PROJECT#3(b) Astrocyte Analysis BY Bhimanathini Venkatsai Sai Kumar Maddula

1. Astrocyte Analysis 2. Algorithm for Converting into 8-bit 3. Algorithm for Thresholding 4. Sample code for Thresholding 5. Overlapping/Touching of cell structure ◦Watershed Algorithm ◦Analyze Particles And Future workContents

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

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

Algorithm for Threshold: 1. start set THRESHOLD = 30 red = 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

Code for Threshold import java.awt.image.BufferedImage; public class ImageOperations { public static BufferedImage Threshold(BufferedImage img,int requiredThresholdValue) { int height = img.getHeight(); int width = img.getWidth(); BufferedImage finalThresholdImage = 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 int mixColor(int red, int green, int blue) { return red<<16|green<<8|blue; } public static int getRed(int color) { return (color & 0x00ff0000) >> 16; } public static int getGreen(int color) { return(color & 0x0000ff00) >> 8; } public static int getBlue(int color) { return (color & 0x000000ff) >> 0; }

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

Watershed algorithm: # 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()

Analyze particles: # 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)

Expected Results:

Expected Results

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

Thank you..!!