Basic AnalySIS tutorial
Contents
1. brightness and contrast slides to make background as bright as possible while losing as little as possible data from the image Useful values are between 60 70 for both brightness and contrast Functions like Square root and Hyperbola also works well in this image Try some of the functions while checking the results Click Change when done The result Hvperbola function Examples pore wa Tae tle st re oa te Logarthim function J Paaa will end up in selected destination image buffer As you can see these images have lost some info but background and More advanced users are advised to read the AnalySIS manual for additional tips 11 marked fibres have now been separated Consider for yourself whether the result is acceptable or not for the task you are trying to solve e Second attempt trying once again to improve image while losing as little data as possible Starting off with a slight adjustment in brightness and contrast approx 65 for each gives this image to work further with Some data is lost and the background can still give some problems The next part will therefore be to use filters to enhance the image The filters are available under the Oper menu Some filters have interactive features 1 e they allow the user to change their variables These filters also have a useful preview function The preset filters are located in Oper gt Filter the interactive filters are found in2. Find overlay in the button bar list and select it Note when you exit AnalySIS after the session choose yes when it asks you to save the configuration E Fa b L E R a La Cs EA Part 2 Overlay bar icons at a glance l 16 17 18 Part 3 Arrange object order bring to front back etc Text Edit overlay L h Edit the overlays Select none Deselects currently selected object s If no objects are selected it changes function to select all Object properties Opens a dialog box which enables you to change object properties text size line weight colour etc Layer Changes which layers that are visible image overlay both etc Burn overlay i Burns overlays into the image merges the original image and the overlay to a bitmap i e it will no longer be possible to edit the overlays but it will be possible to share the images in your database including overlays with people who don t have access to AnalySIS Delete layers e Deletes selected objects Load objects i Loads previously saved objects Save object 5i Saves objects for later use Cut copy and paste objects d J saci abl Creates a text overlay Rectangle Draws a rectangle Ellipse Draws an ellipse Line 5 Draws a line Arrow b Draws an arrow Use the Object properties button to change the direction of the arrow Polyline
3. Oper gt Define filters Try some of the filters to find one that separates the background as much as possible from the marked fibres Useful ones are Edge enhance and DCE 2 Second step Thresholds Now the grey values in the background should differ enough from the marked fibres to separate fibres and background Go to Image menu gt Set threshold to set grey values that separate background and fibres This opens up a window Choose the Manual tab then press Delete until it turns grey AnalySIS window should now look something like this re a re 7 ee wr h p i eS v a ig a E Pe ae es lt mi y gt R Poe 5 n s j a Sy vr T ee gt gt x 2 n pe i m om gt te s pe mer Qa tall 4 P s en et OD r ha et a d 5 D a fa 7 Ser an 2 PS sy p E 3 i as 7 A Phase is a range of grey values You can create as many ranges as you want by pressing the new button It is however a good idea to keep the number of phases as The Preview radio buttons allows small as possible you to change what area AnalySIS colours artificially Manual Auto Settings m Preview Phase Cluster v Low 60 C None Color m High 1354 2 C Curent RRE ic Include Pixel C Background File Z Transparent Diagram e Using thresholds Chose All in Preview subwindow Then change the high and low values of the sel
4. want to collect and display in your database Database menu gt Arrange fields Generally this dialog box allows you to choose what data to collect and display Use Add gt gt and lt lt Remove to choose fields Use the up and down arrows to arrange the order of selected fields Insert tab Here you select what info you want to enter manually into the database and it s order Info window tab The info window is a small Post it note Max 7 fields of data Form View tab The form view displays a single image with a list of fields from the database Table view tab The table view gives you a list of database fields without images in a table Print tab what to print when using the print database function File menu gt print choose database in the page layout field Query by example field A list fields for searching the database The database setup should now be complete Hit Ok to finish You can now add records to the database Part 3 Add view edit search records l Add a record e From image buffer simply drag an image from the buffer to the database window A dialog box will open and prompt you to fill out the fields you previously selected Insert tab under arrange fields e From a different database Drag records from one database to the other Unless the fields in the two databases match you will have to fill out the fields required for the target database e From a file Rig
5. 2 Draws a polyline Polygon S Draws a polygon Highlighter Highlights parts of an image 1 e draws a rectangle with a transparent colour Also useful to invert colours in an image Editing a The drawing tools ee See e Rectangle and ellipse said Click on the shape you want to create the mouse pointer will then be moved to the image Use the mouse to place the object then hold down the left mouse button to change object size Use Object properties to change line weight colour and fill colour Line tools os Click on the shape you want to create the mouse pointer will then be moved to the image Place mouse where you want the line to start left click and move the mouse to the ending point then left click again Right click to end a polyline Use Object properties to change line weight and colour and optionally arrow direction Polygon Click on the shape you want to create the mouse pointer will then be moved to the image Place mouse where you want the object to start move mouse to next position left click again Right click to finish The software will then draw a line connecting start and endpoint Use Object properties to change line weight colour and fill colour 2 Text 2 Creates a text overlay Usage is quite clumsy When you click on the text tool it opens a red rectangle in the image This rectangle is your text box Move mouse around to place the rectangl
6. AnalySIS Tutorial part 1 Written by Daniel yan Thanks to Sveinung Lillehaug and Jan Byjalie for input AnalySIS basics Click on the table of contents to jump to selected page Laporan Noles cscs ctoseriact cceutas chaste as seaeeds E E osmeeeesensaen toes l TATPOGUCTION TO ANALY SIS O eaa a ai 2 Analys Sse Mera e at GO aCe oa nr A A 2 Getting around the Interi aCE oiio trir Eir EE ON 2 Using AnalySIS to acquire an image from the MICrOSCOPE cceeeeeeeeeeeeeeeees 4 Part 1 Calibration of microscope Koehler ilumination cc eeeeceeceeeeeeeees 4 Part 2 Camera calibration and image ACQUISITION csseeeesseeeeeececeeeeeeeeeaaees 4 IMU ple 1a Sa CMI IN ois en te tiinne ts wan teehehts a a Bands ween a saeco 5 Part 1 Acquire an image series without using a motorized table 5 Part 2 Acquisition using a motorized table cssseseeseeseeeeeeeceeeeeeeeeeeeaeaaaes 5 Parto Aen acquired tia gOS nena tls eaeaeteaeeee ees 6 Add text arrows and lines Overlay ccccccccccssssssssssssssssseeeeeccccceeeeeessaaaeeeasssseeees 7 Part 1 Possible necessary configuration 0 0 cccccsseseeseeeeeeeeeeeeeeeeeseeeeeeeaeaaaas 7 Part 2 Overlay Daricons alao lahte ouen a N 7 Pareo E MUTI annn E TE ee E E E ET di Creamin ANG USING a Cala DASS neina ae AS 9 Patt 1 Create database and define ficldSrenniniesine t a eee 9 Part 2 Arrange fields See part 3 for usage 0000000oooonennnnennnsnssssssssssssee
7. awing overlay button bar to remove the pixel value numbers and crosses If you don t these may create troubles later on 5 Enhance image to strengthen signal select relevant threshold values then binarize the image Particles should now be white on a black background 6 Add a grid to separate particles then burn the overlay into the image 7 Finally detect particles Take a look at the result if necessary use classification criterions to improve results and measurement options Analysis gt Define measurements to exclude artefacts and add useful measurements such as coordinates size shape etc 8 Select Particle results in the Analysis menu to get a result sheet No conversion filters here so save this too as in Excel and SIS formats Notes This pilot had the advantage of previously plotted data and this was useful for aligning data and comparing manual and automatic approaches Some ideas to improve results 1 Acquisition Use the Manually adjust fixed scaling option ath to improve contrast See manual for details of this tool 2 Thresholds and ROIs This pilot tried to acquire data from two ROIs at once marked areas of the pontine nuclei and peduncle As long as the various ROIs are relatively similar when it comes to data and colour they can use the same threshold and be analysed together In this pilot I tried to analyse the pontine nuclei and the peduncle at the same time This is not ideal as the unmark
8. ck to see that 75 of visible aperture is filled with light BENEFITS e Evenly illuminated image e Brilliant image without reflection or glare e Minimum heating of specimen iS ee Part 2 Camera calibration and image acquisition 1 After the microscope has been properly calibrated select an empty image buffer and open this image preferably on the 2 screen if the computer is a dualscreen system Then press the Livecam Acquire button T which will give live images in the selected viewport Refocus microscope for optimum screen sharpness When done press the snapshot button to t save an image to the image buffer e Note when you press the livecam snapshot buttons a dialog box may prompt you to select magnification 2 Other camera controls e Select camera Allows you to choose between several different inputs Useful if the computer is connected to different cameras Also allows you to change camera setup add a new camera e Camera control ee Opens a dialog box that allows you to change the cameras exposure time colour etc You also have access to a sharpen button which enhances image contrast and detail during live acquisition Multiple image alignment Part 1 l Part 2 Acquire an image series without using a motorized table Define image series gt How many images do you need Image menu gt Image series gt Define Select a folder to save the images in then fill in fields in
9. e While keeping the left mouse button down you can move the mouse to change rectangle size When text rectangle size and placement is correct right click Right clicking will bring up the object properties dialog box Enter your text then use the options under Font and Colors and Lines to change font size font colour background colour etc Click Apply to preview then change values if necessary Click Ok Close when satisfied 3 Edit overlay h When you click this icon the mouse pointer is transported to the image To change size and placement of an object click on the object To change the objects properties double click on the object As long as this tool is active it is not possible to move the mouse pointer outside the image To deselect this tool right click To undo actions press Esc on keyboard before you right click Object properties Opens a dialog box which enables you to change object properties text size line weight colour etc Creating and using a database Why create a database There are several reasons to create a database It helps organizing images it also allows you to search a set of images according to various search criteria etc Part 1 l 2 Part 2 Create database and define fields New database Database menu gt New Enter database name and if necessary where you want to save the database and files Define fields AnalySIS will automatically add technical info about the i
10. ected phase either by using the red and blue lines To use this function press the Window button in the Preview subwindow then select a part of the image you are working on Useful values bandwidth around 20 enhancement around 65 quality on 12 in the histogram subwindow or by entering values in the high and low fields Try to select high and low values so that the phase colour covers the parts of the image that you are interested in while covering as little as possible of the surroundings Try changing between A and Background in the Preview field to select ideal threshold values Click Ok when done This will cause the artificial colours to disappear Set the destination image buffer to an empty buffer then binarize the image Oper menu gt Binarize This will create an image that only contains two colours black and white and this is the ideal image for the software to analyse It should look something like this Analysing Vocabulary and tools 1 AnalySIS analyses an image by looking for particles A particle can be virtually anything a red blood cell a neuron or whatever else you want the program to detect in an image It s your job to specify what the particle looks like Part of your job involves separating the background from the particles by using the techniques described earlier Other parts of the separating job will be described later 2 The tools e Define classification It is not necessary to use
11. ed area in the pontine nuclei is grey while the peduncle background is nearly white The fibres in the peduncle were generally thin while the marked fibres in the pontine nuclei were clustered Clusters tend to be black while fibres often end up dark grey Attempting to get data from both areas at once resulted in artefacts in the pontine nuclei as 16 the background here had the same grey value as single marked fibres in the peduncle
12. erreeeeees 9 Parto Add view edit SCALCIN TEC OLS asoa a eia 10 Usine themace analysis 1eatures so 350d aesieveedawveboneat uae AT darabemesensiane 11 Image enhancement tools Improve signal strength and reduce noise 11 Analysing Vocabulary and tools seinir i EE E E 13 Parnclendivistom toO Sereen whist anata ee eas 15 Wsmme results tn MICO Digre nee n E A tees 16 Example 1C MSi onien a o a Cen ae 16 Important notes l Zi Some notation menus are underlined dialog box options in italics The undo function doesn t work very well remember to protect important images in the image buffer to prevent deletion right click on the image in the buffer and select protect image a padlock icon should appear It s also a good idea to check destination buffer and to save regularly to prevent data loss Introduction to AnalySIS AnalySIS interface at a glance early STs P a ih ile Cdt Database image Oper M me Anas C Module Spada window 7 Qe HAG OSM DO amp oi WM eo gu I MEOW ae E O Mm 4 Te st rl et BSR BBR HOSCN ws HE SZ 3 y E Ri imaga 18 Cytoplasma 32 Sal Screen tools 14 gt d 20 mipi 2i w Image 21 Image a2 pa image 3 buffers l 2D m Database window 25 peee w database toolbar a fa wlan 27 i oe Pal wa oe Image 3 vas Ea DE fo Gag S a Rect G amp iaze tee mady ae Ecen are mag o gt N S
13. ext in all fields then click search A e Sort database Click 24 in the database window toolbar and select the sort order you want 10 Using the image analysis features Time to start image analysis The first parts of this chapter will teach you some of the used tools to analyse the image starting from an acquired image The rest will list examples of previous current project pilots and show how AnalySIS was used to solve various tasks Note spend some time getting used to image acquisition before you start this chapter Final results depend very much on the image quality of the source image Images shown in this tutorial are far from perfect and this will lead to artefacts during image preparation and analysis Image enhancement tools Improve signal strength and reduce noise Histological material from Rat somatosensory cerebropontocerebral pathways by Trygve B Leergaard et al 2000 Ca a Ti doai ai k PE Ba ann ee a ee ee EE L A selection of the starting image shows part of pons and peduncle The dark cluster to the right is a group of fibres labelled with the antegrad tracer BDA 1 First step improve quality e First attempt As the background and the marked fibres have different grey values grey gradients in the background vs black and near black marked fibres the simples way to differentiate is to increase brightness and contrast Oper menu gt Intensity gt Modify grey values Use the
14. ful one Particle filter set minimum size to remove small artifacts Pixel connectivity Describes how pixels are connected in order to consider them a particle Consider the following three pixels ME Adjacent borders option will describe this as two particles one that is 2x1 pixels large the second 1x1 pixels The Include diagonals option will consider this a single particle however as they are connected diagonally Finally Define detection I dialog box has three tabs Detection tab Define Detection 1 Detection Classification Results Particle filter Fill holes MW Use ranges In range Total count Fisel conmectivity Measurements Minimum 3 Fisel 0 Adjacent borders 4 Include diagonals 8 File Border particles f Truncate C Include C Include 50 Exclude Search area 0 Frame C Als C Mask Execute Draw Particles Delete All Load and save buttons It s a good idea to save the ROI when done as this allows you to use the same ROI in different images and to use the ROI the next time you start analysis Analyse menu gt Define detection This Border particles How are particles located at the border of the search area treated Truncate Particles are counted but only the part inside the search area Area size and shape are thus reduced Include All particles at the border are
15. ht click in the database window and chose Insert Document File from the pop up menu chose the image files you want to add then fill out the fields View record e Post it info select the image whose info you want to view then press Alt Enter on the keyboard To make the info stay visible use the button in the post it window e Other views Icons from database window toolbar 1 Thumbnail view 2 Gives a thumbnail list of images in the database the only additional info shown is the Record name 2 Form view gt displays a single image with a list of fields from the database 3 Table view a list of database fields without images ordered in a table Edit record e Select the image whose record you wish to change then press Enter on keyboard or use the Edit record icon in the database window toolbar Search database Database menu Query There are several ways to search the database the simplest is to use the Query by example option It is also possible to search database by SQL or Free filter e Query by example right click in the database window or use the query button in the toolbar i fill out fields to match your search criteria Then click search Data matching your criteria should now be displayed e Free filter SQL Consult the AnalySIS manual pages 173 and 177 if you want to use these query options e To restore the full list of images in the database Query again and remove t
16. included their shape and size left unchanged Include 50 particles on the top and left are counted bottom and right are excluded Exclude excludes border particles Limits the search area Frame limits search to the area inside the red and white frame ROIs limits search to ROIs etc 14 Allows you to label Measurement label particles over a minimum pixel size Click this button to Color a F Hame select particle measurements Gives you the ability to exclude particles depending on their shape size etc See manual for details Classification criterion Area size x y coordinate etc How are particles shown Filled outline only labelled Classification tab Define Detection Detection Classification Results Classify Criterion Ho classification Classification Particle outline Style Filled Type IF area larger thar None fag Pixel I Zoom text with image M Unit Measurements Execute Cancel Help Results tab has some options on how results are described This is just a rough outline of AnalySIS particle detection function Consult Example 2 for an example of usage and consult the user manual for detailed information Particle division tools Divide particles Sometimes particles are clustered and this makes it difficult for the program to differentiate between particles To separate cl
17. ke personalizing your version Just remember to save the old configuration before you do any changes and then save the new configuration under a new filename afterwards The save load reset configurations menu is found here Special gt Configuration e Window standard window tools change window remove tool windows etc e Why they use instead of writing Help is beyond me Anyway you ll find some help files As analysis 1s a piece of software that 1s under development the help files are far from complete though You can also access updated manuals from the web with this menu Menu gt SIS on the web gt Product news This will open a web browser Select the analySIS documentation download page link then software version 3 Toolbars Some of these are standard but several are AnalySIS specific To get a glimpse of their function let the mouse hover over the icon for a few seconds For more info on what they do read the manual 4 Status bar Shows currently selected input i e camera some info about currently selected tool etc 5 The image buffer Images are put in buffers while you use them Shows current buffer source destination buffer i e the buffer in FAET a E where the processed image will end up secondary source and A A TE mask Read the manual for in depth info no how to use these functions Catoplasma m Woki ia ae Image buffers The folder shows buffer number the co
18. ltiple image alignment gt Arrange Image tiles Select the number of horizontal and vertical tiles you recorded Arrangement Use these options to arrange the images Correlation How well does the image tiles correlate Useful values 0 8 1 0 If necessary it is possible to change arrangement manually Hit Ok and wait for the software to align images e Short description on the various types of arrangement Combing Vertical Combing horizontal Menader Vertical Bo Meander Horizontal Tje Next dialog box Check if the software has managed to align the images manual corrections are possible by dragging images to desired position If manual corrections are necessary the original size checkbox is useful The Overlap area option allows you to chose how the program mages overlaps from one sub image to another side by side gives a sharp line between the images if they don t match well the other options tries to blend the images Hit ok the image 1s now ready e Note If the image sequence has a staircase look i e the images in a horizontal sequence are not aligned vertically it will look something like this CL If so the camera is not properly aligned with the microscope gt manual realigning of camera microscope angle is necessary Add text arrows and lines Overlay Part 1 Possible necessary configuration 1 The overiay l bar ao ometre rs ii If it bar isn t t available a menu Edit baoi bars
19. mages you put into the database Other info must be defined first Database menu gt Define fields Hit the New button to add a new field e g staining method specimen etc and fill out the Current field field From the dropdown list beneath Data type select the type of info e g Text a short text Long amp Double numbers memo text of any length Use the required checkbox for mandatory fields It might be useful to simplify data entry by making a list the user can choose from when entering new data If you want this functionality hit Edit in the picklist subwindow and enter the values a user may select from Hit Add after each value entered Hit Ok when finished After this is done use the options in the picklist subwindow to choose how you want the picklist to work Expand picklist on input is generally the best choice When you have defined all the fields you need hit Ok A dialog box will appear and ask if you want to arrange fields now Choose yes and continue to next part Notes e Remember to hit New before you define a new field If you receive weird error messages like Item not found in this collection there is something wrong with the user defined fields e Ifyou need to add fields to the database and the new button is greyed out close the database open it again Database menu gt Open and select Exclusive in the dialog window Arrange fields see part 3 for usage To start arranging the info you
20. mputer screen symbolizes image type true color grayscale etc The text shows buffer name and image resolution Divide screen into subscreens Image window Shows the current buffer s Display properties TA Note the thin red frame around the image You i i can reduce the size of this frame with the ee mouse and use the frame for zooming Display config s a Toggle navigator These tabs allow you to change currently active screen if you are using a dual screen system Using AnalySIS to acquire an image from the microscope Part 1 Calibration of microscope Koehler illumination Courtesy of Carl Zeiss www zeiss com micro For optimum results in light microscopy precise control of the light path should start before the light reaches the specimen Prof A Koehler of Carl Zeiss was the first to apply exact control of the light path in the illuminating beam a method known as Koehler illumination Equipment specifications The microscope must have a vertically adjustable centerable condenser and iris diaphragm Procedure 1 Rack up condenser with top lens swung in Focus on specimen with 10X objective Close down lamp field stop diaphragm in base while viewing Lower condenser slightly until diaphragm image is in focus Center image using condenser centering screws Open diaphragm to edge of field fine focus and open further to just clear field Adjust contrast using condenser diaphragm Remove eyepiece and che
21. o an hourglass within a couple of seconds and the stage is ready to use 1f not click cancel and check the following e Is the stage controller turned on There is a green light on the front that lights up if the equipment is on If not turn it on the on off button is conveniently hidden on the backside e Check the position of the front switch it should be A Auto e Check position of dipswitches on the back of the stage controller set both in the lower position e Check connections is the right cable connected to the stage e Try the procedure again if it still doesn t work turn off stage and reboot the PC Start AnalySIS turn on the stage and try the procedure again Click the Limits button to set stage limits Image menu gt Multiple image alignment gt Acquire This opens up a dialog where you set the number images you want to acquire Use the joystick to move the specimen it s position should be roughly in the centre of the area Number of images amp Number acquisition Unless you know what you are doing use identical values Useful if using a computer controlled tablet Part 3 you want to acquire Click Acquire to start acquisition and then click Align to start image alignment You can skip the first paragraph of part 3 if you use this method Align acquired images The images should now be in the image buffer Left click on the start image in the buffer then Image menu gt Mu
22. overlay from the overlay button bar 15 Using results in Micros D Create outlines Creating outlines of various anatomical features helps aligning the results in Micro3D If you want to create an outline select pixel value from the Measure menu and set points to mark necessary features This will result in a sheet with X and Y coordinates colour greyscale information Currently there is no easy way to convert this sheet to something Micro3D can read so save the table as both Excel xls and SIS sheet sfs Example 1 Cluster A pilot using old histological material from Rat somatosensory cerebroponto cerebral pathways by Trygve B Leergaard et al 2000 Fibres in the pontine nuclei were labelled with the antegrad tracer BDA The first problem Differentiate labelled fibres from the background The second problem Divide the resulting particles allowing density and population analysis using micro3D Having show some of the steps from acquisition to analysed image and the necessary tools it s time to get on with the real job of image analysis 1 Acquire an image image series 2 If you want to create an outline of various anatomical features to help aligning later on it s a good idea to do it now It s also a good idea to save your image either as a single file or in a database 3 Select your Regions of Interest ROI and save these 4 If you have saved your image select Delete overlay found in the dr
23. tatus bar Currently selected Input Getting around the interface l While parts of AnalySIS functions like standard windows programs the overall interface is different and complex for a newcomer We ll start with the parts of the interface that resembles a standard windows interface A bit about the menus contents hidden inside them etc File contains some standard file tools here in addition options to send images databases etc via email and create reports Edit standard editing tools including a dysfunctional undo function Also some tools to edit sheets and diagrams add bookmarks some search replace functions Database Open create edit query database Image Acquire copy delete protect align image Also tools to configure camera Additional tools for staging fluorescence etc may be added Oper Loads of tools to enhance the image change brightness contrast add various filters change image bit depth separate colours edit image etc Measure measuring tools horizontal vertical arbitrary length angles area pixel value histogram etc Analysis Image analysis tools C module add remove create modules Pretty much an experts only menu e Special Contains all the setup preferences menus you need to streamline analysis for your needs record edit macros add remove modules edit create menus button bars etc Very useful menu to return to when you get the hang of the program and feel li
24. the right side of the window e Begin at series image What is the number of first image in this series Set to 1 if it is the first image of a series 1f you are continuing an image series set to the appropriate value e Number of images how many images are stored when series are saved e Number acquisition of images acquired e Delay acquisition Time between each acquisition Ready to acquire Image menu gt Image series gt Acquire The camera will now acquire the images Depending of camera setup additional dialog boxes may appear e Remember to calibrate microscope and camera before you start acquiring an image series as there is no elegant way to calibrate the camera during acquisition e When using a non motorized table must be moved manually and it is necessary to perform a first pass aligning manually 1 e look in the microscope and at the screen and move the tablet so that the sequence overlap each other partially Load images into buffer Image menu gt Image series gt Load Select the folder you chose in step 1 images will now be loaded into the image buffer Acquisition using a motorized table The first and in many ways most difficult part is to initialise the equipment Special menu gt Preferences select the stage tab Choose your stage from the list click the Properties button A new window opens click the Search button and cross your fingers With some luck the pointer changes int
25. this tool It will however often improve the final result as it enables you to differentiate detected particles according to size shape etc The tool is located here Measure menu Define classification Define Classification m Classificati Uri r Gee Name of new classification assittcatiors rit ew classihicatiors Currently selected Ll F Enter a name and press New classification Hew j Delete A table where you enter sizes or other Unit values to Seb colar differentiate the Compute particles Click here and enter classification unit e g um if you are going to differentiate particles after their area Sheet Show sample objects Help ud gl e Set range of interest ROI Again a tool that isn t really necessary but it is nonetheless useful as it enables you to divide particles into various groups depending on their location e g create groups as pontine nuclei trigeminus nuclei etc It can also limit the area where AnalySIS will search for particles The tool is located here Analysis menu gt Define 13 Name of current ROI To start using a ROI enter a name here then choose a drawing tool This will cause this box to disappear Draw your ROI rightclick when done This box will then reappear Click close when done ROIs Define ROIs Label CeSP42Pons Aol Active AUls The drawing tools The upper left tool is the most use
26. usters there are several tools Dividing large particles marked neurons etc I advise trying the morphological filters first The manual gives in depth information concerning the various filters and how they can be defined to enhance their functionality further This is beyond the scope of this manual so I ll only briefly discuss the filters used so far The filters are here Oper gt Morphological filter 1 Separate particles Separates connecting particles in a binary image This is the primary particle separation filter Try this first and check the results If this filter creates too many particles try using morph open and morph close first They will smooth the structures and give better results If the filter creates too few particles you can try a grid 2 Morph open Erosion gt Dilation removes noise smoothes edges some particle separation 3 Morph close Dilation gt Erosion Fills holes and separates particles Dividing small clustered particles entwined fibres etc The morphological filters are not ideal tools to separate a tight cluster of small particles and a different approach can be used a grid To use a grid select Grid from the Measure menu and enter a grid size To break up a cluster of several small particles use the smallest possible grid size AnalySIS will then add the grid as an overlay but this wont be visible for the particle detection tools To make it visible for these tools select burn
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