// This macro has been designed to process image stacks of a two-channel image of FRET and CFP channels in alternating order 
	// It is possible to edit this code to account for additional channels 
	// The images used to test the macro were acquired using Andor IQ2 Software 
		// Created by Daniel Shiwarski (c) 2013 Carnegie Mellon University 

// Begin the processing of the EPAC Ratio Analysis by following these steps:

	//1.) Open the image for analysis (making sure it is the only image open)

	//2.) Draw an ROI for the Background Region and save to ROI manager - Remove Slice Info from ROI

	//3.) Draw an ROI for the Whole Image Region and save to ROI manager - Remove Slice Info from ROI

	//4.) Proceede by following the onscreen prompts built into the macro 

	//5.) Clicking OK during a prompt will continue; cancel during a prompt with abort the analysis

// If you would like to use the Hotkeys to start the macro [F3] and close all of the open images [F4] you must first install // the macro upon imagej startup. 



macro "EPAC Analysis [F3]" {
	
	rename("EPAC Analysis");


waitForUser("Draw the Background ROI and \n an ROI for the whole image")
	//Dialog.create("ROI Background")
		//Dialog.addString("Draw a BG ROI", "")
		//Dialog.show()
	//Dialog.create("ROI Image")
		//Dialog.addString("Draw a Whole Image ROI", "")
		//Dialog.show()
	
	roiManager("Select", 0);
	roiManager("Remove Slice Info");
	roiManager("Select", 1);
	roiManager("Remove Slice Info");

		for (n= 1; n<=nSlices; n++) {
			setSlice(n);
			roiManager("Select", 0);
			run("Measure");
			BG = getResult("Mean");
			roiManager("Select", 1);
			run("Subtract...", "value=BG slice");
			run("Next Slice [>]");
		}

	Dialog.create("Channels")
		Dialog.addNumber("How many Channels?", 2)
		Dialog.show()
		ch=Dialog.getNumber()

	Dialog.create("Slices")
		Dialog.addNumber("How many Slices?", 1)
		Dialog.show()
		sl=Dialog.getNumber()

	Dialog.create("Frames")
		Dialog.addNumber("How many Frames?", 240)
		Dialog.show()
		fr=Dialog.getNumber()

	run("Stack to Hyperstack...", "order=xyczt(default) channels=ch slices=sl frames=fr display=Grayscale");

	selectWindow("EPAC Analysis")
		run("Split Channels");
		selectWindow("C1-EPAC Analysis");
			rename("EPAC FRET");
			run("Enhance Contrast", "saturated=0.35");
		selectWindow("C2-EPAC Analysis");
			rename("EPAC CFP");
			run("Enhance Contrast", "saturated=0.35");


	imageCalculator("Divide create 32-bit stack", "EPAC CFP","EPAC FRET");
		selectWindow("Result of EPAC CFP");
			rename("EPAC CFP/FRET");

	selectWindow("EPAC FRET");

		run("Duplicate...", "title=[EPAC FRET-1] duplicate range=1-fr");
		selectWindow("EPAC FRET-1");
		rename("EPAC FRET Mask");		
		setAutoThreshold("Default dark");
		//run("Threshold...");
		setOption("BlackBackground", false);
		run("Convert to Mask", "method=Default background=Dark calculate black");

	selectWindow("EPAC FRET Mask");
		run("Divide...", "value=255 stack");

	selectWindow("EPAC FRET");
		run("Duplicate...", "title=[EPAC FRET for Ratio] duplicate range=1-fr");
		selectWindow("EPAC CFP");
		run("Duplicate...", "title=[EPAC CFP for ratio] duplicate range=1-fr");
		selectWindow("EPAC FRET for Ratio");
		run("Gaussian Blur...", "sigma=2 stack");
		selectWindow("EPAC CFP for ratio");
		run("Gaussian Blur...", "sigma=2 stack");

		imageCalculator("Divide create 32-bit stack", "EPAC CFP for ratio","EPAC FRET for Ratio");
		selectWindow("Result of EPAC CFP for ratio");
		rename("EPAC CFP/FRET Ratio");



	imageCalculator("Multiply create 32-bit stack", "EPAC CFP/FRET Ratio","EPAC FRET Mask");
		selectWindow("Result of EPAC CFP/FRET Ratio");
		rename("Final CFP/FRET Image");
		run("01 Jet");

	selectWindow("Final CFP/FRET Image");	
		run("Clear Results");
		setMinAndMax(0.5677, 0.7702);
		

	selectWindow("EPAC FRET Mask");		
		run("Close");
	selectWindow("EPAC CFP/FRET Ratio");
		run("Close");
	selectWindow("EPAC CFP/FRET");
		run("Close");
	selectWindow("EPAC FRET for Ratio");
		close();
	selectWindow("EPAC CFP for ratio");
		close();

	run("Tile");
		selectWindow("EPAC FRET");
		selectWindow("EPAC CFP");
		selectWindow("Final CFP/FRET Image");
}




macro "Close All Windows [F4]" {
		while (nImages>0) {
		selectImage(nImages);
		close();
	}
	}