// Jan Wisniewski, EIB/NCI/NIH, Bethesda, MD

//Detects cell colonies, counts  and measures their average size
//gathers results into a custom table



print("Colony Identification and Counting Macro\nJan Wisniewski, Experimental Immunology Branch\nNational Cancer Institute, NIH, Bethesda, Maryland");
getDateAndTime(year, month, dayOfWeek, dayOfMonth, hour, minute, second, msec);
print("");
print("run: ", month, "/", dayOfMonth, "/", year, "   at ", hour, ":", minute);
print("");


output=getDirectory("Choose a Destination Folder");
input=getDirectory("Select a Source Directory");
list=getFileList(input);

title1 = "Analysis_Results"; 
title2 = "["+title1+"]"; 
f=title2; 
run("New... ", "name="+title2+" type=Table"); 
print(f,"\\Headings:Image\tChannel\tName\tMin._Detection_Limit(um2)\t#_of_Detected_Colonies\tAverage_Size"); 

run("Bio-Formats (Windowless)");
rename("x");
getDimensions(width, height, channels, slices, frames);
chn=channels;
if(chn>1) {run("Split Channels");
run("Tile");
selectWindow("C1-x");
run("Enhance Contrast", "saturated=0.10");
selectWindow("C2-x");
run("Enhance Contrast", "saturated=0.10");	
if(chn==3) {selectWindow("C3-x");
run("Enhance Contrast", "saturated=0.10");	}

Dialog.create("Channel setup");
itemx=newArray("C1-x", "C2-x", "C3-x");
Dialog.addRadioButtonGroup("Which channel will you use?", itemx, 1, 3, "C1-x")
Dialog.addString("Name", "");
items=newArray("Grays", "Blue", "Cyan", "Green", "Yellow", "Red", "Magenta");
Dialog.addChoice("Display color", items, "Red");
//Dialog.addNumber("Size reduction factor:", 2);
Dialog.addNumber("ROI diameter(fraction of whole image)", 0.90);
Dialog.show();
chnid=Dialog.getRadioButton();
chname=Dialog.getString();
chcol=Dialog.getChoice();
chres=2;
diam=Dialog.getNumber();
selectWindow(chnid);
run("Duplicate...", "title=x");
close("C1-x"); 
close("C2-x"); 
if(chn==3) {close("C3-x");	}	}
else {run("Enhance Contrast", "saturated=0.10");
Dialog.create("Channel setup");
Dialog.addString("Name", "");
items=newArray("Grays", "Blue", "Cyan", "Green", "Yellow", "Red", "Magenta");
Dialog.addChoice("Display color", items, "Red");
//Dialog.addNumber("Size reduction factor:", 2);
Dialog.addNumber("ROI diameter(fraction of whole image)", 0.90);
Dialog.show();
chnid="x";
chname=Dialog.getString();
chcol=Dialog.getChoice();
chres=2;
diam=Dialog.getNumber();	}
redsz=width/chres;
roisz=redsz*diam;
strt=(redsz-roisz)/2;
print(width, chnid, chname, chcol, chres, diam, redsz, roisz, strt);
run("Resample", "factor=chres factor_0=chres");
run("Enhance Contrast", "saturated=0.10");
rename(chname);
run(chcol);
close("x");

myDir = output+"temporary"+File.separator;
File.makeDirectory(myDir);
saveAs("Tiff", myDir+"temp1.tif");
rename(chname);
run("Duplicate...", " ");
rename("mask");
run("Multiply...", "value=0");
selectWindow(chname);
makeOval(strt, strt, roisz, roisz);
run("Copy");
run("Select None");
selectWindow("mask");
run("Paste");
run("Select None");
setAutoThreshold("Default dark");
run("Threshold...");
waitForUser("Adjust threshold");
getThreshold(lower, upper);
thrlow=lower;
thrup=upper;
setOption("BlackBackground", true);
run("Convert to Mask");
run("Maximum...", "radius=15");
run("Fill Holes");
run("Watershed");
saveAs("Tiff", myDir+"temp2.tif");
rename("mask");

Dialog.create("Select minimum size limit");
Dialog.addMessage("Minimum colony size (um2) = factor x 100000");
Dialog.addNumber("Factor value:", 1);
Dialog.show();
lmt1=Dialog.getNumber();
szlimit=100000*lmt1; 
print(szlimit);
run("Analyze Particles...", "size=szlimit-Infinity circularity=0.33-1.00 show=Outlines summarize in_situ");
run("Green");
run("Maximum...", "radius=2");
selectWindow(chname);
run("RGB Color");
selectWindow("mask");
run("RGB Color");
run("Images to Stack", "name=Stack title=[] use");
run("Z Project...", "projection=[Sum Slices]");

try1=getBoolean("Were colonies selected properly?", "Yes, proceed to analysis", "No, try different value");
close("Stack");
close("SUM_Stack");
if(try1==1) {tlist=getFileList(myDir);
		for (h=0; h<tlist.length; h++)	
		ok = File.delete(myDir+tlist[h]);
		ok = File.delete(myDir);	}
else {open(myDir+"temp1.tif");
	rename(chname);
	open(myDir+"temp2.tif");
	rename("mask");
Dialog.create("Select minimum size limit");
Dialog.addMessage("Minimum colony size (um2) = factor x 100000");
//itemy=newArray(50000, 70000, 100000, 140000, 200000, 280000, 400000, 560000, 800000, "specify");
//Dialog.addRadioButtonGroup("Minimum size of colony:", itemy, 2, 5, 200000);
Dialog.addNumber("Factor value:", 1);
Dialog.show();
lmt2=Dialog.getNumber();
szlimit=100000*lmt2; 
print(szlimit);
run("Analyze Particles...", "size=szlimit-Infinity circularity=0.33-1.00 show=Outlines summarize in_situ");
run("Green");
run("Maximum...", "radius=2");
selectWindow(chname);
run("RGB Color");
selectWindow("mask");
run("RGB Color");
run("Images to Stack", "name=Stack title=[] use");
run("Z Project...", "projection=[Sum Slices]");	

try2=getBoolean("Were colonies selected properly?", "Yes, proceed to analysis", "No, try different value (last time!)");
close("Stack");
close("SUM_Stack");
if(try2==1) {tlist=getFileList(myDir);
		for (h=0; h<tlist.length; h++)	
		ok = File.delete(myDir+tlist[h]);
		ok = File.delete(myDir);	}
else {open(myDir+"temp1.tif");
	rename(chname);
	open(myDir+"temp2.tif");
	rename("mask");
Dialog.create("Select minimum size limit");
Dialog.addMessage("Minimum colony size (um2) = factor x 100000");
//itemy=newArray(50000, 70000, 100000, 140000, 200000, 280000, 400000, 560000, 800000, "specify");
//Dialog.addRadioButtonGroup("Minimum size of colony:", itemy, 2, 5, 200000);
Dialog.addNumber("Factor value:", 1);
Dialog.show();
lmt3=Dialog.getNumber();
szlimit=100000*lmt3; 
print(szlimit);
run("Analyze Particles...", "size=szlimit-Infinity circularity=0.33-1.00 show=Outlines summarize in_situ");
run("Green");
run("Maximum...", "radius=2");
selectWindow(chname);
run("RGB Color");
selectWindow("mask");
run("RGB Color");
run("Images to Stack", "name=Stack title=[] use");
run("Z Project...", "projection=[Sum Slices]");

try3=getBoolean("Were colonies selected properly?", "Yes, proceed to analysis", "No (Abort)");
close("Stack");
close("SUM_Stack");
tlist=getFileList(myDir);
		for (h=0; h<tlist.length; h++)	
		ok = File.delete(myDir+tlist[h]);
		ok = File.delete(myDir);
	if(try3==1)	{	}
	else {end; }	}	}
close("Summary");
print("selected limit:  ", szlimit);

for (i = 0; i < list.length; i++) {
	open(input+list[i]);
	ttl=File.nameWithoutExtension;
	rename("x");
if(chn>1)	{run("Split Channels");
selectWindow(chnid);
run("Duplicate...", "title=x");
close("C1-x");
close("C2-x");
if(chn==3) {close("C3-x");	}	}
run("Resample", "factor=chres factor_0=chres");
run("Enhance Contrast", "saturated=0.10");
run(chcol);
rename(chname);
close("x");
run("Duplicate...", "mask");
makeOval(strt, strt, roisz, roisz);
run("Clear Outside");
run("Select None");
setThreshold(thrlow, thrup);
run("Convert to Mask");
run("Maximum...", "radius=15");
run("Fill Holes");
run("Watershed");
rename(ttl);
run("Analyze Particles...", "size=szlimit-Infinity circularity=0.33-1.00 show=Outlines summarize in_situ");

Table.rename("Summary", "Results");
colnumb=getResult("Count");
avesz=getResult("Average Size");
close("Results");

selectWindow("Analysis_Results");
print(f,ttl+"\t"+chnid+"\t"+chname+"\t"+szlimit+"\t"+colnumb+"\t"+avesz);

run("Green");
run("Maximum...", "radius=2");
run("RGB Color");
selectWindow(chname);
run("RGB Color");
run("Duplicate...", "circle");
run("Select All");
setBackgroundColor(0, 0, 0);
run("Clear", "slice");
run("Select None");
makeOval(strt, strt, roisz, roisz);
run("Flatten");
run("Maximum...", "radius=2");
close("circle");

run("Images to Stack", "name=Stack title=[] use");
run("Z Project...", "projection=[Max Intensity]");
saveAs("Tiff", output+ttl+"_detected_colonies");
close();
close();
close("Stack");	}

getDateAndTime(year, month, dayOfWeek, dayOfMonth, hour, minute, second, msec);
//Table.rename("Summary", "Results");
//saveAs("Results", output+"Results"+"_"+month+"_"+dayOfMonth+"_"+year+"_"+hour+"_"+minute+".csv");
//close("Results");
selectWindow("Analysis_Results");
saveAs("Text", output+"Analysis_Results"+"_"+month+"_"+dayOfMonth+"_"+year+"_"+hour+"_"+minute+".csv");
close("Analysis_Results");
//selectWindow("Log");
//saveAs("Text", output+"Log"+"_"+month+"_"+dayOfMonth+"_"+year+"_"+hour+"_"+minute+".txt");
close("Log");