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==How to use Matlab to identify features in microscope images?== *This is a short tutorial on using [http://arep.med.harvard.edu/kzhang/polHap/DataAnalysis.html PISA] for feature identification under Matlab. *Here we start with a [[Media:Series021_20xobj-Zstack-2kx2k_MaxProject_Cy5.tif|FISSEQ rolony image generated by Hosuk]]. The goal is to find how many rolonies there are in this image using an algorithm. Here we use the PISA tool that was previous developed for polony haplotyping. *To run this tutorial, you need to download two files ([[Media:PISA7.m|PISA7.m]] [[Media:PISA7.fig|PISA7.fig]]) and place them in your Matlab default or path. % Load the raw image img_raw=imread('Series021_20xobj-Zstack-2kx2k_MaxProject_Cy5.tif','tiff'); % Perform median filtering to remove some speckles img_flt=medfilt2(img_raw,[3 3]); % Find out the dimension of the image [xsize ysize]=size(img_raw); % Find out the near saturated intensity of the image for adjusting the contrast hi=prctile(double(reshape(img_flt,1,xsize*ysize)),99.9); % Prepare the image for segmentation img_double=double(img_flt)/hi; % Create a mask for analyzing a specific region of interest (There are all ones in this matrix, so the wholge image is processed.) mask=ones(xsize,ysize); % Identification of polonies using PISA. When adjusting parameters, click "Process ROI" first to find out how the new parameters work. Once you find the best parameters, click "Process All". % bw_img is a black/white image of all polonies identified; % imgsegdata contains all morphological information of all polonies found. [bw_img imgsegdata]=PISA7(img_double,mask); [[Image: PISA_gui.png|400px]] % Check how many polonies were identified (bw_count). [bw_count dummy]=size(imgsegdata); % Create a composite image so that you can visually check how many polonies were correctly or incorrectly identified. comp_img=zeros(2048,2048,3); comp_img(:,:,1)=img_double; comp_img(:,:,3)=bw_img; imwrite(comp_img,'test_composite_img.png','png'); % Using an image editor, you can manually identify missing polonies by painting them in white circles. [[Image: PISA_feature_inspection_editing.png|400px]] *Further analyses can be perform on imgsegdata or the edited composite image.
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