" + "The requirements for this plugin are twofold: that the image be 8-bit
" + "grayscale, and that the pixels constituting part of the neuron are all the
" + "same gray value, while the rest of the pixels are some other gray value.
" + "To use the plugin, first select a point on the image to be the center of
" + "analysis, then run the plugin. After setting the parameters and clicking OK,
" + "the plugin will produce a graph of # of intersections vs. distance, and the
" + "results can then be exported to a spreadsheet program.
" + "This plugin uses the scale information embedded in the image to perform its
" + "analysis. If the scale information needs to be set, this may be done by going to the
" + "\"Analyze\" menu and selecting the \"Set Scale...\" item while the image is open.
" + "Starting Radius: The smallest radius of the analysis circles, i.e., the
" + "radius of the first circle on which the # of intersections are measured.
" + "Ending Radius: The cutoff limit for how large the radii of the analysis
" + "circles can get. There is no guarantee that there will be a measurement
" + "for this radius value, unless the difference between this and the starting
" + "radius is a multiple of the step size (below).
" + "Radius Step Size: The interval between radii of analysis circles. Can be
" + "set to zero to enable continuous Sholl analysis.
" + "Radius Span: The margin around each radius in which continuous intersection
" + "measurements are made, which are then combined to become the value for the
" + "# of intersections at that radius length. Can be set to zero to disable span.
" + "Span Type: The statistical function used to combine measurements in a span.
" + "Current options are mean and median." + ""; public void run(String arg0) { // Should we display the help message? if (IJ.shiftKeyDown() || (arg0 != null && arg0.equalsIgnoreCase("HELP"))) { // Show the help message showHelp(); // Unset the shift key if it is set if (IJ.shiftKeyDown()) IJ.setKeyUp(java.awt.event.KeyEvent.VK_SHIFT); return; } // Get the current image ImagePlus image = IJ.getImage(); // Make sure the image is the right type if (image.getType() != ImagePlus.GRAY8) { IJ.showMessage("Error", "8-Bit Grayscale Image Required"); return; } // Get the image calibration Calibration cal = image.getCalibration(); // Set default dimensions info String scalestring = "Pixels"; double scale = 1.0; // Get image dimension info from the calibration if (cal != null && cal.pixelHeight == cal.pixelWidth) { scalestring = cal.getUnits(); scale = cal.pixelHeight; } // Get the ImageProcessor for the image ImageProcessor ip = image.getProcessor(); // Get the current ROI Roi roi = image.getRoi(); int x, y; if (roi != null && roi.getType() == Roi.LINE) { //Recast the ROI into its true self Line line = (Line)roi; // Get the center coordinates x = line.x1; y = line.y1; // Get the length of the line UnitEnd = line.getRawLength()*scale; } else if (roi != null && roi.getType() == Roi.POINT) { //Recast the ROI into its true self PointRoi point = (PointRoi)roi; // Get the center coordinates Rectangle rect = point.getBoundingRect(); x = rect.x; y = rect.y; } else { // Not a proper ROI type IJ.showMessage("Error", "Point or Line Selection Required"); return; } // Get the target color int color = ip.getPixel(x, y); // Set the initial values UnitStart = (UnitStart > UnitEnd) ? 0 : UnitStart; UnitStep = (UnitStep > UnitEnd - UnitStart) ? 0 : UnitStep; // Setup the help button Button helpbutton = new Button("Click For Additional Help"); helpbutton.setActionCommand("OPENHELP"); helpbutton.addActionListener(this); // Setup the help panel Panel helppanel = new Panel(); helppanel.add(helpbutton); do { // Create the plugin PluginDialog box PluginDialog = new GenericDialog("Sholl Analysis Options"); // Add the parameter fields PluginDialog.addMessage("Unit Scale: 1 Pixel = " + scale + " " + scalestring); PluginDialog.addMessage("Analysis Parameters (in " + scalestring + ")"); PluginDialog.addNumericField("Starting Radius", UnitStart, 2); PluginDialog.addNumericField("Ending Radius", UnitEnd, 2); PluginDialog.addNumericField("Radius Step Size", UnitStep, 2); PluginDialog.addNumericField("Radius Span", UnitWidth, 2); PluginDialog.addChoice("Span Type", BIN_TYPES, BIN_TYPES[BinChoice]); // Add the help button PluginDialog.addPanel(helppanel, GridBagConstraints.SOUTH, new Insets(5,0,0,0)); // Display the PluginDialog PluginDialog.showDialog(); // Stop if the user pressed cancel if (PluginDialog.wasCanceled()) { PluginDialog = null; return; } // Get the values from the PluginDialog UnitStart = Math.max(0, PluginDialog.getNextNumber()); UnitEnd = Math.max(0, PluginDialog.getNextNumber()); UnitStep = Math.max(0, PluginDialog.getNextNumber()); UnitWidth = Math.max(0, PluginDialog.getNextNumber()); BinChoice = PluginDialog.getNextChoiceIndex(); // Check the Start and End Points if (UnitStart > UnitEnd) IJ.showMessage("Error", "Invalid Start and End Parameters"); else break; // Keep presenting the dialog until the user gets it right } while (true); // Set the lower bound for these variables double unitstep = Math.max(scale, UnitStep); double unitwidth = Math.max(scale, UnitWidth); // Calculate how many samples we're taking int size = (int)((UnitEnd - UnitStart) / unitstep) + 1; // Create an x-values and pixel radii array int[] radii = new int[size]; double[] xvalues = new double[size]; // Populate the arrays for (int i = 0; i < size; i++) { xvalues[i] = UnitStart + i*unitstep; radii[i] = (int)Math.round(xvalues[i]/scale); } // Analyze the data double[] yvalues = analyze(x, y, radii, (int)Math.round(unitwidth/scale), BinChoice, color, ip); // Create a plot of the results Plot plot = new Plot("Sholl Analysis for " + image.getTitle(), "Distance (" + scalestring + ")", "# of Intersections", xvalues, yvalues); double max = 0; // Figure out the maximum y value for (int i = 0; i < yvalues.length; i++) if (yvalues[i] > max) max = yvalues[i]; // Fix the plot range and add tick marks prettyPlot(plot, xvalues[xvalues.length-1], (int)Math.round(max)); // Show the plot window plot.show(); PluginDialog = null; } /* This function processes all custom button clicks*/ public void actionPerformed(ActionEvent e) { // The button to show the help dialog was clicked if (e.getActionCommand().equals("OPENHELP")) showHelp(); // The "OK" button on the help dialog was clicked else if (e.getActionCommand().equals("CLOSEHELP")){ JDialog helpdialog = (JDialog)((JButton)e.getSource()).getClientProperty("DIALOG"); helpdialog.setVisible(false); helpdialog.dispose(); } } /* Shows the help dialog box */ public void showHelp() { JDialog helpdialog; // Create the help dialog box if (PluginDialog != null) helpdialog = new JDialog(PluginDialog, HelpTitle, false); else helpdialog = new JDialog(IJ.getInstance(), HelpTitle, false); // Set up the dialog's contents ij.util.Java2.setSystemLookAndFeel(); Container container = helpdialog.getContentPane(); container.setLayout(new BorderLayout()); JPanel panel = new JPanel(); panel.setLayout(new FlowLayout(FlowLayout.CENTER, 15, 15)); JLabel label = new JLabel(HelpMessage); panel.add(label); container.add(panel, "Center"); panel = new JPanel(); JButton button = new JButton("OK"); button.setActionCommand("CLOSEHELP"); button.addActionListener(this); button.putClientProperty("DIALOG", helpdialog); panel.add(button); container.add(panel, "South"); helpdialog.pack(); // Show the help dialog ij.gui.GUI.center(helpdialog); helpdialog.setVisible(true); } /* Does the work of the analysis. Accepts an array of radius values, and takes the appropriate measurements for each */ static public double[] analyze(int x, int y, int[] radii, int binsize, int bintype, int targetColor, ImageProcessor ip) { int i, j, k, r, rbin, sum, size; int[] binsamples, pixels; int[][] points; double[] data; // Create an array to hold the results data = new double[size = radii.length]; // Create an array for the bin samples binsamples = new int[Math.max(binsize, 0)]; // Outer loop to control the analysis bins for (i = 0; i < size; i++) { // Set the progress bar IJ.showProgress(i, size); // Get the radius we are sampling r = radii[i]; // Set the first radius for this bin rbin = r - (int)Math.round((double)binsize / 2.0); j = 0; // Cut any analysis below a 0 radius if (rbin < 0) {j = -rbin; rbin = 0;} // Inner loop to gather samples for each bin for (; j < binsize; j++, rbin++) { // Get the circle pixels for this radius points = getCircumferencePoints(x, y, rbin, LineWidth); pixels = getPixels(ip, points); // Count the number of intersections binsamples[j] = countTargetGroups(pixels, points, targetColor, ip); } // Now perform the proper function on the bin data if (bintype == BIN_MEDIAN && binsize > 0) { // Sort the bin data Arrays.sort(binsamples); // Pull out the median value data[i] = (binsize % 2 == 0) // Average the two middle values if no center exists ? ((double)(binsamples[binsize/2]+binsamples[binsize/2-1]))/2.0 // Pull out the center value : (double)binsamples[binsize/2]; } else if (binsize > 0) { /* Average bin samples by default */ // Find the sum of the samples for (sum = 0, k = 0; k < binsize; k++) sum += binsamples[k]; // Divide the sum by the number of samples data[i] = ((double)sum) / ((double)binsize); } else data[i] = 0; } // Set the progress bar IJ.showProgress(0, 0); return data; } /* Counts how many groups of targetColor are present in the given data. * A group consists of a formation of adjacent pixels, where adjacency * is true for all eight neighboring positions around a given pixel. */ static public int countTargetGroups(int[] pixels, int[][] rawpoints, int targetColor, ImageProcessor ip) { int i, j; int[][] points; // Count how many targetcolors we have for (i = 0, j = 0; i < pixels.length; i++) if (pixels[i] == targetColor) j++; // Create an array to hold the targetpixels points = new int[j][2]; // Copy all targetpixels into the array for (i = 0, j = 0; i < pixels.length; i++) if (pixels[i] == targetColor) points[j++] = rawpoints[i]; return countGroups(points, 1.5, targetColor, ip); } /* For a set of points in 2d space, counts how many groups there are * such that for every point in each group, there exists another * point in the same group that is less than threshold units of * distance away. If a point is greater than threshold units of * distance away from all other points, it is in its own group. */ static public int countGroups(int[][] points, double threshold, int tc, ImageProcessor ip) { double distance; int i, j, k, target, source, dx, dy, groups, len; // Create an array to hold the point grouping data int[] grouping = new int[len = points.length]; // Initialize each point to be in a unique group for (i = 0, groups = len; i < groups; i++) grouping[i] = i+1; for (i = 0; i < len; i++) for (j = 0; j < len; j++) { // Don't compare the same point with itself if (i == j) continue; // Compute the distance between the two points dx = points[i][0] - points[j][0]; dy = points[i][1] - points[j][1]; distance = Math.sqrt(dx*dx + dy*dy); // Should these two points be in the same group? if ( (distance <= threshold) && (grouping[i] != grouping[j]) ) { // Record which numbers we're changing source = grouping[i]; target = grouping[j]; // Chance all targets to sources for (k = 0; k < len; k++) if (grouping[k] == target) grouping[k] = source; // Update the number of groups groups--; } } /* Our next task is to throw out 1-pixel groups satisfying the condition that they * exist solely on the edge of a "stair" of target pixels. These such groups seem * to significantly contribute to the number of "spikes" in the Sholl Analysis * results, and although we cannot get all of them, this should suppress a great * deal of the spikes. */ if (DoSpikeSupression) { boolean multigroup; int[] pixels; int[][] testpoints = new int[8][2]; byte pixelmask; for (i = 0; i < len; i++) { // Check for other members of this group for (multigroup = false, j = 0; j < len; j++) { if (i == j) continue; if (grouping[i] == grouping[j]) { multigroup = true; break; } } // If not a single-pixel group, try again if (multigroup) continue; // Save the coordinates of this point dx = points[i][0]; dy = points[i][1]; // Calculate the points surrounding this point testpoints[0][0] = dx-1; testpoints[0][1] = dy+1; testpoints[1][0] = dx ; testpoints[1][1] = dy+1; testpoints[2][0] = dx+1; testpoints[2][1] = dy+1; testpoints[3][0] = dx-1; testpoints[3][1] = dy ; testpoints[4][0] = dx+1; testpoints[4][1] = dy ; testpoints[5][0] = dx-1; testpoints[5][1] = dy-1; testpoints[6][0] = dx ; testpoints[6][1] = dy-1; testpoints[7][0] = dx+1; testpoints[7][1] = dy-1; // Pull out the pixel values for these points pixels = getPixels(ip, testpoints); // Now perform the stair checks if ( (pixels[0]==tc && pixels[1]==tc && pixels[3]==tc && pixels[4]!=tc && pixels[6]!=tc && pixels[7]!=tc) || (pixels[1]==tc && pixels[2]==tc && pixels[4]==tc && pixels[3]!=tc && pixels[5]!=tc && pixels[6]!=tc) || (pixels[4]==tc && pixels[6]==tc && pixels[7]==tc && pixels[0]!=tc && pixels[1]!=tc && pixels[3]!=tc) || (pixels[3]==tc && pixels[5]==tc && pixels[6]==tc && pixels[1]!=tc && pixels[2]!=tc && pixels[4]!=tc) ) groups--; } } return groups; } /* Returns the pixel values for a given set of points */ static public int[] getPixels(ImageProcessor ip, int[][] points) { int x, y; // Create the array to hold the pixel values int[] pixels = new int[points.length]; // Put the pixel value for each circumference point in the pixel array for (int i = 0; i < pixels.length; i++) { // Pulls the coordinates out of the array x = points[i][0]; y = points[i][1]; // Check if the coordinates are valid for the image pixels[i] = (x < 0 || x >= ip.getWidth() || y < 0 || y >= ip.getHeight()) // Use -1 to indicate an invalid pixel location ? -1 // Get the pixel value in the image : ip.getPixel(points[i][0], points[i][1]); } return pixels; } /* Return the location of along a circumference of a given width */ static public int[][] getCircumferencePoints(int cx, int cy, int r, int width) { int i, j, x; // Shortcut for unit width if (width == 1) return getCircumferencePoints(cx, cy, r); // Create an array to store all the points int[][][] points = new int[width][][]; // Choose which radius to start the circumference at x = Math.max(0, r - (int)Math.round((double)width/2.0)); // Get the points for each individual circumference for (i = 0; i < width; i++, x++) points[i] = getCircumferencePoints(cx, cy, x); // Figure out how many points there are in total for (i = 0, x = 0; i < width; i++) x += points[i].length; // Create a new array to hold all the points int[][] result = new int[x][]; // Copy all the points into the combined array for (i = 0, x = 0; i < width; i++) for (j = 0; j < points[i].length; j++) result[x++] = points[i][j]; return result; } /* Return the location of pixels clockwise along the circumference of the given circle */ static public int[][] getCircumferencePoints(int cx, int cy, int r) { /* Implementation of Bresenham's Circle Algorithm */ // Initialize Algorithm Variables int i = 0, x = 0, y = r, err = 0, errR, errD; // Array to store first 1/8 of points relative to center int[][] data = new int[++r][2]; do { // Add this point as part of the circumference data[i][0] = x; data[i++][1] = y; // Calculate the errors for going right and down errR = err + 2*x + 1; errD = err - 2*y + 1; // Choose which direction to go if (Math.abs(errD) < Math.abs(errR)) { // Go down y--; err = errD; } else { // Go right x++; err = errR; } } while (x <= y); // Create an array to hold the absolute coordinates int[][] points = new int[r*8][2]; // Loop through the relative circumference points for (i = 0; i < r; i++) { // Pull out the point for quick access; x = data[i][0]; y = data[i][1]; // Convert the relative point to an absolute point points[i][0] = x + cx; points[i][1] = y + cy; // Use geometry to calculate the other 7/8 of the circumference points points[r*4-i-1][0] = x + cx; points[r*4-i-1][1] = -y + cy; points[r*8-i-1][0] = -x + cx; points[r*8-i-1][1] = y + cy; points[r*4+i] [0] = -x + cx; points[r*4+i] [1] = -y + cy; points[r*2-i-1][0] = y + cx; points[r*2-i-1][1] = x + cy; points[r*2+i] [0] = y + cx; points[r*2+i] [1] = -x + cy; points[r*6+i] [0] = -y + cx; points[r*6+i] [1] = x + cy; points[r*6-i-1][0] = -y + cx; points[r*6-i-1][1] = -x + cy; } // Create a new array to hold points without duplicates int[][] refined = new int[Math.max(points.length-8, 1)][2]; // Copy the first point manually refined[0] = points[0]; // Loop through the rest of the points for (i = 1, x = 1; i < points.length; i++) { // Duplicates are always at multiples of r if ((i+1) % r == 0) continue; // Copy the non-duplicate refined[x++] = points[i]; } // Return the array without duplicates return refined; } /* Sets the viewing range of the plot with 0 at the origin and xmax * and ymax as the maximum values for the respective axes, as well * as drawing tick marks on the y axis at no less than 1 unit intervals. */ public static void prettyPlot(Plot plot, double xmax, double ymax) { // First set the plot viewing range plot.setLimits(0, xmax, 0, ymax); // Get the processor of the plot ImageProcessor ip = plot.getProcessor(); /* Add tick marks to y axis */ int skip = 1; // Figure out what interval ticks will be drawn at while ((ymax)/skip >= 50) skip++; // Figure out the length of one unit in pixels double unitlength = 200.0 / ymax; // Set drawing variables int value, bottom = ip.getHeight() - 30; // Draw tick marks for (int i = 0; i <= ymax; i += skip) { // Compute where we will draw the mark value = bottom - (int)(unitlength * i); // Draw the tick mark ip.putPixel( 49, value, 0 ); ip.putPixel( 48, value, 0 ); } /* Add tick marks to x axis */ skip = 1; // Figure out what interval ticks will be drawn at while ((xmax)/skip >= 50) skip*=10; // Figure out the length of one unit in pixels unitlength = 450.0 / xmax; // Draw tick marks for (int i = 0; i <= xmax; i += skip) { // Compute where we will draw the mark value = 50 + (int)(unitlength * i); // Draw the tick mark ip.putPixel( value, bottom + 1, 0 ); ip.putPixel( value, bottom + 2, 0 ); } } }