stained_memb_segmentation.m

close all
clear all
clc

currentfolder=string(pwd)
srcFile = dir(currentfolder+'/*.tif');
image_folder = currentfolder+'/'

golgi_mean=[];
memb_mean=[];
file_name_arr=[];

%%
for n=1:length(srcFile)        
        FileName = strcat(image_folder,srcFile(n).name);
        file_name_arr=[file_name_arr, string(srcFile(n).name)];
        path=strcat(image_folder, srcFile(n).name);

        % read image and split channels
        I1=imread(FileName,1); % channel 1 = HRAS
        figure
        imshow(I1,[])
        I1=medfilt2(I1);
        imshow(I1,[])
        close

        I2=imread(FileName,2); % channel 2 = Golgi
        imshow(I2,[])
        I2=medfilt2(I2);
        imshow(I2,[])
        close
     
        I3=imread(FileName,2); % channel 3 = Membrane
        imshow(I3,[])
        I3=medfilt2(I3);
        imshow(I3,[])
        close
        
%% GOLGI SEGMENTATION
        [level,~] = multithresh(I2); %multi level thresholding of golgi channel
        seg_I2 = imquantize(I2,level);
        RGB_golgi = label2rgb(seg_I2); 	 

        figure;
        imshow(seg_I2,[])
        imshow(RGB_golgi)
        close
        
        imwrite(RGB_golgi, FileName+'_golgi_segment.png','png'); 
        seg=seg_I2-1;
        golgi = immultiply(uint16(seg),I1);

%% MEMBRANE SEGMENTATION

        [level,~] = multithresh(I3); %multi level thresholding of membrane channel
        seg_I3 = imquantize(I3,level);
        RGB_memb = label2rgb(seg_I3); 	 

        figure;
        imshow(seg_I3,[])
        imshow(RGB_memb)
        close
        
        imwrite(RGB_memb, FileName+'_memb_segment.png','png'); 
        seg=seg_I3-1;
        memb = immultiply(uint16(seg),I1);

%%  MEMBRANE HISTOGRAM 

        figure 
        h_memb=histogram(memb,'BinWidth',5, 'BinLimits',[1,4090]); %plot histogram of membrane
        title(string(srcFile(n).name) + ' histogram of membrane')
        data_memb = h_memb.Values;
        counts_memb = data_memb;
        centerBinGrayLevels_memb = (h_memb.BinEdges(1:end-1) + h_memb.BinEdges(2:end)) / 2;
        meanBinnedGrayLevel_memb = sum(centerBinGrayLevels_memb .* counts_memb) / sum(counts_memb);
        memb_mean=[memb_mean,meanBinnedGrayLevel_memb];
        xline(meanBinnedGrayLevel_memb, 'Color', 'g', 'LineWidth', 2);
        saveas(gcf, string(path)+'_memb_mean.png')
        close

%% GOLGI HISTOGRAM 
        figure
        h_golgi=histogram(golgi,'BinWidth',5, 'BinLimits',[1,4090]); %plot histogram of golgi
        title(string(srcFile(n).name) +' histogram of golgi')
        data_golgi = h_golgi.Values;
        counts_golgi = data_golgi;
        centerBinGrayLevels_golgi = (h_golgi.BinEdges(1:end-1) + h_golgi.BinEdges(2:end)) / 2;
        meanBinnedGrayLevel_golgi = sum(centerBinGrayLevels_golgi .* counts_golgi) / sum(counts_golgi);
        golgi_mean=[golgi_mean,meanBinnedGrayLevel_golgi];
        xline(meanBinnedGrayLevel_golgi, 'Color', 'r', 'LineWidth', 2);
        saveas(gcf, string(path) + '_golgi_mean.png')
        close

%% Uncomment to save histogram data
%         data_golgi_filename=string(path_golgi)+'.csv';
%         data_memb_filename=string(path_memb)+'.csv';

%         csvwrite(string(path)+'_golgi_data.csv', transpose(data_golgi));
%         csvwrite(string(path)+'_memb_data.csv', transpose(data_memb));

end

%% Save R_mean data (for Method 1)

csvwrite('memb_mean_data_4090.csv', transpose(memb_mean));
csvwrite('golgi_mean_data_4090.csv', transpose(golgi_mean));
writematrix(transpose(file_name_arr), 'file_names.csv');

% GOLGI/MEMBRANE
mean_ratio=golgi_mean./memb_mean;
csvwrite('mean_ratio_4090.csv', transpose(mean_ratio));