//by Louisiane Perrin from Gligorijevic Lab
//2020.06.23
//This macro measures the area of the spheroid over time and fits an ellipse.
//To save individual images (i.e. timepoints) within a stack (i.e. timelpase x,y movie), save your image as an image sequence: File>Save As>Image Sequence
//Utilize the TIFF format and select the directory to save the images. This directory will be then utilized by the macro.

//Select Directory
   requires("1.33s"); 
   run("Set Measurements...", "area centroid fit display redirect=None decimal=6");
   roiManager("Reset");
   dir = getDirectory("Choose a Directory");
   Dialog.create("Type parent folder");
   Dialog.addString("Parent folder", "Parent folder");
   Dialog.show();
   parentname=Dialog.getString;
   count = 0;
   countFiles(dir);
   n = 0;
   parentindex=indexOf(dir, parentname);
   processFiles(dir, parentindex);

   
//Count files in Directory
function countFiles(dir) {
      list = getFileList(dir);
      for (i=0; i<list.length; i++) {
          if (endsWith(list[i], "/"))
              countFiles(""+dir+list[i]);
          else
              count++;
      }
  }

//Process images
function processFiles(dir, parentindex) {
      list = getFileList(dir);
      initialrow=0;
      for (i=0; i<list.length; i++) {
          if (endsWith(list[i], "/"))
              processFiles(""+dir+list[i], parentindex);
          else {
             showProgress(n++, count);
             path = dir+list[i];
             processFile(path, n, initialrow, parentindex);
             initialrow++;
             }
      }
  }

function processFile(path, n, initialrow, parentindex) {  
       if (endsWith(path, ".tif")) {
       	    run("Bio-Formats Macro Extensions");
       		run("Bio-Formats Importer", "open=&path color_mode=Default view=Hyperstack stack_order=XYCZT use_virtual_stack");
           imagedir=getDirectory("image");
           imagetitle=getTitle();

           setLocation(0,23,700,700);
           run("Grays");
           	run("Enhance Contrast...", "saturated=0.3");//Optional
           	run("Auto Threshold", "method=RenyiEntropy white");//Update the thresholding method
           	run("Fill Holes");
           	setTool("polygon");
           	waitForUser("Select area for analysis or click OK if whole image can be used");
           	run("Analyze Particles...", "size=30-Infinity include summarize add");//Update the particle size
           	roiManager("Combine");
           	roiManager("Measure");
           	saveAs(".tif", imagedir + imagetitle + "_SpheroidArea.tif");
           	roiManager("Delete");
           	close();
       }
       run("Close All");
}