//macros for image quantifications

//macro1 
//dapi measurement immunofluorescence

dir1 = getDirectory("Choose folder where the images are located");

dir2 = getDirectory("Choose folder where the results will be saved");

list=getFileList(dir1);
//setBatchMode(true);
for(i=0; i<list.length; i++){
	
showProgress(i+1, list.length);
open(dir1+list[i]);

Title=getTitle();           
//dapi

run("Set Scale...", "distance=1 known=0.3225 pixel=1 unit=micron");
run("Subtract Background...", "rolling=50");
run("8-bit");
run("Threshold...");
setAutoThreshold("Otsu");
setThreshold(12, 255);
run("Despeckle");
run("Despeckle");
run("Convert to Mask");
run("Analyze Particles...", "size=3-Infinity circularity=0.01-1.00 show=Outlines include summarize");
run("Close All");}

selectWindow("Summary");
saveAs("text", dir2 + "Summary.text");
exit("ANALYSIS FINISHED");



//macro 2
//sma measurement immunofluorescence

dir1 = getDirectory("Choose folder where the images are located");

dir2 = getDirectory("Choose folder where the results will be saved");

list=getFileList(dir1);
//setBatchMode(true);
for(i=0; i<list.length; i++){
	
showProgress(i+1, list.length);
open(dir1+list[i]);

Title=getTitle();           


run("Set Scale...", "distance=1 known=0.3225 pixel=1 unit=micron");
run("8-bit");
run("Threshold...");
setAutoThreshold("Default");
setThreshold(71, 255);
run("Convert to Mask");
run("Set Measurements...", "area mean integrated area_fraction display redirect=None decimal=3");
run("Measure");
run("Close All");}
selectWindow("Results");
saveAs("text", dir2 + "Results.text");
exit("ANALYSIS FINISHED");



//macro 3
//collagen measurement immunofluorescence

dir1 = getDirectory("Choose folder where the images are located");

dir2 = getDirectory("Choose folder where the results will be saved");

list=getFileList(dir1);
//setBatchMode(true);
for(i=0; i<list.length; i++){
	
showProgress(i+1, list.length);
open(dir1+list[i]);

Title=getTitle();           

run("Set Scale...", "distance=1 known=0.3225 pixel=1 unit=micron");
run("Remove Outliers...", "radius=20 threshold=100 which=Bright");
run("Subtract...", "value=10");
run("8-bit");
setAutoThreshold("Default dark");
setThreshold(25, 255);
run("Convert to Mask");
run("Set Measurements...", "area mean integrated area_fraction display redirect=None decimal=3");
run("Measure");
run("Close All");}
selectWindow("Results");
saveAs("text", dir2 + "Results9bis.text");
exit("ANALYSIS FINISHED");



//macro 4
//sma quantification in the mouse tumours

//subtract background
selectWindow("T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3");
run("Stack to RGB");
run("Subtract Background...", "rolling=10 light");
selectWindow("T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3");
close();
selectWindow("T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3 (RGB)");
run("Duplicate...", " ");

// colour deconvolution
selectWindow("T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3 (RGB)");
run("Colour Deconvolution", "vectors=[H DAB] hide");
selectWindow("T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3 (RGB)-(Colour_3)");
close();
//cellular area on hematoxylin
selectWindow("T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3 (RGB)-(Colour_1)");
run("Duplicate...", " ");
//make manual selection excluding necrotic area
waitForUser("Select Area for analysis using freehand");
waitForUser("Press Ok");
roiManager("Add")
run("Set Measurements...", "area mean integrated area_fraction display redirect=None decimal=3");
// sma and hematoxylin visualization
selectWindow("T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3 (RGB)-(Colour_2)");
run("Duplicate...", " ");
run("Threshold...");
setThreshold(0, 180);
run("Convert to Mask");
selectWindow("T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3 (RGB)-(Colour_1)");
setThreshold(0, 234);
run("Convert to Mask");
// sma-hematoxylin in the cellular area
imageCalculator("Subtract create", "T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3 (RGB)-(Colour_2)-1","T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3 (RGB)-(Colour_1)");
selectWindow("Result of T1Rcage1_CTR-1.czi - T1Rcage1_CTR-1.czi #3 (RGB)-(Colour_2)-1");
roiManager("Select", 0);
run("Measure");