//Macro to generate Kymos from MT snaps and GFP streams.
//Chris Gell 21-05-2018
//Version 2

//macro to make kymos for gfp tirf data
//assumes 2 images are loaded and that 
//these are a stream and a mt.
//go to either and add lines to the 
//ROI manager. Then run the macro.

//to do the next one clear the ROI manager
//and close all open windows.
//load the next strem and snap.



// to add 
// DONE- have it work out which is the stream
// reduce thickness or transparency of the lines in the mt map
//DONE - name directory in an intelegent way - not wanted, not flexible.
//DONE, not wanted - cope with multiple streams (of the same MT FOV)?




//environment variables
envMakeFullOverlay=0;
envAskForPNGs=0;





//see if any images are open.
if (nImages!=2) {
	//
	Dialog.create("");
	Dialog.addMessage("Too many or too few images open, open 2 images, the MTs and stream.");
	Dialog.show();
	exit; 
}



roiManager("show all with labels")


//ask for an identifier for this analysis
expName=getString("Enter an indetifier for this experiment", "");




 //create an array with a list of open window names and then work out which image is which.
 n = nImages; 
    list = newArray(n); 
    for (i=1; i<=n; i++) { 
        selectImage(i); 
        list[i-1] = getTitle; 
    } 

selectWindow(list[0]);
getDimensions(width, height, channels, slices, frames);
if (slices==1) {
	gfpImageName=list[0];
	mtImageName=list[1];
}

if (slices>=1) {
	gfpImageName=list[1];
	mtImageName=list[0];
}



//get the number of frames in the stream
selectWindow(gfpImageName);
getDimensions(width, height, channels, slices, frames);
 gfpImageID=getImageID();

  n = frames;


//make the MT image the same length
 selectWindow(mtImageName);
 mtImageID=getImageID();
 //make a blurred mt image, a little better look for the kymos
 run("Gaussian Blur...", "sigma=1");
   
  run("Copy");
  for (i=0; i<n-1; i++) {
      run("Add Slice");
      run("Paste");
  }


//if wanted make a full overlay


if (envMakeFullOverlay==1) {
setSlice(1);
run("Select None");
//create mt id tools
run("Merge Channels...", "c2=["+gfpImageName+"] c6=["+mtImageName+"] create keep");
run("Z Project...", "projection=[Average Intensity]");
run("Red");
run("Enhance Contrast", "saturated=0.35");
}

//the user needs to add the ROI
waitForUser("Please make sure you have selected ROI's as required.");


//loop throught the ROI manager to create each of the kymos
n = roiManager("count");
  for (i=0; i<n; i++) {
      


//Make the GFp channel kymo
selectWindow(gfpImageName);
roiManager("select", i);
Roi.setStrokeWidth(3);
run("Multi Kymograph", "linewidth=3");
selectWindow("Kymograph");
rename("GFP - Kymograph" + (i + 1) +" "+expName);
run("Green");
run("Enhance Contrast", "saturated=0.35");

//Make the MT kymo
selectWindow(mtImageName);
roiManager("select", i);
Roi.setStrokeWidth(7);
run("Magenta");
run("Multi Kymograph", "linewidth=7");
selectWindow("Kymograph");
rename("MT - Kymograph" + i+1+" "+expName);
selectWindow("MT - Kymograph" + (i+1)+" "+expName);
run("Magenta");

//make the merged kymo
run("Merge Channels...", "c2=[GFP - Kymograph"+ (i+1)+" "+expName+"] c6=[MT - Kymograph"+ (i+1)+ " "+expName+"] create");
selectWindow("Composite");
rename("MT - GFP - Kymograph" + (i+1)+" "+expName);


  }// end of ROI loop


//make record image
selectWindow(mtImageName);
run("Duplicate...", " ");
rename("MTs");
run("Invert");
run("Grays");
run("Enhance Contrast", "saturated=0.35");
roiManager("Show All");

//save everything
dir = getDirectory("Choose a Directory");
//dirTemp = getDirectory("Choose a Directory");
//File.makeDirectory(dir+expName); 
//newDir=dir + "/"+expName+"/";
newDir=dir;

 roiManager("deselect"); 
 roiManager("save", newDir +expName+" "+gfpImageName+".zip")
 roiManager("Show None"); 


//close orignals
selectWindow(mtImageName);
close(mtImageName);
selectWindow(gfpImageName);
close(gfpImageName);


ids=newArray(nImages); 
for (i=0;i<nImages;i++) { 
        selectImage(i+1); 
        title = getTitle; 
        //print(title); 
        ids[i]=getImageID; 

        saveAs("tiff", newDir + expName + title + " " + "MT-" + mtImageName+ "Stream-"+gfpImageName); 
}



if (envAskForPNGs==1) {

 //ask user if wants kymo and mt 'map' saving as png.
//getBoolean("Save PNGs of everything?");
 if (getBoolean("Save PNGs of everything?")) {
 
ids=newArray(nImages); 

for (i=0;i<nImages;i++) { 
        selectImage(i+1); 
        title = getTitle; 
        //print(title); 
        ids[i]=getImageID; 
		//run("Flatten");
        //saveAs("png", dir+title+" "+gfpImageName); 
        saveAs("png", newDir+expName+title); 
}
} 
}


//End of code