EMBL Course

code/imagej-macro/CellAndSpotDetection.ijm

+open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+run("Gaussian Blur...", "sigma=15");
+run("Find Maxima...", "noise=50 output=[Segmented Particles]");
+run("Analyze Particles...", "exclude add");
+open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+rename("raw");
+run("Duplicate...", "title=median_bg");
+run("Median...", "radius=5");
+imageCalculator("Subtract", "raw","median_bg");
+run("Find Maxima...", "noise=50 output=[Single Points]");
+run("Divide...", "value=255");
+rename("spots.tif")
+selectWindow("spots.tif");
+roiManager("Measure");
+saveAs("Results", "/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/spot_count.csv");
+
+close("*");
+
+list = getList("window.titles"); 
+for (i=0; i<list.length; i++) { 
+	winame = list[i]; 
+    selectWindow(winame); 
+    run("Close"); 
+} 
\ No newline at end of file

code/imagej-macro/CellAndSpotDetection_Batch.ijm

+dirIn = getDirectory("Choose input directory!");
+imFileExtension = getString("What is the ending of your image files?",".tif");
+files = getFileList(dirIn);
+
+setBatchMode(true); 
+		
+for(i=0; i<lengthOf(files); i++) {
+
+	if(endsWith(files[i],imFileExtension)) { 
+		
+		closeEverything();
+		
+		print("Working on: "+dirIn+files[i]);
+		
+		open(dirIn+files[i]); // opens the image
+		rename("inputImage"); 
+		
+		///////////////////////////////////////////
+		// Copy and paste your macro code BELOW! //
+		/////////////////////////////////////////// 
+		
+		// find cells
+		selectWindow("inputImage"); 
+		run("Duplicate...", "title=GaussianBlur"); 	
+		run("Gaussian Blur...", "sigma=10");
+		run("Find Maxima...", "noise=50 output=[Segmented Particles]");
+		rename("Cells");
+		selectWindow("GaussianBlur");
+		setThreshold(230, 100000); // changed: set upper threshold super high
+		setOption("BlackBackground", false);
+		run("Convert to Mask");
+		rename("Foreground");
+		imageCalculator("AND create", "Foreground","Cells");
+		run("Analyze Particles...", "size=500-Infinity exclude add"); // changed: put minimum cell size higher
+		
+		// find spots
+		selectWindow("inputImage"); 
+		run("Duplicate...", "title=median_bg");
+		run("Median...", "radius=5");
+		imageCalculator("Subtract create", "inputImage", "median_bg");
+		run("Find Maxima...", "noise=50 output=[Single Points]");
+		run("Divide...", "value=255");
+		rename("spots.tif");
+		
+		// measure spots and save results
+		selectWindow("spots.tif");
+		roiManager("Measure");
+		saveAs("Results", dirIn+files[i]+"--spot_count.csv"); // changed: used dirIn to construct path
+		
+	
+		///////////////////////////////////////////
+		// Copy and paste your macro code ABOVE! //
+		///////////////////////////////////////////
+		 	
+	}
+
+}
+
+///////////////
+// functions //
+///////////////
+
+function closeEverything() {
+	
+  // closes all image windows 
+  close("*");
+
+  // closes all non-image windows
+  list = getList("window.titles"); 
+  for (i=0; i<list.length; i++) { 
+	winame = list[i]; 
+    selectWindow(winame); 
+    run("Close"); 
+  } 
+	
+}

code/imagej-macro/CellAndSpotDetection_ChooseImage.ijm

+closeEverything(); // close all windows
+
+////////////////
+// load image //
+////////////////
+
+// get, open and rename the input file
+path = File.openDialog("Select a File");
+open(path);
+rename("inputImage"); 
+
+// hide windows during run (much faster!!)
+//setBatchMode(true);
+
+////////////////
+// find cells //
+////////////////
+
+// removed: open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+selectWindow("inputImage"); // added
+run("Duplicate...", "title=GaussianBlur"); // added (to avoid that input image is 'lost' as we need it later)		
+run("Gaussian Blur...", "sigma=10");
+run("Find Maxima...", "noise=50 output=[Segmented Particles]");
+rename("Cells");
+selectWindow("GaussianBlur");
+setThreshold(230, 100000); // changed: set upper threshold super high
+setOption("BlackBackground", false);
+run("Convert to Mask");
+rename("Foreground");
+imageCalculator("AND create", "Foreground","Cells");
+run("Analyze Particles...", "size=100-Infinity exclude add");
+
+
+////////////////
+// find spots //
+////////////////
+
+// removed: open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+selectWindow("inputImage");
+run("Duplicate...", "title=median_bg");
+run("Median...", "radius=5");
+imageCalculator("Subtract create", "inputImage", "median_bg");
+run("Find Maxima...", "noise=70 output=[Single Points]");
+run("Divide...", "value=255");
+rename("spots.tif");
+
+////////////////////////////
+// measure and save spots //
+////////////////////////////
+
+selectWindow("spots.tif");
+roiManager("Measure");
+// removed: saveAs("Results", "/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/spot_count.csv");
+saveAs("Results", path+"--spot_count.csv"); // added (to have a flexible output filename)
+
+
+///////////////
+// functions //
+///////////////
+
+function closeEverything() {
+	
+  // closes all image windows 
+  close("*");
+
+  // closes all non-image windows
+  list = getList("window.titles"); 
+  for (i=0; i<list.length; i++) { 
+	winame = list[i]; 
+    selectWindow(winame); 
+    run("Close"); 
+  } 
+	
+}
\ No newline at end of file

code/imagej-macro/CellAndSpotDetection_Improved.ijm

+// close all windows
+closeEverything();
+
+// hide windows during run (much faster!!)
+//setBatchMode(true);
+
+// find cells
+open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+run("Gaussian Blur...", "sigma=10");
+rename("blurredImage");
+run("Find Maxima...", "noise=50 output=[Segmented Particles]");
+rename("cells");
+selectWindow("blurredImage");
+setThreshold(230, 714);
+setOption("BlackBackground", false);
+run("Convert to Mask");
+rename("foreground");
+imageCalculator("AND create", "foreground","cells");
+run("Analyze Particles...", "size=100-Infinity exclude add");
+
+// find spots
+open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+rename("raw");
+run("Duplicate...", "title=median_bg");
+run("Median...", "radius=5");
+imageCalculator("Subtract create", "raw","median_bg");
+run("Find Maxima...", "noise=70 output=[Single Points]");
+run("Divide...", "value=255");
+rename("spots.tif");
+
+// measure spots and save results
+selectWindow("spots.tif");
+roiManager("Measure");
+saveAs("Results", "/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/spot_count.csv");
+
+
+// functions ////////////////////////////////////////////////
+
+function closeEverything() {
+	
+  // closes all image windows 
+  close("*");
+
+  // closes all non-image windows
+  list = getList("window.titles"); 
+  for (i=0; i<list.length; i++) { 
+	winame = list[i]; 
+    selectWindow(winame); 
+    run("Close"); 
+  } 
+	
+}
\ No newline at end of file

code/imagej-macro/CellDetection.ijm

+open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+run("Gaussian Blur...", "sigma=10");
+run("Find Maxima...", "noise=100 output=[Segmented Particles]");
+run("Analyze Particles...", "size=100-Infinity exclude add");
\ No newline at end of file

code/imagej-macro/CellDetection2.ijm

+open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+run("Gaussian Blur...", "sigma=10");
+rename("blurredImage");
+run("Find Maxima...", "noise=100 output=[Segmented Particles]");
+rename("cells");
+selectWindow("blurredImage");
+setThreshold(230, 714);
+setOption("BlackBackground", false);
+run("Convert to Mask");
+rename("foreground");
+imageCalculator("AND create", "foreground","cells");
+run("Analyze Particles...", "size=100-Infinity exclude add");
+open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+rename("raw");
+run("Duplicate...", "title=median_bg");
+run("Median...", "radius=5");
+imageCalculator("Subtract create", "raw","median_bg");
+run("Find Maxima...", "noise=100 output=[Single Points]");
+run("Divide...", "value=255");
+rename("spots.tif")

code/imagej-macro/SpotDetection.ijm

+open("/Users/tischi/Dropbox/Presentations_Courses/2015--Tischi--Kopenhagen/0000--PracticalImagej/data/autophagosomes/autophagosomes_raw.tif");
+rename("raw");
+run("Duplicate...", "title=median_bg");
+run("Median...", "radius=5");
+imageCalculator("Subtract create", "raw","median_bg");
+run("Find Maxima...", "noise=100 output=[Single Points]");
+run("Divide...", "value=255");
+rename("spots.tif")

code/imagej-macro/closeEverything.ijm

+// some options:
+// selectWindow("Results"); 
+// run("Close"); 
+// selectWindow("ROI Manager"); 
+// run("Close"); 
+
+// closes all image windows (found on: http://rsb.info.nih.gov/ij/developer/macro/functions.html) 
+close("*");
+
+// closes all non-image windows (found on:http://imagej.1557.x6.nabble.com/closing-non-picture-windows-from-a-macro-td3693084.html)
+list = getList("window.titles"); 
+for (i=0; i<list.length; i++) { 
+	winame = list[i]; 
+    selectWindow(winame); 
+    run("Close"); 
+} 
+
+// make one function of it:
+function closeEverything() {
+	
+  // closes all image windows 
+  close("*");
+
+  // closes all non-image windows
+  list = getList("window.titles"); 
+  for (i=0; i<list.length; i++) { 
+	winame = list[i]; 
+    selectWindow(winame); 
+    run("Close"); 
+  } 
+	
+}

code/imagej-macro/closing-2d.ijm

+radius = 3;
+rename("input");
+run("Duplicate...", "title=closed");
+run("Maximum...", "radius="+radius);
+run("Minimum...", "radius="+radius);
+

code/imagej-macro/create-noisy-spots-uneven-background.ijm

+// create empty image
+newImage("test", "8-bit black", 400, 400, 1);
+
+// add offset
+run("Add...", "value=10");
+
+//
+// draw uneven background
+//
+
+background  = 100;
+makeOval(150, 150, 100, 100);
+run("Add...", "value=&background");
+
+// remove selection (otherwise all following actions will only be executed in the selected region)
+run("Select None");
+
+// blur backgroud
+run("Gaussian Blur...", "sigma=20");
+
+//
+// draw spots
+//
+
+diameter = 5;
+spots = 40;
+
+// draw spot
+makeOval(170, 170, diameter, diameter);
+run("Add...", "value=&spots");
+
+// draw spot
+makeOval(350, 350, diameter, diameter);
+run("Add...", "value=&spots");
+
+// remove selection (otherwise all following actions will only be executed in the selected region)
+run("Select None");
+
+// blur spots
+run("Gaussian Blur...", "sigma=2");
+
+//
+// add noise
+//
+
+// add noise
+run("Add Specified Noise...", "standard=5");

code/imagej-macro/create-test-image.ijm

+// create empty image
+newImage("test", "8-bit black", 400, 400, 1);
+
+// add offset
+run("Add...", "value=25");
+
+// draw object
+setForegroundColor(164, 164, 164);
+makeOval(72, 88, 14, 14);
+run("Fill", "slice");
+
+// draw object
+setForegroundColor(210, 210, 210);
+makeOval(168, 90, 14, 13);
+run("Fill", "slice");
+
+// remove selection (otherwise all following actions will only be executed in the selected region)
+run("Select None");
+
+// blur
+run("Gaussian Blur...", "sigma=5");
+
+// add noise
+run("Add Specified Noise...", "standard=5");

code/imagej-macro/deep-conv.ijm

+selectWindow("line-of-dots.tif smallest structure eigenvalues");
+close();
+selectWindow("line-of-dots.tif largest structure eigenvalues");
+close();
+selectWindow("line-of-dots.tif");
+run("FeatureJ Structure", "largest smallest smoothing=1 integration=1");
+selectWindow("line-of-dots.tif largest structure eigenvalues");
+close();
+run("Bin...");
+run("Duplicate...", "title=[line-of-dots.tif smallest structure eigenvalues-bin]");
+run("Bin...", "x=3 y=3 bin=Average");
+selectWindow("line-of-dots.tif smallest structure eigenvalues");
+selectWindow("line-of-dots.tif smallest structure eigenvalues-bin");
+run("Bin...", "x=3 y=3 bin=Average");
+selectWindow("line-of-dots.tif");
+selectWindow("line-of-dots.tif smallest structure eigenvalues");
+selectWindow("line-of-dots.tif smallest structure eigenvalues-bin");
+selectWindow("line-of-dots.tif smallest structure eigenvalues");
+run("Duplicate...", "title=[line-of-dots.tif smallest structure eigenvalues-bin]");
+run("Bin...", "x=3 y=3 bin=Average");
+selectWindow("line-of-dots.tif smallest structure eigenvalues-bin");
+run("FeatureJ Hessian", "largest smallest smoothing=1");
+selectWindow("line-of-dots.tif smallest structure eigenvalues-bin largest Hessian eigenvalues");
+selectWindow("line-of-dots.tif smallest structure eigenvalues-bin smallest Hessian eigenvalues");
+selectWindow("line-of-dots.tif smallest structure eigenvalues-bin largest Hessian eigenvalues");
+close();
+run("Close");

code/imagej-macro/distance-measurement-2d.ijm

+#@File reference
+#@File objects
+
+run("Close All");
+
+open(reference);
+rename("reference");
+
+open(objects);
+rename("objects");
+
+// select the binary image with the reference structure to which we want to compute the distances to 
+selectWindow("reference");
+run("Duplicate...", "title=invert");
+
+// we need to invert this, because the distance map measures distances to background pixels
+run("Invert");
+
+// run the distance map computation
+run("Chamfer Distance Map", "distances=[Chessknight (5,7,11)] output=[32 bits] normalize");
+rename("distances");
+
+// measure "intensity" ( = distance ) of objects in the distance map image 
+run("Intensity Measurements 2D/3D", "input=distances labels=objects mean");
+
+// show reference and objects as composite image
+run("Merge Channels...", "c2=reference c4=objects create ignore");
+
+
+

code/imagej-macro/dots-verticalLine--convolution.ijm

+run("Close All");
+
+// Load image
+open("/Users/tischi/Documents/imagej-courses/data/convolution/dots-verticalLine/input01.tif");
+input = getTitle();
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+run("Duplicate...", "title=dup");
+run("Convolve...", "text1=[-1 +2 -1\n-1 +2 -1\n-1 +2 -1\n]");
+rename("VerticalLineConvolution");
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+run("Duplicate...", "title=dup");
+setThreshold(4, 255);
+setOption("BlackBackground", false);
+run("Convert to Mask");
+run("Invert LUT");
+rename("Result");
+run("Maximize");

code/imagej-macro/dots-verticalLine-horizontalLine--convolution.ijm

+run("Close All");
+
+// Load image
+open("/Users/tischi/Documents/imagej-courses/data/convolution/dots-verticalLine-horizontalLine/input01.tif");
+input = getTitle();
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+selectWindow(input);
+run("Duplicate...", "title=dup");
+run("Convolve...", "text1=[-1 +2 -1\n-1 +2 -1\n-1 +2 -1\n]");
+rename("VerticalLineConvolution");
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+selectWindow(input);
+run("Duplicate...", "title=dup");
+run("Convolve...", "text1=[-1 -1 -1\n+2 +2 +2\n-1 -1 -1\n]");
+rename("HorizontalLineConvolution");
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+imageCalculator("Add create", "VerticalLineConvolution","HorizontalLineConvolution");
+rename("HorizontalLineConvolution_Add_VerticalLineConvolution");
+
+selectWindow("HorizontalLineConvolution_Add_VerticalLineConvolution");
+run("Duplicate...", "title=dup");
+setThreshold(5, 255);
+setOption("BlackBackground", false);
+run("Convert to Mask");
+run("Invert LUT");
+rename("Result");
+run("Maximize");

code/imagej-macro/edge-of-dots--deep-convolution.ijm

+run("Close All");
+
+// Load image
+open("/Users/tischi/Documents/imagej-courses/data/deep-convolution/edge-of-dots/input-01.tif");
+input = getTitle();
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+run("Duplicate...", "title=dup");
+run("Convolve...", "text1=[-1 -1 -1\n-1 +2 -1\n-1 -1 -1\n]");
+rename("SpotConvolution");
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+run("Duplicate...", "title=dup");
+run("Bin...", "x=2 y=2 bin=Max");
+rename("SpotConvolution-MaxBin");
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+selectWindow("SpotConvolution-MaxBin");
+run("Duplicate...", "title=dup");
+run("Convolve...", "text1=[-1 -1 -1\n+2 +2 +2\n-1 -1 -1\n]");
+rename("SpotConvolution-MaxBin-HorizontalLine");
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+selectWindow("SpotConvolution-MaxBin");
+run("Duplicate...", "title=dup");
+run("Convolve...", "text1=[-1 +2 -1\n-1 +2 -1\n-1 +2 -1\n]");
+rename("SpotConvolution-MaxBin-VerticalLine");
+run("Enhance Contrast", "saturated=0.35");
+run("Maximize");
+
+selectWindow("SpotConvolution-MaxBin-HorizontalLine");
+run("Duplicate...", "title=dup");
+run("Bin...", "x=5 y=5 bin=Max");
+rename("SpotConvolution-MaxBin-HorizontalLine-MaxBin");
+run("Maximize");
+
+selectWindow("SpotConvolution-MaxBin-VerticalLine");
+run("Duplicate...", "title=dup");
+run("Bin...", "x=5 y=5 bin=Max");
+rename("SpotConvolution-MaxBin-VerticalLine-MaxBin");
+run("Maximize");
+
+imageCalculator("Add create", "SpotConvolution-MaxBin-HorizontalLine-MaxBin","SpotConvolution-MaxBin-VerticalLine-MaxBin");
+run("Enhance Contrast", "saturated=0.35");
+rename("Addition");
+run("Maximize");
+
+run("Duplicate...", "title=dup");
+setThreshold(17, 255);
+setOption("BlackBackground", false);
+run("Convert to Mask");
+run("Invert LUT");
+rename("Result");
+run("Maximize");

code/imagej-macro/median-subtraction.ijm

+#@File image
+#@Integer radius
+
+// open
+run("Close All");
+open(image);
+rename("input");
+
+// median filter
+run("Duplicate...", "title=median");
+run("Median...", "radius=31");
+
+// image subtraction
+imageCalculator("Subtract create 32-bit", "input","median");

code/imagej-macro/object-intensity-splitting-workflow.ijm

+#@File image
+#@Integer threshold
+#@Double blurHalfWidth
+
+// Notes
+// - this only works for 8-bit images, because of 'max = 255 - threshold'
+
+// open image
+run("Close All");
+open(image);
+
+// smooth
+run("Duplicate...", "title=blur");
+run("Gaussian Blur...", "sigma="+blurHalfWidth);
+
+// invert
+run("Duplicate...", "title=invert");
+run("Invert");
+run("Enhance Contrast", "saturated=0.35");
+
+// watershed
+max = 255 - threshold; // this is because we inverted the 8-bit input image
+run("Classic Watershed", "input=invert mask=None use min=0 max="+max);
+run("16_colors");

code/imagej-macro/object-segmentation.ijm

+run("Close All"); // close all images
+
+// get file from user (see: https://imagej.net/Script_Parameters)
+#@File (label="Please select file") file
+
+// open file
+open(file);
+rename("input");
+
+// threshold
+run("Duplicate...", "duplicate title=binary");
+setThreshold(21, 255);
+run("Convert to Mask", "method=Default background=Dark black");
+
+// connected components labeling
+run("Connected Components Labeling", "type=[8 bits]");
+rename("labels");
+
+// TODO: remove very small objects
+// Hint: Record [ Plugins > MorphoLibJ > Label images > Label Size Opening ]
+
+// TODO: remove objects touching image boundary
+// Hint: Record [ Plugins > MorphoLibJ > Label images > Remove Border Labels ]
+
+// display labels on input
+run("Merge Channels...", "c2=labels c4=input create ignore keep");
+run("glasbey_inverted"); // change LUT
+rename("overlay");
+
+// clean up
+selectWindow("binary");
+close();

code/imagej-macro/object-shape-measurement.ijm

+// Get input from user
+#@File image (label="input image")
+#@File (style="directory", label="output directory") outputDirectory
+
+// Open image
+run("Close All");
+open(image);
+
+// Binarize (Threshold)
+setThreshold(24, 255);
+setOption("BlackBackground", true);
+run("Convert to Mask");
+
+// Connected components
+run("Connected Components Labeling", "connectivity=4 type=[16 bits]");