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script-and-macro-examples/Segment_nuclei_count_golgi.ijm

+// Select file directory and one image from the dialog box
+//#@ File (label = "input file directory", style="directory") inputDir
+//#@ File (label = "input image file", style="file") inputFile
+//run("Bio-Formats", "open=[inputFile] color_mode=Default rois_import=[ROI manager] view=Hyperstack stack_order=XYCZT use_virtual_stack");
+
+//operate on black background:
+run("Options...", "iterations=1 count=1 black");
+
+fileName   = getTitle();
+fileName   = File.nameWithoutExtension;
+//savename   = inputDir + File.separator + fileName + "_labeling_watershed";
+//resultname = inputDir + File.separator + fileName + "_overlay_watershed";
+
+
+// parameters
+runInteractive = false;
+closeIntermediateImages=true;
+nucleusDilationPixels = 30;
+
+nucleusMinimalSizePixels = 2000;
+nucleusMaximalSizePixels = 7000;
+
+minGolgiCount = 1;
+maxGolgiCount = 200;
+
+
+// open image (could be commented for adaptive feedback)
+//imagePath = "/Users/tischer/Documents/joanna-zukowska-golgi-morphology/src/test/resources/image-data/frag.czi";
+//run("Bio-Formats Importer", "open=[&imagePath] color_mode=Default rois_import=[ROI manager] view=Hyperstack stack_order=XYCZT");
+
+run("Set Scale...", "distance=0 known=0 unit=pixel");
+// store calibration
+getPixelSize(unit, pixelWidth, pixelHeight);
+distance = 1 / pixelWidth
+rename("origin");
+
+closeAllExceptOrigin()
+run("Duplicate...", "duplicate");
+rename("raw");
+
+
+
+run("Split Channels");
+selectWindow("C1-raw");
+rename("nuclei");
+selectWindow("C2-raw");
+rename("golgiChannel");
+selectWindow("C3-raw");
+close();
+
+// segment nuclei 
+selectWindow("nuclei");
+//run("Command From Macro", "command=[de.csbdresden.stardist.StarDist2D], args=['input':'nuclei', 'modelChoice':'DSB 2018 (from StarDist 2D paper)', 'normalizeInput':'true', 'percentileBottom':'1.0', 'percentileTop':'99.8', 'probThresh':'0.479071', 'nmsThresh':'0.3', 'outputType':'Label Image', 'nTiles':'1', 'excludeBoundary':'2', 'roiPosition':'Automatic', 'verbose':'false', 'showCsbdeepProgress':'false', 'showProbAndDist':'false'], process=[false]");
+//workaround for Stardist issue in batch mode
+//run("Command From Macro", "command=[de.csbdresden.stardist.StarDist2D], args=['input':'nuclei', 'modelChoice':'DSB 2018 (from StarDist 2D paper)', 'normalizeInput':'true', 'percentileBottom':'1.0', 'percentileTop':'99.8', 'probThresh':'0.479071', 'nmsThresh':'0.3', 'outputType':'ROI Manager', 'nTiles':'1', 'excludeBoundary':'2', 'roiPosition':'Automatic', 'verbose':'false', 'showCsbdeepProgress':'false', 'showProbAndDist':'false'], process=[false]");
+
+
+
+setOption("ScaleConversions", true);
+run("8-bit");
+run("Enhance Contrast...", "saturated=1");
+run("Smooth");
+run("Auto Threshold", "method=Otsu white");
+run("Fill Holes");
+run("Dilate");
+run("Fill Holes");
+run("Dilate Labels", "radius=5");
+//run("Watershed");
+run("Analyze Particles...", "size=100-10000 show=Masks display exclude add");
+
+Table.rename("Results", "111");
+
+newImage("Labeling", "16-bit black", getWidth(), getHeight(), 1);
+
+for (index = 0; index < roiManager("count"); index++) {
+    roiManager("select", index);
+    setColor(index+1);
+    fill();
+}
+
+resetMinAndMax();
+run("glasbey");
+//saveAs("Tiff", savename + ".tif");
+
+
+//
+
+run("Label Size Filtering", "operation=Greater_Than size=&nucleusMinimalSizePixels");
+//run("Label Size Filtering", "operation=Greater_Than size=500");
+
+run("Label Size Filtering", "operation=Lower_Than size=&nucleusMaximalSizePixels");
+//run("Label Size Filtering", "operation=Greater_Than size=500");
+
+rename("nuclei-labels");
+//run("Set Scale...", "distance=&distance known=1 unit=&unit"); // https://github.com/stardist/stardist-imagej/issues/10
+
+// segment cells
+run("Dilate Labels", "radius=&nucleusDilationPixels");
+run("Remove Border Labels", "left right top bottom");
+rename("cells-labels");
+//run("Set Scale...", "distance=&distance known=1 unit=&unit"); // https://github.com/ijpb/MorphoLibJ/issues/55
+run("Analyze Regions", "area centroid");
+
+// segment golgi
+selectWindow("golgiChannel");
+run("Duplicate...", "golgi-binary");
+run("Gaussian Blur...", "sigma=1");
+run("Auto Threshold", "method=Otsu white");
+run("Connected Components Labeling", "connectivity=4 type=[16 bits]");
+run("glasbey_on_dark");
+rename("golgi-labels");
+
+// measure golgi shape features
+selectWindow("golgi-labels");
+run("Analyze Regions", "area perimeter circularity euler_number equivalent_ellipse ellipse_elong. convexity max._feret geodesic tortuosity max._inscribed_disc average_thickness geodesic_elong.");
+Table.rename("golgi-labels-Morphometry", "golgi-shapes");
+
+// measure golgi parent cell index
+run("Intensity Measurements 2D/3D", "input=cells-labels labels=golgi-labels max");
+Table.rename("cells-labels-intensity-measurements", "golgi-parent-labels");
+
+// merge golgi "intensity" and shape measurement tables
+run("Merge Results Tables Columns", "tablenamea=golgi-shapes tablenameb=golgi-parent-labels outputtablename=golgi");
+
+// measure mean golgi features for each cell and create new cell based table
+run("Merge Child and Parent Results Tables", "parenttablename=cells-labels-Morphometry childtablename=golgi childname=Golgi parentlabelcolumn=Max aggregationmode=Mean outputtablename=cells-with-mean-golgi-features");
+
+// inteactively inspect the output
+if ( runInteractive )
+{
+	run("Open Intensity and Label Mask Image and MorpholibJ Results Table...", "intensityimage=golgi labelimage=cells-labels resultstabletitle=cells-with-mean-golgi-features");
+}
+
+// TODO: add derived table column: total golgi area
+// TODO: what to save?
+
+//filter rows based on Golgi Counts
+Table.rename("cells-with-mean-golgi-features", "Results");
+nCells=Table.size;
+for (i = (nCells-1); i >=0; i--) {
+    nFragments=parseInt(Table.get("Golgi_Count", i));
+    if((nFragments<minGolgiCount)||(nFragments>maxGolgiCount)){
+    	
+        Table.deleteRows(i, i)
+    }
+}
+
+//add filtered rois to RoiManager
+roiManager("reset");
+selectWindow("cells-labels");
+
+nCells
+ = Table.size;
+for (i = 0; i < nCells; i++) {
+    label=parseInt(Table.getString("Label", i));
+    setThreshold(label, label);
+    run("Create Selection");
+    roiManager("Add");
+}
+resetThreshold();
+
+selectWindow("origin");
+roiManager("Show All");
+//saveAs("Tiff", resultname + ".tif");
+
+if(closeIntermediateImages){
+    closeAllImagesExceptOrigin();
+}
+function closeAllExceptOrigin()
+{
+    //run("Close All");
+    images=getList("image.titles");
+    for (i = 0; i < images.length; i++) 
+    {
+        
+        if(images[ i ]!="origin"){
+            close( images[ i ] );
+        }
+    }
+
+    roiManager("reset");
+    windows = getList("window.titles");
+    for (i = 0; i < windows.length; i++) 
+    { 
+    
+         if(windows[ i ]!="ROI Manager"){
+            close( windows[ i ] );
+         }
+    }
+}
+
+function closeAllImagesExceptOrigin()
+{
+    //run("Close All");
+    images=getList("image.titles");
+    for (i = 0; i < images.length; i++) 
+    {
+        
+        if(images[ i ]!="origin"){
+            close( images[ i ] );
+        }
+    }
+}