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Ilastik_Nuc_V9_20181203.m
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Ilastik_Nuc_V9_20181203.m
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%Name: Ilastik_Nuc_V9
%Date last modified: 2018/12/03, debugging
%Author: Gautam Dey
%Collaborators: Scott Curran
%Goal: Extract information from Ilastik segmentations; with nuclear mask; includes binning
%%%%%%%Notes%%%%%%
%Note for future: combination of Canny and Prewitt edge detectors
%actually do a great job of picking out both internal and external
%features- in case we need to replace Ilastik with deterministic
%segmenting at any point
%initialise
clear; close all
%load files
namefrag='sc317_cdc25-mNG';
indir='/Users/Gautam/Dropbox/Collaborations/Pombe Segmentation files/Nuclei Segmentation/';
%filenames
fname_bf=[namefrag '_BF.tif'];
fname_ng=[namefrag '_MAX3-7.tif'];
fname_mask1=[namefrag '_seg.tif'];
fname_mask2=[namefrag '_nuc.tif']; %nuclear mask
fname_mask_output=[namefrag '_seg_split.tif'];
%number of frames in stack
numframes=27;
%analysis parameters
solidity_thresh=0.959; %for kinked cells
large_disk=20; small_disk=12; %septation filter
minsize=4000; %minimum cell size
pixelsize=0.0651;
bin_min=5; bin_max=25; bin_step=0.25;
background_value=0; %change if not wildtype
splitornot=0; %1 for split, 0 for not
botperc=2; topperc=98; %plotting percentiles
%open file for writing
fileID1=fopen([indir namefrag '_raw_data.txt'],'w'); %15 columns, currently
fprintf(fileID1,'%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\n','Frame','Cell','Frame_Cell','Length','Area','Intensity','Mean','Num_Nuclei','Nuc_Area','Nuc_Int','Nuc_Mean','Cyto_Area','Cyto_Int','Cyto_Mean','Nuc/Total');
%loop through stack
totdata_cell=[]; totdata_nuc=[]; cntr1=0;
for f=1:(numframes)
%input
%mask
cellmask1=tiffread([indir fname_mask1],f);
cellmask1=cellmask1.data; cellmask1=~cellmask1;
%nuclear mask
nucmask=tiffread([indir fname_mask2],f);
nucmask=nucmask.data; nucmask=~nucmask;
nucmasklbl=bwlabel(nucmask);
%fluorescence
cellng=tiffread([indir fname_ng],f);
cellng=cellng.data;
%background subtraction
cellng_sub=cellng-background_value; %negative values eliminated
%Remove edge objects 2018/07/11
cellmask1=imclearborder(cellmask1);
%thresholding
if splitornot == 1
%First identify kinked cells
kinkedcells=bwpropfilt(cellmask1,'Solidity',[0 solidity_thresh]);
kinkmarker1=imerode(kinkedcells,strel('disk',large_disk));
cellmarker1=cellmask1; cellmarker1(kinkedcells==1)=0;
%marker-based watershed
cellmarker1=imerode(cellmarker1,strel('disk',small_disk));
cellmarker1=im2bw(cellmarker1+kinkmarker1); %#ok<*IM2BW>
cellwat1=watershed(bwdist(cellmarker1));
cellmask1split=cellmask1; cellmask1split(cellwat1==0)=0;
%clear edges and any remaining noise
cellmask1split=bwareaopen(cellmask1split,minsize);
cellmask1split=imclearborder(cellmask1split);
else
cellmask1split=cellmask1;
end
%save masks to multipage tiff
cellmasklbl=bwlabel(cellmask1split); %2018/07/11
cellmasklbl=uint16(cellmasklbl);
%cytoplasm only
cellmask1cyto=cellmasklbl;
cellmask1cyto(nucmask>0)=0;
if f==1
imwrite(cellmasklbl,[indir fname_mask_output],'Compression','none');
else
imwrite(cellmasklbl,[indir fname_mask_output],'Compression','none','WriteMode','append');
end
%cell length + intensity measurements
celldata=regionprops(cellmask1split,'Extrema','PixelIdxList','Area');
%cytoplasm measurements
cytodata=regionprops(cellmask1cyto,'PixelIdxList','Area');
%nuclear measurements
nucdata=regionprops(nucmask,'PixelIdxList','Area');
for c=1:length(celldata)
%identifying matching nuclei
nucids=unique(nucmasklbl(cellmasklbl==c));
nucids=nucids(nucids>0);
%Finding longest axis
cext=celldata(c).Extrema;
D=pdist(cext); clength=max(D);
%intensity in cell (nucleus + cytoplasm)
cint=cellng_sub(celldata(c).PixelIdxList);
cint=sort(cint,'Descend');
%intensity in cytoplasm
cytoint=sum(cellng_sub(cytodata(c).PixelIdxList));
cytoarea=cytodata(c).Area;
%looping through nuclei
if ~isempty(nucids)
%track
cntr1=cntr1+1;
nucint=[]; nucarea=[];
for n=1:length(nucids)
nucarea=[nucarea; nucdata(nucids(n)).Area];
nucidx=nucdata(nucids(n)).PixelIdxList;
nucint=[nucint; sum(cellng_sub(nucidx))];
end
nucint=sum(nucint); nucarea=sum(nucarea); %combining nuclei
%storing values;
%totdata_cell=[totdata_cell; 1:f 2:cntr1 3:c 4:clength.*pixelsize 5:celldata(c).Area 6:sum(cint) 7:mean(cint) 8:length(nucids) 9:nucarea 10:nucint 11:nucint./nucarea 12:cytoarea 13:cytoint 14:cytoint./cytoarea 15:nucint./sum(cint)];
totdata_cell=[totdata_cell; f cntr1 c clength.*pixelsize celldata(c).Area sum(cint) mean(cint) length(nucids) nucarea nucint nucint./nucarea cytoarea cytoint cytoint./cytoarea nucint./sum(cint)]; %#ok<*AGROW>
fprintf(fileID1,'%6.2f\t%6.2f\t%6.2f\t%6.2f\t%10.2f\t%12.3f\t%12.3f\t%4.0f\t%10.2f\t%12.3f\t%10.2f\t%10.2f\t%12.3f\t%10.2f%12.3f\n',totdata_cell(cntr1,:));
disp([f c])
end
end
end
fclose(fileID1);
%%
%%%% plotting routines %%%%%
%total data binning
fileID2=fopen([indir namefrag '_binned_data_singlenuc.txt'],'w');
fprintf(fileID2,'%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\n','Bin_Centre','Bin_Count','Intensity','Avg_Intensity','Nuc_Intensity','Nuc_Mean_Intensity','Nuc/Total_Ratio','Intensity_SD','Avg_Intensity_SD','Nuc_Intensity_SD','Nuc_Mean_Intensity_SD','Nuc/Total_Ratio_SD');
fileID3=fopen([indir namefrag '_binned_data_multinuc.txt'],'w');
fprintf(fileID3,'%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\n','Bin_Centre','Bin_Count','Intensity','Avg_Intensity','Nuc_Intensity','Nuc_Mean_Intensity','Nuc/Total_Ratio','Intensity_SD','Avg_Intensity_SD','Nuc_Intensity_SD','Nuc_Mean_Intensity_SD','Nuc/Total_Ratio_SD');
%plot vectors
%order: [1:f 2:cntr1 3:c 4:clength.*pixelsize 5:celldata(c).Area 6:sum(cint)
%7:mean(cint) 8:length(nucids) 9:nucarea 10:nucint 11:nucint./nucarea 12:cytoarea 13:cytoint 14:cytoint./cytoarea 15:nucint./sum(cint)]
singlenuc=find(totdata_cell(:,8)==1);
multinuc=find(totdata_cell(:,8)>1);
lvec1=totdata_cell(singlenuc,4);
ivec1=totdata_cell(singlenuc,6);
ivec_mn1=totdata_cell(singlenuc,7);
ivec_nuc1=totdata_cell(singlenuc,10);
ivec_nuc_mn1=totdata_cell(singlenuc,11);
ivec_ratio1=totdata_cell(singlenuc,15);
lvec2=totdata_cell(multinuc,4);
ivec2=totdata_cell(multinuc,6);
ivec_mn2=totdata_cell(multinuc,7);
ivec_nuc2=totdata_cell(multinuc,10);
ivec_nuc_mn2=totdata_cell(multinuc,11);
ivec_ratio2=totdata_cell(multinuc,15);
%binning by length
%binvec=(1.1*min(lvec)):0.25:(0.9*max(lvec));
binvec=bin_min:bin_step:bin_max;
binmat_mn1=zeros(length(binvec)-1,5); binmat_sd1=zeros(length(binvec)-1,5); bcent=zeros(length(binvec)-1,1);
binmat_mn2=binmat_mn1; binmat_sd2=binmat_sd1;
for b=1:(length(binvec)-1)
%bin centres
bcent(b)=binvec(b)+((binvec(b+1)-binvec(b))./2);
%binning each vector
bin_ind1=find(lvec1<binvec(b+1) & lvec1>=binvec(b));
binmat_mn1(b,:)=[mean(ivec1(bin_ind1)) mean(ivec_mn1(bin_ind1)) mean(ivec_nuc1(bin_ind1)) mean(ivec_nuc_mn1(bin_ind1)) mean(ivec_ratio1(bin_ind1))];
binmat_sd1(b,:)=[std(ivec1(bin_ind1)) std(ivec_mn1(bin_ind1)) std(ivec_nuc1(bin_ind1)) std(ivec_nuc_mn1(bin_ind1)) std(ivec_ratio1(bin_ind1))];
bin_ind2=find(lvec2<binvec(b+1) & lvec2>=binvec(b));
binmat_mn2(b,:)=[mean(ivec2(bin_ind2)) mean(ivec_mn2(bin_ind2)) mean(ivec_nuc2(bin_ind2)) mean(ivec_nuc_mn2(bin_ind2)) mean(ivec_ratio2(bin_ind2))];
binmat_sd2(b,:)=[std(ivec2(bin_ind2)) std(ivec_mn2(bin_ind2)) std(ivec_nuc2(bin_ind2)) std(ivec_nuc_mn2(bin_ind2)) std(ivec_ratio2(bin_ind2))];
%print to file
fprintf(fileID2,'%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\n',[bcent(b) length(bin_ind1) binmat_mn1(b,:) binmat_sd1(b,:)]);
fprintf(fileID3,'%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\t%10.2f\n',[bcent(b) length(bin_ind2) binmat_mn2(b,:) binmat_sd2(b,:)]);
end
fclose(fileID2);
%%
%colormaps
c1=([211 224 237])./255;
c2=([45 98 173])./255;
c3=([44 162 95])./255;
c4=([153 216 201])./255;
%set x axis
xmin=bin_min; xmax=bin_max;
%plotting
figure;
subplot(5,1,1); hold on
plot(lvec1,ivec1,'.','MarkerFaceColor',c1,'MarkerEdgeColor',c1); errorbar(bcent,binmat_mn1(:,1),binmat_sd1(:,1),'.','Color',c2,'MarkerFaceColor',c2,'MarkerEdgeColor',c2);
plot(lvec2,ivec2,'.','MarkerFaceColor',c3,'MarkerEdgeColor',c3); errorbar(bcent,binmat_mn2(:,1),binmat_sd2(:,1),'.','Color',c4,'MarkerFaceColor',c4,'MarkerEdgeColor',c4);
axis([xmin xmax prctile(ivec1,botperc) prctile(ivec1,topperc)]);
title(namefrag); xlabel('Cell length'); ylabel('Intensity');
subplot(5,1,2); hold on
plot(lvec1,ivec_mn1,'.','MarkerFaceColor',c1,'MarkerEdgeColor',c1); errorbar(bcent,binmat_mn1(:,2),binmat_sd1(:,2),'.','Color',c2,'MarkerFaceColor',c2,'MarkerEdgeColor',c2);
plot(lvec2,ivec_mn2,'.','MarkerFaceColor',c3,'MarkerEdgeColor',c3); errorbar(bcent,binmat_mn2(:,2),binmat_sd2(:,2),'.','Color',c4,'MarkerFaceColor',c4,'MarkerEdgeColor',c4);
axis([xmin xmax prctile(ivec_mn1,botperc) prctile(ivec_mn1,topperc)]);
title(namefrag); xlabel('Cell length'); ylabel('Average intensity');
subplot(5,1,3); hold on
plot(lvec1,ivec_nuc1,'.','MarkerFaceColor',c1,'MarkerEdgeColor',c1); errorbar(bcent,binmat_mn1(:,3),binmat_sd1(:,3),'.','Color',c2,'MarkerFaceColor',c2,'MarkerEdgeColor',c2);
plot(lvec2,ivec_nuc2,'.','MarkerFaceColor',c3,'MarkerEdgeColor',c3); errorbar(bcent,binmat_mn2(:,3),binmat_sd2(:,3),'.','Color',c4,'MarkerFaceColor',c4,'MarkerEdgeColor',c4);
axis([xmin xmax prctile(ivec_nuc1,botperc) prctile(ivec_nuc1,topperc)]);
title(namefrag); xlabel('Cell length'); ylabel('Nuclear Intensity');
subplot(5,1,4); hold on
plot(lvec1,ivec_nuc_mn1,'.','MarkerFaceColor',c1,'MarkerEdgeColor',c1); errorbar(bcent,binmat_mn1(:,4),binmat_sd1(:,4),'.','Color',c2,'MarkerFaceColor',c2,'MarkerEdgeColor',c2);
plot(lvec2,ivec_nuc_mn2,'.','MarkerFaceColor',c3,'MarkerEdgeColor',c3); errorbar(bcent,binmat_mn2(:,4),binmat_sd2(:,4),'.','Color',c4,'MarkerFaceColor',c4,'MarkerEdgeColor',c4);
axis([xmin xmax prctile(ivec_nuc_mn1,botperc) prctile(ivec_nuc_mn1,topperc)]);
title(namefrag); xlabel('Cell length'); ylabel('Avg Nuclear Intensity');
subplot(5,1,5); hold on
plot(lvec1,ivec_ratio1,'.','MarkerFaceColor',c1,'MarkerEdgeColor',c1); errorbar(bcent,binmat_mn1(:,5),binmat_sd1(:,5),'.','Color',c2,'MarkerFaceColor',c2,'MarkerEdgeColor',c2);
plot(lvec2,ivec_ratio2,'.','MarkerFaceColor',c3,'MarkerEdgeColor',c3); errorbar(bcent,binmat_mn2(:,5),binmat_sd2(:,5),'.','Color',c4,'MarkerFaceColor',c4,'MarkerEdgeColor',c4);
axis([xmin xmax prctile(ivec_ratio1,botperc) prctile(ivec_ratio1,topperc)]);
title(namefrag); xlabel('Cell length'); ylabel('Nuclear/Total Ratio');
%print figure to folder
print([indir namefrag '_plots'],'-dpdf','-r300','-fillpage')