%To determine the territory of HC and quantify the territory area and the overlap between every two adjacent HCs

%Images are numbered as time (e.g. 16) directly followed by 0 (whole image) or cell number (e.g. 2).
%Examples: 30 means the time point 3 and the whole image; 161 means the time point 16 and the cell image 1.
%Variable "result" is for each image; Variable "resultAll" accumulates all images.

close all;
clear variables;
clc;

%Input file directory and partial name (leaving the number part for looping purpose)
directory = ;
name1 = ;
name2 = ;

%Input a region as tissue background for background removal; Make sure this region does not include any HC during the whole movie
backgroundDepthStart = ;
backgroundDepthEnd = ;
backgroundTransverseStart = ;
backgroundTransverseEnd = ;

%Input the number of HC and time lapse images
numberHC = ;
numberImage = ;

%Input parameters for the territory radius in pixel;
%Calculated based on pixel interval and the territory regin size;
%Example: radius of 12 for a target of 10 um diameter disk region size and a 0.42 um pixel interval.
territorySize = ; %Diameter; unit um 
pixelInterval = ; %Unit um
radius = round(territorySize/pixelInterval/2);

%Go through each image
for imageNumber = 1:numberImage
    
    %Load the whole image containing all cells
    rawImageWhole = imread(strcat(directory,name1,int2str(imageNumber),'0',name2));
    %Show the image
    figure(1); imshow(rawImageWhole,[]); title(strcat('Original Image Time',num2str(imageNumber))); 
    
    %Set the background threshold as %95 CI value (mean+1.96std)
    backgroundRegion = rawImageWhole(backgroundDepthStart:backgroundDepthEnd,backgroundTransverseStart:backgroundTransverseEnd);
    backgroundRegionVector = double(reshape(backgroundRegion,[(backgroundDepthEnd-backgroundDepthStart+1)*(backgroundTransverseEnd-backgroundTransverseStart+1) 1]));
    backgroundThreshold = mean(backgroundRegionVector(backgroundRegionVector~=0))+1.96*std(backgroundRegionVector(backgroundRegionVector~=0));
    
    %Get the size of the whole image
    [imageSizeY,imageSizeX] = size(rawImageWhole);

    %Initialize variables
    territoryImageAll(1:imageSizeY,1:imageSizeX,1:numberHC) = 0;
    territoryAreaIndividual(1:numberHC) = 0;
    territoryOverlapImage(1:imageSizeY,1:imageSizeX) = 0;
    territoryArea = 0;
    [columnsInImage,rowsInImage] = meshgrid(1:imageSizeX,1:imageSizeY);

    %Go through each cell that was manually selected and seperated using ImageJ
    for cellNumber = 1:numberHC
    
        %Initialize the variable
        territoryImage(1:imageSizeY,1:imageSizeX) = 0;
        
        %Load the particular cell image
        rawImage = imread(strcat(directory,name1,int2str(imageNumber),num2str(cellNumber),name2));
        %Show the image
        figure(2+cellNumber-1+numberHC*(cellNumber-1));imshow(rawImage,[]); title(strcat('HC Cell No.',int2str(cellNumber),' Time',num2str(imageNumber))); 
        
        %Background removal
        imageNoBackground = rawImage;
        imageNoBackground(imageNoBackground<=backgroundThreshold) = 0;
   
        %Filter the image to further remove tissue background
        filteredImage = medfilt2(imageNoBackground,[2 2]);
        figure(3+cellNumber-1+numberHC*(cellNumber-1)); imshow(filteredImage,[]); title(strcat('Background-Removed and Filtered HC Image',' HC Cell No.',int2str(cellNumber),' Time',num2str(imageNumber))); 

        %Binarize the HC image
        binaryThreshold = ; %Input the threshold. This threshold is set to further remove the remaining tissue background; The value should be established based on filteredImage.
        binaryImage = filteredImage;
        binaryImage(:,:) = 0;
        binaryImage(filteredImage>binaryThreshold) = 1;
        figure(4+cellNumber-1+numberHC*(cellNumber-1)); imshow(binaryImage,[]); title(strcat('Binarized HC Image',' HC Cell No.',int2str(cellNumber),' Time',num2str(imageNumber))); 
        
        %Generate the territory centered at the HC cell positive pixels
        for x = 1:imageSizeX
            for y = 1:imageSizeY
                if binaryImage(y,x) == 1
                   territoryImageTemp = (rowsInImage - y).^2 + (columnsInImage - x).^2 <= radius.^2;
                   territoryImage = territoryImage | territoryImageTemp;
                end
            end
        end
        %Perform morphological closing with a dilation followed by an erosion
        se = strel('disk',radius); %using the same diameter disk.
        territoryImageClosed = imclose(territoryImage,se);
        %Show the territory image
        figure(5+cellNumber-1+numberHC*(cellNumber-1)); imshow(territoryImageClosed,[]); title(strcat('Territory Image',' HC Cell No.',int2str(cellNumber),' Time',num2str(imageNumber))); 
        
        %Calculate the individual territory area and collect the territory image
        territoryAreaIndividual(cellNumber) = sum(sum(territoryImageClosed(:,:)==1));
        territoryImageAll(:,:,cellNumber) = double(territoryImageClosed);
    
    end

    %Calculate the true area and store individual territory area
    result(1,1:numberHC) = territoryAreaIndividual*pixelInterval*pixelInterval;
    resultAll(imageNumber,1:numberHC) = result(1,1:numberHC);
    
    %Calculate the overlap region area and percentage between every two adjacent cells
    for i = 2:numberHC
        
        %Calculation and show the overlap image
        territoryAreaSum = sum(territoryAreaIndividual(i-1:i));
        territoryOverlapImage = territoryImageAll(:,:,i-1) + territoryImageAll(:,:,i);
        territoryOverlapArea = sum(sum(territoryOverlapImage(:,:)==2));
        overlapPercentage = territoryOverlapArea/territoryAreaSum;
        %Show the overlap image
        figure(5+numberHC+i-2+numberHC*(numberHC-1)); imshow(territoryOverlapImage,[0 2]); title(strcat('Overlap between',' HC Cell No.',int2str(i-1),' and',' HC Cell No.',int2str(i),' Time',num2str(imageNumber))); 

        %Calculate the true area and store the results
        result(i,1) = territoryOverlapArea*pixelInterval*pixelInterval;
        result(i,2) = territoryAreaSum*pixelInterval*pixelInterval;
        result(i,3) = overlapPercentage;
        
        resultAll(imageNumber,(numberHC+1)+(3*(i-2)):numberHC+3+(3*(i-2))) = result(i,1:3);

    end
    
end