  PK
     ^.Nz¯/ß  ß  A   10xSM22forCosV Nuclei Maxima UserInputGreenwithROImanagerJune.ijm//Macro to identify overlaps in 3 channel images, and output the positive cells as a new channel.
//Dale Moulding and Carlotta Camilli June 2016
//User defined thresholding for highly variable input images

 macro "SM22 10x analysis"{
  
  dir1 = getDirectory("Input folder: images at any magnification from a single microscope"); //select an input folder
  dir2 = getDirectory("Choose a folder to save to"); //select an output folder. 
  list = getFileList(dir1); //make a list of the filenames
  //setBatchMode(true); 

for (i=0; i<list.length; i++) {
 	showProgress(i+1, list.length);
 	filename = dir1 + list[i];
 	if (endsWith(filename, "tif")||endsWith(filename, "lsm")) {
open(filename);	
Imagename = File.nameWithoutExtension;
h=getHeight();
w=getWidth();
run("Colors...", "foreground=black background=white selection=yellow");
roiManager("Reset");

filename=getTitle();
run("Duplicate...", "title=Copy duplicate");
selectWindow(filename);
//run("Subtract Background...", "rolling=10 stack");
run("Median...", "radius=1 stack");
run("Split Channels");
selectWindow("C1-"+filename);
run("Find Maxima...", "noise=45 output=[Maxima Within Tolerance]"); // find all maxima and make a new black and white image of all signals around each maxima i.e nuclei
run("Dilate"); run("Watershed");
run("Analyze Particles...", "size=5-200 show=Masks include summarize");
rename("DapiMask");// makes a new window called "DapiMask" the dapi channel
run("Duplicate...", "title=DapiMaskCopy duplicate");//duplicates the DapiMask
selectWindow("C2-"+filename);
run("Threshold...");
waitForUser("Set Threshold, press 'Apply' then press 'OK");
//setThreshold(50, 255);
setOption("BlackBackground", false);
run("Convert to Mask");
//run("Dilate"); 
run("Close-"); 
run("Watershed"); //join up closely stained parts of a cell, then run a watershed to divide into cells.
run("Analyze Particles...", "size=15-Infinity pixel show=Masks include summarize");// makes a new window called "Mask of C2-filename" the green cytoplasm channel
rename("CytoMask");// makes a new window called "CytoMask" the green cytoplasm channel
run("Duplicate...", "title=CytoMaskCopy duplicate");//duplicates the CytoMask
selectWindow("C3-"+filename);
run("Median...", "radius=1 stack");
run("Find Maxima...", "noise=35 output=[Maxima Within Tolerance]"); // find all maxima and make a new black and white image of all signals around each maxima i.e nuclei called "Mask of C3-"+filename+" Maxima"                                                                           
run("Dilate"); run("Watershed");
run("Analyze Particles...", "size=5-200 show=Masks include summarize");// makes a new window called "Mask of C3-filename" the red nuclei channel
rename("RedNucMask");// makes a new window called "RedNucMask" the red nuclei channel
run("Duplicate...", "title=RedNucMaskCopy duplicate");//duplicates the RedNucMask
imageCalculator("AND create", "CytoMask","RedNucMask");
rename("RedNucinGreen");// makes a new window of the overlap between green cytoplasm and red nuclei called "RedNucinGreen"
run("Clear Results");
run("Find Maxima...", "noise=250 output=List light"); // this section counts the red nuclei in green cells, records the xy coordinates and highlights each nuclei with the magic wand

selectWindow("RedNucMask");
          for(n=0; n<nResults;n++) {
	        x=(getResult("X", n));
	        y=(getResult("Y", n));
        
            doWand(x, y, 10.0, "Legacy");
            roiManager("Add");
           }
newImage("RedNucinGreenCells", "8-bit white", w, h, 1);
roiManager("Show All");
roiManager("Fill");
run("Duplicate...", "title=RedNucInGreenCells2 duplicate"); // end of section counting red nuclei in green cells.

selectWindow("Copy");
run("Split Channels");
run("Merge Channels...", "c1=C1-Copy c2=C2-Copy c3=C3-Copy c4=DapiMask c5=CytoMask c6=RedNucMask c7=RedNucInGreenCells2 create");
selectWindow("Copy");
Stack.setChannel(1); //choose the colour of the first channel:
          run("Blue");
          Stack.setChannel(2); //choose the colour of the second channel:
          run("Green");
          Stack.setChannel(3); //choose the colour of the third channel:
          run("Red");
          Stack.setChannel(4); //choose the colour of the identified Red Nuclei channel:
          run("Grays");
          Stack.setChannel(5); //choose the colour of the masked Dapi channel:
          run("Cyan");
          Stack.setChannel(6); //choose the colour of the maksed green channel:
          run("Magenta");
          Stack.setChannel(7); //choose the colour of the masked red nuclei channel:
          run("Yellow"); run("Invert LUT");
selectWindow("Copy");
saveAs("Tiff", dir2+Imagename+"-masks&RedNucinGreenCells");//saves an image of the original plus masked Dapi, Green, Red Nuclei and Red nuclei in Green cells.

//this section selects Dapi nuclei that aren't red, then makes a new count and image of these non Red Dapi cells that are +ve for green cytoplasm
selectWindow("RedNucMaskCopy");//select the red nuclei mask window
run("Clear Results");
roiManager("Reset");
run("Find Maxima...", "noise=250 output=List light"); // this section counts the red nuclei in green cells, records the xy coordinates and highlights each nuclei with the magic wand
selectWindow("DapiMaskCopy");// dapi mask window to draw rois on and delete
run("Duplicate...", "title=DapiOnly duplicate"); 
           for(n=0; n<nResults;n++) {
	        x=(getResult("X", n));
	        y=(getResult("Y", n));
                if (getPixel(x, y)==255){
                    doWand(x, y, 10.0, "8-connected");
            roiManager("Add");
           }
           }
roiManager("Show All");
run("Colors...", "foreground=white background=white selection=yellow");
roiManager("Fill");
run("Colors...", "foreground=black background=white selection=yellow");

run("Select None");
run("Analyze Particles...", "size=10-Infinity show=Nothing include summarize"); 

imageCalculator("AND create", "DapiOnly","CytoMaskCopy");
rename("DapiOnlyinGreenCellsTemp"); // makes a new window of the overlap between green cytoplasm and Dapi nuclei that aren't red
run("Clear Results");
run("Find Maxima...", "noise=250 output=List light"); // this section counts the dapi only nuclei in green cells, records the xy coordinates and highlights each nuclei with the magic wand.
//
roiManager("Reset");
selectWindow("DapiOnly");
          for(n=0; n<nResults;n++) {
	        x=(getResult("X", n));
	        y=(getResult("Y", n));
        
            doWand(x, y, 10.0, "Legacy");
            roiManager("Add");
           }
newImage("DapiNucinGreenCells", "8-bit white", w, h, 1);
roiManager("Show All");
roiManager("Fill");
run("Select None");
run("Analyze Particles...", "size=10-Infinity show=Nothing include summarize"); 

selectWindow("DapiOnly");
run("Select None");
//selectWindow("DapiNucinGreenCells");
open(dir1+filename);
rename("OrigImage");	
run("Split Channels");
run("Merge Channels...", "c1=C1-OrigImage c2=C2-OrigImage c3=C3-OrigImage c4=DapiOnly c5=DapiNucinGreenCells create");
selectWindow("OrigImage");
Stack.setChannel(1); //choose the colour of the first channel:
          run("Blue");
          Stack.setChannel(2); //choose the colour of the second channel:
          run("Green");
          Stack.setChannel(3); //choose the colour of the third channel:
          run("Red");
          Stack.setChannel(4); //choose the colour of the identified Red Nuclei channel:
          run("Cyan");
          Stack.setChannel(5); //choose the colour of the masked Dapi channel:
          run("Grays"); run("Invert LUT");
saveAs("Tiff", dir2+Imagename+"+DapiOnlyinGreenCells");//saves an image of the nuclei that only have Dapi as Ch4 and inside green cells as Ch5.
run("Close All");
roiManager("Reset");
selectWindow("Summary"); saveAs("Results", dir2+Imagename+"-summary.csv");
run("Close");
 	}
}

exit("Macro finished "+i+" images processed");
 }PK? 
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