Leica INM-100 Microscope Extended Depth of Focus Instructions
Contents
1. bu Network File name M vExample P aCe Save as lype TIFF TIF TIFF Cancel Compression Output BPP None DA 18 For Image Pro Plus to store all of the separate images 7 in this example in one file the APPLY TO setting has to be ALL see below o Apply To If you attempt to save a multiple image window without this setting in the ALL position Image Pro will prompt you 19 If you want to combine your multiple images using Image Pro Plus EXTENDED DEPTH OF FIELD tool do the following Make sure the image you want to process 1s open 14 Wimage Pro Plus MyExample tif 7 7 File Edit Acquire Sequence Enhance Process Measure Macro Window Help SHHeC EGER 2 ROOCCaA ALS H H S Ea i i i BALE BS amp 2 MyExample tif 7 7 oa RGB 24 14 745 600 bytes Zoom 25 14041308 5133 WH 256 7 Select PROCESS ENDED DEPTH OF FIELD 15 e er dackaround Sofrfecton Tinheshold PSEUOO GIOT Extended Depth of Field Select the image you want to process on the left side of the IMAGE LIST tab and then press ADD to put it on the SELECTED FRAMES list Extended Depth of Field Extended Dena Remove lt Moye Up Move Down 16 Change to the FOCUS SETTINGS tab Extended Depth of Field f X Image List Focus Settings Output options Topographic Map TT Generate topographic map C Return best focus frame D2. 3238 um GoTo Bottom Minimum Z Set 23215 3856 um Gola Thickness 29520000 um i Depth ot Field B5 um Number of Images 7 Cycle Focus Image No 3 Je Pause 1000 ma Hoving to Image No 3 When the cycle finishes the following message box will be displayed INM Focus ne The Cycle Focus routine has reached the end If for some reason you need to stop the CYCLE FOCUS routine you can press the 11 STOP button Within a couple of seconds the routine should stop and you should see the following message INv Focus NN The Cycle Focus routine has been stopped 15 If you want to make the computer and scope loop through the focus zone that you have set select the LOOP radio button and press the START button The software will drive the scope from the bottom to the top of the focus zone pausing the user selected time at each image After the top is reached the software sends the stage directly back to the bottom This loop will continue until you press the STOP button 16 Once the software has captured your images in Image Pro Plus you should see something like the following 2 Image Pro Plus untitled000 7 7 Ble xj File Edit Acquire Sequence Enhance Process Measure Macro Window Help sii Cc Ra2 D00CcO7 r 8 amp Hie HH to RA Le Bee AMET ey _ ol gt a E Sequencer Toolbar 7 Fra nes suene U E DI EE sn cbr e e a Flay Apply To
3. GO TO buttons in the TOP Maximum Z and BOTTOM Minimum Z sections of the dialog box to confirm your settings You can also manually enter numbers into these sections of the dialog box PLEASE NOTE There is no error checking on the numbers you enter It is possible for you to request a stage position which would cause the sample to smash into contact with the microscope objective possibly causing serious damage It is MUCH better to position the stage using the focus knob update the software and then set the limits of travel 13 Once the TOP and BOTTOM locations have been set you can use buttons shown below to manually step through the focus levels Image Ho I Go To ou 14 When you are satisfied with the settings you can capture all of the images using the CYCLE FOCUS section of the dialog box With the radio button labeled CAPTURE selected press the START button and the software will Travel to the point you designated as BOTTOM Pause for the amount of time designated Capture the first image into a new image frame in Image Pro Plus Travel to next focus point Pause for the amount of time designated Capture the next image into the frame created for the first image Continue until all images are captured While images are being captured the dialog box will look something like this 10 lu INM Focus Yer 1 0 34 Current Position Update 23215 3856 Uni Top Maximum lt det 29218
4. the appropriate SET button i INM Focus Yer 1 0 34 Ioj x M Preview Image On Current Position Update 23215 3856 Uni Top Maximum det 29218 3238 um Goto Bottom Minimum Z 29215 386 um GoTo Thickness 23520000 um i Depth ot Field ara lo um SSS ae GG g x EIE O Number of Images 4 Cycle Focus Image No Go Ta ENE Pause 1000 ms o Stat f Capture step Loop 11 The dialog box automatically calculates the thickness of the sample by subtracting the BOTTOM from the TOP Note the arithmetic symbols to the left of the dialogue The THICKNESS 1s displayed in um The dialog box defaults to assuming that the DEPTH OF FIELD for the microscope objective is lum In the example shown above with a THICKNESS of 2 95 um and a DOF of um the system calculates that the NUMBER OF IMAGES as 4 One at 0 um at 1 um at 2 um and 1 at 3 um You can override this number In the following example I have changed the DOF to 5 um and you can see that the NUMBER OF IMAGES has changed to 7 w INM Focus Yer 1 0 34 e _ iol x M Preview Image On Current Position Update 23215 3856 Uni Top Maximum lt O ose 29218338 um GoTo Bottom Minimum Z Set 23215 3856 um Goto Thickness Es 25520000 um Depth of Field ae zi of Images 7 Cycle Focus Image No 12 You can press the
5. Leica INM 200 Microscope Extended Depth of Focus Instructions Version 1 0 0 34 Matthew Batchelor Meyer Instr 8 18 2005 Roger Robbins UTD C MyDocuments Teaching RogerPapers INM Focus doc Purpose This instruction paper describes a satellite program that works with the Leica INM 100 microscope and the Image Pro software to automate the collection of component images required for the Extended Depth of Focus feature Concept This feature is a software means of obtaining a high magnification image over topology deeper than the depth of focus of the objective lens It achieves this by taking multiple images through the depth of the topology and then recombining all these images into a single image composed of just the in focus portions of all the component images This automation software requires that the operator find the feature of interest and input the upper and lower stage positions associated with the bottom and top of the image focus range declaring the lens depth of focus given later in a table and then pushing the acquire button The software then automatically steps the z axis stage height through the topology range and captures images at the appropriate focus These images are then fed to the Image pro software and composed into a single image using just the in focus portions of all the component images A clever routine allows the user to view all the component images in a timed series movie just like an operator would
6. Start Current End Options I Synchronize play of all sequences Oey AGE 24 14 745 600 bytes Zoon 25 24161340 119 54 wi H 2561 7 12 In the example above we have an image frame titled UNTITLED000 which contains 7 images Currently the 7 of 7 images is being displayed Play through these images using the controls in the SEQUENCE TOOLBAR Extra options are behind the OPTIONS button in this dialog box 17 To store these image on your disk select FILE SAVE or FILE SAVE AS File Edit Acquire Sequence Enhance Process Measure Macro Window Help Mew Ckrl M Open Chrl 0 Image Database Reload Close i Save iCtrl 5 Save 4s Archive Batch Conversion File Signature Image Signature Send Mail Remote File Transfer H Remote Conference Screen Capture Print Screen F Print Ctrl P Mosaic Image Exit 1 CAINM Focus Robbins Samplest001 20x Lum tif 2 CHINM FocusiExample Images 100 Sum spacing Local Contrast Composite tif SC AINM Focus Example Images100 Sum spacing tif 4 CAINM Focus Example Images 50 S00nm spacing No 2 tif When the dialog box appears direct the operating system to store the file in your desired location with your desired name In this example MyExample TIF will be stored in My Documents 13 Save in E My Documents 1 My Recent Documents Deskto n My Documents ra My Computer
7. aming for your sample The image should look something like the following 2151 x File Edit Acquire Sequence Enhance Process Measure Macro Window Help SUH PCSHEA2 O00C S728 fx tet n El ORANGE eIBeB lolx NM Preview Image On Current Z Position Update 29112 264 um Top Maximum Z Set um GoTo Bottom Minimum Z Set um GoTo Thickness LL m Depth of Field le UL m Number of Images LIL Cycle Focus Image No Go To EEn Focus 35 Max 35 1 84 fps 7 Pause 1000 ms _ Stat_ Capture sep Loop Image lt none gt System 10x um 4 If the image is too dark as it is here right click on the image select AUTO EXPOSURE DYNAMIC shown below and the computer will automatically find an appropriate exposure Auto Set A uto Contrast Auto Exposure REY Dynamic Suto White Balance Reset ADI Live Histogram Set Area Calculate Transparency After the proper exposure is found turn off dynamic auto exposure or your results will be affected by the changes in the exposures during your image capture 8 Adjust the focus on your microscope so that the stage is at the highest max z position and then press the UPDATE button to capture the stage z position Note that the highest stage z position occurs when you are focused on the bottom portion of your image 9 FE Image Pro Pl
8. isplay calibrated height STO Generate surface plat Normalize illumination Activate selected image Select Focus regions for Focus analysis description Maximum local contrast Pixels are analyzed in the area around the current pixel location The piel from the plane with the largest variance or local contrast is selected Magimum through depth contrast I Maximum intensity C Minimum intensity Teststrips Generate topographic map Generate sequence gallery 1 image s frame s in stack Lancel Select the settings as they are shown above and press the CREATE button The resulting image will be displayed and will be given a name based on your original image s name and the EDOF algorithm used to create the image 17 2 Image Pro Plus MyExample tif Local Contrast Composite 1 1 File Edit Acquire Sequence Enhance Process Measure Macro Window Help bi Cage ROOLAAN A Hi ti Hi MEHR 1 cn ee 2 MyExample tif Local Contrast Composite 1 1 All i lis KA a oO Fi RGB 24 14 745 600 bytes Zoom 25 1636 16 33 28 MH 256 This image has not been saved to the disk yet so please do so 20 The size of these Images on disk can be very large so please take this into account fia My Documents File Edit View Favorites Tools Help CE Li gt Bi j Search gt Folders Sy Music Roger MyExample Local Contrast Composite tif 14 419 KB Micro
9. see if he were to move the stage through the range of focus Procedure 1 Turn on all microscope and computer equipment 2 Start the Image Pro Plus software by clicking on the icon below in the lower left corner of the Windows screen Image Pro Plus 3 Start the INM Focus Software by clicking on the INM Focus exe icon also located in the lower left hand corner of the Windows screen INM Focus exe 4 Your computer screen should look something like this a 3 Sr TA Enhance Process Measure Macro Window Help bi i C R2 2 DO 682 38 ate gt i COHN Gk 4 Ba amp Cycle Focus ImageNo To jee Jes Pause 1000 ms Lst Capire E Loop mE i Image lt none gt System 10x um A start 6 aa how to make an ico file ra vicon ico The IES B Image Pro Plus 5 1 ir age Pro Plus W INM Focus Manual doc sl a 28 9 13 AM O IPWinS1 oe sktop INM Focus Ver 1 0 34 SU thursday 5 If the INM Focus software is communicating properly with the INM200 microscope there should be a number displayed in the CURRENT Z POSITION frame in the dialog box If this is not showing properly see the next step 6 If you do not see a position on the OPERATE tab in the CURRENT Z POSITION section and if pressing UPDATE does not produce an updated position then communications with the scope is not working properly Switch to the SETUP tab There are a number of settings which change the
10. soft Office Do MyExample tif 100 925 EB Microsoft Office Do 18 File Folder File Folder File Folder 7 20 2005 9 00 AM 3 3 2005 10 40 4M 8 12 2005 2 36 PM 8 18 2005 10 11 AM 8 18 2005 10 03 AM Note that the file named MyExample Local Contrast Composite tif contains the composite image of all the in focus regions of all the contributing images through the depth of focus The really large file named MyExample tif is a file containing each of the individual images making up the composite Conclusion This document has outlined the procedure required to utilize the Extended Depth of Focus feature which automatically acquires the composite images of a substrate having topology deeper than the depth of focus of the objective lens Note that the software cannot know the critical z stage position of a collision so care must be taken in setting the z stage limits 1 e carefully follow the procedures outlined in this document 19
11. us File Edit Acquire Sequence Enhance Process Measure Macro Window Help SHE MPSSHERILHOocez aea He toeHetrern bE oBAab Zhe amp w INM Focus Yer 1 0 34 IY Preview Image On Current Position 29218 338 um SS rea E Top Maximum Gel um GoTo Bottom Minimum lt Set um GoTo Thickness la Depth of Field l um Number of Images Cycle Focus Image Mo _GoTo 0460 Focus 100 Max 133 1 04 fps B Pause 1000 me Stat Capture Dust Loop While the software 1s communicating with the microscope there will be a YEILD symbol in the upper right corner of the INM FOCUS dialog box Please do not push any buttons while this symbol is being displayed Sending multiple commands to the microscope simultaneously could yield unexpected results When the Maximum Z value has been captured press the SET button to record the Current Z Position as the TOP MAXIMUM Z ox M Preview Image On Current Position Update 29218 338 Uni Top Maximum lt i 29218 338 um GoTo Bottom Minimum 1 Set um GoTo Thickness 29218 2338 um p of Images i 29219 Cycle Focus Image No 10 Change the focus on the scope so that the stage 1s at its minimum position highest point in the image update the current position with the UPDATE button then transfer that position to the BOTTOM MINIMUM Z section of the dialog box with
12. way that INM Focus interacts with the COMM Serial Port the operating system and Image Pro If changes are made to these settings press the STORE SETUP IN INM FOCUS INI button close the INM FOCUS software and restart it This allows the changes to be stored on disk and then used by the next run of the software a INM Focus Yer 1 0 34 Oo x Preview Image On Comm Serial Port Number Comm Serial Port Settings 3600 n 8 1 Baud Party Data Bits Stop Bits No spaces Keep INM Focus on top of all other windows I O NMo 1 es Path to IPCI2 DLL C MPWINET Normally CSIP YT ST Store setup in INK FOCUS INI Changes made here will not apply until the program is restarted The content of the INM FOCUS INI file that is stored by the settings shown above is shown in the following screen INM FOCUS ini Notepad File Edit Format View Help Comm Port Number 1 Settings Baud Parity Data Bits Stop Bits No spaces J 9600 0 8 1 IMNM FOCUS Top Most window Setting 0 Yes i Path to IPC32 DLL Normally CiMIPWINSIA CiMIPWINSIA File Edit View Favorites Tools Help sack ki Q 4 po Search Folders INM Focus Manual doc INM Focus exe INM FOCUS ini 7 When the INM FOCUS software is properly communicating with the microscope check the PREVIEW IMAGE ON checkbox and Image Pro Plus will turn on its PREVIEW so that you can set the focus exposure and fr
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