder 7 Click OK While in Image Output Settings 8 Check that the Play Movie Files Using window points to a suitable application 9 Change it by clicking on the Browse button and navigating to the required application See Movie Disk Space allocation Hardware Setup Firmwares Update Full Sereen LAS User Manual 4 36 Movie Setup While Preferences is open check that the disk space allocated for movies is sufficient 1 Click on the Movie Settings tab Two disk space options are available 2 Maximum movie size as a percentage of available free disk space and 3 Limit move size in MBytes which is enabled by clicking the checkbox The maximum value is 1000MBytes or 1GByte Close the Preferences dialog by clicking on OK Select Acquisition Mode 4 Click on the Acquisition Mode button and then No Sequence Image Overlay Montage 5 Select Movie by clicking on the icon Continued 4 37 2007 Leica Microsystems Switzerland Ltd Movie Setup continued 6 If it is not already selected click on the Acquire Workflow tab The Movie tab should now have been added to the Acquire panels 7 Click on the Camera tab The number of frames that can be captured each second and therefore the overall real time detail of the film depends upon the Exposure time the Live Format and the Came
145. e Stack Definition complete click the Options Save button to save the settings See Acquire Z Stack Images EI Create Multfocus after stack acquire eo re yer Ge 4 59 2007 Leica Microsystems Switzerland Ltd MultiFocus Acquire Z Stack Images Automatic Microscopes 1 Click on the Acquire MultiFocus button to start the acquisition 2 At each step position the microscope will automatically move to the next step and focus A progress message is displayed 3 To cancel the sequence click on the Cancel button 4 When all of the images have been captured the Browse Workflow opens with the MultiFocus Image Set control panel displayed 5 Crante Mpttfocan image LAS User Manual 4 60 MultiFocus Define Z Stack Manual Microscopes 1 If necessary click on the Acquire Z tab to reveal the MultiFocus control panels Defining the stack consists of setting the first Z position on the specimen called the Start setting the last Z position called the End and then deciding the number of slices or steps to capture between Start and End Start and End may encompass either the whole specimen from bottom to top or any part of the specimen The positions chosen are displayed on a graphical stack 2 with the Start position 3 shown as an arrow on the left hand side and the End position as an arrow on the right hand side Windows attached to the arrows display the microscope focus
146. e With a SD 4 Click Yes to confirm the deletion copy Selected magas TO and the thumbnails and images will wi a Import From File be deleted They cannot be zogi DIN Send To Leica IM restored Rename Current image Refresh Thumbnail Hide Selected Images To make the Gallery less cluttered and simplify image comparison hide some of the thumbnails by 5 Use Control and click to select K Are you sure you want to delete the image s multiple thumb nails to hide gt aD Unselect 5 Delete Selected Images Hide Selected images 6 Right click on any of the selected thumbnails to reveal the Gallery Control menu 7 Click Hide Selected Images A Remove Images from Gallery Open Current image With confirmation panel appears Copy Selected Images To 8 Click Yes and the selected Import From File thumbnails will be removed from Send To Leica IM the Gallery The images remain intact in the folder Rename Current Image Refresh Thumbnail fi Remove Images from Gallery YD Ara you sure you want to remove the imagels from the gallery Cem eem LAS User Manual 3 120 Browse Open Image with Application In the Preferences dialog an program other than Leica Application Suite was selected to view images It is shown under Open Image using Select All images To view the i in the Vi Unselect All Images o view the image in the Viewer in the Preferences application De
147. e accessory is not fitted Or Select the appropriate accessory 6 Enabling other accessories may also reveal other options In this example enabling the External Shutter also required that it is assigned to a computer port 7 Check the manual and the microscope s delivery manifest before making selections 8 Click the Test button This will perform a connection integrity test Incorrect or faulty connections will result in the Error message 9 Hardware Setup Please select the connected Microscege and Image Soerce Pease select the connected microscope meratramegrabber you want to use as image scurce Cenrector Oe nenn GED Eternal Foo controlled via DASS Bord ee E igs Se es p mg Error Connection Test on COM failed serial H communication not initialized OK LAS User Manual 2 8 Hardware Configuration Cameras Hardware selection Camera A few models do not have a camera option so there will not be a model list For those microscopes that may be Please select the connected Microscopes and Image Source fitted with a camera Microsecce 1 Click on the arrows to the right of the Image Source window Please select tha connected microscope Connection 2 Use the up down arrows to scroll imp CR through the list External Shutter 16000 on Port 3 If a camera is fitted to the GR Etema Focus controlled via OASIS Boord microscope find the model number on
148. e and then click on the Start Stop button a Current movie clip fo Recording frame rate 5 Click on the Save Image button 6 The frame is stored in the current ge a as capture folder not in the movie folders with the name of the movie the name of the capture folder and a sequential number which is automatically generated 7 The file name extension represents the capture format jpg etc a amp x FA TA ts FA TA Bp Move Wome zoomy mm0 mm mu zoos z006 x AS TSX ESN TA TA Sta ONS RONG LOT LOTS ma e e a a Da Lt LAS User Manual 4 46 Movie Play Existing Movies 1 On the Browse Workflow click the Image tab 2 Reveal the Directory Browser by clicking on the arrows to the right of the header bar 3 Navigate to the folder in which the movie was stored If it is the current capture folder click on the Go To Current Capture Folder icon 4 Double click on the Movie folder that contains the required movie 5 A thumbnail representing the movie s appear in the Gallery 6 Click on the Browse Movie tab to reveal the Play Movie controls See Movie play controls See Movie play and frame capture 4 47 2007 Leica Microsystems Switzerland Ltd Images obtained from microscopes have a known and limited depth of focus For specimens with varying surface height when the Z position of the specimen is adjusted different regions of the specimen appear in focus
149. e change Total Area mmr psss XReds P YRelds E i X Step size um IT Step size um Ir X eld size ym oe Y Field size pm a Total Fields Sean Width Imm _ ScanHeightimm E LAS User Manual 4 192 Power Mosaic Save Configuration Save Configuration With the Power Mosaic configuration complete it is possible to save the settings for immediate use in the future To save the current settings 1 On the Scan Pattern panel click the Save As button 2 On the Save As dialog 3 type a file name and 4 click the Save button Files are automatically saved with the pat extension The Save As dialog defaults to the folder selected in Preferences or Browse See Preferences Save in Folder See Browse Select Default Folder 5 The Save As folder may be changed on the Save As dialog using Windows navigation 4 193 2007 Leica Microsystems Switzerland Ltd Power Mosaic Scan Before starting a Power Mosaic scan the thumbnails and tiles from previous scans if not required should be deleted This is not mandatory keeping previous information can be a vital part of an ongoing session in which the specimen is in several parts for instance Thumbnails of each tile are stored in RAM to make access fast and immediate They are used to paint the mosaic on the Viewer Keeping thumbnail size as small as possible See Advanced Options Thum
150. e ex Hardware Setup Firmware Update Registration Info Leica Application Suite Enter Licence Ke To Authorise purchased Leica Application Suite modules please send the following site code to Leica Sales Support and enter the licence key returned Register LAS User Manual 2 10 Registration Obtaining a Licence Key continued Obtaining a License Key continued Applications for a Licence Key are made with a standard on screen form See Getting a Licence Key 1 Complete the user details The items marked with an must be completed 2 To select a country click in the Country text box and type the first few letters of the country name or Click on the arrows to the right of the Country text box From the drop down menu choose a country 3 The computer Site Code is automatically imported from the previous screen Do not alter it 4 Carefully type the Module Licence Number provided when the module s was purchased in the Licence Number text box 5 Click the Save button Use the Windows navigation dialogs to name and save the form To fax the form click on the Print button to obtain a printout which must then be sent to the fax number at the top of the form 6 Clicking the E mail button will automatically launch the resident e mail software set the recipient to the Leica Configuration Centre and attach the form ready for sending Important Having sent the form
151. e for all but individual images use a different filter and exposure to optimise contrast for a specific part of the image Additional enhancement and separation is achieved with pseudo colour a digital staining technique all controlled within Leica Application Suite The final step is to bring all of the images together in a single combined overlay in which individual parts can still be easily identified to illustrate their place in the whole ka a LAS User Manual Image Overlay Select Capture Folder Select or create a folder in which to store the Overlay images 1 Click on Options on the header bar Acguira Time tspsa d Hordwore Setur 2 From the drop down menu select Preferences Firnware Update Full Screen 3 On the Save Images panel check that the path in the In this folder window is correct or change it 4 Click on the Browse button and 5 Navigate to the required folder and Click it to select Alternatively 6 Click on the Make New Folder button to name and create a new folder 7 Click OK Continued See Auto Create Folders 4 79 2007 Leica Microsystems Switzerland Ltd Image Overlay Enable Image Overlay The Image Overlay module must be installed and enabled If it is not the icon will not appear on the Acquisition menu 2 ci WC a y ELA 3 sa pp i NoSequence t Overlay Meer MlykiSta
152. e of 1 0 and gradually increase the value Too high a Gain setting will bleach the image cause a loss of fine detail and may introduce noise 3 Make sure the Image Controls Automatic Exposure icon is de selected white graphic on a black background to enable the manual controls See Further information about 3 Manual Exposure Ee LAS User Manual 3 86 Camera Linking Exposure Exposure Linking associates a specific microscope setup with a specific camera setup With Exposure Linking enabled the Application Suite automatically checks the major microscope settings against a previously stored list and if there is a match will retrieve and load the camera settings associated with it The Linking list is created by the user it is not a preset part of the Leica Application Suite The microscope settings checked are SG Objective or Zoom level for stereo microscopes SG Mag changer SG Camera and port SG Filter and Contrast method A Link may be created for all of these items in any combination making it a really powerful tool for precise repetition To turn Exposure Linking on or off 1 Check the Exposure Link button 3 87 2007 Leica Microsystems Switzerland Ltd Camera Linking Create Link Exposure Create link To create an Exposure Link the microscope must be setup for the image required 1 Check the Exposure Link button 2 The status
153. e speed of movement so that the motors move quickly without stalling Drive Speed Stage Speed 40 10 00mmvsec a Focus Speed 50 10 00nm sec 2 On the Automation Settings dialog adjust the Focus Speed by moving the slider Determining the actual value will depend upon the microscope and the imaging setup and may require some trials 3 Click on the Initialise Focus button 4 Set the Upper Focus Limit relative to the current position by clicking on the text box and entering a new value For example to limit the focus position to 100um above the current position type in 100 Typical values to enter here are 5000 and 5000 Take care not to move the focus position too quickly during manual adjustment to avoid damaging the specimen Initialise Focus Focus the Image Type in the Upper and Lower Limits Then press OK 5 Set the Lower Focus Limit in the same way Positive numbers will be automatically converted to a negative value so there is no need to type the leading sign 6 Click OK g Settings Not Changed 7 Click Apply to apply the settings AN and Close to save them Changes have not been applied Are you sure 8 If Apply is not clicked the Settings TD CH GED Not Changed message appears Click No to apply the new settings Go to Autofocus Setup 4 169 2007 Leica Microsystems Switzerland Ltd Power Mosaic Auto Focus Setup Initialise Auto Focus Auto Focus
154. e with the image Merged lines cannot be edited 3 131 2007 Leica Microsystems Switzerland Ltd Annotate Hide Autosize and Merge The displayed annotations may be hidden removed or merged with the image by clicking the buttons in the Actions box 1 To hide the annotations click Hide click again to reinstate 2 To resize text to default size click Auto Size 3 Merge will merge annotations and graphics with the image After merging text and graphics editing is not possible 4 Clear will uncheck all chosen options and delete the annotations and graphics 5 To move an annotation or a line click on the item to be moved and when the cursor changes to a target click hold and drag the item to its new position REMEMBER to save your work click Save on the side toolbar before moving on LAS User Manual 3 132 Calibrate The Calibrate feature is used for images that have already been acquired and need to be re calibrated perhaps due to a mistake in the original calibration or if an image has been imported without calibration The default measurement unit for the Scale Bar and Distance Line is pixels but in most cases this is not an appropriate value Calibration is the process of calculating the ratio of a known distance between two points on the image to the pixel value The known distance units may be nanometres microns millimetres centimetres or inches
155. ed Text Capture Date 8 Print Time AvantGarde Bk BT D AvantGadeMdBT x Ettects Sample S l Shaeou Capture Time wep User Name Computer Name Calibration File Name Bit Depth Image Size Image Size calibrated File Size kb Description LAS User Manual 3 116 Browse Copy Paste an Image The image in the Viewer may be copied to the Windows Clipboard and from there imported into other applications as well as back into the Viewer To copy to the clipboard 1 Click the Copy icon on the side bar or 2 Right click in the Viewer and select Copy from the Image Control menu The copied image will remain on the clipboard until overwritten by copying again Paste an Image An image copied to the Windows Clipboard either from the Viewer or another application may be pasted into the Viewer as follows 3 Click the Paste icon on the side bar or right click on the Viewer and select Paste from the Image Control menu The copied image appears in the Viewer but does not have an associated thumbnail in the Gallery Save As The Save As function will save the image in the Viewer to a folder and file name of your choice It is a copy the original image remains intact 4 Save As is only available by right clicking the Viewer and then selecting Save As from the Image ve I peony Control menu A Windows emm Navigation pane opens 5 Navigate to the folder and click Open Type a File
156. eeeee The Acquire Workflow ccccccccsceeesssccseeeessesssssneeseeeeesseessenes 3 25 Preferences ae 3 26 Save Images To Folde urisi aea EEE EEEN AE EAEAN 3 27 Save Images Image Name 3 28 Save Images Zero Paddimg AAA 3 29 Save Images Bitdeptht As igaeg oder see siana aeons 3 30 Save Images Image e E 3 31 After Capture Options siiis netacies Mahood mead 3 32 After Capture Options sicoptinued 3 33 Play Movie Using Lock calbratton 3 34 Movies Disk Ee EE 3 35 Store and Recall Store eiiean neen aianei ea aiaa da ee aes 3 36 Store and Recall RecaliModelndo 3 37 DM Microscope Operation een 3 38 Contrast Methods Control ASSEN BA dieing aar NS 3 39 Fluorescence Controls AEN venice aes aes 3 40 ll ee ut EE 3 41 Klee ue NEE 3 42 Magnification Changer Cotro 3 43 Ree lu Ne uge EE 3 44 Motorized Tube Controlicis aeianc lai eiei ane as caida AER 3 45 Memory e Cult NEE 3 46 ee E e tre WEE 3 47 Stage e vr WEE 3 48 Multi UseriPackage riede lege ae en ee es 3 49 Integrated Filter Wheel c 2 cccte codec eGiieie tine aa aaia 3 50 Stereo Microscope Operation seen 3 51 LAS Startup caenndai filed dead dea gaa adidexa tcc 3 52 The SMS Nee ue EE 3 53 Fluorescence Shutter Control 3 54 Filter Wheel Control cassis nau EEN EEN cannes Ges 3 55 Motorised Zoomi DIVE csecsenc naanin iene oe E 3 56 Zoom Magnification EE 3 57 Motorised TC 3 58 Ee VE 3 59 Zoom Iris Aperture Gool ieee A eee ee 3 60 External Illumination Co
157. eeesesenstenesteeeaesees 4 167 Stage Imitialisation ooo eessssessssesessceeseseeseseeseeeeatseeseseeaeseeneseeatseeeesteanateess 4 168 Focus Initialisation Autofocus Setup Calibration Camera Rotation GET UO GE 4 173 Load Configuration c cescssssessesesssscsssseassscasseeaeseeaesesanseeeeseseeatsanseenestsenatsess 4 174 select Scan Patten zveris anA aede a anes aaia adaa arais 4 175 A to Step Siz saisissait asia aaia eaaa 4 176 Scan Direction ege a aa a Eaa a Eana a a EE 4 177 Advanced Options csssssssssssssssessssssssssssseassenssecnarsenseausneaussearsesaussearsesaness 4 178 Rectangular Scan Pattern sescssesessssesessesssseeseeseseeaesteeeseceeatsenaeeeesteenaesees 4 179 Circular Scan Pattern ccssssssesccsssssesssseseececscsessssesseeacseseessscesaracseseseeeseearans 4 180 Cross amp CrossX Scan Pattern ccssssssccscesssssssssssessecscssseessseesaraeeeseeseeseearans 4 181 Random Scan Pattern ccsssssscccsssssssssssssececsessssssseeaeacsesesssssesaracsnsesnesseeanans 4 182 Annular Scan Patten 5s icsscccscdescccaseddkicvasvestnesentt iaa aia aai 4 183 Focus Methods sccicissticavenstecestesisessavecrsvnnsteneieeadeevicietsiaed antvisenctindbdnaiens 4 184 Predictive FOCUSS sirsenis eonan ana darea Ea ae E AEE 4 185 Predictive Focus Continued s ssaseseseesesnunnununununurnunununennunununununnnnunununennnnnnnn 4 186 Predictive Focus Continued
158. efined by the user Click on the coloured box to open the LAS colour picker and select the desired colour Q Save reticule as an overlay E Marge reticule into the image When Saving an Image Select one of e Save Reticule as an overlay The reticule is saved together with the image Reloading the image in the Browser allows the reticule to be displayed if required e Merge reticule into the image The reticule is permanently burnt into the image Subsequent viewing of the image in any application will show the reticule as part of the image 5 5 2007 Leica Microsystems Switzerland Ltd Reticule Available Reticules This panel lists all the reticules available to the LAS once they have been loaded Click on Add to open a file browse window and browse to the required location Select the reticule files to add To remove a file select it from the list and click on Remove A preview of the selected reticule is displayed in the lower panel You can find example reticules by browsing to the folder Leica Application Suite that is under the Documents and Settings All User Shared documents folder on Windows XP or in the Public folder for Windows Vista Creation of Reticules requires a graphics program that can manipulate SVG images An example is called Inkscape Scale Horiz 500um Bar Horiz 001mm Bar Horiz 002mm Bar Horiz 005mm Bar Horiz 010mm CAAA XScale Horiz S00um svg LAS User
159. efore the printing takes place Very small changes in Gamma can have dramatic effects the examples show a range of 0 35 to 1 50 with the original in the centre Generally avoid altering the Gamma settings unless really necessary Continued LAS User Manual 3 140 Enhance Contrast and Gamma 3 All three controls may be applied to the image simultaneously To reset the changed values and start with the original image click Discard on the Confirm panel To keep the changes click Apply 4 If the changes are applied the Save Changes panel appears Click to Replace and replace image Save As under a new name or hs E E EEE LENER Discard clear the changes See Brightness See Hue and Saturation 3 141 2007 Leica Microsystems Switzerland Ltd Enhance Hue and Saturation Hue Saturation and Intensity control the actual colours the amount of colour and the vibrancy As each is adjusted the lower of two Spectrum Bars 1 Change Hue Seturatioa intensity changes as a comparison to the static spectrum above it a Eeer 2 Hue is another word for colour As the slider is moved the colours sien shift from the dominant red in the S N a middle illustration which is the Intensity original toward green on the left or blue on the right The Spectrum _ Bar shifts to reflect the change in dominances Use Hue to correct any perceived E colour imbalance espec
160. els to contrast and gamma each image can be adjusted to your requirements The basic annotation tools mean that a file name the time of acquisition and a brief description can be included on the image providing concise information Even scale bars and lines can be added and individually customized Annotations can either be saved with the image or merged with it so that the data is still visible when exported 3 127 2007 Leica Microsystems Switzerland Ltd Digital images can be annotated with graphic and text objects to provide information or to indicate features of interest in the image The annotations are stored in file along with the image and recalled whenever the image is displayed and can be printed or modified if required The annotation may optionally modify etch burn the stored image The Basic mode provides annotation with a scale bar image name description and time date A single line can be drawn as a pointer or to indicate a point to point distance Annotations appear on top of a selected underlying image independent of the colour depth of the image However when annotation is merged into a monochrome image the annotation colours are lost and become shades of grey To overcome this it is possible to convert the monochrome image to a colour image before performing the merge operation Where images are larger than the window and it is possible to scroll the image the annotation
161. em is correctly aligned and adjusted some of the steps will not need to be repeated again It is assumed that the system is already installed according to the installation procedure detailed in the LAS Install Guide to be found on the LAS software disk 1 Prepare the specimen Check the following e The specimen is as flat as possible to minimise the need for focus changes e The slide surface is clean e The specimen is firmly fixed to the stage loose slides are a common problem 2 Setup the microscope To gain experience with Power Mosaic start with an x5 objective as this magnification probably does not require focus compensation during the scan This makes it easier to check that the scanning is working correctly e Ensure the condenser is in focus and apertures are set for Koehler illumination With an automatic microscope this should be checked for all of the objectives expected to be used 3 Initialise the Stage and Focussing This checks the stage travel limits finishing at the centre point The small green target in the stage area indicates current position The hatched border around the periphery indicates the soft limit switch area e Lower the condenser and check that stage will not collide with objectives e Check adjust the stage speed and initialise See Stage Initialisation e Initialise adjust the focus limits See Focus Initialisation e Check the Autofocus range and threshold See Autofocus Setup
162. en 1 Click on the Camera Live button to halt the Camera Check the Viewer top bar The message should read Paused 2 Click on the Capture Single Image button 3 Click on the arrows to the right of the Working Gallery bar and the Working Gallery will appear 4 The snapshot should be in the gallery as a thumbnail Take as many snapshots as needed 5 Click on the Remove Selected button to delete the selected image from the Gallery About to permanently delete all images that are shown in the gallery 6 Clear the Gallery completely by clicking on the AU button Are you sure Continued LJ 4 87 2007 Leica Microsystems Switzerland Ltd Image Overlay Snapshot and Working Gallery continued To view snapshots 7 Click on Browse Workflow 8 If necessary click on the arrows to the right of the Directory Browser bar to reveal and load the Browser 9 A thumbnail for each snapshot is shown in the Viewer gallery Click a thumbnail to display the image in the Viewer LAS User Manual 4 88 Image Overlay Auto Create Folders continued 4 The folder name comprises the prefix C_ denoting image Overlay the Default Sequence Name and a sequential 4 digit number Creation and naming are fully automatic 5 Save the configuration including the Channel sequence by clicking on the Save button and 6 Entering an appropriate name for the configuration and 7 Clicking the
163. equired Motorised microscopes will automatically turn the filter turret to the correct position but non motorised machines will have to be set manually The filtered image will appear in the Viewer GR AUO 7815m3 24 293 3 1m 243 3 If necessary alter the Exposure time by clicking and holding the Exposure slider and drag it left to decrease exposure or right to increase it Continued 4 85 2007 Leica Microsystems Switzerland Ltd Image Overlay Adjust Channel Exposure continued 4 The Gamma value is normally set to best suit the viewing medium in this case a colour monitor Small changes can have a dramatic effect on colour density but in some cases can help to improve contrast Click and hold the Gamma slider and drag left to reduce the Gamma value or right to increase it 5 Gain will brighten or darken the image without affecting the Exposure time Try to keep Gain to low values to avoid introducing noise into the images Click and hold the Gain slider and drag it left to darken the image or right to lighten it See Adjust Channel Exposure LAS User Manual 4 86 Image Overlay Snapshot and Working Gallery The Capture Single Image function provides a simple snapshot method of saving and checking images from individual Channels after making changes to the Exposure settings If necessary click on the Channel to snap Wait for the Viewer to settle and th
164. er Ader Capture 3 Click on the up down arrows to the right of the Open In window A drop down with the Workflow options appears With tis name Zaro Padding B 4 Click to select the Workflow option required T Bier Movie F es Using See After Capture Options 2 LAS User Manual 3 32 Preferences After Capture Options continued This option will run an application not included in the Leica Application Suite software such as Microsoft Paint or Adobe Photoshop To select an application 5 Click on the Open Image Using button 6 Click on the Browse button A Windows navigation pane appears 7 Navigate to the program required click to select it and 8 Click Open to load it to the Open Image Using window See After Capture Options 3 33 2007 Leica Microsystems Switzerland Ltd Preferences Play Movie Using Lock calibration A third party program such as Windows Movie Player wmplayer exe may be selected as the default to run movies To select the application 1 Click on the Browse button to the right of the Play Movie Files Using window A Windows navigation pane appears 2 Navigate to the application required and click to select it 3 Click Open and the path to the application is stored and displayed in the Play Movie Files Using window Output Settings Administrator 4 Check checkbox Lock calibrations to ensure that only
165. er delay of 2 hours has been selected When the delay has elapsed each of the sequences will be executed in turn The example sequences are 2 10 images with a 30 minute delay between each 3 100 images with a short 1 second delay between each image and finally 4 25 images with 1 minute between Each sequence was added to the overall program by clicking on the Add Sequence icon Changing the Sequence order To move a sequence up or down the execution order 4 Click on the sequence to be moved 5 Click on the up down order icons Deleting a sequence 4 Click on the sequence to be deleted 6 Click on the Trash Can icon Starting the Multi Time sequence 7 To start a sequence or sequence program click on the Acquire button Manwal Ardate ond time Sequence 3 steps 00 3540 Number of images acquired D Interval between images 00 01 00 ry 2 10 st 00 01 00 00 10 00 PT 2 100 at 0 00 01 herr Test Acquire Time Test feica Acquire Time lapse 4 29 2007 Leica Microsystems Switzerland Ltd Time Lapse Project names A sequence or sequence program may be saved as a file for future use as follows 1 Click on the arrows to the right of the Options header to reveal the panel 2 Click in the Configuration text box and type a name for the sequence 3 Click Save Project name When a Multi Time sequence starts a new folder is created int
166. essenseesass 5 7 Reticules on Captured Images iviccscssccescsscessessecsseesessesseseeseeseesesseesenseneens 5 8 Ju ts Del CIE TEE 5 9 Category ET TE 5 10 Template Management sscsssssscssssessssssssseesseeersteeestesnsteeeatseeaesenanseearseeetate 5 11 Seapchbanel _ sa csssseavescsusadsassasansevuvauenssuaviacacscesssosasnceacvcngsacnsuseusondsvacanonssaers 5 12 Device Settings arassa aaar a a A aiea 5 13 Image Data posisnnii inn a A 5 14 Chapter 1 Leica Application Suite Introduction Leica Application Suite Introduction Leica Application Suite LAS integrates Leica automated microscopes macroscopes digital cameras and software into a common micro imaging environment to provide a convenient and consistent imaging solution with unrivalled performance The versatility of the Leica Application Suite means that it is suitable for a diverse range of life science and industrial applications such as materials quality control biotechnology analysis pharmaceutical testing and many other laboratory tasks LAS accelerates the visualization enhancement measurement and documentation of digital images through the total integration of microscopes and digital cameras This powerful software solution can control all functions of the Leica DM range of upright and inverted Compound Microscopes and motorised Stereo microscopes and Macroscopes An essential feature of LAS is its unique user interfa
167. ession when the image is retrieved it will be identical to the original To select the Captured Format 2 Click on the arrows to the right of the Captured Format header bar 3 From the drop down menu click on the option required To save an image with 16 bit colour depth a HQ High Quality option must be selected Camera Input Options Live Format amp Captured Form Live Format 22 Color Binning 1044 x 772 Full Frame 2088 x 1550 Full Frame HO 2088 x 1550 ne eli MA Captured Format 22 Color Binning 1044 x 772 3 79 2007 Leica Microsystems Switzerland Ltd Camera Captured Colour Depth amp Configure Came Captured Colour Depth The Bitdepth is a digital value which determines the colour range and precision of the saved image A value of 8 bits provides a spectrum of 256 separate colours whereas 16 bits yields 65536 colours Greyscale or mono images are captured and saved as either 8 or 16 bits per pixel Colour images require three times the number of bits per pixel to store Golo each of the three primary colours red Captured Colour Depth green and blue So colour images are stored either as 3 x 8 bits or 3 x 16 bits the latter being the HQ High Quality format 22 Color Binning 1044 x 772 The Bitdepth setting has a considerable effect on the disc file size so generally 8 bit should be chosen unless more colour subtlety and variation are needed The 16 bit option is not
168. evel No stack images White represents high confidence and therefore an accurate depth value Black represents low z multiiocus11 confidence and an uncertain depth om gine 0 0 Sec value Low confidence will often represent areas of low contrast where several planes will appear to be in focus Stack Images The confidence image is the same KEE size as the source images used to generate it but is always r monochrome Values are Depth Map expressed as percentages ranging Se from 0 no confidence that depth d Confidence Map map is correct for that pixel to SE 100 complete confidence Anaglyph 2 The Anaglyph image when viewed through suitable red and green lenses offers a 3 dimensional monochrome view of the montage image with depth information synthesized from the depth map Stereo Pair Colour Relief 3D Model LAS User Manual 4 108 1 A Stereo Pair image which when viewed correctly offers a 3 dimensional colour view of the montage image with depth information synthesized from the depth map No stack images To view a stereo pair image COC position your head about 250mm from the screen The technique requires that you view the left hand SEN pris 0 0 Sec image with your left eye and the right hand image with your right 3 eye Try to relax your eye muscles and allow your eyes to diverge as though they were viewing a distant z multifocus11 Stack Images object
169. experience it is possible to produce excellent mosaic images In particular the accurate calibration of the camera and motorised stage is essential and is convenient by performing automatically Good results depend however on the system being well configured and set up and on the user becoming reasonably familiar with the available facilities and functionality The principal purpose of this manual is to provide practical guidance on configuration set up and use of LAS Power Mosaic WARNING High illumination levels are used with Turboscan Switch 100 light to the camera DO NOT look a the specimen through the eyepieces 4 153 2007 Leica Microsystems Switzerland Ltd Power Mosaic Features Integration with LAS Power Mosaic integrates seamlessly within LAS LAS Power Mosaic is selected from the Mode Selection menu bar as are other multiple image acquisition modules High resolution mosaic images can be acquired and reviewed Images from LAS Power Mosaic can be saved and measured in LAS Specimen movement is performed by a stepper stage A Leica DFC camera acquires images It is integrated with the LAS environment with an acquire and a browse tab The image window can show the live image the stage plan or the mosaic Power Mosaic Scanning Uses triggered image capture for fast continuous scan and acquire Standard scan using step and acquire for low light applications Image streaming for mosaic sizes limited only by free disk spac
170. f the Coverage text box and 3 select either Full Coverage 4 or Inside only 5 from the drop down menu Full Coverage covers the entire circle with overlap where necessary Inside only places tiles within the circle 6 Click Apply to apply the values to the image Repeat from Step 1 if necessary to adjust the pattern Click Close to save the pattern and exit Power Mosaic Circular Scan Pattern If an area enabled for editing is modified Press Apply to implement the change Dismeterimm 525 AN Totalel s T Width trum KL Total Area tm P P Sg LAS User Manual 4 180 Cross and CrossX Pattern Types The options are X and Y tiles for the Cross pattern Tile count only for the CrossX pattern 1 For the Cross pattern 2 Click on the X or Y text boxes and enter a value The X value represents the horizontal tile count and the Y value the vertical count 3 Click Apply to apply the settings Repeat from Step 2 to change the pattern Click Close to save and exit 4 For the CrossxX pattern which is a regular X 5 Click on the X text box to enter a new total tiles value 6 Click Apply to apply the setting Repeat from Step 5 to change the pattern Click Close to save and exit Power Mosaic Cross amp CrossX Scan Patterns X Step size ym fT TSS Y Step size tem JI X Reald size um Ji beid size ipm Total fields _ Scan Width mm 1
171. f an incorrect Licence Key re enter it or the Site Code changing due to software being removed or installed If that is the case repeat the application for a Key Purchasing a module also provides access to e mail help from Leica by contacting DI support leica microsystems com Or MQM Hotline leica microsystems com Leica Application Suite File tp Hardware Setup Firmware Update Registration Info Leica Application Suite Enter Licence Ke To Authorise purchased Leica Application Suite modules please send the following site code to Leica Sales Support and enter the licence key returned Site Code 8815 1010 1918 6209 CA Ucence Key 4162 C3D4 ESF6 G7H8 JIKO LAS User Manual 2 12 Registration Information Enable and disable modules On the Framework screen it is possible Leica Application Sute File to enable and disable modules and applications 1 Click Options on the tool bar Firmware Updete Registration 2 From the drop down click on the Registration Info entry Registration Information x 3 The Registration Information panel appears with all of the installed Uceace Number No Licence Number modules listed The Status column shows whether Sits Coa G09A DEEN F353 F208 E1 the module is licensed or not and if it is still in the evaluation period Licence Key bs tGeeneeke the number of days remaining under Time Left The Enabled colu
172. for each colour 4 131 2007 Leica Microsystems Switzerland Ltd Measurement Tools Circle Angle and Count 1 Circle Draws a circle around an object and measures the area within Select the Circle tool and either Click three points on the perimeter of the object to be measured After the third click a circle is drawn to intersect all three points or Specify a diameter or Specify a radius Right click on the Circle icon to select a method Parameter Diameter is the Circle diameter Parameter Radius is the Circle radius Parameter Mean Red Green and Blue are the average colour values of the area within the outline Each value is shown separately on the result together with the highest and lowest value for each colour 2 Angle Measures the angle between two axes Click hold and drag a line along the first axis Release the mouse button Click on the end point of the first axis hold and drag a line along the second axis Release and click once to end The angle is displayed Parameters none 3 Count This is a click and count tool Click on items on the image to count them Each click increments the count and displays it The target marker is available in several sizes Parameter Mean Intensity represents the average brightness of the sum of the target points compared with the overall image brightness or intensity Parameter Mean Red
173. g Wizard button Shading Link does not have to be enabled The Linked Shading E E Wizard panel appears a Conhgeratces already ext for these microscope sengs Do you ward to overwrite them 3 If shading links have already been created for the current microscope settings a warning will appear Click Yes to overwrite the existing link or No to cancel the Wizard K Are you mart you want to delete ail fhe Linked Shading configurations tor the currant camera 4 If you decide to overwrite existing p ae files the Confirm Delete prompt appears Click Yes to delete ALL of the selected links or No to cancel Deleted links cannot be retrieved LAS User Manual 3 92 Camera Linking Shading Wizard Reference 1 The Shading Wizard requires a reference for a blank area for every objective so Either move the microscope stage to view an empty field on the specimen Or Remove the specimen slide and fit a blank slide and cover slip if necessary of the same type as the specimen If necessary carefully adjust Exposure and Gain on the empty field or blank specimen slide to achieve a very small amount of over exposure Refer to Camera Histogram for details of how to turn on over under exposure indication 2 Click on the objective to be processed It becomes high lighted The first objective is selected automatically for motorised microscopes but can be changed by clicking on another To prevent cont
174. ght of its header bar The Image Controls panel comprises four icons 3 Automatic Exposure 4 Automatic White Balance Leica GD 5 DFC Twain launch and 6 Link current camera and microscope configurations To capture an image 7 Click on the Acquire Image button which is always available LAS User Manual 3 74 Camera Fast Track Exposure Fast Track Exposure Very often a very acceptable image can be achieved in minutes by making basic settings to the exposure using Auto Exposure and fine tuning the result with white balance Exposure Adjust KM Make the basic exposure settings 1 Set the Saturation to 1 75 2 Set the Gamma to 0 6 On the Histogram 3 Set the black level to 0 and 4 the white level to 255 by clicking and dragging the sliders Run Auto Exposure 5 Click on the Auto Exposure icon and then click again to turn Auto Exposure off Set the white balance 6 Click and drag a Region of Interest around a white area 7 Select White Balance from the menu and that is it Done WhiteBalance Spot Exposure Find Foos Region of Interest 3 75 2007 Leica Microsystems Switzerland Ltd Camera Input Options amp Select Camera The Input Options panel provides the main controls for setting up the camera From Input Options E Seen Select the active camera Load and use a camera Select Camera configuration Select a colour o
175. gle on nu Mosaic Image Name Power Mosaic 1 Click on the arrow to the right of the Image Handling header to reveal the panel 2 Using the arrows to the right of the Reduction Factor text box increase or decrease the Reduction Factor The illustrations show the considerable difference in image size with factors of 1 and 4 3 Click in the Mosaic Image Name Width x Height text box and type a name for the Total fle size mosaic The image will be stored with this name in the Capture Folder Large image files are not shown in the Gallery because they take too long to display Generally large files are saved to be used in third party applications Continued on the next page LAS User Manual 4 200 Power Mosaic Save Mosaic Continued Save the Mosaic Continued It is not necessary to save the entire mosaic Very often only one part is of real interest and the remainder can be discarded It is possible to crop the image to a selected region and save that alone The individual tiles are not affected and they remain intact To save a selected part of the mosaic 1 Click on the Saved Marked Region check box to enable it 2 Click on the edge of the region to be saved hold and drag diagonally to the right to encompass the region Release the mouse button For clarity the illustration shows a red dotted line however on screen it is black 3 Save the mosaic by clicking Send
176. he image Specimen To add a Description 3 Click in the Description text box and type a description The text will scroll if it reaches the right hand edge of the text box Do not use the keyboard Enter key to finish click outside the text box Description Le Observations Report Publish in Word amp Create Report To add general Observations 7 4 Click in the Observations text box and type The text will scroll if it reaches the right hand edge of the text box Do not use the keyboard Enter key to finish click outside the text box To Select a Report type 5 Click on the arrows to the right of the Report header bar 6 From the drop down menu click to select the application in which to publish 7 Click on the Create Report button 4 149 2007 Leica Microsystems Switzerland Ltd Output Results Output to Word Publishing to Microsoft Word will display the Leica Application Suite template LASMeasurements dot which has been specially designed to quickly and easily layout and print your data 1 Click to select the template and 2 click Open 3 Microsoft Office Word is launched as a new document and your data including the Specimen name Details and Observations are displayed All of the usual Word facilities such as font change colour fills and basic graphics are available to you Save and print the document in the usual way
177. he Scale Bar end strokes 4 Click on the small arrow to the right of the Scale Bar Ends window and from the drop down menu select either None Plain bar Small Medium or Large To change the Scale Bar thickness 5 Click on the up down arrows to the right of the Scale Bar Thickness window to increase decrease the bar thickness To change the Scale Bar colour 6 Click on the Scale Bar Colour window 7 From the Select Colour dialog select a colour from the swatches or from the wheel and slider Click OK To change the Scale Bar display length 8 The Scale Bar length may be adjusted by entering either a value in Pixels or in the selected Measurement Units in this case Microns Click and type a value into either window followed by return The correct scale value is displayed against the bar f Scale Bar Scale Bar Show Colour Pixel Length E B LLL II Custom LUC LL Currente a ER ef B cen KH teen PR mf g rer RH CED 3 101 2007 Leica Microsystems Switzerland Ltd Camera Processing The Processing panel provides tools to improve quality and orientation primarily for a captured image but may be used for live images as well Shading corrects light level variations that often occur due to bright spots caused by the microscope light source and the optics If Linking Shading is ON it takes precedence and Processing Shading is disabled 1
178. he Sharpening window 2 From the drop down menu select either Off to turn off sharpening or the level required BB Ap Horizontal BB Rip Vertical 3 Click on the Apply to Live button to check it and apply sharpening Repeat the process choosing another level if the result is not suitable The illustrations show the original image left and Medium sharpening right 3 103 2007 Leica Microsystems Switzerland Ltd Camera Processing Flip Flip Horizontal and Flip Vertical These two options re orientate the image To flip the image from side to side 1 Click on the Flip Horizontal button Click again to return it to its original position To flip the image from top to bottom 2 Click on the Flip Vertical button Click again to return the image to its original position Acquire Camera Colour Balance Colour Balance is a feature which allows the user to emulate the image seen through the microscope eyepiece It may be applied to both live and captured images by 3 Click on the Camera Settings icon The Camera Interface appears showing the image on a small Viewer 4 Click on the Check Colour check box to enable Colour Balance The Colour Correction wheel appears 5 Click and hold on the small rectangle in the centre of the wheel Drag the rectangle to a graduated area on the wheel to emulate the overall colour cast seen through the eyepiece LAS User Manual 3 10
179. he icon on the Linking header bar is RED 1 Click the Shading Snapshot button 2 When the Linked Shading prompt appears make sure the microscope and camera exposure settings are suitable and then Either move the stage to view an empty field on the specimen or Remove the specimen slide and replace it with a slide and cover slip if necessary of the same type and quality A very small amount of microscope de focus may be helpful to prevent contaminants affecting the Shading reference 3 Click Yes 4 A shading link will be created and when complete the Configuration Stored message will appear Click OK 3 91 2007 Leica Microsystems Switzerland Ltd Camera Linking Shading Wizard The Shading Wizard creates a Shading Link for each microscope objective in turn starting with the one selected Having completed one it automatically moves to the next Because it can be stopped after completing an objective groups rather than the entire collection may be processed For a single objective use Snapshot The mag changer camera and contrast method remain the same for each link 1 Turn Shading Linking on or off by clicking the Shading Link button Before starting the Wizard the specimen image must be properly exposed and focused The Shading process requires either a blank area of the specimen slide for a reference or a blank slide and cover slip if used on the stage 2 Click the Shadin
180. hods for transmitted and incident light axis are displayed in the control window The current contrast method is highlighted on the control Each contrast method can be selected by a left mouse click Appropriate contrast methods for the current objective in the light path are marked with a triangle If the selected contrast method is not valid Pseudo Bright Field is applied Light rings dark stops DIC prisms polarizer mot and analyzer mot are addressed automatically if necessary Mechanical polarizer and analyser have to be inserted manually NB For Combi Contrast FLUO DIC a manual analyser has to be inserted in the appropriate slot on the upper left side of the stand Each contrast method button shows a small status icon d Triangle marks a contrast method if valid for the currently selected objective Please use the right mouse button to get a context menu for additional functions Call the Online Help DM Operation overview 3 39 2007 Leica Microsystems Switzerland Ltd DM Microscope Operation Fluorescence Control All learned in Fluo Cubes are listed in the control window The current fluo cube in the light path is highlighted on the control Fluo Cubes can be selected with a left mouseclick The IL Shutter can be closed to protect the sample from bleaching To learn in a new Fluo Cube please go to the Setup workflow Please use the right mouse button to get a context menu for additional functions
181. ially on printed images 3 Saturation determines the amount of each colour that is present At the highest setting each colour will be at its most vibrant The right hand illustration is the high setting and the colours cannot be more prominent without combining to make white Use Saturation to make powerful if a little bit unnatural images Reducing Saturation is a convenient way of turning a colour image into a monochrome image essentially just shades of grey without losing detail or becoming a black solid See Intensity LAS User Manual 3 142 Enhance Intensity 1 Intensity is close to Brightness in the way it affects the image Itis a measure of the power of each colour swinging from solid black to solid white Use small increases in Intensity to help differentiate between colours too much and detail begins to disappear All three controls Hue Saturation and Intensity may be used together to achieve a desired effect and they may be combined with Brightness Contrast and Gamma to fine tune an image 2 Discard return to the original or Apply changes on the Confirm panel 3 If Accept is clicked the Save Changes panel appears Click Replace to overwrite the original image Click Save As to write the altered image to another file name or Discard to revert to the original 3 143 2007 Leica Microsystems Switzerland Ltd The Analysis Workflow
182. ic Microscopes Define Z Stack End Automatic Microscopes u cccseesssesseeteseeseeeeeeeeeee Defiine Z Stack End Continued uu ccscsscsseeseesseseesseseeesessessessesseseeseuseees Set Z Stack Steps Automatic Microscopes Acquire Z Stack Images Automatic Microscopes csssssseeeeeseeeees 4 60 Define Z Stack Manual Microscopes cccscscssssesesscessessessessessesseseeseeeesees Set Start Position Manual Microscopes Set End Position Manual Microscopes ccccccssscssesssseeseeseeesesseeeeseeeeeeeeee 4 63 Set Z steps Manual Microscopes uu cccccetsssssessseseesseseeseevsssessesessesesesenee 4 64 Set Z steps continued sscessscscssesssssssscessesesssorsessesseorsessracsrsecsecaestrecseaseass 4 65 Acquire Z Stack Images Manual Microscopes ssssscssessseeesteeeseeeees 4 66 Show Image Set E 4 67 Show Image Set continued eeeseesssseseeseeestestseeeestesestereatseeatseeatseeatseeetats 4 68 MultiStep Is II OH ET GC 4 70 Scan Position Control ecssssssssssesseecsessssssssescecasscsesesesssacacseassesssssacanseseseesens 4 71 III USR 4 72 Define Sequence continued seccssesessesessesestsseatseeaesteaestesestentaesetsteeeansees 4 73 Opti ns sesseiviecuesestidebsasisscdhvistveseiverasdcast ddatedevsesaiceseucesixiscevervesaseeedaees 4 74 Options continued oon esesessesessesessesessseesesreeesteaestenestsauseeeeaeseeaeseeatsteaeseeetae
183. icon on the Linking header bar will show RED This indicates there is no stored camera information for the current microscope settings 3 The Linking icon on the Image Controls panel will be enabled white against a black ground 4 To create a link click on the Image Controls Linking icon The Image Controls Linking icon becomes disabled grey on a black ground 5 The status icon the Linking header bar will turn GREEN to indicate an established link LAS User Manual 3 88 Camera Update amp Delete Link Exposure Update link 1 If changes are made to the camera settings in the example the Exposure value has been reduced the status icon will become YELLOW 2 The Image Controls Linking icon also becomes enabled again Click it to incorporate the change into the link 3 Undo changes to the link by right clicking the Image Controls Linking icon and then left clicking on the Revert tag On stereo microscopes an additional warning may occur the status icon on the Linking header bar changes to BLACK indicating that the zoom level has changed but is being ignored All other settings are correct Update the link to include the revised zoom by clicking the Image Control Linking icon 2 All of the Links for the current camera may be removed by clicking the Exposure Delete button 4 This is not reversible Use with care 3 89 2007 Leica Microsystems Switzerland Ltd Camera
184. icrons fi 000000 Invert deection Nudge size microns 1 00 Software Joystick Color Speeds Color Set Standard Colors v Software Joystick Coker Speeds Hormat 4 163 2007 Leica Microsystems Switzerland Ltd Power Mosaic Imaging Setup Imaging setup If Power Mosaic Turboscan is going to be used the exposure times must be very short typically less than 200us microseconds The highlight levels make some contrast methods unsuitable If the required exposure times cannot be achieved choose Standard scan instead for which light levels and exposure times are not critical WARNING Switch 100 light to the camera DO NOT look at the specimen through the eyepieces 1 Click on the appropriate Mic roscope tab and 2 select the required Objective Magnification and 3 Contrast method 4 Set the Aperture and 5 Intensity to suit specimen and scan type Often a good image can be achieved quickly by setting basic exposure parameters using Auto Exposure and then fine tuning the result with white balance This is especially suitable for Standard scan Turboscan will probably require closer attention to exposure 6 Click on the Camera tab to reveal the exposure controls 7 Set the Saturation to 1 75 by clicking on the slider and dragging it to the left to reduce the value and to the right to increase it 8 Set the Gamma to 0 6 1 0 for
185. image of approx 640 x 480 It displays the image size and amount of MB storage that will be used Final image 4176 x 3875 46MB Create MultiStep Image NB Memory requirements can only be shown accurately if the image is to be saved as in BMP or TIF formats JPGs will show the maximum size for the image Disk space requirements are additional because field images need disk space too Even if system is set up to acquire 16 bit images mosaic images will be acquired in 8 bits 4 77 2007 Leica Microsystems Switzerland Ltd Image Overlay Image Overlay is an LAS module that enables the acquisition of Channel images with a LAS compatible microscope and the creation of a composite image from a sequence of these images The Channels are typically Fluorescent channels but can also be any other available contrast method Sequences of channels may be defined and for each channel the microscope and camera settings set and saved Once a sequence is acquired a coloured overlay image may be made using a variety of methods If using a colour camera the Image Type should be set to greyscale See Input Options Camera Options Fully motorised microscopes will automatically select filters but users of manual machines may still use Image Overlay simply by physically turning the filter turret when prompted Between 2 and 8 images are captured during an Overlay sequence The subject and magnification are the sam
186. in each stack tile Continued on the next page Enable ZStack Scan Method Z Range microns has B Step Size foo BR Number of steps hb PR Set Step Size To Depth of Focus Z Range n Step Size Minimum Projection 4 195 2007 Leica Microsystems Switzerland Ltd Power Mosaic Z Stacks Continued Z Stacks Continued The Z Range Step Size and Number of Steps are inter related Change one value and the others will be calculated and changed automatically Actual settings will depend upon the specimen 1 2 and 3 Use the up down arrows to the right of each text box to enter a value For large values click on the appropriate text box and type a value 4 The Set Step Size check box if enabled will automatically load a step distance based upon the depth of focus and adjust the Z Range and Number of Steps accordingly Changing the Step Size manually will turn off Set Step Size 5 Click on the Clear Tiles button to discard any previous scans The Clear Mosaic warning will appear 6 click Yes to clear N Click the Acquire Power Mosaic Acquire PowerMosaic button Z Stacks are only available in Standard Scan mode If Turboscan has been selected it will be ignored Continued on the next page LAS User Manual 4 196 Power Mosaic Z Stacks Continued Z Stacks Continued When the scan is complete the entire image is displayed with the tile grid overlaid
187. ine caption to select it 6 Click on the Text colour window The Select Colour dialog appears 7 Click on a colour swatch or select a colour from the wheel and slider Click OK Extended Annotations Edit a Distance Line LAS User Manual 4 16 Extended Annotations Insert Image It is quick and simple to display smaller images over the main image In the example a highly magnified section of a paint flake is to have a smaller scaled image alongside as an indication of the location of a split in the paint Inserted images should be typically low resolution 640 x 480 pixels or less so that they do not slow loading To insert an image 1 Click on the Extended tab and on the Fit to Screen icon on the Side Bar This will ensure that the entire 21102005 image can be seen z 0014 jpe 2 Click on the Insert Image icon 200 rm 3 On the Viewer position the cursor on the point at where the top left hand point of the inserted image is ga to appear Click hold and drag an i Image Box to suit the size of the Lock x O Pot Ines i O Te Tosso new image An outline of the Image E Zen 2017 99 Box appears EE en mme Ei Ste rte ge eine DR pa 4 Release the mouse button and the Folder and File navigation pane appears Navigate to the folder in which the new image is stored locate the image file and click to select it 5 Click Open 6 The new image s
188. ing drawn Either click the Finish button if the mask is satisfactory or click Cancel to clear the mask and start again Continued 3 135 2007 Leica Microsystems Switzerland Ltd Enhance Crop continued 9 To keep the masked area click Apply on the Confirm panel or click Discard to start again 10 If Apply is clicked the Save Changes prompt appears Click Replace to replace the original image Save As to save the cropped image to anew name or Discard to restore the image and start again EN E SEE E A EE QS reeet cnt tent LAS User Manual 3 136 Enhance Orientation The image may be flipped top to bottom or side to side and rotated about its central axis 1 Click on the arrow to the right of the Orientation bar to reveal it To flip the image top to bottom 2 Click on the Flip Vertical button To flip the image side to side 3 Click on the Flip Horizontal button To rotate image about its central axis 4 Click on the Rotate Up arrow to rotate clockwise by one degree increments or on the Rotate Down arrow to rotate anti clockwise by one degree increments or 5 Double click the Rotate by window to highlight the existing value Type the number of degrees to rotate and press the Enter key on the keyboard The image will rotate to the required position clockwise To rotate anti clockwise precede the number with the negative sign _ Save Chan Al
189. inside the selected folder 3 Click on the folder within which the new folder is to be created It will be highlighted 4 Right click on the selected folder The Browser menu appears Select Create New Folder 5 When the Enter new folder name prompt appears type the new name in the entry box 6 Click OK and the new folder is created and displayed on Directory Browser VY Heyward Analytica P Create New Folder Rename Getimages from sub directories Delete v J Hayward ona ai 2 Project Alpha ect Be eg Vd Refresh C Project Create New Folder Rename Getimages from sub directories Delete C Project Th 1 Norfolk and 9 ackard Enter new folder name ee toiect_Beta_Resuts LAS User Manual 3 122 G Browse Rename and Get Images To rename a folder 1 Click on the folder to be renamed It should be highlighted 2 Right click on the folder and from the Browser menu select Rename The Enter new folder name prompt appears _ Project_Alpba el Refresh Create New Folder Ko 3 Type a new name in the textbox and 4 Click OK Get Images from sub directories It is possible to display all of the thumbnails and images from several folders simultaneously providing they are all contained within a parent folder 5 Click on the parent folder that contains the sub folders containing images 6 Right click on the
190. ion 5 Click in the Focus Position text box and press the keyboard Delete key to clear the existing entry Type a new value and press the keyboard Enter key The default units are micrometers um but to move in millimeters type mm after the value Mouse Wheel 4 Click on the Focus Control 6 Rotate the mouse wheel to move the motorised focus up or down The focus step for each indent of the mouse wheel depends on the depth of focus and the zoom magnification Continued LAS User Manual 3 58 Stereomicroscope Operation Memory Current microscope settings may be saved in any one of five separate memory locations represented by a button and numbered 1 to 5 1 Place the mouse cursor over a numbered memory button to reveal the microscope settings Motorised focus position Zoom position Filter selected Iris diaphragm setting Internal and external light source settings and Motorised stage X and Y values Left click on the button to drive the microscope to the memorised values 2 Empty memory locations are denoted by on the button To save the current microscope settings 3 Select a memory location either Set or Clear and right click the button From the drop down menu click on Set Memory The button will display the location number to indicate that the settings have been saved 4 To clear a memory location right click on the button and from the drop down menu c
191. ion Delete Current To delete the current configuration 2 Click on the arrows to the right of the of the Configuration header bar 6 From the drop down menu select Delete Current 7 Confirm the deletion on the message panel by clicking Yes Input Options Select Camera DFC 320 004123603 Configuration Delete Current Save Current Configuration OMX Enter a name for the new configuration 22 Color Binning 1044 x 772 Configure Camera maximum length 15 characters Detta_3 x Ga D High Sensitivity Binning Input Options Select Camera DFC 320 004123803 Delete Current KEE s 2 J Do you want to delete the current configuration Delta_3 CZE GID 3 81 2007 Leica Microsystems Switzerland Ltd Camera Exposure Adjust Exposure Adjust There are two options for adjusting the exposure Automatic with some post fine tuning and Manual with a wide range of precision controls Before starting work on a live image using the Automatic Exposure is a good option because it could produce a perfectly acceptable image Combine this with Automatic White Balance and the result may not need any further adjustment at all Very fast and very efficient LAS User Manual 3 82 Camera Exposure Adjust Automatic Exposure Description Automatic Exposure Powerful software uses current light levels to establish best values for Brightne
192. ion and is ideal for electronic transmission Some detail and colour subtlety may be lost in complex images Bitmaps are the most faithful to the original image but result in the largest Kamna sotoa files Not as good for electronic A sd transmission because of the size but if Zero Padding local high quality printing is required k B and disc space is not a consideration use bitmaps The Image Format may be changed at any time Avoid mixing formats in any one collection The Image Format appears as an extension to the saved image file as tif Jpg or bmp To set the Image Format 1 Click on the up down arrows to the right of the Image Format window A drop down of the available options appears 2 Click to select the format required 3 31 2007 Leica Microsystems Switzerland Ltd Preferences After Capture Options There are three buttons on the After Capture panel only one at a time may be selected They provide the options for image handling after it has been saved Do Nothing 1 Click on this button if no further action is required after the image has been saved Open In This option opens the image in one of the Workflows available Browse and Process are always available and gt a Opa kaag was Analysis will be an option if it has been With ia same Ce installed Please see specific Help entries for details To select the Workflow option 2 Click on the Open In button Save imag
193. ir Colour Relief 3D Model LAS User Manual 4 106 Montage Depth Map s Show Multfocus Image Set o No stack images oE se 1 As part of the Montage operation a Depth Map image is also generated The Depth Map is a record of which source image provided the in focus region for the montage image at each pixel location expressed as a grey level The depth map image is the same size as the source images used to generate it but is always monochrome The grey levels depend on the particular Montage mode used to generate the depth map If fixed depth is used the pixel values will be integer and represent the stack image which has best focus If blended depth or weighted depth is used the pixel values will be fractional and represent the interpolated best focus position Note that the weighted depth mode may generate completely meaningless depth map values in certain cases where there is more than one plane with a good focus The depth map image is displayed in the depth map image window The depth map image will be saved in the dataset file itself but may also be exported to an image file if required Delay between Multifocus Image 4 107 2007 Leica Microsystems Switzerland Ltd Montage Confidence Map Anaglyph 1 A Confidence Map is also generated for each montage operation It is an estimate of accuracy of the depth map image at each pixel location expressed as a grey l
194. ire Z tab to reveal the MultiFocus control panels Defining the stack consists of setting the first Z position on the specimen called the Start setting the last Z position called the End and then deciding the number of focussed slices or steps to capture between Start and End Start and End may encompass either the whole specimen from bottom to top or any part of the specimen The positions chosen are displayed on a graphical stack 2 with the Start position 3 shown as an arrow on the left hand side and the End position as an arrow on the right hand side Windows attached to the arrows display the microscope focus values The Z Range window 5 will show the numerical difference between the Start and the End positions Z Position 6 is the current microscope focus position Z Step 7 represents the distance between each of the Z steps and Steps 8 is the number of images that will be captured The Optimise step size button 9 will determine the best step size based upon the microscope and the optics Limit buttons 10 will automatically move the objective to either the Start or End position 4 55 2007 Leica Microsystems Switzerland Ltd MultiFocus Define Z Stack Start Automatic Microscopes Generally the Start position is the part of the specimen closest to the stage It provides the opportunity to place the specimen on a textured background which when in sharp focus is a precise
195. is is especially true for specimens that have irregular focus 1 Click on the Focus Method Predictive checkbox to enable it 2 Click on the Setup button to display 3 the Predictive Focus dialog 4 Three pre determined points are automatically placed on the image Their positions are displayed in the X Y columns on the dialog Normally the set points will be coloured red to indicate that they require focussing If they are green and have a value in the Z column denoting that they are already focussed possibly as the result of a previous scan the points should be focussed again Continued on the next page 4 185 2007 Leica Microsystems Switzerland Ltd Power Mosaic Predictive Focus Continued Predictive Focus Continued Reset Focus Point values 1 To clear all focus point values click on the Reset button The values will clear and will be displayed in the Z column of the dialog Delete a Focus Point To delete a focus point 2 Click on the point on the dialog list 3 Click the Delete button The list entry and the point disappear To create a New Point 4 Click on the Move Stage To Point button 5 Select a new point on the image and click The point appears on the image and is coloured red to indicate that it requires focussing 6 Click on the Add button Delete all Points All of the points may be deleted by 7 clicking on the Delete AN button The dia
196. is used for Turboscan Perform a Standard Scan emm aD 4 175 2007 Leica Microsystems Switzerland Ltd Power Mosaic Auto Step Size Auto Step Size The Step Size represents the distance between adjacent tiles in um micro meters Normally Auto Step Size is enabled to allow mosaic creation and the program calculates the number of tiles required to cover the image on the basis that they will all abut as adjacent tiles 2 Turning off Auto Step Size allows the step to be adjusted so that tiles may be overlapped or spaced apart 8 Focus Method BB Predictive 1 Enable Auto Step Size The check box will be coloured red with a tick Auto Step Size must be enabled for a mosaic to be created 2 The tiles are butting and arranged to cover the image In these examples the Cross Scan Pattern has been used for clarity 3 Click to disable Auto Step Size The check box becomes grey EE implement the change 4 Click the Details button 2 E ee 6 5 The Details Dialog appears with X Step size lum Jos oa YStep sze tum Je X Beld size tum IT YReldsizetpm o 6 the tile X and Y co ordinates Total Fields er Scan Width imm por enabled for editing To change the step size click on either the X or Y tere Soan Heh san E text box and type a new value Qara GED 7 Click Apply If necessary re enter the step values to get the required spacing or overlap 8
197. it File Name z DOUD po Acquired Time 2371172006 10 54 24 bits per pixel Colour Image Size Wath 1360 x 1024 Image Size calibrated 3 125 2007 Leica Microsystems Switzerland Ltd Browse Device Settings Providing the Store and Recall module is registered and enabled the Device Setting panel details the mechanical information camera and microscope setup relevant to the displayed image 1 Click on the expand collapse arrow to the right of the Device Settings bar to display the settings 2 If the system has an automatic Image Source microscope attached clicking the Camera DFC 300 FX Recall button will automatically Chipsize X 6 976400 return the microscope and camera Chipsize Y 6 708000 to the image settings Exposure Time 0 001846 Gain 1 000000 3 A Recall settings panel appears ey eel after the Recall button is clicked Colour Enabled 1 Select Yes to confirm Auto Brightness Enabled 0 Brightness Level 00 000000 Microscope Stand DM60008 Method TL BF Nosepiece 1090 30 11508507 Ports 2 Magnification Changer Dic Turret Dic Turret Fine 0 Lamp 46 Sveltchable Condenser Top t Condenzer Turret SF TI Field Ofaphragm 32 Tl Aperture Diaphragm Do you want to recall the selected device settings LAS User Manual 3 126 By using the Process Workflow images can be further enhanced and annotated From brightness and saturation lev
198. it can be disabled by clicking the check box 6 Click the Close button on the dialog 2500 07 N PLAN DRY 4 173 2007 Leica Microsystems Switzerland Ltd Power Mosaic Load Configuration Load Configuration A Scan Pattern configuration may be saved for future recall as a fast and accurate way of loading settings To retrieve and automatically load a saved configuration 1 If necessary click on the arrow to the right of the Scan Pattern panel to reveal it 2 Click on the Load button 3 On the Open dialog click to select the configuration required The selected file name appears in the window 4 Scan Pattern configuration files have the file extension Dat 5 Click on the Open button Only the Scan Pattern settings are loaded Optics and Z Stack settings must be adjusted for each session 6 The Folder defaults to the selection made either in Preferences or Browse Use Windows navigation to open an alternative folder See Preferences Save in Directory See Browse Select Default Directory utoscnn Standard og e Auto Step Size ron put Pattern pat LAS User Manual 4 174 Select Scan Pattern There are two scan pattern options which govern the stage tracking as the image tiles are collected bi directional and uni directional The bi directional pattern scans left to right moves down to the next row and reverses and sca
199. itional panel the On screen Joystick is available by clicking the Show Joystick Tools button Click and hold on the Joystick header to drag and dock it on any part of the screen Turboscan EI Auto Step Size Stoners Focus Method BB Predictive WW Autofocus 4 159 2007 Leica Microsystems Switzerland Ltd Power Mosaic User Interface Map The User Interface is shown in the illustration with the Acquire Workflow selected and the Power Mosaic tab active 1 Workflow tabs 2 Control panels and function tabs 3 Acquire Mosaic button 4 Scan viewing area with on screen Joystick shown 5 Scan grid pattern 6 Live image field Split screen view is selected 7 Tool bar The tools are explained on the following page Continued on the next page LAS User Manual 4 160 The Toolbar is located on the right hand Power Mosaic User Interface Tools side of the screen The tools descriptions are 1 9 Live Screen The entire screen is devoted to the live image Split Screen Divided between viewer part of live screen Grid Pattern View Entire screen is devoted to the scan grid No Tool selected Stage Area displayed on Viewer Display Grid Pattern Hide display Grid Pattern Zoom n Zoom Out Note double click the mouse wheel to set Zoom 1 Pan 10 Create a New Scan Grid 11 Extend Cont
200. its label appears on a pale yellow background Each tool is itemised in detail on the Measurement panel 1 To reveal the Measurement panel click on the arrows to the right of the Measurement header The measurements are grouped together under the Image Name To reveal them 2 If necessary click on the right facing arrow to the left of the Image Name and all of the measurements will appear An icon indicates the type of tool used in the measurement followed by its sequential number and the Class it belongs to if any Measurement 3 To reveal the parameters for each tool click on the right facing arrow E ri alongside its icon In the example the Area tool parameter which was enabled on the Setup tab were Mean Red Green and Blue and each of those is displayed as a value 4 GB Show default values only The actual count 18 in this case is the main parameter of Count so Ares 7350 08 will always be displayed Perimeter 1908 00 Height 337 00 Width 418 00 4 141 2007 Leica Microsystems Switzerland Ltd To delete a measurement both from the image and from the Measurement panel 1 Click on the measurement result to highlight it and then 2 Click on the Delete button Previous Next 3 4 The Previous and Next buttons move to the previous or next image and display it in the Viewer Hide Measurements 5 The Hide button conceals all of the measurements on the image Click ag
201. ity C Min intensity LAS User Manual 4 146 Interactive Measurements Results Results Summary To determine which parameters to display on the Results Summary table 1 Click the Summary checkbox to hide or reveal the Results Summary table 2 Click on the right facing arrow to reveal the Results Summary table parameters Each of the parameters and its summary may be turned on or off as appropriate by 3 Check or clear the checkbox alongside the parameter to display or hide it 4 To display all of the parameters click the Show All checkbox See Results Summary Continued 4 147 2007 Leica Microsystems Switzerland Ltd Interactive Measurements Results Results Summary conti 1 The parameter descriptions are Total Is the total for all tools beneath that heading For example Total Line Length is the total measurement for all the tools Distance and Line that have length as a parameter Mean Represents the Total divided by the number of measurements made Total Count Mode The most frequently occurring value in the set of data Median The actual mid value in a list of values For example 676 is the Median of 214 676 and 1031 For an even numbered list of values the values either side of the mid point are averaged Standard Deviation A measure of the spread of a set of data using all of the results Standard Error Uses the convention Standard Deviation Squa
202. ive in the light path is highlighted on the control Objectives which are valid for the selected contrast method are marked with a triangle Immersion objectives are marked with a black drop Objectives which have been learned in as combi objectives module Fine tuning are marked with a clear drop The selected objective blinks if you are changing the mode from DRY to IMMersion and vice versa The stage is lowered and you have to confirm the change of mode with an additional mouse click Parfocality can be adjusted using the context menu of the right mouse button This will start the parfocality wizard It is recommended that the parfocality of all listed objectives is adjusted if new objectives are learned in To learn in new objectives please go to the workflow Setup Each objective button shows small status icons 4 Marks an objective if it is valid for the currently selected contrast method 6 Marks Immersion Objectives Oil Water Glycerine 6 Marks Combi Objectives for use in both modes Immersion and Dry Mode Please use the right mouse button to get a context menu for additional functions Lill Starts the Parfocality Wizard Call the Online Help DM Operation overview LAS User Manual 3 42 DM Microscope Operation Magnification Changer Control The current value of Magnification Changer is highlighted in the control window Since the Magnification Changer is not motorized remote control is not possible NB The va
203. l save the typed text and remove the cursor To change the size of a text box 6 Click on the Select tool and then click the text box to be altered The cursor changes to a white arrow and square handles appear at the corners of the text box 7 Click on one of the handles and drag the box to the required size Text will wrap to fit the new box size keet typing spaces until the 43 required position on the line is N keai Oo LAS User Manual 4 6 Extended Annotations Change Text properties To change the text 1 To change the text click on the Select tool and double click on the text box The text will be selected in a white text box To add text click on the position to insert new text and begin typing Delete text by clicking on the text to delete and then using the Delete or Rubout keys To change the font Font Style or Font Size 2 Click on the annotation to be changed with the Select tool 3 Click on the Font button The Font dialog will appear 4 Change the Font properties and click OK The change is immediately reflected in the annotation 4 7 2007 Leica Microsystems Switzerland Ltd Extended Annotations Change Text colour To change the Font colour 1 Click on the annotation to be changed with the Select tool 2 Click on the Text window The Select Colour dialog appears 3 Select a new colour either from the swatches or from the colour
204. left mouse button Open dialog with right mouse button and use select LAS User Manual 3 14 DM Microscope Configuration IL Fluo Turret The current fluo cubes reflector cubes and their position on the turret are displayed in the status list The cubes can be removed exchanged and or renamed Learn in a new cube from the selection list e Mark position in the status list with the left mouse button e Mark new cube in the selection list with left mouse button and exchange cubes with a double click of the left mouse button Alternatively e Mark new cube in the selection list with left mouse button Open dialog with right mouse button and use select NB At least one position of the fluo turret has to be learned in as EMPTY to perform bright field contrast BF in transmitted light axis EMP BF has to be selected in case the stand is capable of a fully motorized IC contrast in transmitted light axis If the microscope is capable of a partial manual IC contrast the EMP DIC has to be selected Alternatively in case a A cube is used select A TL to perform contrast methods of the transmitted light axis with this fluo cube Continued 3 15 2007 Leica Microsystems Switzerland Ltd DM Microscope Configuration IL Fluo Turret continued Continued Register a new user defined cube in the selection list e Go to selection list e Open dialog window with right mouse button e Go to user define
205. lete Selected Images 1 Right click on the selected Hide Selected Images thumbnail The Gallery Control mengappears Copy Selected Images To 2 Click on Open Current Image Import From File With ge Send To Leica IM If the selected program exists it Unselect All Images Rename Current Image will load run and display the Delete Selected Images Refresh Thumbnail current image Hide Selected Images Copy Multiple Images 3 If more than the current image OPy selecte needs to be copied select multiple Import From File images by holding down the Control Send To Leica IM key on the keyboard and clicking the thumbnails of the images to copy Open Current Image With Rename Current image 4 Right click on any of the selected thumbnails to reveal the Gallery Control menu 5 Click Copy Selected Images To A Windows Navigation pane appears 6 Navigate to the folder in which to copy the images Click to select it and click OK 3 121 2007 Leica Microsystems Switzerland Ltd Browse Refresh and Create New Folder ki Refresh maintains the Directory Tree especially after folders have been created or deleted To re load and display the folder tree 1 Right click on the selected folder The Browser menu appears The menu options may vary with the type of folder selected 2 Click on Refresh The folder tree is re loaded and displayed Create New Folder This function creates a new folder
206. lick to select Clear Memory The button number will be replaced with the empty symbol Continued Filter Name Empty Fluorescence Shutter Closed Visual Magnification 10 1x Tris Diaphragm 0 Set Memory Got Si Clear Memory Slot3 Help mmv CY o Set Memory Slot3 j Clear Memory Slot3 4 3 59 2007 Leica Microsystems Switzerland Ltd Stereomicroscope Operation Zoom Iris Aperture control There are three methods for changing the Zoom Iris aperture 1 Click hold and drag the Slider to the left to close the iris and right to open it The aperture value is shown as a percentage of fully open on the header bar The minimum value is limited to 20 2 Click on the Slider Bar and the Slider will track to the selected position 3 Click in the Iris Setting text box to select the existing value and press the keyboard Delete key to clear it Type a new aperture value and press the Enter key on the keyboard The maximum value is 100 and the minimum value 20 4 Click on the closed iris icon to close the iris to 20 of full or 5 Click on the open iris to fully open the aperture Continued LAS User Manual 3 60 Stereomicroscope Operation External Illumination control _ The CLS 150XD and 150LS external light sources may be controlled remotely from SMS 1 The Brightness Control may be External ilumination 310k 9 rot
207. llows you to test before committing Make changes to the image then either 1 Copy it to the clipboard and paste into another application like Powerpoint or 2 Simply print it to check how it looks Both functions are available on the Side Bar 3 If the image is satisfactory only then Apply and keep it a g e e Intensity H seet Convert 4 16 Bit to M Bit colour a D a Cal ey KJ KJ LAS User Manual 3 138 Enhance Brightness The image in the Viewer comprises tiny individual dots called pixels Each pixel is a mixture of three primary colours red green and blue RGB and each colour is represented by a value For 8 bit colour the values are in the range 0 to 255 All of the colour controls manipulate the values of the three colours to produce a certain effect If all three colour values are set to 0 0 0 0 then black is the result If they are all set to 255 255 255 255 white is the result On both the Brightness Contrast Gamma and Hue Saturation Intensity colour panels click and hold the slider and move it to the left to decrease or right to increase values The displayed numbers are not a reflection of the colour byte values but rather a scale associated with the parameter being changed KL A 4 1 Brightness increases or decreases V e the value of all three colours ae simultaneously The illustrations na A W show
208. log list will clear and the points will disappear from the image Update ij Having added or deleted points update the list and the displayed points by oe 8 clicking on the Update button Continued on the next page LAS User Manual 4 186 Power Mosaic Predictive Focus Continued Predictive Focus Continued The Grid feature automatically creates eee a regular pattern of points across the image The number of points can be established by typing a value 1 Click on the Grid text box to highlight the existing value Press the keyboard delete key to remove the value Type a new value The value represents the number of points that will be created along the top row of the grid On the second row the number of points is reduced by one The next row is increased by 1 and so on across the entire grid In the illustration a value of 3 has been typed 2 Click on the Grid button and the new points will be created and listed on the dialog Points sitting on completely clear areas of the image 3 will result in a failed Autofocus because there is no detail to focus on To avoid a failure either delete the points See Delete a focus set point or adjust the Focus Threshold Setting upward to include values closer to white clear However setting the threshold to a very high value can affect the precision of Autofocus See Initialisation Focus Continued on the next page 4 187
209. lose shutter when not acquiring Yes Compression MJPEG Overall File Sue Timestamp 7 Skip to the end of the movie 8 Skip to the start of the movie 9 Skip to the next or previous clip by clicking on the left right arrows to the right of the Current movie clip window Play Movie bei i 10 The playback speed may be adjusted in the text box 10 The default is 100 which is playback speed recorded speed Increase or decrease the speed by clicking in the text box and typing a speed as a of the recorded speed or clicking on the up down arrows to the right of the text box 4 45 2007 Leica Microsystems Switzerland Ltd Movie Play and Frame Capture To play a movie 1 Click on the Start Stop button The button icon changes to Pause on a brown background Click the button again to halt the movie 2 The Progress Indicator moves from left to right as a graphical indication of running time Click and hold the slider and then move it left or right to re position the movie playing position Time elapsed Current movie clip 3 Running Time elapsed as Recording frame rate 21 hh mm ss is displayed in the window Perte mm 7 Aut var Frame Capture Individual movie frames may be captured and saved for future examination 4 Click on the Start Stop button to stop the movie at the frame required Alternatively use the Progress Indicator to position the movie at the fram
210. lour dialog appears Select a new colour either from the swatches or from the colour wheel and slider Click OK If necessary click on hold and drag to move the caption to a new position Properties BI Auto Size Qum EI Transparent CT Background LAS User Manual 4 10 Extended Annotations Scale Bar Add a Scale Bar to the image by 1 Click on Scale Bar The Scale Bar appears in the default top left hand position Change the style and orientation by clicking the arrows to the right of the Scale Bar Style bar and from the menu choosing Horizontal Vertical or Mixed which displays both horizontal and vertical lines Change the Scale Bar ends by clicking on the arrows to the right of the ends bar and selecting the size from the menu Create a custom length Scale Bar by clicking the User Length checkbox and typing the required length in the text box 5 To change the Scale Bar and Scale Annotation colour click on the Scale Bar with the Select tool Click on the Text window Although the Scale Bar is a line it is treated as text for the purposes of colour changing The Select Colour dialog appears Select a new colour either from the swatches or from the colour wheel and slider Click OK To delete a line click on the Select tool then on the shape to be deleted Press the Delete key on the keyboard Properties E Auto Size
211. lso checks for camera rotation i e the angle of rotation from the Stage X and Y axes 1 Select the Live Screen view 2 Click on the Set Calibration button When the Calibration Dialog appears 3 a small rectangle is displayed on the live image Use the on screen joystick or stage controls to navigate to a well defined and detailed region of the image lying within the rectangle Avoid uniform patterns 4 Click on the Calibrate button 5 If there is insufficient detail in the selected region a warning will appear Repeat the process from Step 3 If the camera image needs to be flipped do this in the Camera tab Continued on the following page ov u s 77 CC geng 10x 0 3 HCX PL FLUOTAR DRY Calibration Please choose a stage postion that shows a kt of detal on the specimen Then cick E the Calibrate button x Y d 1 032000 Dose A libration Insufficant image Detail Please select a differant area on the specimen 4 171 2007 Leica Microsystems Switzerland Ltd Power Mosaic Camera Rotation In Detail Calibration Camera Rotation The values for camera rotation returned by the Calibration relate to the stage X and Y axis If the rotation is excessive a warning will be displayed 2 Rotation for both axis should not exceed 0 10 degrees 1 Check that the camera mount is secure Loosen the camera cl
212. lues of the Magnification Changer are used for the calculation of the total magnification see LeicaScreen Please use the right mouse button to get a context menu for additional functions Call the Online Help DM Operation overview 3 43 2007 Leica Microsystems Switzerland Ltd DM Microscope Operation Focus Drive Control The current position of the Z Focus is displayed in the control window Lower threshold and focus position are displayed as icons in the control window and can be recalled Press the appropriate button until the threshold or focus point is reached Lower threshold and focus position can be deleted or set in the Advanced Settings The control window uses the following status icons Focus Position Lower Threshold Please use the right mouse button to get a context menu for additional functions Advanced Settings define clear the lower threshold and the focus position Call the Online Help DM Operation overview LAS User Manual 3 44 DM Microscope Operation Motorised Tube Control The current beam ratio is displayed is highlighted in the control window The beam ratio for a motorized tube can be selected NB After turning on the microscope the light path is automatically set to 100 visual exit eyepieces Each button on the control shows a small status icon The light path is set to 100 visual exit eyepieces rg The light path is set to 100 camera port G
213. mines the manner in which the Channel images are combined to create the Overlay 5 The Image Groups and Overlay Method settings can be saved as the ongoing default Continued Channel Colour 330 nm BW Apply False Colour LAS User Manual 4 94 Image Overlay Creating and Viewing Overlays continued The Adjust Appearance panel provides the controls for 6 Adding false pseudo colour to monochrome images 7 Adjusting colour balance with Histogram controls 8 Changing the Gamma value of the Channel images and the Overlay and 9 Varying the mix strength or dominance of a Channel image Images captured in colour will not have all of the colour controls available The Overlay Image panel 10 determines which of the Channel images are to be included in the Overlay At least 2 Channels must be selected before an Overlay can be created 11 The Create Overlay button creates a new Overlay based upon the selected settings Once the Channel images have been captured they may be altered at will and almost any number of different Overlays created 4 95 2007 Leica Microsystems Switzerland Ltd Image Overlay Selecting Image 1 Click on the arrows to the right of the Image Groups and image A 2 from the drop down list of existing folders click to select the required folder and images The Channel images will appear in the Gallery and the last image to be capt
214. mn shows a Y Yes if the module is active or N ere Status rime Lett No if not 5 Demo Period Au Days remaining Use the slider or up down arrows to Manual Measure De aE scroll through the list Software licence information 4 To enable or disable a module Geet Cer e EECH click on it so that it becomes highlighted 5 Click on either the Enable or Disable buttons 6 The Y N code changes accordingly 7 All of the modules may be enabled disabled en masse by clicking either the Enable All or Disable All buttons 8 Click OK to save the settings See The Framework 2 13 2007 Leica Microsystems Switzerland Ltd Chapter 3 Using the Core Features Using the Core Features This chapter describes the intuitive Workflow organization of the unique Leica Application Suite user interface and its basic capabilities Workflow of LAS describes the order and grouping of tasks for image documentation and analysis While the Workflow suggests an order for the tasks versatility is retained so that the operation of the software is not constrained to fixed steps The grouping of the tasks into related operations makes it easy for the user to become adept in achieving their goals The Workflows are SG Setup Acquire Browse Process and optionally Analysis Each is selected by clicking on the Workflow bar Selecting a Workflow displays the appropriate controls arranged on o
215. mouse button Open dialog with right mouse button and use select Continued LAS User Manual 3 12 DM Microscope Configuration Microscope Data continued Register a user defined item in the selection list e Go to selection list e Open dialog window with right mouse button e Go to user defined item The newly defined item is displayed instantly in the selection list and can be selected Rename an existing item and changing parameters e Go to selection list e Open dialog window with right mouse button e Go to copy item name e Define name and parameters The newly defined item is displayed instantly in the selection list and can be selected 3 13 2007 Leica Microsystems Switzerland Ltd DM Microscope Configuration IC Turret The current IC prisms and their position in the turret are displayed in the status list New prisms can be added existing prisms can be removed or exchanged The system automatically consider for each objective the new prisms if reasonable Remove one prism from the IC turret e Mark prism which has to be deleted in the status list with left mouse button e Use right mouse button and select delete prism Learn in a new prism from the selection list e Mark position in the status list with the left mouse button e Mark new prism in the selection list with a double click of the left mouse button Alternatively e Mark new cube in the selection list with
216. multiple images will rapidly consume disk space Always carry out an Auto White Balance Check for a Shading Link or create a new one Regularly backup Multi Time projects to CD or DVD and free up disk space 4 23 2007 Leica Microsystems Switzerland Ltd Time Lapse Set up The Multi Time module must be installed and enabled See Registration 1 Click on the Acquisition Mode selector 2 From the menu click on the Multi Time icon 3 Click on the Acquire Workflow if it is not already visible 4 An additional tab marked T will be displayed Click on it to reveal the Multi Time controls The main panel Define Time Sequence is further divided into 3 smaller panels Start Sequence and Test Acquire Time There is an additional panel Options which can be revealed by o Manal 5 Clicking on the arrows to the right dat and time of the header bar ecember AYE Number of images acquired B Interval between images 000010 Total time eo o LAS User Manual 4 24 Time Lapse Set up Options The Start panel provides three start up options for the Multi Time sequence Manual The Sequence starts as soon as the Acquire Time Lapse button is clicked At date and time Starts the capture on a specific date and at a specified time After delay Waits for a specified time before starting the capture Manual start 1 Click on the Start Manual button
217. n a position to prevent collision with the support in SMS specimen and that all cables have sufficient slack to allow the carrier to travel to the top of the stand MZ 16F 10 447 064 MZ 16 10 447102 MZ 12 5 10 446 370 MZ 9 5 10 446 272 MZ 7 5 10 446 371 MZ 6 10 445 614 MS 5 10 445 613 S6D 10 446 297 S8 APO 10 810 038 Z16 APO 10 447 173 Z6 APO 10 447 174 Macrofluo Fluocombi 3 51 2007 Leica Microsystems Switzerland Ltd Stereomicroscope Operation LAS Start up If at start up LAS detects a motorised focus it will ask to initialise the focus _Init Motor Focus Position position by displaying the message 1 SMS Control ia going to move the motocfocus drive to its topmost position Make sure that this travel range is free and cables are long enough If you skip this reference movement all stored 2 Click OK to send the carrier to the reference positions cannot be reused top of the stand and automatically mm initialise the focus position z a 3 Click Cancel to skip the initialisation If initialisation is skipped the exact focus position may not be reliable and will not be saved with other settings 4 Click the Acquire Workflow to reveal the SMS control panels If focus was initialised the Focus Drive position 5 will display the current focus position If focus was NOT initialised the value will be 0 Previous settings that were saved may be recalled and loaded by
218. n area using the mouse The selected area is outlined with a yellow line Double clicking will close the polygon and complete the editing operation Edge Style There are 4 possibilities for the edge style the left most selection uses a hard edge with no blending of the edited area and the background image Each of the other 3 edge style add progressively heavier blurring of the compound image and the selected layer while still remaining within the selected outline LAS User Manual 4 118 Montage Measure Tools Z Position Measure Tools allows the user to read depth information from a point or line on a Montage Multifocus Image and Position Readout display a profile of the surface as a graph on a chart Measurements may Enable be absolute or relative to a reference point set by the user This mode is controlled by several checkboxes on the panel The panel is Distance inactive if no depth map is available for the Multifocus image The depth map Line Mode is created when Montage Multifocus MB Profile makes a new multifocus image Enable Z Position Readout Checking this checkbox gives a continuous readout in the table of the current mouse position when the mouse is moved over the image The X and Y positions are given in calibrated units relative to the top left corner of the image The Z position is taken from interpreting the depth map and is displayed in microns Distance is measured from the top left c
219. nce completely or No to continue the sequence LAS User Manual 4 32 Time Lapse Replay When the capture sequence finishes the Browse Workflow opens 1 with the name of the sequence in LAS J File Options Help the View Image Set window mm Browse 2 Normally the Multi Time images Bee a are played continuously but an interval may be set and inserted between the images by clicking on the up down arrows to the right of the Delay window The delay interval is in seconds 3 To emulate the interval between images precisely click on the arrows to the right of the Options panel to reveal timing details of the sequence 4 Replicate the interval in the Delay window 5 Start and stop the replay by clicking the Start Stop button 6 Skip to the next or previous image by clicking the Forward Back buttons Start option Manual 7 The Beginning and End buttons skip to the first or last image in the sequence Close shutter when not acquiring selected 1 5 at 0000 03 00 00 12 8 The name of the current image being displayed is shown in the Image window 9 Replay progress is displayed on the ViewimageSet OI Progress Bar When the sequence is finished it will repeat loop again until stopped Move to specific parts of the sequence by clicking and holding the Progress Bar Indicator and sliding it to the left go back or right go forward Previously captured sequences E in
220. nd with its associated image becomes a Channel Continued 4 83 2007 Leica Microsystems Switzerland Ltd Image Overlay Channel Dialog continued 5 Select a Channel by 7 Clicking anywhere on a filter entry 8 Clicking to enable the checkbox At least two and up to the total filters available may be chosen for a complete Channel sequence Changing sequence order 9 Select an enabled Channel by clicking the Next Filter button 10 Click either the Move Up or Move Down buttons to change the order in which images will be captured IA 5 BGR AUO 785m3 24 Ce an m 293 19 17 80 sept 3091ms 439 Saving a Channel sequence When all of the required filters have been selected 11 Click on the Create new sequence button to save the Channel sequence TLBF 6293 10 FLUO 280 4m3s 23 2 AA 642 9 ms 5 BGR FLUO 324 5ms 54 4 N2 FLUO 5037ms 10 le Ana TLDIC 29s 10 2BD BREF aam 4 LAS User Manual 4 84 Image Overlay Adjust Channel Exposure Each Channel may have Exposure time Gamma and Gain values set individually to achieve the best image 1 Check that the Camera is in live mode The message on the top bar of the Viewer indicates the camera state If it reads Paused then click the freeze button to return to live The button icon should have a small square in the centre not an arrow 583 1 ms 2 Click on the Next Filter button to move to the Channel r
221. ne or more panels that allow the user to perform the selected action Because the Workflow arrangement is so versatile in contrast to many Windows programs LAS does not employ a menu bar for the main operation of the software 3 3 2007 Leica Microsystems Switzerland Ltd Core Starting Leica Application Suite Launching Leica Application Suite In most cases the Application Suite can be launched by double clicking on the desktop icon Alternatively 1 Click the Start button on the Windows Task Bar bottom left 2 From the popup click on All Programs 3 Locate Leica Application Suite on the All Programs list and 4 Click on Leica Application Suite from the flyout LAS should load and run LAS User Manual 3 4 The Setup Workflow The Setup Workflow provides the method of specifying the components of the Leica microscope connected to Leica Application Suite Components such as objective types and filter descriptions can be readily selected saved and subsequently recalled In addition fine tuning of some of the microscope features can be performed such as parfocality setting the focus step size for each lens It is expected that the Setup workflow will be used infrequently as once the microscope settings are established they are seldom changed When leaving the Workflow a dialog window appears to allow you to affirm the changes If the modifications are confi
222. necessary by clicking its thumbnail See Browse 4 123 2007 Leica Microsystems Switzerland Ltd Interactive Measurements Setup 1 Click on the Setup tab Three panels are available Settings in which the Configuration and Results files are either selected or created 2 Define Classes in which similar objects are associated so that their measurements may be summarised by Class in addition to overall statistics Create new or use existing Classes here 3 Specify Measurements is a complete list of the measuring tools available and their options 4 To reveal a panel click on the arrows to the right of its header bar Click again to collapse the panel LAS User Manual 4 124 Interactive Measurements Setup Configuration Options Two preset options are available for the Configuration name Default which does not have any Classes and uses a small pre defined range of tools and measurement options and Last Used which simply reverts to the last Configuration used when the application is re started or the previous Last Used configuration is recalled This could be Default Create and save a new Configuration for every Class list so yward Theta that it is immediately available for Hayword Aoemum new images Additionally any Configuration names that have been previously created and saved will also be available These will probably contain at least one Class
223. ns Optional Applications Applications operate within the LAS framework and share many of the configuration facilities of LAS However they operate independently of the LAS optional imaging modules For example one or more optional modules can be loaded and used within LAS However only one application can be loaded and used at a time In the following descriptions of the operation of the Applications features that they share with LAS is not repeated In the current version of LAS the following Applications are available SG LAS Image Organiser LAS Reticule 5 3 2007 Leica Microsystems Switzerland Ltd ESE This Chapter contains the following topics Introduction LU e Setup Reticule e Available Reticules LU J Reticules on Live Images Reticules on captured images Reticule Introduction The LAS Reticule application is intended as a replacement for the glass reticules that are commonly placed in the optical path of a microscope or other optical inspection system that is used for routine manual inspection This electronic reticule will provide greater flexibility in terms of reticule design and selection and lower cost to the end user There are two types of reticule e Fixed Reticule Fixed reticules cannot be changed and are always centred in the image window Fixed reticules are defined in units of pixels Example A cross hair displayed across the centre of the image window When
224. ns right to left The uni directional pattern scans left to right returns to the left moves to the next row and scans from left to right again Depending upon the specimen shape the scan direction may be from top to bottom 1 Choose bi directional for speed 2 Choose uni directional for greater accuracy The selected check box is coloured red Select Scan Speed Turboscan is the fastest way to capture a image but it does require a very fast exposure time 200us micro seconds or less and a progressive scan camera with trigger facility During Turboscan the stage moves continuously It does not stop to make an exposure which is why a short exposure is needed During Standard scan the stage halts at every tile position so the exposure time is immaterial Standard scan can also operate with non progressive cameras and the trigger facility must be connected 3 Choose Turboscan for speed but only if the exposure time is less than 200us Trying to scan with a higher exposure time will display the warning message 4 5 Click to select Standard scan speed for exposure times greater than 200us and non progressive cameras Power Mosaic Select Scan Pattern e Rectangular H k Z d s 3 Turboscan J Auto Step Size 5 Standard Direction Automatic Focus Method Predictive WB Autofocus Stace Turboscan Information itis recommended a Camera Exposure lt 200 uSecs
225. ntrol 3 61 Internal Illumination contre 3 62 XY Stageicontrols vasa E aati nena aes 3 63 X Y Stage control continue 3 64 Wiring Diagrams 1 lt 0iitasiaie nia a a ee kA a 3 65 Wiring Diagrams EE 3 66 FS Operation Introduction seen 3 67 Objective Magnifications sed shed cia edhe dass eae 3 68 Comparison Bridge Copie luede acne 3 69 Illumination IMEnSity sssini aita 3 70 Ee lte ie Ee ue EE 3 71 Tube and Photo Portis ssh ade keete eee eee 3 72 Camera feed ege 3 73 image ei EE 3 74 FastTrack gimna a cee tata eateries cates 3 75 Input Options amp Select Camera 3 76 E ele ie ONE 3 82 MERU DEE ele TC RE Calibration Settings teste tecnica eben el cael Le let enatls scaleiBar veel ea ee ees tee ee Region of Interest siari mestaan anea reset neta The Browse Workflow Opening BROWSE sci eege ees Saarai aiara seas seactcesepentves atapata aaas tE Directory Broweer zsciczccsssccicecscscesessdescccasiec ctscstcceceeeteccesssteende atenda aoda ceatcscscios Selectan Image EE Pan Window aaraa Eed Fit to Screen and ZOOM is heehee Aiba diana 3 114 Openiimage tte 3 115 Printian Image ve baer EES ES 3 116 Copy Paste an Mage E 3 117 Fast Es E Ce EE 3 118 Thumbnail EE 3 119 DeletelHide Tu EE 3 120 Open Image with Appltcation 3 121 Refresh and Create New Foder 3 122 Rename and Get Images s oesseeeseesseseeesrnerrststnernstntntrsrnsrntnnrnennersnnennnnene 3 123 Delete Folder Aeddi SEENEN eens 3 1
226. ntrols allow the user to go to the beginning or end of the sequence or step through images one at a time forward or back or play the sequence at the specified frame rate The red image or bar in the sequence visualization can also be grabbed using the mouse like using a scroll bar and the sequence navigated by moving this back or forth through the image sequence The image being displayed is listed in the text box Continued Showing stack image 1 of 20 E MultiStep image Q Image Set Reduction Factor Final image 4176 x 3875 46MB Create MultiStep Image LAS User Manual 4 76 MultiStep Browse MultiStep Images continued MultiStep This control allows the user to select the image to be displayed MultiStep Image Selecting this automatically loads the MultiStep image from the currently selected image set in the gallery The View Image Set controls will be disabled as the mosaic image is being displayed Image Set Selecting this option loads the first image from the image set The Create MultiStep Image button allows the user to re create the MultiStep Image from the current image set Reduction Factor Auto provides a final image at a size comparable with an individual field image size None provides an image at max size size of final image is sum of sizes of individual images Values e g x2 indicate an image of size Max Size value value Highest value is capped to give an
227. nual 4 48 MultiFocus Enable MultiFocus MultiFocus is an optional module which must be installed and operational before use To enable MultiFocus 1 Click on the Module Select icon 2 From the drop down menu click on the MultiFocus Montage icon to enable it The icon will only be available if MultiFocus is installed 3 The Z logo appears on a new tab in the Acquire Workflow and 4 Also in the Browse Workflow 4 49 2007 Leica Microsystems Switzerland Ltd MultiFocus Select Image Format 1 On the Acquire Workflow click on the Camera tab 2 Click on the arrow to the right of the Options panel to reveal it 3 Click on the arrow to the right of the Captured Format header bar and from the list of options 4 Click to select the appropriate format Thick specimens may require a large number of steps so consider a more compact format that does not compromise quality but speeds the capture time reduces disk space and makes processing quicker Captured Colour Depth 8 bit channe LAS User Manual 4 50 MultiFocus Select or Create Capture Folder 1 Click Options on the LAS header bar 2 Select Preferences from the drop down menu 3 On the Preferences dialog if necessary click on the Image Output Settings tab to reveal the Save Images panel 4 Click on the Browse button and on the Browse For Folder dialog navigate to the required folder 5
228. o wet the mouse button is released click on the Spot Exposure option The rectangle outline will be red The automatic exposure function calculates and applies the new settings 3 To move the Region of Interest click and hold on the red border not a handle and drag it toa new position 4 To resize the Region click hold and drag on any one of the eight handles 5 In this example the Region of Interest has been moved and resized and as a result the overall exposure has altered 6 Click on the arrow to the right of the Region of Interest header to reveal the panel 7 Clicking the Spot Exposure button will hide the red crop mask Click again to reveal it It will retain its size and position 3 107 2007 Leica Microsystems Switzerland Ltd Camera Region of Interest Find Focus Find Focus This feature displays the focus precision for the area of the live image contained within the Region of Interest The Region may be moved around the image to compare different areas with a focus bar indicating the best level achieved 1 Click on the arrows to the right of the Region of Interest header to reveal the panel BB Crop to RO WW Spot Exposure 2 Click and drag on the image to create a rectangle around the Point of Focus 3 From the menu revealed when the mouse button is released click on 5 the Find Focus option The a Ly rectangle outline will
229. o which all of the images and their data files will be loaded By default these folders are given the name T MultiTime n where n is a sequential number if other folders of the same name exist The prefix T is always present because it denotes a Multi Time project but change the folder name to one of your choice by 4 Clicking in the Folder Name text box and delete by backspacing the word MultiTime Type in the new name In the example the word VeeGon the name of the test has been typed so the folder will be named T VeeGon To retrieve an existing configuration 5 Click on the arrows to the right of the Configuration text box 6 From the drop down list click to select a configuration To delete a configuration 6 Select a configuration from the list 7 Click on the Delete button oO Show Acquire Panel Show Browse Pane EI Show images during acquire Show Acquire Panel Show Browse Panel Show images during acquire LAS User Manual 4 30 Time Lapse Capture Display Options Three options are available to determine the state of the Viewer during the capture sequence 1 Click the Show Acquire Panel to remain in the Acquire Workflow and watch the images in real time To momentarily freeze the image as it is captured But there must be a sufficient interval between images to allow the Viewer to refresh 2 Enable the Show images during capture checkbox This op
230. oard while drawing the line 3 The line colour can be changed by clicking on the Line box The Select Colour dialog will appear Choose a colour from the swatches or wheel and click OK 4 To increase or decrease the line width click on the Up Down arrows in the Width window 5 To change the line style simple dotted dashed etc click on the arrows to the right of the Style window and select a style from the drop down menu 6 To move a line click on the Select tool and position the cursor on the line Click hold and drag the line to the new position 7 To lengthen or shorten a line click on the line end hold and drag to the required length 4 9 2007 Leica Microsystems Switzerland Ltd Extended Annotations Annotations Single line annotations or captions may be added to lines and arrows After drawing the line 1 2 Click on the Select tool Click on the line that will have an annotation added Click in the Edit Label text box on the Properties panel and type the caption The text appears next to the line as it is typed To change the Font Font Style or Font Size click on the Font button The Font dialog will appear Change the Font properties and click OK The change is immediately reflected in the annotation To change the Font colour click on the annotation to be changed with the Select tool Click on the Text window The Select Co
231. odule 3 35 2007 Leica Microsystems Switzerland Ltd Preferences Store and Recall Store The Store and Recall feature allows the microscope and camera setting to be stored with an image so that precisely the same conditions may be repeated at a later date It can also provide consistency across a range of different specimens Fully automated microscopes can automatically adjust to the settings The settings display can be used to adjust manual models Of the four functions available Store Recall and Image modes each have options selected by clicking a button The buttons are mutually exclusive only one may be active within a function Undo Recall is switched on or off by clicking a checkbox This is a toggle action 1 To reveal the Store and Recall options click on the tab Store 2 This function has three setting to determine if and when camera and microscope settings are stored with the image Always will always store the settings Ask will prompt you to store or not when the image is saved Never switches off settings store The store facility adds a very short delay to storing images and the files are longer Only use Store and Recall if really necessary LAS User Manual 3 36 Preferences Store and Recall Recall Mode Undo 3 Recall 1 Recall determines if stored settings are also recalled when an image is retrieved from disk Always will always recall any settings store
232. of the camera sensing elements are reset they have no usable image information at all Then they are exposed to the specimen and each begins to charge to a value that numerically represents the light falling upon it Each element is designed to respond to one of the three primary colours red green or blue At the end of the exposure period each element is read and its value used in combination to create a pixel on the Viewer For any image there will be an optimum period of exposure for the elements to reach values that truly represents the image Too short an exposure and the elements will not have sufficient time to reach the proper value the image will be dark and muddy under exposed Too long and the image will be washed out and lacking detail over exposed The time scale for exposure will depend upon the camera and may be measured in either micro us or milli ms seconds A typical value for a bright field image would be 10 to 50 msec See Further information about Manual Exposure 3 85 2007 Leica Microsystems Switzerland Ltd Camera Manual Exposure Continued Manual Exposure Description Continued 1 Saturation and Gamma See Automatic Exposure 2 Gain is a function for changing the brilliance of an image without changing the exposure The examples shown are centre a Gain of 1 5 To the left a Gain of 1 0 and to the right a Gain of 6 0 Start with a Gain valu
233. of the montage image 3 Select the color scheme in which to plot the color relief image 4 Adjust the Saturation Saturation varies from purely monochrome intensity left most position to full colored depth map with no intensity information right most position Hint If your mouse has a wheel for scrolling click on the slider and use the wheel to adjust the parameter value 5 Check Preview to see the effect of your settings in the Preview window er Click Create Colour Relief to see the finished Stereo Pair image in the Image viewer I Montage Colour Retief Kc Apply Confidence Feiere Colours Saturation E Preview eS ee Create Colour Relief 4 117 2007 Leica Microsystems Switzerland Ltd Montage Edit Mode continued Select Layer Select the layer from the source images stack which will be used as the source Edit Mode for the editing It is also possible to select the layer from the gallery and Select Layer define the selected area directly on that fe RH layer image This will have the same result as defining the area on the compound image E Brush Brush Gd This mode allows a line to be drawn which defines the path followed by a Edge Style brush during the processing The visible line defines the centre line followed by RAA A the brush There are 3 widths of brush available Polygon Selecting this option allows the easy selection of a
234. on B E Project Gamen Desktop Project Lambda E Project Theta My Documents My Computes My Network Place File name Save at pe LAS User Manual 4 152 Power Mosaic amp Power Mosaic Plus LAS Power Mosaic software integrates high performance specimen scanning into the Leica Application Suite to provide an easy to use application for creating viewing and saving ultra high resolution mosaic images LAS Power Mosaic is used in conjunction with a Leica microscope Leica DFC digital camera and a stepping stage With LAS Power Mosaic you can scan a selected area or an entire slide quickly and accurately and then effortlessly relocate to areas of interest with a simple click Additionally with the LAS Power Mosaic Plus software it is possible to acquire images at multiple focus positions and view the complete mosaic while scrolling through the focus The ability to generate high resolution mosaic images provides a powerful and novel method of visualizing a specimen The specimen overview is an aid to understanding the relationships between microscopic features and the overall structure Once you have created a scanned mosaic you can save your specimen s details as a workspace or export the full resolution mosaic for use with image publishing and analysis packages The software is designed to offer a comprehensive range of facilities with a strong emphasis on versatility and ease of use With minimal
235. on drop down and then select Save This entry defines the mosaic name and the sub folder name if sub images are saved To remove a previously saved configuration the user must select it from the drop down list and then press the Delete button The Create Mosaic image if checked means that a mosaic image will be created after the last image in the Scan Pattern has been acquired This will be displayed and stored in the Gallery If Save sub Images is selected all acquired images will be saved as an image set If de selected these images will not be saved Show During Acquisition e Mosaic Shows the dynamic build up of the mosaic on the screen as each image is acquired Single Images Shows each acquired image on the main screen as it is captured in turn Continued AContiguraion gt Create Mosaic image Save sub images S Mosaic2 Show During Acquisition OH Mosaic Reduction Factor ei Ei A e Single images Final image 4176 x 3875 46MB LAS User Manual 4 74 MultiStep Options continued Reduction Factor e Auto provides a final image at a size comparable with an individual field image size None provides an image at max size size of final image is sum of sizes of individual images Values e g x2 indicate an image of size Max Size value value Highest value is capped to give an image of approx 640 x 480 It displays the image size and amount of MB storage that will
236. oo large volatile RAM becomes clogged and the speed advantage is lost 1 Click on the Advanced button 2 On the dialog click to enable or disable tile buffering 3 If tile buffering is required click on the Browse button to reveal the Browse for Folder dialog 4 Click to select a folder or 5 create a new folder Use Windows navigation to reach other levels or directories 6 Click OK 7 If necessary use the arrows to the right of the Thumbnail Reduction Factor text box The larger the number the greater the thumbnail detail and also the amount of disc space occupied 8 Click Apply and 9 click Close gt 4 gt era lt Auto Step Size CH Turboscan Standard 5 CH Hayward_Analytical Project_Alpha E Project_Beta Project_Delta E Project_Epsiion Q omm GammawsS_1 G Gammaws_s E Gammaws_5 Gammaws_6 O Gammaws_11 E Gammaws_12 Garmmaws_13 LAS User Manual 4 178 Power Mosaic Rectangular Scan Pattern Select Pattern Type There is a wide range of scan patterns available designed to provide total flexibility and efficiency in the capture and storage of images Each pattern type can be configured to best suit the task in hand On the following illustrations each small rectangle represents a separate tile 1 Click on the arrows to the right of the Pattern Type header 2 From the drop down menu select the pattern
237. opriate immersion medium For this purpose the stage is lowered 3 mm LAS User Manual 3 20 DM Microscope Configuration Fine Tuning Node Fine Tuning Dry IMM e Each learned in objective is automatically assigned as dry or immersion objective For dry objectives with a long free working distance gt 2mm it is possible to monitor the specimen through a layer of oil for survey purposes In this case it may make sense to learn them in as combi objectives with a mark in the click box NB If an objective is assigned to combi the stage is not lowered when switching to this objective Fine Tuning Node Stage and Z Step e The step width sensitivity of movement of electronic focus and the velocity of the stage movement are correlated and predefined for each objective e The node stage and z step size allows to change both parameters e The step sizes are SO very fine up to SC coarse They can be selected in the pull down list under the displayed objectives NB On Leica Screen and in the module DMOperation the user can switch between Fine and Coarse focus or Precise and Fast stage respectively The values for fine and precise refer to the values set in the node stage and z step size of Fine tuning module 3 21 2007 Leica Microsystems Switzerland Ltd DM Microscope Configuration Fine Tuning The module Fine Tuning is only
238. orner of the image or from a Reference Point placed on the image This Distance follows a path constrained to the surface prescribed by the depth map Distance along the surface will be longer than the straight line distance between the start and end point It is sensible to use calibrated images for Distance measurement A nominal one micron per pixel will be used as a default for uncalibrated images Z depth is given in microns 4 119 2007 Leica Microsystems Switzerland Ltd Montage Measure Tools Reference Line Mode Set Reference In order to place a reference point on the image click the Set Reference button move the mouse pointer to the desired position and click Once a reference point has been set Z position and Distance are computed relative to this reference position and the values are displayed in the table Set Reference can be repeated at any time but at most one reference point will be on the image Clear Reference In order to clear the reference point click the Clear Reference button Line Mode The Line Mode section allows the placing of a line segment on the image This gives a profile of the surface along the line segment The profile is shown on a floating panel see right LAS User Manual 4 120 Montage Measure Tools Profile and Edit Line Profile and Edit Line Two checkboxes control the production Measure Tools Z 4 69 um v d of the profile Position Readout
239. ory Crnate a zw category 1 Prasan select the catagory you wart ta copy 2 Please dafine name ter the new cemgory 3 Please insert a shor escription optoralh 5 11 2007 Leica Microsystems Switzerland Ltd Image Organiser Search Panel The Search panel allows searching in the desired category according to various criteria to quickly find images and data Enter text for a search in the box Previous search text in the same session is remembered in the pull down Patient Data control at the right of the text The Search can be conducted in all AJ Hice details fields or in the selected fields The search will start automatically when a Activate category autosave Search button or an enter key is pressed The Search results are displayed in the Gallery If you want to see all the ir i Image C Documents and ay eat es ade click on the Path Settings Administrator My Documents My ImageOrganiser UserTemplate Created 10 12 2005 Modited 10 12 2005 No of lmajes 2 Enter textfor a search in the box Got BED Description Search in LE S LAS User Manual 5 12 Image Organiser Device Settings The panel Device Settings shows the camera and microscope data which have been saved together with the image By clicking on the button Recall the camera and microscope settings can be restored Image Source Camera DFC 320 R2 Chipsize X 7 203600 Chipsize Y 5 347500 Exposure Time 0 03
240. ows the current fill colour From the Select Colour dialog choose a colour and click OK LAS User Manual 4 12 Extended Annotations Edit Shapes All shapes can be rotated by 1 Click on the Select tool then press and HOLD DOWN the Shift key on the keyboard 2 Click on the shape Small square handles appear at each corner 3 Click and hold on one of the handles and drag to rotate the shape clockwise or anti clockwise as required To add an annotation to a shape 4 Click on the Select tool and then on the shape to be annotated 5 Type the text in the Edit Label Text a box The text appears in the centre of the shape O fens i SE E is S uch AaBbYv2 Co em 6 Change font style size and colour by clicking on the Font button and then selecting from the Select Font dialog we To move a shape 7 Click on the Select tool then click and hold on the shape to be moved and drag it to the new position To delete a shape 8 Click on the Select tool then click on the shape to be deleted It should be surrounded by handles Press the Delete key on the keyboard 4 13 2007 Leica Microsystems Switzerland Ltd Extended Annotations Boxed Captions For longer captions an attractive option is to create a coloured rectangle or square with or without a coloured outline and to place text within it The text is typed into a transparent text box
241. p MuntTime Movie d To start Image Overlay 1 Click on the Select Acquisition icon and from the menu 2 Click to select Image Overlay After it is selected an additional tab marked with the Lambda A symbol 3 appears in both the Browse and Acquire Workflows LAS User Manual 4 80 Image Overlay Setup Tools 1 Select the Acquire Workflow by clicking on its tab 2 Click on the Image Overlay tab marked with the Lambda A symbol to reveal the Channel Setup panel comprising 3 The Setup Tools and 4 The Channel Dialog Using the Setup Tools 5 Camera Live freeze halts the camera activity leaving the latest image on the Viewer Click again to resume taking live images Use the freeze button in combination with 6 Capture Single Image which will TLDIC save the current image to the C 7 8D BEBE sims 49 capture folder The filename is TL BF created automatically and a thumbnail of the image is displayed in the Working Gallery Continued See Channel Dialogue 4 81 2007 Leica Microsystems Switzerland Ltd Image Overlay Setup Tools continued 7 The Next Filter button moves the focus of interest to the next available filter on the Channel dialog The focus rolls over from bottom to top automatically 8 Click the Move Up button to move the selected channel up in the filter sequence 9 Click the Move Down button to move the selected channel down in
242. p Define Delete To delete an individual Class name 1 Click to select the Class name to be deleted in the Name window 2 Click on the Remove button The Class name will be removed from the Class list To delete all Classes 3 Click on any entry in the Class name list The Remove and Clear All buttons become enabled 4 Click on the Clear All button A warning message appears 5 Click Yes to clear all of the Class names or No to exit without clearing Deleted Classes cannot be retrieved Use with care 6 If an image is displayed which used classes now deleted a query message appears The options are Yes Use the new Classes or No Continue to use the previous Classes Change Class Display Sequence The order in which Classes appear in the window may be changed by using the Change Sequence buttons 7 Click on the Class name to be moved 8 Click on either the move up or move down button as required Display Class Names on an Image To display the Class name on the image 9 Enable the Add Classes to Measurements button The Class list is saved in the Configuration file so if a new image without Classes is selected it will use the current Configuration until another is chosen Define Classes v Add Classes to measurements Rame 3 Ree ate fode Lender Node Runner Bud Adventitious k Class definition has changed ei if you want to use the new class definition Click Yes
243. parent folder and from the Browser menu select Get Images from sub directories Images from all of the sub folders will be loaded to the Gallery This can be a long process and should be used sparingly 3 123 2007 Leica Microsystems Switzerland Ltd Browse Delete Folder E The delete option is only available for single level folders those which do not have sub folders within them 1 Click on the folder to be deleted It should be highlighted Refresh tE Create New Folder 2 Right click on the selected folder If Project_6 Rename the Browser menu has the Delete ro Get images from sub directories option the folder may be deleted ec 3 Select Delete from the Browser menu A Delete Folder Confirm panel appears 4 Click Yes to proceed with the e deletion or No to cancel The folder a on a COTO SAY PNTA tenn Spaeth will be deleted and the folder tree ee C emm LAS User Manual 3 124 Browse File Information Precise details of the image in the Viewer are available on the File Information panel To reveal the panel 1 Click on the expand collapse arrow to the right of the File Information bar Any other open panels will collapse and the image details will be displayed 2 If needed type a short description of the image in the Description text box 3 More detailed information may be entered in the Notes window When the image is closed the new information is stored with
244. pears 3 To enable tile skipping click on the Autofocus every check box 4 Set the number of tiles to skip by either clicking on the text box deleting the existing value and typing another or clicking on the increase decrease arrows to the right of the text box 5 To Retry on Failure click the check box to enable 6 Click on the Apply button and 7 Click on the Close button Go to Create the Pattern Grid 4 189 2007 Leica Microsystems Switzerland Ltd Power Mosaic Create Pattern Grid Create the Pattern Grid This step draws a pattern grid representing the scan tiles over the image Power Mosaic will use this grid as a scanning map As the specimen has yet to be scanned its precise position on the stage is unknown so the first grid drawing locates it 1 Click on the Split View button and navigate to an appropriate part of the specimen using the on screen joystick 2 Click on the Pattern View button 3 Click on the arrows to the right of the Pattern Type header and from the menu 4 select by clicking the Rectangular option A different pattern may be selected later 5 Click on the Draw Pattern tool and 6 positioning the cursor close to the stage marker small green crosshair click and hold 7 and drag diagonally to the right An arbitrary grid pattern should appear If it does not 8 click on the Show Grid Lines b
245. ple in focus planes at any one pixel location This is particularly effective on transparent biological samples Note that the resulting depth map will contain inaccurate depth values Weighted Exponentially Similar to Weighted but biased more strongly towards the planes of best focus Compound Weighted A combination of Exponentially Weighted and special post processing It is particularly effective on biological samples under incident illumination e Max Projection The image is based on the brightest point at each pixel location Min Projection The image is based on the darkest point of each pixel location LAS User Manual 4 102 Montage Optimize Preview Optimize Select the optimization method from the drop down box e Speed uses an extremely fast calculation of best focus e Precision is slower than Speed but tends to generate cleaner depth maps e Patch Size sets the size of equally focused regions constructed into a montage image in the range 1 to 200 The optimal value should be found by experiment with typical samples Preview Rapidly generate a preview image which changes immediately as any parameter is changed Select Montage Image Depth Map Image or Confidence Image The region of the original image shown on the preview display may be changed by holding down the left mouse button over the preview display and dragging the preview image within the display The size of the preview image ma
246. pplication Suite Release Notes pdf See Release Notes Leica pplication Suite oO installation information GB install Leica Application Suite pl Feedback and Hotline Details gt Install Example Images eo Install DEC TWAIN Drivers eo install E240 Drivers Leica pplication Suite About LAS gt System requirements for usang LAS LD How to install this software Release notes K Deke this file ir zip e A tk za Ex om Mier LKD Regstration Eraries hrectib exe app Appkation OQ Star Bich Tent Format 458 Application Extension D SaveRestoreModule dil 156K Application Extersion S sequence d zeg ation Extension K geg LAS User Manual 1 10 Chapter 2 Installing Configuring and Licensing Installing Configuring and Licensing This chapter covers the topics required to prepare Leica Application Suite for use For example You will need to install the software Inform the software what type of z gr er microscope and camera is Leica Application Suite connected License any Modules or instalation information install Leica Application Suite Applications you have purchased e Opt to evaluate Modules or D CR ee AE en Sey IERES Applications before committing i install DEC TWAIN Drivers all before LAS is ready for use D Fait EZ4D Orivers It is assumed that you have already assembled the microscope camera and feica other accessories and are now rea
247. pre focussed use the table to predict a focus position without going through the time consuming process of an Autofocus on every field Autofocus should be used for irregular specimens at any magnification Focussing is carried out at regular intervals across the specimen with options to set the repetitiveness Both Predictive and Autofocus may be used in combination to benefit from the speed of Predictive and the precision of Autofocus Using Predictive focus e Select the focus points manually or use the automatic Grid e Select and focus each point manually or choose Autofocus on all points e Run Predictive focus See Predictive Focus Using Autofocus e Set the repeat pattern for fields to be focussed e Enable disable field skipping on focus failure See Focus Methods LAS User Manual 4 158 Power Mosaic Starting Power Mosaic Loading the module 1 Click on the Select Acquisition button to reveal the available modules If a module is already loaded and running the button icon will differ from the illustration 2 The Power Mosaic icon will only be 3 B L present if it is installed However it MultiTime Movie Power Mosaic will also need to be enabled See Installing Configuration and Licensing Registration Information Click on the icon 3 Click on the Acquire Workflow tab 4 When Power Mosaic is loaded and running the PM tab will be present with the main panels displayed 5 An add
248. r from the dialog 3 The Framework will load The utilities are available from the Options drop down menu Only users with Administrator privileges will be able to use the utilities Go to Optional module evaluation Go to Enable and disable modules Go to Hardware selection Go to Licensing optional modules 2 5 2007 Leica Microsystems Switzerland Ltd Module and Application Evaluation Optional Modules and Applications are available for a 60 day free evaluation period After that they have to be Leica Application Suite File Help purchased and licensed to continue Hordware Setup working When Leica Application Suite is installed the modules are also installed but remain disabled until either Demo mode is started or the module is purchased and licensed Firmware Update Registration Info Leica Application Suite Enter Licence Ke the D tion i lected EE To Authorise purchased Leica Application Suite modules please send it cannot be stopped and is available the following site code to Leica Sales Support and enter the licence once only regardless of whether key returned individual modules are enabled or not The 60 day evaluation period begins Demo mode may be deferred until the user is ready to evaluate the modules To start the evaluation period 1 Click on Options on the tool bar 2 From the drop down menu choose Ge ee lt 3 On the Licence Key dialog click th
249. r grayscale Ce eae Choose the bit depth image Type Select the live and captured CE image format Captured Colour Depth SG Apply the configuration to the camera and e E SE j aplurec rommaa SG Enable or disable high sensitivity 22 HO Col Binning 1044x772 gt binning ihe Fara z ve a Select a Camera Occasionally more than one camera each with its own FireWire connection may be fitted to the computer They appear as a drop down menu with model names and serial numbers The current active camera appears at the top of the menu list High Sensitivity Binning To select a camera 1 Click on the arrow to the right of the Input Options Select Camera header bar From the drop down menu click to select the camera required LAS User Manual 3 76 Camera Configuration Image Type amp Live Format Configuration Input Options values may be saved and retrieved later to quickly and precisely replicate settings When the Viewer opens Configuration defaults to the last used Input Options To select a saved Configuration 1 Click on the arrows to the right of the Configuration window and from the drop down menu select a configuration appropriate to the selected camera Image Type If a colour camera is active both colour and grayscale monochrome options are available 2 Click on the arrows to the right of the Input Options Image Type header bar and from the d
250. ra 8 Make sure the image quality is good and the Exposure time is as short as possible commensurate with a good image Carry out an Auto White Balance if necessary 9 Consider using VGA 696 x 516 preferred or 4x4 Binning for the Live Format because although High Quality HQ formats may be used they can result in very large file sizes and very long exposure times For smooth playback aim for 20 to 25 frames per second Regularly backup Movies to CD or DVD and free up hard drive space LAS User Manual 4 38 Movie Select Recording Mode 1 In the Acquire Workflow click on the Movie tab Two recording options are available Continuous results in a single clip lasting as long as frames can be captured within the constraints of the hard disk space available See Preferences Movie Settings See Estimating Movie times Define a sequence of clips creates a single file for each clip Clip duration and interval may be set individually When the clips are replayed in Browse they are assembled into a single continuous movie The movie starts and records for the duration of the first clip It then stops until the first interval has passed The next clip then runs for its designated duration recording stops and the second interval starts and so on until all the clips have been recorded To select the Recording Mode 2 Click on the Continuous button and then go to Options See Recording Options
251. ra being used Three binning formats 2 x 2 3 x 3 and 4 x 4 are available each with a HQ High Quality option The format numbers describe how the pixel values are grouped 2 x 2 4 pixel group 3 x 3 9 pixel group and 4 x 4 16 pixel group Progressive Red Blue or Green use only the value of the selected colour The Viewer displays a grayscale image representing the intensity of the chosen colour Even if the Image Type is set to colour the image will appear monochrome 22 Color Binning 1044 x 772 Full Frame 2088 x 1 Full Frame HQ 2 rogressive VGA 696 x f Binning 44x 77 ol Binning 1044 x 772 Binning 696 x 514 Binning 696 x 514 or Binning 520 x 384 LAS User Manual 3 78 Live Format To select the Live Format 1 Click on the arrows to the right of the Input Options Live Format header bar and from the menu click to select a format As a guide and depending upon the camera model the 2 x 2 Colour Binning 1044 x 772 format is a good choice for most situations Avoid saving images using the 16 bit format They will be slow to expose and process and if captured and saved in the same format may be unusable in third party image processing applications Captured Format Captured Format determines how the image is finally captured and saved In many cases the Captured Format will be identical to the Live Format so providing the image is saved as a bitmap on a bit by bit basis without compr
252. ract existing scan grid 12 Go to Stage point 13 Move Scan Pattern 14 Clear scanned Tiles Continued on the next page 4 161 2007 Leica Microsystems Switzerland Ltd Power Mosaic User Interface Views Viewing Options There are three viewing options available each selected by clicking the appropriate button 1 View Stage Map fills the viewing area with 2 the scan pattern grid and scanned tiles scaled to fit 3 Split View displays the pattern grid and scanned tiles on the left 5 and a live image on the right 4 6 The Live Image option fills the viewer with a live image LAS User Manual 4 162 Power Mosaic Onscreen Joystick On screen Joystick setup With the On Screen Joystick visible it can be customised to suit individual preferences 1 Right click on the Joystick to reveal the Speed and Properties menu 2 Click to select the speed at which the stage will be driven when the joystick is moved Three options are available Normal Fast or Slow Actual speed will depend upon the stage type so select and test to find the most appropriate 3 Click on Properties to reveal the Properties Dialog Three tabs provide Speed Similar to Step 2 above but on this tab X and Y speeds may be selected individually and the travel direction can be inverted Z Axis allows the Mouse Wheel if fitted to act as a focussing control and sets the focus
253. re root n where n is the number of measurements made Maximum and Minimum The largest and smallest measurements made regardless of the tool used Confidence Interval A range of values within which 95 of true results are likely to fall Total Sample Area s the area selected units 2 of the entire image Total Count The number of measurements made with this parameter See Results Summary pr Line Length Width 264 00 1301 00 1433 00 1301 00 650 50 254 00 650 50 477 57 650 50 325 235 254 00 88200 882 00 832 00 441 09 264 00 190 26300 419 00 1447680 00 1447680 00 2 LAS User Manual 4 148 Interactive Measurements Results Output Results There are four easy flexible ways of publishing your results Output to Microsoft Word providing it is installed and available Create a new Microsoft Excel spreadsheet again if it is installed and available Append to an existing Microsoft Excel spread sheet or Save the data as an ASCII coded text txt file A Specimen Name Description and general Observations may be added to complete the document 1 Click on the arrows to the right of the Output Results header bar to reveal the panel To add a Specimen Name 2 Click in the Specimen text box and type a name or identifier The text will scroll if it reaches the right hand edge of the text box Do not use the keyboard Enter key to finish click anywhere on t
254. required 3 Click on the Details button This opens the appropriate pattern dialog 4 5 For the Rectangle Pattern 7 the X and Y tile counts are available for editing Click in the X or Y text box and enter a new value 6 Click on the Apply button and the new configuration is applied to the image Repeat steps 5 and 6 until the pattern is suitable For clarity the tile layouts on the following pages are shown neatly abutting each other However if the camera rotation is not perfect the tiles may appear at an angle and possibly overlapping 8 The software has been designed to accommodate these variations See Create the Pattern Grid Scan Pattern ETD Rectangular Scan Q x lf an area enabled for editing is modified Press Apply to implement the change X Step size ium Stap size tum 1 _XField size um _Y Feld size tym Total Reds Scan Width imm 1 Total Area mm Scan Height imm j b E CAD Y Fields weng 4 t t 4 179 2007 Leica Microsystems Switzerland Ltd The following pages illustrate the Pattern Types available and the options for each Circular Pattern Type The options are Full Coverage and Inside only 1 Click on the Diameter text box and enter a value for the circle diameter in millimetres The circle does not have to cover the entire image 2 Click on the arrows to the right o
255. rmed the new items settings are permanently stored There are three major types of Leica microscope that are used with LAS and these are described in separate sections STEET 2 WON 3 5 2007 Leica Microsystems Switzerland Ltd DM Microscope Configuration Configuration is accessed from the Setup Workflow and serves as convenient means for configuring the microscope It allows to learn in permanently new devices or components of the microscope e g new filter cubes or new objectives In addition it serves to adjust the microscope to personal preferences e The new components are fully integrated in the automation system When exiting the Setup Workflow step a message window appears to affirm the changes If the modifications are confirmed the new items settings are permanently learned in A profile sheet can be printed for documentation This profile sheet displays the current configuration of the microscope To print the sheet select FILE in the main menu bar and press print identification sheet This option is only available when Configuration module is activated LAS User Manual 3 6 DM Microscope Configuration Overview The Configuration module consists of 3 windows 1 Microscope tree which represents graphically all physical parts of the microscope 2 The status list which displays the current devices or settings respectively 3 A selection list with all
256. rop down menu select the type required Live Format Live Format determines the quality and resolution of the displayed image in the Viewer The active camera will determine the extent of the format options available To ensure that all of the possible options are available 3 Right click on the Live Format text box 4 Left click on the Show All Modes label to check it High Sensitivity Binning Select Camera DFC 320 004123603 Configuration DFC_320_Delta_2 Last Used DFC_320_ Deits_1 DFC_320 Beta MENE Save Current Delete Current SA Col Binning 1044x772 Select Camera DFC 320 004123603 Full Frame 2088 x 1550 V Show All Modes Configure Camera 3 77 2007 Leica Microsystems Switzerland Ltd Camera Input Options Live Format Options Live Format options Full Frame options display each of the camera element pixels individually Resolution and quality is very high especially if the HQ High Quality option is selected and 16 bit depth is enabled See Captured Colour Depth Progressive VGA produces the lowest resolution images but they are suitable for very fast exposure rates See Manual Exposure Adjust Colour Binning is a process of grouping adjacent camera element pixels to create Super pixels Each group is then used to drive a single display pixel The feature improves overall sensitivity and speed Available binning options depend upon the came
257. rowse and Process Workflows Go there SG LAS Modules and Applications each described in a dedicated chapter Go to Modules Go to Applications LAS User Manual 1 6 Introduction Combined User Manual and Help User documentation for LAS is provided by an on line help file that is accessed using function key F1 when LAS is operating This same file is also supplied in pdf format that can be opened from Windows Start menu All Programs Leica Application Suite but Adobe Acrobat Reader must be installed on the computer This file is provided for those users who wish to make a printed copy of the User Manual 1 7 2007 Leica Microsystems Switzerland Ltd Introduction Other Important Documents In addition to this LAS User Manual and Help a number of other documents contain important information concerning the installation operation and restrictions on the use of Leica Application Suite Please consult them before using LAS They can be found in the root folder of the LAS installation media and also installed under Windows Start Leica Application Suite once LAS is installed Recent information about the version of LAS is described in the document LAS Release Notes pdf This describes features of the software that have recently changed operational limitations and other technical information The supported hardware that is the microscopes macroscopes and cameras tha
258. rrows to reveal other daco Binong ee 4x4 Color Binning 520 x 334 options 6 x2 Live Format Live format is not used during Power Mosaic but for the sake of completeness keep the Live Format the same as the Captured Format 7 Click on the arrows to the right of the Live Format header and from the drop down menu 8 Select the same format as the Captured Format See Acquire Camera for more detailed information 4 167 2007 Leica Microsystems Switzerland Ltd Power Mosaic Stage Initialisation Stage Initialise and Set Speed The Stage must be initialised the first time an objective is selected and again if the stage stalls for any reason Initialising determines the limits of travel in both the X and Y directions and matches the stage speed to the objective Warning Ensure that there are no obstructions before initialising Turn to an empty turret position and lower the sub stage condenser Speed Settings x To compensate for different stage Sed Initialising the stage or focus will automatically move to the types ages slackness and stiffness in limits in order to determine the range of travel Make sure the mechanisms as well as various EE imaging setups the stage speed can be Adjust the speed of movement so that the motors move de rated from its maximum Lowering quickly without stalling stage speed also helps prevent stalling initiative Drive Speed which results in
259. rther modification of images already acquired for example to change the colour or contrast of the image or to crop it to a different size The modified image may be saved as a new image or can replace the original image LAS User Manual 3 134 Enhance Crop Cropping is the process of removing unwanted parts of the image leaving only the area of interest 1 A a This step is not mandatory but strongly recommended Right click on the image The Viewer menu appears Select Save As navigate to the current folder and save a copy of the image under a new name In case cropping or colour adjustment go wrong this is a pristine backup Click the Fit To Screen icon on the Side Bar to display the entire image Click on the Enhance tab Click on the arrow to the right of the Crop header to reveal the panel The area of the image to be kept is enclosed in a rectangular mask which is created by click hold and drag As long as the mouse button is held down the mask may be re sized to enclose just the area of interest Click Start on the Crop panel Click and hold on a point on the image where the top left hand corner of the mask will be Drag to the right and down The mask appears as a black outlined rectangle When the area of interest is enclosed by the mask release the mouse button The position and size of the mask in pixels is displayed in the Crop panel as the mask is be
260. rther option to add a line to the image is available using the Line panel 1 To add a line click on the Line Show checkbox Click on the arrows to the right of the Line Style bar The line style menu appears Select a line style by clicking on it Line only will draw a plain straight line Arrow will draw a line with an arrow head at the start end Distance line will draw a line measure the length of the line and display the distance as a caption Edit allows any of the lines to be moved or rotated Adjust the line width by clicking on the up down arrows on the Line Width window The new width will also be reflected in the Scale Bar Change the line colour by clicking on the Line Colour button On the Select Colour pane choose a colour as described before To draw the line click and hold on the image and drag the line to the required length Arrow lines have the facility to add a label Click on the Line Label text box and type the words appear at the end of the Arrow line To move a line click on the arrows to the right of the Line Style bar and select Edit from the menu Click and hold on the central bar of a line to select it and then drag it to the required position Rotate a line by clicking and holding on one of the end handles Drag and the line will rotate around the opposite handle Before drawing another line first click on the Merge button to integrate the lin
261. s A measurement can be subtracted from another by using the Negative check box 9 Select Area or Circle 10 Click on the Group check box Draw the first shape 11 Click on the Negative check box and draw the second shape 12 In the example the area of the smaller circle will be subtracted from that of the larger and the result displayed as a single item on the Measurements panel Measurement Actions Ci Measurement oO m Gam Show default values only I Node Runner Prior to budbur eS EE DEB 5 Node Runner Prior to budbu PIB 6 Node Runner Prior to budbur DEA 8 Node Runner Prior bo 4 143 2007 Leica Microsystems Switzerland Ltd The Results tab provides you with all of the tools you will need to organise display and print your measurement and analysis results Results are displayed as two tables on the screen Results Table on the upper part of the screen shows precise details of the measurement parameters The parameters may be turned on and off so that the table displays only pertinent data Results Summary is shown in the lower part of the screen It summarises the details from the Results Table to give a comprehensive overview of all of the measurements and their parameters Each table may be turned on or off Organise Results The Organise Results panel determines the Classes and measurement parameters that will be displayed and printed To re
262. s 4 75 Browse MultiStep Images wooo ccc ccscescscsseesessessessessessessesseesessessesaesaeseesoeseses 4 76 Browse MultiStep Images Continued ooo csessssessceseessessessessesseseseseesees 4 77 Image Overlay EE Select Capture Folder oo sesssesssssessssessssesesseseseceestesestseeseseeatseeaeseeaeseeaeseeneats Enable Image Overlay uo i cccccccccssscescessessesseusessessessevseusesseesesaessessesaesaesoasesses Setup Tools sa Setup Tools continued Channel Dialog RE Channel Dialog continued Adjust Channel Exposure scssssssssessssssessesesssseatsseasseeatseeaestearseenesteatateeeas Adjust Channel Exposure Continued sssescsssessssesssseeeseeaeseeeesteansteeeas Snapshop and Working Gallery Snapshot and Working Gallery Continued ccssesssseessesesssseseereeeseeeeaes Auto Create Folders Continued ou ccc ccscscssssesssessessesssssessessesseseesaessesnsenees Auto Create Overlay Auto Overlay Without Auto Folder csesssessssssessssessssssssesesseaestenesteaesteeeas Auto Overlay With Delete Channels ecsessssessssesessssesssseeesteaestenesteanseeeeas Recall Configurations and Manual Acquire Creating and Viewing Overlays cssessssesssssessssesesseessteaestesesteesaeeeesseesansees Creating and Viewing Overlays continued Selecting Image Group and Overlay Method e EI der LO GN Colour Control continued eseNER
263. scrolls to retain its position on the image If the image is zoomed the annotation is also zoomed If the image is reduced annotation is reduced but lines are drawn at the minimum size that is still visible on the display rather than disappearing from or appearing broken on the display To further enhance this functionality the optional Leica Application Suite Extended Annotation module can be added to your system LAS User Manual 3 128 Annotate Font and Text Colour To select a Font 1 To select a font Click on the Font Select button The Font Selector appears Choose a Font Font Style and Size Click OK Click on the Font Text button The Select Colour pane appears Choose a colour for the text by clicking on one of the basic colours or by clicking and dragging on the colour wheel and then using the slider to select a hue Click OK The Font Text button will change to reflect the selected colour To choose a colour for the text background click on the Font Back button and use the same procedure as for text colour Click OK The Font Back button will change to reflect the chosen colour If no background colour is required check the Font Transparent button Text without a background colour appears directly on the image Font line and background settings affect all of the graphics simultaneously They may be altered at any time using the steps above Lie
264. se it Include timestamp Arial Narre 4 41 2007 Leica Microsystems Switzerland Ltd Movie Time Stamp and Saving Configuration To display a progress time stamp on the movie 1 2 Click on the Font button and from the Font dialog 3 select a font type style and size and click OK 4 Click on the Colour button select a font colour from the Select Colour dialog and click OK 5 Click on the Include timestamp check box to enable the time stamp Saving the Configuration The current settings may be saved with a unique name and recalled for another movie at a later date include timestamp Microsoft Sans Serif 48pt To save the current setting 6 Click in the Configuration text box to highlight the text type a unique name for the current configuration and press Enter on the keyboard 7 Click on the Save button which will save all of the current settings Allarntac Ir ne AvantGaide Bk BT Select Color Green b B Saturation bss B Blue ba RH Brightness ba BR LAS User Manual 4 42 Movie Existing Configuration and Testing Previously saved configurations These may be retrieved and used again to load the settings for a new movie 1 Click on the arrows to right of the Options bar to reveal the Options panel 2 Click on the arrows to the right of the Configuration text box and from the drop down list 3 Select the existing configuration to use
265. sing images each with only a shallow depth of field in focus acquired with the Montage module may be processed to generate a single completely focused montage image The Montage Z tab of the Process Workflow allows the manipulation of the image stack which images to include or exclude from the final result and the generation of the Multifocus image by selecting one of a variety of methods The other results of the Montage operation are the depth map image which contains depth information for all points on the image and the confidence map image which contains an estimate of the accuracy of the depth map at all points on the image Leica Microsystems LAS v File Options Help The results of the Montage operation may be processed to create a number of different visual representations of the image 1 Click on the Z tab right to open the Montage control panel The Montage tab has the following panels Source Images Montage Multifocus Montage Enhance Montage Anaglyph Montage Stereo Pair Montage Colour Relief Measure Tools Montage Edit 4 111 2007 Leica Microsystems Switzerland Ltd Montage Source Images An image stack must be selected from the Browse View Montage tab The stack images will be listed in the Source Images panel Clicking on an image in the list will select it and display it in the Image Viewer Once selected clicking on the line will switch between IGNORE and Include in Multifocus An
266. specimen See Testing Multi Time 4 27 2007 Leica Microsystems Switzerland Ltd Time Lapse Testing With the number of images required and the interval between each capture set and the sequence loaded to the Sequence window it is easy to check Saquance step 00 0005 that the exposure and data capture will fit within the interval Number ofimages acquired BR 1 Click the Test button Interval between images 0 00 01 2 The result in seconds of exposing sier ZS the image and emulating a write to 1 6atonoooi ooooos disk together with the necessary data files is displayed beneath the Test button In the example the interval between images is set to 1 second The test capture and write amounted to 0 846 seconds so this sequence would have succeeded with a small margin to spare 3 This test fails because either the exposure is too long or the captured image format results in a Time takon very large file or both The test result here is 3 446 seconds much longer than the 1 second interval so most images are going to be missed ran See Image Exposure and Capture Close shutter when not acquiring Format Close shutter when not acquiring LAS User Manual 4 28 Time Lapse Configuration To achieve complete flexibility Multi Time can be configured with any number of capture sequences 1 The Start option is executed first in the example Aft
267. ss Saturation and Gamma 1 Brightness is a measure of the power of each colour in the image It can swing from solid black to solid white Use small increases in brightness to help differentiate between colours too much and detail begins to disappear 2 Saturation determines the amount of each colour that is present At the highest setting each colour will be at its most vibrant The right hand illustration is the high setting and the colours cannot be more prominent without combining to make white Use Saturation to achieve colour subtlety in the image Reducing Saturation is a convenient way of turning a colour image into a monochrome image essentially just shades of grey without losing detail or becoming a black solid 3 Gamma is a value applied to colour levels to compensate for different ways in which the image is viewed Liquid crystal displays LCDs have a specific Gamma setting cathode ray tube CRT monitors will have another and printers yet another Changes in Gamma are applied automatically so when an image is printed for example the printer software will make adjustments before the printing takes place Very small changes in Gamma can have dramatic effects the examples show a range of 0 35 to 1 50 with the original in the centre Use Gamma to achieve a contrast match to the specimen 3 83 2007 Leica Microsystems Switzerland Ltd Camera Run Auto Exposure amp White Bal
268. step size To change it click on the Step size window press the Delete key to clear the existing value and type a new value in ums Limit Alarm There is a small indicator at each of the joystick quadrants and also top and bottom of the focus slider Normally they are green when the stage and the focus are within limits If the Show Limit Alarm LEDs box is checked the indicators will turn red as the travel limits are approached Nudge size Each joystick quadrant also has a small arrow displayed Clicking an arrow nudges the stage in that direction It is a very useful facility for precise positioning Set the nudge value in ums by clicking the window deleting the existing value and typing a new one Color The area around the joystick defaults to pale grey silver but may be changed by clicking to select a required color Various color sets are available Speeds Calibration The three speed options may be calibrated individually by clicking and dragging the appropriate slider Again actual speeds will vary with the stage type Change and test to achieve the best settings X 300 v Normal Fast Slow Properties Software Joystick Properties Software Joystick Color Speeds Seed T Invert direction E ZAss je Focus control using Mouse Wheel IV Show fmt alarm LEDs amp Properties BackColot Software Joystick Properties X Asis Y Ass Speed Step size m
269. steps continued The illustrations show two images 1 The Start Z layer with part of the specimen in focus together with a sharp textured background and 2 The End Z layer with a different part of the specimen sharp but the background out of focus 3 The Z Range window shows the distance between the Start and End Z layers and 4 The number of steps and the size of each step in the Z Step window Adjust 2 Stack Oe Start a d L ig pass lyum WB Optimize step size 4 65 2007 Leica Microsystems Switzerland Ltd MultiFocus Acquire Z Stack Images Manual Microscopes 1 Click on the Acquire MultiFocus button to start the acquisition Acquire MultiFocus 2 At each step position the prompt appears Re focus the microscope using the Z Step value always focussing in the same direction 3 Click OK to capture the image To D Acquiring image 1 2 cancel the sequence if it is believed J sufficient slices have been captured click on the Cancel Click Concel if you want to complete stack acquisition button The number of captured images does not have to correspond with the calculated or entered number of steps Planse set the first Z position and then click OK 4 When all of the images have been captured the Browse Workflow opens with the MultiFocus Image Set control panel displayed 5 Granta Motttocag image LAS User Manual 4 66 Multi
270. sure that it remains accurate e Check microscopes with a manual turret so the selected objective matches that which is selected on the Acquire Mic1 tab The same applies to the Mag Changer if it is fitted e Set calibration See Calibration e If necessary adjust the camera rotation See Camera rotation Continued on the next page 4 157 2007 Leica Microsystems Switzerland Ltd Operating Steps Continued 7 Create and perform a test scan e Select the Split Screen view The crosshair shows the current stage position e Move the stage so that the specimen can be seen in the live image window e Select the New Pattern tool click close to the stage crosshair and drag a small scan region e Click Acquire Power Mosaic e Use Zoom and Pan to check that the mosaic is formed correctly See Create Pattern Grid 8 Extend the scan pattern to the required region To include parts of the specimen not included in the test scan e Click on the Create Expand tool e Click on the stage in an area outside the boundary of the test scan and drag to include the required parts of the specimen See Create Pattern Grid 9 Select the Focus Method During scanning two focussing methods are available Predictive Focus is best suited to uniform specimens and low magnification up to x10 Focussing is performed either manually or automatically on a number of points across the specimen to create a table of values Points which have not been
271. t M Spent F Unine Cutten Sea See ea S LRSL se ER Zeg rei Zo B om gt H Seege E H ma E H tosa Ex RH 3 129 2007 Leica Microsystems Switzerland Ltd With text colour style and background selected return to the Information panel to add Image Name Description Date and Time annotations which appear in the default positions 1 Click on the Information lmage Name checkbox and the image file name will appear bottom left 2 Click on the Information Description checkbox and then type in a description of the image which is displayed bottom right 3 Click on the nformation Date checkbox to display the date top right 4 Click on the Information Time checkbox to display the time which is added to the date string At this stage an optional Scale Bar may be added and will appear on the image in the default position top left 5 To add a Scale Bar click on the Scale Bar Show checkbox 6 To adjust the length of the Scale Bar click on the Scale Bar User Length checkbox and type ina value Make it a round number such as 100 150 or 200 The line will be drawn to scale 7 Select the Scale Bar Line Ends style by clicking the arrows to the right of the Scale Bar Show window and selecting None Short end Medium end or Long end from the drop down menu Annotate Information and Scale Bar LAS User Manual 3 130 Annotate Add a Line A fu
272. t can be used with LAS is described in the document System Requirements pdf This document also describes the appropriate computer specification Please ensure that your computer specification corresponds to the recommendations made Other factors that influence the performance of LAS are also noted in the same document A detailed description of the installation procedure for Leica Application Suite is in the LAS Install Guide pdf For Leica DM microscopes refer to the operator s manual supplied with the microscope for detailed guidance on configuring and operating the microscope LAS User Manual About LAS Leica pplication Suite o System requirements for using LAS gt How to install this software gt Release notes Eer et oe RI Release Notes User Manual Install Guide BI System Requirements The Release Notes contain information about changes and revisions that came too late to be included in this documentation The Notes may be found on the original installation CD or on the computer hard drive Release Notes on the CD Load the CD to the CD or DVD drive on the computer In most cases it should start up automatically If it does not 1 Click on the My Computer icon on the computer desktop 2 From the dialog right click on the CD or DVD drive The disk name will reflect the LAS Software Version and may not be the same as the illustration Other Documents Release Notes
273. t light increase the camera gain by small amounts to give the correct exposure Check that this does not significantly increase image noise WARNING Switch 100 light to the camera DO NOT look a the specimen through the eyepieces Adjusting the camera for Standard scan Standard scan is used when the conditions for Turboscan are not met e Select the camera image format to suit the specimen detail required Choose the lowest resolution without compromising image quality to save disk space e Set the lamp voltage to give a comfortable image in the eyepiece e Adjust lamp voltage and camera exposure until red flecks appear on a white region of the image e Set Exposure to less than 50 ms 5 Set shading Shading correction has to be set in Power Mosaic LAS settings are not used Shading correction must be repeated every time the objective is changed e Move the specimen so a clear region without artefacts is visible over the whole image e Set the shading correction See Shading 6 Calibration for each objective LAS Power Mosaic derives its calibration from the stage movement to accurately align the tile edges Calibration must be carried out for every objective on first use Calibration tests that the values returned are reasonable If they are not a warning is displayed Camera rotation must be less than 0 1 Conditions can alter over time and systems are susceptible to dirt heat and vibration Periodically check the calibration to en
274. tablish sharpness Because it is a continuous process of focussing and checking Autofocus is slower than Predictive but yields extremely good results especially over irregular specimens Predictive and Autofocus in combination uses the speed of Predictive and the quality of Autofocus to achieve very good results quickly Use this for specimens that are predominantly uniform but also have local irregularities Predictive will come close to focus and the Auto will fine tune it Both Predictive and Autofocus methods and the combination are selected by enabling the check boxes 1 When a method is enabled its setup button becomes active 2 Setup is explained on the following pages For perfectly flat specimens both methods can be turned off Continued on the next page gt San Seno O Pattern Type a mm en Standard Auto Step Size Direction Focus Method Prodictive LAS User Manual 4 184 Power Mosaic Predictive Focus Predictive Focus The Predictive Focus function focuses on a number of predetermined points on the image and creates a table of their focus values The focus position for any other point can be predicted by interpolating the table values ane OH E v Auto Step Size Turboscan Sindari The greater the number of pre determined points then the greater the accuracy of the prediction and better the overall focus Th
275. the y Acquire button being clicked and O Apparentimage shift the acquisition of the first layer Click in the text box and type a Zayer3 value Z layer2 Z laye Stage T 0 ms Wait before acquire 4 53 2007 Leica Microsystems Switzerland Ltd MultiFocus Configuration The Configuration facility allows MultiFocus Z step settings and options to be saved retrieved and used again Opbons K Create a new Configuration 1 Click in the Configuration text box and type an appropriate file name for the MultiFocus session 2 Click on the Save button The settings are saved to disk and the file name added to existing Configurations After the Z stack has been set up the Configuration will be saved again to include the new stack settings Perform manual focus 9 ms Wait before scquire Retrieve an existing Configuration 3 Click on the arrows to the right of the Configuration text box 4 From the drop down list click to select the Configuration required The Last Used option will automatically load the settings used during the last MultiFocus session Delete a Configuration 5 Click the Delete button to remove the Configuration displayed in the text box The settings cannot be retrieved The Last Used option may not be deleted Align images before combining WW Perform manuel focus 0 ms Waitbefore acquire LAS User Manual 4 54 1 If necessary click on the Acqu
276. the list and click to select it For microscopes without a camera select the None option fmage Source Please select he camera framegrabter you want to use as image source 4 Click Save to save the microscope and camera settings and exit 2 9 2007 Leica Microsystems Switzerland Ltd Registering Modules and Applications The Module or Application Licence Number Optional Modules and Applications are purchased through local Leica Sales centres which will provide a Module Licence Number when the sale is complete The Module Licence Number can be found in the packaging A single Module Licence Number can relate to one or several modules The user sends the Module Licence Number and the Site Code to the Leica Configuration Centre either by e mail or by fax They return a Licence Key usually within a few hours The License Key is a code which will allow Optional Modules to perform normally The Licence Key is then entered into the computer and the modules are enabled without restriction Getting a Licence Key 1 From the Licensing tool bar click to select Options 2 Select Registration from the drop down menu The Licence Key dialog appears 3 The computer Site Code is automatically displayed The Site Code is unique and appropriate for this computer only 4 Ignore the License Key window and 5 Click on Register Go to Getting a Licence Key continued Leica Application Suite Fil
277. ties panel by clicking on the arrows to the right of the Properties header bar To make the Properties panel visible all of the time click hold drag and release it on another part of the screen to To change text attributes 2 Auto Size scales the text size to suit the image size Turn Auto Size off to quickly scale down a large label 3 Clicking the Font button reveals the Font dialog 4 to change font font style italic bold etc and size 5 After selecting a new font click OK to apply the change Any subsequent tool labels will reflect the change To alter existing labels click on the Picker tool click on the label to be changed and then click on the Font button 6 The text colour can be changed by clicking the Text window which shows the current colour and when the Select Colour dialog 7 appears clicking on either a swatch or the colour wheel 8 Click OK to apply the new colour To alter existing labels click on the Picker tool click on the label to be changed and then click on the Text window Properties el Auto Size D Text Transparent Background sE with E ire Marker Size Comment Set Default Apply Class Label measurement with Pie Custom Col WH LUC CU UL Current Color rei bs nek BR Green le B Saturation ba B ue Je RH Brightness pss E LAS User Manual 4 136 Interactive Measurements Measure To change a Label background 1 Click on
278. tion Slide is properly focused with the correct objective and Mag lens in position 1 Click on the arrows to the right of the Type window A drop down menu appears Selecting the None option will turn off calibration 2 Select the Calculated option to calibrate 3 Click on the New button 4 On the Configuration Name dialog type an appropriate name for the settings to be saved under Click OK 5 The Scale Bar which appears over the Calibration Slide image must be positioned click and drag on the bar and resized click and drag on the end handles to fit perfectly between two known points on the Calibration Slide image in the example 3 0millimetres 6 Click on the arrows to the right of the Measurement Units window and from the drop down menu select the units of the known distance millimetres in this case 7 In the Actual Length window type the known distance 8 Click the Save button and a Calibration Link will be created The value in the Actual Length window may change as a result of conversion to display settings made previously on the Scale Bar panel Actual length of line shown on image ez CED af Enter new configuration name Beta_Mould 100 Calibration Settings Type Culruiated E Configuration Za a ZS Actual length of line shown on image Q Millimeters Nanometers Microns Millimeters Centimeters Meters inches Mils 3 99 2007 Leica
279. tion works only if the Show Acquire Panel is selected 3 Click Show Browse Panel to watch the sequence on the Browse Workflow This has the advantage that the captured images are displayed on the Viewer as they are made and the Gallery 4 is progressively populated Ze r ae Continued 4 31 2007 Leica Microsystems Switzerland Ltd Time Lapse Capture Display Options continued Progress Pause and Stop panel During the capture sequence progress is displayed as 5 The number of images acquired so far and the number remaining to be captured 6 The current time Next image acquire time sequence time remaining and estimated time of completion If there are several sequences in a project the Confirm Sequence Termination completion time is for the entire project not just for the current sequence The Time lapse requence iz about to be terminated betore Miz complete Yes to terminate sequence Ne to continue with seqeence 7 Graphical representation of mine H progress both as a bar and a percentage Time remaining 001 609 8 To suspend the sequence click on Completion expected 13 06 56 the Pause button The button label ma changes to Resume Click again to SE continue the capture Note d mm C x however that images may be lost during Pause 9 Stop the sequence by clicking the Stop button The Confirm Sequence Termination message appears 10 Click Yes to stop the seque
280. tion expected at the next if there is one click on the Pause button 8 To halt the entire movie click on the Stop button Time elapsed Overall Progress is detailed in the Time to completion upper panel wn 9 Time now is the real time clock Completion expected at 10 Time elapsed for the entire movie all clips so far 11 Computed Time to completion as hh mm ss 12 Real time Completion expected at There is also a graphical display of time elapsed so far 13 Dileica Mainstreem_ProjectMainstream_2O0SMaywerd_AnabytcelProject_Alpha Movies M Movied2 bmp When the movie recording is complete a sample reference image 14 is placed in the capture directory with a thumbnail Placing the cursor over the thumbnail reveals the path and name 15 LAS User Manual 4 44 Movie Play Controls After capture the display will open in the Browse Workflow 1 in the specified movie capture folder The movie will normally be played in the Viewer but the playback application can be changed by clicking the Play with button 2 which will launch the program specified in Preferences i Image Output Settings Play Movie SM Bey with The playback controls 3 on the Browse Viewer are located on the Play Movie panel Click the icons 4 To Start Stop the movie 00 00 00 Time elapsed Current movie clip 5 Skip a frame forwards 6 Skip a frame backwards Recording mode C
281. tion of digital images at specified resolution e A thumbnail gallery of acquired images that can be reviewed quickly and easily e Basic tools that annotate an image with text scale bar and distance measurement LAS User Manual Introduction Optional Modules The underlying capabilities of the core functions can be enhanced with a range of advanced modules and applications Each LAS module provides the flexibility to tailor a system solution to fulfill individual needs with upgrade options available for future requirements Some examples include Extended depth of focus Movie recording SG Image measurements Autofocus SG Image Overlay SG Power Mosaic and many more that you will find described in this manual and help 1 5 2007 Leica Microsystems Switzerland Ltd Introduction Manual and Help Organisation In addition to this introductory chapter you will find the following chapters Installing Configuring and Licensing covering the topics required to prepare Leica Application Suite for use For example you need to install the software tell the software what type of microscope and camera is connected and to license any LAS Modules you have purchased or wish to evaluate Go there SG Using the Core Features describes the intuitive Workflow organization of the unique LAS user interface and its basic capabilities This is done by describing the Setup Acquire B
282. to their original filter colour values click on a thumbnail and then on the Restore To Default button LAS User Manual 4 98 Image Overlay Fine Tuning False colour is applied to a Channel image electronically by association only the original remains intact and unchanged This means that colour may be removed completely or fine tuned at any time after capture and a new Overlay created Individual colours in overlays cannot be altered but the Histogram 1 and the Gamma 2 controls are available to lighten or intensify overall effect and in the process enhance a particular feature It is not necessary nor always desirable to include all of the Channel images in an Overlay The Overlay Image panel provides the means to include or exclude individual Channels 3 A Channel is included when the check box to its left is checked Click to un check and exclude At least two Channels must be selected in order to create an Overlay If only two are still selected the program will prevent any more exclusions The colour bar to the right of a Channel name indicates the filter colour used in its capture 4 Click the Show Overlay button to select and display the last overlay created or 5 Click on the Create Overlay button to produce a new overlay using the revised Channel images Overlay image Overlay Addition 4 99 2007 Leica Microsystems Switzerland Ltd Montage
283. tween display of stage overview and pattern view Drag to create pattern scan or enter exact details Expand scan pattern to specimen position Point and click relocation of stage to indicated position Real time graphic display of current stage position on specimen map Zoom and pan over entire mosaic Calibration Automatically measures calibration and updates scan patterns according to selected magnification on microscope using a correlation based calibration procedure Camera rotation and stage skew is measured to allow convenient adjustment Small rotation of camera is compensated for in the mosaic creation Automatic stage backlash correction Automatic Focus Multi point predictive focus for continuous focus tracking Predictive focus setup by combined autofocus pre scan with user review Blank fields are ignored in autofocus pre scan Autofocus can be used for specimens that are not flat Predictive and Autofocus can be combined LAS Power Mosaic Plus This is an extended version of Power Mosaic and includes all the features of Power Mosaic and in addition Z Scan optional at each field Definition of Z Scan positions step number and step width Extended focus image can be created Mosaic image can be reviewed while sweeping through all focus positions 4 155 2007 Leica Microsystems Switzerland Ltd Operating Steps E The following procedure lists the steps required to obtain high quality mosaic images Once the syst
284. ued The illustrations show two images 1 The Start Z layer with part of the specimen in focus together with a sharp textured background and 2 The End Z layer with a different part of the specimen sharp but the background out of focus Using the Go To buttons 3 The Go To Start button when clicked will drive the microscope to the Start position with the value displayed in the Z Position window 5 for a focussing check 4 The Go To End button drives the microscope to the End position for a focussing check Again the microscope value is displayed in the Z Position window 5 6 The Z Range window shows the distance between the Start and End positions Adjust 2 Stack Get E tart Geff lym Z Position Z Range Steps Z Step 3810 2 LAS User Manual 4 58 MultiFocus Set Z Stack Steps Automatic Microscopes With the Start and End points set the distance between the two is calculated and displayed in the Z Range and Z Step windows There are two methods of setting the number of layers or steps 1 Click in the Steps text box and type the number of steps required The distance between each is calculated and shown in the Z Step text box 2 Click in the Z Step text box and type the distance required between each step The range value is divided by the entered figure and the resulting number of steps displayed in the Steps text box 3 With th
285. ured displayed in the Viewer Genera image Groups C_PE_Overiay 0009 C_PE_ Overlay 0010 C_PE_Overlay 0011 3 To select the method of combining Sones the Channel images to create the E EE Overlay oot Overlay click on the arrows to the C PE Overlay 0015 right of the Overlay Method text C_PE Overlay Gm General box and from the drop down list C_PE_Overlay 0017 Eeer i i 4 Click to select the required method Se In the illustrations images 5 6 Overlay Method and 7 are the original common e Channel images that have had Maximum false pseudo colour applied Addition Average 8 Represents the Overlay combining the three common Channel images using the Maximum option The highest pixel value from the same location in the three common images is used to create the Overlay 9 The same process using the Addition option The pixel value from the same location in all three Channel images are added together 10 Shows the result with the Average option The pixel values in the same location in all three Channel images are added together and then averaged 11 To save the settings as default click the Set as Default button wreney Method E a K Maximum gt LAS User Manual 4 96 Image Overlay Colour Control Monochrome Channel images can have false or pseudo colour applied to them before they are combined into a single overlay to improve contrast and clarity of the final im
286. utton to reveal it 9 Click on the Clear Tiles button to clear any previous images 10 Confirm clearing the images on the warning message by clicking Yes 11 Click on the Acquire Power Mosaic button The specimen will be scanned with the tiles filled in sequence The first drawing usually encloses only part of the image as shown in the illustration If this is the case go to the next page If none of the specimen is found 9 Click on the Clear Tiles button and repeat the process starting the drawing at a different location Continued on the next page Scan Pattern Rectangular Clear Mosaic LAS User Manual 4 190 Power Mosaic Create Pattern Grid Continued Create the Pattern Grid Continued With the pattern grid enclosing only part of the specimen it can be extended so that the specimen is fully covered 1 Click on the Create Expand button This feature allows the existing grid to be expanded The Create Expand button may also be used to reduce the pattern grid if it is too large 2 Click on the top left corner of the existing pattern grid hold and drag diagonally to the right A dotted rectangle follows the cursor to indicate the extent of the grid Release the mouse button when the specimen is considered to be completely enclosed 3 4 Click on the Clear Tiles button and confirm the clear on the warning message 5 Re scan by clicking the Acquire
287. veal the Organise Results panel 1 Click on the arrows to the right of the Organise Results header bar Configure Results Three controls allow you to configure the Results Table and Results Summary in the most appropriate way 2 Classes to show selects the Classes to be included in the results 3 Details defines the measurement parameters to display as headers on the Results Table and 4 Summary selects the headers for the Summary Table Interactive Measurements Results 26400 264 00 264 00 264 00 DO DO 4 00 24 00 254 00 264 329 66 971 0 1447680 00 H Er 1301 00 690 50 419 00 680 50 231 50 163 70 82 00 413 00 0 1447680 00 2 Hei 1433 00 477 67 283 00 288 00 286 03 185 17 82 00 253 00 D gt D 1301 00 650 50 41900 650 50 271 50 163 70 88200 41300 Radis 65050 FBS 203 50 325 25 11575 EES A0 20350 15393 00 32965 971 16483 1447680 3 D 1447690 00 2 0 1447690 00 2 LAS User Manual 4 144 To determine Classes to display 1 Click on the arrows to the right of the Class to Show window and 2 Click to display No Class No Class information will appear in the tables All Classes To display Class descriptions for all of the classes or Class name Display only the Class selected and to ignore any others Interactive Measurements Results Select Classes 4 145 2007 Leica Mi
288. vides all of the tools for making interactive measurements on the image To expand the tab 1 Click on the Measure tab to reveal 2 The Measurement Tools panel To use a tool click on its icon 3 Select Class panel which allows the Class and its related tools to be applied to a measurement 4 Measurement panel shows all of the default and selected measurements and parameters for each tool 5 The Properties panel which formats the tools and also controls the items displayed on a measurement label Measurement Tools Each tool is represented by an icon Click on the icon to activate and use the tool 6 Select Use the Select tool to select objects such as distance lines and labels to modify or move them When an object is moved or modified the associated measurements are immediately updated 7 Distance tool 8 Line tool 9 Area tool 10 Ellipse and Circle tool 11 Angle tool 12 Count tool 13 Cross tool 14 Parallel Lines tool 15 Undo tool reverses the last action 16 Delete tool erases the selected item Use the keyboard Delete key as an alternative 17 18 Several tools have Group and Negative facilities They are available only when the appropriate tool is selected and the button is checked See Measurement Actions Interactive Measurements Measure Marker Size foult Set Defaul Label measurement with Z Num LIZIO ff LAS User Manual
289. w Zoe Ei T rmage_Crop_t p9 image0004 pg AN Images Ta gg bop gt File rome Files of ype 3 115 2007 Leica Microsystems Switzerland Ltd Browse Print an Image 1 Click on the Print icon on the side bar or 2 Right click on the image and select Print from the Image Control menu The Print Properties dialog appears Select a Printer 3 If several printers are available click Select and choose a printer Headers and Footers 4 If Page header or Page footer are required click the appropriate Edit button The Header or Footer Text Setup dialog appears Font Style Size and Colour 5 To change the font size colour or weight click the Select button The Font dialog appears 6 Select the font and attributes and click OK Text Layout 7 Click on the selection arrows to the right of either Left Justified Centre Justified or Right Justified text windows The Print Item Selection menu appears 8 Click on the items required to appear in the header or footer The selected data is imported automatically 9 Click OK Page Layout Preview and Print 10 Click Page Setup to change the paper size and Preview to see how the image will be positioned on the paper 11 Click Print to print the image or Cancel to exit Header Text Setu Lelt Justified Text lt Descriptions lt CaptureD ate gt lt PrintD ates D Centre Justii
290. wheel and slider Click OK 4 When Transparent is unchecked the text sits upon a coloured background The background extends across the text only and does not fill the text box To add more background colour at the beginning or end of a line pad out with spaces Text alignment 5 Typed text can be aligned left right or centre within its text box To align text select the Picker tool and click on the text to be re aligned Click on the arrows to the right of the Align bar The Align menu appears Select Centre Left or Right The text moves to the selected position Col RSR ACIC IC IIL d SS d Mai Sat ef STS y Ka and this is aligned centre l ay 2 roe oe LAS User Manual 4 8 Extended Annotations Lines Arrows and Distances The Extended drawing tools can be used to add lines boxes ellipses and circles to the image Line types are plain arrowed and distance A Scale Bar is available also To draw a Plain Arrow or Distance line 1 Click on the line tool required on the Tools panel 2 Position the cursor which changes from an arrow to a white cross on the image in the position where the line is to begin Arrows always start with the arrow head Distance lines automatically display the distance between the two end points Click hold and draw the line in the direction and to the length required To constrain a line in increments of 45 degrees hold down the Shift key on the keyb
291. xchange cubes with a double click of the left mouse button Alternatively e Mark new cube in the selection list with left mouse button e Open dialog with right mouse button and use select Continued 3 23 2007 Leica Microsystems Switzerland Ltd DM Microscope Configuration Tube continued Continued Documentation port e This node allows to learn in c mounts with fixed magnification factor and vario mounts with a predefined magnification factor e The proper definition and selection of the mounts is necessary for autocalibration of the microscope NB The symbol S which is displayed on the LeicaScreen refers to the total magnification with respect to the current objective and eyepieces The selected c mount or vario mount does not influence this value The c mounts and vario mounts can be removed or exchanged e Remove one c mount vario mount from the documentation tube e Mark c mount vario mount which has to be deleted in the status list with left mouse button e Use right mouse button and select delete c mount Learn in a new c mount vario mount from the selection list e Mark position in the status list with the left mouse button e Mark new c mount vario mount in the selection list with a double click of the left mouse button Alternatively e Mark new c mount vario mount in the selection list with left mouse button e Open dialog with right mouse button and use select
292. y be enlarged by resizing the whole dialog 4 103 2007 Leica Microsystems Switzerland Ltd Montage Browse Montage Images Click on the Z tab to open the Montage control panel The Montage tab has two panels e Show Multifocus Image Set e 3D Options Show Multifocus Image Set Show Multifocus image Set DI Select the required Z Stack from the drop down box The Z Stack images will be loaded into the gallery together with any associated EE Q multifocus image and maps Clicking on a thumbnail in the gallery will display the relevant image From this panel it is possible to review the following image Stack Images CEA Multifocus Image between Depth Map Se images 00 Sec Confidence Map Anaglyph Stereo Pair Colour relief Stack Images 3D Model d d Multifocus Image Depth Map Confidence Map Anaglyph Stereo Pair Colour Relief 3D Model LAS User Manual 4 104 Montage Stack Images 1 To view the Z Stack sequence click on the Stack Images button This will display the first image in the stack and enable the controls to view the stack Enter a delay time between images and then use the control buttons to scan the images 2 Plays the image sequence continuously with the specified delay time between each image Click to play click again to stop 3 Moves to first and last image in the sequence 4 Steps forward or back in the sequence Multifocus Image 5 Click on red frame to
293. y clicking ona sequence and then on the up down icon as appropriate LAS User Manual 4 40 Movie Name and Compression To give the movie a name 1 Click in the Movie name text box and type an appropriate name 2 The first time a movie is captured a common folder named Movies is created in the capture directory 3 Within this common folder a new folder is created for every new movie with the name specified in the Movie name text box 4 The movie avi file s together with several control files are stored in this folder Select the Compression option Compression results in smaller movie files but can affect image quality The compression is set on the Quality slider Values up to about 50 on the slider have little or no noticeable reduction in quality Higher figures represent a greater level of compression and a smaller file size but may result in a noticeable loss of quality Start with a value of 80 and then experiment by gradually decreasing the to achieve acceptable quality 5 Click on the arrows to the right of the Compression type window and 6 from the drop down menu select either None for no compression or MJPEG to apply compression 7 If MJPEG is selected the Quality slider appears This controls the degree of compression applied to the images and therefore the display quality Click and hold the slider and drag to the left to decrease the quality or to the right to increa
294. y has been created automatically This is an excellent housekeeping arrangement for very high throughput where there is no requirement to add pseudo colour to the images 1 Check the Auto create overlay check box and 2 e Delete channels after overlay creation UW Always create new overlay folder 2 the Delete channels after Overlay creation check box 3 Click on the Acquire Overlay Pa ee button The Channel images are deleted feica after the Overlay is created and saved in the current Capture Folder 4 not in a newly created folder or the last newly created folder 5 Each Overlay is automatically given a name comprising the prefix Overlay Maximum together with a sequential number LAS User Manual 4 92 Previously saved configurations stored in the current capture folder can be re called by clicking on the arrows to the right of the Configuration window 1 and then choosing from the list 2 Changes may then be made to the exposures and to the Options with the altered settings saved as a new configuration 3 Manual Acquire Primarily designed for non motorised microscopes the Manual acquire facility prompts for the filter turret to be moved manually to the correct position before the image is captured 4 Click to enable the Manual acquire option 5 Any or all of the other options may be selected in Manual acquire mode 6 Click on the Acquire Overlay button 7
295. y image that is ignored will not be used in the generation of the best multifocus image If the Align Images before combining box was checked in the Acquire Z Options panel then relative alignment data will be shown here Generate the Montage image from the Montage Multifocus panel First Image top of Z Stack v z 0021it vV z DI mt v z 004 1 Sz _008 tif w z DOE dee oe ES Huese LAS User Manual 4 112 Montage Create Anaglyph Image j l The Montage Anaglyph operation generates the anaglyph image which when viewed through suitable red and green blue lenses offers a 3 dimensional monochrome view of the montage image with depth information synthesized from the depth map It is the same size as the montage image used to generate it but always comprises 3 RGB planes The anaglyph image will be saved as one of the result image files momma O To create an Anaglyph image Q 1 Select Method from dropdown box Red Green Choose Red Green or Red Blue to match your particular glasses both Kei Canu are commonly available Choose Red Cyan for better printed results the effect depends on the particular montage image Separation KH T Preview 2 Check Colorize to add a hint of the original montage image s color n back to the anaglyph image the effect depends on the particular montage image 3 Set the amount of Separation the maximum amount in pixels by which left

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