Dialog.create("Image stained nuclei");
 Dialog.addMessage(" Open image with DAPI Channel: C2        ");
 Dialog.show();
open();
 // make result directory 
      dir1 = getDirectory("current");
      dir2 = dir1+"results"+File.separator;
      File.makeDirectory(dir2);
      if (!File.exists(dir2))
      exit("Unable to create directory");
     filename_Nuc=getTitle();  
     	index_ext=lastIndexOf(filename_Nuc,".tif");
	index_basename=(index_ext-9);
	index_channel=(index_ext-1);
	filename_sh=substring(filename_Nuc,0,index_basename);  
     Stack.getDimensions(width, height, channels, slices, frames);
                channels= channels;
                zslices= slices;
                timeframes=frames;
                Zreduction=1;     //set z stack reduction factor 

//--------------------------------automated z-slice reduction\
//new_slnr=floor(zslices/Zreduction);
//new_slicenr=new_slnr*Zreduction;
//run("Make Substack...", "  slices=1-"+(new_slicenr)+"");rename("temp_substack");
//run("Grouped Z Project...", "projection=[Sum Slices] group="+(Zreduction)+"");
//run("Subtract...", "value=50 stack");
//selectWindow("temp_substack");close();
//selectWindow(filename_Nuc);close();
//selectWindow("SUM_temp_substack");
//-------------------------------automated z slice reduction\
//pre-process nuclei// DAPI stack=================================================
//setMinAndMax(800, 4095);
rename("Nuc_orig");
//setBatchMode(true);
run("Duplicate...", "title=Nuc_Mask duplicate range=1-zslices");
run("Gaussian Blur...", "sigma=7 stack");
imageCalculator("Subtract create stack", "Nuc_orig","Nuc_Mask");
run("Smooth", "stack");

// -------------------------------------threshold binary mask
setThreshold(80, 3500);
run("Convert to Mask", "method=IJ_IsoData background=Dark black");
run("Median...", "radius=3 stack");
run("Watershed", "stack");
//==================================================
//--------------------------------open and automated z-slice reduction EdU
filename_EdU=filename_sh+".lif - C1.tif";
open(filename_EdU);
new_slnr=floor(zslices/Zreduction);
new_slicenr=new_slnr*Zreduction;
run("Make Substack...", "  slices=1-"+(new_slicenr)+"");rename("temp_substack");
run("Grouped Z Project...", "projection=[Sum Slices] group="+(Zreduction)+"");
//run("Subtract...", "value=50 stack");
selectWindow("temp_substack");close();
selectWindow(filename_EdU);close();
selectWindow("SUM_temp_substack");
//pre-process EdU nuclei// stack=================================================
rename("EdU_orig");
//setBatchMode(true);
run("Duplicate...", "title=EdU_Mask duplicate range=1-zslices");
run("Gaussian Blur...", "sigma=7 stack");
imageCalculator("Subtract create stack", "EdU_orig","EdU_Mask");
run("Smooth", "stack");
// -------------------------------------threshold binary mask
setThreshold(100, 3500);
run("Convert to Mask", "method=IJ_IsoData background=Dark black");
run("Median...", "radius=3 stack");
run("Watershed", "stack");

// FOR EdU: clean up roi set for double centroids close to z------------------------------------------
roiManager("Reset");
run("Set Measurements...", "  centroid stack");
run("Analyze Particles...", "size=75-1000 pixel circularity=0.30-1.00 show=Nothing clear add stack");
r02=roiManager("count"); 
k2=0;t=0;
n=nResults;
for(i=0; i<n; i++) { x1= getResult('X', i); y1=getResult('Y', i);s1=getResult('Slice',i);
for(j=i+1; j<n; j++) {		 
  x2= getResult('X', j); y2=getResult('Y', j);s2=getResult('Slice',j);
  dx=abs(x2-x1); dy=abs(y2-y1); dist=sqrt(dx*dx+dy*dy);ds=abs(s1-s2);
if (ds<=3) {	//restricts z position for deleting ROI 
  if (dist<=4) //px = 0.36 um // 9 px ~ 3x3 or ~1 um pix shift of centroid 
  {roiManager("Select",j);roiManager("Rename", "mark");k2=k2+1;}}
} //j-loop
} //i-loop
//delete marked ROIs----------------------------------
post_roi_list=r02-k2; r_updated=r02; roiManager("Sort");
for (t=post_roi_list; t<r_updated; t+1){  roiManager("Select", t); roiManager("Delete");r_updated=r_updated-1;}
print(filename_sh+": EdU: Of isolated "+r02+" ROIs, "+post_roi_list+" were approved, "+k2+" rejected"); 
N_EdU_masked=post_roi_list;
roiManager("Save", dir2+filename_sh+"_EdU_Masked RoiSet.zip");

//----------------------------------------------------------
// measure total nuclei: nuc and EdU images are added since EdU seems to quench DAPI stain
imageCalculator("Max create stack", "Nuc_orig","EdU_orig");
run("Duplicate...", "title=nuc_Outline duplicate range=1-"+(new_slicenr)+"");
run("Remove Outliers...", "radius=5 threshold=30 which=Bright stack");
run("Remove Outliers...", "radius=5 threshold=0 which=Dark stack");		//smooths background
//run("Difference of Gaussians", "  sigma1=20 sigma2=2 stack");
run("Sharpen", "stack");
// threshold binary mask
setThreshold(100, 3500);
run("Convert to Mask", "method=IJ_IsoData background=Dark black");
run("Median...", "radius=2 stack");
run("Watershed", "stack");

// FOR total nuclei: clean up roi set for double centroids close to z------------------------------------------
roiManager("Reset"); run("Clear Results");
run("Set Measurements...", "  centroid stack");
run("Analyze Particles...", "size=75-1000 pixel circularity=0.30-1.00 show=Nothing clear add stack");
r01=roiManager("count"); 
k1=0;t=0;
n1=nResults;
for(i=0; i<n1; i++) { x1= getResult('X', i); y1=getResult('Y', i);s1=getResult('Slice',i);
for(j2=i+1; j2<n1; j2++) {		 
  x2= getResult('X', j2); y2=getResult('Y', j2);s2=getResult('Slice',j2);
  dx=abs(x2-x1); dy=abs(y2-y1); dist=sqrt(dx*dx+dy*dy);ds=abs(s1-s2);
if ((ds<=2) && (dist<=4))	//restricts z position for deleting ROI && px = 0.36 um // 9 px ~ 3x3 or ~1 um pix shift of centroid 
  {roiManager("Select",j2);roiManager("Rename", "mark");k1=k1+1;}
} //j-loop
} //i-loop
//delete marked ROIs----------------------------------
post_roi_list=r01-k1; r_updated=r01; roiManager("Sort");
for (t=post_roi_list; t<r_updated; t+1){  roiManager("Select", t); roiManager("Delete");r_updated=r_updated-1;}
print(filename_sh+": Total Nuc: Of isolated "+r01+" ROIs, "+post_roi_list+" were approved, "+k1+" rejected"); 
N_nuc=post_roi_list;
roiManager("Save", dir2+filename_sh+"_Nuc_Masked RoiSet.zip");
//----------------------------------------------------------
//calculate % EdU pos cells---------------------------------------------------------
Perc_pos_Nuc=100*((N_EdU_masked)/(N_nuc));
// print result in xls compatible results log, and show current result in table
// append in result table when already open
  title1 = "EdU positive nuclei counts";
  title2 = "["+title1+"]";
  f = title2;
  if (isOpen(title1)){
  print(f, filename_sh + "\t" + N_nuc + "\t" + N_EdU_masked +"\t" +Perc_pos_Nuc+"\n");}
 else {
     run("Table...", "name="+title2+" width=500 height=200");
  print(f, "\\Headings:filename\t total nuc\t EdU nuc\t%EdU nuc ");
  print(f, filename_sh + "\t" + N_nuc + "\t" + N_EdU_masked +"\t" +Perc_pos_Nuc+"\n");}
setBatchMode(false);
selectWindow("Nuc_orig"); run("8-bit");
selectWindow("EdU_orig"); run("8-bit");
run("Merge Channels...", "green=EdU_orig blue=Nuc_orig create");
//saveAs("Tiff", dir2+filename_sh+"G_EdU B_nuc"+".tif");
//run("Channels Tool...");
run("Stack to RGB", "frames");
run("Z Project...", "start=1 stop="+(new_slicenr)+" projection=[Max Intensity]");rename("preview_projection");
selectWindow("nuc_Outline");close();
run("Tile");
run("ROI Manager...");
selectWindow("preview_projection");
roiManager("Show All without labels");
waitForUser("ROI manager show all to reveal total nuc ROIs");
//run("Flatten");
saveAs("Tiff", dir2+filename_sh+"merge with Selected ROIs"+".tif");
roiManager("Delete");
run("Clear Results");
run("Close All");