//WH_NJ macro
 
//initial post-processing, doesn't require modification
run("8-bit");
run("Sharpen", "stack");
run("Find Edges", "stack");
setAutoThreshold("Default");
//run("Threshold...");
setAutoThreshold("Huang");
setOption("BlackBackground", true);
run("Convert to Mask", "method=Default background=Dark calculate black");
 
//post-processing required to remove any holes that may exist outside the main scratch. User may need to adjust the counts and iterations, according the sizes of undesirable holes
run("Options...", "iterations=4 count=3 do=Open stack");
run("Options...", "iterations=1 count=1 do=[Fill Holes] stack");
 
//standard attributes to calculate the main scratch. May vary according the size (0-infinity) of image. For our usage we calibrate ImageJ using "mm" as unit of length (ratio of 280 pixels / mm) and the user can increase or decrease this value by 0.05 to 0.05 points
run("Analyze Particles...", "size=0.1-Infinity show=[Overlay Outlines] display summarize add stack");
 
//this command request the user to open the last used image, for overlaying the image with the calculated area
open();
run("Sharpen", "stack");
run("From ROI Manager");
run("Overlay Options...", "stroke=black width=3 fill=none apply set");
 
//choose the destination directory to save all overlapped images
run("Image Sequence... ", "format=JPEG use save=[]");