Matlab code

Distribution code

clear all;

close all;

date = 'G:\XX'; %file path

sample = 'XX';%folder

MChan = 2;

Nmppix = 130;

OrigDim = 256;

MedFiltM = 1;

FiltSizeM = 100;

ThreshMtoP = 100;

direct1 = sprintf('%s\\%s',date,sample);

cd(direct1)

imgin2 = sprintf('XX',date,sample,sample);

imginM = imread(imgin2);

imM = imginM(:,:,MChan);

Zoom = (length(imM)) / OrigDim;

TrueNumppix = Nmppix / Zoom;

%%

imMMed = medfilt2(imM, [MedFiltM MedFiltM]);

ThreshM = graythresh(imMMed);

imMMedThresh = zeros(size(imMMed));

imMMedThresh = im2bw(imMMed, ThreshM);

TrueFiltSizeM = FiltSizeM / TrueNumppix;

TrueFiltSizeM = round(TrueFiltSizeM);

imMMedThreshFilt = bwareaopen(imMMedThresh, TrueFiltSizeM, 8);

SE = strel ('square', 3);

imMMedThreshFiltD = imdilate (imMMedThreshFilt, SE);

figure(1)

subplot(2, 2, 1);

imshow(imM);

drawnow;

caption = sprintf('Original Image');

title(caption);

axis square;

subplot(2, 2, 2);

imshow(imMMed);

drawnow;

caption = sprintf('Median Filtered Image');

title(caption);

axis square;

subplot(2, 2, 3);

imshow(imMMedThresh);

drawnow;

caption = sprintf('Median & Thresholded Image');

title(caption);

axis square;

subplot(2, 2, 4);

imMBd = bwperim(imMMedThreshFiltD, 8);

imshow(imMBd);

drawnow;

caption = sprintf('Identified boundaries, dilated 1 pixel');

title(caption);

axis square;

saveas(figure (2), 'XX.jpg');

%%

ccM = bwconncomp(imMMedThreshFiltD, 8);

Lm1 = labelmatrix(ccM);

RpM1 = regionprops(Lm1, 'all');

[DM, LmM2] = bwdist(imMMedThreshFiltD);

TrueThreshMtoP = ThreshMtoP/TrueNumppix;

DM2 = DM <= TrueThreshMtoP;

PUR = imMMedThreshFilt;

FN = numel(RpM1);

overlay = zeros(size(imginM));

overlay(:,:,2) = imMMedThreshFilt;

overlay(:,:,1) = imMMedThreshFiltD;

overlay(:,:,3) = PUR;

[DM, LmM2] = bwdist(imMMedThreshFiltD);

TrueThreshMtoP = ThreshMtoP/TrueNumppix;

DM2 = DM <= TrueThreshMtoP;

OL1 = DM2 & imMMedThreshFilt;

ccOL = bwconncomp(OL1, 8);

LOL1 = labelmatrix(ccOL);

RpOL1 = regionprops(LOL1, 'all');

OLIdx = RpOL1(1,1).PixelIdxList;

for l = 2:numel(RpOL1)

OLId = RpOL1(l,1).PixelIdxList;

OLIdx = [OLIdx; OLId];

end

figure(2)

imshow(overlay);

drawnow;

caption = sprintf('OverlapImage');

title(caption);

axis square;

saveas(figure (2), 'OverlapImage.jpg');

ccFinal = bwconncomp(PUR, 8);

LFinal = labelmatrix(ccFinal);

RpFinal = regionprops(LFinal, 'all');

ColocPercent = (numel(RpFinal)/numel(RpM1))*100;

OverlapAreaFract = (sum([RpOL1.Area])/sum([RpM1.Area]))*100;

%%

imcomb = or(imM, imM);

blurcomb = imfilter(imcomb,fspecial('gaussian',2,1),'same','replicate');

%imshow(blurcomb)

cellbound = roipoly(blurcomb);

[nucbound, xi, yi] = roipoly(blurcomb & cellbound);

centx=mean(xi);

centy=mean(yi);

%[centx, centy] = centroid(xi, yi);

nuccent = [centx centy];

cellmask = cellbound & ~nucbound;

figure(3);

imshow(cellmask)

saveas(figure (1), 'cellmask.jpg');

%%

figure(4)

cellmaskdouble=double(cellmask);

bw = imfill(cellmaskdouble,'holes');

cc = bwconncomp(bw);

[out,LM] = bwferet(cc,'MaxFeretProperties');

maxLabel = max(LM(:));

h = imshow(LM,[]);

axiss = h.Parent;

for labelvalues = 1:maxLabel

xmax = [out.MaxCoordinates{labelvalues}(1,1) out.MaxCoordinates{labelvalues}(2,1)];

ymax = [out.MaxCoordinates{labelvalues}(1,2) out.MaxCoordinates{labelvalues}(2,2)];

imdistline(axiss,xmax,ymax);

a=imdistline(axiss,xmax,ymax);

dist = getDistance(a);

diameter=dist*TrueNumppix/1000;

end

title(axiss,'Maximum Feret Diameter of Objects');

figure(5)

Halfdiameter=ceil(diameter)/2;

bins = 0:0.1:Halfdiameter;

ER= cellmask & Lm1;

ER2 = bwconncomp(ER, 8);

ER2mask = labelmatrix(ER2);

ER2mask = regionprops(ER2mask, 'all');

ER22 = cat(1,ER2mask.PixelList);

ER2 = pdist2 (ER22, nuccent);

ER2 = (ER2.*TrueNumppix)/1000;

hMER = hist(ER2, bins);

hist(ER2, bins);

caption = sprintf('ER distribution');

title(caption);

xlabel('Distance to nuclear center')

ylabel ('#Positive pixels')

axis square;

xlim([0 Halfdiameter]);

figure(6)

Halfdiameter=ceil(diameter)/2;

bins = 0:0.1:Halfdiameter;

cellmask= cellmask;

cellmask2 = bwconncomp(cellmask, 8);

cellmask2mask = labelmatrix(cellmask2);

cellmask2mask = regionprops(cellmask2mask, 'all');

cellmask22 = cat(1,cellmask2mask.PixelList);

cellmask2 = pdist2 (cellmask22, nuccent);

cellmask2 = (cellmask2.*TrueNumppix)/1000;

hMcellmask = hist(cellmask2, bins);

%hMNorm = hist(ER2, binsNorm);

hist(cellmask2, bins);

caption = sprintf('cellmask distribution');

title(caption);

xlabel('Distance to nuclear center')

ylabel ('#Positive pixels')

axis square;

xlim([0 Halfdiameter]);

figure(7)

dist=hMER./hMcellmask ;

nbin=0:0.1:Halfdiameter;

bar(nbin,dist);

caption = sprintf('ER distribution');

title(caption);

xlabel('Distance to nuclear center')

ylabel ('#Positive pixels')

axis square;

xlim([0 Halfdiameter]);