//Full fluorescent counter macro by Anthony Joshua, with contributions from Tony Collins, Christopher Mei and thansk to Wayne Rasband for the Paintbrush tool macro incorporated herein

macro "Setup [F1]" {

//Ask for file containing raw flourescent image
image=getBoolean("Do you want to open the composite image");
if (image==1) {open();}
else if (image==0) {}

//Ask for need for copy
copy=getBoolean("Do you want to copy the composite image");
if (copy==1) {run("Duplicate...", "title=CopyofComposite");}
else if (image==0) {}

//Ask for file containing DAPI image
image2 = getBoolean("Do you want to open the DAPI image");
if (image2==1) {open();}
else if (image2==0) {}

//Select the fluorescent image
selectImage(1);

//Ask for ROIs
ROI=getBoolean("Can you open the region of interest file");
if (ROI==1){open();}
else if (ROI==0){showMessage("ROI","Please define ROIs,save them, then press F1 to restart");
run("ROI Manager...");
exit;}

//Carry out rolling ball
rb=getNumber("Rolling ball radius[recommended]", 5);
run("Subtract Background...", "rolling="+rb);

//Ask user to remove lipofuscin and colour lumens black
showMessage("Removing Noise", "Please remove luminal noise and autoflourescence then press F2 OR after thresholding visually press F3");
	stage = 2;
//open paintbrush tool

//   This is a tool macro similar to the paint brush tool in NIH Image.
//   It draws in the current foreground color, which can be set by
//   double-clicking on the eye dropper tool. Double-click on 
//   the paintbrush tool icon to set the brush width.
 
   macro "Paintbrush Tool - C0aao33aa" {
	var brushWidth = 10;
        leftClick=16;
        //setupUndo(); // requires 1.32g or later
        getCursorLoc(x, y, z, flags);
        setLineWidth(brushWidth);
        moveTo(x,y);
        x2=-1; y2=-1;
        while (true) {
            getCursorLoc(x, y, z, flags);
            if (flags&leftClick==0) exit();
            if (x!=x2 || y!=y2)
                lineTo(x,y);
            x2=x; y2 =y;
            wait(10);
        }
   }

  // ImageJ runs this macro when user double-clicks on the paint brush tool icon
  macro 'Paintbrush Tool Options' {
      brushWidth = getNumber("Brush Width:", brushWidth);
  }


macro "Process [F2]" {

showMessage("Thresholding", "Ready for Otsu Thresholding- please wait");

//Carry out Otsu Thresholding
run("OtsuThresholding 16Bit");

//Check the threshold
getThreshold(lower,upper);
print("your thresholds are;"+ lower,"to"+ upper);

//Run through the ROIs
run("Set Measurements...", "area mean standard min limit redirect=None decimal=3");
run("Set Scale...", "distance=1 known=1 pixel=1 unit=pixel global");
n = roiManager("count");
dir = getDirectory("Select a directory for output files");
name = getString("Name of file:", "output file name");
  for (i=0; i<n; i++) {
      	roiManager("select", i);
	run("Analyze Particles...", "minimum=1 maximum=999999 bins=256 show=Nothing display clear");
  	updateResults();
	j=i+1;
	saveAs("text", dir+name+j+".txt");

  }
macro "Analysis-only [F3]" {

showMessage("Thresholded Image", "Your image should already be thresholded");

//Check the threshold
getThreshold(lower,upper);
print("your thresholds are;"+ lower,"to"+ upper);

//Run through the ROIs
run("Set Measurements...", "area mean standard min limit redirect=None decimal=3");
run("Set Scale...", "distance=1 known=1 pixel=1 unit=pixel global");
n = roiManager("count");
dir = getDirectory("Select a directory for output files");
name = getString("Name of file:", "output file name");
  for (i=0; i<n; i++) {
      	roiManager("select", i);
	run("Analyze Particles...", "minimum=1 maximum=999999 bins=256 show=Nothing display clear");
  	updateResults();
	j=i+1;
	saveAs("text", dir+name+j+".txt");

  }
}
selectWindow("Results");
run("Close");
roiManager("deselect");