Automatic Nuclei Count tif stacks

I am using this borrowed code to automatically count DAPI stained nuclei:

Hs.dirPath = pwd;
[Hs.dataFiles,Hs.dataNums] = getDataFiles(Hs.dirPath);
[Hs.foundChannels,Hs.fileNums,Hs.imgExts] = getImageFiles(Hs.dirPath);
for i = 1:length(Hs.fileNums)
    Hs.fileNum = Hs.fileNums(i);    
    fileName = ['dapi' sprintf('%03d',Hs.fileNum) '.tif'];    
    Hs.DI = readmm(fileName);
    Hs.DI = Hs.DI.imagedata;
    Hs.DI = scale(max(Hs.DI(:,:,round(linspace(10,size(Hs.DI,10),50))),[],3));
    binDapi = Hs.DI>adaptthresh(Hs.DI);
                masktmp = imclearborder(binDapi | ~Hs.DI);
                masktmp = bwareaopen(masktmp,50); 
                if any(masktmp(:))
                    binDapi = masktmp;
                end      
    L = bwlabel(binDapi);
    numObjects = max(L,[],1:2);
    Hs.currObjs = [];
    Hs.allMasks = [];
    for j = 1:numObjects
        fnumStr = sprintf('%03d',Hs.fileNum);
        objMask = L == j;
        newObj = improc2.buildImageObject(objMask, fnumStr, Hs.dirPath);
        Hs.currObjs = [Hs.currObjs, newObj];
        Hs.allMasks = cat(3,Hs.allMasks,objMask);
    end

    objects = Hs.currObjs;
    save(sprintf('%s%sdata%03d.mat',Hs.dirPath,filesep,Hs.fileNum),'objects');
    [Hs.dataFiles,Hs.dataNums] = getDataFiles(Hs.dirPath);
    clear objects;
    Hs.currObjs = [];
end

It ends up fusing nuclei that are on different planes. In the image shown here (Matlab Output on the left and original on the right)there should be 14 nuclei but 1 and 6, 2 and 5 and 3 and 7 end up being counted as the same.

I have tried changing the threshold etc. but it does not help with the stacks.

Any suggestions to modify the code? Thanks.

Answer 1

[Abhinav Aravindan]

I understand that you are encountering an issue with counting the number of nuclei in a TIF image, specifically when two nuclei are mistakenly counted as one. I recommend referring to the example in the “Image Processing Toolbox” on how to “Detect and Count Cell Nuclei in Whole Slide Images”.

I hope this helps solve your problem!

https://www.mathworks.com/help/images/detect-and-count-cell-nuclei-in-wsi-using-blocked-image.html