This macro is listed in the appendix of chapter 15 by Merrill et al. “Measuring mitochondrial shape with ImageJ” in the upcoming book “Techniques to investigate mitochondrial function in neurons” Eds.: Strack & Usachev, Series: Neuromethods, Springer, Berlin, 2017

var ch = 0; /* channel to be analyzed for RGB images */ /* * Measure mitochondrial morphology in the current selection * Ctrl+Shift+O closes current and opens next image */

macro “Simple Morphometry [F7]” {

title = getTitle();
morphometry(title, false); // not batch mode

} /* * Batch-apply a set of “named” ROIs to analyze images with that file name */

macro “Batch Morphometry [F8]” {

dir = getDirectory("Select an image directory");
while (roiManager("Count") == 0)
	waitForUser("Please open named ROIs into ROI manager");
prevName = imgName = "";
n = roiManager("Count");
for (i = 0; i < n; ++i) { // loop through the ROI Manager table
	prevName = imgName;
	imgName = call("ij.plugin.frame.RoiManager.getName", i);
	if (isOpen(imgName)) { // named image is open
		selectWindow(imgName);
	} else { // done with current image, close and open next
		if (isOpen(prevName)) {
			selectWindow(prevName);
			close();
		}
		open(dir + imgName);
	}
	roiManager("Select", i); 
	morphometry(imgName, true); // batch mode
}

}

function morphometry(title, batchMode) {

while (ch < 1 || ch > 3) { // RGB channel not yet selected, initialize
	ch = getNumber("Analyze RGB channel(1-3):", 1);
	run("Set Measurements...", "decimal=5 area perimeter fit");
	print("image\t n\t area2\t area-weighted ff\t form factor\t aspect ratio\t length"); // header for results table
}
if (bitDepth == 24) // RGB image
	run("Make Composite");
if (isOpen("Binary")) {
	selectWindow("Binary");
	close();
} // close previous working image
if (isOpen("Skeleton")) {
	selectWindow("Skeleton");
	close();
} // close previous working image
selectWindow(title);
if (selectionType() == -1) // no selection
	run("Select All");
if (!batchMode) {
	roiManager("Add"); // save selection to ROI Manager and rename to image name (for batch processing)
	last = roiManager("Count") - 1;
	roiManager("Select", last);
	roiManager("Rename", title);
	/* roiManager("Save", File.directory + "named_ROIs.zip"); */ // un-comment to save ROIs automatically
}
// copy selection to new window and clear outside
setSlice(ch); // ignored if grayscale
run("Duplicate...", "title=Binary");
run("Make Inverse");
if (selectionType != -1) { // outside of ROI is selected
	run("Duplicate...", " "); // make a mask of the background
	run("Convert to Mask");
	run("Create Selection");
	run("Make Inverse");
	roiManager("Add");
	close();
	n = roiManager("Count");
	roiManager("Select", n - 1);
	getRawStatistics(_area, backG); // mean is background
	setColor(backG);
	run("Restore Selection"); // fill outside of selection with background
	fill();
	run("Gaussian Blur...", "radius=64"); // smooth abrupt background transition
	roiManager("Delete"); // delete masking selection (ROI manager accumulates cell selections)
}
run("Select None");
// subtract background and threshold
run("Subtract Background...", "rolling=50"); // non-destructive filter even if already applied
run("Make Binary"); 
// also try other threshold methods included with Fiji, e.g.: run("Auto Threshold", "method=Li white");
// create Results table of metrics, one line/particle
run("Analyze Particles...", "size=9-Infinity circularity=0.00-1.00 show=Masks pixel clear");
awff = ff = ar = sum_a = a2 = len = 0;
for (i = 0; i < nResults; i++) { // for every particle in table
	a = getResult("Area", i);
	p = getResult("Perim.", i);
	ar += getResult("Major", i) / getResult("Minor", i); // aspect ratio = length / width
	sum_a += a;
	a2 += a * a; // area2 = a2 / (sum_a * sum_a)
	awff += b = (p * p) / (4 * 3.14159265358979); // awff = ff * (a / sum_area)
	ff += b / a; // ff = p^2 / (4 * pi * a)
}
nParticles = nResults;
// skeletonize to get length
selectWindow("Mask of Binary"); // created by Analyze Particles .., excludes noise (< 9 pixels)
rename("Skeleton");
run("Skeletonize");
run("Analyze Particles...", "size=0-Infinity show=Nothing pixel clear");
for (i = 0; i < nResults; i++)
	len += getResult("Area", i);
// average and output
a2 /= sum_a * sum_a;
awff /= sum_a;
ff /= nParticles;
ar /= nParticles;
len /= nResults;
print(title + "\t " + nParticles + "\t " + a2 + "\t " + awff + "\t " + ff + "\t " + ar + "\t " + len);
selectWindow(title);

}