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BIL_Huntington_2D_v10.ijm
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BIL_Huntington_2D_v10.ijm
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/* BIL_Huntinton_2D_v10.ijm
-----------------------
Author: Marlies Verschuuren -- [email protected]
Building Blocks -- Winnok H. De Vos: CellBlocks_v12.ijm -- [email protected]
Date Created: 2020 - 07 - 07
Date Last Modified: 2022 - 02 - 08
*/
/* Plugins needed (Help > Update > Manage update sites):
Bio-Formats: https://sites.imagej.net/Bio-Formats/
ImageScience: https://sites.imagej.net/ImageScience/
Stardist: https://sites.imagej.net/StarDist/
CSBDeep: https://sites.imagej.net/CSBDeep/
*/
/* VERSION SUPPORT:
V1: - Nuclei, vessel and spot segmentation
+ morphotextural measurements in all channels
+ overlap with Marker+ mask
V2: - Spot segmentation: circularity filter
- Measurements for each channel mask
.1: Fix bug export mvd2 files
V3: - Nuclei segmentation on substacks
V4: - Substacks with slides in between
- Median filter nuclei
- Multi scale vessel detection
- Max finding in spot detection
v5: - Include spot detection in region vessel
- Include measure region around nuclei
- Diameter estimation vessel
v6: - Multi-scale vessel detection: No multiplication with index
v7: - Option multi-scale spot segmentation and max finding spot detection
v8: - Add action tools and validation tools to test settings nuclei, spot and marker detection
- Measure channel intensities
v9: - Add index to log verification stack
- Add length as parameter for vessel exclusion
-.2 Fix bug loading verification stacks
v10:- Save branch skeleton information
- Verification tool skeleton
*/
//------------------------- Variables -------------------------//
//General --------------
var dirInput = "";
var dirOutput = "";
var suffix = ".tif";
var order = "xyczt(default)";
var micron = getInfo("micrometer.abbreviation"); // micro symbol
var threshMethods = getList("threshold.methods");
var threshMethods = Array.concat(threshMethods,"Fixed");
var filetypes = newArray(".tif",".tiff",".nd2",".ids",".jpg",".mvd2",".czi");
var dimensions = newArray("xyczt(default)","xyctz","xytcz","xytzc","xyztc","xyzct");
var filters = newArray("Median","Gaussian");
var analysisStack = true;
var analysisSubStack= true;
//Settings -------------
var pixelSize = 0.1785703;
var imgWidth = 1000;
var imgHeight = 1000;
var nChannels = 4;
var chNuc = 1;
var chMarker1 = 2;
var chMarker2 = 3;
var chSpot = 4;
var bg = true;
var zproj = true;
var bgRadius = 100;
//Mask ids
var idOriginal = -100;
var idMax = -100;
var idSubMax = -100;
var idMaskMarker1 = -100;
var idMaskMarker2 = -100;
var idMaskNuclei = -100;
var idMaskSpots = -100;
var idMaskExclude = -100;
var idMaskCell = -100;
//Nuclei detection
var claheNuc = true;
var filterNuc = "Gaussian";
var filterNucScale = 8;
var probNuc = 0.70;
var overlapNuc = 0.40;
var minAreaNuc = 25;
var maxAreaNuc = 250;
var minCircNuc = 0.8;
//Cytoplasm detection
var itCellMicron = 5;
var exclude_nuclei = true;
//Spot detection
var multiScaleSpot = true;
var maxFindingSpot = true
var minScaleSpot = 1;
var maxScaleSpot = 4;
var threshSpot = 30;
var minAreaSpot = 0.2;
var maxAreaSpot = 6;
var minCircSpot = 0.6;
var excludeVesselStructures = true;
var gausExclude = 2;
var tubeExclude = true;
var minScaleExclude = 0.5;
var maxScaleExclude = 4;
var threshExclude = "Fixed";
var threshExcludeFix = 0.15;
var minAreaExclude = 50;
var minVesselLengthExclude = 15;
//Marker 1 detection
var gausMarker1 = 2;
var tubeMarker1 = true;
var minScaleTubeMarker1 = 0.5;
var maxScaleTubeMarker1 = 4;
var threshMarker1 = "Fixed";
var threshMarker1Fix = 0.15;
var minAreaMarker1 = 50;
var skeletonMarker1 = true;
//Marker 2 detection
var gausMarker2 = 2;
var tubeMarker2 = true;
var minScaleTubeMarker2 = 0.5;
var maxScaleTubeMarker2 = 4;
var threshMarker2 = "Fixed";
var threshMarker2Fix = 0.2;
var minAreaMarker2 = 50;
var skeletonMarker2 = false;
//------------------------- Macro -------------------------//
macro "[I] Install Macro"{
// Only works on my personal drive
run("Install...", "install=[/data/CBH/mverschuuren/ACAM/CF-BIL_TamaraVasilkovska/200401_Huntington/GitHub/Huntington/BIL_Huntington_2D_v10.ijm]");
}
macro "Split Files Action Tool - Cf88 R0077 C888 R9077 R9977 R0977"{
setBatchMode(true);
splitRegions();
setBatchMode("exit and display");
}
macro "Setup Regions Action Tool - C888 T5f16S"{
setup();
}
macro "Segment Nuclei Action Tool -C999 H11f5f8cf3f181100 C999 P11f5f8cf3f18110 Ceee V5558"{
erase(0);
setBatchMode(true);
//Get image id and title
id = getImageID;
title = getTitle;
prefix = substring(title,0,lastIndexOf(title,suffix));
//Get directory
dirInput = getInfo("image.directory");
dirMax = dirInput+"Max"+File.separator;
dirSubMax = dirInput+"SubMax"+File.separator;
dirOutput = dirInput+"Output"+File.separator;
if(!File.exists(dirMax)){
print("No Max Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirSubMax)){
print("No SubMax Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirOutput)){
File.makeDirectory(dirOutput);
}
//Select substack
nrSub = getNumber("Nr Substack",3);
//Open substack
name=prefix+"_"+nrSub;
print("Image: "+name);
path=dirSubMax+"SubMax_"+name+".tif";
open(path);
idSubMax=getImageID();
getPixelSize(unit, pixelWidth, pixelHeight);
if(unit!=micron){
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
}else{
pixelSize = pixelWidth;
}
//NucleiDetection
countNuc = segmentationNuclei2D(idSubMax,name);
if(countNuc>0){
countCell = segmentationCell2D(name);
}
//Visualisation
selectImage(idSubMax);
if(File.exists(dirOutput+name+"_CH"+chNuc+"_ROI_Cell.zip")){
roiManager("Open",dirOutput+name+"_CH"+chNuc+"_ROI_Cell.zip");
countCell=roiManager("count");
roiManager("Set Color", "yellow");
roiManager("Show All without labels");
}
if(File.exists(dirOutput+name+"_CH"+chNuc+"_ROI_Nuc.zip")){
roiManager("Open",dirOutput+name+"_CH"+chNuc+"_ROI_Nuc.zip");
countNuc=roiManager("count")-countCell;
for(i=0; i<countNuc; i++){
roiManager("Select", countCell+i);
roiManager("Set Color", "cyan");
}
roiManager("Show All without labels");
}
selectImage(idMaskNuclei); close();
selectImage(idSubMax);
Stack.setChannel(chNuc);
setBatchMode("exit and display");
run("Tile");
}
macro "Spot Detection Action Tool - C999 H11f5f8cf3f181100 C999 P11f5f8cf3f18110 Ceee V3633 V4b33 V7633 Va933"{
erase(0);
setBatchMode(true);
//Get image id and title
id = getImageID;
title = getTitle;
prefix = substring(title,0,lastIndexOf(title,suffix));
//Get directory
dirInput = getInfo("image.directory");
dirMax = dirInput+"Max"+File.separator;
dirSubMax = dirInput+"SubMax"+File.separator;
dirOutput = dirInput+"Output"+File.separator;
if(!File.exists(dirMax)){
print("No Max Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirSubMax)){
print("No SubMax Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirOutput)){
File.makeDirectory(dirOutput);
}
//Select substack
nrSub = getNumber("Nr Substack",3);
//Open substack
name=prefix+"_"+nrSub;
print("Image: "+name);
path=dirSubMax+"SubMax_"+name+".tif";
open(path);
idSubMax=getImageID();
getPixelSize(unit, pixelWidth, pixelHeight);
if(unit!=micron){
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
}else{
pixelSize = pixelWidth;
}
//Spot detection
countSpots = segmentationSpots2D(idSubMax,name,true);
//Visualisation
if(File.exists(dirOutput+name+"_CH"+chSpot+"_ROI.zip")){
roiManager("Open",dirOutput+name+"_CH"+chSpot+"_ROI.zip");
}
selectImage(idMaskSpots); close();
selectImage(idSubMax);
Stack.setChannel(chSpot);
roiManager("Show All without labels");
setBatchMode("exit and display");
run("Tile");
}
macro "Segment Markers Action Tool -C999 Hff1f1cfcff00"{
erase(0);
setBatchMode(true);
//Get image id and title
id = getImageID;
title = getTitle;
prefix = substring(title,0,lastIndexOf(title,suffix));
getPixelSize(unit, pixelWidth, pixelHeight);
if(unit!=micron){
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
}else{
pixelSize = pixelWidth;
}
//Get directory
dirInput = getInfo("image.directory");
dirMax = dirInput+"Max"+File.separator;
dirSubMax = dirInput+"SubMax"+File.separator;
dirOutput = dirInput+"Output"+File.separator;
if(!File.exists(dirMax)){
print("No Max Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirSubMax)){
print("No SubMax Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirOutput)){
File.makeDirectory(dirOutput);
}
//get SubStack
nrSub = getNumber("Nr Substack (0 = Max Proj. entire stack)",0);
//Open Image
if(nrSub==0){
name=prefix;
path=dirMax+"Max_"+name+".tif";
open(path);}
else {
name=prefix+"_"+nrSub;
path=dirSubMax+"SubMax_"+name+".tif";
open(path);
}
idProj=getImageID();
getPixelSize(unit, pixelWidth, pixelHeight);
if(unit!=micron){
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
}else{
pixelSize = pixelWidth;
}
//Detection
idMaskMarker1 = segmentationMask2D (idProj,name,chMarker1,gausMarker1,tubeMarker1,minScaleTubeMarker1, maxScaleTubeMarker1,threshMarker1,threshMarker1Fix, minAreaMarker1,skeletonMarker1,true);
idMaskMarker2 = segmentationMask2D (idProj,name,chMarker2,gausMarker2,tubeMarker2,minScaleTubeMarker2, maxScaleTubeMarker2,threshMarker2,threshMarker2Fix, minAreaMarker2,skeletonMarker2,true);
//Visualisation
roiManager("reset");
if(File.exists(dirOutput+name+"_CH"+chMarker1+"_ROI.zip")){
roiManager("Open",dirOutput+name+"_CH"+chMarker1+"_ROI.zip");
currentCount=roiManager("count");
roiManager("Select", (currentCount-1));
roiManager("Set Color", "green");
roiManager("Show All without labels");
}
if(File.exists(dirOutput+name+"_CH"+chMarker1+"_Skeleton.zip")){
roiManager("Open",dirOutput+name+"_CH"+chMarker1+"_Skeleton.zip");
currentCount=roiManager("count");
roiManager("Select", (currentCount-1));
roiManager("Set Color", "yellow");
roiManager("Show All without labels");
}
print(dirOutput+name+"_CH"+chMarker2+"_ROI.zip");
if(File.exists(dirOutput+name+"_CH"+chMarker2+"_ROI.zip")){
roiManager("Open",dirOutput+name+"_CH"+chMarker2+"_ROI.zip");
currentCount=roiManager("count");
roiManager("Select", (currentCount-1));
roiManager("Set Color", "RED");
roiManager("Show All without labels");
}
selectImage(idProj);
Stack.setChannel(chMarker1);
if(File.exists(dirOutput+name+"_CH"+chMarker1+"_ROI.zip") || File.exists(dirOutput+name+"_CH"+chMarker2+"_ROI.zip")){
roiManager("Show All without labels");
run("From ROI Manager");
}
selectImage(idMaskMarker2); close();
selectImage(idMaskMarker1); close();
setBatchMode("exit and display");
}
macro "Analyse Single Image Action Tool - C888 T5f161"{
start = getTime();
run("ROI Manager...");
setBatchMode(true);
setOptions();
//Get Directory
dirInput = getInfo("image.directory");
dirMax = dirInput+"Max"+File.separator;
dirSubMax = dirInput+"SubMax"+File.separator;
dirOutput = dirInput+"Output"+File.separator;
if(!File.exists(dirMax)){
print("No Max Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirSubMax)){
print("No SubMax Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirOutput)){
File.makeDirectory(dirOutput);
}
//Get Image Properties
title = getTitle;
prefix = substring(title,0,lastIndexOf(title,suffix));
idOriginal = getImageID;
selectImage(idOriginal);
imgWidth = getWidth();
imgHeight = getHeight();
//Open image
path=dirMax+"Max_"+prefix+".tif";
open(path);
idMax=getImageID();
getPixelSize(unit, pixelWidth, pixelHeight);
if(unit!=micron){
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
}else{
pixelSize = pixelWidth;
}
//Intensity measurements
selectImage(idMax);
run("Select All");
run("Set Measurements...", "mean standard min redirect=None decimal=2");
run("Clear Results");
for(c=1; c<=nChannels;c++){
selectImage(idMax);
Stack.setChannel(c);
run("Measure");
}
saveAs("Measurements",dirOutput+prefix+"_ChannelInt.txt");
run("Clear Results");
if(analysisStack){
//Marker Detection
idMaskMarker1 = segmentationMask2D (idMax,prefix,chMarker1,gausMarker1,tubeMarker1,minScaleTubeMarker1, maxScaleTubeMarker1,threshMarker1,threshMarker1Fix, minAreaMarker1,skeletonMarker1,true);
selectImage(idMaskMarker1);
rename("maskMarker1");
idMaskMarker2 = segmentationMask2D (idMax,prefix,chMarker2,gausMarker2,tubeMarker2,minScaleTubeMarker2,maxScaleTubeMarker2,threshMarker2,threshMarker2Fix, minAreaMarker2,skeletonMarker2,true);
selectImage(idMaskMarker2);
rename("maskMarker2");
//Analyze masks
analyzeMasks(idMax,idMaskMarker1,prefix,chMarker1);
analyzeMasks(idMax,idMaskMarker2,prefix,chMarker2);
//Visualisations
selectImage(idMax);
for(c=1; c<=nChannels; c++){
if(File.exists(dirOutput+prefix+"_CH"+c+"_ROI.zip")){
roiManager("Open",dirOutput+prefix+"_CH"+c+"_ROI.zip");
currentCount=roiManager("count");
roiManager("Select", (currentCount-1));
if(c==chNuc){
roiManager("Set Color", "cyan");
}else if(c==chMarker1){
roiManager("Set Color", "green");
}else if(c==chMarker2){
roiManager("Set Color", "red");
}else if(c==chSpot){
roiManager("Set Color", "magenta");
}
roiManager("Show All without labels");
}
}
if(File.exists(dirOutput+prefix+"_CH"+chMarker1+"_Skeleton.zip")){
roiManager("Open",dirOutput+prefix+"_CH"+chMarker1+"_Skeleton.zip");
currentCount=roiManager("count");
roiManager("Select", (currentCount-1));
roiManager("Set Color", "yellow");
roiManager("Show All without labels");
}
run("From ROI Manager");
selectImage(idMaskMarker2); close();
selectImage(idMaskMarker1); close();
}
//Analysis substacks
if(analysisSubStack){
it=1;
while(File.exists(dirSubMax+"SubMax_"+prefix+"_"+it+".tif")){
name=prefix+"_"+it;
print("--Analysis Substack: "+it);
path=dirSubMax+"SubMax_"+name+".tif";
open(path);
getStatistics(area, mean, min, max, std, histogram);
//Run if standard deviation of intensities > 2
if(std>2){
//Image properties
idSubMax=getImageID();
getPixelSize(unit, pixelWidth, pixelHeight);
if(unit!=micron){
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
}else{
pixelSize = pixelWidth;
}
//Marker detection
idMaskMarker1 = segmentationMask2D (idSubMax,name,chMarker1,gausMarker1,tubeMarker1,minScaleTubeMarker1, maxScaleTubeMarker1,threshMarker1,threshMarker1Fix, minAreaMarker1,false,true);
selectImage(idMaskMarker1);
rename("maskMarker1");
idMaskMarker2 = segmentationMask2D (idSubMax,name,chMarker2,gausMarker2,tubeMarker2,minScaleTubeMarker2,maxScaleTubeMarker2,threshMarker2,threshMarker2Fix, minAreaMarker2,false,true);
selectImage(idMaskMarker2);
rename("maskMarker2");
//Nuclei and spot detection
countNuc = segmentationNuclei2D(idSubMax,name);
if(countNuc>0){
countCell = segmentationCell2D(name);
roiManager("reset");
countSpots = segmentationSpots2D(idSubMax,name,false);
}else{
print("No Nuclei detected");
newImage("maskCell", "8-bit black", imgWidth, imgHeight, 1);
idMaskCell = getImageID();
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
newImage("maskSpots", "8-bit black", imgWidth, imgHeight, 1);
idMaskSpots = getImageID();
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
}
//Summarise results
readout = analyzeRegions(name);
if(readout){
summarizeResults(name);
}
analyzeMasks(idSubMax,idMaskMarker1,name,chMarker1);
analyzeMasks(idSubMax,idMaskMarker2,name,chMarker2);
//Visualisation
selectImage(idSubMax);
if(File.exists(dirOutput+name+"_CH"+chNuc+"_ROI_Cell.zip")){
roiManager("Open",dirOutput+name+"_CH"+chNuc+"_ROI_Cell.zip");
countCell=roiManager("count");
roiManager("Set Color", "yellow");
roiManager("Show All without labels");
}
if(File.exists(dirOutput+name+"_CH"+chNuc+"_ROI_Nuc.zip")){
roiManager("Open",dirOutput+name+"_CH"+chNuc+"_ROI_Nuc.zip");
countNuc=roiManager("count")-countCell;
for(i=0; i<countNuc; i++){
roiManager("Select", countCell+i);
roiManager("Set Color", "cyan");
}
roiManager("Set Color", "red");
roiManager("Show All without labels");
}
if(File.exists(dirOutput+name+"_CH"+chSpot+"_ROI.zip")){
roiManager("Open",dirOutput+name+"_CH"+chSpot+"_ROI.zip");
roiManager("Deselect");
countSpot=roiManager("count")-countNuc-countCell;
for(i=0; i<countSpot; i++){
roiManager("Select", countNuc+countCell+i);
roiManager("Set Color", "magenta");
}
roiManager("Show All without labels");
}
if(File.exists(dirOutput+name+"_CH"+chMarker1+"_ROI.zip")){
roiManager("Open",dirOutput+name+"_CH"+chMarker1+"_ROI.zip");
currentCount=roiManager("count");
roiManager("Select", (currentCount-1));
roiManager("Set Color", "green");
roiManager("Show All without labels");
}
if(File.exists(dirOutput+name+"_CH"+chMarker2+"_ROI.zip")){
roiManager("Open",dirOutput+name+"_CH"+chMarker2+"_ROI.zip");
currentCount=roiManager("count");
roiManager("Select", (currentCount-1));
roiManager("Set Color", "red");
roiManager("Show All without labels");
}
run("From ROI Manager");
selectImage(idMaskMarker2); close();
selectImage(idMaskMarker1); close();
selectImage(idMaskNuclei); close();
selectImage(idMaskSpots); close();
}else{
print("Standard deviation in image to low to detect nuclei");
}
it=it+1;
}
}
print((getTime()-start)/1000,"sec");
print("Analysis Done");
setBatchMode("exit and display");
run("Tile");
run("Synchronize Windows");
}
macro "Batch Analysis Action Tool - C888 T5f16#"{
erase(1);
run("ROI Manager...");
setBatchMode(true);
//Get Directory
dirInput = getDirectory("Choose a Source Directory With Raw Images");
dirMax = dirInput+"Max"+File.separator;
dirSubMax = dirInput+"SubMax"+File.separator;
dirOutput = dirInput+"Output"+File.separator;
logPath = dirOutput+"Log.txt";
if(!File.exists(dirMax)){
print("No Max Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirSubMax)){
print("No SubMax Projections Found. Run 'SplitAction' Tool");
}
if(!File.exists(dirOutput)){
File.makeDirectory(dirOutput);
}
//Scan files
list = getFileList(dirInput);
prefixes = scanFiles();
fields = prefixes.length;
setup();
start = getTime();
for(i=0;i<fields;i++){
prefix = prefixes[i];
print(i+1,"/",fields,":",prefix);
//Marker Detection
path=dirMax+"Max_"+prefix+".tif";
open(path);
idMax=getImageID();
//Image properties
getPixelSize(unit, pixelWidth, pixelHeight);
if(unit!=micron){
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
}else{
pixelSize = pixelWidth;
}
//Intensity measurements
selectImage(idMax);
run("Select All");
run("Set Measurements...", "mean standard min redirect=None decimal=2");
run("Clear Results");
for(c=1; c<=nChannels;c++){
selectImage(idMax);
Stack.setChannel(c);
run("Measure");
}
saveAs("Measurements",dirOutput+prefix+"_ChannelInt.txt");
run("Clear Results");
if(analysisStack){
//Marker detection
idMaskMarker1 = segmentationMask2D (idMax,prefix,chMarker1,gausMarker1,tubeMarker1,minScaleTubeMarker1, maxScaleTubeMarker1,threshMarker1,threshMarker1Fix, minAreaMarker1,skeletonMarker1,true);
selectImage(idMaskMarker1);
rename("maskMarker1");
idMaskMarker2 = segmentationMask2D (idMax,prefix,chMarker2,gausMarker2,tubeMarker2,minScaleTubeMarker2,maxScaleTubeMarker2,threshMarker2,threshMarker2Fix, minAreaMarker2,skeletonMarker2,true);
selectImage(idMaskMarker2);
rename("maskMarker2");
//Analyze masks
analyzeMasks(idMax,idMaskMarker1,prefix,chMarker1);
analyzeMasks(idMax,idMaskMarker2,prefix,chMarker2);
//Close images
selectImage(idMaskMarker2); close();
selectImage(idMaskMarker1); close();
selectImage(idMax);close();
}
//Analyse substacks
if(analysisSubStack){
it=1;
while(File.exists(dirSubMax+"SubMax_"+prefix+"_"+it+".tif")){
name=prefix+"_"+it;
print("--Analysis Substack: "+it);
path=dirSubMax+"SubMax_"+name+".tif";
open(path);
idSubMax=getImageID();
//Image Properties
getPixelSize(unit, pixelWidth, pixelHeight);
if(unit!=micron){
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
}else{
pixelSize = pixelWidth;
}
//Run if standard deviation of intensities > 2
getStatistics(area, mean, min, max, std, histogram);
if(std>2){
//Marker detection
idMaskMarker1 = segmentationMask2D (idSubMax,name,chMarker1,gausMarker1,tubeMarker1,minScaleTubeMarker1, maxScaleTubeMarker1,threshMarker1,threshMarker1Fix, minAreaMarker1,false,true);
selectImage(idMaskMarker1);
rename("maskMarker1");
idMaskMarker2 = segmentationMask2D (idSubMax,name,chMarker2,gausMarker2,tubeMarker2,minScaleTubeMarker2,maxScaleTubeMarker2,threshMarker2,threshMarker2Fix, minAreaMarker2,false,true);
selectImage(idMaskMarker2);
rename("maskMarker2");
//Nucleus and spot detection
countNuc = segmentationNuclei2D(idSubMax,name);
if(countNuc>0){
countCell = segmentationCell2D(name);
roiManager("reset");
countSpots = segmentationSpots2D(idSubMax,name,false);
}else{
print("No Nuclei detected");
newImage("maskCell", "8-bit black", imgWidth, imgHeight, 1);
idMaskCell = getImageID();
run("Properties...", " unit="+micron+" pixel_width="+pixelSize+" pixel_height="+pixelSize);
newImage("maskSpots", "8-bit black", imgWidth, imgHeight, 1);
idMaskSpots = getImageID();
}
//Summarise results
readout = analyzeRegions(name);
if(readout){
summarizeResults(name);
}
analyzeMasks(idSubMax,idMaskMarker1,name,chMarker1);
analyzeMasks(idSubMax,idMaskMarker2,name,chMarker2);
//Close Images
selectImage(idMaskMarker2); close();
selectImage(idMaskMarker1); close();
selectImage(idMaskNuclei); close();
selectImage(idMaskSpots); close();
}else{
print("Standard deviation in image to low to detect nuclei");
}
selectImage(idSubMax); close();
it=it+1;
erase(0);
}
erase(0);
}
}
print((getTime()-start)/1000,"sec");
print("Analysis Done");
if(isOpen("Log")){
selectWindow("Log");
saveAs("txt",dirOutput+"Log.txt");
}
setBatchMode("exit and display");
}
macro "Verification Nuclei and Spot Stack Action Tool - C888 T1f16V Taf07N Tff07S"{
erase(1);
setBatchMode(true);
dirInput = getDirectory("Choose a Source Directory With Raw Images");
list = getFileList(dirInput);
dirMax = dirInput+"Max"+File.separator;
dirSubMax = dirInput+"SubMax"+File.separator;
dirOutput = dirInput+"Output"+File.separator;
if(!File.exists(dirOutput) | !File.exists(dirOutput) |!File.exists(dirOutput) ){
print("No Output-, Max- or SubMax- Directory found");
}else{
logPath = dirOutput+"Log.txt";
prefixes = scanFiles();
n=prefixes.length;
it=0;
for (i = 0; i < n; i=i+100) {
if(i+99>n){
max=n-(it*100);
}else{
max=100;
it=it+1;
}
prefixesSub=newArray(prefixes[i]);
for (k = 1; k < max; k++) {
prefixesSub=Array.concat(prefixesSub,prefixes[i+k]);
}
createOverlayNucleiSpots(prefixesSub);
setBatchMode("exit and display");
run("Tile");
run("Channels Tool... ");
Stack.setDisplayMode("composite");
Stack.setActiveChannels("100111");
waitForUser("Next 100 images?");
setBatchMode(true);
run("Close All");
run("Collect Garbage");
}
}
}
macro "Verification Marker Stack Action Tool - C888 T1f16V Taf07M "{
erase(1);
setBatchMode(true);
dirInput = getDirectory("Choose a Source Directory With Raw Images");
list = getFileList(dirInput);
dirOutput = dirInput+"Output"+File.separator;
dirMax = dirInput+"Max"+File.separator;
dirSubMax = dirInput+"SubMax"+File.separator;
dirOutput = dirInput+"Output"+File.separator;
if(!File.exists(dirOutput) | !File.exists(dirOutput) |!File.exists(dirOutput) ){
print("No Output-, Max- or SubMax- Directory found");
}else{
logPath = dirOutput+"Log.txt";
prefixes = scanFiles();
createOverlayMarkerStack(prefixes);
}
setBatchMode("exit and display");
run("Tile");
run("Channels Tool... ");
Stack.setDisplayMode("composite");
Stack.setActiveChannels("0110111");
}
macro "Verification Marker SubStack Action Tool - C888 T1f16V Taf07M Tff07S"{
erase(1);
setBatchMode(true);
dirInput = getDirectory("Choose a Source Directory With Raw Images");
list = getFileList(dirInput);
dirMax = dirInput+"Max"+File.separator;
dirSubMax = dirInput+"SubMax"+File.separator;
dirOutput = dirInput+"Output"+File.separator;
if(!File.exists(dirOutput) | !File.exists(dirOutput) |!File.exists(dirOutput) ){
print("No Output-, Max- or SubMax- Directory found");
}else{
logPath = dirOutput+"Log.txt";
prefixes = scanFiles();
n=prefixes.length;
it=0;
for (i = 0; i < n; i=i+100) {
if(i+99>n){
max=n-(it*100);
}else{
max=100;
it=it+1;
}
prefixesSub=newArray(prefixes[i]);
for (k = 1; k < max; k++) {
prefixesSub=Array.concat(prefixesSub,prefixes[i+k]);
}
createOverlayMarkerSubStack(prefixesSub);
setBatchMode("exit and display");
run("Tile");
run("Channels Tool... ");
Stack.setDisplayMode("composite");
Stack.setActiveChannels("011011");
waitForUser("Next 100 images?");
setBatchMode(true);
run("Close All");
run("Collect Garbage");
}
}
}
macro "Toggle Overlay Action Tool - Caaa O11ee"{
toggleOverlay();
}
macro "[t] Toggle Overlay"{
toggleOverlay();
}
// ----- FUNCTIONS ----- //
function setup(){
//erase(1);
setOptions();
Dialog.createNonBlocking("Huntington 2020: General settings");
Dialog.setInsets(0,0,0);
Dialog.addMessage("-------------------------------------------------- General Parameters --------------------------------------------------", 14, "#ff0000");
Dialog.setInsets(0,0,0);
Dialog.addChoice("Image Type", filetypes, suffix);
Dialog.addNumber("Pixel Size", pixelSize, 3, 5, micron);
Dialog.addNumber("Number of Channels", nChannels, 0, 5, " ");
Dialog.setInsets(0,0,0);
Dialog.addMessage("Channels:\n",12,"#ff0000");
Dialog.setInsets(0,0,0);
Dialog.addNumber("Nuclear Channel", chNuc, 0, 4, "");
Dialog.addToSameRow();
Dialog.addNumber("2B4 Spot Channel", chSpot, 0, 4, "");
Dialog.addToSameRow();
Dialog.addNumber("Marker1 Channel ", chMarker1, 0, 4, "");
Dialog.addToSameRow();
Dialog.addNumber("Marker2 Channel", chMarker2, 0, 4, "");
Dialog.setInsets(0,0,0);
Dialog.addMessage("Background subtraction:\n",12,"#ff0000");
Dialog.setInsets(0,100,0);
Dialog.addCheckbox("Bg subtraction", bg);
Dialog.addToSameRow();
Dialog.addNumber("Radius filter", bgRadius, 2, 5, "px");
Dialog.setInsets(0,0,0);
Dialog.addMessage("Analysis blocks:\n",12,"#ff0000");
Dialog.setInsets(0,100,0);
Dialog.addCheckbox("Stack: marker1/2", analysisStack);
Dialog.addToSameRow();
Dialog.addCheckbox("SubStack: Nuclei, 2B4 and marker1/2", analysisSubStack);
Dialog.setInsets(0,0,0);
Dialog.addMessage("-------------------------------------------------- Nuclei Segmentation --------------------------------------------------", 14, "#ff0000");
Dialog.setInsets(0,0,0);
Dialog.addMessage("PreProcessing:\n",12,"#ff0000");
Dialog.setInsets(0,100,0);
Dialog.addCheckbox("CLAHE (Local Contrast)", claheNuc);
Dialog.addToSameRow();
Dialog.addChoice("Filter",filters,filterNuc);
Dialog.addToSameRow();
Dialog.addNumber("Filter radius", filterNucScale, 2, 4, "");
Dialog.setInsets(0,0,0);
Dialog.addMessage("Stardist:\n",12,"#ff0000");
Dialog.setInsets(0,0,0);
Dialog.addNumber("Probability", probNuc, 2, 4, "");
Dialog.addToSameRow();
Dialog.addNumber("Overlap", overlapNuc, 2, 4, "");
Dialog.setInsets(0,0,0);
Dialog.addMessage("Nuclei filter:\n",12,"#ff0000");
Dialog.setInsets(0,0,0);
Dialog.addNumber("Min Area ", minAreaNuc, 2, 5, micron+"2");
Dialog.addToSameRow();
Dialog.addNumber("Max Area ", maxAreaNuc, 2, 5, micron+"2");
Dialog.addToSameRow();
Dialog.addNumber("Min Circ. ", minCircNuc, 2, 5, "");
Dialog.setInsets(0,0,0);
Dialog.addMessage("Cell detection:\n",12,"#ff0000");
Dialog.setInsets(0,0,0);
Dialog.addNumber("Dilation", itCellMicron, 2, 5, micron+"2");
Dialog.setInsets(0,0,0);
Dialog.addMessage("-------------------------------------------------- Spot Segmentation --------------------------------------------------", 14, "#ff0000");
Dialog.setInsets(0,0,0);
Dialog.addMessage("Spot enhancement:\n",12,"#ff0000");
Dialog.setInsets(0,100,0);
Dialog.addCheckbox("Multi-scale detection", multiScaleSpot);
Dialog.addToSameRow();
Dialog.addNumber("Min Scale laplace", minScaleSpot, 2, 4, "");
Dialog.addToSameRow();
Dialog.addNumber("Max Scale laplace", maxScaleSpot, 2, 4, "");
Dialog.setInsets(0,100,0);
Dialog.addCheckbox("Max finding", maxFindingSpot);
Dialog.setInsets(0,0,0);
Dialog.addMessage("Spot detection:\n",12,"#ff0000");
Dialog.setInsets(0,0,0);
Dialog.addNumber("Fixed Threshold", threshSpot, 2, 4, "");
Dialog.setInsets(0,0,0);
Dialog.addMessage("Spot filter:\n",12,"#ff0000");
Dialog.setInsets(0,0,0);
Dialog.addNumber("Min Area ", minAreaSpot, 2, 5, micron+"2");
Dialog.addToSameRow();
Dialog.addNumber("Max Area ", maxAreaSpot, 2, 5, micron+"2");
Dialog.addToSameRow();
Dialog.addNumber("Min Circ. ", minCircSpot, 2, 5, "");
Dialog.setInsets(0,0,0);
Dialog.addMessage("Exclude vessel structures:\n",12,"#ff0000");
Dialog.setInsets(0,100,0);
Dialog.addCheckbox("Exclude vessel", excludeVesselStructures);
Dialog.addToSameRow();
Dialog.addNumber("Gaussian Blur Radius", gausExclude, 2, 4, "");
Dialog.setInsets(0,100,0);
Dialog.addCheckbox("Tube enhancement", tubeExclude);
Dialog.addToSameRow();
Dialog.addNumber("Min Scale", minScaleExclude, 2, 4, "");
Dialog.addToSameRow();
Dialog.addNumber("Max Scale", maxScaleExclude, 2, 4, "");
Dialog.addChoice("Threshold Algortihm", threshMethods, threshExclude);
Dialog.addToSameRow();
Dialog.addNumber("Fixed Threshold", threshExcludeFix, 2, 4, "");
Dialog.addNumber("Minimum Area ", minAreaExclude, 2, 5, micron+"2");
Dialog.addToSameRow();
Dialog.addNumber("Minimum Length ", minVesselLengthExclude, 2, 5, micron);
Dialog.setInsets(0,0,0);
Dialog.show();
print("-------Settings-------");
suffix = Dialog.getChoice();
pixelSize = Dialog.getNumber(); print("Pixel size:",pixelSize);
nChannels = Dialog.getNumber(); print("Channels:",nChannels);
chNuc = Dialog.getNumber(); print("Channel Nuclei:", chNuc);
chSpot = Dialog.getNumber(); print("Channel 2B4 Spot:", chSpot);
chMarker1 = Dialog.getNumber(); print("Channel Marker 1:", chMarker1);
chMarker2 = Dialog.getNumber(); print("Channel Marker 2:", chMarker2);
bg = Dialog.getCheckbox(); print("Background subtraction:", bg);
bgRadius = Dialog.getNumber(); print("Radius background subtraction:", bgRadius);
analysisStack = Dialog.getCheckbox(); print("Analysis Stack Marker1/2: ", analysisStack);
analysisSubStack= Dialog.getCheckbox(); print("Analysis SubStack DAPI, 2B4, Marker1/2: ", analysisSubStack);
claheNuc = Dialog.getCheckbox(); print("CLAHE nculei:",claheNuc);
filterNuc = Dialog.getChoice(); print("Filter nuclei:",filterNuc);
filterNucScale = Dialog.getNumber(); print("Filter radius nuclei:",filterNucScale);
probNuc = Dialog.getNumber(); print("Stardist Probability Threshold:",probNuc);
overlapNuc = Dialog.getNumber(); print("Stardist Overlap Threshold:",overlapNuc);
minAreaNuc = Dialog.getNumber(); print("Min area nuclei:",minAreaNuc);
maxAreaNuc = Dialog.getNumber(); print("Max area nuclei:",maxAreaNuc);
minCircNuc = Dialog.getNumber(); print("Max area nuclei:",minCircNuc);
itCellMicron = Dialog.getNumber(); print("Dilation Cell Detection:", itCellMicron);
multiScaleSpot = Dialog.getCheckbox(); print("Multiscale spot detection:",multiScaleSpot);
minScaleSpot = Dialog.getNumber(); print("Min Laplace radius spots:", minScaleSpot);
maxScaleSpot = Dialog.getNumber(); print("Max Laplace radius spots:", maxScaleSpot);
maxFindingSpot = Dialog.getCheckbox(); print("Max finding spot detection:", maxFindingSpot);
threshSpot = Dialog.getNumber(); print("Fixed threshold spots:", threshSpot);
minAreaSpot = Dialog.getNumber(); print("Min area spots:",minAreaSpot);
maxAreaSpot = Dialog.getNumber(); print("Max area spots:",maxAreaSpot);
minCircSpot = Dialog.getNumber(); print("Min circ spots:",minCircSpot);
excludeVesselStructures = Dialog.getCheckbox(); print("Exclude vessel structures:",excludeVesselStructures);
gausExclude = Dialog.getNumber(); print("Blur radius Exclude:",gausExclude);
tubeExclude = Dialog.getCheckbox(); print("Edge enhancement Exclude:",tubeExclude);
minScaleExclude = Dialog.getNumber(); print("Min Scale Tube enhancement Exclude:",minScaleExclude);