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ER_mobility_analysis_part1.m
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ER_mobility_analysis_part1.m
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%% Script to analyze local ER mobility around IBs
% Part 1 of 3
% Felix JB Baeuerlein
% Baeuerlein et al. Cell 2017
%% Input Filenames:
% [Filename]_1.tif : Reference GFP-image before 10Hz imaging
% [Filename]_2.tif : 10Hz ER-timeseries
% [Filename]_3.tif : Reference GFP-image after 10Hz imaging
% [Filename]_4.tif : 5Hz ER-timeseries (not necessary - only experimental)
%% Output Filenames:
% Output files are cutouts of the IB with local neighbourhood +-5um around IB
% IB[IB#]_[Filename]_2.tif_Average_IB_[# of IB in FOV].tif :
% Time series average of red ER-channel combined with green IB-GFP channel
% IB[IB#]_[Filename]_2.tif_GFP_[# of IB in FOV].tif :
% green IB-GFP channel
% IB[IB#]_[Filename]_2.tif_RadialAverage_IB_[# of IB in FOV].tif :
% Radial average of the ER and the variance of the ER signal around the IB
% IB[IB#]_[Filename]_2.tif_Timeseries_BC_IB_[# of IB in FOV].tif :
% Full time series of ER-channel - after bleach correction
% IB[IB#]_[Filename]_2.tif_Timeseries_IB_[# of IB in FOV].tif :
% Full time series of ER-channel - before bleach correction
% IB[IB#]_[Filename]_2.tif_Variance_IB_[# of IB in FOV].tif :
% Variance of the full time series of the ER-channel
%% script body
clear all
close all
tic
[FileName,PathName] = uigetfile('*.tif','Select one file in the correct folder');
cd(PathName)
D=dir;
mkdir('Analysis')
IB_count = 0;
fid = fopen('IB_FIJI_Macro.txt','wt');
%% load TimeLapse of 10Hz Imaging
Extension = '2.tif'; % for 10Hz: 2.tif For 5Hz 4.tif
for n=3:size(D,1)
tic
if isempty(strfind(D(n).name, '_4.tif'))==0 && (D(n).isdir==0) % loop checking all time-series
fname_series = [D(n).name(1:size(D(n).name,2)-5) Extension];
fname_GFP1 = [D(n).name(1:size(D(n).name,2)-5) '1.tif']; % Reference GFP-image before 10Hz imaging
info = imfinfo(fname_series);
Series_10 = [];
numberOfImages = length(info);
for k = 1:numberOfImages
currentImage = imread(fname_series, k, 'Info', info);
Series_10(:,:,k) = currentImage;
end
GFP1.original = imread(fname_GFP1);
disp(['-- File: ' fname_series ' loaded --'])
toc
%% Identification of IB's in the image
GFP1.IB = GFP1.original.*sign(sign(GFP1.original - 12*median(median(GFP1.original)))); % thresholds the GFP image to detect IB's
GFP1.props = regionprops(logical(GFP1.IB),'centroid','Area','BoundingBox','Eccentricity','MajorAxisLength', 'MinorAxisLength'); % measures location and properties of IB's
disp(['- ' num2str(size(GFP1.props,1)) ' IBs found in image'])
center = round(cat(1,GFP1.props.Centroid)); % center coordinates of all IB's in the image
%% calculate per IB
xSize = 70; % with OPS 102nm = +-5um around IB
ySize = 70; % with OPS 102nm = +-5um around IB
Length = size(Series_10,3); % Length of time series
for m = 1:size(center,1)
IB.original(:,:,:,m) = Series_10(center(m,2)-xSize:center(m,2)+xSize,center(m,1)-ySize:center(m,1)+ySize,:); % cut out IB's from ER series
IB_count = IB_count + 1;
%% calculate average of ER time series and GFP
GFP_shift_y = 0;
GFP_shift_x = 0;
IB.average(:,:,1,m) = mean(IB.original(:,:,:,m),3); % red channel - ERmCherry
IB.average(:,:,2,m) = GFP1.original(center(m,2)-xSize+GFP_shift_x:center(m,2)+xSize+GFP_shift_x,center(m,1)-ySize+GFP_shift_y:center(m,1)+ySize+GFP_shift_y,:); % green channel - GFP
% IB.average(:,:,3,m) blue channel in next section
GFP1.IBcut(:,:,m) = IB.average(:,:,2,m); % cut out IB's from GFP image
%% define IB boarder
GFP_decay = 0.4; % Threshold of max GFP-Fluorescence where the IB boarder is defined
GFP1.IB_Boarder(:,:,m) = sign((GFP1.IBcut(:,:,m) - GFP_decay*max(max(GFP1.IBcut(:,:,m))))); % defines the boarder pixels of the IB
GFP1.IB_Boarder(GFP1.IB_Boarder<0) = 0;
GFP1.IB_Boarder(:,:,m) = bwperim(GFP1.IB_Boarder(:,:,m),4);
IB.average(:,:,3,m) = GFP1.IB_Boarder(:,:,m); % blue channel - IB boarder
%% --------- Calculate the ER-Mobility -----------------
% will be calculated in part2
%% save files
for k = 1:size(IB.original,3)
imwrite(uint16(IB.original(:,:,k,m)),['Analysis/IB' num2str(IB_count,'%03i') '_' fname_series '_Timeseries_IB_' num2str(m) '.tif'],'writemode','append')
end
imwrite(uint16(IB.average(:,:,:,m)),['Analysis/IB' num2str(IB_count,'%03i') '_' fname_series '_Average_IB_' num2str(m) '.tif'])
imwrite(uint16(GFP1.IBcut(:,:,m)),['Analysis/IB' num2str(IB_count,'%03i') '_' fname_series '_GFP_IB_' num2str(m) '.tif'])
toc
disp(['- files written'])
%% write FIJI-Macro to BleachCorrect there
fprintf(fid, ['open("' strrep(PathName,'\','\\\\') 'Analysis\\\\IB' num2str(IB_count,'%03i') '_' [D(n).name(1:size(D(n).name,2)-5) Extension] '_Timeseries_IB_' num2str(m) '.tif");\n']);
fprintf(fid, ['selectWindow("IB' num2str(IB_count,'%03i') '_' [D(n).name(1:size(D(n).name,2)-5) Extension] '_Timeseries_IB_' num2str(m) '.tif");\n']);
fprintf(fid, ['run("Bleach Correction", "correction=[Exponential Fit]");']);
fprintf(fid, ['selectWindow("y = a*exp(-bx) + c");\n']);
fprintf(fid, ['close();\n']);
fprintf(fid, ['selectWindow("IB' num2str(IB_count,'%03i') '_' [D(n).name(1:size(D(n).name,2)-5) Extension] '_Timeseries_IB_' num2str(m) '.tif");\n']);
fprintf(fid, ['close();\n']);
fprintf(fid, ['selectWindow("DUP_IB' num2str(IB_count,'%03i') '_' [D(n).name(1:size(D(n).name,2)-5) Extension] '_Timeseries_IB_' num2str(m) '.tif");\n']);
fprintf(fid, ['saveAs("Tiff", "' strrep(PathName,'\','\\\\') 'Analysis\\\\IB' num2str(IB_count,'%03i') '_' [D(n).name(1:size(D(n).name,2)-5) Extension] '_Timeseries_BC_IB_' num2str(m) '.tif");\n']);
fprintf(fid, ['close();\n']);
end
end
end
fclose(fid)
toc
% run FIJI-Macro before starting part 2 of the ER_mbilitly_analysis script!