//(c)Jan Wisniewski, Experimental Immunology Branch, NCI, NIH, Bethesda, MD
//This Macro identifies spots across whole image(including stacks) or in selected cells/regions
//It will process all images in the initial folder
 and all slices in the stack
//Measures dot intensity and corrects it for local background, estimates SNR of each spot
//Outputs spreadsheet with results and images showing outlined measured spots



print("Photometry of Diffraction-Limited Spots for ImageJ\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("");
mac=getBoolean("Are you using Mac?");

//specify folders
//if(mac==1) {waitForUser("Choose a Temporary Directory for Image Series"); }
//output=getDirectory("Choose a Temporary Directory for Image Series");
if(mac==1) {waitForUser("_Choose a Results Folder"); }
rsts=getDirectory("_Choose a Results Folder");
if(mac==1) {waitForUser("Select a Source Directory"); }
input=getDirectory("Select a Source Directory");
print("source: ", input);
print("results: ", rsts);
print("");
NAMES=getFileList(input);

run("Set Measurements...", "area mean standard center median display redirect=None decimal=2");

//open test image
run("Bio-Formats (Windowless)");
ttt=File.name;
rename("x");
getDimensions(width, height, channels, slices, frames);
chns=channels;
stks=slices*frames;
if(chns==1) {run("Enhance Contrast", "saturated=0.10");
		Dialog.create("Name the target");
		Dialog.addString("Name of the Feature: ", " ");
		itemx=newArray("Grays", "Blue", "Cyan", "Green", "Yellow", "Red", "Magenta");
		Dialog.addChoice("Display color: ", itemx, "Grays");
		Dialog.show();
		CN1=Dialog.getString();
		CC1=Dialog.getChoice();
		C1="single";
		rename(CN1);
		run(CC1);	}

if(chns>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(chns>2) {selectWindow("C3-x");
		run("Enhance Contrast", "saturated=0.10");	}
		if(chns>3) {selectWindow("C4-x");
		run("Enhance Contrast", "saturated=0.10");	}

	if(chns==2) {Dialog.create("Name the target(s)");
		Dialog.addString("Name of the 1st Feature: ", " ");
		items=newArray("C1-x", "C2-x");
		Dialog.addRadioButtonGroup("Choose a Channel: ", items, 1, 2, "C1-x");
		itemx=newArray("Grays", "Blue", "Cyan", "Green", "Yellow", "Red", "Magenta");
		Dialog.addChoice("Display color: ", itemx, "Grays");
		Dialog.addMessage("  *  *  *  *  *");
		Dialog.addString("Name of the 2nd Feature: ", " ");
		items2=newArray("C1-x", "C2-x", "none");
		Dialog.addRadioButtonGroup("Choose a Channel: ", items2, 1, 3, "none");
		Dialog.addChoice("Display color: ", itemx, "n/a");
		Dialog.show();
				CN1=Dialog.getString();
				C1=Dialog.getRadioButton();
				CC1=Dialog.getChoice();	
				CN2=Dialog.getString();
				C2=Dialog.getRadioButton();
				CC2=Dialog.getChoice();
		selectWindow(C1);
		run("Duplicate...", "duplicate");
		rename(CN1);
		run(CC1);
		if(C2=="none") { }
		else {selectWindow(C2);
		run("Duplicate...", "duplicate");
		rename(CN2);
		run(CC2);	}
		close("C1-x");
		close("C2-x");
		run("Tile");	}
		
	if(chns==3) {Dialog.create("Name the target(s)");
		Dialog.addString("Name of the 1st Feature: ", " ");
		items3=newArray("C1-x", "C2-x", "C3-x");
		Dialog.addRadioButtonGroup("Choose a Channel: ", items3, 1, 3, "C1-x");
		itemx=newArray("Grays", "Blue", "Cyan", "Green", "Yellow", "Red", "Magenta");
		Dialog.addChoice("Display color: ", itemx, "Grays");
		Dialog.addMessage("  *  *  *  *  *");
		Dialog.addString("Name of the 2nd Feature: ", " ");
		items4=newArray("C1-x", "C2-x", "C3-x", "none");
		Dialog.addRadioButtonGroup("Choose a Channel: ", items4, 1, 4, "none");
		Dialog.addChoice("Display color: ", itemx, "n/a");
		Dialog.addMessage("  *  *  *  *  *");
		Dialog.addString("Name of the 3rd Feature: ", " ");
		Dialog.addRadioButtonGroup("Choose a Channel: ", items4, 1, 4, "none");
		Dialog.addChoice("Display color: ", itemx, "n/a");
		Dialog.show();
				CN1=Dialog.getString();
				C1=Dialog.getRadioButton();
				CC1=Dialog.getChoice();	
				CN2=Dialog.getString();
				C2=Dialog.getRadioButton();
				CC2=Dialog.getChoice();
				CN3=Dialog.getString();
				C3=Dialog.getRadioButton();
				CC3=Dialog.getChoice();
		selectWindow(C1);
		run("Duplicate...", "duplicate");
		rename(CN1);
		run(CC1);
		if(C2=="none") { }
		else {selectWindow(C2);
		run("Duplicate...", "duplicate");
		rename(CN2);
		run(CC2);	}
		if(C3=="none") { }
		else {selectWindow(C3);
		run("Duplicate...", "duplicate");
		rename(CN3);
		run(CC3);	}
		close("C1-x");
		close("C2-x");
		close("C3-x");
		run("Tile");	}
	if(chns==4) {Dialog.create("Name the target(s)");
		Dialog.addString("Name of the 1st Feature: ", " ");
		items5=newArray("C1-x", "C2-x", "C3-x", "C4-x");
		Dialog.addRadioButtonGroup("Choose a Channel: ", items5, 1, 4, "C1-x");
		itemx=newArray("Grays", "Blue", "Cyan", "Green", "Yellow", "Red", "Magenta");
		Dialog.addChoice("Display color: ", itemx, "Grays");
		Dialog.addMessage("  *  *  *  *  *");
		Dialog.addString("Name of the 2nd Feature: ", " ");
		items6=newArray("C1-x", "C2-x", "C3-x", "C4-x", "none");
		Dialog.addRadioButtonGroup("Choose a Channel: ", items6, 1, 5, "none");
		Dialog.addChoice("Display color: ", itemx, "n/a");
		Dialog.addMessage("  *  *  *  *  *");
		Dialog.addString("Name of the 3rd Feature: ", " ");
		Dialog.addRadioButtonGroup("Choose a Channel: ", items6, 1, 5, "none");
		Dialog.addChoice("Display color: ", itemx, "n/a");
		Dialog.addMessage("  *  *  *  *  *");
		Dialog.addString("Name of the 4th Feature: ", " ");
		Dialog.addRadioButtonGroup("Choose a Channel: ", items6, 1, 5, "none");
		Dialog.addChoice("Display color: ", itemx, "n/a");
		Dialog.show();
				CN1=Dialog.getString();
				C1=Dialog.getRadioButton();
				CC1=Dialog.getChoice();	
				CN2=Dialog.getString();
				C2=Dialog.getRadioButton();
				CC2=Dialog.getChoice();
				CN3=Dialog.getString();
				C3=Dialog.getRadioButton();
				CC3=Dialog.getChoice();
				CN4=Dialog.getString();
				C4=Dialog.getRadioButton();
				CC4=Dialog.getChoice();
		selectWindow(C1);
		run("Duplicate...", "duplicate");
		rename(CN1);
		run(CC1);
		if(C2=="none") { }
		else {selectWindow(C2);
		run("Duplicate...", "duplicate");
		rename(CN2);
		run(CC2);	}
		if(C3=="none") { }
		else {selectWindow(C3);
		run("Duplicate...", "duplicate");
		rename(CN3);
		run(CC3);	}
		if(C4=="none") { }
		else {selectWindow(C4);
		run("Duplicate...", "duplicate");
		rename(CN4);
		run(CC4);	}
		close("C1-x");
		close("C2-x");
		close("C3-x");
		close("C4-x");
		run("Tile");	}	}

clls=getBoolean("Are you going to analyze whole image?", "Yes", "No - Individual cells will be selected");
if(clls==1) {cllch="whole image";
	Dialog.create("Margin to crop?");
	Dialog.addNumber("How wide is margin to be cropped (pixels)?", 0);
	Dialog.show();
	marg=Dialog.getNumber();		}
else {cllch="selected cells"; }
print("analysis method:", cllch);
print("");

selectWindow(CN1);
fou1=getBoolean("Is the dot superimposed on a diffuse (e.g. nuclear) background?");
if(fou1==1) {run("Bandpass Filter...", "filter_large=4 filter_small=1 suppress=None tolerance=5");
	print("      FFT-filtered to suppress strong diffuse local background");	}
run("Point Tool...", "type=Circle color=Red size=[Extra Large] label counter=0");
run("Find Maxima...", "prominence=500 output=[Point Selection]");
dtn1=getBoolean("Are dots selected properly?");
if(dtn1==1) {prom=500; }
if(dtn1==0) {Dialog.create("Set detection level");
	Dialog.addNumber("Enter coefficient to adjust detection sensitivity / If too many dots detected: enter 1.5 or more / If too few: 0.66 or less ", 1);
	Dialog.show();
	coef=Dialog.getNumber();
	prot1=coef*500;
	run("Select None");
	run("Find Maxima...", "prominence=prot1 output=[Point Selection]");
	dtn2=getBoolean("Are dots selected properly?");
		if(dtn2==1)	{prom=prot1;	}
		if(dtn2==0)	{Dialog.create("Set detection level - last try");
		Dialog.addNumber("Enter coefficient to adjust detection sensitivity (with regard to the previous try!) If too many dots detected: enter 1.5 or more / If too few: 0.66 or less ", 1);
		Dialog.show();
		coeg=Dialog.getNumber();
		prot2=coeg*prot1;
		run("Select None");
		run("Find Maxima...", "prominence=prot2 output=[Point Selection]");
		dtn3=getBoolean("Are dots selected properly?");
			if(dtn3==1)	{prom=prot2;	}
			if(dtn3==0) {waitForUser("Aborting run");
			close();
			end }	}	}
run("Select None");
print("test image: ", ttt);
print("   channel:", C1, " target name:", CN1, " display:", CC1);
setTool("zoom");
waitForUser("Zoom-in on a representative dot until pixels are clearly visible");
setTool("rectangle");
waitForUser("Draw a generous rectangle around a single representative dot");
run("Find Maxima...", "prominence=prom output=List");
run("Select None");
	xxs=getResult("X");
	yys=getResult("Y");
	close("Results");
makeOval(xxs-3, yys-3, 7, 7);
phbk=5;
run("Make Band...", "band=0.5");
apert1=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes (use defaults)", "No - modify in the next Dialog box");
if(apert1==1) {phap=7;
				ap=3;	}
if(apert1==0) {Dialog.create("Photometry parameters");
	Dialog.addMessage("Change default values below if needed");
	Dialog.addNumber("Diameter of central aperture in pixels (min=3, max=10)", 7);
	Dialog.show();
phap2=Dialog.getNumber();
ap2=phap2/2-1;
makeOval(xxs-ap2, yys-ap2, phap2, phap2);
run("Make Band...", "band=0.5");
apert2=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes - Proceed to photometric measurements", "No - modify on more time");
if(apert2==1) {phap=phap2;
				ap=ap2;	}
if(apert2==0) {Dialog.create("Photometry parameters - Last Try");
	Dialog.addMessage("Adjust one more time");
	Dialog.addNumber("Diameter of central aperture in pixels (min=3, max=10)", phap2);
	Dialog.show();
phap3=Dialog.getNumber();
ap3=phap3/2-1;
makeOval(xxs-ap3, yys-ap3, phap3, phap3);
run("Make Band...", "band=0.5");
apert3=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes - Proceed to photometric measurements", "No - Macro will abort!");
if(apert3==1) {phap=phap3;
				ap=ap3;	}
if(apert3==0) {close();
				end;	}	}	}

if(phap<7) {if(phap<3) {waitForUser("Aperture too small !!!");
			end }
		if(phap==3) {apar=9; }
		else {apar=phap*phap-4; }	}
if(phap>6) {if(phap>10) {waitForUser("Aperture too large !!!");
			end }
		if(phap==10) {apar=80; }
		else {apar=phap*phap-12; }	}

print("      detection limit:", prom, "  aperture size:", phap, "  aperture area: ", apar, "  width of local background band:", phbk);
close(CN1);

if(chns>1) {if(C2=="none") {	}
	else {selectWindow(CN2);
	fou2=getBoolean("Is the dot superimposed on a diffuse (e.g. nuclear) background?");
	if(fou2==1) {run("Bandpass Filter...", "filter_large=4 filter_small=1 suppress=None tolerance=5");
		print("      FFT-filtered to suppress strong diffuse local background");	}
run("Point Tool...", "type=Circle color=Red size=[Extra Large] label counter=0");
run("Find Maxima...", "prominence=500 output=[Point Selection]");
dto1=getBoolean("Are dots selected properly?");
if(dto1==1) {promo=500; }
if(dto1==0) {Dialog.create("Set detection level");
	Dialog.addNumber("Enter coefficient to adjust detection sensitivity / If too many dots detected: enter 1.5 or more / If too few: 0.66 or less ", 1);
	Dialog.show();
	coefo=Dialog.getNumber();
	proto1=coefo*500;
	run("Select None");
	run("Find Maxima...", "prominence=proto1 output=[Point Selection]");
	dto2=getBoolean("Are dots selected properly?");
		if(dto2==1)	{promo=proto1;	}
		if(dto2==0)	{Dialog.create("Set detection level - last try");
		Dialog.addNumber("Enter coefficient to adjust detection sensitivity (with regard to the previous try!) If too many dots detected: enter 1.5 or more / If too few: 0.66 or less ", 1);
		Dialog.show();
		coego=Dialog.getNumber();
		proto2=coego*proto1;
		run("Select None");
		run("Find Maxima...", "prominence=proto2 output=[Point Selection]");
		dto3=getBoolean("Are dots selected properly?");
			if(dto3==1)	{promo=proto2;	}
			if(dto3==0) {waitForUser("Aborting run");
			close();
			end }	}	}
run("Select None");
print("   channel:", C2, " target name:", CN2, " display:", CC2);
setTool("zoom");
waitForUser("Zoom-in on a representative dot until pixels are clearly visible");
setTool("rectangle");
waitForUser("Draw a generous rectangle around a single representative dot");
run("Find Maxima...", "prominence=promo output=List");
run("Select None");
	xxo=getResult("X");
	yyo=getResult("Y");
	close("Results");
makeOval(xxo-3, yyo-3, 7, 7);
phbk=5;
run("Make Band...", "band=0.5");
aperto1=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes (use defaults)", "No - modify in the next Dialog box");
if(aperto1==1) {phapo=7;
				apo=3;	}
if(aperto1==0) {Dialog.create("Photometry parameters");
	Dialog.addMessage("Change default values below if needed");
	Dialog.addNumber("Diameter of central aperture in pixels (min=3, max=10)", 7);
	Dialog.show();
phapo2=Dialog.getNumber();
apo2=phapo2/2-1;
makeOval(xxo-apo2, yyo-apo2, phapo2, phapo2);
run("Make Band...", "band=0.5");
aperto2=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes - Proceed to photometric measurements", "No - modify on more time");
if(aperto2==1) {phapo=phapo2;
				apo=apo2;	}
if(aperto2==0) {Dialog.create("Photometry parameters - Last Try");
	Dialog.addMessage("Adjust one more time");
	Dialog.addNumber("Diameter of central aperture in pixels (min=3, max=10)", phapo2);
	Dialog.show();
phapo3=Dialog.getNumber();
apo3=phapo3/2-1;
makeOval(xxo-apo3, yyo-apo3, phapo3, phapo3);
run("Make Band...", "band=0.5");
aperto3=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes - Proceed to photometric measurements", "No - Macro will abort!");
if(aperto3==1) {phapo=phapo3;
				apo=apo3;	}
if(aperto3==0) {close();
				end;	}	}	}

if(phapo<7) {if(phapo<3) {waitForUser("Aperture too small !!!");
			end }
		if(phapo==3) {aparo=9; }
		else {aparo=phapo*phapo-4; }	}
if(phapo>6) {if(phapo>10) {waitForUser("Aperture too large !!!");
			end }
		if(phapo==10) {aparo=80; }
		else {aparo=phapo*phapo-12; }	}

print("      detection limit:", promo, "  aperture size:", phapo, "  aperture area: ", aparo, "  width of local background band:", phbk);
close(CN2);	}	}
if(chns>2) {if(C3=="none") {	}
	else {selectWindow(CN3);
	fou3=getBoolean("Is the dot superimposed on a diffuse (e.g. nuclear) background?");
	if(fou3==1) {run("Bandpass Filter...", "filter_large=4 filter_small=1 suppress=None tolerance=5");
		print("      FFT-filtered to suppress strong diffuse local background");	}
run("Point Tool...", "type=Circle color=Red size=[Extra Large] label counter=0");
run("Find Maxima...", "prominence=500 output=[Point Selection]");
dtq1=getBoolean("Are dots selected properly?");
if(dtq1==1) {promq=500; }
if(dtq1==0) {Dialog.create("Set detection level");
	Dialog.addNumber("Enter coefficient to adjust detection sensitivity / If too many dots detected: enter 1.5 or more / If too few: 0.66 or less ", 1);
	Dialog.show();
	coefq=Dialog.getNumber();
	protq1=coefq*500;
	run("Select None");
	run("Find Maxima...", "prominence=protq1 output=[Point Selection]");
	dtq2=getBoolean("Are dots selected properly?");
		if(dtq2==1)	{promq=protq1;	}
		if(dtq2==0)	{Dialog.create("Set detection level - last try");
		Dialog.addNumber("Enter coefficient to adjust detection sensitivity (with regard to the previous try!) If too many dots detected: enter 1.5 or more / If too few: 0.66 or less ", 1);
		Dialog.show();
		coegq=Dialog.getNumber();
		protq2=coegq*protq1;
		run("Select None");
		run("Find Maxima...", "prominence=protq2 output=[Point Selection]");
		dtq3=getBoolean("Are dots selected properly?");
			if(dtq3==1)	{promq=protq2;	}
			if(dtq3==0) {waitForUser("Aborting run");
			close();
			end }	}	}
run("Select None");

print("   channel:", C3, " target name:", CN3, " display:", CC3);
setTool("zoom");
waitForUser("Zoom-in on a representative dot until pixels are clearly visible");
setTool("rectangle");
waitForUser("Draw a generous rectangle around a single representative dot");
run("Find Maxima...", "prominence=promo output=List");
run("Select None");
	xxq=getResult("X");
	yyq=getResult("Y");
	close("Results");
makeOval(xxq-3, yyq-3, 7, 7);
phbk=5;
run("Make Band...", "band=0.5");
apertq1=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes (use defaults)", "No - modify in the next Dialog box");
if(apertq1==1) {phapq=7;
				apq=3;	}
if(apertq1==0) {Dialog.create("Photometry parameters");
	Dialog.addMessage("Change default values below if needed");
	Dialog.addNumber("Diameter of central aperture in pixels (min=3, max=10)", 7);
	Dialog.show();
phapq2=Dialog.getNumber();
apq2=phapq2/2-1;
makeOval(xxq-apq2, yyq-apq2, phapq2, phapq2);
run("Make Band...", "band=0.5");
apertq2=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes - Proceed to photometric measurements", "No - modify on more time");
if(apertq2==1) {phapq=phapq2;
				apq=apq2;	}
if(apertq2==0) {Dialog.create("Photometry parameters - Last Try");
	Dialog.addMessage("Adjust one more time");
	Dialog.addNumber("Diameter of central aperture in pixels (min=3, max=10)", phapq2);
	Dialog.show();
phapq3=Dialog.getNumber();
apq3=phapq3/2-1;
makeOval(xxq-apo3, yyq-apo3, phapq3, phapq3);
run("Make Band...", "band=0.5");
apertq3=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes - Proceed to photometric measurements", "No - Macro will abort!");
if(apertq3==1) {phapq=phapq3;
				apq=apq3;	}
if(apertq3==0) {close();
				end;	}	}	}

if(phapq<7) {if(phapq<3) {waitForUser("Aperture too small !!!");
			end }
		if(phapq==3) {aparq=9; }
		else {aparq=phapq*phapq-4; }	}
if(phapq>6) {if(phapq>10) {waitForUser("Aperture too large !!!");
			end }
		if(phapq==10) {aparq=80; }
		else {aparq=phapo*phapq-12; }	}

print("      detection limit:", promq, "  aperture size:", phapq, "  aperture area: ", aparq, "  width of local background band:", phbk);
close(CN3);	}	}
if(chns>3) {if(C4=="none") {	}
	else {selectWindow(CN4);
	fou4=getBoolean("Is the dot superimposed on a diffuse (e.g. nuclear) background?");
	if(fou4==1) {run("Bandpass Filter...", "filter_large=4 filter_small=1 suppress=None tolerance=5");
		print("      FFT-filtered to suppress strong diffuse local background");	}
run("Point Tool...", "type=Circle color=Red size=[Extra Large] label counter=0");
run("Find Maxima...", "prominence=500 output=[Point Selection]");
dtr1=getBoolean("Are dots selected properly?");
if(dtr1==1) {promr=500; }
if(dtr1==0) {Dialog.create("Set detection level");
	Dialog.addNumber("Enter coefficient to adjust detection sensitivity / If too many dots detected: enter 1.5 or more / If too few: 0.66 or less ", 1);
	Dialog.show();
	coefr=Dialog.getNumber();
	protr1=coefr*500;
	run("Select None");
	run("Find Maxima...", "prominence=protr1 output=[Point Selection]");
	dtr2=getBoolean("Are dots selected properly?");
		if(dtr2==1)	{promr=protr1;	}
		if(dtr2==0)	{Dialog.create("Set detection level - last try");
		Dialog.addNumber("Enter coefficient to adjust detection sensitivity (with regard to the previous try!) If too many dots detected: enter 1.5 or more / If too few: 0.66 or less ", 1);
		Dialog.show();
		coegr=Dialog.getNumber();
		protr2=coegr*protr1;
		run("Select None");
		run("Find Maxima...", "prominence=protr2 output=[Point Selection]");
		dtr3=getBoolean("Are dots selected properly?");
			if(dtr3==1)	{promr=protr2;	}
			if(dtr3==0) {waitForUser("Aborting run");
			close();
			end }	}	}
run("Select None");
print("   channel:", C4, " target name:", CN4, " display:", CC4);
setTool("zoom");
waitForUser("Zoom-in on a representative dot until pixels are clearly visible");
setTool("rectangle");
waitForUser("Draw a generous rectangle around a single representative dot");
run("Find Maxima...", "prominence=promo output=List");
run("Select None");
	xxr=getResult("X");
	yyr=getResult("Y");
	close("Results");
makeOval(xxr-3, yyr-3, 7, 7);
phbk=5;
run("Make Band...", "band=0.5");
apertr1=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes (use defaults)", "No - modify in the next Dialog box");
if(apertr1==1) {phapr=7;
				apr=3;	}
if(apertr1==0) {Dialog.create("Photometry parameters");
	Dialog.addMessage("Change default values below if needed");
	Dialog.addNumber("Diameter of central aperture in pixels (min=3, max=10)", 7);
	Dialog.show();
phapr2=Dialog.getNumber();
apr2=phapr2/2-1;
makeOval(xxr-apr2, yyr-apr2, phapr2, phapr2);
run("Make Band...", "band=0.5");
apertr2=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes - Proceed to photometric measurements", "No - modify on more time");
if(apertr2==1) {phapr=phapr2;
				apr=apr2;	}
if(apertr2==0) {Dialog.create("Photometry parameters - Last Try");
	Dialog.addMessage("Adjust one more time");
	Dialog.addNumber("Diameter of central aperture in pixels (min=3, max=10)", phapr2);
	Dialog.show();
phapr3=Dialog.getNumber();
apr3=phapr3/2-1;
makeOval(xxr-apo3, yyr-apo3, phapr3, phapr3);
run("Make Band...", "band=0.5");
apertr3=getBoolean("Are apertures OK? (Dot is completely inside red square)", "Yes - Proceed to photometric measurements", "No - Macro will abort!");
if(apertr3==1) {phapr=phapr3;
				apr=apr3;	}
if(apertr3==0) {close();
				end;	}	}	}

if(phapr<7) {if(phapr<3) {waitForUser("Aperture too small !!!");
			end }
		if(phapr==3) {aparr=9; }
		else {aparq=phapq*phapq-4; }	}
if(phapq>6) {if(phapr>10) {waitForUser("Aperture too large !!!");
			end }
		if(phapr==10) {aparr=80; }
		else {aparr=phapr*phapr-12; }	}

print("      detection limit:", promr, "  aperture size:", phapr, "  aperture area: ", aparr, "  width of local background band:", phbk);
close(CN4);	}	}

//prepare image
for (i=0; i<NAMES.length; i++) {
	open(input+NAMES[i]);
ttl=File.nameWithoutExtension;
print("");
print("analyzed image:", ttl);
getDimensions(width, height, channels, slices, frames);
print("size:", width, "x", height, " channels:", channels, " slices", slices, " frames:", frames);
rename("x");

//create custom table
title1 = "Analysis_Results_"+ttl; 
title2 = "["+title1+"]"; 
f=title2; 
run("New... ", "name="+title2+" type=Table"); 
print(f,"\\Headings:Image\t#\tX\tY\tAperture_Mean\tMedian_Background\tSignal(ADU)\tSNR"); 

if(chns==1) {rename(CN1);
	run(CC1);
	zts=nSlices();
	run("Reverse");
	
//create temporary directory
myDir1 = rsts+"temporary_"+CN1+"_images"+File.separator;
File.makeDirectory(myDir1);

	if(clls==1)	{setTool("rectangle");
		makeRectangle(marg, marg, width-marg*2, height-marg*2);
		run("Crop");
		run("Stack to Images");
		for (j = 0; j < zts; j++) {run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width-marg*2+20, height-marg*2+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
			run("Enhance Contrast", "saturated=0.10");
			saveAs("Tiff", myDir1+ttl+"_"+CN1+"_"+j+1);
			close();
			close();	}	}

	if(clls==0) {Dialog.create(" ");
		Dialog.addNumber("How many cells/regions will you analyze?", 0);
		Dialog.show();
		clln=Dialog.getNumber();
		print("number of selected cells:", clln);
		print("");
		if(clln==0) {close(CN1);	}
		else {for (l = 0; l < clln; l++) {setTool("freehand");
			setSlice(zts);
			waitForUser("Select cell/region - ensure it is within selected region throughout the stack\nand none of dots gets near selection boundary!");
			run("Duplicate...", "duplicate");
			run("Clear Outside", "stack");
			run("Select None");
			run("Stack to Images");
		for (j = 0; j < zts; j++) {getDimensions(width, height, channels, slices, frames);
			run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width+20, height+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
			run("Enhance Contrast", "saturated=0.10");
			saveAs("Tiff", myDir1+ttl+"_cell_"+l+1+"_"+CN1+"_"+j+1);
			close(); 
			close();
		run("Select None");	}	}
		close(CN1);	}	}	}

if(chns>1) {if(clls==1)	{setTool("rectangle");
		makeRectangle(marg, marg, width-marg*2, height-marg*2);
		run("Crop");
	run("Split Channels");
	selectWindow(C1);
	run("Duplicate...", "duplicate");
	rename(CN1);
	run(CC1);
	run("Reverse");
//create temporary directory
myDir1 = rsts+"temporary_"+CN1+"_images"+File.separator;
File.makeDirectory(myDir1);	
	zts=nSlices();
	run("Stack to Images");
	for (j = 0; j < zts; j++) {run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width-marg*2+20, height-marg*2+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
			run("Enhance Contrast", "saturated=0.10");
			saveAs("Tiff", myDir1+ttl+"_"+CN1+"_"+j+1);
			close();
			close();	}
	if(C2=="none") {	}
	else {selectWindow(C2);
		run("Duplicate...", "duplicate");
		rename(CN2);
		run(CC2);
		run("Reverse");
//create temporary directory
myDir2 = rsts+"temporary_"+CN2+"_images"+File.separator;
File.makeDirectory(myDir2);
		run("Stack to Images");
		for (j = 0; j < zts; j++) {run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width-marg*2+20, height-marg*2+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
			run("Enhance Contrast", "saturated=0.10");
			saveAs("Tiff", myDir2+ttl+"_"+CN2+"_"+j+1);
			close();
			close();	}	}
	if(chns>2) {if(C3=="none") {	}
	else {selectWindow(C3);
	run("Duplicate...", "duplicate");
	rename(CN3);	
	run(CC3);
	run("Reverse");
//create temporary directory
myDir3 = rsts+"temporary_"+CN3+"_images"+File.separator;
File.makeDirectory(myDir3);
	run("Stack to Images");
	for (j = 0; j < zts; j++) {run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width-marg*2+20, height-marg*2+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
			run("Enhance Contrast", "saturated=0.10");
			saveAs("Tiff", myDir3+ttl+"_"+CN3+"_"+j+1);
			close();
			close();	}	}	}
	if(chns>3) {if(C4=="none") {	}
	else {selectWindow(C4);
	run("Duplicate...", "duplicate");
	rename(CN4);	
	run(CC4);
	run("Reverse");
//create temporary directory
myDir4 = rsts+"temporary_"+CN4+"_images"+File.separator;
File.makeDirectory(myDir4);
	run("Stack to Images");
	for (j = 0; j < zts; j++) {run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width-marg*2+20, height-marg*2+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
			run("Enhance Contrast", "saturated=0.10");
			saveAs("Tiff", myDir4+ttl+"_"+CN4+"_"+j+1);
			close();
			close();	}	}	}
	close("C1-x");
	close("C2-x");
	if(chns>2) {close("C3-x");	}
	if(chns>3) {close("C4-x");	}	}

if(clls==0) {myDir1 = rsts+"temporary_"+CN1+"_images"+File.separator;
	File.makeDirectory(myDir1);
	myDir2 = rsts+"temporary_"+CN2+"_images"+File.separator;
	File.makeDirectory(myDir2);
	if(chns>2) {myDir3 = rsts+"temporary_"+CN3+"_images"+File.separator;
	File.makeDirectory(myDir13);	}
	if(chns>3) {myDir4 = rsts+"temporary_"+CN4+"_images"+File.separator;
	File.makeDirectory(myDir4);	}

		Dialog.create(" ");
		Dialog.addNumber("How many cells/regions will you analyze?", 0);
		Dialog.show();
		clln=Dialog.getNumber();
		print("number of selected cells:", clln);
		print("");
	if(clln==0) {close("x");	}
	else {for (l = 0; l < clln; l++) {setTool("freehand");
			rename("y");
			waitForUser("Select cell/region - ensure it is within selected region throughout the stack\nand none of dots gets near selection boundary!");
			run("Duplicate...", "duplicate");
			rename("x");
			run("Clear Outside", "stack");
			run("Select None");
		run("Split Channels");
		selectWindow(C1);
		getDimensions(width, height, channels, slices, frames);
		rename(CN1);
		run(CC1);
		zts=nSlices();
		run("Reverse");
		run("Stack to Images");
	for (j = 0; j < zts; j++) {run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width+20, height+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
		run("Enhance Contrast", "saturated=0.10");
		saveAs("Tiff", myDir1+ttl+"_cell_"+l+1+"_"+CN1+"_"+j+1);	
		
		close();
		close();	}	
		
	if(C2=="none") {	}
	else {selectWindow(C2);
		rename(CN2);
		run(CC2);
		run("Reverse");
		run("Stack to Images");
	for (j = 0; j < zts; j++) {run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width+20, height+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
		run("Enhance Contrast", "saturated=0.10");
		saveAs("Tiff", myDir2+ttl+"_cell_"+l+1+"_"+CN2+"_"+j+1);	
		close();
		close();	}	}
		
	if(chns>2) {if(C3=="none") {	}
	else {selectWindow(C3);
		rename(CN3);
		run(CC3);
		run("Reverse");
		run("Stack to Images");
	for (j = 0; j < zts; j++) {run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width+20, height+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
		run("Enhance Contrast", "saturated=0.10");
		saveAs("Tiff", myDir3+ttl+"_cell_"+l+1+"_"+CN3+"_"+j+1);	
		close();
		close();	}	}
		
	if(chns>3) {if(C4=="none") {	}
	else {selectWindow(C4);
		run("Duplicate...", "duplicate");
		rename(CN4);
		run(CC4);
		run("Reverse");
		run("Stack to Images");
	for (j = 0; j < zts; j++) {run("Select All");
			run("Copy");
			newImage("Untitled", "16-bit black", width+20, height+20, 1);
			wait(200);
			run("Paste");
			run("Select None");
		run("Enhance Contrast", "saturated=0.10");
		saveAs("Tiff", myDir4+ttl+"_cell_"+l+1+"_"+CN4+"_"+j+1);	
		close();
		close();	}	}	}	}	}	}
		close("y");	}	}

LIST1=getFileList(myDir1);
for (h=0; h<LIST1.length; h++) {
	open(myDir1+LIST1[h]);
ttl2=File.nameWithoutExtension;
rename("xx");
run(CC1);
	if(fou1==1) {run("Bandpass Filter...", "filter_large=4 filter_small=1 suppress=None tolerance=5");	}
run("Find Maxima...", "prominence=prom output=List");
Table.sort("X");
rs=Table.size();
Table.rename("X_Y_list");

for (q = 0; q < rs; q++) {m=q+1;
	selectWindow("X_Y_list");
	xxt=Table.get("X", q);
	yyt=Table.get("Y", q);
		xxs=xxt-ap;
		yys=yyt-ap;	
	makeOval(xxs, yys, phap, phap);
	run("Measure");
	cx=getResult("XM");
	cy=getResult("YM");
	cav=getResult("Mean");
	run("Make Band...", "band=0.5");
	run("Measure");
	makeOval(xxs, yys, phap, phap);
run("Overlay Options...", "stroke=cyan width=0 fill=none");
run("Add Selection...");
	run("Select None");
wait(100);
	bck=getResult("Median");
	nois=getResult("StdDev");
	adu=(cav-bck)*apar;
	snr=adu/nois;
	close("Results");
	selectWindow(title1);
	print(f,ttl2+"\t"+m+"\t"+cx+"\t"+cy+"\t"+cav+"\t"+bck+"\t"+adu+"\t"+snr);	}
run("Flatten");
run("RGB Color");
saveAs("Tiff", rsts+ttl2+"_finished");
close();
close("xx");
close("X_Y_list"); }

for (h=0; h<LIST1.length; h++)
ok = File.delete(myDir1+LIST1[h]);
ok = File.delete(myDir1);
 
if(chns>1) {if(C2=="none") { }
else {LIST2=getFileList(myDir2);
	for (h=0; h<LIST2.length; h++) {
	open(myDir2+LIST2[h]);
ttl2=File.nameWithoutExtension;
rename("xx");
run(CC2);
	if(fou2==1) {run("Bandpass Filter...", "filter_large=4 filter_small=1 suppress=None tolerance=5");	}
run("Find Maxima...", "prominence=promo output=List");
Table.sort("X");
rs=Table.size();
Table.rename("X_Y_list");

for (q = 0; q < rs; q++) {m=q+1;
	selectWindow("X_Y_list");
	xxt=Table.get("X", q);
	yyt=Table.get("Y", q);
		xxo=xxt-apo;
		yyo=yyt-apo;	
	makeOval(xxo, yyo, phapo, phapo);
	run("Measure");
	cxo=getResult("XM");
	cyo=getResult("YM");
	cavo=getResult("Mean");
	run("Make Band...", "band=0.5");
	run("Measure");
	makeOval(xxo, yyo, phapo, phapo);
run("Overlay Options...", "stroke=cyan width=0 fill=none");
run("Add Selection...");
	run("Select None");
wait(100);
	bcko=getResult("Median");
	noiso=getResult("StdDev");
	aduo=(cavo-bcko)*aparo;
	snro=aduo/noiso;
	close("Results");
	selectWindow(title1);
	print(f,ttl2+"\t"+m+"\t"+cxo+"\t"+cyo+"\t"+cavo+"\t"+bcko+"\t"+aduo+"\t"+snro);	}

run("Flatten");
run("RGB Color");
saveAs("Tiff", rsts+ttl2+"_finished");
close();
close("xx");
close("X_Y_list"); }

  for (h=0; h<LIST2.length; h++)
      ok = File.delete(myDir2+LIST2[h]);
  ok = File.delete(myDir2);	}	}

if(chns>2) {if(C3=="none") { }
	else {LIST3=getFileList(myDir3);
	for (h=0; h<LIST3.length; h++) {
	open(myDir3+LIST3[h]);
ttl2=File.nameWithoutExtension;
rename("xx");
run(CC3);
	if(fou3==1) {run("Bandpass Filter...", "filter_large=4 filter_small=1 suppress=None tolerance=5");	}
run("Find Maxima...", "prominence=promq output=List");
Table.sort("X");
rs=Table.size();
Table.rename("X_Y_list");

for (q = 0; q < rs; q++) {m=q+1;
	selectWindow("X_Y_list");
	xxt=Table.get("X", q);
	yyt=Table.get("Y", q);
		xxq=xxt-apq;
		yyq=yyt-apq;	
	makeOval(xxq, yyq, phapq, phapq);
	run("Measure");
	cxq=getResult("XM");
	cyq=getResult("YM");
	cavq=getResult("Mean");
	run("Make Band...", "band=0.5");
	run("Measure");
	makeOval(xxq, yyq, phapq, phapq);
run("Overlay Options...", "stroke=cyan width=0 fill=none");
run("Add Selection...");
	run("Select None");
wait(100);
	bckq=getResult("Median");
	noisq=getResult("StdDev");
	aduq=(cavq-bckq)*aparq;
	snrq=aduq/noisq;
	close("Results");
	selectWindow(title1);
	print(f,ttl2+"\t"+m+"\t"+cxq+"\t"+cyq+"\t"+cavq+"\t"+bckq+"\t"+aduq+"\t"+snrq);	}

run("Flatten");
run("RGB Color");
saveAs("Tiff", rsts+ttl2+"_finished");
close();
close("xx");
close("X_Y_list"); }

  for (h=0; h<LIST3.length; h++)
      ok = File.delete(myDir3+LIST3[h]);
  ok = File.delete(myDir3);	}	}

if(chns>3) {if(C4=="none") { }
	else {LIST4=getFileList(myDir4);
	for (h=0; h<LIST4.length; h++) {
	open(myDir4+LIST4[h]);
ttl2=File.nameWithoutExtension;
rename("xx");
run(CC4);
	if(fou4==1) {run("Bandpass Filter...", "filter_large=4 filter_small=1 suppress=None tolerance=5");	}
run("Find Maxima...", "prominence=promr output=List");
Table.sort("X");
rs=Table.size();
Table.rename("X_Y_list");

for (q = 0; q < rs; q++) {m=q+1;
	selectWindow("X_Y_list");
	xxt=Table.get("X", q);
	yyt=Table.get("Y", q);
		xxr=xxt-apr;
		yyr=yyt-apr;	
	makeOval(xxr, yyr, phapr, phapr);
	run("Measure");
	cxr=getResult("XM");
	cyr=getResult("YM");
	cavr=getResult("Mean");
	run("Make Band...", "band=0.5");
	run("Measure");
	makeOval(xxr, yyr, phapr, phapr);
run("Overlay Options...", "stroke=cyan width=0 fill=none");
run("Add Selection...");
	run("Select None");
wait(100);
	bckr=getResult("Median");
	noisr=getResult("StdDev");
	adur=(cavr-bckr)*aparr;
	snrr=adur/noisr;
	close("Results");
	selectWindow(title1);
	print(f,ttl2+"\t"+m+"\t"+cxr+"\t"+cyr+"\t"+cavr+"\t"+bckr+"\t"+adur+"\t"+snrr);	}

run("Flatten");
run("RGB Color");
saveAs("Tiff", rsts+ttl2+"_finished");
close();
close("xx");
close("X_Y_list"); }

  for (h=0; h<LIST4.length; h++)
      ok = File.delete(myDir4+LIST4[h]);
  ok = File.delete(myDir4);	}	}

if(clls==1) {selectWindow(title1);
saveAs("Text", rsts+title1+".csv");
close(title1);	}

if(clls==0) {if(clln>0) {selectWindow(title1);
		saveAs("Text", rsts+title1+".csv");
		close(title1);	}
		if(clln==0) {close(title1);	}	}	}	

selectWindow("Log");
saveAs("Text", rsts+"Log"+"_"+month+"_"+dayOfMonth+"_"+year+"_"+hour+"_"+minute+".txt");
close("Log");