REGENT INSTRUMENTS INC. wwrw.regentinstruments.com tinstruments.comABOUT THIS MANUAL Menu commands are described as Menu name/Command name. For e: command of the “Image” menu is written Image /Save image mple, the “Save image FOR TECHNICAL SUPPORT Technical support is available exclusively by e-mail (we answer rapidly). Ifyou need help to install or use this product, send an e-mail to the tech sup. email address below. We answer within one hour when received on the working hours of North America eastern time. If it takes longer it can be because of a national holiday or problems with the Internet network. If the message bounces back to you, contact our sales department to verify if the technical support address has changed. Do not forget to identify yourself, the product you need assistance with (Ex: WinCELL Pro 2007a) and to give your customer number written on the protection key, invoice and this manual. This number is very important. It allows us to answer more precisely and rapidly to your request (it tell us who purchased the system and which components you have and since when). Please give your message a pertinent subject (such as “WinCELL question”) to help us differentiate it from unsolicited spam or The technical support email address changes every year and has the year number in it (which is the only part which changes over time). The email address is made of the word “tech” followed by the year (such as “2007") and the domain name “@regentinstruments.com*. As example, send technical emails to: tech2007@regentinstruments.com if you send your message in year 2007 or to: tech2008@regentinstruments.com if you send your message in year 2008 (and so on), For help, see also the FAQ section at the end of this manual and the following web pages: www.regentinstruments.com/pages/TechSupport FAQS, html for the late jon of the most Frequently Asked Questions and www-regentinstruments,com/products/cell/CellMostRecent.html for the latest information about WinCELL’s most recent version (features, bugs and shipping date FOR SALES OR UPGRADE INFORMATION sales@regentinstruments.com THANKS ‘To Dr. Jean Poliquin, Wood Science Dept., Laval University, Quebec Qe., Canada whos was essential to th development of this product in the early stages. ‘To Annie Deslauriers, Fundamental Sciences Dept., Université du Québec a Chicoutimi, Chicoutimi Qe., Canada for guiding us during the development of path measurements, To Dr. Alain Cloutier, Wood Science Dept., Laval University, Quebee Qe., Canada for providing us with some images used in this manual To Jacques Tardif, Centre for Forest Interdisciplinary Research (C-FIR), University of Winnipeg, Winnipeg, Manitoba, for help in testing WinCELL 2004, Canada for providing us with some images used in this manual an1.0 20 3.0 34 3.2 33 34 35 40 4at 42 43 44 45 CONTENTS INSTALLATION PACKING LIST AND IMPORTANT NOTICES PROTECTION KEY WINCELL AND THE DEMONSTRATION IMAGES INTRODUCTION WINCELL PRESENTATION THE MAIN WINDOW THE GRAPHIC AREA ANALYSING A DEMONSTRATION IMAGE 2.4.1. IMAGE ACQUISITION 2.4.2 SAMPLE IDENTIFICATION 2.4.3 CHOOSE THE REGION TO ANALYSE 2.4.4 THE ANALYSIS 2.4.5 THE ANALYSIS DATA 2.4.5.1 DATA IN THE COMMAND AREA 2.4.5.2 WHERE THE ANALYSIS DATA ARE STORED AND WHEN 2.4.5.3 YOU CAN CHOOSE WHICH DATA ARE SAVED IN DATA FILES 2.4.5.4 DATA VISUALIZATION AND ANALYSIS 24.6 TO ANALYSE ANOTHER IMAGE MORE ABOUT THE ANALYSIS IMAGES USED FOR AN ANALYSIS WINCELL'S WORKING MODES 3.2.1 THE ANALYSIS WORKING MODE 3.2.2. THE INTERACTIVE MEASUREMENTS WORKING MODE 3.2.3. THE IMAGE EDITION WORKING MODE ANALYSED AND EXCLUSION REGIONS 3.3.1 RECTANGULAR IMAGE REGION 3.3.2 CIRCULAR IMAGE REGION 3.3.3 FREE STYLE IMAGE REGION 4 REGION RESIZING 3.3.5. REGION MOVING THE ANALYSES 9.4.1 GLOBAL ANALYSIS AND GLOBAL COLOR ANAL YSIS. 3.4.2 INDIVIDUAL ANALYSIS AND INDIVIDUAL COLOR ANALYSIS: A THIRD CELL TYPE, PARENCHYMA 3.5.1 CELLS GROUPING 3.52 CELLS SPLITTING 3.5.3 ANALYSED CELLS AND REGIONS' BOUNDARIES INTERACTIVE ANALYSES (PATHS) MANUAL LENGTH PATHS MANUAL LENGTH & WIDTH PATHS INTERSECT PATHS TOUCH/CENTRE PATHS (FOR WOOD CELL ANALYS YEAR BEGINNING AND ENDING5.0 COLOR PRI TO 6.1 CAMERA INSTALLATION ON THE MICROS\ 62 1E-REQUI GREY LEVELS VS COLOR ANALYSIS ANALYSING THE ANALY’ \SE OF COLOR ANALYSIS TE FOR COLOR ANALYSIS COLOR DEMO IMAGES R CLASSES AND GROUPS CREATE CC ACQUIRING YOUR OWN IMAGES ;HTING AND FILTERING SYSTEM 6.3 SELECT THE IMAGE ORIGIN AND TWAIN SOURCE 64 TO ACQUIRE IMA FROM A SCANNER 0 REFERENCE 7.1. THE MISC MENU 714 12 13 14 15 7.1.6 747 LOAD CONFIGURATION SAVE CONFIGURATION PRINT GRAPHIC PRINT IMAGE AREA PRINT WHOLE IMAGE PRINTER SET UP EXIT 7.2 _THE DATA MENU 72.2 72.3 72.4 73.2 7. 3.4 7.3.5 7.3.6 37 7. 74 THE NEW FILE OPEN FILE CLOSE FILE DATA SAVE OPTIONS THE IMAGE MENU ORIGIN ACQUISITION PARAMETERS 3.2.1 REGENT’S SCANNER INTERFACE ACQUIRE IMAGE SAVE DISPLAYED IMAGE ‘SAVE ORIGINAL IMAGE WITH ANALYSIS SAVE IMAGE REGION BACKGROUND UNEVENNESS COMPENSATION 3.7.1. METHOD PARAMETERS 3.7.2 LOAD REFERENCE IMAGE 3.7.3 CLEAR REFERENCE IMAGE 3.7.4 DO COMPENSATION NOW DISPLAY MENU INFORMATION COLORS IMAGE AREA COMMAND AREA UNDO EDITION CHANNEL 7.5 THE ANALYSIS MENU CANCEL ANALYSIS RESTART ANALYSIS DUPLICATE LAST REGION EDIT SAMPLE IDENTIFICATION PREFERENCES PARAMETERS FILTERS 58PIXELS CLA 7.5.8.1 HOW THRI 75.8.2 ADJU: D MANUALLY 7.5.8.3 ADJUSTING THE PIXELS CLASSIFICATION FOR A PART OF THE 7.5.9 DELETE CELLS/VESSELS GROUPING 7.5. 10DELETE CELLS/VESSELS SPLITTING HE EXCLUSION MENU 1 DEFINE REGION 7.6.2 DELETE ACTIVE REGION 6.3 DELETE ALL REGIONS 4 EXPORT REGIONS IMPORT REGIONS 7.7 THE PATHS MENU 7.1. PATH CREATION PARAMETERS 7.2 DELETE ACTIVE PATH DELETE ALL PATHS RECREATE LAST DELETED PATH DELETE SEGMENT CONTINUE ACTIVE PATH TERMINATE PATH 7.8 THE COLOR MENU 7.8.1 NEW CLASS 7.8.2 EDIT CLASSES 7.8.2.1 TO CHANGE THE COLOR OF A GROUP FOR VISUALIZATION 7.8.3 LOAD CLASSES 7.8.4 SAVE CLASSES 7.8.5 DISPLAY GROUPS 7.9 THE BATCH MENU 7.9.1 ACQUISITION PARAMETERS 7.9.2 START ACQUISITION 7.9.3 ANALYSIS PARAMETERS 7.9.4. START ANALYSIS 7.10 THE CALIBRATION MENU 7.10.1 METHOD 7.10.1.1 THE OBJECT OF KNOWN DIMENSIONS CALIBRATION METHOD 7.10.2 NEW CALIBRATION (WITHOUT REGENT'S TARGET) 7.10.3 NEW CALIBRATION (WITH REGENT'S TARGET) 7.10.4LOAD CALIBRATION 7.10.5 SAVE CALIBRATION 7.10.6 CANCEL CALIBRATION 7.10.7 VIEW CALIBRATION 7.10.8 DO WHITE BALANCE 7.11, THE WINDOWS MENU 7.11.1 CASCADE 7.11,2TILE 7.11.3NEW 12. THE HELP MENU 7.12.1 ABOUT WINCELL T APPENDIX A: WINCELL DATA FILE FORMAT APPENDIX B: FAQS AND ERROR MESSAGES APPENDIX C: COMMANDS SUMMARY 85 927 INSTALLATION‘The WinCELL program comprises the following items; 1) one or more color manuals, 2) a CD-ROM (in a pocket at the end of this manual) containing the program, the demonstration images and the protection key drivers, 3) registration information and last minute information on isolated sheets, and 4) very important, a protection key (required to run the WinCELL program IMPORTANT: Do not lose the protection key, it is worth the same as a second license Regent Instruments programs are not copy protected, but are execution controlled by the proteetion key: Copies of the program can be made for your internal use, but only one cop allowed to run at any given time. During its execution, the program looks for the prote ch key has a sticker on it indicat war customer identification number (itis very important to give this number to and, if none is found, it aborts which software model it activates and our technical support staff when you contact them), ou you received a calibration file called on the WinCELL CD. Thi file is unique to your scanner and increases the measurement precision (see “THE SCANNER.CAL FILE” on page 9 This file will not be sent with WinCELL's updates so keep this disk in a safe plac VERY IMPORTANT: Do not lose the protection key. Replacing a lost key costs the same amount as purchasing a second license. PROTECTION KEY ‘The protection key must be connected to a computer USB port or, for older keys, to a parallel (printer) port. The installation procedure is given below. If, The beige at image acquisition (when you click the Acquisition Icon or activate Im: protection Acquire Image), you get the following error message: “ “ ia ”, that means WinCELL cannot locate the protection key required for its proper functioning. The key must be connected The USB to the computer on which WinCELL is running. If, after its installation you **¥ CE still get the above error message, refer to Appendix B (or our internet site) for potential causes of the problem and suggestions to remedy th Do not connect a scanner to the key. Parl you experience any problems w key, try removing any external peripheral like printers) connected toi. You must install the protection key driver for the key to be effective. There is a single driver for all Windows operating systems (98, ME, 2000, XP and Vista 32 and 64 bits) and for all Regent’s pro} with an installation instructions file (Protection KeyDriverInfo pdf). This driver was the latest when your WinRHIZO was shipped. If you later reinstall the program and have problems with the key, it is worth downloading the latest drivers from the key manufacturer web site at www.safenet-inc.com support/index.asp (the protection key model is Sentinel SuperProWinCELL comes on a CD-ROM labelled with the product name and its version number (WinCELL 2007a for example). On the CD, there are two folders. One bears the product name (WinCELL Pro 2007a) and contains the program (WinCELL.exe), its calibration file (Scannercal, if you bought a scanner from Regent) and its demonstration images. Copying this folder to your hard disk is all that is required to install our programs for the first time. If you are installing an update, you must also install the scannercal file (if you bought a scanner from us) a explained in next section. The second folder contains the drivers for the protection key (see previous section 1.4 THE s If you purchased a scanner from Regent Instruments, you also received a scanner calibration file on the WinCELL CD that came with the scanner. This file is unique to your scanner and will not be sent again with WinCELL's updates. The scanner calibration file is named Scannercal and is in the folder that contains the file WinCELL.exe. If you are installing an update or an upgrade, you should copy the Scanner-cal file into WinCELL’s folder. When the file is properly installed and recognized by WinCELL, its name is written in the Information area above the image (see “” on page 13) and in Calibration / Method. Each scanner has a unique Scanner.cal file. The file can be copied into many of Regent's program folders (WinRHIZO, WinFOLIA...). ‘The Scanner.cal calibration file allows WinCELL to compensate for differences in the dpi reported by the scanner and the real dpi measured with a high-precision calibration target. It is used only with the Intrinsic calibration method (see “THE CALIBRATION MENU” on page 78 If you analyse images with a scanner not calibrated by Regent Instruments, you shot Seannercal file from the WinCELL directory. remove the 5 NEXT Install the camera or scanner and its programs if you have purchased such components, The TWAIN driver is the only software that is required by WinCELL to acquire images directly from a camera or scanner, See their manufacturer documentation for installation instructions. If you purchased these components from Regent Instruments see also the additional manual that we provide (ex: The 5MP Camera Installation And Utilisation With Regent’s Products) You can now run WinCELL and try to analyse the demot that you try the We strongly recommend tration imag lemonstration images before acquiring and analysing your own samples, Our images are easy to analyse and it is easier for our technical support people to help if you have difficulties.2 INTRODUCTION This section presents WinCELL and shows how to analyse an image and obtain data without discussing the program a tings and image acquisition (these topics will be covered in the next chaptersStart the WinCELL application by double-clicking its icon When WinCELL is launched, the Select Source window i displayed if any TWAIN compatible scanner or camera driver has been installed on your computer (more information i provided later in this manual). This window allows you to seleet a scanner or a digital camera from which to obtain images. In this tutorial we will analyse a demonstration image from a disk, s0 cli After a few seconds, you should see WinCELL’s welcome message which identifies the version running. Click Ok to continue, The model of WinCELL that The year it was released and the version (a=first -=second,.) WinCELL’s main window is displayed next. The different areas of this window and their purposes are described in the following sections. At this time you might simply look at them briefly as we refer to these sections all along this manual. WinCELL aftTHE MAIN WINDOW The ( + B camera ce disk BS) sire an image fom th activating Image See IMAGE ACQUISITION” ON PAGE 1 " the ertor mi Protection hey nt ound” when you click 7 welectore.T | working mode that WinCELL Bic i 1 (a Interactive measurement Analyse “WINCELLS WC "To selo shape of analysed and exclusion py Rectangular Be ose Sve “ANALYSED AND EXCLUSION REGIONS" ON PAGE are visi Instructions tothe operator and partial analysis data, More data can be found in WinCELL data fles. Soo “APPENDIX A WINCELL DATA PILE FORMAT” ON PAGE 85:The information displayed ther and the size ofthe characters can be selected in Display! Command Area. image when heed the lower left corner See “THE [MAGE EDITION WORKING MODE" ON RKING MODES" ON IMAGES USED FOR AN ANALYSIS Horizontal mouse postion in the image relative tothe top et corn rial mouse positon in the image relative tothe top let corn Data file open measurements data goto this file until tis clase The name of the calibration file loaded or saved from the current working session. Name of the open eolor la for color analysis). Ifa star is appendk 1 P wi that means the classes have heen edited since they were load i 4 he menu bar I eibod in REFERENC om pa 4 | {rh Tho | Click it to hide or | irectly 1 J | jshow the cll distribution | from ome magna 3 following keyboard key if hie above the mage! to another cick it wb ixrllip, | y(t - button and select th i # toscrott | yi | sired vale from th Wm, Home or vo acroll | | Sein Pop-up ment. A val DE ete V1 zor in the imag 1 Usthemagniteation at 1 Jj more deca | whichone pel inthe alg J. cick the smi" button tol | image corresponds ti nd | asmout he image tase lone plonacren, Chane rel sera othe tp | ait nage so that itp bottom, left or right iu SNE La isons are tiThe Gr contains the distribution graphic (number of cel or width or the area distribution in function of color (Pro version the Show / Hide Gi in function of cell length, area To hide or show this graphic, click ‘hic button fey in the lower left corner of the image area (see previous page). The height of the olor bars represents one of types: 1) area, 2 length or 3) width in each cass, aegis ae When the mouse is moved pera last ata numberof elo atest oe Ip ‘he _ =F case the gr bs — —eo gem |. click this two The cl be in function of measurement units or color class or group. Click the horizontalaxis move it until tochange the classification, When measurement unit is selected, the rightmost class will contain data the grae! for sizes that do not fall into of the classes on its lef ‘Tochange the scale ofthe vertical axis or the setting of Sh clase Al anos have the same with he ick the vores sae Enter the numberof classes you want and hee with rp Thy ca be any wit and be nou Ok Stu ander he lasses boundary enter the new active, WinCELL displays the area data type per color loweranduprer Seba clas he graphic and its name ro) When this classification method active, WinCEL To change the grid major and minor divisions ofthe grid enter new values here. Maj u divisions are drawn « while minor divis p of the graphie and its name is drawn wit the group color (a mix ofall color el that group : «only: (Pro) When this item is active, the gr displays the color area data (per cla 3 cells), wh 1 is inactive it is for the analy background, 4 ANALY ATION IMAGE. This introductory section shows how to use WinCELL without image acquisition in order to facilitate learning. Images which normally come from a camera or scanner are replaced by demonstration images from the hard disk. We strongly recommend that you use the demonstration images that are supplied with WinCELL to familiarize yourself with the program ‘This example only works with the default configuration of WinCELL. If someone has worked with it and has changed the default settings, you can retrieve them by deleting the WinCELL WinCELLexe program at rting WinCELL. When the file is deleted, WinCELL starts in its default recreates a new WinCELLefe file. If yo file, restart it and reenter your setti fg file that is in the same folder as th e and it later encounter problems with the program, we recommend to delete the cfitWinCE 's analysis process is started by clicking the cig, By default, inc alepays i fl \equisition icon [BS] in the Command area or by ‘** ay You ean cha activating Image/Acquire Image. If you get the w following error message Protection ke foun ition, see Appendix B, Question 6. and selecting the desired type WinCE LL then displays a standard window for opening a document. Select the Conifer2.tif demo image by clicking its name in the list, and click Open Notes fi ts WinCELL can only load the following images stored in tiff, jpeg or bmp files; * 1 bit per pixel black and white images. These im intain only 2 light intensities, black and white. If you choos: to work with this type of image, the analysis will be faster if you set the threshold method to Manual and enter a threshold value of 125 in Analysis/Pi ssificato + 8 bits per pixel grey levels images (256 grey levels), This is the recommended format unless a color analysis i required, 24 bits per pixel color images (millions of colors) If you encounter problems after loading an image (if it is not displayed correctly on screen) verifying in another program (like Adobe Photoshop) that is has one of the three format. caved from a program other than WinCELL, make sure these are saved as “uncompress worth opening it and ed above. If you analyse . WinCELL e ges stored in ti Win format. Jpeg is not the ideal format for scientific image analysis because when imag compressed with a lossy method. When such images are subsequently opened by any pro 1s are saved to file, they a am, the image content is not rocess, Measurements made identical to what it was b ore compression. Their content is altered by the compre ‘on modified images are less precise. The tiff (non-compressed) file format is strongly recommended, WinCELL can only save images in the latter format When you click Open, WinCELL loads the image and displays it in the Image area. A warning is shown in a window in front of it to indicate that no calibration has been done or loaded. You must load a calibration file (with a .cal extension) or make a calibration before analysing the image. Usually the calibration is loaded or made right at startup so you don't get this error message. Click Ok, welll get the opportunity to load the calibration later. a Ifyou load an image that was previously analysed with WinCELL and saved to a tiff file (with Image/Save Original Image With Analysis), the analysis will be recreated identical as to what it was when saved if L alysis & settings is active in Analysis / Preferences. If L ysis & settings is selected instead, the image will be loaded without the analysis. For this tutorial it is best to load a demo image that w unanalysed). not analysed (all our demo images are shipped2.4.2. SAMPLE IDEN’ WinCELL then displays the Sample Identification window which is used to enter information about the analysed sample. Enter the information as per the figure below and click Ok to continue the analysis or Cancel to stop it. If you click Cancel, you will have to load another image or activate Analysis / Restart Analysis before going any step further. The information you enter in this window is wwed with the measurements data. The Sample Identification window. "The year the cells belong to s he : . =} Image #: The image number for the entered year above. I the entire ee annual ring is analysed in one image enter 1, If it is ana os 3 more images, enter 1 when you analyse the first im: e second and 30 on, These numbers are very important for data nalysis in XLCel. They permit data from many images to be merg into a single year to compute earlywood and ring width for exampl -ginning: Activate this control ithe cell paths that you will defin in this image bogins on the annual ring boundary. In suc wall before the fist cell contains a mix of the previous year's latewood and the current year earlywood and are treated differently than other cells. Refer to"YBAR BEGINNING AND ;DING" on page 34 for more detail. sev onccall Se "VEAR Ending: Activate this control ifthe cell paths that you will define in BEGINNING AND ENDING" on this image ends on the annual ring boundary: In such case, the wall bh page 34 or deta after the last cell contains a mix of this year’s atewood and the next year earlywood and are treated differently than other cel Except for 8 and Ending (which have an effect on paths) the other values entered in this window have no effect. on measurements done in WinCELL. They influence only the data processing done after in XLCell or other program: After you click jn the Sample identification window, WinCELL makes a pause. This allows you to verify or edit the image content (see next figure) and to choose the region to analyse, During that time, it displays a message in the command area indicating that it is waiting for you to click or make an image selection to start the analysis process. It is recommended that you look at the image to verify that it does not contain artefacts or defects to remove. ick the Z buttons to zoom in Use the scrollbars (or the mouse wheel) to change the or out the image. visible part of the image If no calibration has been loaded before, it is the time to do so now. For this tutorial, activate Calibration /Load Calibration, select the demonstration calibration file Demo.cal and click Ok. If you analyse other images after, you can use the same calibration. When a calibration is loaded, applied to other images if the optical setup used to acquire them is identical.After an image and/a calibration have been loaded, WinCELL is in Analysis mode ready to analyse the image. This mode is used to create or modify the analysed region (the part of the image in which cells are analysed). You have two choices regarding the analysed region; If you click the image, WinCELL will analyse the whole image (left figure below). With the WinCELL Pro version, you can analyse a part of any shape of the image. To do this, click the image, hold the mouse button down and drag the mouse to outline the area you wish to analyse. For examples and details about how to create them, see “ANALYSED AND EXCLUSION REGIONS” on page 24 A free style region has been defined in this image de only the eells of an annual ring. Analysed regions are surrounded by a i the whole image, i s important to click and release the ‘mouse button without moving the 4.4 THE ANALYSIS After you click the image or make a selection as explained in the previous s WinCELL starts the analysis and displays a progress bar that shows the remaining analysis time which is function of image size and the analysis Parameters). It can be stopped by pressing the keyboard § long amount of time, it is probably due to some wrong analysis settings for a particular sample. At the end of the analy inCELL is ready to save data but it cannot because no data file is open. It displays a window asking what to do with the data. For this tutorial, click Create and enter a file name (with the éx¢ extension) ttings (in Analysis key. If the analysis takes an abnormally Click Open one to append measurements to an existing file When a Click Save nothing ityou don’t want to save any measurements data fi Click Create one to ereate a new file and save data init open, its Iyou click Create or enna written in WinCELL will present you the Save |= ie as or Open window. It allows to click eee the filename to open or to enter a ed name to create a file, then it wil yee open the file and save data into To open a WinCELL data file from Excel see Appendix A. Once data are saved, the distribution graphic above the image (see Cai length “" on page 13) is updated and measurements are drawn over the Cell width cells in the image and in the Command area. The information drawn depends on the settings in Analysis / Parameters), Display Image Area and Display /Command Area “differentiate cellsTHE ANALYSIS DATA 4.5.1 DATA INT After an analysis, global measurements data (related to the whole image like total lumen or wall area) are displayed in the Command area. You can also view measurements on an individual cell basis by clicking a cell in the image (the information will be displayed below the global data). Th selection of which measurements are displayed is done in Display /Command Area. More data and information can be found in WinCELLs data files. the information is not visib displayed, increase the main window vertical size. You can also modif r the text size and choo what is displayed in th The number fou defined The average Command arva in The mumbo fects sped The vcr uber of ec pet Display /Command Ar Vessel informa 3 displayed only if Analysis data, in their entirety, can only be viewed at the end of the analys saved in a file created by or opened from WinC after they have been LL. Data are automatically saved to a text file at the end of an analysis. Software programs like Microsoft Excel can be used to view data from these file in a tabular form (refer to Appendix A). WinCELL can produce two types of files, Image and Data The commands for each file type can be found in their respective Image and Data menus. Image files are saved in tiff format which is supported by many software programs and have the .tif extensionData files are in ASCII text format that many programs can read and have the .txt extension. These files contain measurements but not the image. To save an image acquired directly from WinCELL to a tiff file, use Image /Save Displayed Image. To save the image with its analysis use Image/Save Original Image With Analysis (the analysis will be accessible only to WinCELL) or Misc / Print Whole Image (the analysis will be visible in other programs but not in WinCELL) E WHICH DATA ARE SAVED IN DATA FIL! You can select which data are saved into WinCELL data files with Data /Data Save Options. If no options are selected, only global information about the analysis will be saved. The data file will contain one line of global data for each analysed image or region. If options are active in Data / Data ave Options, one or more lines of data will also be saved. These data lines contain morphological measurements on a cell basis rather than on an analysed region basis. Because they produce significantly more data lines, we suggest that you activate these options only if you need the information they generate 2.4.5.4 DATAV UALIZATION AND ANALYSIS When the time comes to visualize and analyse data produced by WinCELL, XLCell is greatly appreciated. This is a utility program that has been designed to run in Microsoft Excel. It allows you to manipulate, reorganize and display measurement data graphically. It can also analyse path data (interactive measurements) on an annual ring basis (i.e. calculate conductive area, earlywood latewood and ring widths) for years analysed in one or more images per year. 2.4.6 TO ANALYS| ANOTHER IMAGE At the end of an analysis, another image can be analysed by clicking the Acquisition icon (or by activating Image / Acquire Image). If you do so, the previous analysis cycle is repeated (you choose another image, open it and click or make a selection in it) except that this time data will be saved into the previously opened data file (WinCELL won't ask for another file unless you have closed the previous one ond ’o save data into another file, close the open one (Data / Close File) before the analysis and open or create another file. Tf you have analysed an image but do not want to save the measurements in the open data file, activate Analysis /Cancel Analysis. This will delete the current anal: over the image and remove the associated saved data from the data file if they was the last saved. To reanalyse the image (after you have changed some parameters, for example) activate Analysis / Restart Analysis (you will have to click in the image or define a region) ‘The analysis cycle that was described in this section is called interactive. In WinCELL it is also possible to analyse images in batch. These are described in “THE BATCH MENU” on page 75. We recommend that you become very familiar with WinCELL before using batch analysis. ‘There are many types of analyses available in WinCELL, some of which are specific to dendrometry or dendrochronology while others are more generic. These analyses are covered in detail in “MORE ABOUT THE ANALYSIS” on page 21, “INTERACTIVE ANALYSES (PATHS)” on page 31 and “THE PATHS MENU” on page 68.3 MORE ABOUT THE ANALYS/S There are many types of measurements and ways to do them in WinCELL, Some are done automatically while others require the operator intervention (interactive analyses). An overview of both is given in this chapter along with more in-depth information about the concepts that were introduced in the previous chapter. More details about two analyses, interactive and color based, are presented in the next two chapters.WinCELL uses up to four types of image to make an analysis and present the results. After an analysis, you can view these images by clicking their corresponding Image selectors in the Command rea. You should take a look at them to verify that the analysis is done correctly. YW ‘This is the image which comes from the camera, scanner or disk and is displayed by default. It is madi displ the Oteither back and white Ibivpixly236 grey levels Sbtsptcly, rina ima or millions of olor (24 bitsipisel Ie contains a reall reproduction of the cells and walls. The deseribed ge by image processing, The nal image can be edited before the analysis to remove defect: i artefacts, See “THE IMAGE EDITION WORKING MODE" ot an example of image edition page 23 fo This image has only tw intensities, black for pixels belonging to the cells or vessels and white for pixels belonging to the walls. This ima, result ofthe pixel classification into these two alternatively displaying the Pixels el nal image, you can verify ifthe cla illustrates the Classification ups BY image, click the ation image and th fication has been 'S CLASSIFICATION’ oN Pace 61 for more information ‘ about the importance ofthe Piels classification image and how it nthe Comm is created. The Pixels classification image can be edited after the rea. Click it ag analysis to force specific image areas to take on the classification 0 redisplay th -ll vs, wall) you want. The analysis is updated after the edition Original imag: ‘See “THE IMAGE EDITION WORKING MODE" on page 23 for an example of image e os Analysed image, 3 This image indicates which objects have beer click the 4 analysed. See “ANALYSED CELLS AND REGIONS’ ‘ BOUNDARIES" on page 29 for explanations about the colors used in this imag Click it again t redisplay the ‘ (Prov color classified during color analysis (to whi ANALYSIS" on page 35. This image is only created when a color analysis has been done. Ie shows how pixels were ‘lor class or group the pixels belongs). This image is described in “COLOR, To display th lick the Co inactive, the classes pixels classification i displayed, when it 3s pixels Ori Jisplayed insteadINCELL’S WORKING $ WinCELL has three working modes; 1-) Analysis, 2) Interactive measurements and 3-) Image edition, that can be activated by clicking their Working mode selector R[XEA in the Command area This mode must be active to make an analysis as described in “ANALYSING A DEMONSTRATION IMAGE” on page 14 or to create an exclusion region (see “ANALYSED AND EXCLUSION REGION! 24). WinCELL enters this mode; 1-) after image acquisition, 2-) after activation of Analysis /Cancel Analysis or Analysis / Restart Analysis and 3-) when it is launched. To manually enter the Analysis mode, click the Analysis mode selector in the Command area VTSWORKING MODE ‘This mode must be active to make interactive cell length and walls thickness measurements with paths. Paths allow to measure objects manually or semi-automatically as explained in “INTERACTIVE ANALYSES (PATHS)” on page 31. To enter the Interactive measurements mode, click the Interactive measurements mode selector Z]in the Command area. To exit this mode and return to the Analysis mode, click the Analysis mode selector [J You can set WinCELL to automatically enter the Interactive measurements mode after the analysis by turning on Analysis, Parameters /Switch to interactive measurement mode after analysis. EDITIONWORKING MOD The Image edition working mode allows to modify the image content. It is activated by clicking the Image tor (| in the Command area. Image edition can be done on the Original image before the analysis or on the Pixels classification image after the analysis. There are three pre-defined colors to draw with and you can also define your own (strongly recommended). The next two figures illustrate two applications of image edition, one to remove artefacts, the other to close vessel boundaries. See also “ADJUSTING THE PIXELS CLASSIFICATION FOR A PART OF THE IMAGE” on page 64 for another application of image edition. When you have terminated editing the image, do not forget to switch back to the Analysis mode (click the Analysis mode se in the Command area). ‘The cur The mouse isch 3s moved to B trom draw ree to small by clicking the smallest cursor touching the Command ‘An example of edition with the lasso tool. this rogion has been filed with the red background colorThe n the Command arva are visible only when WinCELL is in Imag mode. They allow t the color the image edition will be done with. Edited pixels will be replaced by this colo ne Black color. Click it to select a perfectly black colo The White color. Click it to select a perfectly white color, The Grey color. Click it to select a middle grey color. This color can be changed (see below ET iy default you can edit with black, white or grey. When you select the Unfortunately edition with these colors may influen image edition mode for | * the analysis if they are not present in the image the first time, the before edition. Because of their presence, WinCEL dium pen size has more difficulty distinguishing the object to and black color are analyse (lumen, fibre) and the background (walls) and selected | hence the selected automatic threshold might not be jptimal or the pixels eolor Then press the s Jose to the abject to The grey color is by the color of the selected and i visible in the lower | a left corner first click one of the three F ee eee rust first click is the C the image by outlining an area Move the mouse to one end of th region, click the mouse button and hold itd and area. You To draw, move the mouse to the place in the image edit, lick the mouse and hold the button down. As you move the mouse, pixels covered by the wish t wn, drag the mouse brush are drawn sith the pean ones ioe oa selected colo, Release the mouse region you wish to modi ane When yon slease the button, the region sill wth the selected to modify have been changed tor Not: Tis possible to cancel or undo the last edition stroke with Display/ Undo Euition 3.3 ANALYSED AND EXCLUSION REGIONS Regions can be used to analyse a specific part of an the analysis (Exclusion regions, see “THE EXCLUSION MENU” on page 66). Measurements are always associated with an analysed region, one global data line is saved for each one. Regions’ shape can be rectangular, circular or irregular. They can be created while WinCELL is in Analysis mode. An Analysed region can only be created when no analysis exists while Exclusion regions can be created before or after the analysis. If they are done after the analysis, the latter is updated (redone) so it is faster if you create them before. The next sections illustrate how to create regions of different shapes and how to resize and move them. nage (Analysed regions) or to exclude them fromThe 4 arva are visible only when WinCE} will be done with. Edited pixels will be replaced by this o The Black 1a perfectly black colo The White color. Click it to select a perfectly white col The Grey color. Click it to select a middle grey color. This color can be changed (see below z When you select t rtunately, edition with these colors may influen mage edition mode for he first time, the the analysis if they are not present in the imag. before edition, Because of their presence, WinCEL has more difficulty distinguishing the dium pen size tt analyse (lumen, fibre) and the background (walls) and hence the selected automatic threshold might not be and black color are selected ‘optimal or the pixels color classification might be ——— ie f ross the shift key le and click the background [gz background c Jose to the object to. | present in the rave from the image age pet the The grey color is replaced tov by the color ofthe The grey eri background at th visible inthe lower | naa left corner first click one ofthe three P rust frst click its ieon the Command area. You ca edi the image by outlining an area, Move the mouse to one end of the region, click the mouse button and hold it down, drag the mou: ind in order to enclose th To draw, move the mouse to the place in the image you wish to cit, click the mi button down. As ‘and hold the ou move the mouse, pixels covered by the brush are drawn with the region you wish to modify When you release the button, the region is filled with the selected ected color. Release the mouse button when all pixels you wish to modify have been changed Note: Ibis possible to cancel or undo the last edition stroke with Display /Undo Edit ALYSED AND EXCLUSION REGIONS Regions can be used to analys the analysis (Exclusion a specific part of an image (Analysed regions) or to exclude them from gions, see “THE EXCLUSION MENU" on page 66). Measurements are always associated with an analysed region, one global data line is saved for each one. Regions’ shape can be rectangular, circular or irregular. They can be created while WinCELL is in Analysis mode. An Analysed region can only be created when no analysis exists while Exclusion regions can be created before or after the analysis. If they are done after the analysis, the latter is updated (redone) so it is faster if you create them before. The next sections illustrate how to create regions of different shapes and how to resize and move them3.1 RECTANGULAR.IMAGE REGION ‘To create a rectangular region, follow the steps given below. 1) Click the F in the Command area 2) position the mouse at a corner of the rectangle to be define 3) lick and hold down the mouse button, 4) drag the mouse to the diagonally opposite corner and 5) release the mouse button, When the button is released, the region is analysed CIRCULAR IMAGE REGION To create a circular region, follow the steps given below. 3) click and hold down the mouse button. A circle with the same diameter as the previously defined region is displayed 4) while holding down the mouse button, press the upper (¢ ) or ower (+) keyboard arrow key to resize the boundary circle 7 until it has the desired diameter, or drag the mouse to change the circle position, 5) release the mouse bution. The a ~ ee ar Seer When defining a circular regions, you must either resize or move the circle in the image once you have clicked an: are holding the mouse button down, otherwise the whole image will be selected. 3.3.3 FREE STYLE IMAGE REGION ‘To create an irregular shape (free-style) region, follow the steps given below 1) Click the 4) release the ‘mouse button, Upon releasing, 2) click at mouse button, 3) move the mouse to region boun between the and the last point and analyses the outline the region boundaryREGION RESIZING An Analysed region (but not an Exclusion region) can be resized after it has been created. 3.5 REGION MOVING (but not an exclusion region) can be moved after it has been created.The most basic and the fastest analysis is the global analysis. It consists in measuring the total lumen and wall areas in units (ym* for example) and in percentage of the Analysed reg Pixels are not classified per cell as in individual analysis described next, so it is not possible to filter out debris. A global analysis is done when Analysis / Parameters / Indiv Analysis /Pixels classification / Based jevels is active off. It is computed also when other analyses are turned on. a sis is inactive and The global analysis cannot be turned A global color analysis is similar to the global analysis described above. It performs measurements plus total area in function of color classes and groups. Objects filtering is not possib! It is done when Analysis /Parameters/ Individual analysis is inactive and that Analysis / Pixels classification / Based on color is selected. To learn how to make a color analysis and to see some applications, refer to “COLOR ANALYSIS" on page 35 and “THE COLOR MENU” on page 70. the same 3.4.2 INDIVIDUAL ANALYSIS AND INDIVIDUAL COLOR ANALYSIS. When an Individual analysis is done, pixels are grouped into objects (cell lumen) and their morphology measurements (area, length, width...) are computed in addition to those of the global analysis. This analysis is done when Analysis / Parameters / Individual analysis and Analysis / Pixels classification | Based on gr Is are active. You can choose which morphological measurements are done and how in Analysis/Parameters. When an individual analysis is done, more data are produced in the global data also. For example, average cell area, length and width are also computed An Individual color analysis comprises all the measurements of the individual above plus area in function of color for each cell and for the analysed nalysis described gion (as in global color analysis). An Individual color analysis is done when both Analysis / Parameters / Inc analysis and Analysis / Pixels classification /Based on color are active. For more information about color analysis, refer to “COLOR ANALY on page 35 and “THE COLOR MENU” on page 70. ATHIRD C LL TYPE, PARENCHYMA In addition to cells and vessels, it is possible to manually measure a third cell type that we call parenchyma. This is done manually and interactively. Global and individual information about parenchyma cells is available in the Command area (below vessels data) and in data files. Parenchyma are identified by a different color (grey) in the Analysed image. Tod keys and click the cell until iis dra manually change a cell type form cel to vessel and the reverse. Each time you clic a cell in trl and Shift keys, the cell xt type inthe following ing ctr and shift, the operator has clicked t fy them from cll ime it will be classified as parenchCells of all typ Is, Vessels and Parenchyma) can be grouped and analysed as a whole. Dat about grouped cells are available as individual components (for each clicked cell) in OBJECT date lines (when / ual data and Sat y In are active in Data/Data Save Options) and as a group in GROUP data lines (when Save in GROUF s active in Data Data Save Options). The number of groups and the average number of objects per group can bi displayed in the Command area and saved in global data lines of data fil To an ne four vesels as ifthe 1) Press the keyboard @ key and 2) While the G key is held down, click other ob rer n the keep it pressed) then click toinclude in the group. The "box is exten th st object encompass al ofthe group. When all ob isdrawn around of the group you wish to create have b red offin release the @ key a a ndicates to WinCELL. thatthe group is complet Image Area. is drawn betwoe re ofthe objects of hen Lines to the rig mato ts active in Display Image Area To remove acellivessel from a group that isnot completed (still under definition), press the G key and click th sel. Note that if the group is. pleted) this command will delete the erour up. press the G key and click any’ cell ofthe group grouped cls, activa An e Cells Vessels Grouping. This will work ifno group is under defini jonally the analysis might merge cells which a nsider them a single one. These objects can be splitted to get the exact number of objects and measurements data closer to reality. Note that measurements will be more precise if the image is edited instead. , ~) ~~ serdrawninatead = | } is to th ; | Themanterof ier ican Il Lice is inten ha mers te la E —— an n,2) click the object to spit reles are drawn around its to the rit isa Area).The number of cites indicates in how many par ects has been spit into 2 fis split fe measurements data foreach part oft separately: Their data \area length width... isthe larger objects data amber of parts it has been splitted into, each part having the same data (Ex: The area of each part of an object sp riginal objects area) They have an identification nuraber with a decimal fraction which is made of the original objWinCELL analyses the objects differently in function of their position in the analysed region and it boundaries. This is to avoid that incomplete cells affect the measurements. The figure below illustrates the possible situations. The analyse Cells rejected by the operator with etrl-eliek are drawn in blue. They an not included in any analysis. They a cell ‘onsidered as bel completely z a backgrouni analysed region Cells included in the analysis and included in lassified a drawn in the analysi green. Th nchym and NOT are draw in 'A THIRD CELL classified as TYPE, PARENCHYMA’ on page 2 Lr are drawn in (I DT ey These calls fs tharentiety fb tt 7 1 inal Cells partly in the analysed region Cells completely are drawn in They are not outside the analysed in the individual data. Their’ analysed region ar Colls truncated by th area INSIDE the analysed region are drawh'in image boundary are drawn counted in the total ell area and They are not nal They are ot inluded percentage cll are cluded in any any analyse When you analyse the whole image, WinCELL can make the analysed Cells filtered out by the area filter a slightly smaller than the image to avoid analy pen in dark yc, They are not included in runcated by the im: any analysis. They are considered as debri d othe background. incomplete cel we boundary). You can change the thickness sis/ Parameters by entering the desired valu and as such belonging Example of a shape4 INTERACTIVE ANALYSES (PATHS) Interactive analyses are done by tracing lines called paths over the objects to analyse. They allow se and analy Creation Paran any object or a row of cells depending on fers. There are four types of pa path type selected in in this chapter They are d4.1 MANU Manual Length paths can be used to measure the length of any objects, including those WinCELL cannot isolate from the background. As its name implies, this method is completely manual, which means WinCELL does not interpret the contents of the image during measurement You simply click the paths beginning and ending in the image and WinCELL measures the distance between these points (the path length). The values that are saved in the data file is this length. The operator has clicked the two ends of a 2 emcasure its length. The number \ « segment in Paths Path C Pare a 4) "The operator has clicked many places al from one end to the ather! to measure it len umber of 2 Paths) Path Creation Parameters. The path length isthe sum of the lengths of its sem 4.2 MANUAL LENGTH & WIDTH PATHS Manual Length & Width paths can be 4 manual lngth & width path When a munca! used to measure an object (like a ismade fa pairof length length & width vessel) length and width manually. As_ ts. One presents the path is complet with manual length paths, WinCELL l=pith me directin © teens does not interpret the contents of the perpendicular or any direction ‘The two length image during measurement You decide the vessel width. Vessel area paths ends a where the paths begin and end and §ih/tterteesimate fom linked by dated WinCELL simply measures _ the distance between these points (two for the path length and two for the width). 4.3 INTERSECTPATHS When the path type is set to Intersect in Paths /Path Creation Parameters, WinCELL automatically detects and measures lengths separately in the objects (lumen) and the background (wall) along lines that you trace in the image. This method can be used for wood cell analysis but is not specific to it. Tt can be used to measure many types of objects s emi-automatically. aven between the two points jor to ilustrate that its in a fina wall (os). Le ents in cells or wal along the path line and correspon length ofthe different colors ements, "The operator has clicked at the two ‘ In this image, the operat The length measurements are the thre has cliched at th calls lumen lengths and wall len . w squares to create a and after each of them, path for ibustrat The place the path is created is very important in this method becaus purposes only as itis along this path line that the measurements are made. For example if you unlikely that conifer cells trace a line at an angle o rees across a cll, the cell length wil b horizontally asin Touch Centre paths Tn the versions before WinCE 04a, all measurements ofthe walls in inte 7 paths were not divided by two, In 2 more, they ae. Its advised to click in the cell proveding the first wall and the cell following the last wall wanted in the path so that th entire walls are counted in the analysis (asin the 's example). If not possible, the frst liek ofthe path should be at the very beginning ofthe fist wall and the last click at the very end ofthe last wall. The reason is thatthe first and last walls are also divided t two so you need them in their entirety if you want ta avoid erroneous values. Intermediary points of multi-segment paths should be made in the walls andi is strongly advised to create paths horizontally in both In sect and Touch Ce4.4 TOUCH/CENTRE PATHS (FOR WOOD CELL ANALYSIS) Touch / Centre paths are provided to study cell anatomy on an annual ring basis. They allow to measure earlywood, latewood and ring width in addition to the variations in proportions of cell to wall (which is related to wood color, density and other chemical or mechanical properties). A Touch Centre path has some knowledge about wood cells. Ir knows far example that Ils surrounded by two double walls. I ‘measures the lumen length horizontally or vertically (you choose) and it associates to th Cellhalfthe walls’ thickness infront and ater it. This allows to measure cll le A double wall It is divided in ton. One half Belongs to ‘he col to is eft and the other ta the ell tits right longi smen length i then 1-) Lumens touched by the path are detected and their identification number are stored for analysis, L ‘measured horizontally or vertically depending on the path orientation at the lumen gentre of gravity position With the Touch /Centre method, the postion of the path line is less critical than with the Intersect method. The line i used only to identify on which cell th measurements are done illustrates the postions where th erator has clicked to begin or end 2.) WinCELL then measures the wall thickness between land its two neighboring cells by prolonging the lmen measurement line unt it reaches another vl lumen Thess Gletances are then divided by two ts produc two half Walls Measurement lines are drawn ao ag oT cher the cells and walls toilustrate where the measurements have been done, Because hey hi Mid fre made a the centre of gravity height pnlton foreach men, they might not be pefely signa as with the Inert method fd d+ for lumen Tumen length plus adjacent walls length (i++ for first cell or c+!+!for second cell rea ofthe lumen (real area measured by counting lumen pixels, not an estimation) ‘The criteria for this classification is from Mork 1928 or its interpretation by Denne 1988, A latewood cell i a cell for which two times the total wall thickness (ae? is larger or equal to lumen length (2"avev2>b) b "oS St 1 Asora on 9) TAWA Bolts Vl 10 (1, 988 588 1) Each cell must be surrounded by a fs measured first. Ie does wall infront of and after ion the path, not include the distanc Ta path begins or ends in alumen as from the \ to the wall. 7is illustrated below, that lumen is skipped then divided by 2t ‘not analysed). Measurements begin on the half wall nse pst the first encountered wall on the path from that lumen, ) The first wall of path is divided by two to estimate the wall thickness belonging to the st cel Simla he st wall path i divided by two toestimate the wall thickness belonging to the last ¢/ Intermediary points of multi-segment paths must be in a wall, They do not need tobe in the middie ofa wall, WinCELL adds the distances and «on each side ofthe intermediary point to the fist cell ofeach segment then divides that distance into two equal parts that become the wall thicknesses of the neighboring cells andThe Begin: vg and Ending controls have an effect on Intersect and Touch /Centre paths only. Activate this control ifthe cll paths that you wil wall bfore te frst cell th first wall encountered on the Tenath Activate this control i paths that you wil define int eee (ee reall ater hela all he lst wal o ae st measured but is given the sa has the wall before the cel In this example, if cell isthe § is active, wall thickn Do not activate both B nd when definitions cannot be asthe abow sly, You might be tempted todo soto analyse ical parameters of annual rings with only one eel thick. These cells should be analysed with Intersect or Manual path types. In both cas {ou wil have to estimate where the current year walls before and after the o divided by 2) or measure them with their snd that it wll Walls and apply a special process5 COLOR ANALYS/S This section illustrates the purpose and how to do a color analysis with the WinCELL Pro versionIn WinCELL Pro, color analysis can be used for two reasons; 1) to get a more precise pixel: classification for a certain type of images and 2) to differentiate and classify objects or parts objects based on their color and get measurements in function of color. At the beginning of the color analysis, the image colors are classified into classes that you choose from those you see on the computer screen. You must define color classes and groups in order to detect and quantify the colors of interest. This process is illustrated in the following sections. It is complemented by “THE COLOR MENU” on page 70. PRE-RI mn D In order to do color analysis, your image files must meet the following requirements 8 must be in 24 bits RGB (Red, Groen, Blue) color format (8 bits per pixel per color channel) stored in tif a RGB Color in th ige/ Mode menu. If you encounter problems after loading an image (if it is not displayed on screen), it is wor Photoshop) that itis in the above mentioned format, npressed) or jpeg files. In the Adobe Photoshop program, these images are referred ‘opening it and verifying in another program (like A\ 3. GREY LEVEL Compared to color images, grey levels images take one quarter the memory and time to analyse and store. Unless they are too difficult to analyse, it is recommended that you use analyse grey levels images. To be analysed precisely, a grey levels image must have a good contrast between the cells and the walls. The cells must be bright, without dark areas and the walls must be dark and uniform. If this is impossible to achieve, or if you want to analyse objects other than wood cells with lower contrasts, then color analysis might be the answer: 4 ANALYSING TF 10 IMAGES WinCELL comes with two color demo images in the folder “WinCELL Color Demo images”. The image named Red.tif must be analysed with the set of color classes in the Red.cac file. Green.tif must be analysed with the set of color classes in the Green.cac file. CELL and activate Based on color in Analysis / Pixels Classification and Load a calibration file (1x.cal) with Calibration /Load Calibration. Click the disk icon in the command area to load an image (set the image origin to Disk if nec ary), choose the Red.tif file (in WinCELL Color Demo images) and click Open. Enter the sample identification information and click Ok. A message is displayed telling you not to forget to load or define color classes. Click Ok Activate Color/ Load Classes and select the directory where the color images are stored (WinCELL Color Demo images). Choose the Red.cac file and click Open. This, file contains the color classes that we have defined for the image Red.tif. You can see those classes, and the groups they belong to by activating Color /Edit Classesoups of colors have been defined, ane for the walls and one forthe lumens, One group must be identified as backround by he button below its name cells the background group is the one that contains the wall color Theluncn :.0up hs) TT! We hve chosen thr 0 represent th mnly one eolo, grey Ser] al colors dark es pink and ht pink aa Colors perception change with the devie Click OF for the itis looked at. For example, a monitor will moment, this window TESS} display the color differently th int will be explained later sama] used for this manu: s should be we recommend that you turn off the display of complete. In order to better visualize what follow Cell number and Cell width in Display /Image Area. When a color analysis has been done successfully, the Col is displayed in the Command area. When you click it, the Color image is displayed. Click it a second time to display the Original image again. The Color image allows you to verify that the pixels belonging to the walls and lumens have been properly classified into these respective classes. The 1 color The color classes im (RGB) imag thas four colors (pixels It has millions of are drawn with one of the colors four colors of the defined classes). It shows into which class the pie have been classified Sometimes it is not easy to verify that pixels have been classified into the color classes correctly. This can happen when there are many color classes or when two or more classes have similar colors. To see the pixels classified by color groups rather than classes, activate Color/Display Groups. After this when you click the Color image selector in the Command area, you will see the color groups classification. This image has only two colors (one per group) so it is easy to verify if the pixels have been classified correctly The color group i The original color thas two colors (pixels RGB) ima are drawn in one of the Tt has millions of two colors defined for colors the groups). It sh into which group the pixels have been classified, You can try to analyse the other demonstration image that is supplied with WinCELL (Green.tif). Do not forget to load its corresponding color classes file (Green.cac). Because the image has good contrasts, only one color per group has been defined.To clear all color classes from the previous examples, we recommend that you quit WinC restart the program. Make sure that Analysis / Pixels Classification / Based is active calibration file (1x.cal) with Calibration / Load Calibration Click the Acquisition icon in the Command area to load an image (set the image origin to Disk if necessary), choose the Red.tif file (in WinCELL Color Demo images) and click Open. Enter the sample identification information and click Ok. A me: load or define color classes. Click Of ge is displayed telling you not to forget to Activate Color / New Class. WinCELL display. Make an image selection” in the Command srea. The first step of defini o click on a specific color of interest, a lumer area for example or to make a selection in it.I you click, only the color ofthe pixel at the click sition is considered. If you make an imag election, WinCELL will mix all the colors nncluded in i After the elick or selection, the played. This window shows the average colo the mix ofall isd he Enter the Color Class name (Grey You must specify which g1 licking the button te mn, in this case p the class belongs 6 left of the group name L Enter the names of the groups. There can be up to three groups, in this example the groups are named and Not Use In this image, the lumen areas we do not define any other colo or this group. The new class window also contains controls to adjust the color class definition parameters, this adjustment all you to include colors that differ slightly by hue, saturation or intensity in the same class. Most of the time you w 1 no have to adjust these settings. In this example we have used the default settings, For more information, please refer t “EDIT CLASSES’ od with Color/ Edit Classes, page 72, Class and group parameters ean be laterrequired to defin a group is hard to determine withou trials. Images with greater variability in or hue will require more dea is to have a class for each color which is part of that object wall or lumen| A color class has been defined in ight pink area of the wall other color through selections in darker parts of the walls. They are also identified as being aria ie seer ee | rte group. ; iene | a4 By default the first group is selected as the background group. In this example, the backgr group should be changed to the Wail group. Once all the classes have been defined, they can be saved to afile so they can be reused later on other images, It also allow to experiment with modifications and to come back to the actual set of classes if the modifieatic the expected results, activate Co do not produc save the color classes to a file ‘Save Classes and enter the file name (with tension), Activate Analysis /Restart A) analyse the image. Click the color el verify the class the fication. Do not -get to tur off Color Display Groups if you want to see the classes rather than thé groups. After the classes and groups creation is complete, click the image to analyse it or make an image selection. The first step of the analysis is the pixels classification into the defined color classes. Pixels that meet the criteria for more than one class are classified into the closest color Color similarity is determined by the program mathematically. If a pixel’s color does not fall into any class (an orphan pixel), it will be classified into the closest class or into a class that you specify according to the settings in Color/Edit Classes. The result of the previous classification is stored in the Color image. The pixels are drawn in the colors of their respective class or group in this image In data files produ d by WinCELL two kinds of data related to color analysis are saved. The global color information indicates the total area occupied by each class and group for each analysed image. The individual data gives the same information for each object in the image. Refer to Appendix A for a description of WinCELL’s data format and content6 ACQUIRING YOUR OWN IMAGES a camera or scanner is exactly the same as with the ns except for the image juisition step.Cameras sold by Regent have a standard C-mount thread to screw a lens in or to allow them to be attached to an adapter. To install the camera on the microscope, you unt adapter for your microscope. These adapters are sold by microscope manufacturers. Your microscope must have an opening on which the camera will be installed. On some microscope it is offered as an option while on others it is built-in. At each point in the image, WinCELL looks at the original light intensity to decide if it belongs to a cell lumen or a wall. Because light intensities are important in this decision, care must be taken when setting and using the illumination system. Light distribution must be uniform and must provide a good contrast between lumen and walls. Greater contrasts can be obtained by staining the samples with Safranine O (v«:\) and lighting it with its complementary color which (place a green filter between the light and the sample). Color image taken without green filter Color image converted to grey levels, The walls have or image above converted to bright areas which can be misclassified as cells s are opaque, dar contrasts between o and of uniform inter and walls are pronounced.SELECT THE DRIGIN AND TWAIN SOURCE Start the WinCELL program by double-clicking its icon il If a TWAIN driver has been installed on your yj, is shows the sompatibe computer, the Select Source window will be TWAIN sanmors asl ceners displayed. Select the source name corresponding installed on your «ystem. ther: | —T// to your camera (or scanner) and click Select. This is a TWAIN source for each name can be found in the camera or scanner 4°viee manufacturer manual and in Regent's manuals shipped with our cameras or scanners (Ex: 5 MP Installation and Utilization...). If you acquire images with a camera, make sure that Use TWAIN Interface is active in Image! Acquisition Parameters (it is by default). If you acquire images from a scanner it can be active or not (see next section) Itis very important to =§ —_______——] never select a source which names begins with W WAIN’ TWAIN is a standard set of commands and drivers that allows software programs to acquir from scanners and cameras without a-priori kn das y become available without having to modify exist their |AIN drivers in the operating system. When a TWAIN installation is successful, a source is installed on your system for each device that is TWAIN compatible. Sources are installed by running the installation disks that come wledge about them. New scanners or cam: with a device When the camera or scanner is On, properly installed and a TWAIN source has been selected, WinCELL selects it at start-up as the image origin and draws a scanner in the Acquisition icon instead of a disk. Clicking this icon while a scanner is drawn will start the image acquisition proces rather than the image loading process described in previous sections. You can manually set the origin to the scanner or camera in Image /Origin (if a source was selected at start-up as described above). When Use TWAIN Interface is active, the camera or scanner TWAIN interface is displayed before each image acquisition. This allows you to set the image parameters (size, dpi, grey vs color...). When you trigger the image acquisition according to your camera or scanner instructio the image is transferred to WinCELL’s main window and is displayed in its Image area. When the image transfer is complete, you can analyse the image as with our demo images If, using a camera, you cannot see an image live in the TWAIN window it may be because * lighting system is off or not strong enough, * light goes to the eyepiece but not to the camera (some microscopes have a mechanical switch), * light is too strong for the camera (which becomes saturated), * the camera is not turned on. WinCELL can only acquire the following thre * 1 bit per pixel black a s. These images contain only 2 light intensities, black and white. If you choose to work with this type of image, the analysis will be faster if you set the threshold method to Manual and enter a threshold value of 125 in Ay pes of images; * 24 bits per pixel If you encounter problems after acquiring an image (if iti not displayed correctly on screen), it is worth verifying that is has one of the three formats listed. The grey levels is the recommended type of image unless your applic: needs color. For examples of color applications see “COLOR ANALYSIS" on page 35 To ensure the validity of the measurements, a calibration must be done or loaded at the beginning of each work session. The calibration method that must be used with cameras is the Object of known dimensions (see “THE OBJECT OF KNOWN DIMENSIONS CALIBRATION METHOD” on page 79). This calibration can be used as long as you do not change the optical setup (magnification of the microscope.Very high resolution scanners (+4800 true optical dpi or more) can be used to acquire images fron well prepared wood surfaces to measure vessels area. Before acquiring images with a scanner in WinCELL, you must choose the si latter determines how and when you enter the image specifications (ar grey levels...). WinCELL has two such interfaces which can be s Parameters anning interface. The can, resolution, color ato lected in Image/Acquisitior Regent’s simple interface can be selected if you purchased a scanner from Regent. This interface, described in “ACQUISITION Parameters” on page 50, is optimized for simplicity rather than flexibility. To manually select it, deactivate Use TWAIN Interface in Image /Acquisition Paran When this scanning method is selected, you set the scanning parameters once in Image / Acqui Parameters and no window is displayed before each scan. Each time you click the Acquisition icon to acquire an image, the scan area, image resolution, image type and other parameters entered in Image /Acquisition Parameters are used. This method is appreciated by novice users and also for repetitive scans of similar samples placed at the same position in the scanner The second interface is the standard TWAIN scanner manufacturer's interface. It is used when | TWAIN Interface is active in Image/Acquisition Parameters. When you select that option, th scanner manufacturer's interface is displayed before each scan. This allows you to set the imag parameters for each scan. Because that interface gives you more choices it is more difficult to learn for a novice user. On the other hand it allows you to optimize the image contrast and quality. If you choose to work with that interface, make sure that you scan in one of the image types supported and described in the previous section. When the sample is in place, start the analysis cycle by clicking the Acquisition icon or by activating Image / Acquire Image. Enter the sample identification information (if it is asked) and click Ok. Wait for the scan to be completed and the image to be displayed in WinCELL’s main window, then analys: the image as you did in the previous sections. Images from a scanner can be analysed with th Intrinsic calibration method (see “METHOD” on page 78)?TEFERENCE This section presents WinCELL’s menu commands in the order they are en b juntered on the menu‘This command loads WinCELL's working settings from a configuration file (*.cfg) created with Misc Save Configuration. Only those made with the same version of WinCELL can be loaded (others ar ignored). At startup, WinCELL automatically loads the working configuration that was used the time it was run, ‘This command saves WinCELLs settings to a configuration file (*.cfg). These can later be reactivated with the Misc /Load Configuration command (see previous section Use this command to print the cells distribution graphic above the image. Refer to your printer documentation for printing instructions. ‘This command prints or sends to a Windows bitmap bmp file, the part of the image displayed in the Image area including its analysis marks and the graphic above it, if it is displayed (to turn its display off, click the Show / Hide Graphic button in the Command area), To print a title (caption) above the Select Prinwr to print the imag Sele to ereate a Window: graphic, enter the text to be printe Bitmap “mp fil Click to change the font of the Select toget the title i printed text. P the upper left corner or select to centre the tle horizontally Select Justify Left i you After you have entered a name and image to be printed start clicked Save, the file name (including left side of the sheet or selec ts directory) is shown here to have it centered both horizontally and vertically in the you can give it a name by clicki middle of the page Choose the directory an enter the file name in the Save A Turn on A to get a larger window white margin surrounding the image .If you have chosen to send the image to the printer, the window of the default (active) printer driver will be shown after you click Of. Refer to your printer documentation for in ructions about th window. The Save As window will be shown if you decided to send the image to a bmp fil Use this command to print the entire image (rather than the just the portion of the image that i displayed on sereen as in the previous command). See “PRINT DISPLAYED IMAGE" on previou page for printing instructions as both commands are similar This command it used to set the printing parameters for the two previous image printing commands. The choice of parameters available is governed by the printer which is installed in your system. These parameters usually include page size, orientation, zoom factor and more. Refer to your printer documentation for further information Activate this command to exit WinCEThe analysis’ data and information are automatically saved right after it is done. There is nc command to activate to save them but a data file must be opened or created before with the menu commands. WinCELL's data files are text files (*txt) that have the format given in Appendix A Choose New File to create a data file. Analysis data (but not the image) are rues) saved in this file until it is closed or another file is opened or created. When you ae activate New File, a standard window for creating a document is displayed. Enter the name of the new file (with the .txt extension) and click Save. Ifa file with the same name already exists, WinCELL displays a warning message that asks if you want to replace it or not. Click Yes or Replace and the contents = he new measurements; otherwise another opportunity to give the file is open, its name appears in the Information area above the of the old file will be replaced by file a name is offered. When a da Choose Open File to open and add the next analyses measurements to an —_——— existing file. Data are saved in the open file until it is closed or another file is opened or created. When you activate Open File, a standard window for opening a document is displayed. Select the file name to open and click Q; When a data file is open, its name appears in the Information area above the image Use this command to close a data file when you no more want to save data in it. It is activated automatically when you quit WinCELL.This command allows to select which c A for a detailed description of how data are matted in those files, Append When this tem is ative, one individual data line is saved for ce “INDIVIDUAL ANALYSIS AND INDIVIDUAL COLOR ANALYSIS Beware that when this option is selected, data files ean be quite large, so turn n you need the data i produc When this itom is active, interactive © ibaa nts like cell length, lumen length, wall thickness) are saved for each path} mmucrernt la) actively (see “INTERACTIVE ANALYSES (PATHS)" on page 31 ray Activate ths item when you want each object of a grou >be saved individually in CELL data lines as if they were not grouped. Turning on his item and the next one allows you to get information about the group and its members separately. When ths item is not active, grouped cells are not saved in CELL s Activate this tem when you want information about grouped lls to be saved on a group basis. Each group is treated like a regular cell and its total area, length width ete. are saved on a single GROUP data linveto omtes ‘The Origin command indicates to WinCELL, the source (image acquisition device) of the images for analysis. The image origin or source can be a scanner, digital camera or a disk aining or when im ed in tiff or jpeg files. Images from files can be useful for e acquisition and analysis are done at two different moments, Seleet S; to acquire and analyse images from a scanner or a TWAIN compatible camera. If no scanner or digital camera is connected or if its TWAIN alled, the scanner button is inactive. It might als be disabled when no source has been selected in the Select Sow ‘SELECT THE IMAGE ORIGIN AND TWAIN SOURCE” ON ‘oftware drivers are not correctly in ve window at start up (see AGE i PARAMETERS This command can be used only when an optical scanner is the image input device. It does not work with cameras Acquisition parameters is active only when a TWAIN compatible scanner or digital camera has been detected by WinCELL, and that you selected its source in the Select Source window when WinCELL was launched. It allows you to choose the image acquisition interface, the image type and quality and the sample's positioning scheme on the scanner. WinCELL gives you the choice of two scanning interfaces; by default, Regent's simple i selected. This interface is very easy to use and works only with some scanners not with cameras. It allows to choose basic settings like image resolution, type (grey levels or color) and sean area position on the scanner glass. It does not permit to do sophisticated adjustments like contrasts enhancement To use Regent's simple scanning interface, turn off Use TWAIN interfi The other interface is the TWAIN interface made by the scanner manufacturer. It allows complete control over the scanner capabilities, but requires more expertise. This approach to scanning is documented in the manual supplied by the scanner manufacturer (in printed form or on CD-ROM When Use TWAIN interface is active, all items of Acquisition Parameters are inactive (scanning parameters are set at image acquisition in the TWAIN window that is displayed before each scan). The remaining part of this section describes Regent when Use TWAIN inter inactive. simple interface for scanners which is useduse Regent's simple scanning interface turn off . ution image to scan. higher resolu precise measurements but requi and t re time to sean ter the dpi value. This valu ecified (Bx: [2x < 1200 m between 12 and 120 increases, the pixel sia measurements are always a mul which means they are either 1.2, sample, at 400 dpi, an object is Regent's positioning system Tum on scan with Regent's sitioning system, With it, scanning is faster and easier reause you know exactly where to place the obj ne scanner. Then choose the scan area (see below Regent's positioning system ar to avoid seeing the tra tioning system has up to five predefined nning widths (horizontal dimension ofthe imag 10,15, and 20 em and for large area seanners, 25 and 30 om. Select the width by clicking its number hen tical dimension ofthe image’: 5, 10, i 40cm is available only whe ng system on some scanners). Some LC seann do not allow the 30 em scanning len eva ne pre-defined ‘de of th anned by WinCELL u an) before each sea ond set the image off F ner glass origin, rigin is indicate by a mark a triangle) or the numbers 0, th ga Tom: JS week fnActivate Image/Acquire Image to acquire an image from the selected image origin (disk, camera or scanner) and display it in WinCELL’s main window. This image can then be analysed. This command as clicking the Acquisition icon in the command area. has the same effe Note I: Ifyou get an error message (Protection key not found...), see Appendix C, Question 6, Note 2: WinCELL can only load the following three types of images stored in tif. jpeg or bmp * 1 bit per pixel black and white images, These images contain only 2 light intensities, black and white. If you choose 10 work with this type of image, the analysis will be faster if you set the threshold method to and enter threshold value of 128 in Analysis/ Pixels Classification + 8 bits per pixel grey levels images (256 grey levels). This is the recommended format. ‘© 24 bits per pixel color images (millions of colors) If you encounter problems after loading an if it is not displayed correctly on screen) it is worth opening it an verifying in another program (like Adobe Photoshop) that is has one of the three formats listed. The Is is the recommended type of image unless your application needs color. For examples of color applications see “COLO! ANALYSIS" on page 35, SAVE DISPLAYED IMAGE You can save in the standard tiff format (readable by many programs); 1.) the Pixels classification image, 2-) the Color image and 3-) the Original image. To save an image, display it on screen first (click its corresponding Image selector in the Command area), then activate Image / Save Displayed Image. A standard window for creating a document is displayed. Enter the name of the image file with the .tif extension) and click Save. Note t: WinCELL can save images only in tiff’ uncompressed format. If you need to compress them for storage use a lossless compression program like zip, which allows to decompress th 1 as they were b Note 2: You ean also save the image with its analysis marks over it and/or with its distribution “PRINT IMAGE AREA” on page 46. The latter allows other programs to load and display the analysis marks. See al the next command to save an image with its analysis for later retrieving in WinCELL sm identica fore compressi aphic as explained i 3.5 SAVE O! WITH ANALYSIS An image which has been analysed can be saved with its complete analysis by using this commar When such images are loaded back in WinCELL, the latter can load the analysis and take on the settings that were effective when the image was analysed or discard the analysis and retail current settings (see “PARAMETERS” on page 59). The analysis saved in tiff files can only b xccessed by WinCELL, other programs will not see and preserve it if you open such images wit them. Beware that if you save an analysed tiff image from WinCELL, open it in another program and save it back to a tiff file from that program, the analysis will be lost. To view the image and the analysis in other programs, refer to “PRINT WHOLE IMAGE” on page 47 ‘To save an image with its analysis, activate Image /Save Original Image With Analysis. A standard window for creating a document is displayed. Enter the name of the image file (with the .ti/ extension) and click Save AGE REGION ‘This command allows to save to a (tif) file the image area delimited by an analysed region bounding box. Note that the analysis is not saved with this command.It is difficult to accurately classify the pixels of an image which has spatial light variations. The Background Unevenness Compensation menu commands allow to modify the lighting of the image to uniformise it so that pixels classification is more easily and precisely done Lighting uniformisation is usually not required with images acquired from a scanner and is sometime required with images from a camera. Although WinCELL has functions to overcome lighting non-uniformity problems, it is best to try to uniformise light at image acquisition (on the microscope or with the camera TWAIN driver) rather than after by image processing (you will save a lot of analysis time 40D PARAMETERS You set how and when background unevenness compensation is done in Method Parameters There are three lighting non-uniformity compensation meth s se is the fastest and safest meth It modifies less the image content than the other methods, Uniformisation is achieved by increasing light where itis darker and reducing it where it is brighter while preserving fine light variations which objects are made of, The reference image is used to identify the regions which are unusually darker or brighter. Turn on this item if you want ound compensation t one each time an image i backs: be achground with acquired. If the method ted requires a referen must be aded before compensation an be done W ige is similar to si but process the image so that contrasts are increased in the areas where lighting variations are adjusted (because these areas tend to be over or under-saturated Test it on your images before using it extensively si is a method invented by one of Regent's employee. The reference image that you provide with the other methods is replaced by an image of the background light variations estimated from the image to analyse (made of objects and background). This method is much slower than the other two method: an be used however in situations where it is impossible to acquire and im. ference or when the image siz changes between each acquired image. Test it on your images before using it extensivelyActivate this command to load a reference image before using one of the two methods of background unevenness compensation that require such image (see previous section for an example of reference image and information about the compensation methods). The reference image must have the same dimensions as the images to analyse (they must be acquired in the same conditions). The referenc image can be applied to as many images you wish and will be effective for as long as the imag acquisition conditions are not modified (same region, aperture, field of view...) ‘The reference image remains visible in the main window until you acquire an image for analysi After that it remains in memory until you quit WinCELL or activate Image / Background Unevenness Compensation / Clear Reference Image but it cannot be displayed again This command deletes the reference image loaded with Image/ Background Compensation | Load Reference Image. 7.4 DO COMPENSATION NOM When Do Compensation Now is activated, the lighting unevenness compensation method selected in Image/Background Unevenness Compensation /Method Parameters is applied to the image displayed in the main window. Original image with uneven background Many subtile tonal variations of these images cannot bbe seen due to the low fidelity of the printing process (compared to a Background compensated image Background compensated image with with the S fi the W ge method, meth:HE DISPLAY fh 4.1 INFORMATION COLORS Information Colors allows to change the colors used by WinCELL to display inf length, number or width over the images and data in the Command area. Select the color to change by clicking it A white rectangle is drawn around the olor to show that it has been selected. 2) lick B Anew window i 5.) To change th active color enter a Red, Green and Blue numerical value or double click one ofthe predefined colors abov ‘mation such as cell he active colori shown her shows the color you hanging, then you modify the RGB Jor, move these scroll bars 4.2 IMAGE AREA This command if for selecting which information is displayed in the Image area and the text size used for it. Note that the colors used for drawing can be changed with Display Information Colors see the preceding command) These items allow to select what s displayed over the image ee colors used to displ image have bee the lumen width line (blue to contrast nthe red walls and the green lumen in ells & ovn in walls) made with paths, the paths name if name has been entered at creati the bout ell (blue), More likely to be us The cell's perimet Note that it will be visible only i Analysts Parometers)is active during the Glick Sma Medion ot ona ofthe tre po analysis. fined toxt sizes forthe information shown over the imag Choose Other and enter the size value (in points) if none of th the bounding rectangle that three pre-define fencompasses the analysed reg a N\lines connect Av" bounding: meter are drawn in <0, whe passing all is active (in Analysis /Paramet. objects of a group and 2) play (previous figur Circles (in pink) spread across the object cent elects the method The number ofcireles indicate in how many sub-object ntify the merged the object has been splitted (2 in this exampl Lines (in pink) spread across the object cent The number of lines indicate in how many sub-objec the object has been splitted (2 in this examp This command allows to select which information (global and individual) is displayed in the Command area and the text size used for it ‘Todisplay a measurement in the C activate its item (see also “DATA IN THE COMMAND AREA on page on (without the exclusion region The valid cells' lumen total area in calibration unit oo ‘et ir) The valid els amen teal area in perentage of anal «| \Bemesenrs (2 Sea Aru: The valid ell lumen average area cimeee |e tain ‘ The valid els lumen average Tength z Meer, | © Wives Width The valid cells lumen average maximum with 4 ge os 4 fray peer a The valid cell’ average perimeter N The number of valid and Parenchyma The number of valid cells which have 28) or splitted (“Cells SPLITTING + group or split GROUPING on fell rouped (see “CEI & P: the cells identification number (blue) and their type (C=Cell, V and P=Parenchyma Ara: the lumen a Click M 3F Large to choose on the lumen the three pre-defined text sizes fi W the walls length aformation shown in the Command Width: the lumen width Choos and enter the size valu c gif: the cell length (lumen plus walls’ length points) if none of the three pre-defined siz ponds to your need: the lumen perimeter (boundary length ation is displayed, you can increase WinCELLs main window ribed ahove or turn of the display of some information not neededtivate this command to cancel the last drawing stroke of image edition. You can also press th Backspace key. An edition stroke can only be deleted right after it has been crea another one or before returning to the Ana before doing sis working mode dition. After activatir After one edition ‘This command allows to 1o0se which color channel(s) of a color image are used for visualization and analysis. A color image has brightness information in three wavelength bands; red, green and blu which are combined so that we perceive color as if we were looking at the scene directly. Depending on the scene, some channels might exhibit more contrasts between the background and the objects to be measured, hence making pixels classification easier. You can also display, analyse and save to a tiff file any of these image channels. Color channel selection should always be used with a Grey levels based pixels classification because once a channe ther than RGB is selected, the image reverts t levels image (containing only the light intensities of the selected channel The ems and is active only before the analysis. Color (RGB) is se fault for a color in Below are three images viewed with the five available cha iThis command cancels the current analysis. The image is kept in memory and can be reanalysed Data from the cancelled analysis are removed from the data file (if it is still open and the analy was the last saved) This command cancels the current analysis (see previous section above) and starts a new one. This, command can be used for example after WinCELL has interrupted the analysis after a warning or error. If for example you turn on color analysis and forget to load or define color classes and try to analyse an image WinCELL will inform you and stop the analy classes, activate Analysis / Restart Analysis s. After you have loaded the color This command allows you to create an analysed region at the same position and of the same shape and size as the last region created. The parameters for the last region are saved in the configuration file. This allows you to recreate the region even after quitting and restarting WinCELL. 4 EDIT SAMPLE IDENTIFICATION This command allows you to review and edit the sample identification information entered when th image was loaded. When Analysis / Edit Sample Identification is activated, the sample identification window used to identify the sample is displayed. Edit the entry field(s) you want to modify and click Ok to save the modifications or Cancel to leave the identification unchanged ‘The Sample identification window. IBN These two items are explained in “YEAR BEGINNING AND ENDING” on patThis command allows to set some generic behaviors of WinCELL These items determine what WinCELL does when an i nalysed and saved is loaded. If « is active, it will chang ge was analysed and will redo the ions to the analysis, IF ettings to those that were active when the i analysis. You can then view or make modifica active, WinCELL will ignore the analysis and simply load the image and will keep When Ast to sition is active, WinCELL offers the opportunity to save th out eh analysis) after their acquisition, ARer each acquisition, a standard window for creating a document is displayed Enter the image name and click Sav (to save the image otherwise cli AMETERS The Parameters command, allows you to choose which measurements will be done during the analysis and to set other analysis parameters, When this item is active, individual cells are measured (see “INDIVIDUAL ANALYSIS AND INDIVIDUA! OR ANALYSIS" on page 27) along with global analysis (“THE ANALYSES” on p don to activate other measurements like cell length and width and some cell paths (see “INTERACTIVE ANALYSES Indiv tasting and non-touching objects, By default WinCELL mages with a dark background (the walls) and white or light objects (cells). Ifyou analyse other objects you the proper setting in Analysis/ Pixels classific cou PATHS" on pa expects to analyse I ‘analysis must be analysis works only with e i. Activate this control to automatically measure cell ‘Anais Parameters length and width. Ifyou don't need these measurements, it is recommended hat this item be turned off as it slows down the analysis Eee Sorat va F When this item is active, the cells perimeter Canty tending tin OE we and form coefiicient are calculated. The form coefficient is; 4piA/P? wher Azcell area, P= cell perimeter, Itcan take values between 0 and 1, 1 being perfect circle a When this control is activ WinCELL will separate cells and vessels based on their areas. All objects with an area smaller than this value will be classified cells and those with area larger than it will be classified a B ‘The value you enter here sets vessels, fan analysed image is displayed when this value is, the width of the excluded ni border of the image to the n 1 (in pixels) From the mn boundary. A value of ‘The mplete cell hanged, the image must be reanalysed for the classification to be effective. Cells are displayed in n the analysed imag zero is equivalent to analysing the whole in while vessels are displayed in green. The classification of acell or , resselcan also be changed interactively (see Appendix C). which are truncated by the image boundary) from the Activate when analysed region. This value is used only when you rou want that cell trune by the image boundar nalyse the whole im: re not analysed (drawn in red) or deactivate it include them. th too Activate this item if you want WinCELL to activate seni to Interactive measurement mode INTERACTIVE er ANALYSES (PATHSY' on pe after the imag! Bs has been analysed automatically nrrect i See “ANALYSE LS AND REGIONS’ BOUNDARIES" on mation about the border width.Wher a w length and ingth and width width are measured as the norizontal and vertical sz vertical size ofthe Lin its centre of he bounding box on t right is delimited by points s the vertical distance between ( and |) at ght, tum on Dioplaytmage Area i vik satal postion the and th methods, \ and I! doesn't have pe at the same vertical position and ( and 1) doesn’t have to b Wher the same horizontal positon active, the length When porizontal distanc distance hetween the tw tween the tw points on the cell boundar arthest cell that are the farthest apar boundary points of and !), The automat ame vertical width is measured position, For perpendicular tot ‘example, in th automatic length at th image on the right, the length measured is the distance position where the width J between point \and point |, which areon the same measurement is the largest vertical position. The width is the vertical distan and |)). This method is very general in the sense that it eaz between point ( and point |), which are on the same analyse any object and the returned length will always be in the horizontal position, objects main axis direetion. Walls are not calculated in this meth With the and the M thods, Length is always in the horizontal direction and Width is always in the vertical direction. Depending on the orientation of the wood sample in the image, these can correspond to radial and longitudinal cell size. These 3 methods also calculate the left and right walls of each cell Filtering is available to prevent image defects to be considered as cells. Objects with an area larger or smaller than a specific value can be filtered out. Objects rejected from the analysis with filters or manually by clicking them are considered to belong to the background (they are counted in th background area). If you prefer to exclude them completely from the analysis (so that they are not counted in objects and background area or any measurement data), you should use exclusion region instead (see “THE EXCLUSION MENU” on page 66 and “ANALYSED AND EXCLUSION REGIONS on page 24 To turn on small objects filtering, activate nd enter the threshold value, Objects smaller than the threshold will ‘dered as debris and will not be analysed as cells, To activate large objects filtering, select f and enter the thre alue on its right, Objects larger than the threshold will not be analysed as cells, Re i lisplayed in dark red in the analysed image. In the example to the right, an isolated white spot in thy on filtered out. An object ca \y be rejected from the sis (and later reincluded) by pressing the Ctr! key and clickingPIXELS CLASSIFICATION In order to measure cell area or morphology, WinCELL must fir: surrounding background (walls). This s parate the cells from their ep is done according to the settings in Pixels Classification (see “COLOR ANALYSIS’ on page 35 This command also allows you to turn on or off color analy h is displayed to help you he visualize how li nsities are distributed among cells and the _/ shows how many pixels there are per grey background. The 0 line represents the selected threshold valu level, The numbers of pixels are normalised divided by the total number of pixels in the image). Classes with longer lines (toward the top of the graphic) have more pixels. This bar shows the relative intensities of the grey levels (0 is black and 255 or the maximum histogram value is white) The minimum and maximum grey levels present in the image are displayed here and an estimation of how many bits per pixels are required to store this range (left side). The latter is provided for the Pro version which can open files with more than 8 bits per pixels images. I indicates the grey levels range used in the image The Thresh ‘The Log button toggles the button is for histogram vertical seale (number visualizing the result of of pixels) between a linear (figure the application of the under) and logarithmic (above Coxpressed a8 8,9, 10, 12,14, 0F 16 bits per selected pixels figure) representation. The log re classification method scale allows to more easily view When the grey levels pixels classification method is and parameters to the small and very large values active, itis important to indicate WinCELL the original image relative grey levels ofthe cells and their The Manual button background, Itean be Dark cs on W allows to select the ‘ or F a ° default Pale ces on a Black background is acti threshold value manually analyse dark cells on a pale background, select Dark cells on White background instead. You might need this setting if you decide to change the default background behind cells, MANUALLY” on page 63 for more information, For image, the histogram can display separately or simultaneously * The grey levels distribution of the color image transformed into a grey lew and grey. To view it, activate * The red channel displays the light intensity distribution of the red sensors of the camera. Thi data line is shown in red. To view it, activate The green channel displays the light intensity distribution of the green sensors of the camera. This data line is shown in green. To view it, * The blue channel displays the light intensity distribution of the blue sensors of the camera. TRIS data line is shown in blue. To view it, activate B Pro version) When this method is active, color information is used to classify pixels into cells and background. Unlike the method based on grey levels, this method uses many criteria to elassify pixels. Color classification in WinCELL is more than just a color threshold, itis a multilevel pixels segmentation. This method is deseribed in “COLOR ANALYSIS" on page 35. Bass shold When this method is active, WinCELL looks at the intensity of each pixel to decide if they belong to the cells or the background, All pixels brighter than a threshold value are classified as background, the others as This is best described in “HOW THRESHOLDING WORKS" on page 62