// This macro is designed to determine the amount of FKBP within the Golgi versus the ER. It employes the use of a Golgi Marker to mask the Golgi region. 
// The first channel of the image stack has been designed to be the Golgi Marker, or any ,marker being used for the pass through filter. 
// The second channel of the image stack has been designed to be the FKBP channel, or any protein marker whos location is being determined. 
// If more than 2 images are present within the stack they will be split, but not discarded. 
// The final result will be a tiled series of images with the original channels present and the thresholded channels to measure. 
 
//Designed by Dan Shiwarski version 1.0 November 25, 2013. Validated and used by Collin Bachert.




// This is the start of the macro loop.
macro "FKBP Analysis" {
	
	rename("FKBP Analysis");

	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()


// This is a background subtraction loop to minimize the noise within the images.
	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 [>]");
		}


// This is a series of steps to rename the images after the background has been removed. 
	selectWindow("FKBP Analysis")
		run("Split Channels");
			selectWindow("C1-FKBP Analysis");
			rename("Golgi Channel");
			run("Enhance Contrast", "saturated=0.35");
			selectWindow("C2-FKBP Analysis");
			rename("FKBP Channel");
			run("Enhance Contrast", "saturated=0.35");


// This series of steps removes the spekle noise from the image and creates a mask of the Golgi to use as a passthrough filter. 		
	selectWindow("Golgi Channel")
		run("Duplicate...", "title=[Golgi Channel -1]");
			run("Despeckle");
			run("Despeckle");
			run("Despeckle");
		setAutoThreshold("MaxEntropy dark");
		run("Convert to Mask");
		run("Divide...", "value=255.000");
		run("Enhance Contrast", "saturated=0.35");


// This series of steps removes the spekle noise from the image and mutiplies it by the Golgi filter to obatin FKBP within the Golgi. 
	selectWindow("FKBP Channel");
			run("Despeckle");
			run("Despeckle");
			run("Despeckle");
		imageCalculator("Multiply create", "FKBP Channel","Golgi Channel -1");
			selectWindow("Result of FKBP Channel");
			rename("FKBP in Golgi");
			run("Duplicate...", "title=[FKBP in Golgi-Threshold]");
				setAutoThreshold("Triangle dark");
				
// Final Steps run auto thresholding procedures, ,clears any current results and Tiles the images. 
	selectWindow("FKBP Channel");
		run("Duplicate...", "title=[FKBP Channel-Threshold]");
		setAutoThreshold("Triangle dark");
	

	run("Clear Results");	
	selectWindow("Golgi Channel -1")
	run("Tile");
}



// This macro enabels the F4 key to be used to close all open images if the macro has been imstalled prior to running the analysis. 
	macro "Close All Windows [F4]" {
		while (nImages>0) {
		selectImage(nImages);
		close();
	}
	}