// This version uses the built-in selection Convex Hull (for Start Mask) and Fit Ellipse (for centering Start) features of ImageJ.
// It is based on v7, which uses the ImageJ Fit Spline for the start mask and convex hull for centering.

     requires("1.43s");
     run("Tile");
     Dialog.create("Input Filenames");
     Dialog.addMessage("Type the names of\nthe start and end\nimages below.");
     Dialog.addString("Start =:", "?.tif");
     Dialog.addString("End =:", "?.tif");
     Dialog.show();
     startimage = Dialog.getString();
     endimage = Dialog.getString();;
	selectImage(startimage);
	run("Duplicate...", "title=Start");
	selectImage(endimage);
	run("Duplicate...", "title=End");

//setBackgroundColor(0, 0, 0);
setForegroundColor(0, 0, 0);
roiManager("Centered", "false");
selectImage("Start");
     run("Subtract Background...", "rolling=50");
     run("Brightness/Contrast...");
     run("Enhance Contrast", "saturated=0.5");
     run("8-bit");
     run("Make Binary");

//Or, the following may be sufficient for brighter images...
//     run("Subtract Background...", "rolling=50");
//     run("8-bit");
//     run("Make Binary");

//The following eliminates far-outliers in the Start image by dilating the central array of 
//nuclei to a 'singularity', then erasing everything outside the bounds of this cluster.
     run("Duplicate...", "title=Start_Dilated");
     run("Options...", "iterations=10 black count=1");

// Note that originally, we used iterations=10. The lower value here results in exclusion of more distant nuclei.
// Indeed, if the 'End' image is such that cells along the periphery have moved considerable distances away 
// from their nearest neighbor, the iteration value of 15 in the section below could increased empirically.
     run("Dilate");
     run("Fill Holes");
     w=getWidth(); h=getHeight();
     wandx=w/2; wandy=h/2;
     //setTool(8);
     doWand(wandx, wandy);
     run("Add to Manager ");
     roiManager("Add");
     selectWindow("Start");
     roiManager("Select", 0);
     run("Clear Outside");

// The following finds the minimum bounding circle of the nuclei, adds the region to the
// Manager, uses it to find the center of the image, then crops the image so as to
// center the nuclei in the frame.

     run("Convex Hull");
     roiManager("Add");
     run("Fit Ellipse");
     roiManager("Add");
     roiManager("Select", 3);     
     smx = 0; smy = 0; 
     getSelectionBounds(x, y, w, h);
     smx=x+(w/2); smy=y+(h/2); 
     scanvasw=getWidth(); scanvash=getHeight;
     snw=0; snh=0;
     if (smx < scanvasw/2)
	snw = 2*smx;
     else snw=2*(scanvasw-smx);
     if (smy < scanvash/2)
	snh = 2*smy;
     else snh=2*(scanvash-smy);
     run("Specify...", "width="+snw+" height="+snh+" x="+smx+" y="+smy+" centered");
     run("Crop");


selectImage("End");
     run("Subtract Background...", "rolling=50");
     run("Brightness/Contrast...");
     run("Enhance Contrast", "saturated=0.5");
     run("8-bit");
     run("Make Binary");

//The following eliminates far-outliers in the End image by dilating the central array of 
//nuclei to a 'singularity', then erasing everything outside the bounds of this cluster.
// Note that if the 'End' image is such that cells along the periphery have moved considerable 
// distances away from their nearest neighbor, the number of iterations - currently 15 - below 
// could increased empirically.
     run("Duplicate...", "title=End_Dilated");
     run("Options...", "iterations=15 black count=1");
     run("Dilate");
     run("Fill Holes");
     w=getWidth(); h=getHeight();
     wandx=w/2; wandy=h/2;
     //setTool(8);
     doWand(wandx, wandy);
     run("Add to Manager ");
     roiManager("Add");
     selectWindow("End");
     roiManager("Select", 4);
     run("Clear Outside");

//The following centers the End image, 'substracts' the Start image particles, 
// and counts the remaining particles.
     run("Fit Ellipse");
     roiManager("Add");
     emx = 0; emy = 0; 
     roiManager("Select", 6);
     getSelectionBounds(x, y, w, h);
     emx=x+(w/2); emy=y+(h/2); 
     ecanvasw=getWidth(); ecanvash=getHeight;
     enw=0; enh=0;
     if (emx < ecanvasw/2)
	enw = 2*emx;
     else enw=2*(ecanvasw-emx);
     if (emy < ecanvash/2)
	enh = 2*emy;
     else enh=2*(ecanvash-emy);
     run("Specify...", "width="+enw+" height="+enh+" x="+emx+" y="+emy+" centered");
     run("Crop");
     run("Duplicate...", "title=End-unmasked");
     selectWindow("End");
     roiManager("Centered", "true");
     roiManager("Select", 2);
     run("Clear");
     run("Select None");
     run("Make Binary");
     run("Analyze Particles...", "size=1.50-Infinity circularity=0.00-1.00 show=Masks exclude clear summarize");

//Data presentation and window clean-up
selectWindow("B&C");
run("Close");
//selectWindow("ROI Manager");
//run("Close");
if (isOpen("Mask of End")==1) {
    selectWindow("End");
    run("Set... ", "zoom=25");
}
selectWindow("End");
run("Set... ", "zoom=25");
selectWindow("End-unmasked");
run("Set... ", "zoom=25");
selectWindow("End_Dilated");
run("Close");
selectWindow("Start");
run("Set... ", "zoom=25");
selectWindow("Start_Dilated");
run("Close");
run("Tile");
if (isOpen("Summary")==1) 
    selectWindow("Summary");
else
    print("Number of migrated cells = 0 for this image pair.");