//This is designed for analyzing the colocalized points for GFP-LC3 and mCherry on 
  confocal   images 
//Creator: Dan Shiwarski, Duvvuri Lab 101410
  Co-author: Ruben K. Dagda, Ph.D., University of Pittsburgh
macro "Background subtraction [F5]" {
 print(" RFP-GFP-LC3 Colocalization macro, Dan Shiwarski and Ruben Dagda 10/19/10");
wait(100); 
open();
run("Split Channels");
run("Window/Level...");
run("Enhance Contrast", "saturated=0.35");
setMinAndMax(25, 96);
saveAs("Tiff", "C:\\Blue.tif");
close();
run("Window/Level...");
run("Enhance Contrast", "saturated=0.35");
setMinAndMax(25, 96);
saveAs("Tiff", "C:\\Green.tif");
close();
run("Enhance Contrast", "saturated=0.35");
setMinAndMax(25, 96);
saveAs("Tiff", "C:\\Red.tif");
close();
  open();
open();
selectWindow("Red.tif");
run("Subtract Background...", "rolling=14");
selectWindow("Green.tif")
run("Subtract Background...", "rolling=14");
selectWindow("Red.tif");
//run("Threshold...");
setAutoThreshold("RenyiEntropy");
setAutoThreshold("RenyiEntropy dark");
run("Convert to Mask");
selectWindow("Green.tif");
setAutoThreshold("RenyiEntropy");
//run("Threshold...");
setAutoThreshold("RenyiEntropy dark");
run("Convert to Mask");
     run("Merge Channels...", "red=Red.tif green=Green.tif blue=*None* gray=*None* create");
run("Stack to RGB");
run("Split Channels");
selectWindow("Composite (RGB) (blue)");
close();

run("Colocalization ", "channel_1=[Composite (RGB) (red)] channel_2=[Composite (RGB) (green)] ratio=30 threshold_channel_1=15 threshold_channel_2=15 display=255  colocalizated");
selectWindow("Colocalizated points (8-bit) ");
run("Make Binary");
run("Analyze Particles...", "size=3-1000 circularity=0.00-1.00 show=Outlines display summarize");

selectWindow("Composite (RGB) (green)");
run("Make Binary");
run("Analyze Particles...", "size=3-1000 pixel circularity=0.00-1.00  show=Outlines display summarize");

selectWindow("Composite (RGB) (red)");
run("Make Binary");
run("Analyze Particles...", "size=3-1000 pixel circularity=0.00-1.00  show=Outlines       display summarize"); 
    s = getString("Do you want to analyze and count nuclei? [y/n+comment]","n");
     if (fromCharCode(charCodeAt(s,0)) == "n") exit;   // end of program, updated on May 15-2010
   else { open();
   selectWindow("Blue.tif");
 //run("Threshold...");
 macro "Count Nuclei [F10]"{
run("Make Binary");
run("Analyze Particles...", "size=15-1000000 pixel circularity=0.00-1.00 show=Outlines   display summarize");
  }  
   macro "Close All Windows [F4]" 
  { 
    while (nImages>0) 
   { selectImage(nImages); close(); 
   }