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% the goal of this script is to determine cluster information starting from
% the results extracted from IMARIS 3D segmentation
clear all
clast_par=1.3;
% filename = '20241107 1457 18h_0002-1.tif DAPI.xls';
% filename = '202406 1457dsbp pRB473_1.xls';
% filename = '1457 1h 2.xls';
%filename = '20231214 1457dsbp pRB473 sbp alfa 508_003-1.xls';
filename = '20240321 1457dsbppRB473sbp ALFA508 0 1.xls';
%filename = '202406 1457dsbp pRB473_1.xls';
%The only two input necessary are the name of the file to be analized i.e file name and parameter that control how far away two
% segmented volumes should be. clast_par < 1 means the distance between two segmented volume should be inferior to the sum of their mean radius
% clast_par =1 the distance between two segmented volumes should be equal to the sum of their mean radius
% clast_par >1 the distance between two segmented volumes should be bigger than the sum of their mean radius
% the data to be quantified is stored in an excel multipage file
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% segmented volumes radius determination
%read_volume=xlsread(filename,32);% opens page where the volumes are listed and extracts the volume information for each cluster
read_volume=readtable(filename, 'Sheet', 32);
siz_point=size(read_volume); %it extracts the information about how many volumes were quantified
radius_nn(1:siz_point(1))=0; %it creates a vector that will contain the estimated radius of all the detected volumes
%radius_nn=1000*( read_volume(:,1)*3/(4*pi)).^(1/3);
radius_nn = 1000 * (read_volume.Volume * 3 / (4 * pi)).^(1 / 3);
% the radius is calculated approximating the quantified volumes with be the volumes of a sphere
Mean_radius_nm = mean(radius_nn) % displays the mean radius of the segmented volumes
%please note that the radius of the segmented volumes is a good
%approximation of the full width at half maximum of the fluorescence object
Variance_radius_n = std(radius_nn) % displays the variance of the radius of the segmented volumes
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% determination of
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%% the distance between the segmented volumes and sorting them between volumes belonging to the same cluster and volumes not belonging to the same cluster.
% center_of_mass=xlsread(filename,5);% open page where the coordinate of the center of mass of the segmented volumes are listed
%
% total_num=size(center_of_mass); % again number of detected spots just to check if it match with the previous values (not necessary)
% total_resolved_spots = total_num(1) % displays the total number of volumes quantified
% %center_of_mass=xlsread(filename,5); % open the page of the file where the values of the centre of mass of the detected volumes are stored and write them into a matrix
% distances(1:siz_point(1),1:siz_point(1))=0;% produce the matrix were the distances between detected volumes will be stored
% cluster(1:siz_point(1),1:siz_point(1))=0;%produce a matrix where will be stored the information wether two segmented volumes are close together (distance fulfil cluster parameter) cluster (i,j)=1
% %or not cluster(i,j)=0;
%
% %%calculates the matrix of the distance between segmented volumes i and j as the distance between their centre of mass
% %the factor 1000 converts the distance from micronmeter to nanometer
% for i=1:siz_point(1)
% for j=1:siz_point(1)
%
% distances(i,j)= sqrt( (center_of_mass(i,1)-center_of_mass(j,1))^2 + (center_of_mass(i,2)-center_of_mass(j,2))^2 + (center_of_mass(i,3)-center_of_mass(j,3))^2 )*1000;
% end
% end
%
% %Here is determined if two volumes are clustering together i.e. if their distance <= the sum of their radius multiplied for clust_par defined above, or not
% for i=1:siz_point(1)
% for j=1:siz_point(1)
%
% if i==j
% cluster(i,j)=0;
% else
% if distances(i,j)<=clast_par*(radius_nn(i)+radius_nn(j))
% cluster(i,j)=1;
% end
% end
%
% end
% end
% cluster3=cluster; %%%here the cluster matrix is doubled for keep avaliable its information at the end of the script
% Read the data from Sheet 5 (Center of Homogeneous Mass)
center_of_mass = readtable(filename, 'Sheet', 5);
% Display the modified variable names (these are the names used by MATLAB)
disp('Modified Variable Names:');
disp(center_of_mass.Properties.VariableNames);
% Display the original column headers (as stored in the Excel file)
disp('Original Column Names:');
disp(center_of_mass.Properties.VariableDescriptions);
% Define the starting row (based on the repeating headers)
%start_row = 3; % Adjust this according to your actual data structure
% Read the table, starting from the correct row
%center_of_mass = readtable(filename, 'Sheet', 5, 'Range', sprintf('A%d', start_row));
% Optional: Remove rows that are still unwanted (if any)
% For example, if there are rows with unwanted headers in the middle of the data:
%center_of_mass = center_of_mass(~ismember(center_of_mass{:, 1}, {'CenterofHomogeneousMass'}), :);
%center_of_mass = center_of_mass(~ismember(center_of_mass{:, 1}, {'CenterofHomogeneousMassX'}), :);
% Use the modified column names to extract the data
x_coords = center_of_mass{:, 'CenterOfHomogeneousMass'}; % Extract X coordinates
y_coords = center_of_mass{:, 'Var2'}; % Adjust as needed for Y coordinates
z_coords = center_of_mass{:, 'Var3'}; % Adjust as needed for Z coordinates
% Determine the number of detected spots (volumes)
siz_point = size(center_of_mass); % Size of the table, number of rows (spots)
total_resolved_spots = siz_point(1); % Total number of volumes quantified
disp(['Total number of volumes quantified: ', num2str(total_resolved_spots)])
% Create a matrix to store the distances between detected volumes
disp('Initializing distance matrix...');
distances = zeros(total_resolved_spots, total_resolved_spots); % Initialize distance matrix
% Create a matrix to store cluster information (1 for close volumes, 0 for far volumes)
disp('Initializing cluster matrix...');
cluster = zeros(total_resolved_spots, total_resolved_spots); % Initialize cluster matrix
% Loop to calculate the distance matrix between detected volumes
% The factor 1000 converts the distance from micrometers to nanometers
disp('Calculating distance matrix...');
for i = 1:total_resolved_spots
for j = 1:total_resolved_spots
distances(i,j) = sqrt( (x_coords(i) - x_coords(j))^2 + (y_coords(i) - y_coords(j))^2 + (z_coords(i) - z_coords(j))^2 ) * 1000; % Calculate distance in nm
end
end
% Loop to calculate whether two volumes belong to the same cluster
% Check if distance between volumes is less than or equal to the sum of their radii
disp('Determining cluster assignments...');
% ERROR_NAME_SHOULD_BE_clast_par. clust_par = 1.5; % Define your clustering parameter (this is an example, adjust as necessary)
for i = 1:total_resolved_spots
for j = 1:total_resolved_spots
if i == j
cluster(i,j) = 0; % No clustering with itself
else
if distances(i,j) <= clast_par * (radius_nn(i) + radius_nn(j)) % Check if the distance is less than the sum of radii
cluster(i,j) = 1; % Assign to the same cluster
end
end
end
end
cluster3 = cluster; % Store the cluster matrix for further use
%disp('Cluster Matrix (1 indicates volumes in the same cluster, 0 otherwise):');
%disp(cluster3);
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
% determination of the number of clusters the cluster size i.e. how many
% volumes are contained into one cluster and other cluster information
numclast=0;
numclast2(1:siz_point(1),1)=1;
numclast3(1:siz_point(1),1:siz_point(1))=0;
num=0; % initial number of clusters
cluster_record(1:siz_point(1),1:siz_point(1))=0;% matrix where the label of the cluster members should be stored i.e the label
%of the volumes of cluster members. Please note that the column number of cluster_record matrix is also the label of the first volume find for a given
%cluster if this column is not made only by zero values
% searching and isolating clusters
for i=1:siz_point(1)
count=0; % how many volumes are clustering with an individual volume
count3=0; % how many volumes are belonging to the same cluster
if sum( cluster(i,:))>=1
num=num+1;
% ind= sum( cluster(i,:));
for j=1:siz_point(1)
if cluster(i,j)==1
count=count+1;
count3=count3+1;
cluster(i,j)=0;% every time if a point on the cluster matrix is found to belong to a specific cluster it is set to
%zero in order to not choose this point twice. At the end of the cluster search the cluster matrix should be a full zeros matrix
cluster(j,i)=0;% everytime if a point on the cluster matrix is found to belong to a specific cluster is set to
%zero in order to the choose this point twice at the end the cluster matrix should be a full zeros matrix
vect(count)=j;% here are listed the label of the volumes clastering with the volume under analysis
cluster_record(count3,i)=j;
end
end
end
while count > 0;%%%% in this recursive loop are searched the volumes that were clustering with the first one and in case
%they are found clustering with other volumes these are stored and this cycle is repeated until when no more clustering volume are found
count2=count;
count=0;
vect2=vect;% saving the information contained in vect (is a variable size vector so overwriting is not to suggest)
clear vect % delete this variable in a way it can be defined newly in the following part of the loop
for ss=1:count2
for j=1:siz_point(1)
if cluster(vect2(ss),j)==1
count=count+1;
cluster(vect2(ss),j)=0;
cluster(j,vect2(ss))=0;
vect(count)=j;
ll=0; %%ll=0 a volume is for the first time found to be member of a given cluster; ll=1 the volume was already found to be part of the cluster
% and it will be not counted twice
for ms=1:count3
if cluster_record(ms,i)==j
ll=1;
end
end
if ll==0
count3=count3+1;
cluster_record(count3,i)=j;
end
ll=0;
end
end
end
clear vect2 % this variable vector will be redefined at the beginning of the loop
count;
end
if num > 0 % if a cluster is found
if count3>0
numb(num)=count3+1;% 1 comes from the fact that a cluster is always found starting from a volume that is not included
%in the counting routine
end
end
end
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%dysplaying the information
sit=size(numb);
number_of_cluster=sit(2)% the number of columns of the variable numb gives the total number of clusters
total_number_of_spot_in_cluster= sum(numb)% the total number of volumes belonging to the one of the clusters (it must be inferior to the total number of volumes quantified!!!)
average_number_of_spot_in_cluster= mean(numb)% mean number of volumes belonging to a cluster
dispersion_number_of_spot_in_cluster= std(numb)% variance of the number of volumes belonging to a cluster
frequent_number_of_spot_in_cluster= median(numb)% median of the number of volumes belonging to a cluster
indd = 0;
% Preallocate dist and disti arrays with a sensible size limit
max_cluster_size = siz_point(1); % Max number of volumes per cluster, assuming worst case
% Preallocate clust_info for efficiency
clust_info = zeros(siz_point(1), 8); % Preallocate space for cluster info
for l = 1:siz_point(1)
% Display progress
fprintf('Processing cluster %d of %d\n', l, siz_point(1));
% Initialize variables
cluster_member = [];
if sum(cluster_record(:, l)) >= 1
indd = indd + 1;
cluster_member = [l; cluster_record(cluster_record(:, l) >= 1, l)]; % All labels in the cluster
end
if ~isempty(cluster_member)
got = numel(cluster_member); % Number of volumes in the cluster
rel = 0; % To keep track of valid distances
xx = 0; yy = 0; zz = 0;
% Sum x, y, z positions (in nm, factor 1000)
cluster_coords = [center_of_mass{cluster_member, 'CenterOfHomogeneousMass'}, ...
center_of_mass{cluster_member, 'Var2'}, ...
center_of_mass{cluster_member, 'Var3'}] * 1000; % Coordinates in nm
xx = sum(cluster_coords(:, 1));
yy = sum(cluster_coords(:, 2));
zz = sum(cluster_coords(:, 3));
% Vectorized distance calculation between all points in the cluster
% We calculate the pairwise distance matrix for all points in the cluster
dist_matrix = pdist2(cluster_coords, cluster_coords); % pdist2 is highly optimized
% Extract non-zero distances and store them in disti
disti = dist_matrix(dist_matrix > 0); % Convert to vector and remove zeros
% Collect statistical information
clust_info(indd, 1) = numb(indd); % Number of volumes in the cluster
clust_info(indd, 2) = 0.5 * mean(disti); % Mean distance between volumes
clust_info(indd, 3) = std(disti / 2); % Estimate of variance
clust_info(indd, 4) = max(disti); % Max distance
clust_info(indd, 5) = min(disti); % Min distance
clust_info(indd, 6) = xx / got; % X coordinate of the cluster center of mass
clust_info(indd, 7) = yy / got; % Y coordinate of the cluster center of mass
clust_info(indd, 8) = zz / got; % Z coordinate of the cluster center of mass
% Clear temporary variables for next iteration
clear dist_matrix disti cluster_coords
end
end
% --------- Preparing the final results and save them in the Excel files ---------
%%% the labels of cluster members are saved in a two column matrix the
%%% second column contains the label of the first member of the cluster and
%%% it repeats its value until when the same cluster is under consideration.
%%% The first column displays the label of the other volumes belonging to
%%% the clusters
a=0;
for i=1:siz_point(1)
if sum(cluster_record(:,i))>=1
for j=1:siz_point(1)
if cluster_record(j,i)>=1
a=a+1;
position(a,1)=cluster_record(j,i);
position(a,2)=i;
end
end
end
end
position2= position-1; % since the labelling in Imaris starts from number 0 and not from one in order to find the same cluster in Imaris to the
% to the label values must be substructed 1
%
%%%%%%%% printing in the output file the data quantified till now
%%%%%%
names={'number of particles', 'mean distance', 'variance of distance', 'max distance', 'min distance', 'center_x', 'center_y', 'center_z' };
names2={'Mean_radius_nm', 'Variance_radius_n ', 'Total_resolved_spots' };
values=[Mean_radius_nm , Variance_radius_n total_resolved_spots];
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'],names2,1,'A1')
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'],values,1,'A2')
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'],clust_info,2,'A2')
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'],names,2,'A1')
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'],position2,3,'A1')
% Write 'names2' and 'values' to the first sheet
%DEBUG: writetable(table(values), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 1, 'WriteRowNames', false, 'WriteVariableNames', false);
%DEBUG: writetable(table(names2), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 1, 'WriteRowNames', false);
% Create a table where the first row is the header and the second is the data
output_table = [names2; num2cell(values)];
disp(output_table);
% Write the table to the Excel file, starting at cell A1
writetable(cell2table(output_table), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 1, 'WriteRowNames', false, 'WriteVariableNames', false);
% % Saving as an older .xls format (Excel 97-2003 format)
% writetable(cell2table(output_table), [num2str(clast_par) '_output_' filename '.xls'], 'Sheet', 1, 'WriteRowNames', false, 'WriteVariableNames', false);
% Save as CSV file
writetable(cell2table(output_table), [num2str(clast_par) '_output_' filename '_Sheet1.csv'], 'WriteRowNames', false, 'WriteVariableNames', false);
% Convert 'clust_info' into a table if it's not one already and write to the second sheet
clust_info_table = array2table(clust_info, 'VariableNames', names); % Convert 'clust_info' to table with proper column names
writetable(clust_info_table, [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 2, 'WriteRowNames', false);
% Assuming position2 is a matrix that needs to be written to the third sheet
writetable(array2table(position2), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 3, 'WriteRowNames', false, 'WriteVariableNames', false);
%%%% quantification of the distance between different clusters
%%%%% the distance between clusters is defined to be the distance between their centre of
%%%%% mass
dist_clust(1:sit(2),1:sit(2))=0;
cc1=0;
for i=1:sit(2)
for j=1:sit(2)
if i==j
dist_clust(i,j)=100000;% the fact that the distance of one cluster respect itself is zero would make more complicate
%to determine the minimum distance between a cluster and it neighbours
else
cc1=cc1+1;
dist_clust(i,j)=sqrt( (clust_info(i,6)-clust_info(j,6))^2 + (clust_info(i,7)-clust_info(j,7))^2 + (clust_info(i,8)-clust_info(j,8))^2 );
dist_clust2(cc1)=dist_clust(i,j);
end
end
end
mindist_cluster=min(dist_clust);% the minimum distance between one cluster and the other ones
mean_dist_cluster=mean(mindist_cluster);% the mean distance between closer clusters
%%% exporting the latest information
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'], dist_clust,4,'A1')
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'],mean_dist_cluster,5,'A1')
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'],mindist_cluster',5,'B1')
% names3={'number_of_cluster', 'total_number_of_spot_in_cluster ', 'average_number_of_spot_in_cluster', 'dispersion_number_of_spot_in_cluster','frequent_number_of_spot_in_cluster' }
% values3=[number_of_cluster total_number_of_spot_in_cluster average_number_of_spot_in_cluster dispersion_number_of_spot_in_cluster frequent_number_of_spot_in_cluster];
% values3=values3';
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'],names3,6,'A1')
% xlswrite([num2str(clast_par) '_output_' filename '.xlsx'],values3',6,'A2')
% Exporting the latest information
writetable(table(dist_clust), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 4, 'WriteRowNames', true);
writetable(table(mean_dist_cluster), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 5, 'WriteRowNames', true);
writetable(table(mindist_cluster'), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 5, 'WriteRowNames', true, 'WriteVariableNames', false);
% Save other information about clusters
names3 = {'number_of_cluster', 'total_number_of_spot_in_cluster', 'average_number_of_spot_in_cluster', ...
'dispersion_number_of_spot_in_cluster', 'frequent_number_of_spot_in_cluster'};
values3 = [number_of_cluster, total_number_of_spot_in_cluster, average_number_of_spot_in_cluster, ...
dispersion_number_of_spot_in_cluster, frequent_number_of_spot_in_cluster];
%DEBUG: writetable(table(values3), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 6, 'WriteRowNames', true, 'WriteVariableNames', false);
%DEBUG: writetable(table(names3), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 6, 'WriteRowNames', true);
% Create a table where the first row is the header and the second row is the data
output_table3 = [names3; num2cell(values3)];
disp(output_table3);
% Write the combined table to the Excel file, starting from cell A1 of Sheet 6
writetable(cell2table(output_table3), [num2str(clast_par) '_output_' filename '.xlsx'], 'Sheet', 6, 'WriteRowNames', false, 'WriteVariableNames', false);
% % Saving as an older .xls format (Excel 97-2003 format)
% writetable(cell2table(output_table3), [num2str(clast_par) '_output_' filename '.xls'], 'Sheet', 6, 'WriteRowNames', false, 'WriteVariableNames', false);
% Save as CSV file
writetable(cell2table(output_table3), [num2str(clast_par) '_output_' filename '_Sheet6.csv'], 'WriteRowNames', false, 'WriteVariableNames', false);
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