foci_and_coloc_counting_wholedir_20x_moments_thresh.txt

//Assumes the image is a MIP'ed tif with PML in channel1, H2AX in channel2 
//(although these can be the other way round and it doesn't matter too much except for labelling in results)
//and DAPI in channel3.  It looks for pure overlap and counts respective spots.
//All results get stored in a concatenating results file.
//Plus the mask images are stored (all 3 original images converted to masks plus the coloc maskin the 4th channel)
// so you can see what it did.

var tolerance = 30;
run("Set Measurements...", "area integrated limit display redirect=None decimal=3");

dir1 = getDirectory("Choose folder with tif files ");
list = getFileList(dir1);

setBatchMode(true);

// create folders for the tifs
	dir1parent = File.getParent(dir1);
	dir1name = File.getName(dir1);
	dir2 = dir1parent+File.separator+dir1name+"_masks";
	if (File.exists(dir2)==false) {
				File.makeDirectory(dir2); // new directory for mask images
		}

for (i=0; i<list.length; i++) {
	showProgress(i+1, list.length);
	print("processing ... "+i+1+"/"+list.length+"\n         "+list[i]);
	filename = dir1 + list[i];
	if (endsWith(filename, "tif")) {
		path=dir1+list[i];
		run("Bio-Formats Importer", "open="+filename+" autoscale color_mode=Default view=Hyperstack stack_order=XYCZT"); 
		originalImage = getTitle();
		roiManager("Reset");
		selectWindow(originalImage);
		run("Duplicate...", "duplicate channels=1"); //Change this to the DAPI channel slice*******
//		run("8-bit");
		setAutoThreshold("Moments dark");
		run("Threshold");
		run("Watershed");
//		run("Entropy Threshold");
		run("Analyze Particles...", "size=120-2500 circularity=0.04-1.00 exclude add");
		rename("nuc");
//nuclei now added to ROI manager

//next find pml bodies
		selectWindow(originalImage);
		run("Duplicate...", "title=PML duplicate channels=3");
//		run("8-bit");
		setAutoThreshold("Triangle dark");
		run("Threshold");
//		rename("PML");

//next find h2ax foci
		selectWindow(originalImage);
		run("Duplicate...", "title=h2ax duplicate channels=2");
		wait(1000);
//		run("8-bit");
		setAutoThreshold("MaxEntropy dark");
		run("Threshold");
//		rename("h2ax");

//Next find colocalised PML-H2AX foci
		imageCalculator("Multiply create", "h2ax","PML");
		selectWindow("Result of h2ax");
		rename(originalImage+":coloc");
		run("Grays");
		for(j=0; j<roiManager("count"); j++) {
			roiManager("select", j);
			run("Find Maxima...", "noise="+tolerance+" output=[Count]");
//	run("Find Maxima...", "noise="+tolerance+" output=[Point Selection]");
			run("Add Selection...");
			}
//		saveAs("Results", dir2+File.separator+originalImage+"_nuc_coloc.xls"); 

//next find H2AX foci per nuc
		selectWindow("h2ax");
		rename(originalImage+":h2ax");
		for(j=0; j<roiManager("count"); j++) {
			roiManager("select", j);
			run("Find Maxima...", "noise="+tolerance+" output=[Count]");
//	run("Find Maxima...", "noise="+tolerance+" output=[Point Selection]");
			run("Add Selection...");
			}
		saveAs("Results", dir2+File.separator+originalImage+"_nuc_coloc_and_h2ax.xls"); //This is savign as it goes along in case it crashes
		selectWindow(originalImage+"h2ax");
		rename("h2ax");
		selectWindow(originalImage+"coloc");
		rename("coloc");
		selectWindow(originalImage);
		close();

		run("Merge Channels...", "c1=PML c2=h2ax c3=nuc c4=coloc create");
		rename("masks");

		saveAs("TIFF", dir2+File.separator+originalImage+"_colocmasks.tif");


	close();
	}
setBatchMode(false);
}

showMessage(" Well, I think we gave them a damn good thrashing there, what what??");

// macro