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Copy pathCellRaQ_Main.m
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CellRaQ_Main.m
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%%Script for calculating the RNA expresion levels and nascent RNA site
%%distances.
%P. Aurelio, 2019.
%Matlab R2016a.
clear all
close all
warning off
clc
%% Some inputs:
%%----------------------------------------------------------
%Define starting directory and data types----------------------------------------
%--------------------------------------------------------------
startlocn = {"\\pasteur\SysBC-Home\schuerca\Desktop\ANALYSIS\Data\RPL3\SC\MAX"};
Condition = 'RPL3_SC';
%----------------------------------------------------------------
%Binary inputs------------------------------------------------------
%-----------------------------------------------------------------------
% Save_Results = 0; %Binary: 1 for save the results.
Save_Figures = 0; %Binary: 1 for save ALL the figures automatically in the specified folder.
Save_Results = 1; %Binary: 1 for save calculated variabled in a ,mat file.
Plot_Images = 0; %Binary: 1 for plot all the images.
subROI = 0; %1 for Analyzing subregions (This requires to input the corresponding subROI from ImageJ). If 0, this analysis won't be performed.
%----------------------------------------------------------------------------
%Some Numerical Inputs-------------------------------------------------------------
%-------------------------------------------------------------------------
Pixel_size = 65; %Effective Pixel size in nm
PSF_roi = 5; %Roi size in pixels in order to extract the intensity from the PSFs (PSF_roi x PSF_roi). It must be an odd number.
PSF_bckg_roi = 7; %Roi size in pixels in order to take the background around the PSFs
polyScale_Init = 1; %Initial scaling factor for the polygons of around the condensates. This value is used to start the iteration.
Max_polyScale = 3; %Maximum scaling factor for the polygons of around the condensates.
Bckg_Int_thr = 1.15; %This value multiplied by the average of the minimum intensity values of each PSF of single RNAs will be used to segment the Condensate regions.
Condensates_Intensity_thr = 600; %Intensity threshold for identifying the P-Bodies and perform the colocalization step.
RNA_Condensates_dist_thr = 8; %Distance in pixels in order to recognize that an RNA localization corresponds to a Condensate. Distance between the center of mass of the condensate and the center of the RNA localization.
edges=0:0.1:12; %Histogram of Normalized Intensities edges. (Intensity/MeanIntensity)
%-----------------------------------------------------------------------------
%Initialize Variables----------------------------------------
%%----------------------------------------------------------
%--------------------------------------------------------------
PSF_length = (PSF_roi-1)/2;
PSF_bckg_length = (PSF_bckg_roi-1)/2;
%% Load the Data:
files_RNA_images = Select1DataGroup('RNA Images','.tif',startlocn{1});
nimages = size(files_RNA_images.data,1);
%files_PBodies_images = Select1DataGroup('P-Bodies Images','.tif',startlocn{1});
% if subROI == 1;
% files_ROI{1} = Select1DataGroup('subROI (Fiji)','*.zip;*.roi',startlocn{1});
% end
%Load the files with the list of localizations and the Images
%P-Bodies images and RNA images must have the same number of frames (i.e, the same number of Z stacks)
for c=1:nimages;
if subROI == 1;
[sROI{c}] = ReadImageJROI(strcat(files_ROI{1}.data{c,2},files_ROI{1}.data{c,1}));
end
%Localizations{c} = readtable(strcat(files_localizations.data{c,2},files_localizations.data{c,1}));
Localizations{c} = readtable(strcat(files_RNA_images.data{c,2},files_RNA_images.data{c,1}(1:end-4),'.csv'));
Localizations_Array{c} = table2array(Localizations{c}(:,:));
Localizations_Array{c}(:,2:4) = Localizations_Array{c}(:,2:4)/Pixel_size; %Transform units to pixels.
Number_of_Localizations(c) = size(Localizations_Array{c},1); %Compute the total number of localizations for each image
% Localizations_Array{c}(:,[2,3])= Localizations_Array{c}(:,[3,2]); %Swap X and Y columns (from ImageJ to Matlab).
Im_info{c} = imfinfo(strcat(files_RNA_images.data{c,2},files_RNA_images.data{c,1}));
numberOfFrames = length(Im_info{c});
for k = 1:numberOfFrames;
currentImage = imread(strcat(files_RNA_images.data{c,2},files_RNA_images.data{c,1}), k, 'Info', Im_info{c});
Image_RNA{c}{k} = currentImage;
%currentImage = imread(strcat(files_PBodies_images.data{c,2},files_PBodies_images.data{c,1}), k, 'Info', Im_info{c});
currentImage = imread(strcat(files_RNA_images.data{c,2},files_RNA_images.data{c,1}(1:end-15),'P-Bodies_MaxProj.tif'), k, 'Info', Im_info{c});
Image_PBodies{c}{k} = currentImage;
end
if Plot_Images == 1;
figure()
imagesc(Image_RNA{c}{1});
title(strcat(files_RNA_images.data{c},' -- RNA Channel'),'Interpreter', 'none')
axis square
figure()
imagesc(Image_PBodies{c}{1});
title(strcat(files_RNA_images.data{c},' -- P-Bodies Channel'),'Interpreter', 'none')
axis square
end
end
%% Create Color variable
Color =lines(nimages);
%% Calculate some preliminary numbers
Loc2Bckg_numPX_Ratio = (PSF_roi*PSF_roi)/(PSF_bckg_roi*PSF_bckg_roi);
%% Load the subROI and extract the localizations Inside and Outside them
% if subROI == 1;
%
% for c=1:nimages;
%
% if size(sROI{c},2) == 1;
%
% [Locs_INSIDE{c}, Locs_OUTSIDE{c}] = spotsINROI(Localizations_Array{c}(:,2:3),sROI{c}); %Do the first filtering: discard nuclear localizations
% Localizations_inROI{c} = Localizations_Array{c}(Locs_INSIDE{c}{1},:);
% Localizations_outROI{c} = Localizations_Array{c}(Locs_OUTSIDE{c},:);
%
% figure()
% imagesc(Image{c}{Channel_RNA});
% hold on
% scatter(sROI{c}.mnCoordinates(:,1),sROI{c}.mnCoordinates(:,2),'+');
% hold on
% scatter(Localizations_inROI{c}(:,2),Localizations_inROI{c}(:,3),'r');
% hold on
% scatter(Localizations_outROI{c}(:,2),Localizations_outROI{c}(:,3),'g');
% title(strcat(files_images.data{c},' -- RNA Channel, ROI and segmented localizations (Green acepted; Red rejected)'));
% axis square
%
% else if size(sROI{c},2) == 2;
%
% [Locs_INSIDE_temp{c}, Locs_OUTSIDE_temp{c}] = spotsINROI(Localizations_Array{c}(:,2:3),sROI{c}{1}); %Do the first filtering: discard nuclear localizations
% Localizations_inROI_temp{c} = Localizations_Array{c}(Locs_INSIDE_temp{c}{1},:);
% Localizations_outROI_temp{c} = Localizations_Array{c}(Locs_OUTSIDE_temp{c},:);
%
% [Locs_INSIDE{c}, Locs_OUTSIDE{c}] = spotsINROI(Localizations_outROI_temp{c}(:,2:3),sROI{c}{2}); %Do the second filtering: discard localizations outside the cells
% Localizations_inROI{c} = Localizations_outROI_temp{c}(Locs_OUTSIDE{c},:);
% Localizations_outROI{c} = Localizations_outROI_temp{c}(Locs_INSIDE{c}{1},:);
%
% figure()
% imagesc(Image{c}{Channel_RNA});
% hold on
% scatter(sROI{c}{1}.mnCoordinates(:,1),sROI{c}{1}.mnCoordinates(:,2),'y','+');
% hold on
% scatter(sROI{c}{2}.mnCoordinates(:,1),sROI{c}{2}.mnCoordinates(:,2),'y','+');
% hold on
% scatter(Localizations_inROI{c}(:,2),Localizations_inROI{c}(:,3),'r');
% hold on
% scatter(Localizations_outROI{c}(:,2),Localizations_outROI{c}(:,3),'g');
% hold on
% scatter(Localizations_inROI_temp{c}(:,2),Localizations_inROI_temp{c}(:,3),'r');
% title(strcat(files_images.data{c},' -- RNA Channel, ROI and segmented localizations (Green acepted; Red rejected)'))
% axis square
%
%
% end
%
% end
% end
%
% else
% for c=1:nimages;
% Localizations_outROI{c} = Localizations_Array{c};
% end
%
% end
%% Reorganize the localizations in z
for c=1:nimages;
for z=1:size(Image_PBodies{c},2);
idx = find((Localizations_Array{c}(:,1)==z));
Localizations_outROI{c}{z} = Localizations_Array{c}(idx,:);
Number_of_Localizations_perStack{c}(z) = size(Localizations_outROI{c}{z},1);
end
end
%% Identify the regions ocuppied by Condensates
for c=1:nimages;
for z=1:size(Image_PBodies{c},2);
Mask_idx = Image_PBodies{c}{z}>Condensates_Intensity_thr;
Mask_idx_double = (Image_PBodies{c}{z}>Condensates_Intensity_thr).*1;
Mask{c}{z} = double(Image_PBodies{c}{z}).*Mask_idx_double;
CC{c}{z} = bwconncomp(Mask_idx);
S{c}{z} = struct2cell(regionprops(CC{c}{z},'Centroid'));
Condensate_Boundaries{c}{z} = bwboundaries(Mask_idx');
Condensate_Boundaries{c}{z}(cellfun('length',Condensate_Boundaries{c}{z})<3) = [];
for iiii=1:size(Condensate_Boundaries{c}{z},1);
if ~isempty(Condensate_Boundaries{c}{z});
Condensates_Positions{c}{z}(iiii,1) = round(mean(Condensate_Boundaries{c}{z}{iiii}(:,1)));
Condensates_Positions{c}{z}(iiii,2) = round(mean(Condensate_Boundaries{c}{z}{iiii}(:,2)));
else
Condensates_Positions{c}{z} = [];
end
end
if exist('Condensates_Positions');
else
Condensates_Positions{c}{z} = [];
end
end
if Plot_Images == 1;
figure()
imagesc(Mask{c}{1});
title(strcat(files_RNA_images.data{c},' - Z= ',num2str(z),' -- Mask of the Condensates'),'Interpreter', 'none');
set(gca,'FontSize',18);
axis square
end
end
%% If subROIs, remove Condensates that are outside the specified region
if subROI == 1;
for c=1:nimages;
if size(sROI{c},2) == 1;
[Cond_INSIDE{c}, Cond_OUTSIDE{c}] = spotsINROI(Condensates_Positions{c},sROI{c}); %Do the first filtering: discard nuclear localizations
Cond_inROI{c} = Condensates_Positions{c}(Cond_INSIDE{c}{1},:);
Cond_outROI{c} = Condensates_Positions{c}(Cond_OUTSIDE{c},:);
else if size(sROI{c},2) == 2;
[Cond_INSIDE_temp{c}, Cond_OUTSIDE_temp{c}] = spotsINROI(Condensates_Positions{c},sROI{c}{1}); %Do the first filtering: discard nuclear localizations
Cond_inROI_temp{c} = Condensates_Positions{c}(Cond_INSIDE_temp{c}{1},:);
Cond_outROI_temp{c} = Condensates_Positions{c}(Cond_OUTSIDE_temp{c},:);
[Cond_INSIDE{c}, Cond_OUTSIDE{c}] = spotsINROI(Cond_outROI_temp{c},sROI{c}{2}); %Do the second filtering: discard localizations outside the cells
Cond_inROI{c} = Cond_outROI_temp{c}(Cond_OUTSIDE{c},:);
Cond_outROI{c} = Cond_outROI_temp{c}(Cond_INSIDE{c}{1},:);
end
end
end
else
Cond_outROI = Condensates_Positions;
end
%% Perform Colocalization and remove the localizations corresponding to condensates from the list of RNA localizations
for c=1:nimages;
for z=1:size(Image_PBodies{c},2);
Localizations_outROI_all{c}{z} = Localizations_outROI{c}{z};
if ~isempty(Localizations_outROI{c}{z}(:,2:3)) && ~isempty(Condensate_Boundaries{c}{z});
[Overlapping_locs_boundaries{c}{z}, othr,Positive_Cond_idx{c}{z}] = spotsINROI_V3(Localizations_outROI{c}{z}(:,2:3), Condensate_Boundaries{c}{z}); %Identify Localizations inside Condensate Boundaries
Positive_Condensate_list{c}{z} = unique(nonzeros(Positive_Cond_idx{c}{z}));
Distance_RNA_Cond{c}{z} = pdist2(Cond_outROI{c}{z},Localizations_outROI{c}{z}(:,2:3))'; %Identify Localizations that are closer than the specified distance to the center of the condensates
[Overlapping_locs_idx_dist{c}{z}, othr] = find(Distance_RNA_Cond{c}{z} < RNA_Condensates_dist_thr);
Overlapping_locs_idx{c}{z} = [vertcat(Overlapping_locs_boundaries{c}{z}{:}); Overlapping_locs_idx_dist{c}{z}];
Overlapping_locs_idx{c}{z} = unique(Overlapping_locs_idx{c}{z});
if Plot_Images == 1;
figure()
imagesc(Image_RNA{c}{z});
hold on
scatter(Localizations_outROI{c}{z}(:,2),Localizations_outROI{c}{z}(:,3),'g');
hold on
scatter(Cond_outROI{c}{z}(:,1),Cond_outROI{c}{z}(:,2),'y','filled');
hold on
scatter(Localizations_outROI{c}{z}(Overlapping_locs_idx{c}{z},2),Localizations_outROI{c}{z}(Overlapping_locs_idx{c}{z},3),'r');
hold on
for iii=1:size(Condensate_Boundaries{c}{z},1);
scatter(Condensate_Boundaries{c}{z}{iii}(:,1),Condensate_Boundaries{c}{z}{iii}(:,2),3,'y','+');
hold on
end
axis square
aa = [1:size(Cond_outROI{c}{z},1)]';
bb = num2str(aa);
cc = cellstr(bb);
labelpoints(Cond_outROI{c}{z}(:,1),Cond_outROI{c}{z}(:,2),cc,'S',0.3)
% dx = 0.1; dy = 0.1;
% text(Condensates_Positions{c}(:,1)+dx, Condensates_Positions{c}(:,2)+dy, c);
title(strcat(files_RNA_images.data{c},' - Z= ',num2str(z),' -- RNA Localizations (green); Condensate Boundaries and Center Positions (yellow); Overlapping RNA Localizations (red)'),'Interpreter', 'none')
set(gca,'FontSize',18);
end
Overlapping_Localizations{c}{z} = Localizations_outROI{c}{z}(Overlapping_locs_idx{c}{z},:);
Localizations_outROI{c}{z}(Overlapping_locs_idx{c}{z},:) = [];
Total_number_localizations{c}(z) = size(Localizations_outROI_all{c}{z},1);
Total_number_Overlapping_localizations{c}(z) = size(Overlapping_Localizations{c}{z},1);
Total_number_condensates{c}(z) = size(Cond_outROI{c}{z},1);
Total_number_Positive_condensates{c}(z) = size(Positive_Condensate_list{c}{z},1);
else
Total_number_localizations{c}(z) = nan;
Total_number_Overlapping_localizations{c}(z) = nan;
Total_number_condensates{c}(z) = nan;
Total_number_Positive_condensates{c}(z) = nan;
Positive_Condensate_list{c}{z} = [];
end
end
RNA_Colocalization_Percentage(c) = nansum(Total_number_Overlapping_localizations{c})*100/nansum(Total_number_localizations{c});
Condensate_Positive_Percentage(c) = nansum(Total_number_Positive_condensates{c})*100/nansum(Total_number_condensates{c});
end
%% Print Colozalization Results
for c=1:nimages;
fprintf('\n');
fprintf('---------------------------------');
fprintf('\n');
fprintf(strcat('Image_',num2str(c)));
fprintf('\n');
fprintf(files_RNA_images.data{c});
fprintf('\n');
fprintf('Total percentage of RNAS inside condensates:\n');
fprintf('------');
fprintf('\n');
fprintf(strcat(num2str(RNA_Colocalization_Percentage(c)),' %%'));
fprintf('\n');
fprintf('\n');
fprintf('Total percentage of positive Condensates:\n');
fprintf('------');
fprintf('\n');
fprintf(strcat(num2str(Condensate_Positive_Percentage(c)),' %%'));
fprintf('\n');
fprintf('---------------------------------');
fprintf('\n');
fprintf('\n');
end
%% Obtain the Intensity values of each localization
for c=1:nimages;
for z=1:size(Image_PBodies{c},2);
PSF_x{c}{z} = round(Localizations_outROI{c}{z}(:,3));
PSF_y{c}{z} = round(Localizations_outROI{c}{z}(:,2));
for i=1:length(PSF_x{c}{z});
if (PSF_x{c}{z}(i) - PSF_bckg_length) > 0 & (PSF_x{c}{z}(i) + PSF_bckg_length) <= size(Image_RNA{c}{z},1) & (PSF_y{c}{z}(i) - PSF_bckg_length) > 0 & (PSF_y{c}{z}(i) + PSF_bckg_length) <= size(Image_RNA{c}{z},2);
PSF_Int{c}{z}(i) = sum(sum(Image_RNA{c}{z}(PSF_x{c}{z}(i) - PSF_length:PSF_x{c}{z}(i) + PSF_length, PSF_y{c}{z}(i) - PSF_length: PSF_y{c}{z}(i) + PSF_length)));
PSF_Bckg{c}{z}(i) = sum(sum(Image_RNA{c}{z}(PSF_x{c}{z}(i) - PSF_bckg_length:PSF_x{c}{z}(i) + PSF_bckg_length, PSF_y{c}{z}(i) - PSF_bckg_length: PSF_y{c}{z}(i) + PSF_bckg_length)));
I1 = PSF_Int{c}{z}(i);
I2 = PSF_Bckg{c}{z}(i);
A1 = PSF_roi*PSF_roi;
A2 = PSF_bckg_roi*PSF_bckg_roi;
bckg_level{c}{z}(i) = min(min(Image_RNA{c}{z}(PSF_x{c}{z}(i) - PSF_bckg_length:PSF_x{c}{z}(i) + PSF_bckg_length, PSF_y{c}{z}(i) - PSF_bckg_length: PSF_y{c}{z}(i) + PSF_bckg_length)));
Localization_Normalized_Intensity{c}{z}(i) = I1 - ((I2-I1)*A1/(A2-A1));
else
Localization_Normalized_Intensity{c}{z}(i) = 0;
end
end
end
end
%% Fit the CDF to the Intensities distribution
for c=1:nimages;
for z=1:size(Image_PBodies{c},2);
Localization_Normalized_Intensity_all{c}{z} = nonzeros(Localization_Normalized_Intensity{c}{z}');
Localization_Normalized_Intensity_all{c}{z} = Localization_Normalized_Intensity_all{c}{z}(Localization_Normalized_Intensity_all{c}{z} > 0);
Localization_Normalized_Intensity_all{c}{z} = rmoutliers(Localization_Normalized_Intensity_all{c}{z});
f2_all{c}{z} = fitdist(Localization_Normalized_Intensity_all{c}{z},'Normal');
f4_all{c}{z} = fitdist(Localization_Normalized_Intensity_all{c}{z},'Exp');
Mean_Brightness_Single_RNA{c}{z} = f2_all{c}{z}.mu;
% Std_Brightness_Single_RNA{c} = std(Localization_Normalized_Intensity_all{c});
ci2_all{c}{z} = paramci(f2_all{c}{z}); %Get 95% Confidence Interval
[f1_all,x1_all] = ecdf(Localization_Normalized_Intensity_all{c}{z});
if Plot_Images == 1;
figure()
scatter(x1_all,f1_all,'filled');
hold on
f3_all{c}{z} = cdf(f2_all{c}{z},x1_all);
plot(x1_all,f3_all{c}{z});
title(strcat('Image_',num2str(c),' - Z= ',num2str(z)),'Interpreter','none');
xlabel('Intensity');
ylabel('CDF');
title(strcat(files_RNA_images.data{c},' - Z= ',num2str(z)),' -- Cumulative Density Function of RNA Intensities (Normal Dist Fit)','Interpreter','none')
set(gca,'FontSize',18);
% figure()
% scatter(x1_all,f1_all,'MarkerFaceColor',Color(c,:));
% hold on
% f5_all{c} = cdf(f4_all{c},x1_all);
% plot(x1_all,f5_all{c});
% title(strcat('Image_',num2str(c)),'Interpreter','none');
% xlabel('Intensity');
% ylabel('CDF');
% hold on
% title('Cumulative Density Function of Localization Intensities (Exponential Dist Fit)');
figure()
hist(Localization_Normalized_Intensity_all{c}{z},25);
title(strcat(files_RNA_images.data{c},' - Z= ',num2str(z)),' -- Histogram of RNA Intensities','Interpreter','none');
xlabel('Intensities');
ylabel('Counts');
set(gca,'FontSize',18);
end
fprintf('\n');
fprintf('---------------------------------');
fprintf('\n');
fprintf(strcat('Image_',num2str(c)));
fprintf('\n');
fprintf(files_RNA_images.data{c});
fprintf('\n');
fprintf(strcat('Z plane = ',num2str(z)));
fprintf('\n');
fprintf('Single RNA mean intensity (ADU Counts):\n');
fprintf(num2str(round(Mean_Brightness_Single_RNA{c}{z})));
fprintf('\n');
fprintf('Single RNA mean intensity range - 95%% Confidence Interval (ADU Counts):\n');
fprintf(strcat('[',num2str(round(ci2_all{c}{z}(1,1))),'-',num2str(round(ci2_all{c}{z}(2,1))),']'));
fprintf('\n');
fprintf('---------------------------------');
fprintf('\n');
end
end
%% Plot the distribution of intensities
% for c=1:nimages;
%
% for z=1:size(Image_PBodies{c},2);
%
% if Plot_Images == 1;
% figure()
% scatter(1:1:length(Localization_Normalized_Intensity_all{c}{z}),Localization_Normalized_Intensity_all{c}{z});
% title(strcat(files_RNA_images.data{c},' - Z= ',num2str(z),' -- Normalized intensities of the localizations'),'Interpreter','none');
% xlabel('Localization');
% ylabel('Intensity');
% set(gca,'FontSize',18);
% end
%
% end
%
% end
%% Calculate the intensity of each Condensate and the number of RNAs on each Condensate
polyScale = polyScale_Init;
for c=1:nimages;
for z=1:size(Image_PBodies{c},2);
if ~isempty(Condensate_Boundaries{c}{z});
PSF_Cond_x{c}{z} = round(Cond_outROI{c}{z}(:,2));
PSF_Cond_y{c}{z} = round(Cond_outROI{c}{z}(:,1));
for i=1:length(PSF_Cond_x{c}{z});
polyin{c}{z}{i} = polyshape(Condensate_Boundaries{c}{z}{i});
poly2{c}{z}{i} = scale(polyin{c}{z}{i},polyScale,[Cond_outROI{c}{z}(i,1) Cond_outROI{c}{z}(i,2)]);
polyMASK{c}{z}{i} = poly2mask(rmmissing(poly2{c}{z}{i}.Vertices(:,1)),rmmissing(poly2{c}{z}{i}.Vertices(:,2)),size(Image_RNA{c}{z},1),size(Image_RNA{c}{z},2));
polyPIXELS{c}{z}{i} = find(polyMASK{c}{z}{i}>0);
if length(polyPIXELS{c}{z}{i})>9;
PSF_Cond_Int_Region{c}{z}{i} = Image_RNA{c}{z}(polyPIXELS{c}{z}{i});
idx_Int{c}{z}{i} = find(PSF_Cond_Int_Region{c}{z}{i} > Bckg_Int_thr*mean(bckg_level{c}{z}));
ratio_diff = (length(idx_Int{c}{z}{i})/length(PSF_Cond_Int_Region{c}{z}{i}))-Loc2Bckg_numPX_Ratio;
while (ratio_diff>0.05 && polyScale<Max_polyScale); %Iterate in order to achieve a ratio between the size of the background and the size of the Condensate region that is equivalent to the one specified for the RNA localizations
poly2{c}{z}{i} = scale(polyin{c}{z}{i},polyScale,[Cond_outROI{c}{z}(i,1) Cond_outROI{c}{z}(i,2)]);
polyMASK{c}{z}{i} = poly2mask(rmmissing(poly2{c}{z}{i}.Vertices(:,1)),rmmissing(poly2{c}{z}{i}.Vertices(:,2)),size(Image_RNA{c}{z},1),size(Image_RNA{c}{z},2));
polyPIXELS{c}{z}{i} = find(polyMASK{c}{z}{i}>0);
PSF_Cond_Int_Region{c}{z}{i} = Image_RNA{c}{z}(polyPIXELS{c}{z}{i});
idx_Int{c}{z}{i} = find(PSF_Cond_Int_Region{c}{z}{i} > Bckg_Int_thr*mean(bckg_level{c}{z}));
ratio_diff = abs((length(idx_Int{c}{z}{i})/length(PSF_Cond_Int_Region{c}{z}{i}))-Loc2Bckg_numPX_Ratio);
polyScale = polyScale + 0.05;
end
PSF_Cond_Int{c}{z}(i) = sum(sum(PSF_Cond_Int_Region{c}{z}{i}(idx_Int{c}{z}{i})));
PSF_Cond_Bckg{c}{z}(i) = sum(sum(PSF_Cond_Int_Region{c}{z}{i}));
I1_Cond = PSF_Cond_Int{c}{z}(i);
I2_Cond = PSF_Cond_Bckg{c}{z}(i);
A1_Cond = length(idx_Int{c}{z}{i});
A2_Cond = length(PSF_Cond_Int_Region{c}{z}{i});
Localization_Normalized_Intensity_Cond{c}{z}(i) = I1_Cond - ((I2_Cond-I1_Cond)*A1_Cond/(A2_Cond-A1_Cond));
Number_of_RNAS_per_Condensate{c}{z}(i) = Localization_Normalized_Intensity_Cond{c}{z}(i)/Mean_Brightness_Single_RNA{c}{z};
Std_Sup_Number_of_RNAS_per_Condensate{c}{z}(i) = Localization_Normalized_Intensity_Cond{c}{z}(i)/ci2_all{c}{z}(1,1);
Std_Inf_Number_of_RNAS_per_Condensate{c}{z}(i) = Localization_Normalized_Intensity_Cond{c}{z}(i)/ci2_all{c}{z}(2,1);
polyScale = polyScale_Init; %Re-initialize variable
else
Localization_Normalized_Intensity_Cond{c}{z}(i) = 0;
Localization_Normalized_Intensity_Cond{c}{z}(i) = 0;
Number_of_RNAS_per_Condensate{c}{z}(i) = 0;
Std_Sup_Number_of_RNAS_per_Condensate{c}{z}(i) = 0;
Std_Inf_Number_of_RNAS_per_Condensate{c}{z}(i) = 0;
end
end
Percentage_of_RNAS_inside_Condensates{c}{z} = sum(Number_of_RNAS_per_Condensate{c}{z},'omitnan')/(sum(Number_of_RNAS_per_Condensate{c}{z},'omitnan') + size(Localizations_outROI{c}{z},1));
Std_Sup_Percentage_of_RNAS_inside_Condensates{c}{z} = sum(Std_Sup_Number_of_RNAS_per_Condensate{c}{z},'omitnan')/(sum(Std_Sup_Number_of_RNAS_per_Condensate{c}{z},'omitnan') + size(Localizations_outROI{c}{z},1));
Std_Inf_Percentage_of_RNAS_inside_Condensates{c}{z} = sum(Std_Inf_Number_of_RNAS_per_Condensate{c}{z},'omitnan')/(sum(Std_Inf_Number_of_RNAS_per_Condensate{c}{z},'omitnan') + size(Localizations_outROI{c}{z},1));
else
Percentage_of_RNAS_inside_Condensates{c}{z} = [];
Std_Sup_Percentage_of_RNAS_inside_Condensates{c}{z} = [];
Std_Inf_Percentage_of_RNAS_inside_Condensates{c}{z} = [];
Localization_Normalized_Intensity_Cond{c}{z} = [];
Number_of_RNAS_per_Condensate{c}{z} = [];
Std_Sup_Number_of_RNAS_per_Condensate{c}{z} = [];
Std_Inf_Number_of_RNAS_per_Condensate{c}{z} = [];
end
Localization_Normalized_Intensity_Cond_Positive{c}{z} = nonzeros(Localization_Normalized_Intensity_Cond{c}{z});
fprintf('\n');
fprintf('---------------------------------');
fprintf('\n');
fprintf(strcat('Image_',num2str(c)));
fprintf('\n');
fprintf(files_RNA_images.data{c});
fprintf('\n');
fprintf(strcat('Z plane = ',num2str(z)));
fprintf('\n');
fprintf('Number of RNAS inside condensates:\n');
fprintf('------');
fprintf('\n');
for ii = 1:size(Number_of_RNAS_per_Condensate{c}{z},2);
if ~isempty(find(Positive_Condensate_list{c}{z}==ii));
fprintf(strcat('Condensate_',num2str(ii),' -- Mean: ',num2str(Number_of_RNAS_per_Condensate{c}{z}(ii)),...
' -- Range (95%%CI): [',num2str(Std_Inf_Number_of_RNAS_per_Condensate{c}{z}(ii)),'-',num2str(Std_Sup_Number_of_RNAS_per_Condensate{c}{z}(ii)),']',...
' -- Region size (px): ',num2str(length(PSF_Cond_Int_Region{c}{z}{ii})),' -- Condensate size (px): ',num2str(length(PSF_Cond_Int_Region{c}{z}{ii}(idx_Int{c}{z}{ii}))),...
' -- Density (RNAs/px): ',num2str(Number_of_RNAS_per_Condensate{c}{z}(ii)/length(PSF_Cond_Int_Region{c}{z}{ii}(idx_Int{c}{z}{ii})))));
fprintf('\n');
else
fprintf(strcat('Condensate_',num2str(ii),' -- Mean: ',num2str(Number_of_RNAS_per_Condensate{c}{z}(ii)),...
' -- Range (95%%CI): [',num2str(Std_Inf_Number_of_RNAS_per_Condensate{c}{z}(ii)),'-',num2str(Std_Sup_Number_of_RNAS_per_Condensate{c}{z}(ii)),']'));
fprintf('\n');
end
end
fprintf('\n');
fprintf('Percentage of RNAS inside condensates for each Z Plane:\n');
fprintf('------');
fprintf('\n');
fprintf(strcat('Image_',num2str(c)));
fprintf('\n');
fprintf(files_RNA_images.data{c});
fprintf('\n');
fprintf(strcat('Z plane = ',num2str(z)));
fprintf('\n');
fprintf(strcat('Mean: ',num2str(Percentage_of_RNAS_inside_Condensates{c}{z}*100),'%% ',...
' -- Range: [',num2str(Std_Inf_Percentage_of_RNAS_inside_Condensates{c}{z}*100),'-',num2str(Std_Sup_Percentage_of_RNAS_inside_Condensates{c}{z}*100),']%%'));
fprintf('\n');
fprintf('---------------------------------');
fprintf('\n');
fprintf('\n');
if Plot_Images == 1;
figure()
imagesc(Image_RNA{c}{z});
hold on
for iii=1:size(PSF_Cond_Int_Region{c}{z},2);
if ~isempty(polyin{c}{z}{iii}.Vertices);
polyin_plot{c}{z}{iii}.Vertices(:,:) = [polyin{c}{z}{iii}.Vertices(:,:); polyin{c}{z}{iii}.Vertices(1,:)];
else
polyin_plot{c}{z}{iii}.Vertices = polyin{c}{z}{iii}.Vertices;
end
plot(polyin_plot{c}{z}{iii}.Vertices(:,1),polyin_plot{c}{z}{iii}.Vertices(:,2),'g','LineWidth',1);
hold on
if ~isempty(poly2{c}{z}{iii}.Vertices);
poly2_plot{c}{z}{iii}.Vertices(:,:) = [poly2{c}{z}{iii}.Vertices(:,:); poly2{c}{z}{iii}.Vertices(1,:)];
else
poly2_plot{c}{z}{iii}.Vertices = poly2{c}{z}{iii}.Vertices;
end
plot(poly2_plot{c}{z}{iii}.Vertices(:,1),poly2_plot{c}{z}{iii}.Vertices(:,2),'y','LineWidth',1);
hold on
[row,column] = ind2sub(size(Image_RNA{c}{z}),polyPIXELS{c}{z}{iii});
scatter(column,row,5,'+','k');
hold on
[row,column] = ind2sub(size(Image_RNA{c}{z}),polyPIXELS{c}{z}{iii}(idx_Int{c}{z}{iii}));
scatter(column,row,5,'+','w');
hold on
end
aa = [1:size(PSF_Cond_Int_Region{c}{z},2)]';
bb = num2str(aa);
cc = cellstr(bb);
labelpoints(Cond_outROI{c}{z}(1:size(PSF_Cond_Int_Region{c}{z},2),1),Cond_outROI{c}{z}(1:size(PSF_Cond_Int_Region{c}{z},2),2),cc,'S',0.1);
axis square
colormap default
title(strcat(files_RNA_images.data{c},' - Z= ',num2str(z),' -- Original (green) and Scaled (yellow) Condensate Regions, Condensate Pixels (white), Condensate Background Pixels (black)'),'Interpreter','None')
set(gca,'FontSize',18);
figure()
imagesc(Image_PBodies{c}{z});
hold on
for iii=1:size(PSF_Cond_Int_Region{c}{z},2);
if ~isempty(polyin{c}{z}{iii}.Vertices);
polyin_plot{c}{z}{iii}.Vertices(:,:) = [polyin{c}{z}{iii}.Vertices(:,:); polyin{c}{z}{iii}.Vertices(1,:)];
else
polyin_plot{c}{z}{iii}.Vertices = polyin{c}{z}{iii}.Vertices;
end
plot(polyin_plot{c}{z}{iii}.Vertices(:,1),polyin_plot{c}{z}{iii}.Vertices(:,2),'g','LineWidth',1);
hold on
if ~isempty(poly2{c}{z}{iii}.Vertices);
poly2_plot{c}{z}{iii}.Vertices(:,:) = [poly2{c}{z}{iii}.Vertices(:,:); poly2{c}{z}{iii}.Vertices(1,:)];
else
poly2_plot{c}{z}{iii}.Vertices = poly2{c}{z}{iii}.Vertices;
end
plot(poly2_plot{c}{z}{iii}.Vertices(:,1),poly2_plot{c}{z}{iii}.Vertices(:,2),'y','LineWidth',1);
hold on
[row,column] = ind2sub(size(Image_RNA{c}{z}),polyPIXELS{c}{z}{iii});
scatter(column,row,5,'+','k');
hold on
[row,column] = ind2sub(size(Image_RNA{c}{z}),polyPIXELS{c}{z}{iii}(idx_Int{c}{z}{iii}));
scatter(column,row,5,'+','w');
hold on
end
aa = [1:size(PSF_Cond_Int_Region{c}{z},2)]';
bb = num2str(aa);
cc = cellstr(bb);
labelpoints(Cond_outROI{c}{z}(1:size(PSF_Cond_Int_Region{c}{z},2),1),Cond_outROI{c}{z}(1:size(PSF_Cond_Int_Region{c}{z},2),2),cc,'S',0.1);
axis square
colormap default
title(strcat(files_RNA_images.data{c},' - Z= ',num2str(z),' -- Original (green) and Scaled (yellow) Condensate Regions, Condensate Pixels (white), Condensate Background Pixels (black)'),'Interpreter','None')
set(gca,'FontSize',18);
end
end
Percentage_of_RNAS_inside_Condensates_Mean{c} = mean([Percentage_of_RNAS_inside_Condensates{c}{:}]);
Std_Sup_Percentage_of_RNAS_inside_Condensates_Mean{c} = mean([Std_Sup_Percentage_of_RNAS_inside_Condensates{c}{:}]);
Std_Inf_Percentage_of_RNAS_inside_Condensates_Mean{c} = mean([Std_Inf_Percentage_of_RNAS_inside_Condensates{c}{:}]);
fprintf('\n');
fprintf('Overall percentage of RNAS inside condensates for each Image:\n');
fprintf('------');
fprintf('\n');
fprintf(strcat('Image_',num2str(c)));
fprintf('\n');
fprintf(files_RNA_images.data{c});
fprintf('\n');
fprintf(strcat('Mean: ',num2str(Percentage_of_RNAS_inside_Condensates_Mean{c}*100),'%% ',...
' -- Range: [',num2str(Std_Inf_Percentage_of_RNAS_inside_Condensates_Mean{c}*100),'-',num2str(Std_Sup_Percentage_of_RNAS_inside_Condensates_Mean{c}*100),']%%'));
fprintf('\n');
fprintf('---------------------------------');
fprintf('\n');
fprintf('\n');
end
%% Calculate Normalize Intensity and Plot Histograms of Intensity of RNA in Condensates and Intensity of RNA outside
%%Condensates
figure()
for c=1:nimages;
for z=1:size(Image_PBodies{c},2);
Localization_Normalized_Intensity_RNAs{c}{z} = Localization_Normalized_Intensity_all{c}{z}/Mean_Brightness_Single_RNA{c}{z};
Localization_Normalized_Intensity_Condensates{c}{z} = Localization_Normalized_Intensity_Cond{c}{z}(Positive_Condensate_list{c}{z})/Mean_Brightness_Single_RNA{c}{z}; %Only Positive Condensates!!
Localization_Normalized_Intensity_Condensates{c}{z} = Localization_Normalized_Intensity_Condensates{c}{z}';
Localization_Intensity_RNAs{c}{z} = Localization_Normalized_Intensity_all{c}{z};
Localization_Intensity_Condensates{c}{z} = Localization_Normalized_Intensity_Cond{c}{z}(Positive_Condensate_list{c}{z});
[N,X] = hist(Localization_Normalized_Intensity_RNAs{c}{z},edges);
N = N/sum(N); %Normalize Histogram.Sum of counts equals 1.
bar(X,N,'FaceColor',[0 0.4470 0.7410],'FaceAlpha',0.6);
hold on
[N,X] = hist(Localization_Normalized_Intensity_Condensates{c}{z},edges);
N = N/sum(N); %Normalize Histogram.Sum of counts equals 1.
bar(X,N,'FaceColor',[0.8500 0.3250 0.0980],'FaceAlpha',0.6);
% xlim([])
% ylim([])
end
end
title(strcat(files_RNA_images.data{c},' -- Histogram of Normalized RNA Intensities'),'Interpreter','None');
xlabel('Normalized Intensities');
ylabel('Counts');
legend({'RNA Outside','RNA in P-Bodies'});
set(gca,'FontSize',18);
%% Collapse all images and plot Histograms of Intensity of RNA in Condensates and Intensity of RNA outside
for c=1:nimages;
Localization_Normalized_Intensity_RNAs_Collapse_images{c} = cat(1,Localization_Normalized_Intensity_RNAs{c}{:});
Localization_Normalized_Intensity_Condensates_Collapse_images{c} = cat(1,Localization_Normalized_Intensity_Condensates{c}{:});
Localization_Intensity_RNAs_Collapse_images{c} = cat(1,Localization_Intensity_RNAs{c}{:});
Localization_Intensity_Condensates_Collapse_images{c} = cat(1,Localization_Intensity_Condensates{c}{:});
Percentage_of_RNAS_inside_Condensates_Collapse_images{c} = cat(1,Percentage_of_RNAS_inside_Condensates{c}{:});
Std_Inf_Percentage_of_RNAS_inside_Condensates_Collapse_images{c} = cat(1,Std_Inf_Percentage_of_RNAS_inside_Condensates{c}{:});
Std_Sup_Percentage_of_RNAS_inside_Condensates_Collapse_images{c} = cat(1,Std_Sup_Percentage_of_RNAS_inside_Condensates{c}{:});
end
Localization_Normalized_Intensity_RNAs_Collapse_all = cat(1,Localization_Normalized_Intensity_RNAs_Collapse_images{:});
Localization_Intensity_RNAs_Collapse_all = cat(1,Localization_Intensity_RNAs_Collapse_images{:});
emptyCells = cellfun(@isempty,Localization_Normalized_Intensity_Condensates_Collapse_images);
Localization_Normalized_Intensity_Condensates_Collapse_images(emptyCells) = {0};
Localization_Normalized_Intensity_Condensates_Collapse_all = cat(1,Localization_Normalized_Intensity_Condensates_Collapse_images{:});
Localization_Intensity_Condensates_Collapse_images(cellfun(@isempty,Localization_Intensity_Condensates_Collapse_images)) = {0};
Localization_Intensity_Condensates_Collapse_all = cat(1,Localization_Intensity_Condensates_Collapse_images{:});
%Remove NaNs
Localization_Normalized_Intensity_RNAs_Collapse_all(isnan(Localization_Normalized_Intensity_RNAs_Collapse_all)) = [];
Localization_Normalized_Intensity_Condensates_Collapse_all(isnan(Localization_Normalized_Intensity_Condensates_Collapse_all)) = [];
Localization_Intensity_RNAs_Collapse_all(isnan(Localization_Intensity_RNAs_Collapse_all)) = [];
Localization_Intensity_Condensates_Collapse_all(isnan(Localization_Intensity_Condensates_Collapse_all)) = [];
%Remove Zeros
Localization_Normalized_Intensity_RNAs_Collapse_all = nonzeros(Localization_Normalized_Intensity_RNAs_Collapse_all);
Localization_Normalized_Intensity_Condensates_Collapse_all = nonzeros(Localization_Normalized_Intensity_Condensates_Collapse_all);
Localization_Intensity_RNAs_Collapse_all = nonzeros(Localization_Normalized_Intensity_RNAs_Collapse_all);
Localization_Intensity_Condensates_Collapse_all = nonzeros(Localization_Intensity_Condensates_Collapse_all);
Percentage_of_RNAS_inside_Condensates_Collapse_all = cat(1,Percentage_of_RNAS_inside_Condensates_Collapse_images{:});
Std_Inf_Percentage_of_RNAS_inside_Condensates_Collapse_all = cat(1,Std_Inf_Percentage_of_RNAS_inside_Condensates_Collapse_images{:});
Std_Sup_Percentage_of_RNAS_inside_Condensates_Collapse_all = cat(1,Std_Sup_Percentage_of_RNAS_inside_Condensates_Collapse_images{:});
figure()
[N,X] = hist(Localization_Normalized_Intensity_RNAs_Collapse_all,edges);
N = N/sum(N); %Normalize Histogram.Sum of counts equals 1.
bar(X,N,'FaceColor',[0 0.4470 0.7410],'FaceAlpha',0.6);
hold on
[N,X] = hist(Localization_Normalized_Intensity_Condensates_Collapse_all,edges);
N = N/sum(N); %Normalize Histogram.Sum of counts equals 1.
bar(X,N,'FaceColor',[0.8500 0.3250 0.0980],'FaceAlpha',0.6);
title(strcat(files_RNA_images.data{c},' -- Histogram of Normalized RNA Intensities'),'Interpreter','None');
xlabel('Normalized Intensities');
ylabel('Counts');
legend({'RNA Outside','RNA in P-Bodies'});
set(gca,'FontSize',18);
% xlim([])
% ylim([])
%% Save figures and results
if Save_Results == 1
for c=1:nimages;
for z=1:size(Image_PBodies{c},2);
for ii = 1:size(Number_of_RNAS_per_Condensate{c}{z},2);
if ~isempty(PSF_Cond_Int_Region{c});
if ~isempty(PSF_Cond_Int_Region{c}{z});
if ii>size(PSF_Cond_Int_Region{c}{z},2);
RNAs_Density_in_Condensates{c}{z}(ii) = 0;
Condensate_Region_Size{c}{z}(ii) = 0;
Condensate_Int_Region_Size{c}{z}(ii) = 0;
else
RNAs_Density_in_Condensates{c}{z}(ii) = Number_of_RNAS_per_Condensate{c}{z}(ii)/length(PSF_Cond_Int_Region{c}{z}{ii}(idx_Int{c}{z}{ii}));
Condensate_Region_Size{c}{z}(ii) = length(PSF_Cond_Int_Region{c}{z}{ii});
Condensate_Int_Region_Size{c}{z}(ii) = length(PSF_Cond_Int_Region{c}{z}{ii}(idx_Int{c}{z}{ii}));
end
else
RNAs_Density_in_Condensates{c}{z}(ii) = 0;
Condensate_Region_Size{c}{z}(ii) = 0;
Condensate_Int_Region_Size{c}{z}(ii) = 0;
end
else
RNAs_Density_in_Condensates{c}{z}(ii) = 0;
Condensate_Region_Size{c}{z}(ii) = 0;
Condensate_Int_Region_Size{c}{z}(ii) = 0;
end
end
end
Results{c}{1,1} = 'Image name';
Results{c}{1,2} = 'Mean intensity of single RNA [ADU]';
Results{c}{1,3} = 'Mean intensity of single RNA 95 Confidence Intervals [ADU]';
Results{c}{1,4} = 'Number of RNAs per Condensate';
Results{c}{1,5} = 'Number of RNAs per Condensate Inferior 95 Confidence Interval';
Results{c}{1,6} = 'Number of RNAs per Condensate Superior 95 Confidence Interval';
Results{c}{1,7} = 'Total Condensate Region Size (Condensate and Background) [pixel]';
Results{c}{1,8} = 'Condensate Region Size (Intensity) [pixel]';
Results{c}{1,9} = 'RNAs Density in Condensates [RNAs/pixel]';
Results{c}{1,10} = 'Percentage of RNA Inside Condensates (0 to 1)';
Results{c}{1,11} = 'Percentage of RNA Inside Condensates Inferior 95 Confidence Interval (0 to 1)';
Results{c}{1,12} = 'Percentage of RNA Inside Condensates Superior 95 Confidence Interval (0 to 1)';
Results{c}{1,13} = 'RNAs Intensities (Outside Condensates)';
Results{c}{1,14} = 'RNAs Intensities (Inside Condensates) (Only Positive Condensates)';
Results{c}{1,15} = 'Total number of localizations per image';
Results{c}{1,16} = 'Number of localizations per image per z-stack';
if exist('files_RNA_images')
Results{c}{2,1} = files_RNA_images.data{c};
end
if exist('Mean_Brightness_Single_RNA')
Results{c}{2,2} = cellfun(@round,Mean_Brightness_Single_RNA{c});
end
if exist('ci2_all')
Results{c}{2,3} = cellfun(@round,ci2_all{c}(1,1),'UniformOutput',false);
end
if exist('Number_of_RNAS_per_Condensate')
Results{c}{2,4} = Number_of_RNAS_per_Condensate{c};
end
if exist('Std_Inf_Number_of_RNAS_per_Condensate')
Results{c}{2,5} = Std_Inf_Number_of_RNAS_per_Condensate{c};
end
if exist('Std_Sup_Number_of_RNAS_per_Condensate')
Results{c}{2,6} = Std_Sup_Number_of_RNAS_per_Condensate{c};
end
if exist('Condensate_Region_Size')
Results{c}{2,7} = Condensate_Region_Size{c};