// Jan Wisniewski, Experimental Immunology Branch, NCI/NIH, Bethesda MD

// This is macro uses tif or proprietary image formats
// recognizes Z-stacks and and applies MaxIP or custom projection options
// user can rename channels, specify display color, brightness range, cropping etc.
// it automatically inserts scale bar
// then images are saved as 16-bit tif, RGB color and RGB montage
// scaling and other parameters are saved in the Log file


print("Multichannel Image Display\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("");

run("Set Measurements...", "area mean min bounding integrated median display redirect=None decimal=2");

//functions
function maxStack() {rename("z");
	run("Z Project...", "projection=[Max Intensity]");
	close("z"); }
	
function findCenter() {run("Duplicate...", "title=z duplicate");
	run("Median...", "radius=1 stack");
	run("Measure Stack...");
	selectWindow("Results");
	Table.sort("Max");
	close("z");	}

function getCenter() {rename("z");
	setSlice(maxc);
	run("Duplicate...", " ");
	close("z");	}

function getRange() {zup=slcs-maxc-sup;
	zlo=maxc-slo-1;
rename("z1");
run("Stack to Images");
for (i = 0; i < zup; i++) {close();	}
run("Images to Stack", "name=z2 title=[] use");
run("Reverse");
run("Stack to Images");
for (j = 0; j < zlo; j++) {close();	}
run("Images to Stack", "name=zz title=[] use");	}

function getManual() {mup=slcs-zz-rng;
	mlo=zz-1;
run("Stack to Images");
for (i = 0; i < mup; i++) {close();	}
run("Images to Stack", "name=Stack title=[] use");
run("Reverse");
run("Stack to Images");
for (j = 0; j < mlo; j++) {close();	}
run("Images to Stack", "name=zz title=[] use");	}
	
function autodic() {run("Bandpass Filter...", "filter_large=10 filter_small=3 suppress=None tolerance=5");
	run("Enhance Contrast", "saturated=0.35");
	run("Gamma...", "value=0.99");	}

//  specify folders
Dialog.create("Output and Input Folders");
itv=newArray("No stacks", "Only stacks", "Mix");
Dialog.addRadioButtonGroup("Are Z-stacks present in the source folder?", itv, 1, 3, "Mix");
Dialog.addCheckbox("Crop images", false);
itx=newArray("RGB montage", "16-bit Tiff + RGB montage"); 
Dialog.addRadioButtonGroup("Save Processed Image as:", itx, 2, 1, "RGB montage");
items=newArray("Max Intensity Projection", "Brightest slice only", "A subset of slices around the brightest slice", "Manually-selected subset only");
Dialog.addRadioButtonGroup("Projection method for Z-stacks (if present)", items, 4, 1, "Max Intensity Projection");
Dialog.show(); 
stks=Dialog.getRadioButton();
crp=Dialog.getCheckbox();
tif16=Dialog.getRadioButton();
stp=Dialog.getRadioButton();
if(stks=="No stacks") {mix=0; }
else {mix=1; }


print("General settings:  Z-Stacks=", mix, "   Image cropping+=", crp, "   Saved output=", tif16, "   Z-stack projection=", stp);

res=getDirectory("Choose a Directory to store Results");
myDir1 = res+year+"-"+month+"-"+dayOfMonth+" "+hour+"_"+minute+" Montage"+File.separator;
			File.makeDirectory(myDir1);
myDir3 = res+year+"-"+month+"-"+dayOfMonth+" "+hour+"_"+minute+" RGB sets"+File.separator;
			File.makeDirectory(myDir3);
if(tif16=="16-bit Tiff + RGB montage") {myDir2 = res+year+"-"+month+"-"+dayOfMonth+" "+hour+"_"+minute+" 16-bit tiff"+File.separator;
			File.makeDirectory(myDir2);	}
input=getDirectory("Select a Source Directory");
NAMES=getFileList(input);

//get parameters
//create custom table
title1 = "Parameters"; 
title2 = "["+title1+"]"; 
f=title2; 
run("New... ", "name="+title2+" type=Table"); 
print(f,"\\Headings:Label\tChannel\tLUT\tMin\tMax\tExclusion\tFFT"); 

//open sample image
if(mix==0) {waitForUser("Open sample images");	}
else {waitForUser("Open a Z-stack");	}
run("Bio-Formats (Windowless)");
sttl=File.nameWithoutExtension;
getDimensions(width, height, channels, slices, frames);
chns=channels;
slcs=slices;

print("Setup image:", sttl, "   size=", width, "x", height, "   channels=", chns, "   slices=", slcs, "   frames=", frames);

if(chns==1) {rename("C1-x");
				master="C1-x"; }
else {rename("x");
run("Split Channels");
//run("Tile");
waitForUser("Select master channel for setup");
		master=getTitle();	}
		
print("master channel=", master);

//set up projection
if(slcs>1) {if(stp=="Max Intensity Projection") {	}	
			else {findCenter();
					maxc=getResult("Slice");
					close("Results");
					
				if(stp=="A subset of slices around the brightest slice") {setSlice(maxc);
							waitForUser("Inspect stack to decide how many slices to include on each side of the brightest one");
							Dialog.create("Specify subset");
							Dialog.addMessage("Same number of slices will be used for all images\nbut substack position will depend on the brightest slice in a given stack !");
							Dialog.addMessage("(Enter negative number if you want to move start past the brightest slice)");
							Dialog.addNumber("How many slices do you want to include before center slice:", 0);
							Dialog.addNumber("How many slices do you want to include after center slice:", 0);
							Dialog.show();
							slo=Dialog.getNumber();
							sup=Dialog.getNumber();
							print("substack range=", sup+slo);	}
							
				if(stp=="Manually-selected subset only") {setSlice(maxc);
							waitForUser("Inspect stack to decide how many slices to include");
							Dialog.create("Specify subset");
							Dialog.addNumber("How many slices do you want to include:", 0);
							Dialog.addMessage("(Same number of slices will be used for all images,\nthough you would need to specify start slice in eachcase)");
							Dialog.show();
							rng=Dialog.getNumber();
							print("manual substack range=", rng);	}	}

for (i = 0; i < chns; i++) {j=i+1;
		selectWindow("C"+j+"-x");
		wtl=File.nameWithoutExtension;
	if(slcs>1) 	{if(stp=="Max Intensity Projection") {maxStack(); 	}
				if(stp=="Brightest slice only") {getCenter();	}
				if(stp=="A subset of slices around the brightest slice") {getRange();		}
				if(stp=="Manually-selected subset only") {if(i==0) {waitForUser("Select the first slide to include in a substack\n(any channel will work at this step)");
							zz=getSliceNumber();	}
							getManual();	}	}
					rename("C"+j+"-x");	}

if(stp=="A subset of slices around the brightest slice") {for (i = 0; i < chns; i++) {j=i+1;
																		selectWindow("C"+j+"-x");
																		rename("z");
																		run("Z Project...", "projection=[Max Intensity]");
																		close("z");
																		rename("C"+j+"-x");	}	}
		
if(stp=="Manually-selected subset only") {for (i = 0; i < chns; i++) {j=i+1;
																		selectWindow("C"+j+"-x");
																		rename("z");
																		run("Z Project...", "projection=[Max Intensity]");
																		close("z");
																		rename("C"+j+"-x");	}	}	}

if(crp==1) {selectWindow(master);
			setTool("rectangle");
			waitForUser("Select crop area");
			run("Select None");	
	
for (i = 0; i < chns; i++) {j=i+1;
			selectWindow("C"+j+"-x");
			run("Restore Selection");
			run("Crop");	}	}

//channel setup				
for (i = 0; i < chns; i++) {j=i+1;
		selectWindow("C"+j+"-x");
		Dialog.create(" ");
		Dialog.addString("Enter Dye and/or Target ID", " ");
		items = newArray("Grays", "Blue", "Cyan", "Green", "Yellow", "Red", "Magenta");
		Dialog.addRadioButtonGroup("Display This Channel as:", items, 1, 7, "Grays");
		itemz=newArray("N/A", "Exclude This Channel From Color Composite (It will be still shown separately)", "Include This Channel in Color Composite but do not show separately");
		Dialog.addChoice("Channel exclusion?", itemz)
		Dialog.addCheckbox("Highlight Cell Details in Tramsmitted / DIC Image", false);
		Dialog.addCheckbox("Use same brightness/contrast scaling for all images in this folder (so they can be compared visually)", true)
		Dialog.show();
		chNm=Dialog.getString();
		chCo=Dialog.getRadioButton();
		chEx=Dialog.getChoice();
		chTr=Dialog.getCheckbox();
		chBC=Dialog.getCheckbox();
		if(chBC==1) {run("Brightness/Contrast...");
			waitForUser("Adjust Brightness/Contrast Using B&C Tool Before Proceeding !");
			getMinAndMax(min, max);
			vmin=min;
			vmax=max;	}
		else {vmin=-1;
			vmax="auto";	}

		print("Channel=", "C"+j+"-x", chNm, chCo, "   Exclusion=", chEx, "   FFT=", chTr, "   Fixed brightness/contrast scaling=", chBC);
			
		selectWindow("Parameters");
		print(f,chNm+"\t"+j+"\t"+chCo+"\t"+vmin+"\t"+vmax+"\t"+chEx+"\t"+chTr);
		close("C"+j+"-x");	}

selectWindow("Parameters");
saveAs("Text", res+"Results.csv");
close("Parameters");       

//process images
//separate and adjust channels
for (k = 0; k < NAMES.length; k++) {open(input+NAMES[k]);
			ttl=File.nameWithoutExtension;
			getDimensions(width, height, channels, slices, frames);
			slcs=slices;
	print("   ");
	print(ttl, "   slices=", slcs);
		if(chns==1) {rename("C1-x");	}
		else {rename("x");
			run("Split Channels");
			run("Tile");	}	

if(slcs>1) {if(stp=="Max Intensity Projection") {	}	
			else {selectWindow(master);
					findCenter();
					maxc=getResult("Slice");
					close("Results");
				if(stp=="Manually-selected subset only") {waitForUser("Select the first slide to include in a substack\n(range is already specified!)");
							zz=getSliceNumber();	}	}	

	if(stp=="Max Intensity Projection") {print(stp); 	}
	if(stp=="Brightest slice only") {print(stp, "   slice=", maxc);		}
	if(stp=="A subset of slices around the brightest slice") {print(stp, "   slices=", maxc-slo, "-", maxc+sup);		}
	if(stp=="Manually-selected subset only") {print(stp, "   slices=", zz, "-", zz+rng);	}
			

//project stacks
			for (i = 0; i < chns; i++) {j=i+1;
						selectWindow("C"+j+"-x");
					if(stp=="Max Intensity Projection") {maxStack(); 	}
					if(stp=="Brightest slice only") {getCenter();	}
					if(stp=="A subset of slices around the brightest slice") {getRange();		}
					if(stp=="Manually-selected subset only") {getManual();	}
					rename("C"+j+"-x");		}	

					if(stp=="A subset of slices around the brightest slice") {for (i = 0; i < chns; i++) {j=i+1;
								selectWindow("C"+j+"-x");
								rename("z");
								run("Z Project...", "projection=[Max Intensity]");
								close("z");
								rename("C"+j+"-x");	}	}
					if(stp=="Manually-selected subset only") {for (i = 0; i < chns; i++) {j=i+1;
								selectWindow("C"+j+"-x");
								rename("z");
								run("Z Project...", "projection=[Max Intensity]");
								close("z");
								rename("C"+j+"-x");	}	}	}

if(crp==1) {selectWindow(master);
			run("Restore Selection");
			waitForUser("Adjust size/position of crop box");
			run("Duplicate...", "title=y");
			getDimensions(width, height, channels, slices, frames);
			print("cropped size=", width, "x", height);
			close("y");
			run("Select None");
	
			for (i = 0; i < chns; i++) {j=i+1;
					selectWindow("C"+j+"-x");
					run("Restore Selection");
					run("Crop");	}	}

open(res+"Results.csv");

for (i = 0; i < chns; i++) {j=i+1;
					CC=getResultString("LUT", i);
					vmin=getResult("Min", i);
					vmax=getResult("Max", i);
					CFFT=getResultString("FFT", i);
					selectWindow("C"+j+"-x");
					run(CC);
					if(CFFT==1) {autodic();	}
					else {if(vmin==-1) {scBC="auto";
										run("Enhance Contrast", "saturated=0.10");
													getMinAndMax(min, max);
													vmin=min;
													vmax=max;	}
								else {scBC="fixed";
								setMinAndMax(vmin, vmax);	}	}
								print("C"+j+"-x", CC, scBC, vmin, "-", vmax); 	}

if(tif16=="16-bit Tiff + RGB montage") {if(chns==1) {	}
			if(chns==2) {run("Merge Channels...", "c1=C1-x c2=C2-x create keep");	}
			if(chns==3) {run("Merge Channels...", "c1=C1-x c2=C2-x c3=C3-x create keep");	}
			if(chns==4) {run("Merge Channels...", "c1=C1-x c2=C2-x c3=C3-x c4=C4-x create keep");	}
			if(chns==5) {run("Merge Channels...", "c1=C1-x c2=C2-x c3=C3-x c4=C4-x c5=C5-x create keep");	}
			if(chns==6) {run("Merge Channels...", "c1=C1-x c2=C2-x c3=C3-x c4=C4-x c5=C5-x c6=C6-x create keep");	}
			if(chns==7) {run("Merge Channels...", "c1=C1-x c2=C2-x c3=C3-x c4=C4-x c5=C5-x c6=C6-x C7-x create keep");	}
					saveAs("Tif", myDir2+ttl);
					close();	}
					
excl=0;
incl=0;

for (i = 0; i < chns; i++) {j=i+1;
				CN=getResultString("Label", i);
				CEx=getResultString("Exclusion", i);
				selectWindow("C"+j+"-x");
				run("Apply LUT");
				run("RGB Color");
				if(CEx=="N/A") {rename(CN);	}
					else {if(CEx=="Exclude This Channel From Color Composite (It will be still shown separately)") {rename("excluded");
																			TMP1=CN;
																			excl=1;	}
							if(CEx=="Include This Channel in Color Composite but do not show separately") {rename("included");
																			TMP1=CN;
																			incl=1;	}	}	}

close("Results");

if(chns==1) {saveAs("Tif", myDir1+ttl);
				close();}


if(chns>1) {
          
if(chns==2)	{run("Images to Stack", "name=All title=[] use keep");
							run("Images to Stack", "name=Stack title=[] use");	}
if(chns>2) {run("Images to Stack", "name=All title=[] use keep");	
					if(excl==1) {close("excluded");	}
					run("Images to Stack", "name=Stack title=[] use");
					if(incl==1) {selectWindow("All");
					run("Stack to Images");
					close("included");
					run("Images to Stack", "name=All title=[] use");
					selectWindow"Stack");	}	}
		run("Z Project...", "projection=[Sum Slices]");
		rename(ttl);
		close("Stack");
		selectWindow("All");
		run("Stack to Images");
		run("Images to Stack", "name=ttl title=[] use");
		saveAs("Tif", myDir3+ttl);
		close();	}	}

ok = File.delete(res+"Results.csv");
		
selectWindow("Log");
saveAs("Text", res+year+"-"+month+"-"+dayOfMonth+" "+hour+"_"+minute+" Log"+".txt");
close("Log");

LIST=getFileList(myDir3);

for (h = 0; h < LIST.length; h++) {open(myDir3+LIST[0]);
getDimensions(width, height, channels, slices, frames);
imgwdt=width;
ratio=height/width;
pnls=slices;
close();	}

for (h = 0; h < LIST.length; h++) {open(myDir3+LIST[h]);
ttz=File.nameWithoutExtension;

if(ratio>0.6) {if(imgwdt<600) {
	if(pnls==2) {run("Reverse");
				run("Make Montage...", "columns=2 rows=1 scale=1 border=1 font=25 label"); }
	if(pnls==3) {run("Make Montage...", "columns=3 rows=1 scale=1 border=1 font=25 label"); }
	if(pnls==4) {run("Make Montage...", "columns=2 rows=2 scale=1 border=1 font=25 label"); }
	if(pnls==5) {run("Make Montage...", "columns=3 rows=2 scale=1 border=1 font=25 label"); }
	if(pnls==6) {run("Make Montage...", "columns=3 rows=2 scale=1 border=1 font=25 label"); }
	if(pnls==7) {run("Make Montage...", "columns=4 rows=2 scale=1 border=1 font=25 label"); }
	if(pnls==8) {run("Make Montage...", "columns=4 rows=2 scale=1 border=1 font=25 label"); }
	run("Scale Bar...", "width=10 height=4 font=15 color=White background=None location=[Upper Right] bold");	}

else {if(imgwdt<1200) {
	if(pnls==2) {run("Reverse");
				run("Make Montage...", "columns=2 rows=1 scale=1 border=2 font=35 label"); }
	if(pnls==3) {run("Make Montage...", "columns=3 rows=1 scale=1 border=2 font=35 label"); }
	if(pnls==4) {run("Make Montage...", "columns=2 rows=2 scale=1 border=2 font=35 label"); }
	if(pnls==5) {run("Make Montage...", "columns=3 rows=2 scale=1 border=2 font=35 label"); }
	if(pnls==6) {run("Make Montage...", "columns=3 rows=2 scale=1 border=2 font=35 label"); }
	if(pnls==7) {run("Make Montage...", "columns=4 rows=2 scale=1 border=2 font=35 label"); }
	if(pnls==8) {run("Make Montage...", "columns=4 rows=2 scale=1 border=2 font=35 label"); }	
	run("Scale Bar...", "width=25 height=4 font=20 color=White background=None location=[Upper Right] bold");	}
	
	else {
	if(pnls==2) {run("Reverse");
				run("Make Montage...", "columns=2 rows=1 scale=0.5 border=2 font=50 label"); }
	if(pnls==3) {run("Make Montage...", "columns=3 rows=1 scale=0.5 border=2 font=50 label"); }
	if(pnls==4) {run("Make Montage...", "columns=2 rows=2 scale=0.5 border=2 font=50 label"); }
	if(pnls==5) {run("Make Montage...", "columns=3 rows=2 scale=0.5 border=2 font=50 label"); }
	if(pnls==6) {run("Make Montage...", "columns=3 rows=2 scale=0.5 border=2 font=50 label"); }
	if(pnls==7) {run("Make Montage...", "columns=4 rows=2 scale=0.5 border=2 font=50 label"); }
	if(pnls==8) {run("Make Montage...", "columns=4 rows=2 scale=0.5 border=2 font=50 label"); }
	run("Scale Bar...", "width=50 height=4 font=35 color=White background=None location=[Upper Right] bold");	}	}	}

else {if(imgwdt<600) {
	if(pnls==2) {run("Reverse");
				run("Make Montage...", "columns=1 rows=2 scale=1 border=1 font=25 label"); }
	if(pnls==3) {run("Make Montage...", "columns=1 rows=3 scale=1 border=1 font=25 label"); }
	if(pnls==4) {run("Make Montage...", "columns=2 rows=2 scale=1 border=1 font=25 label"); }
	if(pnls==5) {run("Make Montage...", "columns=2 rows=3 scale=1 border=1 font=25 label"); }
	if(pnls==6) {run("Make Montage...", "columns=2 rows=3 scale=1 border=1 font=25 label"); }
	if(pnls==7) {run("Make Montage...", "columns=2 rows=4 scale=1 border=1 font=25 label"); }
	if(pnls==8) {run("Make Montage...", "columns=2 rows=4 scale=1 border=1 font=25 label"); }
	run("Scale Bar...", "width=10 height=4 font=15 color=White background=None location=[Upper Right] bold");	}

else {if(imgwdt<1200) {
	if(pnls==2) {run("Reverse");
				run("Make Montage...", "columns=1 rows=2 scale=1 border=2 font=35 label"); }
	if(pnls==3) {run("Make Montage...", "columns=1 rows=3 scale=1 border=2 font=35 label"); }
	if(pnls==4) {run("Make Montage...", "columns=2 rows=2 scale=1 border=2 font=35 label"); }
	if(pnls==5) {run("Make Montage...", "columns=2 rows=3 scale=1 border=2 font=35 label"); }
	if(pnls==6) {run("Make Montage...", "columns=2 rows=3 scale=1 border=2 font=35 label"); }
	if(pnls==7) {run("Make Montage...", "columns=2 rows=4 scale=1 border=2 font=35 label"); }
	if(pnls==8) {run("Make Montage...", "columns=2 rows=4 scale=1 border=2 font=35 label"); }	
	run("Scale Bar...", "width=25 height=4 font=20 color=White background=None location=[Upper Right] bold");	}
	
	else {
	if(pnls==2) {run("Reverse");
				run("Make Montage...", "columns=1 rows=2 scale=0.5 border=2 font=50 label"); }
	if(pnls==3) {run("Make Montage...", "columns=1 rows=3 scale=0.5 border=2 font=50 label"); }
	if(pnls==4) {run("Make Montage...", "columns=2 rows=2 scale=0.5 border=2 font=50 label"); }
	if(pnls==5) {run("Make Montage...", "columns=2 rows=3 scale=0.5 border=2 font=50 label"); }
	if(pnls==6) {run("Make Montage...", "columns=2 rows=3 scale=0.5 border=2 font=50 label"); }
	if(pnls==7) {run("Make Montage...", "columns=2 rows=4 scale=0.5 border=2 font=50 label"); }
	if(pnls==8) {run("Make Montage...", "columns=2 rows=4 scale=0.5 border=2 font=50 label"); }
	run("Scale Bar...", "width=50 height=4 font=35 color=White background=None location=[Upper Right] bold");	}	}	}

saveAs("Tiff", myDir1+ttz);
close();
close();	}