PROJECT#3(b) Astrocyte Analysis

1. PROJECT#3(b)Astrocyte Analysis BY BhimanathiniVenkatsai Sai Kumar Maddula

2. Contents • Astrocyte Analysis • Algorithm for Converting into 8-bit • Algorithm for Thresholding • Sample code for Thresholding • Overlapping/Touching of cell structure • Watershed Algorithm • Analyze Particles And Future work

3. Astrocyte Analysis: • Load the stack File into ImageJ File  Open Browse Stack File

4. Converting to 8bit • java.util import Random • imp = IJ.createImage("A Random Image", "8-bit", 512, 512, 1) • Random().nextBytes(imp.getProcessor().getPixels()) • imp.show() Converting to 8bit

6. 1. start set THRESHOLD = 30red = getRedPixelFromImage(row,column) green = getGreenPixelFromImage(row,column) blue = getBluePixelFromImage(row,column) • 2. avg = (red+green+blue)/3 • 3. if(avg<THRESHOLD) paint white else paint black. • 4. end Algorithm for Threshold:

7. import java.awt.image.BufferedImage; public class ImageOperations { public static BufferedImage Threshold(BufferedImageimg,intrequiredThresholdValue) { int height = img.getHeight(); int width = img.getWidth(); BufferedImagefinalThresholdImage = new BufferedImage(width,height,BufferedImage.TYPE_INT_RGB) ; int red = 0; int green = 0; int blue = 0; for (int x = 0; x < width; x++) { // System.out.println("Row: " + x); try { for (int y = 0; y < height; y++) { int color = img.getRGB(x, y); red = ImageOperations.getRed(color); green = ImageOperations.getGreen(color); blue = ImageOperations.getBlue(color); // System.out.println("Threshold : " + requiredThresholdValue); if((red+green+green)/3 < (int) (requiredThresholdValue)) { finalThresholdImage.setRGB(x,y,ImageOperations.mixColor(0, 0,0)); } else { finalThresholdImage.setRGB(x,y,ImageOperations.mixColor(255, 255,255)); } } } catch (Exception e) { e.getMessage(); } } return finalThresholdImage; } private static intmixColor(int red, int green, int blue) { return red<<16|green<<8|blue; } public static intgetRed(int color) { return (color & 0x00ff0000) >> 16; } public static intgetGreen(int color) { return (color & 0x0000ff00) >> 8; } public static intgetBlue(int color) { return (color & 0x000000ff) >> 0; } } Code for Threshold

9. Overlapping/Touching Cell Structures • Here we have to apply Watershed and Analyze Particles. • So we done with some code for Watershed and to Analyze Particles.

10. # 1 - Obtain an image blobs = IJ.openImage(“aaa.tip") # Make a copy with the same properties as blobs image: imp = blobs.createImagePlus() ip = blobs.getProcessor().duplicate() imp.setProcessor("blobs copy", ip) # 2 - Apply a threshold: only zeros and ones # Set the desired threshold range: keep from 0 to 74 ip.setThreshold(147, 147, ImageProcessor.NO_LUT_UPDATE) # Call the Thresholder to convert the image to a mask IJ.run(imp, "Convert to Mask", "") # 3 - Apply watershed # Create and run new EDM object, which is an Euclidean Distance Map (EDM) # and run the watershed on the ImageProcessor: EDM().toWatershed(ip) # 4 - Show the watersheded image: imp.show() Watershed algorithm:

11. # Create a table to store the results table = ResultsTable() # Create a hidden ROI manager, to store a ROI for each blob or cell roim = RoiManager(True) # Create a ParticleAnalyzer, with arguments: # 1. options (could be SHOW_ROI_MASKS, SHOW_OUTLINES, SHOW_MASKS, SHOW_NONE, ADD_TO_MANAGER, and others; combined with bitwise-or) # 2. measurement options # 3. a ResultsTable to store the measurements # 4. The minimum size of a particle to consider for measurement # 5. The maximum size (idem) # 6. The minimum circularity of a particle # 7. The maximum circularity pa = ParticleAnalyzer(ParticleAnalyzer.ADD_TO_MANAGER, Measurements.AREA, table, 0, Double.POSITIVE_INFINITY, 0.0, 1.0) pa.setHideOutputImage(True) if pa.analyze(imp):   print "All ok" else:  print "There was a problem in analyzing", blobs # The measured areas are listed in the first column of the results table, as a float array: areas = table.getColumn(0) Analyze particles:

12. Expected Results:

13. Expected Results

14. Segmentation by 3D Viewer • Combine individual Plugin into one Plugin. Still working on..

15. Thank you..!!