pathwayGran=
pathwayTA=
//where to save

dir = getDirectory("Choose a Directory ");
   setBatchMode(true); //doesn't open image, but processes them without opening - much faster
   count = 0;
   countFiles(dir);
   n = 0;
   processFiles(dir);
   //print(count+" files processed");
   
   function countFiles(dir) {
      list = getFileList(dir);
      for (i=0; i<list.length; i++) {
          if (endsWith(list[i], "/"))
              countFiles(""+dir+list[i]);
          else
              count++;
      }
  }

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

  function processFile(path) {
       if (endsWith(path, ".tif")) {
           open(path);



//Pre-Stage

img=getTitle();

run("Duplicate...", "title=TB1");
run("Duplicate...", "title=Lap1");


//For Granuloma isolation

	run("Mean...", "radius=10");
	setOption("ScaleConversions", true);
	run("8-bit");
	run("Invert");
	run("Duplicate...", "title=Lap2");
	run("FeatureJ Laplacian", "compute smoothing=10");
	run("8-bit");

imageCalculator("Subtract create", "Lap2","Lap2 Laplacian");

selectWindow("Result of Lap2");
	run("Duplicate...", "title=Gran1");
	run("Duplicate...", "title=Gran2");
	run("Duplicate...", "title=Gran3");
	run("Duplicate...", "title=Edge1");


//Stage 1 - Granuloma ROI 
	selectWindow("Edge1");
	run("Invert");
	setAutoThreshold("Li"); //change with Stage 3
	//setThreshold(128, 255);
	setOption("BlackBackground", true);
	run("Convert to Mask");
	run("Create Selection");

type = selectionType();
   if (type==-1) {
      //print("no selection");
      makeRectangle(0, 0, 12000, 12000);
      setBackgroundColor(0, 0, 0);
	  run("Clear", "slice");
   }
   
   else if (type>=0 && type <=10) {
		run("Enlarge...", "enlarge=-5 pixel");
		run("Enlarge...", "enlarge=150 pixel");
		run("Enlarge...", "enlarge=-120 pixel");
		run("Invert");
		run("Select None");
		run("Create Selection");
		roiManager("Add");	
	}

//Stage 2 - Granuloma ROI
		
		selectWindow("Gran1");
		run("Auto Threshold", "method=Otsu"); 
		run("Create Selection");

type = selectionType();
   if (type==-1) {
      //print("no selection");
      makeRectangle(0, 0, 12000, 12000);
      setBackgroundColor(0, 0, 0);
	  run("Clear", "slice");
   }
   
   else if (type>=0 && type <=10) {
		run("Enlarge...", "enlarge=80 pixel");
		run("Enlarge...", "enlarge=-120 pixel"); 
		run("Enlarge...", "enlarge=120 pixel");
		roiManager("Add");
		
   }	
		
//Stage 3 - Granuloma ROI
		selectWindow("Gran2");
		setAutoThreshold("Li"); //change with Stage 1
		run("Convert to Mask");
		run("Create Selection");

type = selectionType();
   if (type==-1) {
      //print("no selection");
      makeRectangle(0, 0, 12000, 12000);
      setBackgroundColor(0, 0, 0);
	  run("Clear", "slice");
   }
   
   else if (type>=0 && type <=10) {
		roiManager("Add");
   }

		selectWindow("Gran3");
		roiManager("Select", 0);
		run("Clear Outside");
		roiManager("Select", 1);
		run("Clear Outside");
		roiManager("Select", 2);
		run("Clear Outside");

run("Select None");

saveAs("TIFF", pathwayGran+"Gran_"+img+"_Granarea.tif");

	for (i=(roiManager("count")-1); i>=0; i--) {
			roiManager("select", i);  // select the ROI 
			roiManager("Delete");  // delete the ROI 	
		} 

//setAutoThreshold("Huang");
		run("Select None");
		setThreshold(1, 255);
		setOption("BlackBackground", true);
		run("Convert to Mask");
		run("Fill Holes");
		run("Create Selection");

		type = selectionType();
   			if (type==-1) {
      		//print("no selection");
      			makeRectangle(0, 0, 12000, 12000);
      			setBackgroundColor(0, 0, 0);
	  			run("Clear", "slice");	  				
   			}
   
   			else if (type>=0 && type <=10) {
				
				run("Enlarge...", "enlarge=-50 pixel");
				run("Enlarge...", "enlarge=5 pixel");
				roiManager("Add");   			  			

// Isolate TB signal
		
		selectWindow("TB1");
		run("8-bit");
		run("Median...", "radius=1");
		run("Duplicate...", "title=TB2");
		imageCalculator("Multiply create 32-bit", "TB1","TB2");
		run("8-bit");
		rename("Img_Sq");
		run("Duplicate...", "title=Img_Sq2");
		run("Remove Outliers", "block_radius_x=100 block_radius_y=100 standard_deviations=1.50");
		imageCalculator("Subtract create", "Img_Sq","Img_Sq2");	

// Clear outside granuloma ROI 

		run("8-bit");
		roiManager("Select", 0);
		run("Clear Outside");
}
	//waitForUser("continue?");	
		saveAs("TIFF", pathwayTA+"TB_"+img+"_TBarea.tif");	
		
		for (i=(roiManager("count")-1); i>=0; i--) {
			roiManager("select", i);  // select the ROI 
			roiManager("Delete");  // delete the ROI 	
		} 
		 
		 run("Close All");


       }
  }

// RenyiEntropy autothreshold on data produced - for Imaris 3D input