Java tutorial
package gdsc.smlm.ij.plugins; /*----------------------------------------------------------------------------- * GDSC Plugins for ImageJ * * Copyright (C) 2011 Alex Herbert * Genome Damage and Stability Centre * University of Sussex, UK * * This program is free software; you can redistribute it and/or modify * it under the terms of the GNU General Public License as published by * the Free Software Foundation; either version 2 of the License, or * (at your option) any later version. *---------------------------------------------------------------------------*/ import gdsc.smlm.fitting.FitConfiguration; import gdsc.smlm.fitting.FitFunction; import gdsc.smlm.fitting.FitResult; import gdsc.smlm.fitting.FitStatus; import gdsc.smlm.fitting.Gaussian2DFitter; import gdsc.smlm.function.gaussian.Gaussian2DFunction; import gdsc.smlm.ij.IJImageSource; import gdsc.smlm.ij.utils.ImageROIPainter; import gdsc.smlm.ij.utils.Utils; import gdsc.smlm.results.MemoryPeakResults; import gdsc.smlm.results.PeakResult; import gdsc.smlm.results.Trace; import gdsc.smlm.utils.Maths; import gdsc.smlm.utils.Statistics; import ij.IJ; import ij.ImageListener; import ij.ImagePlus; import ij.ImageStack; import ij.Menus; import ij.Prefs; import ij.WindowManager; import ij.gui.GUI; import ij.gui.GenericDialog; import ij.gui.Plot2; import ij.gui.PointRoi; import ij.gui.Roi; import ij.plugin.ZProjector; import ij.plugin.filter.GaussianBlur; import ij.plugin.frame.PlugInFrame; import ij.process.ImageProcessor; import ij.text.TextWindow; import java.awt.BorderLayout; import java.awt.Button; import java.awt.Choice; import java.awt.Color; import java.awt.Component; import java.awt.FlowLayout; import java.awt.Frame; import java.awt.GridBagConstraints; import java.awt.GridBagLayout; import java.awt.Insets; import java.awt.Label; import java.awt.Panel; import java.awt.Point; import java.awt.Rectangle; import java.awt.TextField; import java.awt.event.ActionEvent; import java.awt.event.ActionListener; import java.awt.event.ItemEvent; import java.awt.event.ItemListener; import java.awt.event.KeyEvent; import java.awt.event.KeyListener; import java.awt.event.WindowEvent; import java.io.BufferedWriter; import java.io.FileWriter; import java.io.IOException; import java.util.ArrayList; import java.util.Arrays; import java.util.HashMap; import java.util.LinkedList; import java.util.List; import java.util.Scanner; import java.util.TreeSet; import java.util.concurrent.ExecutorService; import java.util.concurrent.Executors; import java.util.concurrent.Future; import javax.swing.DefaultListModel; import javax.swing.JList; import javax.swing.JPanel; import javax.swing.JScrollPane; import javax.swing.event.ListSelectionEvent; import javax.swing.event.ListSelectionListener; import org.apache.commons.math3.analysis.interpolation.LoessInterpolator; import org.apache.commons.math3.analysis.polynomials.PolynomialSplineFunction; /** * Allows analysis of the signal and on/off times for fixed fluorophore spots in an image stack. */ public class SpotAnalysis extends PlugInFrame implements ActionListener, ItemListener, Runnable, ImageListener, ListSelectionListener, KeyListener { private static final long serialVersionUID = 1L; private static String TITLE = "Spot Analysis"; private class Spot implements Comparable<Spot> { int frame; double signal; public Spot(int frame, double signal) { this.frame = frame; this.signal = signal; } @Override public String toString() { //return String.format("%d : %.2f", frame, signal); return String.format("%d : %.2f", frame, getSignal(frame)); } /* * (non-Javadoc) * * @see java.lang.Comparable#compareTo(java.lang.Object) */ public int compareTo(Spot o) { if (o == null) throw new NullPointerException(); return frame - o.frame; } /* * (non-Javadoc) * * @see java.lang.Object#equals(java.lang.Object) */ @Override public boolean equals(Object obj) { if (obj == null || !(obj instanceof Spot)) return false; Spot o = (Spot) obj; return frame == o.frame; } /* * (non-Javadoc) * * @see java.lang.Object#hashCode() */ @Override public int hashCode() { return new Integer(frame).hashCode(); } } private class TraceResult { Spot spot; Trace trace; public TraceResult(Spot spot, Trace trace) { this.spot = spot; this.trace = trace; } } /** * Use a runnable for the image generation to allow multi-threaded operation. Input parameters * that are manipulated should have synchronized methods. */ private class BlurWorker implements Runnable { ImageStack inputStack, outputStack; int slice, slices; Rectangle bounds; double blur; public BlurWorker(ImageStack inputStack, int slice, int slices, Rectangle bounds, double blur, ImageStack outputStack) { this.inputStack = inputStack; this.slice = slice; this.slices = slices; this.bounds = bounds; this.blur = blur; this.outputStack = outputStack; } /* * (non-Javadoc) * * @see java.lang.Runnable#run() */ public void run() { GaussianBlur gb = new GaussianBlur(); for (int i = 0; i < slices && slice <= inputStack.getSize(); i++, slice++) { IJ.showStatus( String.format("Calculating blur ... %.1f%%", (100.0 * ++blurCount) / inputStack.getSize())); ImageProcessor ip = inputStack.getProcessor(slice).duplicate(); ip.setRoi(bounds); ip.snapshot(); gb.blurGaussian(ip, blur, blur, 0.002); outputStack.setPixels(ip.getPixels(), slice); } } } // Image titles private static String rawMeanTitle = TITLE + " Raw mean"; private static String rawSDTitle = TITLE + " Raw SD"; private static String rawSpotTitle = TITLE + " Raw spot"; private static String blurSpotTitle = TITLE + " Blur spot"; private static String avgSpotTitle = TITLE + " Average spot"; private static String[] resultsTitles = new String[] { rawMeanTitle, rawSDTitle, rawSpotTitle, blurSpotTitle, avgSpotTitle }; private static Frame instance; private static TextWindow resultsWindow = null; private Choice inputChoice; private TextField widthTextField; private TextField blurTextField; private TextField gainTextField; private TextField exposureTextField; private TextField smoothingTextField; private Button profileButton; private Button addButton; private Button deleteButton; private Button saveButton; private Button saveTracesButton; private Label currentLabel; private Label rawFittedLabel; private Label blurFittedLabel; private DefaultListModel<Spot> listModel; private JList<Spot> onFramesList; //private final int fontWidth = 12; //private final Font monoFont = new Font("Monospaced", 0, fontWidth); final String OPT_LOCATION = "CT.location"; //private ImageJ ij; private int runMode = 0; private ImagePlus imp, rawImp, blurImp; private double gain, msPerFrame; private double[] xValues; private double[] rawMean; private double[] smoothMean; private double[] rawSd; private double[] smoothSd; private int area; private int currentSlice; private Rectangle areaBounds; private TreeSet<Spot> onFrames = new TreeSet<Spot>(); private ArrayList<Integer> candidateFrames = new ArrayList<Integer>(); private HashMap<Integer, Trace> traces = new HashMap<Integer, Trace>(); private int id = 0; private boolean updated = false; private int blurCount = 0; private Object runLock = new Object(); // Stores the list of images last used in the selection options private ArrayList<String> imageList = new ArrayList<String>(); public SpotAnalysis() { super(TITLE); } public void run(String arg) { if (WindowManager.getImageCount() == 0) { IJ.showMessage(TITLE, "No images opened."); return; } if ("add".equals(arg)) { if (instance != null) ((SpotAnalysis) instance).addFrame(); return; } if (instance != null) { instance.toFront(); return; } instance = this; IJ.register(SpotAnalysis.class); WindowManager.addWindow(this); ImagePlus.addImageListener(this); //ij = IJ.getInstance(); createFrame(); setup(); //addKeyListener(ij); addKeyListener(this); pack(); Point loc = Prefs.getLocation(OPT_LOCATION); if (loc != null) setLocation(loc); else { GUI.center(this); } if (IJ.isMacOSX()) setResizable(false); setVisible(true); // Install shortcut for adding a slice String command = "Spot Analysis (Add)"; String shortcut = "6"; String plugin = "ij.plugin.Hotkeys(" + "\"" + command + "\")"; Menus.installPlugin(plugin, Menus.SHORTCUTS_MENU, "*" + command, shortcut, IJ.getInstance()); } private void setup() { ImagePlus imp = WindowManager.getCurrentImage(); if (imp == null) return; fillImagesList(); } private void fillImagesList() { // Find the currently open images ArrayList<String> newImageList = new ArrayList<String>(); int[] idList = WindowManager.getIDList(); if (idList != null) for (int id : idList) { ImagePlus imp = WindowManager.getImage(id); // Image must be greyscale stacks if (imp != null && (imp.getType() == ImagePlus.GRAY8 || imp.getType() == ImagePlus.GRAY16 || imp.getType() == ImagePlus.GRAY32) && imp.getStackSize() > 2) { // Exclude previous results if (previousResult(imp.getTitle())) continue; newImageList.add(imp.getTitle()); } } // Check if the image list has changed if (imageList.equals(newImageList)) return; imageList = newImageList; // Re-populate the image lists String oldChoice = inputChoice.getSelectedItem(); inputChoice.removeAll(); for (String imageTitle : newImageList) { inputChoice.add(imageTitle); } // Ensure the drop-downs are resized pack(); // Restore previous selection inputChoice.select(oldChoice); } private boolean previousResult(String title) { for (String resultTitle : resultsTitles) { if (title.startsWith(resultTitle)) { return true; } } return false; } /* * (non-Javadoc) * * @see java.awt.event.ActionListener#actionPerformed(java.awt.event.ActionEvent) */ public synchronized void actionPerformed(ActionEvent e) { Object actioner = e.getSource(); if (actioner == null || runMode > 0) return; synchronized (runLock) { if (runMode > 0) return; if (((Button) actioner == profileButton) && (parametersReady())) { runMode = 1; } else if ((Button) actioner == addButton) { runMode = 2; } else if ((Button) actioner == deleteButton) { runMode = 3; } else if ((Button) actioner == saveButton) { runMode = 4; } else if ((Button) actioner == saveTracesButton) { runMode = 5; } } if (runMode > 0) { Thread thread = new Thread(this, TITLE); thread.start(); } super.notify(); } /* * (non-Javadoc) * * @see java.awt.event.ItemListener#itemStateChanged(java.awt.event.ItemEvent) */ public void itemStateChanged(ItemEvent e) { } /* * (non-Javadoc) * * @see ij.plugin.frame.PlugInFrame#windowClosing(java.awt.event.WindowEvent) */ public void windowClosing(WindowEvent e) { Prefs.saveLocation(OPT_LOCATION, getLocation()); close(); } /* * (non-Javadoc) * * @see ij.plugin.frame.PlugInFrame#close() */ public void close() { instance = null; super.close(); } /* * (non-Javadoc) * * @see ij.plugin.frame.PlugInFrame#windowActivated(java.awt.event.WindowEvent) */ public void windowActivated(WindowEvent e) { fillImagesList(); super.windowActivated(e); WindowManager.setWindow(this); } /* * (non-Javadoc) * * @see java.lang.Runnable#run() */ public void run() { synchronized (runLock) { try { switch (runMode) { case 1: createProfile(); break; case 2: addFrame(); break; case 3: deleteFrames(); break; case 4: saveSpot(); break; case 5: saveTraces(); break; } } finally { runMode = 0; } } synchronized (this) { super.notify(); } } private void createProfile() { if (!parametersReady()) { return; } double psfWidth, blur; // Read settings try { psfWidth = Double.parseDouble(widthTextField.getText()); blur = Double.parseDouble(blurTextField.getText()); gain = Double.parseDouble(gainTextField.getText()); msPerFrame = Double.parseDouble(exposureTextField.getText()); } catch (NumberFormatException e) { IJ.error(TITLE, "Invalid numbers in the input parameters"); return; } ImagePlus imp = WindowManager.getImage(inputChoice.getSelectedItem()); // This should not be a problem but leave it in for now if (imp == null || (imp.getType() != ImagePlus.GRAY8 && imp.getType() != ImagePlus.GRAY16 && imp.getType() != ImagePlus.GRAY32)) { IJ.showMessage(TITLE, "Images must be grayscale."); return; } Roi roi = imp.getRoi(); if (roi == null || !roi.isArea()) { IJ.showMessage(TITLE, "Image must have an area ROI"); return; } int recommendedSize = (int) Math.ceil(8 * psfWidth); Rectangle bounds = roi.getBounds(); if (bounds.width < recommendedSize || bounds.height < recommendedSize) { IJ.showMessage(TITLE, String.format("Recommend using an ROI of at least %d x %d for the PSF width", recommendedSize, recommendedSize)); return; } // Check no existing spots are within the ROI if (resultsWithinBounds(bounds)) { GenericDialog gd = new GenericDialog(TITLE); gd.enableYesNoCancel(); gd.hideCancelButton(); gd.addMessage( "The results list contains a spot within the selected bounds\n \nDo you want to continue?"); gd.showDialog(); if (!gd.wasOKed()) return; } createProfile(imp, bounds, psfWidth, blur); } private boolean resultsWithinBounds(Rectangle bounds) { if (resultsWindowShowing()) { float minx = bounds.x; float maxx = minx + bounds.width; float miny = bounds.y; float maxy = miny + bounds.height; for (int i = 0; i < resultsWindow.getTextPanel().getLineCount(); i++) { String line = resultsWindow.getTextPanel().getLine(i); Scanner s = new Scanner(line); s.useDelimiter("\t"); s.nextInt(); float cx = s.nextFloat(); // cx float cy = s.nextFloat(); // cy s.close(); if (cx >= minx && cx <= maxx && cy >= miny && cy <= maxy) { return true; } } } return false; } private boolean resultsWindowShowing() { if (resultsWindow == null || !resultsWindow.isShowing()) return false; return true; } private boolean parametersReady() { if (inputChoice.getItemCount() == 0) { IJ.showMessage(TITLE, "No available images. Images must be 8-bit or 16-bit grayscale."); return false; } if (!onFrames.isEmpty() && updated) { GenericDialog gd = new GenericDialog(TITLE); gd.enableYesNoCancel(); gd.hideCancelButton(); gd.addMessage( "The list contains unsaved selected frames. Creating a new profile will erase them.\n \nDo you want to continue?"); gd.showDialog(); if (!gd.wasOKed()) return false; clearSelectedFrames(); } return (inputChoice.getSelectedIndex() != -1); } private void createProfile(ImagePlus imp, Rectangle bounds, double psfWidth, double blur) { areaBounds = bounds; this.imp = imp; area = bounds.width * bounds.height; clearSelectedFrames(); // Get a profile through the images IJ.showStatus("Calculating raw profile"); final int nSlices = imp.getStackSize(); ImageStack rawSpot = new ImageStack(bounds.width, bounds.height, nSlices); double[][] profile = extractSpotProfile(imp, bounds, rawSpot); // Retain the existing display range double min = 0, max = Double.POSITIVE_INFINITY; if (rawImp != null) { min = rawImp.getDisplayRangeMin(); max = rawImp.getDisplayRangeMax(); } rawImp = showSpot(rawSpotTitle, rawSpot); if (max != Double.POSITIVE_INFINITY) { rawImp.setDisplayRange(min, max); } rawMean = profile[0]; rawSd = profile[1]; // Check if there are fitted results in memory addCandidateFrames(imp.getTitle()); updateProfilePlots(); if (blur > 0) { IJ.showStatus("Calculating blur ..."); ImageStack stack = imp.getImageStack(); ImageStack newStack = new ImageStack(stack.getWidth(), stack.getHeight(), stack.getSize()); // Multi-thread the blur stage ExecutorService threadPool = Executors.newFixedThreadPool(Prefs.getThreads()); List<Future<?>> futures = new LinkedList<Future<?>>(); Utils.setShowProgress(false); blurCount = 0; // TODO - See if this is faster if processing multiple slices in each worker int slices = 5; for (int n = 1; n <= nSlices; n += slices) { futures.add(threadPool.submit(new BlurWorker(stack, n, slices, bounds, blur * psfWidth, newStack))); } IJ.showStatus("Calculating blur ... Finishing"); Utils.waitForCompletion(futures); threadPool.shutdown(); Utils.setShowProgress(false); IJ.showStatus("Calculating blur ... Drawing"); ImageStack blurSpot = new ImageStack(bounds.width, bounds.height, nSlices); extractSpotProfile(new ImagePlus("Blur", newStack), bounds, blurSpot); // Retain the existing display range max = Double.POSITIVE_INFINITY; if (blurImp != null) { min = blurImp.getDisplayRangeMin(); max = blurImp.getDisplayRangeMax(); } blurImp = showSpot(blurSpotTitle, blurSpot); if (max != Double.POSITIVE_INFINITY) { blurImp.setDisplayRange(min, max); } IJ.showStatus(""); } else { blurImp = null; } // Add a z-projection of the blur/original image ZProjector project = new ZProjector((blurImp == null) ? rawImp : blurImp); project.setMethod(ZProjector.AVG_METHOD); project.doProjection(); showSpot(avgSpotTitle, project.getProjection().getImageStack()); if (!candidateFrames.isEmpty()) // Set the first candidate frame rawImp.setSlice(candidateFrames.get(0)); else updateCurrentSlice(rawImp.getCurrentSlice()); IJ.showStatus(""); } private void clearSelectedFrames() { currentSlice = -1; onFrames.clear(); listModel.clear(); candidateFrames.clear(); updated = false; } private double[][] extractSpotProfile(ImagePlus imp, Rectangle bounds, ImageStack rawSpot) { final int nSlices = imp.getStackSize(); IJImageSource rawSource = new IJImageSource(imp); double[][] profile = new double[2][nSlices]; for (int n = 0; n < nSlices; n++) { IJ.showProgress(n, nSlices); float[] data = rawSource.next(bounds); rawSpot.setPixels(data, n + 1); Statistics stats = new Statistics(data); profile[0][n] = stats.getMean() / gain; profile[1][n] = stats.getStandardDeviation() / gain; } return profile; } private ImagePlus showSpot(String title, ImageStack spot) { ImagePlus imp = Utils.display(title, spot); if (Utils.isNewWindow() || imp.getWindow().getCanvas().getMagnification() == 1) { for (int i = 9; i-- > 0;) imp.getWindow().getCanvas().zoomIn(imp.getWidth() / 2, imp.getHeight() / 2); } return imp; } private void addCandidateFrames(String title) { for (MemoryPeakResults r : MemoryPeakResults.getAllResults()) { if (r.getSource() instanceof IJImageSource && r.getSource().getName().equals(title)) { float minx = areaBounds.x; float maxx = minx + areaBounds.width; float miny = areaBounds.y; float maxy = miny + areaBounds.height; for (PeakResult p : r.getResults()) { if (p.getXPosition() >= minx && p.getXPosition() <= maxx && p.getYPosition() >= miny && p.getYPosition() <= maxy) { candidateFrames.add(p.peak); } } } } } private void updateProfilePlots() { final int nSlices = rawMean.length; xValues = new double[nSlices]; for (int i = 0; i < nSlices; i++) xValues[i] = i + 1; smoothMean = interpolate(xValues, rawMean); smoothSd = interpolate(xValues, rawSd); drawProfiles(); } private double[] interpolate(double[] xValues2, double[] yValues) { // Smooth the values not in the current on-frames double[] newX = Arrays.copyOf(xValues, xValues.length); double[] newY = Arrays.copyOf(yValues, yValues.length); for (Spot s : onFrames) { newX[s.frame - 1] = -1; } int c = 0; for (int i = 0; i < newX.length; i++) { if (newX[i] == -1) continue; newX[c] = newX[i]; newY[c] = newY[i]; c++; } newX = Arrays.copyOf(newX, c); newY = Arrays.copyOf(newY, c); double smoothing = 0.25; try { smoothing = Double.parseDouble(smoothingTextField.getText()); if (smoothing < 0.01 || smoothing > 0.9) smoothing = 0.25; } catch (NumberFormatException e) { } LoessInterpolator loess = new LoessInterpolator(smoothing, 1); PolynomialSplineFunction f = loess.interpolate(newX, newY); // Interpolate double[] plotSmooth = new double[xValues.length]; for (int i = 0; i < xValues.length; i++) { // Cannot interpolate outside the bounds of the input data if (xValues[i] < newX[0]) plotSmooth[i] = newY[0]; else if (xValues[i] > newX[newX.length - 1]) plotSmooth[i] = newY[newX.length - 1]; else plotSmooth[i] = f.value(xValues[i]); } return plotSmooth; } private void drawProfiles() { showProfile(rawMeanTitle, xValues, rawMean, smoothMean); showProfile(rawSDTitle, xValues, rawSd, smoothSd); } private void showProfile(String title, double[] xValues, double[] yValues, double[] yValues2) { Plot2 plot = new Plot2(title, "Frame", "Signal", xValues, yValues); double[] limits = Maths.limits(yValues); plot.setLimits(xValues[0], xValues[xValues.length - 1], limits[0], limits[1]); plot.draw(); plot.setColor(Color.red); plot.addPoints(xValues, yValues2, Plot2.LINE); plot.setColor(Color.magenta); // Add the on-frames if (!onFrames.isEmpty()) { double[] onx = new double[onFrames.size()]; double[] ony = new double[onx.length]; int c = 0; for (Spot s : onFrames) { onx[c] = s.frame; ony[c] = yValues[s.frame - 1]; c++; } plot.addPoints(onx, ony, Plot2.CIRCLE); } // Add the candidate frames if (!candidateFrames.isEmpty()) { plot.setColor(Color.cyan); double[] onx = new double[candidateFrames.size()]; double[] ony = new double[onx.length]; int c = 0; for (int i : candidateFrames) { onx[c] = i; ony[c] = yValues[i - 1]; c++; } plot.addPoints(onx, ony, Plot2.BOX); plot.setColor(Color.magenta); } // Overlay current position plot.addPoints(new double[] { rawImp.getCurrentSlice(), rawImp.getCurrentSlice() }, limits, Plot2.LINE); plot.setColor(Color.blue); Utils.display(title, plot); } private void addFrame() { if (rawImp != null) { int slice = rawImp.getCurrentSlice(); double signal = getSignal(slice); Spot s = new Spot(slice, signal); if (onFrames.add(s)) { onFramesList.clearSelection(); // Find the location to insert in order int i = 0; while (i < listModel.size()) { Spot s2 = (Spot) listModel.get(i); if (s.compareTo(s2) < 0) break; i++; } listModel.add(i, s); updateProfilePlots(); updated = true; //pack(); } } } private double getSignal(int slice) { double signal = (rawMean[slice - 1] - smoothMean[slice - 1]) * area; return signal; } private void deleteFrames() { int[] indices = onFramesList.getSelectedIndices(); if (indices.length > 0) updated = true; onFramesList.clearSelection(); for (int i = indices.length; i-- > 0;) { Spot removed = (Spot) listModel.get(indices[i]); onFrames.remove(removed); } if (onFrames.isEmpty()) { listModel.clear(); } else { for (int i = indices.length; i-- > 0;) { listModel.remove(indices[i]); } } //pack(); } private void saveSpot() { if (onFrames.isEmpty() || !updated) return; createResultsWindow(); id++; double signal = 0; Trace trace = null; float psfWidth = Float.parseFloat(widthTextField.getText()); float cx = areaBounds.x + areaBounds.width / 2.0f; float cy = areaBounds.y + areaBounds.height / 2.0f; for (Spot s : onFrames) { // Get the signal again since the background may have changed. final double spotSignal = getSignal(s.frame); signal += spotSignal; //signal += s.signal; float[] params = new float[7]; params[Gaussian2DFunction.X_POSITION] = cx; params[Gaussian2DFunction.Y_POSITION] = cy; params[Gaussian2DFunction.SIGNAL] = (float) (spotSignal); params[Gaussian2DFunction.X_SD] = params[Gaussian2DFunction.Y_SD] = psfWidth; PeakResult result = new PeakResult(s.frame, (int) cx, (int) cy, 0, 0, 0, params, null); if (trace == null) trace = new Trace(result); else trace.add(result); } Statistics tOn = new Statistics(trace.getOnTimes()); Statistics tOff = new Statistics(trace.getOffTimes()); resultsWindow.append( String.format("%d\t%.1f\t%.1f\t%s\t%s\t%s\t%d\t%s\t%s\t%s", id, cx, cy, Utils.rounded(signal, 4), Utils.rounded(tOn.getSum() * msPerFrame, 3), Utils.rounded(tOff.getSum() * msPerFrame, 3), trace.getNBlinks() - 1, Utils.rounded(tOn.getMean() * msPerFrame, 3), Utils.rounded(tOff.getMean() * msPerFrame, 3), imp.getTitle())); // Save the individual on/off times for use in creating a histogram traces.put(id, trace); updated = false; //clearSelectedFrames(); } private void createResultsWindow() { if (!resultsWindowShowing()) { resultsWindow = new TextWindow(TITLE + " Results", "Id\tcx\tcy\tSignal\tt-On (ms)\tt-Off (ms)\tBlinks\tAv.t-On\tAv.t-Off\tSource", "", 600, 200); resultsWindow.setVisible(true); } } private void saveTraces() { if (!onFrames.isEmpty() && updated) { GenericDialog gd = new GenericDialog(TITLE); gd.enableYesNoCancel(); gd.hideCancelButton(); gd.addMessage("The list contains unsaved selected frames.\n \nDo you want to continue?"); gd.showDialog(); if (!gd.wasOKed()) return; } // For all spots in the results window, get the ID and then save the traces to memory if (!resultsWindowShowing()) return; // Create a results set in memory MemoryPeakResults results = new MemoryPeakResults(); results.setName(TITLE); results.begin(); MemoryPeakResults.addResults(results); ArrayList<TraceResult> traceResults = new ArrayList<TraceResult>( resultsWindow.getTextPanel().getLineCount()); for (int i = 0; i < resultsWindow.getTextPanel().getLineCount(); i++) { String line = resultsWindow.getTextPanel().getLine(i); Scanner s = new Scanner(line); s.useDelimiter("\t"); int id = s.nextInt(); s.nextDouble(); // cx s.nextDouble(); // cy double signal = s.nextDouble(); s.close(); Trace trace = traces.get(id); if (trace != null) { results.addAll(trace.getPoints()); traceResults.add(new TraceResult(new Spot(id, signal), trace)); } } results.end(); saveTracesToFile(traceResults); IJ.showStatus("Saved traces"); } private void saveTracesToFile(ArrayList<TraceResult> traceResults) { String resultsDirectory = IJ.getDirectory(TITLE); if (resultsDirectory == null) return; // Save the traces to a single file. // Also save the blinks and on/off times into data files for histogram analysis BufferedWriter[] files = new BufferedWriter[5]; try { files[0] = openBufferedWriter(resultsDirectory + "traces.txt", String.format("#ms/frame = %s\n#Id\tcx\tcy\tsignal\tn-Blinks\tStart\tStop\t...", Utils.rounded(msPerFrame, 3))); files[1] = openBufferedWriter(resultsDirectory + "tOn.txt", ""); files[2] = openBufferedWriter(resultsDirectory + "tOff.txt", ""); files[3] = openBufferedWriter(resultsDirectory + "blinks.txt", ""); files[4] = openBufferedWriter(resultsDirectory + "signal.txt", ""); for (TraceResult traceResult : traceResults) { StringBuffer sb = new StringBuffer(); sb.append(traceResult.spot.frame).append("\t"); sb.append(traceResult.trace.getHead().getXPosition()).append("\t"); sb.append(traceResult.trace.getHead().getYPosition()).append("\t"); sb.append(traceResult.spot.signal).append("\t"); int nBlinks = traceResult.trace.getNBlinks() - 1; sb.append(nBlinks); int[] on = traceResult.trace.getOnTimes(); int[] off = traceResult.trace.getOffTimes(); int t = traceResult.trace.getHead().peak; for (int i = 0; i < on.length; i++) { writeLine(files[1], Double.toString(msPerFrame * on[i])); sb.append("\t").append(t).append("\t").append(t + on[i] - 1); if (off != null && i < off.length) { writeLine(files[2], Double.toString(msPerFrame * off[i])); t += on[i] + off[i]; } } writeLine(files[0], sb.toString()); writeLine(files[3], Integer.toString(nBlinks)); writeLine(files[4], String.format("# Id=%d, Blinks=%d, Signal=%f", traceResult.spot.frame, nBlinks, traceResult.spot.signal)); for (PeakResult r : traceResult.trace.getPoints()) writeLine(files[4], String.format("%d %f", r.peak, r.getSignal())); } } catch (Exception e) { // Q. Add better handling of errors? e.printStackTrace(); IJ.log("Failed to save traces to results directory: " + resultsDirectory); } finally { for (BufferedWriter tracesFile : files) { if (tracesFile != null) { try { tracesFile.close(); } catch (IOException e) { e.printStackTrace(); } } } } } private BufferedWriter openBufferedWriter(String filename, String header) throws IOException { BufferedWriter tracesFile = new BufferedWriter(new FileWriter(filename)); if (header != null && header.length() > 0) { writeLine(tracesFile, header); } return tracesFile; } private void writeLine(BufferedWriter tracesFile, String line) throws IOException { tracesFile.write(line); tracesFile.newLine(); } private void createFrame() { Panel mainPanel = new Panel(); add(mainPanel); inputChoice = new Choice(); mainPanel.add(createChoicePanel(inputChoice, "")); widthTextField = new TextField(); mainPanel.add(createTextPanel(widthTextField, "PSF width", "1.2")); blurTextField = new TextField(); mainPanel.add(createTextPanel(blurTextField, "Blur (relative to width)", "1")); gainTextField = new TextField(); mainPanel.add(createTextPanel(gainTextField, "Gain", "37.7")); exposureTextField = new TextField(); mainPanel.add(createTextPanel(exposureTextField, "ms/Frame", "20")); smoothingTextField = new TextField(); mainPanel.add(createTextPanel(smoothingTextField, "Smoothing", "0.25")); profileButton = new Button("Profile"); profileButton.addActionListener(this); addButton = new Button("Add"); addButton.addActionListener(this); deleteButton = new Button("Remove"); deleteButton.addActionListener(this); saveButton = new Button("Save"); saveButton.addActionListener(this); saveTracesButton = new Button("Save Traces"); saveTracesButton.addActionListener(this); currentLabel = new Label(); mainPanel.add(createLabelPanel(currentLabel, "", "")); rawFittedLabel = new Label(); mainPanel.add(createLabelPanel(rawFittedLabel, "", "")); blurFittedLabel = new Label(); mainPanel.add(createLabelPanel(blurFittedLabel, "", "")); JPanel buttonPanel = new JPanel(); FlowLayout l = new FlowLayout(); l.setVgap(0); buttonPanel.setLayout(l); buttonPanel.add(profileButton, BorderLayout.CENTER); buttonPanel.add(addButton, BorderLayout.CENTER); buttonPanel.add(deleteButton, BorderLayout.CENTER); buttonPanel.add(saveButton, BorderLayout.CENTER); buttonPanel.add(saveTracesButton, BorderLayout.CENTER); mainPanel.add(buttonPanel); listModel = new DefaultListModel<Spot>(); onFramesList = new JList<Spot>(listModel); onFramesList.setVisibleRowCount(15); onFramesList.addListSelectionListener(this); //mainPanel.add(onFramesList); JScrollPane scrollPane = new JScrollPane(onFramesList); scrollPane.getVerticalScrollBarPolicy(); mainPanel.add(scrollPane); GridBagLayout mainGrid = new GridBagLayout(); int y = 0; GridBagConstraints c = new GridBagConstraints(); c.gridx = 0; c.fill = GridBagConstraints.BOTH; c.anchor = GridBagConstraints.WEST; c.gridwidth = 1; c.insets = new Insets(2, 2, 2, 2); for (Component comp : mainPanel.getComponents()) { c.gridy = y++; mainGrid.setConstraints(comp, c); } mainPanel.setLayout(mainGrid); } private Panel createChoicePanel(Choice list, String label) { Panel panel = new Panel(); panel.setLayout(new BorderLayout()); Label listLabel = new Label(label, 0); //listLabel.setFont(monoFont); //list.setSize(fontWidth * 3, fontWidth); panel.add(listLabel, BorderLayout.WEST); panel.add(list, BorderLayout.CENTER); return panel; } private Panel createTextPanel(TextField textField, String label, String value) { Panel panel = new Panel(); panel.setLayout(new BorderLayout()); Label listLabel = new Label(label, 0); //listLabel.setFont(monoFont); //textField.setSize(fontWidth * 3, fontWidth); textField.setText(value); panel.add(listLabel, BorderLayout.WEST); panel.add(textField, BorderLayout.CENTER); return panel; } private Panel createLabelPanel(Label field, String label, String value) { Panel panel = new Panel(); panel.setLayout(new BorderLayout()); Label listLabel = new Label(label, 0); //listLabel.setFont(monoFont); //textField.setSize(fontWidth * 3, fontWidth); field.setText(value); panel.add(listLabel, BorderLayout.WEST); panel.add(field, BorderLayout.CENTER); return panel; } /* * (non-Javadoc) * * @see ij.ImageListener#imageOpened(ij.ImagePlus) */ public void imageOpened(ImagePlus imp) { } /* * (non-Javadoc) * * @see ij.ImageListener#imageClosed(ij.ImagePlus) */ public void imageClosed(ImagePlus imp) { } /* * (non-Javadoc) * * @see ij.ImageListener#imageUpdated(ij.ImagePlus) */ public void imageUpdated(ImagePlus imp) { ImagePlus from = null, to = null; if (imp == rawImp) { from = rawImp; to = blurImp; } else if (imp == blurImp) { from = blurImp; to = rawImp; } if (from != null) { int slice = from.getCurrentSlice(); updateCurrentSlice(slice); if (to != null) { if (to.getCurrentSlice() != slice) { //System.out.println("updating image"); to.setSlice(slice); //to.resetDisplayRange(); } } } } private void updateCurrentSlice(int slice) { if (slice != currentSlice) { currentSlice = slice; double signal = getSignal(slice); double noise = smoothSd[slice - 1]; currentLabel.setText(String.format("Frame %d: Signal = %s, SNR = %s", slice, Utils.rounded(signal, 4), Utils.rounded(signal / noise, 3))); drawProfiles(); // Fit the PSF using a Gaussian float[] data2 = (float[]) rawImp.getImageStack().getProcessor(slice).getPixels(); double[] data = Utils.toDouble(data2); FitConfiguration fitConfiguration = new FitConfiguration(); fitConfiguration.setFitFunction(FitFunction.FIXED); fitConfiguration.setBackgroundFitting(true); fitConfiguration.setSignalStrength(0); fitConfiguration.setCoordinateShift(rawImp.getWidth() / 4.0f); fitConfiguration.setComputeResiduals(false); fitConfiguration.setComputeDeviations(false); Gaussian2DFitter gf = new Gaussian2DFitter(fitConfiguration); double[] params = new double[7]; double psfWidth = Double.parseDouble(widthTextField.getText()); params[Gaussian2DFunction.BACKGROUND] = smoothMean[slice - 1]; params[Gaussian2DFunction.SIGNAL] = (gain * signal); params[Gaussian2DFunction.X_POSITION] = rawImp.getWidth() / 2.0f; params[Gaussian2DFunction.Y_POSITION] = rawImp.getHeight() / 2.0f; params[Gaussian2DFunction.X_SD] = params[Gaussian2DFunction.Y_SD] = psfWidth; FitResult fitResult = gf.fit(data, rawImp.getWidth(), rawImp.getHeight(), 1, params, false); if (fitResult.getStatus() == FitStatus.OK) { params = fitResult.getParameters(); final double spotSignal = params[Gaussian2DFunction.SIGNAL] / gain; rawFittedLabel.setText(String.format("Raw fit: Signal = %s, SNR = %s", Utils.rounded(spotSignal, 4), Utils.rounded(spotSignal / noise, 3))); ImageROIPainter.addRoi(rawImp, slice, new PointRoi(params[Gaussian2DFunction.X_POSITION], params[Gaussian2DFunction.Y_POSITION])); } else { rawFittedLabel.setText(""); rawImp.setOverlay(null); } // Fit the PSF using a Gaussian if (blurImp == null) return; data2 = (float[]) blurImp.getImageStack().getProcessor(slice).getPixels(); data = Utils.toDouble(data2); params = new double[7]; //float psfWidth = Float.parseFloat(widthTextField.getText()); params[Gaussian2DFunction.BACKGROUND] = (float) smoothMean[slice - 1]; params[Gaussian2DFunction.SIGNAL] = (float) (gain * signal); params[Gaussian2DFunction.X_POSITION] = rawImp.getWidth() / 2.0f; params[Gaussian2DFunction.Y_POSITION] = rawImp.getHeight() / 2.0f; params[Gaussian2DFunction.X_SD] = params[Gaussian2DFunction.Y_SD] = psfWidth; fitResult = gf.fit(data, rawImp.getWidth(), rawImp.getHeight(), 1, params, false); if (fitResult.getStatus() == FitStatus.OK) { params = fitResult.getParameters(); final double spotSignal = params[Gaussian2DFunction.SIGNAL] / gain; blurFittedLabel.setText(String.format("Blur fit: Signal = %s, SNR = %s", Utils.rounded(spotSignal, 4), Utils.rounded(spotSignal / noise, 3))); ImageROIPainter.addRoi(blurImp, slice, new PointRoi(params[Gaussian2DFunction.X_POSITION], params[Gaussian2DFunction.Y_POSITION])); } else { blurFittedLabel.setText(""); blurImp.setOverlay(null); } } } public void valueChanged(ListSelectionEvent e) { if (!e.getValueIsAdjusting()) { int index = onFramesList.getSelectedIndex(); //int index = e.getFirstIndex(); if (index >= 0 && index < listModel.size()) { //Utils.log("index = %d, %b", index, e.getValueIsAdjusting()); Spot spot = (Spot) listModel.get(index); rawImp.setSlice(spot.frame); } } } public void keyTyped(KeyEvent e) { System.out.println("keyTyped"); switch (e.getKeyChar()) { case 'a': addFrame(); break; case ',': rawImp.setSlice(rawImp.getSlice() + 1); break; case '.': rawImp.setSlice(rawImp.getSlice() - 1); break; } } public void keyPressed(KeyEvent e) { System.out.println("keyPressed"); } public void keyReleased(KeyEvent e) { System.out.println("keyReleased"); } }