ZebIAT, Zebrafish Image Analysis Tool User's Manual
Contents
1. lmages For visualization purposes registered images with masks are saved into a folder Images_with_masks so that user can see the result of registration Landmarks used in a registration process will be saved into a folder called Points Finally results and figures can be saved into a folder called Results Images both fish and masks should be named in the following way f fish_no _ development_stage d tif file format of the image can be different if MATLAB s imread supports it For example if there are three development stages namely 0 1 and 2 days after injection and for each stage there is four fish images should have names f1_Od tif f2_0d tif 3_Od tif f4_Od tif f1_1d tif f2_1d tif f3_1d tif f4_1d tif f1_2d tif f2_2d tif f3_2d tif f4_2d tif Note that the reference fish and masks should have a names f1_Od tif f1_1d tif and f1_2d tif 3 4 Alignment of reference fish and masks Reference fish and masks are aligned horizontally This is done for visualization purposes as well as minimizing the sizes of images This can be done with a command align_ref_images This function will move the original files into a folder called Old and aligned images will be saved in their place If needed user can then restore the original ones If user does not want to align images horizontally this step is not needed 3 5 Registration Registration is performed by running a command registration registration age fish pick m2. of smaller regions Organs Back Back_labeled Image size 400x2000 Section2 ection4 ectionsS Section6 food fsid fe_2d gt Figure 4 1 E g fish number six at development stage 2 contains fields Organs Back and Back_labeled Organs and back then contain following fields Measurements s x 2 matrix of measurements where s is number of spots First column is the area and second column is the mean intensity of the spot Legend tells the content of the measurements matrix e g Area Intensity Area_organ area of this particular organ section PixelldxList coordinates for each spot Centroid centroid of the spot 4 2 Visualizing the results Zebratool offers several functions to visualize the results of the analysis The obtained data can be visualized with the following commands plot_NumObjects_over_time data age fish plot_area_over_time data age fish plot_intensities data age fish plot_NumObjects data age fish plot_area data age fish plot_heatmap_style_fish data age fish back_lower back_upper The parameter data is the mat file which was saved in the Results folder With the parameters age and fish user can define specific development stage and fish for visualization The last function is meant to visualize the situation when back is divided into smaller sections
3. ZeblAT Zebrafish Image Analysis Tool User s Manual Contents T iNMEROGDUCTION EE E chads doces it ran E E E E E E 1 1 Uses of zebrafish image analysis tOO siisii irisi iseer nsari indera iseis 1 2 Contents of the zebratool program cccccsssecsessecssscessuecssaecseeeecseeseaecseseecsereessaeesssseesenees 2 Running the tool in Matlab ccccescesseceesseeeceeseeeceeseeaeeeeseaaeceeseeaeceseeaaeeeeeeaaeeeeseaaeeecseaueeeeseaas Bs Geine started irar aa A S RIRE A EE EAA EIRA 3 1 Mat rialcnancnnn ea a a A E a EE eee eee 3 2 MaSKS 5 eset a e E E E A E E E E A E eae 3 3 Folder structure and file NAMES 0 0 eee cceeseeceeeeeceeeeeeaaeeeeaeeceeeecsaeeeeaaeeeeaeeceaeeeeaaeeeeaaeeeeaeeee 3 4 Alignment of reference fish and masks ccccsccccessssceseesssceceeseececeesaeeeceesaeeeceesaeeeceeaeeesees 3 5 Registration reee e ERA Eaa Ea Tn EAE EAE Ea EEEa AETR Er aea 3 6 Caudal PESIONS ccenonacais inie aaa A A Results iersi i en i EE iar eislevdnntetaatensgieviasteeeedsbarevaee 4 1 a E E E E E a Sade dab bv aed deewennens 4 2 Visualizing the results ssc sssenetivcstactidareactesszeaaiasssees ces cedaaisuseanteasseaaiebueaetesedaatieteactessteeanivadd 1 Introduction 1 1 Uses of zebrafish image analysis tool Zebrafish image analysis tool is intended to be used to register fluorescent microscopy images and differential interference contrast microcopy images of zebrafish embryos The tool also provides a user interface fo
4. a corresponding point in the moving image bottom By pressing the right mouse button program exits the selection removing mode 3 6 Caudal regions If user wants to divide caudal regions into smaller sections corners of the ventral and dorsal regions must be selected manually This can be done with the function pick_corners age back_label_lower back_label_upper Parameters back_label_lower and back_label_upper are labels for the ventral and dorsal regions in the marked image e g label no 10 and 11 Result mat file containing the corners for the dorsal and ventral regions will then be saved into the folder where the masks are This has to be done only once and only for the reference masks 4 Results After registering procedure images can be analyzed by running a command data get data age fish varargin Age defines the development stage and fish defines what fish are included to analysis Result of the analysis mat file is automatically saved into the Results folder 4 1 Output Result file will be an m x n struct where m is number of fish and n is number of development stages Moreover each fish contains following structure Organs x 1 struct containing information within each organ is number of organs Back k x 1 struct containing information within each region k is number of labeled sections within caudal region Back_labeled a label image where the caudal region has been divided into k number
5. mponents is set to 0 Marking is then done by labeling the organs with a green value 1 n where n is number of the organs This can be done i e using the editor s paint bucket ability Moreover background should be black The resulting mask image should be then same size than the image to be masked and organs must locate in same places File format must be chosen so that MATLAB s imread command supports it e g tif joeg but otherwise it is not limited Result is exemplified in Figure 3 1 If there is e g four development stages each stage should have a reference fish and mask Figure 3 1 Mask of the reference fish organs have been circled with a color value 255 and organs have been labeled with a green color 1 n Background is set to 0 If back is drawn it should be divided into two parts namely into ventral somites and dorsal somites regions like in Figure 3 1 3 3 Folder structure and file names Run create_folder_structure command in your working directory It will create a following folder structure shown in Figure 3 2 in your working directory 4 Di Working directory d Images db Masks 4 di Registered_images ad Images a Images_with_masks L Points a Results Figure 3 2 Folder structure First all images should be copied into a folder called Images and reference masks should be copied into a folder called Masks After thin plate spline procedure registered images are saved into a folder Registered_images
6. ore points DIC Age defines development stages of the fish With parameter fish user can define specific fish for registration Manual selection of landmarks can be enabled by setting pick_more_points to true If user wants to register DIC images D C must set to true For example if user wants to register fluorescence images and specific development stages e g 1 and 3 with specific fish e g 1 2 3 and 5 and with manual selection command would be registration 1 3 1 3 5 1 0 Figure 3 3 shows the user interface when manual selection ability is turned on For more help check the registration m file Bi Figure i o e a H T a i File gdt Yiew Insert Jools Desktop Window Help Istatic pick a point Press RMB to exit from selection remove mode Match amp save Select Remove Next Exit 200 400 600 800 1000 1200 Figure 3 3 User Interface When pressing the Match amp save button the thin plate spline registration is performed and the result is displayed in a popup window If the result is good enough user can click the button Next or Exit and the result will be saved into a folder described in section 3 3 Note that the Exit button will terminate the whole registration process while the Next button proceeds to next image More landmarks can be selected Select or landmarks can be removed Remove Selection of landmarks is done by first selecting a landmark within the reference fish top and then selecting
7. r manual selection of landmarks 1 2 Contents of the zebratool program Zebratool contains several MATLAB functions for registration and data analysis The main program consists of following functions create folder structure align ref images registration age fish pick more points DIC pick corners age back label lower back label upper OPTIONAL data get data age fish varargin There are also several functions for plotting different kind of information Those functions are presented in section 4 2 2 Running the tool in Matlab Zebrafish image analysis tool has been tested on MATLAB R2011b In order for the Matlab version to work Image Processing Toolbox must be installed Extract the contents of the tool archive into a directory e g C zebratool To run the tool change the MATLAB working directory to the directory where the files were extracted and then follow the instructions 3 Getting started 3 1 Material Images should be acquired so that fish s head is on left or they should be rotated so before using the code 3 2 Masks For reference fish relevant organs for a given study must be marked This can be done with numerous photo editing software e g with the Paint NET which is available at http www getpaint net Then open the image to be masked in the editor and draw masks over the organs Borders of the organs should be drawn with a color where one or more components are set to 255 rest of the co
Download Pdf Manuals
Related Search