per_nuc_FISH_modified_Tengfei.txt

//Macro for FISH telomere intensity analysis by Glyn Nelson
//Assumes you input an RGB image with the telomeres in the red channel and the nuclear counterstain in the green channel.
//  This macro will identify bright objects in the red channel of an RGB image that are within the bright objects of the green channel
//It assumes levels for the two thresholds used to determine the object sizes
//would be better with a method of manual adjustment for the nuclear threshold

// inputs for the macro
 
    Dialog.create("Telomere Intensity measurement");
    Dialog.addMessage("This macro measures telomere intensities per nucleus. ");
 Dialog.addMessage("");   
 Dialog.addMessage("It asks you to open an image- please open your image as a merged RGB image with the ");
    Dialog.addMessage("telomeres in the red channel and the nuclear counterstain in the green channel. ");
 Dialog.addMessage("");   
    Dialog.addMessage("Data for telomere fluorescence per nucleus are then saved in an excel file in the same folder as the image file. ");

    Dialog.show();

open();
title = getTitle();
run("Split Channels");
wait(140);
close();
wait(140);
rename("nuc");
run("Put Behind [tab]");
wait(140);
rename("foci");
wait(140);
run("Merge Channels...", "red=[foci] green=[nuc] blue=*None* gray=*None* keep");

selectWindow("foci");
run("Hi Lo Indicator");
run("Brightness/Contrast...");
Stack.setChannel(1);
waitForUser("Please adjust ch1 if necessary, apply, then press OK here.");
Stack.setChannel(2);
waitForUser("Please adjust ch1 if necessary, apply, then press OK here.");
//Dialog.create("User input required");
//Dialog.addMessage("Please adjust minimum signal to remove non telomeric fluorescence if necessary, then press 'Apply'...");   
//    Dialog.show();
//print("Please adjust minimum signal to remove non telomereic fluorescence if necessary, then press 'Apply'...");
//wait(12000);
//run("Apply LUT");
run("Put Behind [tab]");
wait(140);

selectWindow("RGB");

selectWindow("nuc");
//setAutoThreshold();
run("Threshold...");
waitForUser("Now please adjust threshold to highlight nuclear signal, apply the threshold, then press OK here,");
//Dialog.create("User input required");
//Dialog.addMessage("Now please adjust threshold to highlight nuclear signal, then press 'Apply'...");   
//    Dialog.show();
//print("Now please adjust threshold to highlight nuclear signal, then press 'Apply'...");
//wait(12000);
//setThreshold(102, 255);
run("Fill Holes");

setForegroundColor(0, 0, 0);
run("Invert LUT");
setTool(20);
//this is pointing to a specific button in the gui for flood fill.  Had to add in lut inverter to get tit to work- don't understand that.

//Dialog.create("User input required");
//Dialog.addMessage("Please remove unwanted nuclei by clicking on them");
//Dialog.addMessage("- ignore small spots and those on the edge...");   
//    Dialog.show();
waitForUser("Please remove unwanted nuclei by clicking on them- ignore small spots and those on the edge.  Click OK when finished");
//wait(30000);
run("Convert to Mask");
wait(140);

run("Analyze Particles...", "size=20-300 circularity=0.00-1.00 show=Nothing exclude add");

run("Put Behind [tab]");
selectWindow("foci");
wait(140);
rename(title);
run("Set Measurements...", "display area integrated redirect=None decimal=3");
roiManager("Measure");
String.copyResults();
roiManager("Delete");
selectWindow("Results");
saveAs("Measurements", getDirectory("current")+"FISHanalysis_per_nuc"+ substring(title,0) +".xls");

close();
close();
close();

run("Clear Results");