AccPbFRET_Plugin.java | Department of Biophysics and Cell Biology

 /* AccPbFRET - an ImageJ plugin for analysis of acceptor photobleaching FRET images. Written by Janos Roszik (janosr@med.unideb.hu), Janos Szollosi (szollo@med.unideb.hu) and Gyorgy Vereb (vereb@med.unideb.hu) The program is provided free of charge on an "as is" basis without warranty of any kind. http://www.biophys.dote.hu/accpbfret/ */ import ij.*; import ij.gui.*; import ij.io.*; import ij.measure.*; import ij.plugin.*; import ij.plugin.filter.*; import ij.plugin.frame.*; import ij.process.*; import java.awt.*; import java.awt.event.ActionEvent; import java.awt.event.ActionListener; import java.awt.event.WindowEvent; import java.awt.event.WindowListener; import java.awt.image.*; import java.io.*; import java.net.*; import java.text.*; import java.util.*; import javax.swing.*; import javax.swing.text.*; public class AccPbFRET_Plugin extends JFrame implements ActionListener, WindowListener { private final float version = 3.15F; private final String lastModified = "26 Mar 2009"; private final String imageJVersion = "1.42k"; private final String javaVersion = "1.6.0_05"; private int windowWidth = 650; private int windowHeight = 890; private ImagePlus donorBefore, donorAfter, acceptorBefore, acceptorAfter, transferImage = null; private ImageProcessor donorBeforeSave = null, donorAfterSave = null, acceptorBeforeSave = null, acceptorAfterSave = null; private ResultsTable resultsTable; private Analyzer analyzer; private ApplyMaskDialog applyMaskDialog; private CalculateImgRatioDialog calculateImgRatioDialog; private ShiftDialog shiftDialog; private DonorBlCorrDialog donorBlCorrDialog; private AcceptorCTCorrDialog acceptorCTCorrDialog; private AcceptorPPCorrDialog acceptorPPCorrDialog; private JMenuBar menuBar; private HelpWindow helpWindow; private JMenu fileMenu, imageMenu, correctionMenu, helpMenu; private JMenuItem openMenuItem, saveTiffMenuItem, saveBmpMenuItem, splitMenuItem, applyMaskMenuItem, bleachingMaskMenuItem, calculateImgRatioMenuItem, thresholdMenuItem, shiftMenuItem; private JMenuItem lutFireMenuItem, lutSpectrumMenuItem, histogramMenuItem, convertMenuItem, exitMenuItem, helpMenuItem, aboutMenuItem, checkVersionMenuItem; private JMenuItem saveMessagesMenuItem, clearMessagesMenuItem; private JMenuItem semiAutomaticMenuItem, resetImagesMenuItem; private JCheckBoxMenuItem donorBlCorrMenuItem, accCrossTalkCorrMenuItem, accPhotoprCorrMenuItem, partialBlCorrMenuItem; private JCheckBoxMenuItem debugMenuItem; private JButton setDonorBeforeButton, setDonorAfterButton, setAcceptorBeforeButton, setAcceptorAfterButton; private JButton subtractDonorBeforeButton, subtractDonorAfterButton, subtractAcceptorBeforeButton, subtractAcceptorAfterButton; private JButton thresholdDonorBeforeButton, thresholdDonorAfterButton, thresholdAcceptorBeforeButton, thresholdAcceptorAfterButton; private JButton smoothDonorBeforeButton, smoothDonorAfterButton, smoothAcceptorBeforeButton, smoothAcceptorAfterButton; private JButton openImageButton, clearABButton, clearAAButton, resetDBButton, resetDAButton, resetABButton, resetAAButton; private JButton copyRoiButton; private JTextField radiusFieldDB, radiusFieldDA, radiusFieldAB, radiusFieldAA; private JTextField donorBlCorrField, accCrossTalkCorrField, accPhotoprCorrField, partialBlCorrField; private JButton registerButton, createButton, measureButton, nextButton, closeImagesButton; public JButton calculateDBCorrButton, calculateAccCTCorrButton, calculateAccPPCorrButton, calculatePartialBlCorrButton; private JPanel lineDonorBlCorr, lineAccCrossTalk, lineAccPhotopr, linePartialBl; private JLabel donorBlCorrLabel, accCrossTalkCorrLabel, accPhotoprCorrLabel, partialBlCorrLabel; private JCheckBox useLsmImages, useAcceptorAsMask, applyShiftCB; private JTextPane log; private JScrollPane logScrollPane; private SimpleDateFormat format; private File[] automaticallyProcessedFiles = null; private int currentlyProcessedFile = 0; private String currentlyProcessedFileName = null; private String currentDirectory = null; public Color originalButtonColor = null; public Color greenColor = new Color(142, 207, 125); public AccPbFRET_Plugin () { super(); setTitle("AccPbFRET v"+version); IJ.versionLessThan(imageJVersion); Locale.setDefault(Locale.ENGLISH); ToolTipManager.sharedInstance().setDismissDelay(10000); format = new SimpleDateFormat("HH:mm:ss"); createGui(); setDefaultCloseOperation(JFrame.DO_NOTHING_ON_CLOSE); Dimension screen = Toolkit.getDefaultToolkit().getScreenSize(); setSize(windowWidth, windowHeight); setLocation(screen.width - getWidth(), screen.height/2 - getHeight()/2); setVisible(true); currentDirectory = System.getProperty("user.home"); originalButtonColor = setDonorBeforeButton.getBackground(); openImageButton.requestFocus(); } public void createGui() { setFont(new Font("Helvetica", Font.PLAIN, 12)); menuBar = new JMenuBar(); fileMenu = new JMenu("File"); menuBar.add(fileMenu); imageMenu = new JMenu("Image"); menuBar.add(imageMenu); correctionMenu = new JMenu("Corrections"); menuBar.add(correctionMenu); helpMenu = new JMenu("Help"); menuBar.add(helpMenu); openMenuItem = new JMenuItem("Open image"); openMenuItem.setActionCommand("openImage"); openMenuItem.addActionListener(this); fileMenu.add(openMenuItem); saveTiffMenuItem = new JMenuItem("Save image as tiff"); saveTiffMenuItem.setActionCommand("saveImageAsTiff"); saveTiffMenuItem.addActionListener(this); fileMenu.add(saveTiffMenuItem); saveBmpMenuItem = new JMenuItem("Save image as bmp"); saveBmpMenuItem.setActionCommand("saveImageAsBmp"); saveBmpMenuItem.addActionListener(this); fileMenu.add(saveBmpMenuItem); saveMessagesMenuItem = new JMenuItem("Save messages"); saveMessagesMenuItem.setActionCommand("saveMessages"); saveMessagesMenuItem.addActionListener(this); fileMenu.add(saveMessagesMenuItem); clearMessagesMenuItem = new JMenuItem("Clear messages"); clearMessagesMenuItem.setActionCommand("clearMessages"); clearMessagesMenuItem.addActionListener(this); fileMenu.add(clearMessagesMenuItem); semiAutomaticMenuItem = new JMenuItem("Semi-automatic processing"); semiAutomaticMenuItem.setActionCommand("semiAutomaticProcessing"); semiAutomaticMenuItem.addActionListener(this); fileMenu.add(semiAutomaticMenuItem); resetImagesMenuItem = new JMenuItem("Reset all"); resetImagesMenuItem.setActionCommand("resetImages"); resetImagesMenuItem.addActionListener(this); fileMenu.add(resetImagesMenuItem); splitMenuItem = new JMenuItem("Split image"); splitMenuItem.setActionCommand("split"); splitMenuItem.addActionListener(this); imageMenu.add(splitMenuItem); applyMaskMenuItem = new JMenuItem("Apply mask"); applyMaskMenuItem.setActionCommand("applyMask"); applyMaskMenuItem.addActionListener(this); imageMenu.add(applyMaskMenuItem); bleachingMaskMenuItem = new JMenuItem("Create bleached-area mask"); bleachingMaskMenuItem.setActionCommand("bleachingMask"); bleachingMaskMenuItem.addActionListener(this); imageMenu.add(bleachingMaskMenuItem); calculateImgRatioMenuItem = new JMenuItem("Calculate ratio of images"); calculateImgRatioMenuItem.setActionCommand("calculateRatio"); calculateImgRatioMenuItem.addActionListener(this); imageMenu.add(calculateImgRatioMenuItem); convertMenuItem = new JMenuItem("Convert image to 32 bit"); convertMenuItem.setActionCommand("convertto32bit"); convertMenuItem.addActionListener(this); imageMenu.add(convertMenuItem); thresholdMenuItem = new JMenuItem("Set threshold"); thresholdMenuItem.setActionCommand("threshold"); thresholdMenuItem.addActionListener(this); imageMenu.add(thresholdMenuItem); shiftMenuItem = new JMenuItem("Shift image"); shiftMenuItem.setActionCommand("shiftimage"); shiftMenuItem.addActionListener(this); imageMenu.add(shiftMenuItem); histogramMenuItem = new JMenuItem("Histogram"); histogramMenuItem.setActionCommand("histogram"); histogramMenuItem.addActionListener(this); imageMenu.add(histogramMenuItem); lutFireMenuItem = new JMenuItem("LUT Fire"); lutFireMenuItem.setActionCommand("lutFire"); lutFireMenuItem.addActionListener(this); imageMenu.add(lutFireMenuItem); lutSpectrumMenuItem = new JMenuItem("LUT Spectrum"); lutSpectrumMenuItem.setActionCommand("lutSpectrum"); lutSpectrumMenuItem.addActionListener(this); imageMenu.add(lutSpectrumMenuItem); donorBlCorrMenuItem = new JCheckBoxMenuItem("Donor bleaching"); donorBlCorrMenuItem.setSelected(true); donorBlCorrMenuItem.setActionCommand("donorblcorrm"); donorBlCorrMenuItem.addActionListener(this); correctionMenu.add(donorBlCorrMenuItem); accCrossTalkCorrMenuItem = new JCheckBoxMenuItem("Acceptor cross-talk"); accCrossTalkCorrMenuItem.setSelected(false); accCrossTalkCorrMenuItem.setActionCommand("acccrtalkcorrm"); accCrossTalkCorrMenuItem.addActionListener(this); correctionMenu.add(accCrossTalkCorrMenuItem); accPhotoprCorrMenuItem = new JCheckBoxMenuItem("Acceptor photoproduct"); accPhotoprCorrMenuItem.setSelected(false); accPhotoprCorrMenuItem.setActionCommand("accphprcorrm"); accPhotoprCorrMenuItem.addActionListener(this); correctionMenu.add(accPhotoprCorrMenuItem); partialBlCorrMenuItem = new JCheckBoxMenuItem("Partial acceptor photobleaching"); partialBlCorrMenuItem.setSelected(false); partialBlCorrMenuItem.setActionCommand("partialblcorrm"); partialBlCorrMenuItem.addActionListener(this); correctionMenu.add(partialBlCorrMenuItem); exitMenuItem = new JMenuItem("Exit"); exitMenuItem.setActionCommand("exit"); exitMenuItem.addActionListener(this); fileMenu.addSeparator(); fileMenu.add(exitMenuItem); helpMenuItem = new JMenuItem("Help"); helpMenuItem.setActionCommand("help"); helpMenuItem.addActionListener(this); helpMenu.add(helpMenuItem); checkVersionMenuItem = new JMenuItem("Check for latest version"); checkVersionMenuItem.setActionCommand("checkVersion"); checkVersionMenuItem.addActionListener(this); helpMenu.add(checkVersionMenuItem); debugMenuItem = new JCheckBoxMenuItem("Debug mode"); debugMenuItem.setSelected(false); debugMenuItem.setActionCommand("debugmode"); debugMenuItem.addActionListener(this); helpMenu.add(debugMenuItem); aboutMenuItem = new JMenuItem("About"); aboutMenuItem.setActionCommand("about"); aboutMenuItem.addActionListener(this); helpMenu.add(aboutMenuItem); setJMenuBar(menuBar); addWindowListener(this); GridBagLayout gridbaglayout = new GridBagLayout(); GridBagConstraints gc = new GridBagConstraints(); Container container = getContentPane(); container.setLayout(gridbaglayout); JPanel donorBeforeBleachingPanel = new JPanel(new FlowLayout(FlowLayout.LEADING, 0, 0)); donorBeforeBleachingPanel.setBorder(BorderFactory.createEmptyBorder(0,0,0,0)); gc.anchor = GridBagConstraints.WEST; gc.gridwidth = 9; gc.gridx = 0; gc.gridy = 0; gc.insets = new Insets(2,2,2,2); gc.fill = GridBagConstraints.NONE; useLsmImages = new JCheckBox("use LSM", true); useLsmImages.setSelected(false); useLsmImages.setActionCommand("useLsmImages"); useLsmImages.addActionListener(this); useLsmImages.setToolTipText("<html>If this checkbox is checked, the LSM image containing donor and<BR>acceptor channel images (both before and after photobleaching)<BR> are set automatically after opening. Every previously opened image<br>window will be closed. The results window can be left opened.</html>"); useLsmImages.setSelected(false); donorBeforeBleachingPanel.add(new JLabel("Step 1a: open and set the donor before bleaching image ")); donorBeforeBleachingPanel.add(useLsmImages); setDonorBeforeButton = new JButton("Set image"); setDonorBeforeButton.setMargin(new Insets(2,2,2,2)); setDonorBeforeButton.addActionListener(this); setDonorBeforeButton.setActionCommand("setDonorBefore"); container.add(donorBeforeBleachingPanel, gc); gc.fill = GridBagConstraints.HORIZONTAL; gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 0; gc.insets = new Insets(0,0,0,2); openImageButton = new JButton("Open"); openImageButton.setToolTipText("Opens an arbitrary image."); openImageButton.setMargin(new Insets(0,0,0,0)); openImageButton.setFont(new Font("Helvetica", Font.BOLD, 10)); openImageButton.addActionListener(this); openImageButton.setActionCommand("openImage"); container.add(openImageButton, gc); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 0; gc.insets = new Insets(2,2,2,2); container.add(setDonorBeforeButton, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 1; container.add(new JLabel("Step 1b: open and set the donor after bleaching image"), gc); setDonorAfterButton = new JButton("Set image"); setDonorAfterButton.setMargin(new Insets(2,2,2,2)); setDonorAfterButton.addActionListener(this); setDonorAfterButton.setActionCommand("setDonorAfter"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 1; gc.insets = new Insets(2,2,2,2); container.add(setDonorAfterButton, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 2; container.add(new JLabel("Step 1c (optional): open and set acceptor before bleaching image"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 2; gc.insets = new Insets(0,0,0,2); clearABButton = new JButton("Clear"); clearABButton.setToolTipText("Clears the acceptor before bleaching image."); clearABButton.setMargin(new Insets(0,0,0,0)); clearABButton.setFont(new Font("Helvetica", Font.BOLD, 10)); clearABButton.addActionListener(this); clearABButton.setActionCommand("clearAB"); container.add(clearABButton, gc); setAcceptorBeforeButton = new JButton("Set image"); setAcceptorBeforeButton.setMargin(new Insets(2,2,2,2)); setAcceptorBeforeButton.addActionListener(this); setAcceptorBeforeButton.setActionCommand("setAcceptorBefore"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 2; gc.insets = new Insets(2,2,2,2); container.add(setAcceptorBeforeButton, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 3; container.add(new JLabel("Step 1d (optional): open and set acceptor after (partial) bleaching image"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 3; gc.insets = new Insets(0,0,0,2); clearAAButton = new JButton("Clear"); clearAAButton.setToolTipText("Clears the acceptor after bleaching image."); clearAAButton.setMargin(new Insets(0,0,0,0)); clearAAButton.setFont(new Font("Helvetica", Font.BOLD, 10)); clearAAButton.addActionListener(this); clearAAButton.setActionCommand("clearAA"); container.add(clearAAButton, gc); setAcceptorAfterButton = new JButton("Set image"); setAcceptorAfterButton.setMargin(new Insets(2,2,2,2)); setAcceptorAfterButton.addActionListener(this); setAcceptorAfterButton.setActionCommand("setAcceptorAfter"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 3; gc.insets = new Insets(2,2,2,2); container.add(setAcceptorAfterButton, gc); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 4; JPanel line1 = new JPanel(); line1.setPreferredSize(new Dimension(windowWidth-35, 1)); line1.setBackground(Color.lightGray); container.add(line1, gc); JPanel regPanel = new JPanel(); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 5; gc.insets = new Insets(0,0,0,0); gc.fill = GridBagConstraints.NONE; regPanel.add(new JLabel("Step 2: register donor images ")); applyShiftCB = new JCheckBox("apply shift to acceptor image", true); applyShiftCB.setSelected(true); regPanel.add(applyShiftCB); container.add(regPanel, gc); registerButton = new JButton("Register"); registerButton.addActionListener(this); registerButton.setActionCommand("registerImages"); gc.insets = new Insets(4,4,4,4); gc.fill = GridBagConstraints.HORIZONTAL; gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 5; container.add(registerButton, gc); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 6; JPanel line2 = new JPanel(); line2.setPreferredSize(new Dimension(windowWidth-35, 1)); line2.setBackground(Color.lightGray); container.add(line2, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 7; container.add(new JLabel("Step 3a: subtract average of a background ROI of donor before image"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 7; gc.insets = new Insets(0,0,0,2); copyRoiButton = new JButton("Copy"); copyRoiButton.setToolTipText("Sets the same ROI for the donor after and acceptor images."); copyRoiButton.setMargin(new Insets(0,0,0,0)); copyRoiButton.setFont(new Font("Helvetica", Font.BOLD, 10)); copyRoiButton.addActionListener(this); copyRoiButton.setActionCommand("copyRoi"); container.add(copyRoiButton, gc); subtractDonorBeforeButton = new JButton("Subtract"); subtractDonorBeforeButton.addActionListener(this); subtractDonorBeforeButton.setActionCommand("subtractDonorBefore"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.insets = new Insets(2,2,2,2); gc.gridx = 10; gc.gridy = 7; container.add(subtractDonorBeforeButton, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 8; container.add(new JLabel("Step 3b: subtract average of a background ROI of donor after image"), gc); gc.gridwidth = GridBagConstraints.REMAINDER; subtractDonorAfterButton = new JButton("Subtract"); subtractDonorAfterButton.addActionListener(this); subtractDonorAfterButton.setActionCommand("subtractDonorAfter"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 8; gc.insets = new Insets(2,2,2,2); container.add(subtractDonorAfterButton, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 9; container.add(new JLabel("Step 3c (optional): subtract average of a background ROI of acceptor before image"), gc); subtractAcceptorBeforeButton = new JButton("Subtract"); subtractAcceptorBeforeButton.addActionListener(this); subtractAcceptorBeforeButton.setActionCommand("subtractAcceptorBefore"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 9; container.add(subtractAcceptorBeforeButton, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 10; container.add(new JLabel("Step 3d (optional): subtract average of a background ROI of acceptor after image"), gc); subtractAcceptorAfterButton = new JButton("Subtract"); subtractAcceptorAfterButton.addActionListener(this); subtractAcceptorAfterButton.setActionCommand("subtractAcceptorAfter"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 10; container.add(subtractAcceptorAfterButton, gc); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 11; JPanel line3 = new JPanel(); line3.setPreferredSize(new Dimension(windowWidth-35, 1)); line3.setBackground(Color.lightGray); container.add(line3, gc); gc.gridwidth = 9; gc.gridx = 0; gc.gridy = 12; container.add(new JLabel("Step 4a (optional): blur donor before image (Gaussian), radius in pixels:"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 12; radiusFieldDB = new JTextField("2", 4); radiusFieldDB.setHorizontalAlignment(JTextField.RIGHT); container.add(radiusFieldDB, gc); smoothDonorBeforeButton = new JButton("Blur"); smoothDonorBeforeButton.addActionListener(this); smoothDonorBeforeButton.setActionCommand("smoothDBefore"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 12; container.add(smoothDonorBeforeButton, gc); gc.gridwidth = 9; gc.gridx = 0; gc.gridy = 13; container.add(new JLabel("Step 4b (optional): blur donor after image (Gaussian), radius in pixels:"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 13; radiusFieldDA = new JTextField("2", 4); radiusFieldDA.setHorizontalAlignment(JTextField.RIGHT); container.add(radiusFieldDA, gc); smoothDonorAfterButton = new JButton("Blur"); smoothDonorAfterButton.addActionListener(this); smoothDonorAfterButton.setActionCommand("smoothDAfter"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 13; container.add(smoothDonorAfterButton, gc); gc.gridwidth = 9; gc.gridx = 0; gc.gridy = 14; container.add(new JLabel("Step 4c (optional): blur acceptor before image (Gaussian), radius in pixels:"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 14; radiusFieldAB = new JTextField("2", 4); radiusFieldAB.setHorizontalAlignment(JTextField.RIGHT); container.add(radiusFieldAB, gc); smoothAcceptorBeforeButton = new JButton("Blur"); smoothAcceptorBeforeButton.addActionListener(this); smoothAcceptorBeforeButton.setActionCommand("smoothABefore"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 14; container.add(smoothAcceptorBeforeButton, gc); gc.gridwidth = 9; gc.gridx = 0; gc.gridy = 15; container.add(new JLabel("Step 4d (optional): blur acceptor after image (Gaussian), radius in pixels:"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 15; radiusFieldAA = new JTextField("2", 4); radiusFieldAA.setHorizontalAlignment(JTextField.RIGHT); container.add(radiusFieldAA, gc); smoothAcceptorAfterButton = new JButton("Blur"); smoothAcceptorAfterButton.addActionListener(this); smoothAcceptorAfterButton.setActionCommand("smoothAAfter"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 15; container.add(smoothAcceptorAfterButton, gc); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 16; JPanel line4 = new JPanel(); line4.setPreferredSize(new Dimension(windowWidth-35, 1)); line4.setBackground(Color.lightGray); container.add(line4, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 17; container.add(new JLabel("Step 5a: set threshold for donor before image"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 17; gc.insets = new Insets(0,0,0,2); resetDBButton = new JButton("Reset"); resetDBButton.setToolTipText("Resets blur and threshold settings"); resetDBButton.setMargin(new Insets(0,0,0,0)); resetDBButton.setFont(new Font("Helvetica", Font.BOLD, 10)); resetDBButton.addActionListener(this); resetDBButton.setActionCommand("resetDB"); container.add(resetDBButton, gc); thresholdDonorBeforeButton = new JButton("Set threshold"); thresholdDonorBeforeButton.addActionListener(this); thresholdDonorBeforeButton.setActionCommand("thresholdDonorBefore"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 17; gc.insets = new Insets(2,2,2,2); container.add(thresholdDonorBeforeButton, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 18; container.add(new JLabel("Step 5b: set threshold for donor after image"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 18; gc.insets = new Insets(0,0,0,2); resetDAButton = new JButton("Reset"); resetDAButton.setToolTipText("Resets blur and threshold settings"); resetDAButton.setMargin(new Insets(0,0,0,0)); resetDAButton.setFont(new Font("Helvetica", Font.BOLD, 10)); resetDAButton.addActionListener(this); resetDAButton.setActionCommand("resetDA"); container.add(resetDAButton, gc); thresholdDonorAfterButton = new JButton("Set threshold"); thresholdDonorAfterButton.addActionListener(this); thresholdDonorAfterButton.setActionCommand("thresholdDonorAfter"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 18; gc.insets = new Insets(2,2,2,2); container.add(thresholdDonorAfterButton, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 19; container.add(new JLabel("Step 5c (optional): set threshold for acceptor before image"), gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 19; gc.insets = new Insets(0,0,0,2); resetABButton = new JButton("Reset"); resetABButton.setToolTipText("Resets blur and threshold settings"); resetABButton.setMargin(new Insets(0,0,0,0)); resetABButton.setFont(new Font("Helvetica", Font.BOLD, 10)); resetABButton.addActionListener(this); resetABButton.setActionCommand("resetAB"); container.add(resetABButton, gc); thresholdAcceptorBeforeButton = new JButton("Set threshold"); thresholdAcceptorBeforeButton.addActionListener(this); thresholdAcceptorBeforeButton.setActionCommand("thresholdAcceptorBefore"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 19; gc.insets = new Insets(2,2,2,2); container.add(thresholdAcceptorBeforeButton, gc); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 20; container.add(new JLabel("Step 5d (optional): set threshold for acceptor after image"), gc); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 21; gc.insets = new Insets(0,6,0,6); JLabel thInfo = new JLabel("(Threshold setting: set threshold, press apply, select \"Set bg pixels to NaN\", press ok and close threshold window)"); thInfo.setFont(new Font("Helvetica", Font.PLAIN, 10)); container.add(thInfo, gc); gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 20; gc.insets = new Insets(0,0,0,2); resetAAButton = new JButton("Reset"); resetAAButton.setToolTipText("Resets blur and threshold settings"); resetAAButton.setMargin(new Insets(0,0,0,0)); resetAAButton.setFont(new Font("Helvetica", Font.BOLD, 10)); resetAAButton.addActionListener(this); resetAAButton.setActionCommand("resetAA"); container.add(resetAAButton, gc); thresholdAcceptorAfterButton = new JButton("Set threshold"); thresholdAcceptorAfterButton.addActionListener(this); thresholdAcceptorAfterButton.setActionCommand("thresholdAcceptorAfter"); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 20; gc.insets = new Insets(2,2,2,2); container.add(thresholdAcceptorAfterButton, gc); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 22; JPanel line5 = new JPanel(); line5.setPreferredSize(new Dimension(windowWidth-35, 1)); line5.setBackground(Color.lightGray); container.add(line5, gc); // donor bleaching correction gc.gridwidth = 8; gc.gridx = 0; gc.gridy = 23; donorBlCorrLabel = new JLabel("Correction 1: calculate/set donor bleaching correction factor:"); container.add(donorBlCorrLabel, gc); if (!donorBlCorrMenuItem.isSelected()) { donorBlCorrLabel.setVisible(false); } gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 23; donorBlCorrField = new JTextField("1.01", 4); donorBlCorrField.setHorizontalAlignment(JTextField.RIGHT); container.add(donorBlCorrField, gc); if (!donorBlCorrMenuItem.isSelected()) { donorBlCorrField.setVisible(false); } gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 23; gc.insets = new Insets(2,2,2,2); calculateDBCorrButton = new JButton("Calculate"); calculateDBCorrButton.addActionListener(this); calculateDBCorrButton.setActionCommand("calculateDonorBlCorrection"); container.add(calculateDBCorrButton, gc); if (!donorBlCorrMenuItem.isSelected()) { calculateDBCorrButton.setVisible(false); } gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 24; lineDonorBlCorr = new JPanel(); lineDonorBlCorr.setPreferredSize(new Dimension(windowWidth-35, 1)); lineDonorBlCorr.setBackground(Color.lightGray); container.add(lineDonorBlCorr, gc); if (!donorBlCorrMenuItem.isSelected()) { lineDonorBlCorr.setVisible(false); } // acceptor cross-talk correction gc.gridwidth = 8; gc.gridx = 0; gc.gridy = 25; accCrossTalkCorrLabel = new JLabel("Correction 2: calculate/set acceptor cross-talk correction factor:"); container.add(accCrossTalkCorrLabel, gc); if (!accCrossTalkCorrMenuItem.isSelected()) { accCrossTalkCorrLabel.setVisible(false); } gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 25; accCrossTalkCorrField = new JTextField("0.01", 4); accCrossTalkCorrField.setHorizontalAlignment(JTextField.RIGHT); container.add(accCrossTalkCorrField, gc); if (!accCrossTalkCorrMenuItem.isSelected()) { accCrossTalkCorrField.setVisible(false); } gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 25; gc.insets = new Insets(2,2,2,2); calculateAccCTCorrButton = new JButton("Calculate"); calculateAccCTCorrButton.addActionListener(this); calculateAccCTCorrButton.setActionCommand("calculateAccCTCorrection"); container.add(calculateAccCTCorrButton, gc); if (!accCrossTalkCorrMenuItem.isSelected()) { calculateAccCTCorrButton.setVisible(false); } gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 26; lineAccCrossTalk = new JPanel(); lineAccCrossTalk.setPreferredSize(new Dimension(windowWidth-35, 1)); lineAccCrossTalk.setBackground(Color.lightGray); container.add(lineAccCrossTalk, gc); if (!accCrossTalkCorrMenuItem.isSelected()) { lineAccCrossTalk.setVisible(false); } // acceptor photoproduct correction gc.gridwidth = 8; gc.gridx = 0; gc.gridy = 27; accPhotoprCorrLabel = new JLabel("Correction 3: calculate/set acceptor photoproduct correction factor:"); container.add(accPhotoprCorrLabel, gc); if (!accPhotoprCorrMenuItem.isSelected()) { accPhotoprCorrLabel.setVisible(false); } gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 27; accPhotoprCorrField = new JTextField("0.01", 4); accPhotoprCorrField.setHorizontalAlignment(JTextField.RIGHT); container.add(accPhotoprCorrField, gc); if (!accPhotoprCorrMenuItem.isSelected()) { accPhotoprCorrField.setVisible(false); } gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 27; gc.insets = new Insets(2,2,2,2); calculateAccPPCorrButton = new JButton("Calculate"); calculateAccPPCorrButton.addActionListener(this); calculateAccPPCorrButton.setActionCommand("calculateAccPPCorrection"); container.add(calculateAccPPCorrButton, gc); if (!accPhotoprCorrMenuItem.isSelected()) { calculateAccPPCorrButton.setVisible(false); } gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 28; lineAccPhotopr = new JPanel(); lineAccPhotopr.setPreferredSize(new Dimension(windowWidth-35, 1)); lineAccPhotopr.setBackground(Color.lightGray); container.add(lineAccPhotopr, gc); if (!accPhotoprCorrMenuItem.isSelected()) { lineAccPhotopr.setVisible(false); } // correction for partially bleached acceptor gc.gridwidth = 8; gc.gridx = 0; gc.gridy = 29; partialBlCorrLabel = new JLabel("Correction 4: calculate partial acceptor photobleaching correction factor:"); container.add(partialBlCorrLabel, gc); if (!partialBlCorrMenuItem.isSelected()) { partialBlCorrLabel.setVisible(false); } gc.gridwidth = 1; gc.gridx = 9; gc.gridy = 29; partialBlCorrField = new JTextField("0", 4); partialBlCorrField.setHorizontalAlignment(JTextField.RIGHT); container.add(partialBlCorrField, gc); if (!partialBlCorrMenuItem.isSelected()) { partialBlCorrField.setVisible(false); } gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 29; gc.insets = new Insets(2,2,2,2); calculatePartialBlCorrButton = new JButton("Calculate"); calculatePartialBlCorrButton.addActionListener(this); calculatePartialBlCorrButton.setActionCommand("calculatePartialBlCorrection"); container.add(calculatePartialBlCorrButton, gc); if (!partialBlCorrMenuItem.isSelected()) { calculatePartialBlCorrButton.setVisible(false); } gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 30; linePartialBl = new JPanel(); linePartialBl.setPreferredSize(new Dimension(windowWidth-35, 1)); linePartialBl.setBackground(Color.lightGray); container.add(linePartialBl, gc); if (!partialBlCorrMenuItem.isSelected()) { linePartialBl.setVisible(false); } // create fret image JPanel createFretImgPanel = new JPanel(new FlowLayout(FlowLayout.LEADING, 0, 0)); gc.gridwidth = 10; gc.gridx = 0; gc.gridy = 31; gc.insets = new Insets(2,2,2,2); gc.fill = GridBagConstraints.NONE; createFretImgPanel.add(new JLabel("Step 6: create FRET image ")); useAcceptorAsMask = new JCheckBox("use also acceptor before image as mask", true); useAcceptorAsMask.setToolTipText("<html>When calculating intramolecular FRET or intermolecular <br>FRET for one species, the AND of donor before and after <br>images is used as the default mask. If the acceptor label is <br>on another molecular species, the thresholded acceptor <br>image can be AND-ed to this as well.</html>"); useAcceptorAsMask.setSelected(false); createFretImgPanel.add(useAcceptorAsMask); container.add(createFretImgPanel, gc); createButton = new JButton("Create"); createButton.addActionListener(this); createButton.setActionCommand("createFretImage"); gc.fill = GridBagConstraints.HORIZONTAL; gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 10; gc.gridy = 31; container.add(createButton, gc); gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 32; JPanel line7 = new JPanel(); line7.setPreferredSize(new Dimension(windowWidth-35, 1)); line7.setBackground(Color.lightGray); container.add(line7, gc); gc.gridwidth = 8; gc.gridx = 0; gc.gridy = 33; container.add(new JLabel("Step 7: select ROIs and make measurements"), gc); gc.gridx = 9; gc.gridwidth = 1; closeImagesButton = new JButton("Close images"); closeImagesButton.setToolTipText("Closes the source and transfer images and resets button colors"); closeImagesButton.setMargin(new Insets(0,0,0,0)); closeImagesButton.setFont(new Font("Helvetica", Font.BOLD, 10)); closeImagesButton.addActionListener(this); closeImagesButton.setActionCommand("closeImages"); container.add(closeImagesButton, gc); measureButton = new JButton("Measure"); measureButton.setMargin(new Insets(2,2,2,2)); measureButton.addActionListener(this); measureButton.setActionCommand("measureFretImage"); gc.gridx = 10; gc.gridy = 33; container.add(measureButton, gc); nextButton = new JButton("Next"); nextButton.setMargin(new Insets(2,2,2,2)); nextButton.addActionListener(this); nextButton.setActionCommand("nextImage"); gc.gridx = 11; gc.gridy = 33; container.add(nextButton, gc); nextButton.setVisible(false); gc.weighty = 20; gc.gridwidth = GridBagConstraints.REMAINDER; gc.fill = GridBagConstraints.BOTH; gc.gridx = 0; gc.gridy = GridBagConstraints.RELATIVE; log = new JTextPane(); log.setEditable(false); Style defaultStyle = StyleContext.getDefaultStyleContext().getStyle(StyleContext.DEFAULT_STYLE); Style style; style = log.addStyle("RED", defaultStyle); StyleConstants.setForeground(style, Color.red.darker()); style = log.addStyle("BLUE", defaultStyle); StyleConstants.setForeground(style, Color.blue.darker()); style = log.addStyle("BLACK", defaultStyle); StyleConstants.setForeground(style, Color.black.darker()); logScrollPane = new JScrollPane(log); logScrollPane.setBorder(BorderFactory.createTitledBorder("Messages")); container.add(logScrollPane, gc); } public void actionPerformed(ActionEvent e) { try { if (e.getActionCommand().equals("exit")) { exit(); } else if (e.getActionCommand().equals("split")) { if (WindowManager.getCurrentImage() == null) { logError("No open image."); return; } if (WindowManager.getCurrentImage().isHyperStack()) { ij.plugin.HyperStackConverter hc = new ij.plugin.HyperStackConverter(); hc.run("hstostack"); } StackEditor se = new StackEditor(); se.run("toimages"); } else if (e.getActionCommand().equals("applyMask")) { if (applyMaskDialog != null) { applyMaskDialog.setVisible(false); applyMaskDialog.dispose(); } applyMaskDialog = new ApplyMaskDialog(this); applyMaskDialog.setVisible(true); } else if (e.getActionCommand().equals("bleachingMask")) { if (acceptorBefore == null) { logError("No image is set as acceptor before bleaching."); return; } else if (acceptorAfter == null) { logError("No image is set as acceptor after bleaching."); return; } else { ImageProcessor ip1 = acceptorBefore.getProcessor(); ImageProcessor ip2 = acceptorAfter.getProcessor(); float[] ip1P = (float[])ip1.getPixels(); float[] ip2P = (float[])ip2.getPixels(); int width = ip1.getWidth(); int height = ip1.getHeight(); float[][] newImgPoints = new float[width][height]; for (int i = 0; i < width; i++) { for (int j = 0; j < height; j++) { newImgPoints[i][j] = ip1P[width*j+i] - ip2P[width*j+i]; } } FloatProcessor fp = new FloatProcessor(newImgPoints); ImagePlus newImg = new ImagePlus("Acceptor before - acceptor after", fp); newImg.changes = false; newImg.show(); IJ.run("Threshold..."); } } else if (e.getActionCommand().equals("calculateRatio")) { if (calculateImgRatioDialog != null) { calculateImgRatioDialog.setVisible(false); calculateImgRatioDialog.dispose(); } calculateImgRatioDialog = new CalculateImgRatioDialog(this); calculateImgRatioDialog.setVisible(true); } else if (e.getActionCommand().equals("threshold")) { if (WindowManager.getCurrentImage() == null) { logError("No open image."); return; } IJ.run("Threshold..."); } else if (e.getActionCommand().equals("convertto32bit")) { if (WindowManager.getCurrentImage() == null) { logError("No open image."); return; } new ImageConverter(WindowManager.getCurrentImage()).convertToGray32(); } else if (e.getActionCommand().equals("shiftimage")) { if (WindowManager.getCurrentImage() == null) { logError("No open image."); return; } if (shiftDialog == null) { shiftDialog = new ShiftDialog(this); } shiftDialog.setVisible(true); } else if (e.getActionCommand().equals("lutFire")) { if (WindowManager.getCurrentImage() == null) { logError("No open image."); return; } IJ.run("Fire"); } else if (e.getActionCommand().equals("lutSpectrum")) { if (WindowManager.getCurrentImage() == null) { logError("No open image."); return; } IJ.run("Spectrum"); } else if (e.getActionCommand().equals("histogram")) { if (WindowManager.getCurrentImage() == null) { logError("No open image."); return; } IJ.run("Histogram"); } else if (e.getActionCommand().equals("openImage")) { (new Opener()).open(); } else if (e.getActionCommand().equals("saveImageAsTiff")) { ImagePlus image = WindowManager.getCurrentImage(); if (image == null) { logError("No open image."); return; } FileSaver fs = new FileSaver(image); if (fs.saveAsTiff()){ log("Tiff file is saved."); } image.updateAndDraw(); } else if (e.getActionCommand().equals("saveImageAsBmp")) { ImagePlus image = WindowManager.getCurrentImage(); if (image == null) { logError("No open image."); return; } FileSaver fs = new FileSaver(image); if (fs.saveAsBmp()){ log("Bmp file is saved."); } image.updateAndDraw(); } else if (e.getActionCommand().equals("saveMessages")) { JFileChooser jfc = new JFileChooser(currentDirectory); jfc.setFileSelectionMode(JFileChooser.FILES_ONLY); jfc.setDialogTitle("Save messages..."); jfc.showSaveDialog(this); if (jfc.getSelectedFile() == null) { return; } if (jfc.getSelectedFile().exists()) { currentDirectory = jfc.getCurrentDirectory().toString(); int resp = JOptionPane.showConfirmDialog(this, "Overwrite existing file?","Confirmation", JOptionPane.OK_CANCEL_OPTION, JOptionPane.QUESTION_MESSAGE); if (resp == JOptionPane.CANCEL_OPTION) { return; } } try { BufferedWriter out = new BufferedWriter(new FileWriter(jfc.getSelectedFile().getAbsolutePath())); out.write(log.getText()); out.close(); } catch (IOException ioe) { logError("Could not save messages."); } } else if (e.getActionCommand().equals("clearMessages")) { log.setText(""); } else if (e.getActionCommand().equals("openLsmImage")) { JFileChooser jfc = new JFileChooser(currentDirectory); jfc.setFileSelectionMode(JFileChooser.FILES_ONLY); jfc.setDialogTitle("Open LSM image..."); jfc.showOpenDialog(this); if (jfc.getSelectedFile() == null) { return; } if (!jfc.getSelectedFile().exists()) { logError("Selected file does not exist."); return; } try { currentDirectory = jfc.getCurrentDirectory().toString(); boolean close = false; boolean resultsWindow = false; while(WindowManager.getCurrentImage() != null) { WindowManager.getCurrentImage().close(); } resetAllButtonColors(); File imageFile = jfc.getSelectedFile(); (new Opener()).open(imageFile.getAbsolutePath()); WindowManager.putBehind(); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"split")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"setAcceptorAfter")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"lutSpectrum")); WindowManager.putBehind(); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"setDonorAfter")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"lutSpectrum")); WindowManager.putBehind(); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"setAcceptorBefore")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"lutSpectrum")); WindowManager.putBehind(); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"setDonorBefore")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"lutSpectrum")); } catch (Exception ex) { logError("Could not open and set the selected LSM image."); logException(ex.getMessage(), ex); } } else if (e.getActionCommand().equals("setDonorBefore")) { ImagePlus ip = WindowManager.getCurrentImage(); if (ip == null) { logError("No image is selected."); setDonorBeforeButton.setBackground(originalButtonColor); return; } if (ip.getNChannels() > 1) { logError("Current image contains more than 1 channel ("+ip.getNChannels()+"). Please use: Image menu -> Split image."); donorBefore = null; setDonorBeforeButton.setBackground(originalButtonColor); return; } else if (ip.getNSlices() > 1) { logError("Current image contains more than 1 slice ("+ip.getNSlices()+"). Please use: Image menu -> Split image."); donorBefore = null; setDonorBeforeButton.setBackground(originalButtonColor); return; } if (ip != null && donorAfter != null && ip.equals(donorAfter)) { logError("The two donor images must not be the same. Please select and set an other image."); donorBefore = null; setDonorBeforeButton.setBackground(originalButtonColor); return; } donorBefore = ip; donorBefore.setTitle("Donor before bleaching - " + new Date().toString()); new ImageConverter(donorBefore).convertToGray32(); if (automaticallyProcessedFiles == null) { currentlyProcessedFileName = null; } setDonorBeforeButton.setBackground(greenColor); } else if (e.getActionCommand().equals("setDonorAfter")) { ImagePlus ip = WindowManager.getCurrentImage(); if (ip == null) { logError("No image is selected."); setDonorAfterButton.setBackground(originalButtonColor); return; } if (ip.getNChannels() > 1) { logError("Current image contains more than 1 channel ("+ip.getNChannels()+"). Please split it into parts."); donorAfter = null; setDonorAfterButton.setBackground(originalButtonColor); return; } else if (ip.getNSlices() > 1) { logError("Current image contains more than 1 slice ("+ip.getNSlices()+"). Please split it into parts."); donorAfter = null; setDonorAfterButton.setBackground(originalButtonColor); return; } if (donorBefore != null && ip != null && donorBefore.equals(ip)) { logError("The two donor images must not be the same. Please select and set an other image."); donorAfter = null; setDonorAfterButton.setBackground(originalButtonColor); return; } donorAfter = ip; donorAfter.setTitle("Donor after bleaching - " + new Date().toString()); new ImageConverter(donorAfter).convertToGray32(); setDonorAfterButton.setBackground(greenColor); } else if (e.getActionCommand().equals("setAcceptorBefore")) { acceptorBefore = WindowManager.getCurrentImage(); if (acceptorBefore == null) { logError("No image is selected."); setAcceptorBeforeButton.setBackground(originalButtonColor); return; } if (acceptorBefore.getNChannels() > 1) { logError("Current image contains more than 1 channel ("+acceptorBefore.getNChannels()+"). Please split it into parts."); acceptorBefore = null; setAcceptorBeforeButton.setBackground(originalButtonColor); return; } else if (acceptorBefore.getNSlices() > 1) { logError("Current image contains more than 1 slice ("+acceptorBefore.getNSlices()+"). Please split it into parts."); acceptorBefore = null; setAcceptorBeforeButton.setBackground(originalButtonColor); return; } acceptorBefore.setTitle("Acceptor before bleaching - " + new Date().toString()); new ImageConverter(acceptorBefore).convertToGray32(); acceptorBeforeSave = acceptorBefore.getProcessor().duplicate(); setAcceptorBeforeButton.setBackground(greenColor); } else if (e.getActionCommand().equals("setAcceptorAfter")) { acceptorAfter = WindowManager.getCurrentImage(); if (acceptorAfter == null) { logError("No image is selected."); setAcceptorAfterButton.setBackground(originalButtonColor); return; } if (acceptorAfter.getNChannels() > 1) { logError("Current image contains more than 1 channel ("+acceptorAfter.getNChannels()+"). Please split it into parts."); acceptorAfter = null; setAcceptorAfterButton.setBackground(originalButtonColor); return; } else if (acceptorAfter.getNSlices() > 1) { logError("Current image contains more than 1 slice ("+acceptorAfter.getNSlices()+"). Please split it into parts."); acceptorAfter = null; setAcceptorAfterButton.setBackground(originalButtonColor); return; } acceptorAfter.setTitle("Acceptor after bleaching - " + new Date().toString()); new ImageConverter(acceptorAfter).convertToGray32(); acceptorAfterSave = acceptorAfter.getProcessor().duplicate(); setAcceptorAfterButton.setBackground(greenColor); } else if (e.getActionCommand().equals("clearAB")) { if (acceptorBefore == null) { logError("No image is set as acceptor before bleaching."); return; } acceptorBefore = null; acceptorBeforeSave = null; setAcceptorBeforeButton.setBackground(originalButtonColor); } else if (e.getActionCommand().equals("clearAA")) { if (acceptorAfter == null) { logError("No image is set as acceptor after bleaching."); return; } acceptorAfter = null; acceptorAfterSave = null; setAcceptorAfterButton.setBackground(originalButtonColor); } else if (e.getActionCommand().equals("copyRoi")) { if (donorBefore == null) { logError("No image is set as donor before bleaching."); return; } if (donorBefore.getRoi() != null) { if (donorAfter != null) { donorAfter.setRoi(donorBefore.getRoi()); } if (acceptorBefore != null) { acceptorBefore.setRoi(donorBefore.getRoi()); } if (acceptorAfter != null) { acceptorAfter.setRoi(donorBefore.getRoi()); } } else { if (donorAfter != null) { donorAfter.killRoi(); } if (acceptorBefore != null) { acceptorBefore.killRoi(); } if (acceptorAfter != null) { acceptorAfter.killRoi(); } } } else if (e.getActionCommand().equals("subtractDonorBefore")) { if (donorBefore == null) { logError("No image is set as donor before bleaching."); return; } else if (donorBefore.getRoi() == null) { logError("No ROI is defined for donor before bleaching."); return; } int width = donorBefore.getWidth(); int height = donorBefore.getHeight(); double sum = 0; int count = 0; for (int i=0; i < width; i++) { for (int j=0; j < height; j++) { if (donorBefore.getRoi().contains(i, j)) { sum += donorBefore.getProcessor().getPixelValue(i,j); count++; } } } float backgroundAvg = (float)(sum/count); float i = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { i = donorBefore.getProcessor().getPixelValue(x,y); i = i - backgroundAvg; if (i < 0) { i=0; } donorBefore.getProcessor().putPixelValue(x, y, i); } } donorBefore.updateAndDraw(); donorBefore.killRoi(); donorBeforeSave = donorBefore.getProcessor().duplicate(); log("Subtracted background ("+backgroundAvg+") of donor before bleaching."); subtractDonorBeforeButton.setBackground(greenColor); } else if (e.getActionCommand().equals("subtractDonorAfter")) { if (donorAfter == null) { logError("No image is set as donor after bleaching."); return; } else if (donorAfter.getRoi() == null) { logError("No ROI is defined for donor after bleaching."); return; } int width = donorAfter.getWidth(); int height = donorAfter.getHeight(); double sum = 0; int count = 0; for (int i=0; i < width; i++) { for (int j=0; j < height; j++) { if (donorAfter.getRoi().contains(i, j)) { sum += donorAfter.getProcessor().getPixelValue(i,j); count++; } } } float backgroundAvg = (float)(sum/count); float i = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { i = donorAfter.getProcessor().getPixelValue(x,y); i = i - backgroundAvg; if (i < 0) { i=0; } donorAfter.getProcessor().putPixelValue(x, y, i); } } donorAfter.updateAndDraw(); donorAfter.killRoi(); donorAfterSave = donorAfter.getProcessor().duplicate(); log("Subtracted background ("+backgroundAvg+") of donor after bleaching."); subtractDonorAfterButton.setBackground(greenColor); } else if (e.getActionCommand().equals("subtractAcceptorBefore")) { if (acceptorBefore == null) { logError("No image is set as acceptor before bleaching."); return; } else if (acceptorBefore.getRoi() == null) { logError("No ROI is defined for acceptor before bleaching."); return; } int width = acceptorBefore.getWidth(); int height = acceptorBefore.getHeight(); double sum = 0; int count = 0; for (int i=0; i < width; i++) { for (int j=0; j < height; j++) { if (acceptorBefore.getRoi().contains(i, j)) { sum += acceptorBefore.getProcessor().getPixelValue(i,j); count++; } } } float backgroundAvg = (float)(sum/count); float i = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { i = acceptorBefore.getProcessor().getPixelValue(x,y)- backgroundAvg; if (i < 0) { i=0; } acceptorBefore.getProcessor().putPixelValue(x, y, i); } } acceptorBefore.updateAndDraw(); acceptorBefore.killRoi(); acceptorBeforeSave = acceptorBefore.getProcessor().duplicate(); log("Subtracted background ("+backgroundAvg+") of acceptor before bleaching."); subtractAcceptorBeforeButton.setBackground(greenColor); } else if (e.getActionCommand().equals("subtractAcceptorAfter")) { if (acceptorAfter == null) { logError("No image is set as acceptor after bleaching."); return; } else if (acceptorAfter.getRoi() == null) { logError("No ROI is defined for acceptor after bleaching."); return; } int width = acceptorAfter.getWidth(); int height = acceptorAfter.getHeight(); double sum = 0; int count = 0; for (int i=0; i < width; i++) { for (int j=0; j < height; j++) { if (acceptorAfter.getRoi().contains(i, j)) { sum += acceptorAfter.getProcessor().getPixelValue(i,j); count++; } } } float backgroundAvg = (float)(sum/count); float i = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { i = acceptorAfter.getProcessor().getPixelValue(x,y)- backgroundAvg; if (i < 0) { i=0; } acceptorAfter.getProcessor().putPixelValue(x, y, i); } } acceptorAfter.updateAndDraw(); acceptorAfter.killRoi(); acceptorAfterSave = acceptorAfter.getProcessor().duplicate(); log("Subtracted background ("+backgroundAvg+") of acceptor after bleaching."); subtractAcceptorAfterButton.setBackground(greenColor); } else if (e.getActionCommand().equals("thresholdDonorBefore")) { if (donorBefore == null) { logError("No image is set as donor before bleaching."); return; } IJ.selectWindow(donorBefore.getTitle()); IJ.run("Threshold..."); thresholdDonorBeforeButton.setBackground(greenColor); } else if (e.getActionCommand().equals("thresholdDonorAfter")) { if (donorAfter == null) { logError("No image is set as donor after bleaching."); return; } IJ.selectWindow(donorAfter.getTitle()); IJ.run("Threshold..."); thresholdDonorAfterButton.setBackground(greenColor); } else if (e.getActionCommand().equals("thresholdAcceptorBefore")) { if (acceptorBefore == null) { logError("No image is set as acceptor before bleaching."); return; } IJ.selectWindow(acceptorBefore.getTitle()); IJ.run("Threshold..."); thresholdAcceptorBeforeButton.setBackground(greenColor); } else if (e.getActionCommand().equals("thresholdAcceptorAfter")) { if (acceptorAfter == null) { logError("No image is set as acceptor after bleaching."); return; } IJ.selectWindow(acceptorAfter.getTitle()); IJ.run("Threshold..."); thresholdAcceptorAfterButton.setBackground(greenColor); } else if (e.getActionCommand().equals("resetDB")) { if (donorBefore == null) { logError("No image is set as donor before bleaching."); return; } if (donorBeforeSave == null) { logError("No saved image."); return; } donorBefore.setProcessor(donorBefore.getTitle(), donorBeforeSave.duplicate()); donorBefore.updateAndDraw(); thresholdDonorBeforeButton.setBackground(originalButtonColor); smoothDonorBeforeButton.setBackground(originalButtonColor); } else if (e.getActionCommand().equals("resetDA")) { if (donorAfter == null) { logError("No image is set as donor after bleaching."); return; } if (donorAfterSave == null) { logError("No saved image."); return; } donorAfter.setProcessor(donorAfter.getTitle(), donorAfterSave.duplicate()); donorAfter.updateAndDraw(); thresholdDonorAfterButton.setBackground(originalButtonColor); smoothDonorAfterButton.setBackground(originalButtonColor); } else if (e.getActionCommand().equals("resetAB")) { if (acceptorBefore == null) { logError("No image is set as acceptor before bleaching."); return; } if (acceptorBeforeSave == null) { logError("No saved image."); return; } acceptorBefore.setProcessor(acceptorBefore.getTitle(), acceptorBeforeSave.duplicate()); acceptorBefore.updateAndDraw(); thresholdAcceptorBeforeButton.setBackground(originalButtonColor); smoothAcceptorBeforeButton.setBackground(originalButtonColor); } else if (e.getActionCommand().equals("resetAA")) { if (acceptorAfter == null) { logError("No image is set as acceptor after bleaching."); return; } if (acceptorAfterSave == null) { logError("No saved image."); return; } acceptorAfter.setProcessor(acceptorAfter.getTitle(), acceptorAfterSave.duplicate()); acceptorAfter.updateAndDraw(); thresholdAcceptorAfterButton.setBackground(originalButtonColor); smoothAcceptorAfterButton.setBackground(originalButtonColor); } else if (e.getActionCommand().equals("smoothDBefore")) { if (donorBefore == null) { logError("No image is set as donor before bleaching."); return; } else { if (radiusFieldDB.getText().trim().equals("")) { logError("Radius has to be given for Gaussian blur."); return; } else { double radius = 0; try { radius = Double.parseDouble(radiusFieldDB.getText().trim()); } catch (Exception ex) { logError("Radius has to be given for Gaussian blur."); return; } GaussianBlur gb = new GaussianBlur(); if(!gb.blur(donorBefore.getProcessor(), radius)) { return; } donorBefore.updateAndDraw(); smoothDonorBeforeButton.setBackground(greenColor); log("Gaussian blurred donor before bleaching."); } } } else if (e.getActionCommand().equals("smoothDAfter")) { if (donorAfter == null) { logError("No image is set as donor after bleaching."); return; } else { if (radiusFieldDA.getText().trim().equals("")) { logError("Radius has to be given for Gaussian blur."); return; } else { double radius = 0; try { radius = Double.parseDouble(radiusFieldDA.getText().trim()); } catch (Exception ex) { logError("Radius has to be given for Gaussian blur."); return; } GaussianBlur gb = new GaussianBlur(); if(!gb.blur(donorAfter.getProcessor(), radius)) { return; } donorAfter.updateAndDraw(); smoothDonorAfterButton.setBackground(greenColor); log("Gaussian blurred donor after bleaching."); } } } else if (e.getActionCommand().equals("smoothABefore")) { if (acceptorBefore == null) { logError("No image is set as acceptor before bleaching."); return; } else { if (radiusFieldAB.getText().trim().equals("")) { logError("Radius has to be given for Gaussian blur."); return; } else { double radius = 0; try { radius = Double.parseDouble(radiusFieldAB.getText().trim()); } catch (Exception ex) { logError("Radius has to be given for Gaussian blur."); return; } GaussianBlur gb = new GaussianBlur(); if(!gb.blur(acceptorBefore.getProcessor(), radius)) { return; } acceptorBefore.updateAndDraw(); smoothAcceptorBeforeButton.setBackground(greenColor); log("Gaussian blurred acceptor before bleaching."); } } } else if (e.getActionCommand().equals("smoothAAfter")) { if (acceptorAfter == null) { logError("No image is set as acceptor after bleaching."); return; } else { if (radiusFieldAB.getText().trim().equals("")) { logError("Radius has to be given for Gaussian blur."); return; } else { double radius = 0; try { radius = Double.parseDouble(radiusFieldAB.getText().trim()); } catch (Exception ex) { logError("Radius has to be given for Gaussian blur."); return; } GaussianBlur gb = new GaussianBlur(); if(!gb.blur(acceptorAfter.getProcessor(), radius)) { return; } acceptorAfter.updateAndDraw(); smoothAcceptorAfterButton.setBackground(greenColor); log("Gaussian blurred acceptor after bleaching."); } } } else if (e.getActionCommand().equals("registerImages")) { if (donorBefore == null) { logError("No image is set as donor before bleaching."); return; } else if (donorAfter == null) { logError("No image is set as donor after bleaching."); return; } else { DecimalFormat df = new DecimalFormat("#0.000"); FHT fht1 = new FHT(donorBefore.getProcessor().duplicate()); fht1.transform(); FHT fht2 = new FHT(donorAfter.getProcessor().duplicate()); fht2.transform(); FHT res = fht1.conjugateMultiply(fht2); res.inverseTransform(); ImagePlus image = new ImagePlus("Result of registration", res); ImageProcessor ip = image.getProcessor(); int width = ip.getWidth(); int height = ip.getHeight(); int maximum = 0; int maxx = -1; int maxy = -1; for (int i = 0; i < width; i++) { for (int j = 0; j < height; j++) { if (ip.getPixel(i, j) > maximum) { maximum = ip.getPixel(i, j); maxx = i; maxy = j; } } } int shiftX = 0; int shiftY = 0; if (maxx != 0 || maxy != 0){ ShiftDialog sd = new ShiftDialog(this); if (maxy > height/2) { log("Shifting donor after image up " + (height-maxy) + " pixel" + ((height-maxy)>1?"s":"") + "."); sd.shiftUp(donorAfter, height-maxy); if (applyShiftCB.isSelected()){ sd.shiftUp(acceptorAfter, height-maxy); } } else if (maxy != 0) { log("Shifting donor after image down " + maxy + " pixel" + (maxy>1?"s":"") + "."); sd.shiftDown(donorAfter, maxy); if (applyShiftCB.isSelected()){ sd.shiftDown(acceptorAfter, maxy); } } if (maxx > width/2) { log("Shifting donor after image to the left " + (width-maxx) + " pixel" + ((width-maxx)>1?"s":"") + "."); sd.shiftLeft(donorAfter, width-maxx); if (applyShiftCB.isSelected()){ sd.shiftLeft(acceptorAfter, width-maxx); } } else if (maxx != 0) { log("Shifting donor after image to the right " + maxx + " pixel" + (maxx>1?"s":"") + "."); sd.shiftRight(donorAfter, maxx); if (applyShiftCB.isSelected()){ sd.shiftRight(acceptorAfter, maxx); } } actionPerformed(new ActionEvent(registerButton, 1, "registerImages")); } else { double countAll = 0; double count = 0; float db, da = 0; double p = 1.10; log("Registration finished."); Roi roi = donorBefore.getRoi(); if (roi == null) { logWarning("The calculated statistics after registration are more authoritative if there is a ROI defined in the donor before image, and the calculations are based on that."); donorAfter.killRoi(); } else { donorAfter.setRoi(donorBefore.getRoi()); } ImageStatistics isMeanDB = ImageStatistics.getStatistics(donorBefore.getProcessor(), Measurements.MEAN,null); ImageStatistics isMeanDA = ImageStatistics.getStatistics(donorAfter.getProcessor(), Measurements.MEAN,null); ImageStatistics isStdDevDB = ImageStatistics.getStatistics(donorBefore.getProcessor(), Measurements.STD_DEV,null); ImageStatistics isStdDevDA = ImageStatistics.getStatistics(donorAfter.getProcessor(), Measurements.STD_DEV,null); for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { if (roi != null && roi.contains(x,y)) { countAll++; db = donorBefore.getProcessor().getPixelValue(x,y); da = donorAfter.getProcessor().getPixelValue(x,y); if (db != 0 && da != 0 && db / da > p) { count++; } } else if (roi == null) { countAll++; db = donorBefore.getProcessor().getPixelValue(x,y); da = donorAfter.getProcessor().getPixelValue(x,y); if (db != 0 && da != 0 && db / da > p) { count++; } } } } log("Relative dispersion (SD/mean) of donor before bleaching image: " + df.format((float)(isStdDevDB.stdDev/isMeanDB.mean))); log("Relative dispersion (SD/mean) of donor after bleaching image: " + df.format((float)(isStdDevDA.stdDev/isMeanDA.mean))); df.applyPattern("#0.0"); log(df.format(count/countAll*100) + "% of pixels has lower intensity by 10% in the donor after than in the donor before image."); registerButton.setBackground(greenColor); } } } else if (e.getActionCommand().equals("useLsmImages")) { if (useLsmImages.isSelected()) { setDonorBeforeButton.setText("Open & Set LSM"); setDonorBeforeButton.setActionCommand("openLsmImage"); setDonorAfterButton.setEnabled(false); setAcceptorBeforeButton.setEnabled(false); setAcceptorAfterButton.setEnabled(false); } else { setDonorBeforeButton.setText("Set image"); setDonorBeforeButton.setActionCommand("setDonorBefore"); setDonorAfterButton.setEnabled(true); setAcceptorBeforeButton.setEnabled(true); setAcceptorAfterButton.setEnabled(true); } logScrollPane.setPreferredSize(new Dimension(10,10)); } else if (e.getActionCommand().equals("donorblcorrm")) { if (donorBlCorrMenuItem.isSelected()) { donorBlCorrLabel.setVisible(true); donorBlCorrField.setVisible(true); calculateDBCorrButton.setVisible(true); lineDonorBlCorr.setVisible(true); logScrollPane.setPreferredSize(new Dimension(10,10)); } else { donorBlCorrLabel.setVisible(false); donorBlCorrField.setVisible(false); calculateDBCorrButton.setVisible(false); lineDonorBlCorr.setVisible(false); logScrollPane.setPreferredSize(new Dimension(10,10)); if (donorBlCorrDialog != null) { donorBlCorrDialog.setVisible(false); donorBlCorrDialog.dispose(); } } } else if (e.getActionCommand().equals("acccrtalkcorrm")) { if (accCrossTalkCorrMenuItem.isSelected()) { accCrossTalkCorrLabel.setVisible(true); accCrossTalkCorrField.setVisible(true); calculateAccCTCorrButton.setVisible(true); lineAccCrossTalk.setVisible(true); logScrollPane.setPreferredSize(new Dimension(10,10)); } else { accCrossTalkCorrLabel.setVisible(false); accCrossTalkCorrField.setVisible(false); calculateAccCTCorrButton.setVisible(false); lineAccCrossTalk.setVisible(false); logScrollPane.setPreferredSize(new Dimension(10,10)); if (acceptorCTCorrDialog != null) { acceptorCTCorrDialog.setVisible(false); acceptorCTCorrDialog.dispose(); } } } else if (e.getActionCommand().equals("accphprcorrm")) { if (accPhotoprCorrMenuItem.isSelected()) { accPhotoprCorrLabel.setVisible(true); accPhotoprCorrField.setVisible(true); calculateAccPPCorrButton.setVisible(true); lineAccPhotopr.setVisible(true); logScrollPane.setPreferredSize(new Dimension(10,10)); } else { accPhotoprCorrLabel.setVisible(false); accPhotoprCorrField.setVisible(false); calculateAccPPCorrButton.setVisible(false); lineAccPhotopr.setVisible(false); logScrollPane.setPreferredSize(new Dimension(10,10)); if (acceptorPPCorrDialog != null) { acceptorPPCorrDialog.setVisible(false); acceptorPPCorrDialog.dispose(); } } } else if (e.getActionCommand().equals("partialblcorrm")) { if (partialBlCorrMenuItem.isSelected()) { partialBlCorrLabel.setVisible(true); partialBlCorrField.setVisible(true); calculatePartialBlCorrButton.setVisible(true); linePartialBl.setVisible(true); logScrollPane.setPreferredSize(new Dimension(10,10)); } else { partialBlCorrLabel.setVisible(false); partialBlCorrField.setVisible(false); calculatePartialBlCorrButton.setVisible(false); linePartialBl.setVisible(false); logScrollPane.setPreferredSize(new Dimension(10,10)); } } else if (e.getActionCommand().equals("calculateDonorBlCorrection")) { if (donorBlCorrDialog != null) { donorBlCorrDialog.setVisible(false); donorBlCorrDialog.dispose(); } donorBlCorrDialog = new DonorBlCorrDialog(this); donorBlCorrDialog.setVisible(true); } else if (e.getActionCommand().equals("calculateAccCTCorrection")) { if (acceptorCTCorrDialog != null) { acceptorCTCorrDialog.setVisible(false); acceptorCTCorrDialog.dispose(); } acceptorCTCorrDialog = new AcceptorCTCorrDialog(this); acceptorCTCorrDialog.setVisible(true); } else if (e.getActionCommand().equals("calculateAccPPCorrection")) { if (acceptorPPCorrDialog != null) { acceptorPPCorrDialog.setVisible(false); acceptorPPCorrDialog.dispose(); } acceptorPPCorrDialog = new AcceptorPPCorrDialog(this); acceptorPPCorrDialog.setVisible(true); } else if (e.getActionCommand().equals("calculatePartialBlCorrection")) { if (acceptorBefore == null) { logError("No image is set as acceptor before bleaching."); return; } else if (acceptorAfter == null) { logError("No image is set as acceptor after bleaching."); return; } else { ImageProcessor ipAB = acceptorBefore.getProcessor(); ImageProcessor ipAA = acceptorAfter.getProcessor(); int width = acceptorBefore.getWidth(); int height = acceptorBefore.getHeight(); double sum = 0; int count = 0; if (acceptorBefore.getRoi() != null) { acceptorAfter.setRoi(acceptorBefore.getRoi()); for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (acceptorBefore.getRoi().contains(i, j)) { if (!Float.isNaN(ipAA.getPixelValue(i,j)) && !Float.isNaN(ipAB.getPixelValue(i,j))) { sum += ipAA.getPixelValue(i,j) / ipAB.getPixelValue(i,j); count++; } } } } } else { logWarning("No ROI is defined for acceptor before bleaching."); acceptorAfter.killRoi(); for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (!Float.isNaN(ipAA.getPixelValue(i,j)) && !Float.isNaN(ipAB.getPixelValue(i,j))) { sum += ipAA.getPixelValue(i,j) / ipAB.getPixelValue(i,j); count++; } } } } float partialBlCorrFactor = (float)(sum/count); DecimalFormat df = new DecimalFormat("#.###"); partialBlCorrField.setText(df.format(partialBlCorrFactor).toString()); calculatePartialBlCorrButton.setBackground(greenColor); } } else if (e.getActionCommand().equals("createFretImage")) { if (donorBefore == null) { logError("No image is set as donor before bleaching."); return; } else if (donorAfter == null) { logError("No image is set as donor after bleaching."); return; } else if ((useAcceptorAsMask.isSelected() || accCrossTalkCorrMenuItem.isSelected() || accPhotoprCorrMenuItem.isSelected()) && acceptorBefore == null) { logError("No image is set as acceptor before bleaching."); return; } else { if (donorBlCorrMenuItem.isSelected() && donorBlCorrField.getText().trim().equals("")) { logError("Bleaching correction factor has to be given."); return; } else if (accCrossTalkCorrMenuItem.isSelected() && accCrossTalkCorrField.getText().trim().equals("")) { logError("Acceptor cross-talk correction factor has to be given."); return; } else if (accPhotoprCorrMenuItem.isSelected() && accPhotoprCorrField.getText().trim().equals("")) { logError("Acceptor photoproduct correction factor has to be given."); return; } else if (partialBlCorrMenuItem.isSelected() && partialBlCorrField.getText().trim().equals("")) { logError("Partial acceptor photobleaching correction factor has to be given."); return; } else { float donorBlCorr = 1; if (donorBlCorrMenuItem.isSelected()) { try { donorBlCorr = Float.parseFloat(donorBlCorrField.getText().trim()); } catch (Exception ex) { logError("Donor bleaching correction factor has to be given."); return; } if (donorBlCorr < 1) { logWarning("The donor bleaching correction factor should not be lower than 1."); } } float acceptorCTCorr = 0; if (accCrossTalkCorrMenuItem.isSelected()) { try { acceptorCTCorr = Float.parseFloat(accCrossTalkCorrField.getText().trim()); } catch (Exception ex) { logError("Acceptor cross-talk correction factor has to be given."); return; } if (acceptorCTCorr < 0) { logWarning("The acceptor cross-talk correction factor should not be lower than 0."); } } float acceptorPPCorr = 0; if (accPhotoprCorrMenuItem.isSelected()) { try { acceptorPPCorr = Float.parseFloat(accPhotoprCorrField.getText().trim()); } catch (Exception ex) { logError("Acceptor photoproduct correction factor has to be given."); return; } if (acceptorPPCorr < 0) { logWarning("The acceptor photoproduct correction factor should not be lower than 0."); } } float partialBlCorr = 0; if (partialBlCorrMenuItem.isSelected()) { try { partialBlCorr = Float.parseFloat(partialBlCorrField.getText().trim()); } catch (Exception ex) { logError("Partial acceptor photobleaching correction factor has to be given."); return; } if (partialBlCorr < 0) { logWarning("The partial acceptor photobleaching correction should not be lower than 0."); } if (partialBlCorr > 1) { logWarning("The partial acceptor photobleaching correction should not be higher than 1."); } } ImageProcessor ipDB = donorBefore.getProcessor().duplicate(); ImageProcessor ipDA = donorAfter.getProcessor().duplicate(); ImageProcessor ipAB = null; if (acceptorBefore != null) { ipAB = acceptorBefore.getProcessor().duplicate(); } float[] ipDBP = (float[])ipDB.getPixels(); float[] ipDAP = (float[])ipDA.getPixels(); float[] ipABP = null; if (ipAB != null) { ipABP = (float[])ipAB.getPixels(); } if (!partialBlCorrMenuItem.isSelected()) { // acceptor cross-talk correction if (accCrossTalkCorrMenuItem.isSelected()) { for (int i = 0; i < ipABP.length; i++) { if (!Float.isNaN(ipDBP[i]) && !Float.isNaN(ipABP[i])) { ipDBP[i] = ipDBP[i] - ipABP[i] * acceptorCTCorr; } else { ipDBP[i] = Float.NaN; } } } // acceptor photoproduct correction if (accPhotoprCorrMenuItem.isSelected()) { for (int i = 0; i < ipABP.length; i++) { if (!Float.isNaN(ipDAP[i]) && !Float.isNaN(ipABP[i])) { ipDAP[i] = ipDAP[i] - ipABP[i] * acceptorPPCorr; } else { ipDAP[i] = Float.NaN; } } } // donor bleaching correction if (donorBlCorrMenuItem.isSelected()) { for (int i = 0; i < ipDAP.length; i++) { if (!Float.isNaN(ipDAP[i])) { ipDAP[i] = ipDAP[i] * donorBlCorr; } else { ipDAP[i] = Float.NaN; } } } for (int i = 0; i < ipDAP.length; i++) { ipDAP[i] = (float)1 - (ipDBP[i] / ipDAP[i]); } } else { if (accCrossTalkCorrMenuItem.isSelected()) { for (int i = 0; i < ipABP.length; i++) { if (!Float.isNaN(ipDBP[i]) && !Float.isNaN(ipABP[i])) { ipDBP[i] = ipDBP[i] - ipABP[i] * acceptorCTCorr; } else { ipDBP[i] = Float.NaN; } } } for (int i = 0; i < ipDBP.length; i++) { if (accCrossTalkCorrMenuItem.isSelected() || accPhotoprCorrMenuItem.isSelected()) { if (!Float.isNaN(ipDBP[i]) && !Float.isNaN(ipDAP[i]) && !Float.isNaN(ipABP[i])) { ipDAP[i] = (float)((double)donorBlCorr*((double)ipDAP[i] - ((double)partialBlCorr*(double)acceptorCTCorr+(double)acceptorPPCorr*((double)1-(double)partialBlCorr))*(double)ipABP[i]) - (double)partialBlCorr*(double)ipDBP[i]); } else { ipDAP[i] = Float.NaN; } } else { if (!Float.isNaN(ipDBP[i]) && !Float.isNaN(ipDAP[i])) { ipDAP[i] = (float)((double)donorBlCorr*(double)ipDAP[i] - (double)partialBlCorr*(double)ipDBP[i]); } else { ipDAP[i] = Float.NaN; } } } for (int i = 0; i < ipDAP.length; i++) { ipDAP[i] = (float)((double)1 - (((double)1-(double)partialBlCorr)*(double)ipDBP[i] / (double)ipDAP[i])); } } int width = ipDA.getWidth(); int height = ipDA.getHeight(); float[][] tiPoints = new float[width][height]; for (int i = 0; i < width; i++) { for (int j = 0; j < height; j++) { tiPoints[i][j] = ipDAP[width*j+i]; } } float iv = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { iv = tiPoints[x][y]; if (useAcceptorAsMask.isSelected() && (Float.isNaN(ipABP[width*y+x]) || ipABP[width*y+x] == 0)) { tiPoints[x][y] = Float.NaN; } } } FloatProcessor tiFp = new FloatProcessor(tiPoints); if (transferImage != null && transferImage.getProcessor() != null) { ColorModel cm = transferImage.getProcessor().getColorModel(); transferImage.setProcessor("Transfer image", tiFp); transferImage.getProcessor().setColorModel(cm); transferImage.updateAndDraw(); } else { transferImage = new ImagePlus("Transfer image", tiFp); transferImage.show(); } analyzer = new Analyzer(); resultsTable =Analyzer.getResultsTable(); resultsTable.setPrecision(3); resultsTable.incrementCounter(); int widthTi = transferImage.getWidth(); int heightTi = transferImage.getHeight(); if(currentlyProcessedFileName != null) { resultsTable.addLabel("File", currentlyProcessedFileName); } if (transferImage.getRoi() != null) { Roi roi = transferImage.getRoi(); int count = 0; int notNan = 0; for (int i=0; i<widthTi; i++) { for (int j=0; j<heightTi; j++) { if (roi.contains(i, j)) { count++; if (transferImage.getProcessor().getPixelValue(i, j) >= -1) { notNan++; } } } } resultsTable.addValue("Pixels", count); resultsTable.addValue("Not NaN p.", notNan); } else { int notNan = 0; for (int i=0; i<widthTi; i++) { for (int j=0; j<heightTi; j++) { if (transferImage.getProcessor().getPixelValue(i, j) >= -1) { notNan++; } } } resultsTable.addValue("Pixels", widthTi*heightTi); resultsTable.addValue("Not NaN p.", notNan); } ImageStatistics isMean = ImageStatistics.getStatistics(tiFp, Measurements.MEAN,null); resultsTable.addValue("Mean", (float)isMean.mean); ImageStatistics isMedian = ImageStatistics.getStatistics(tiFp, Measurements.MEDIAN,null); resultsTable.addValue("Median", (float)isMedian.median); ImageStatistics isStdDev = ImageStatistics.getStatistics(tiFp, Measurements.STD_DEV,null); resultsTable.addValue("Std. dev.", (float)isStdDev.stdDev); ImageStatistics isMinMax = ImageStatistics.getStatistics(tiFp, Measurements.MIN_MAX,null); resultsTable.addValue("Min", (float)isMinMax.min); resultsTable.addValue("Max", (float)isMinMax.max); if (transferImage.getRoi() != null) { donorBefore.setRoi(transferImage.getRoi()); donorAfter.setRoi(transferImage.getRoi()); if (acceptorBefore != null) { acceptorBefore.setRoi(transferImage.getRoi()); } } else { donorBefore.killRoi(); donorAfter.killRoi(); if (acceptorBefore != null) { acceptorBefore.killRoi(); } } ImageStatistics isMinMaxDB = ImageStatistics.getStatistics(donorBefore.getProcessor(), Measurements.MIN_MAX,null); resultsTable.addValue("Min (DB)", (float)isMinMaxDB.min); resultsTable.addValue("Max (DB)", (float)isMinMaxDB.max); ImageStatistics isDBMean = ImageStatistics.getStatistics(donorBefore.getProcessor(), Measurements.MEAN,null); resultsTable.addValue("Mean (DB)", (float)isDBMean.mean); ImageStatistics isMinMaxDA = ImageStatistics.getStatistics(donorAfter.getProcessor(), Measurements.MIN_MAX,null); resultsTable.addValue("Min (DA)", (float)isMinMaxDA.min); resultsTable.addValue("Max (DA)", (float)isMinMaxDA.max); ImageStatistics isDAMean = ImageStatistics.getStatistics(donorAfter.getProcessor(), Measurements.MEAN,null); resultsTable.addValue("Mean (DA)", (float)isDAMean.mean); ImageStatistics isABMean = null; if (acceptorBefore != null) { ImageStatistics isMinMaxAB = ImageStatistics.getStatistics(acceptorBefore.getProcessor(), Measurements.MIN_MAX,null); resultsTable.addValue("Min (AB)", (float)isMinMaxAB.min); resultsTable.addValue("Max (AB)", (float)isMinMaxAB.max); isABMean = ImageStatistics.getStatistics(acceptorBefore.getProcessor(), Measurements.MEAN,null); resultsTable.addValue("Mean (AB)", (float)isABMean.mean); } else { resultsTable.addValue("Min (AB)", (float)0); resultsTable.addValue("Max (AB)", (float)0); resultsTable.addValue("Mean (AB)", (float)0); } analyzer.displayResults(); analyzer.updateHeadings(); } } donorBefore.changes = false; donorAfter.changes = false; if(acceptorBefore != null) { acceptorBefore.changes = false; } if(acceptorAfter != null) { acceptorAfter.changes = false; } } else if (e.getActionCommand().equals("measureFretImage")) { float donorBlCorr = 1; float acceptorCTCorr = 0; float acceptorPPCorr = 0; if (transferImage == null) { logError("Transfer image required."); return; } resultsTable.incrementCounter(); int width = transferImage.getWidth(); int height = transferImage.getHeight(); if(currentlyProcessedFileName != null) { resultsTable.addLabel("File", currentlyProcessedFileName); } if (transferImage.getRoi() != null) { Roi roi = transferImage.getRoi(); int count = 0; int notNan = 0; for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (roi.contains(i, j)) { count++; if (transferImage.getProcessor().getPixelValue(i, j) >= -1) { notNan++; } } } } resultsTable.addValue("Pixels", count); resultsTable.addValue("Not NaN p.", notNan); } else { int notNan = 0; for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (transferImage.getProcessor().getPixelValue(i, j) >= -1) { notNan++; } } } resultsTable.addValue("Pixels", width*height); resultsTable.addValue("Not NaN p.", notNan); } ImageStatistics isMean = ImageStatistics.getStatistics(transferImage.getProcessor(), Measurements.MEAN,null); resultsTable.addValue("Mean", (float)isMean.mean); ImageStatistics isMedian = ImageStatistics.getStatistics(transferImage.getProcessor(), Measurements.MEDIAN,null); resultsTable.addValue("Median", (float)isMedian.median); ImageStatistics isStdDev = ImageStatistics.getStatistics(transferImage.getProcessor(), Measurements.STD_DEV,null); resultsTable.addValue("Std. dev.", (float)isStdDev.stdDev); ImageStatistics isMinMax = ImageStatistics.getStatistics(transferImage.getProcessor(), Measurements.MIN_MAX,null); resultsTable.addValue("Min", (float)isMinMax.min); resultsTable.addValue("Max", (float)isMinMax.max); if (transferImage.getRoi() != null) { donorBefore.setRoi(transferImage.getRoi()); donorAfter.setRoi(transferImage.getRoi()); if (acceptorBefore != null) { acceptorBefore.setRoi(transferImage.getRoi()); } } else { donorBefore.killRoi(); donorAfter.killRoi(); if (acceptorBefore != null) { acceptorBefore.killRoi(); } } ImageStatistics isMinMaxDB = ImageStatistics.getStatistics(donorBefore.getProcessor(), Measurements.MIN_MAX,null); resultsTable.addValue("Min (DB)", (float)isMinMaxDB.min); resultsTable.addValue("Max (DB)", (float)isMinMaxDB.max); ImageStatistics isDBMean = ImageStatistics.getStatistics(donorBefore.getProcessor(), Measurements.MEAN,null); resultsTable.addValue("Mean (DB)", (float)isDBMean.mean); ImageStatistics isMinMaxDA = ImageStatistics.getStatistics(donorAfter.getProcessor(), Measurements.MIN_MAX,null); resultsTable.addValue("Min (DA)", (float)isMinMaxDA.min); resultsTable.addValue("Max (DA)", (float)isMinMaxDA.max); ImageStatistics isDAMean = ImageStatistics.getStatistics(donorAfter.getProcessor(), Measurements.MEAN,null); resultsTable.addValue("Mean (DA)", (float)isDAMean.mean); ImageStatistics isABMean = null; if (acceptorBefore != null) { ImageStatistics isMinMaxAB = ImageStatistics.getStatistics(acceptorBefore.getProcessor(), Measurements.MIN_MAX,null); resultsTable.addValue("Min (AB)", (float)isMinMaxAB.min); resultsTable.addValue("Max (AB)", (float)isMinMaxAB.max); isABMean = ImageStatistics.getStatistics(acceptorBefore.getProcessor(), Measurements.MEAN,null); resultsTable.addValue("Mean (AB)", (float)isABMean.mean); } else { resultsTable.addValue("Min (AB)", (float)0); resultsTable.addValue("Max (AB)", (float)0); resultsTable.addValue("Mean (AB)", (float)0); } analyzer.displayResults(); analyzer.updateHeadings(); } else if (e.getActionCommand().equals("semiAutomaticProcessing")) { int choice = JOptionPane.showConfirmDialog(this, "Semi-automatic processing of images\n\nOpens and processes FRET images in a given directory. It works with\n"+ "Zeiss LSM images (tested with LSM 510 Version 4.0), which contain two\n"+ "channels:\n"+ "1. donor channel (before and after photobleaching)\n"+ "2. acceptor channel (before and after photobleaching)\n\n"+ "The upper left corner (1/6 x 1/6 of the image) is considered as background.\n"+ "Threshold settings, creation of FRET image and measurements have to be\n"+ "made manually.\n\n"+ "Every previously opened image and result window will be closed when you\n"+ "press \"Ok\".\n\n"+ "Press \"Ok\" to select the directory. To continue with the next "+ "image, do\nnot close any windows, just press the \"Next\" button.\n", "Semi-automatic processing of images", JOptionPane.OK_CANCEL_OPTION); if(choice == JOptionPane.YES_OPTION) { currentlyProcessedFile = 0; automaticallyProcessedFiles = null; currentlyProcessedFileName = null; WindowManager.closeAllWindows(); JFileChooser chooser = new JFileChooser(currentDirectory); chooser.setFileSelectionMode(JFileChooser.DIRECTORIES_ONLY); chooser.setDialogTitle("Select directory"); chooser.setAcceptAllFileFilterUsed(false); if (chooser.showOpenDialog(this) == JFileChooser.APPROVE_OPTION) { log("Processing files in directory: " + chooser.getSelectedFile()); currentDirectory = chooser.getSelectedFile().toString(); } else { log("Semi-automatic processing: no directory is selected."); return; } nextButton.setVisible(true); useLsmImages.setSelected(true); logScrollPane.setPreferredSize(new Dimension(10,10)); automaticallyProcessedFiles = chooser.getSelectedFile().listFiles(); processFile(0); } } else if (e.getActionCommand().equals("nextImage")) { if(transferImage != null) { transferImage.changes = false; transferImage.close(); } if(donorBefore != null) { donorBefore.changes = false; donorBefore.close(); } if(donorAfter != null) { donorAfter.changes = false; donorAfter.close(); } if(acceptorBefore != null) { acceptorBefore.changes = false; acceptorBefore.close(); } if(acceptorAfter != null) { acceptorAfter.changes = false; acceptorAfter.close(); } if(!useAcceptorAsMask.isSelected()) { IJ.selectWindow("Results"); WindowManager.putBehind(); if(WindowManager.getCurrentImage() != null) { WindowManager.getCurrentImage().close(); } } processFile(++currentlyProcessedFile); } else if (e.getActionCommand().equals("closeImages")) { if(transferImage != null) { transferImage.changes = false; transferImage.close(); } if(donorBefore != null) { donorBefore.changes = false; donorBefore.close(); } if(donorAfter != null) { donorAfter.changes = false; donorAfter.close(); } if(acceptorBefore != null) { acceptorBefore.changes = false; acceptorBefore.close(); } if(acceptorAfter != null) { acceptorAfter.changes = false; acceptorAfter.close(); } resetAll(); } else if (e.getActionCommand().equals("resetImages")) { resetAll(); } else if (e.getActionCommand().equals("help")) { if (helpWindow != null) { helpWindow.setVisible(false); helpWindow.dispose(); } helpWindow = new HelpWindow(this); helpWindow.setVisible(true); } else if (e.getActionCommand().equals("checkVersion")) { InputStream is = null; try{ URL url= new URL("http://www.biophys.dote.hu/accpbfret/version.txt"); byte[] buffer = new byte[4]; URLConnection urlCon = url.openConnection(); is = urlCon.getInputStream(); String ver = ""; while (is.read(buffer) != -1) { ver += new String(buffer); } float verf = Float.parseFloat(ver); if(verf > version){ int choice = JOptionPane.showConfirmDialog(this, "There is a newer version on the AccPbFRET homepage.\n" + "Your version: " + version + "\n" + "New version: " + ver + "\n" + "You can download it from: http://www.biophys.dote.hu/accpbfret/ \n" + "Do you want to download it now?", "Checking for latest version", JOptionPane.YES_NO_OPTION); if(choice == JOptionPane.YES_OPTION) { IJ.runPlugIn("ij.plugin.BrowserLauncher", "http://www.biophys.dote.hu/accpbfret/"); } } else { JOptionPane.showMessageDialog(this, "There is no newer version on the AccPbFRET homepage.\n" + "Your version: " + version + "\n" + "Version on the server: " + ver, "Checking for latest version", JOptionPane.INFORMATION_MESSAGE); } } catch (Exception e1) { logException(e1.toString(), e1); } finally { try { if (is != null) } catch (Exception e2) { logException(e2.getMessage(), e2); } } } else if (e.getActionCommand().equals("about")) { JOptionPane optionPane = new JOptionPane(); optionPane.setMessage("AccPbFRET - an ImageJ plugin for analysis of acceptor photobleaching FRET images\n" + "Homepage: http://www.biophys.dote.hu/accpbfret/\n" + "Written by: János Roszik (janosr@med.unideb.hu), János Szöllõsi (szollo@med.unideb.hu),\n" + "and György Vereb (vereb@med.unideb.hu)\n" + "Version: " + version + " (" + lastModified + ")\n" + "The plugin was tested with ImageJ version " + imageJVersion + " using Java " + javaVersion + ".\n\n" + "If you are using the plugin, please cite the following paper:\n" + "Roszik J, Szollosi J, Vereb G: AccPbFRET: an ImageJ plugin for semi-automatic, fully corrected \n" + "analysis of acceptor photobleaching FRET images. BMC Bioinformatics 2008, 9:346"); optionPane.setMessageType(JOptionPane.INFORMATION_MESSAGE); JDialog dialog = optionPane.createDialog(this, "About"); dialog.setVisible(true); } } catch (Throwable t) { logException(t.toString(), t); } } private void processFile(int currentFile) { resetAllButtonColors(); if(currentFile >= automaticallyProcessedFiles.length) { log("Processing files has been finished."); nextButton.setVisible(false); logScrollPane.setPreferredSize(new Dimension(10,10)); IJ.selectWindow("Results"); currentlyProcessedFile = 0; automaticallyProcessedFiles = null; currentlyProcessedFileName = null; return; } if(!automaticallyProcessedFiles[currentFile].isFile() || !(automaticallyProcessedFiles[currentFile].getName().endsWith(".lsm") || automaticallyProcessedFiles[currentFile].getName().endsWith(".LSM"))) { processFile(++currentlyProcessedFile); return; } log("Current file is: " + automaticallyProcessedFiles[currentFile].getName()); currentlyProcessedFileName = automaticallyProcessedFiles[currentFile].getName(); (new Opener()).open(automaticallyProcessedFiles[currentFile].getAbsolutePath()); WindowManager.putBehind(); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"split")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"setAcceptorAfter")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"lutSpectrum")); WindowManager.putBehind(); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"setDonorAfter")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"lutSpectrum")); WindowManager.putBehind(); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"setAcceptorBefore")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"lutSpectrum")); WindowManager.putBehind(); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"setDonorBefore")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"lutSpectrum")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"registerImages")); donorBefore.setRoi(new Roi(0, 0, donorBefore.getWidth()/6, donorBefore.getHeight()/6, null)); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"copyRoi")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"subtractDonorBefore")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"subtractDonorAfter")); if (partialBlCorrMenuItem.isSelected()) { this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"subtractAcceptorBefore")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"subtractAcceptorAfter")); } else if (useAcceptorAsMask.isSelected()) { this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"subtractAcceptorBefore")); } donorBefore.setRoi(new Roi(0, 0, donorBefore.getWidth()/6, donorBefore.getHeight()/6, null)); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"copyRoi")); donorBefore.getProcessor().setValue(0); donorBefore.getProcessor().fill(); donorAfter.getProcessor().setValue(0); donorAfter.getProcessor().fill(); if (partialBlCorrMenuItem.isSelected()) { acceptorBefore.getProcessor().setValue(0); acceptorBefore.getProcessor().fill(); acceptorAfter.getProcessor().setValue(0); acceptorAfter.getProcessor().fill(); } else if (useAcceptorAsMask.isSelected()) { acceptorBefore.getProcessor().setValue(0); acceptorBefore.getProcessor().fill(); } donorBefore.killRoi(); donorAfter.killRoi(); if (partialBlCorrMenuItem.isSelected()) { acceptorBefore.killRoi(); acceptorAfter.killRoi(); } else if (useAcceptorAsMask.isSelected()) { acceptorBefore.killRoi(); } this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"smoothDBefore")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"smoothDAfter")); if (partialBlCorrMenuItem.isSelected()) { this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"smoothABefore")); this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"smoothAAfter")); } else if (useAcceptorAsMask.isSelected()) { this.actionPerformed(new ActionEvent(this,ActionEvent.ACTION_PERFORMED,"smoothABefore")); } } private void resetAll() { donorBefore = null; donorBeforeSave = null; donorAfter = null; donorAfterSave = null; acceptorBefore = null; acceptorBeforeSave = null; acceptorAfter = null; acceptorAfterSave = null; setDonorBeforeButton.setBackground(originalButtonColor); setDonorAfterButton.setBackground(originalButtonColor); setAcceptorBeforeButton.setBackground(originalButtonColor); setAcceptorAfterButton.setBackground(originalButtonColor); registerButton.setBackground(originalButtonColor); subtractDonorBeforeButton.setBackground(originalButtonColor); subtractDonorAfterButton.setBackground(originalButtonColor); subtractAcceptorBeforeButton.setBackground(originalButtonColor); subtractAcceptorAfterButton.setBackground(originalButtonColor); smoothDonorBeforeButton.setBackground(originalButtonColor); smoothDonorAfterButton.setBackground(originalButtonColor); smoothAcceptorBeforeButton.setBackground(originalButtonColor); smoothAcceptorAfterButton.setBackground(originalButtonColor); thresholdDonorBeforeButton.setBackground(originalButtonColor); thresholdDonorAfterButton.setBackground(originalButtonColor); thresholdAcceptorBeforeButton.setBackground(originalButtonColor); thresholdAcceptorAfterButton.setBackground(originalButtonColor); calculateDBCorrButton.setBackground(originalButtonColor); calculateAccCTCorrButton.setBackground(originalButtonColor); calculateAccPPCorrButton.setBackground(originalButtonColor); calculatePartialBlCorrButton.setBackground(originalButtonColor); nextButton.setVisible(false); logScrollPane.setPreferredSize(new Dimension(10,10)); currentlyProcessedFile = 0; automaticallyProcessedFiles = null; currentlyProcessedFileName = null; } private void resetAllButtonColors() { setDonorBeforeButton.setBackground(originalButtonColor); setDonorAfterButton.setBackground(originalButtonColor); setAcceptorBeforeButton.setBackground(originalButtonColor); setAcceptorAfterButton.setBackground(originalButtonColor); registerButton.setBackground(originalButtonColor); subtractDonorBeforeButton.setBackground(originalButtonColor); subtractDonorAfterButton.setBackground(originalButtonColor); subtractAcceptorBeforeButton.setBackground(originalButtonColor); subtractAcceptorAfterButton.setBackground(originalButtonColor); smoothDonorBeforeButton.setBackground(originalButtonColor); smoothDonorAfterButton.setBackground(originalButtonColor); smoothAcceptorBeforeButton.setBackground(originalButtonColor); smoothAcceptorAfterButton.setBackground(originalButtonColor); thresholdDonorBeforeButton.setBackground(originalButtonColor); thresholdDonorAfterButton.setBackground(originalButtonColor); thresholdAcceptorBeforeButton.setBackground(originalButtonColor); thresholdAcceptorAfterButton.setBackground(originalButtonColor); calculateDBCorrButton.setBackground(originalButtonColor); calculateAccCTCorrButton.setBackground(originalButtonColor); calculateAccPPCorrButton.setBackground(originalButtonColor); calculatePartialBlCorrButton.setBackground(originalButtonColor); } public void log(String text) { try{ log.getDocument().insertString(log.getDocument().getLength(), "\n" + format.format(new Date()) + " " + text, log.getStyle("BLACK")); log.setCaretPosition(log.getDocument().getLength()); } catch (javax.swing.text.BadLocationException e) {} } public void logError(String text) { try{ log.getDocument().insertString(log.getDocument().getLength(), "\n" + format.format(new Date()) + " ERROR: " + text, log.getStyle("RED")); log.setCaretPosition(log.getDocument().getLength()); } catch (javax.swing.text.BadLocationException e) {} } public void logWarning(String text) { try{ log.getDocument().insertString(log.getDocument().getLength(), "\n" + format.format(new Date()) + " WARNING: " + text, log.getStyle("BLUE")); log.setCaretPosition(log.getDocument().getLength()); } catch (javax.swing.text.BadLocationException e) {} } public void logException(String message, Throwable t) { try{ if (debugMenuItem.isSelected()) { StringWriter sw = new StringWriter(); PrintWriter pw = new PrintWriter(sw); t.printStackTrace(pw); pw.flush(); log.getDocument().insertString(log.getDocument().getLength(), "\n" + format.format(new Date()) + " ERROR: " + sw.toString(), log.getStyle("RED")); log.setCaretPosition(log.getDocument().getLength()); } else { log.getDocument().insertString(log.getDocument().getLength(), "\n" + format.format(new Date()) + " ERROR: " + message, log.getStyle("RED")); log.setCaretPosition(log.getDocument().getLength()); } } catch (javax.swing.text.BadLocationException e) {} } public void exit() { int choice = JOptionPane.showConfirmDialog(this, "Do you really want to exit?", "Exit", JOptionPane.OK_CANCEL_OPTION); if(choice == JOptionPane.YES_OPTION) { if (shiftDialog != null) { shiftDialog.setVisible(false); shiftDialog.dispose(); } if (donorBlCorrDialog != null) { donorBlCorrDialog.setVisible(false); donorBlCorrDialog.dispose(); } if (acceptorCTCorrDialog != null) { acceptorCTCorrDialog.setVisible(false); acceptorCTCorrDialog.dispose(); } if (applyMaskDialog != null) { applyMaskDialog.setVisible(false); applyMaskDialog.dispose(); } if (calculateImgRatioDialog != null) { calculateImgRatioDialog.setVisible(false); calculateImgRatioDialog.dispose(); } if (helpWindow != null) { helpWindow.setVisible(false); helpWindow.dispose(); } setVisible(false); dispose(); } } public void windowClosing(WindowEvent e){ exit(); } public void windowActivated(WindowEvent e){} public void windowClosed(WindowEvent e){} public void windowDeactivated(WindowEvent e){} public void windowDeiconified(WindowEvent e){} public void windowIconified(WindowEvent e){} public void windowOpened(WindowEvent e){} public ImagePlus getDonorBefore() { return donorBefore; } public ImagePlus getDonorAfter() { return donorAfter; } public void setBleachingCorrection(String value) { donorBlCorrField.setText(value); } public void setCrosstalkCorrection(String value) { accCrossTalkCorrField.setText(value); } public void setPhotoproductCorrection(String value) { accPhotoprCorrField.setText(value); } public static void main(String args[]) { new AccPbFRET_Plugin(); } } class ApplyMaskDialog extends JDialog implements ActionListener{ private AccPbFRET_Plugin accBlWindow; private ImagePlus toMaskImg, maskImg; private JPanel panel; private JButton setToMaskImgButton, setMaskImgButton, createImagesButton; public ApplyMaskDialog(AccPbFRET_Plugin accBlWindow) { setTitle("Apply mask to an image"); this.accBlWindow = accBlWindow; setDefaultCloseOperation(JFrame.DISPOSE_ON_CLOSE); setModal(false); createDialogGui(); Dimension screen = Toolkit.getDefaultToolkit().getScreenSize(); setSize(275, 240); setLocation((screen.width - getWidth())/2, (screen.height - getHeight())/2); } public void createDialogGui() { GridBagLayout gridbaglayout = new GridBagLayout(); GridBagConstraints gc = new GridBagConstraints(); panel = new JPanel(); panel.setLayout(gridbaglayout); gc.insets = new Insets(2,2,6,2); gc.fill = GridBagConstraints.BOTH; gc.gridwidth = 2; gc.gridx = 0; gc.gridy = 0; JLabel infoLabel = new JLabel("<html><center>After setting an image to mask and a mask image (with NaN background pixels), two images will be created. The first one will contain the pixles which are not NaN in the mask, and the second one the others.</center></html>"); panel.add(infoLabel, gc); gc.insets = new Insets(2,2,2,2); gc.gridx = 0; gc.gridy = 1; setToMaskImgButton = new JButton("Set image to be masked"); setToMaskImgButton.addActionListener(this); setToMaskImgButton.setActionCommand("setImageToMask"); panel.add(setToMaskImgButton, gc); gc.gridx = 0; gc.gridy = 2; setMaskImgButton = new JButton("Set mask image (with NaN bg. pixels)"); setMaskImgButton.addActionListener(this); setMaskImgButton.setActionCommand("setMaskImage"); panel.add(setMaskImgButton, gc); gc.gridx = 0; gc.gridy = 3; createImagesButton = new JButton("Create masked images"); createImagesButton.addActionListener(this); createImagesButton.setActionCommand("createImages"); panel.add(createImagesButton, gc); getContentPane().add(panel); } public void actionPerformed(ActionEvent e) { try { if (e.getActionCommand().equals("setImageToMask")) { toMaskImg = WindowManager.getCurrentImage(); if (toMaskImg == null) { accBlWindow.logError("No image is selected. (Masking)"); return; } if (toMaskImg.getImageStackSize() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+toMaskImg.getImageStackSize()+"). Please split it into parts. (Masking)"); toMaskImg = null; return; } else if (toMaskImg.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+toMaskImg.getNSlices()+"). Please split it into parts. (Masking)"); toMaskImg = null; return; } toMaskImg.setTitle("Image to mask - " + new Date().toString()); new ImageConverter(toMaskImg).convertToGray32(); setToMaskImgButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setMaskImage")) { maskImg = WindowManager.getCurrentImage(); if (maskImg == null) { accBlWindow.logError("No image is selected. (Masking)"); return; } if (maskImg.getImageStackSize() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+maskImg.getImageStackSize()+"). Please split it into parts. (Masking)"); maskImg = null; return; } else if (maskImg.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+maskImg.getNSlices()+"). Please split it into parts. (Masking)"); maskImg = null; return; } maskImg.setTitle("Mask image - " + new Date().toString()); new ImageConverter(maskImg).convertToGray32(); setMaskImgButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("createImages")) { if (toMaskImg == null) { accBlWindow.logError("No image to mask is set. (Masking)"); return; } else if (maskImg == null) { accBlWindow.logError("No mask image is set. (Masking)"); return; } ImageProcessor ipTM = toMaskImg.getProcessor(); ImageProcessor ipM = maskImg.getProcessor(); float[] ipTMP = (float[])ipTM.getPixels(); float[] ipMP = (float[])ipM.getPixels(); int width = ipTM.getWidth(); int height = ipTM.getHeight(); float[][] img1Points = new float[width][height]; float[][] img2Points = new float[width][height]; for (int i = 0; i < width; i++) { for (int j = 0; j < height; j++) { if (!Float.isNaN(ipMP[width*j+i])) { img1Points[i][j] = ipTMP[width*j+i]; img2Points[i][j] = Float.NaN; } else { img1Points[i][j] = Float.NaN; img2Points[i][j] = ipTMP[width*j+i]; } } } FloatProcessor fp1 = new FloatProcessor(img1Points); FloatProcessor fp2 = new FloatProcessor(img2Points); ImagePlus img2 = new ImagePlus("Masked image 2 (pixels outside the mask)", fp2); img2.show(); ImagePlus img1 = new ImagePlus("Masked image 1 (pixels in the mask)", fp1); img1.show(); } } catch (Throwable t) { accBlWindow.logException(t.toString(), t); } } } class CalculateImgRatioDialog extends JDialog implements ActionListener{ private AccPbFRET_Plugin accBlWindow; private ImagePlus firstImg, secondImg; private JPanel panel; private JButton setFirstImgButton, setSecondImgButton, createRatioImageButton; private JCheckBox useMainWindowImages; public CalculateImgRatioDialog (AccPbFRET_Plugin accBlWindow) { setTitle("Calculate ratio of two images"); this.accBlWindow = accBlWindow; setDefaultCloseOperation(JFrame.DISPOSE_ON_CLOSE); setModal(false); createDialogGui(); Dimension screen = Toolkit.getDefaultToolkit().getScreenSize(); setSize(275, 250); setLocation((screen.width - getWidth())/2, (screen.height - getHeight())/2); } public void createDialogGui() { GridBagLayout gridbaglayout = new GridBagLayout(); GridBagConstraints gc = new GridBagConstraints(); panel = new JPanel(); panel.setLayout(gridbaglayout); gc.insets = new Insets(2,2,6,2); gc.fill = GridBagConstraints.BOTH; gc.gridwidth = 2; gc.gridx = 0; gc.gridy = 0; JLabel infoLabel = new JLabel("<html><center>After setting the two images and pressing the \"Create ratio image\" button, the ratio of the images (image 1 / image 2) will be calculated pixel-by-pixel and displayed as a new 32 bit image.</center></html>"); panel.add(infoLabel, gc); gc.insets = new Insets(2,2,2,2); gc.gridx = 0; gc.gridy = 1; useMainWindowImages = new JCheckBox("use images of the main window (1a/1b)", false); useMainWindowImages.setActionCommand("useMainWindowImages"); useMainWindowImages.addActionListener(this); useMainWindowImages.setToolTipText("<html>If this checkbox is checked, donor before and after bleaching<BR>images which are set in the main window will be used as<BR>image 1 and image 2.</html>"); panel.add(useMainWindowImages, gc); gc.gridx = 0; gc.gridy = 2; setFirstImgButton = new JButton("Set first image (numerator)"); setFirstImgButton.addActionListener(this); setFirstImgButton.setActionCommand("setFirstImage"); panel.add(setFirstImgButton, gc); gc.gridx = 0; gc.gridy = 3; setSecondImgButton = new JButton("Set second image (denominator)"); setSecondImgButton.addActionListener(this); setSecondImgButton.setActionCommand("setSecondImage"); panel.add(setSecondImgButton, gc); gc.gridx = 0; gc.gridy = 4; createRatioImageButton = new JButton("Create ratio image"); createRatioImageButton.addActionListener(this); createRatioImageButton.setActionCommand("createRatioImage"); panel.add(createRatioImageButton, gc); getContentPane().add(panel); } public void actionPerformed(ActionEvent e) { try { if (e.getActionCommand().equals("setFirstImage")) { firstImg = WindowManager.getCurrentImage(); if (firstImg == null) { accBlWindow.logError("No image is selected. (Ratio)"); return; } if (firstImg.getImageStackSize() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+firstImg.getImageStackSize()+"). Please split it into parts. (Ratio)"); firstImg = null; return; } else if (firstImg.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+firstImg.getNSlices()+"). Please split it into parts. (Ratio)"); firstImg = null; return; } firstImg.setTitle("Image 1 - " + new Date().toString()); new ImageConverter(firstImg).convertToGray32(); setFirstImgButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setSecondImage")) { secondImg = WindowManager.getCurrentImage(); if (secondImg == null) { accBlWindow.logError("No image is selected. (Ratio)"); return; } if (secondImg.getImageStackSize() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+secondImg.getImageStackSize()+"). Please split it into parts. (Ratio)"); secondImg = null; return; } else if (secondImg.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+secondImg.getNSlices()+"). Please split it into parts. (Ratio)"); secondImg = null; return; } secondImg.setTitle("Image 2 - " + new Date().toString()); new ImageConverter(secondImg).convertToGray32(); setSecondImgButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("useMainWindowImages")) { if (useMainWindowImages.isSelected()) { setFirstImgButton.setEnabled(false); setSecondImgButton.setEnabled(false); } else { setFirstImgButton.setEnabled(true); setSecondImgButton.setEnabled(true); } } else if (e.getActionCommand().equals("createRatioImage")) { ImageProcessor ip1 = null; ImageProcessor ip2 = null; if (!useMainWindowImages.isSelected()) { if (firstImg == null) { accBlWindow.logError("No image 1 is set. (Ratio)"); return; } else if (secondImg == null) { accBlWindow.logError("No image 2 is set. (Ratio)"); return; } ip1 = firstImg.getProcessor(); ip2 = secondImg.getProcessor(); } else { if (accBlWindow.getDonorBefore() == null) { accBlWindow.logError("No donor before bleaching image is set. (Ratio)"); return; } else if (accBlWindow.getDonorAfter() == null) { accBlWindow.logError("No donor after image is set. (Ratio)"); return; } ip1 = accBlWindow.getDonorBefore().getProcessor(); ip2 = accBlWindow.getDonorAfter().getProcessor(); } float[] ip1P = (float[])ip1.getPixels(); float[] ip2P = (float[])ip2.getPixels(); int width = ip1.getWidth(); int height = ip1.getHeight(); float[][] ratioImgPoints = new float[width][height]; for (int i = 0; i < width; i++) { for (int j = 0; j < height; j++) { ratioImgPoints[i][j] = ip1P[width*j+i] / ip2P[width*j+i]; } } FloatProcessor fp = new FloatProcessor(ratioImgPoints); ImagePlus ratioImg = new ImagePlus("Ratio of images", fp); ratioImg.show(); } } catch (Throwable t) { accBlWindow.logException(t.toString(), t); } } } class ShiftDialog extends JDialog implements ActionListener{ private AccPbFRET_Plugin accBlWindow; JPanel panel; JButton leftButton, rightButton, upButton, downButton; JButton cancelButton = new JButton("Close"); public ShiftDialog(AccPbFRET_Plugin accBlWindow) { setTitle("32bit image shifter"); this.accBlWindow = accBlWindow; setDefaultCloseOperation(JFrame.HIDE_ON_CLOSE); setModal(false); createDialogGui(); getRootPane().setDefaultButton(cancelButton); Dimension screen = Toolkit.getDefaultToolkit().getScreenSize(); setSize(150, 150); setLocation((screen.width - getWidth())/2, (screen.height - getHeight())/2); } public void createDialogGui() { GridBagLayout gridbaglayout = new GridBagLayout(); GridBagConstraints gc = new GridBagConstraints(); panel = new JPanel(); panel.setLayout(gridbaglayout); gc.insets = new Insets(0,0,0,0); gc.fill = GridBagConstraints.BOTH; gc.gridwidth = 1; gc.gridx = 1; gc.gridy = 0; upButton = new JButton("^"); upButton.addActionListener(this); upButton.setActionCommand("up"); panel.add(upButton, gc); gc.gridx = 0; gc.gridy = 1; leftButton = new JButton("<"); leftButton.addActionListener(this); leftButton.setActionCommand("left"); panel.add(leftButton, gc); gc.gridx = 2; gc.gridy = 1; rightButton = new JButton(">"); rightButton.addActionListener(this); rightButton.setActionCommand("right"); panel.add(rightButton, gc); gc.insets = new Insets(2,2,2,2); gc.gridx = 1; gc.gridy = 2; downButton = new JButton("v"); downButton.addActionListener(this); downButton.setActionCommand("down"); panel.add(downButton, gc); gc.insets = new Insets(0,0,4,0); gc.gridwidth = 3; gc.gridx = 0; gc.gridy = 3; panel.add(cancelButton, gc); cancelButton.addActionListener(this); cancelButton.setActionCommand("cancel"); getContentPane().add(panel); } public void actionPerformed(ActionEvent e) { try { if (e.getActionCommand().equals("cancel")) { setVisible(false); } else if (e.getActionCommand().equals("up")) { ImagePlus image = WindowManager.getCurrentImage(); shiftUp(image, 1); } else if (e.getActionCommand().equals("down")) { ImagePlus image = WindowManager.getCurrentImage(); shiftDown(image, 1); } else if (e.getActionCommand().equals("left")) { ImagePlus image = WindowManager.getCurrentImage(); shiftLeft(image, 1); } else if (e.getActionCommand().equals("right")) { ImagePlus image = WindowManager.getCurrentImage(); shiftRight(image, 1); } } catch (Throwable t) { accBlWindow.logException(t.toString(), t); } } public void shiftUp(ImagePlus image, int value){ if (image == null) { return; } int width = image.getWidth(); int height = image.getHeight(); FloatProcessor fp = (FloatProcessor)image.getProcessor(); ColorModel cm = fp.getColorModel(); float[] fpPixels = (float[])fp.getPixels(); float[][] fpPixels2 = new float[width][height]; for (int i = 0; i < width; i++) { for (int j = 0; j < height-value; j++) { fpPixels2[i][j] = fpPixels[width*(j+value)+i]; } } FloatProcessor newFp = new FloatProcessor(fpPixels2); newFp.setColorModel(cm); image.setProcessor(image.getTitle(), newFp); image.updateAndDraw(); } public void shiftDown(ImagePlus image, int value){ if (image == null) { return; } int width = image.getWidth(); int height = image.getHeight(); FloatProcessor fp = (FloatProcessor)image.getProcessor(); ColorModel cm = fp.getColorModel(); float[] fpPixels = (float[])fp.getPixels(); float[][] fpPixels2 = new float[width][height]; for (int i = 0; i < width; i++) { for (int j = value; j < height; j++) { fpPixels2[i][j] = fpPixels[width*(j-value)+i]; } } FloatProcessor newFp = new FloatProcessor(fpPixels2); newFp.setColorModel(cm); image.setProcessor(image.getTitle(), newFp); image.updateAndDraw(); } public void shiftLeft(ImagePlus image, int value){ if (image == null) { return; } int width = image.getWidth(); int height = image.getHeight(); FloatProcessor fp = (FloatProcessor)image.getProcessor(); ColorModel cm = fp.getColorModel(); float[] fpPixels = (float[])fp.getPixels(); float[][] fpPixels2 = new float[width][height]; for (int i = 0; i < width-value; i++) { for (int j = 0; j < height; j++) { fpPixels2[i][j] = fpPixels[width*j+(i+value)]; } } FloatProcessor newFp = new FloatProcessor(fpPixels2); newFp.setColorModel(cm); image.setProcessor(image.getTitle(), newFp); image.updateAndDraw(); } public void shiftRight(ImagePlus image, int value){ if (image == null) { return; } int width = image.getWidth(); int height = image.getHeight(); FloatProcessor fp = (FloatProcessor)image.getProcessor(); ColorModel cm = fp.getColorModel(); float[] fpPixels = (float[])fp.getPixels(); float[][] fpPixels2 = new float[width][height]; for (int i = value; i < width; i++) { for (int j = 0; j < height; j++) { fpPixels2[i][j] = fpPixels[width*j+(i-value)]; } } FloatProcessor newFp = new FloatProcessor(fpPixels2); newFp.setColorModel(cm); image.setProcessor(image.getTitle(), newFp); image.updateAndDraw(); } } class DonorBlCorrDialog extends JDialog implements ActionListener{ private AccPbFRET_Plugin accBlWindow; private ImagePlus donorCBefore, donorCAfter; private JPanel panel; private JButton setBeforeButton, setAfterButton, registerButton; private JButton setBeforeThresholdButton, setAfterThresholdButton, calculateButton, setButton; private JButton subtractBeforeButton, subtractAfterButton; private JButton resetButton; private ButtonGroup buttonGroup; private JRadioButton averagesButton, quotientsButton; private JLabel mode1ResultLabel, mode2ResultLabel; private JCheckBox showBlCImagesCB; public DonorBlCorrDialog(AccPbFRET_Plugin accBlWindow) { setTitle("Donor bleaching correction factor"); this.accBlWindow = accBlWindow; setDefaultCloseOperation(JFrame.DISPOSE_ON_CLOSE); setModal(false); createDialogGui(); Dimension screen = Toolkit.getDefaultToolkit().getScreenSize(); setSize(300, 410); setLocation((screen.width - getWidth())/2, (screen.height - getHeight())/2); } public void createDialogGui() { GridBagLayout gridbaglayout = new GridBagLayout(); GridBagConstraints gc = new GridBagConstraints(); panel = new JPanel(); panel.setLayout(gridbaglayout); gc.insets = new Insets(2,2,6,2); gc.fill = GridBagConstraints.BOTH; gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 0; JLabel infoLabel = new JLabel("<html><center>This factor is calculated based on images of the donor channel of a donor only labeled sample, before and after photobleaching.</center></html>"); panel.add(infoLabel, gc); gc.insets = new Insets(2,2,2,2); gc.gridwidth = 3; gc.gridx = 0; gc.gridy = 1; setBeforeButton = new JButton("Set donor before bleaching (donor only)"); setBeforeButton.addActionListener(this); setBeforeButton.setActionCommand("setCBefore"); panel.add(setBeforeButton, gc); gc.gridx = 0; gc.gridy = 2; setAfterButton = new JButton("Set donor after bleaching (donor only)"); setAfterButton.addActionListener(this); setAfterButton.setActionCommand("setCAfter"); panel.add(setAfterButton, gc); gc.gridx = 0; gc.gridy = 3; registerButton = new JButton("Register images"); registerButton.addActionListener(this); registerButton.setActionCommand("registerCImages"); panel.add(registerButton, gc); gc.gridx = 0; gc.gridy = 4; subtractBeforeButton = new JButton("Subtract background of donor before"); subtractBeforeButton.addActionListener(this); subtractBeforeButton.setActionCommand("subtractCBefore"); panel.add(subtractBeforeButton, gc); gc.gridx = 0; gc.gridy = 5; subtractAfterButton = new JButton("Subtract background of donor after"); subtractAfterButton.addActionListener(this); subtractAfterButton.setActionCommand("subtractCAfter"); panel.add(subtractAfterButton, gc); gc.gridx = 0; gc.gridy = 6; setBeforeThresholdButton = new JButton("Set donor before threshold"); setBeforeThresholdButton.addActionListener(this); setBeforeThresholdButton.setActionCommand("setCBeforeThreshold"); panel.add(setBeforeThresholdButton, gc); gc.gridx = 0; gc.gridy = 7; setAfterThresholdButton = new JButton("Set donor after threshold"); setAfterThresholdButton.addActionListener(this); setAfterThresholdButton.setActionCommand("setCAfterThreshold"); panel.add(setAfterThresholdButton, gc); gc.gridx = 0; gc.gridy = 8; gc.gridheight = 2; JPanel radioPanel = new JPanel(); GridBagLayout gbl = new GridBagLayout(); GridBagConstraints gcr = new GridBagConstraints(); radioPanel.setLayout(gbl); gcr.insets = new Insets(0,4,4,4); gcr.fill = GridBagConstraints.BOTH; JLabel modeLabel = new JLabel("Mode:"); JLabel resultLabel = new JLabel("Result:"); mode1ResultLabel = new JLabel("", JLabel.CENTER); mode2ResultLabel = new JLabel("", JLabel.CENTER); quotientsButton = new JRadioButton("point-by-point"); quotientsButton.setToolTipText("The factor is the averaged ratio of corresponding pixel values in the donor before and after photobleaching images."); averagesButton = new JRadioButton("average pixels"); averagesButton.setToolTipText("The factor is the ratio of the gated pixel averages in the donor before and after photobleaching images."); quotientsButton.setSelected(true); buttonGroup = new ButtonGroup(); buttonGroup.add(quotientsButton); buttonGroup.add(averagesButton); gcr.gridx = 0; gcr.gridy = 0; radioPanel.add(modeLabel, gcr); gcr.gridx = 1; gcr.gridy = 0; radioPanel.add(quotientsButton, gcr); gcr.gridx = 2; gcr.gridy = 0; radioPanel.add(averagesButton, gcr); gcr.gridx = 0; gcr.gridy = 1; radioPanel.add(resultLabel, gcr); gcr.gridx = 1; gcr.gridy = 1; radioPanel.add(mode1ResultLabel, gcr); gcr.gridx = 2; gcr.gridy = 1; radioPanel.add(mode2ResultLabel, gcr); panel.add(radioPanel, gc); gc.gridx = 0; gc.gridy = 10; gc.gridheight = 1; showBlCImagesCB = new JCheckBox("show correction image (for manual calc.)"); panel.add(showBlCImagesCB, gc); gc.gridwidth = 1; gc.gridheight = 1; gc.gridx = 0; gc.gridy = 11; calculateButton = new JButton("Calculate"); calculateButton.addActionListener(this); calculateButton.setActionCommand("calculate"); panel.add(calculateButton, gc); gc.gridx = 1; gc.gridy = 11; setButton = new JButton("Set selected"); setButton.addActionListener(this); setButton.setActionCommand("setfactor"); panel.add(setButton, gc); gc.gridx = 2; gc.gridy = 11; resetButton = new JButton("Reset"); resetButton.addActionListener(this); resetButton.setActionCommand("reset"); panel.add(resetButton, gc); getContentPane().add(panel); } public void actionPerformed(ActionEvent e) { try { if (e.getActionCommand().equals("reset")) { donorCBefore = null; donorCAfter = null; setBeforeButton.setBackground(accBlWindow.originalButtonColor); setAfterButton.setBackground(accBlWindow.originalButtonColor); setBeforeThresholdButton.setBackground(accBlWindow.originalButtonColor); setAfterThresholdButton.setBackground(accBlWindow.originalButtonColor); calculateButton.setBackground(accBlWindow.originalButtonColor); setButton.setBackground(accBlWindow.originalButtonColor); subtractBeforeButton.setBackground(accBlWindow.originalButtonColor); subtractAfterButton.setBackground(accBlWindow.originalButtonColor); registerButton.setBackground(accBlWindow.originalButtonColor); mode1ResultLabel.setText(""); mode2ResultLabel.setText(""); } else if (e.getActionCommand().equals("setCBefore")) { donorCBefore = WindowManager.getCurrentImage(); if (donorCBefore == null) { accBlWindow.logError("No image is selected. (bl. corr.)"); return; } if (donorCBefore.getNChannels() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+donorCBefore.getNChannels()+"). Please split it into parts. (bl. corr.)"); donorCBefore = null; return; } else if (donorCBefore.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+donorCBefore.getNSlices()+"). Please split it into parts. (bl. corr.)"); donorCBefore = null; return; } if (donorCBefore != null && donorCAfter != null && donorCBefore.equals(donorCAfter)) { accBlWindow.logError("The two images must not be the same. Please select and set an other image. (bl. corr.)"); donorCBefore.setTitle(""); donorCBefore = null; return; } donorCBefore.setTitle("Donor before bleaching (bl. corr.) - " + new Date().toString()); new ImageConverter(donorCBefore).convertToGray32(); setBeforeButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setCAfter")) { donorCAfter = WindowManager.getCurrentImage(); if (donorCAfter == null) { accBlWindow.logError("No image is selected. (bl. corr.)"); return; } if (donorCAfter.getNChannels() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+donorCAfter.getNChannels()+"). Please split it into parts. (bl. corr.)"); donorCAfter = null; return; } else if (donorCAfter.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+donorCAfter.getNSlices()+"). Please split it into parts. (bl. corr.)"); donorCAfter = null; return; } if (donorCBefore != null && donorCAfter != null && donorCBefore.equals(donorCAfter)) { accBlWindow.logError("The two images must not be the same. Please select and set an other image. (bl. corr.)"); donorCAfter.setTitle(""); donorCAfter = null; return; } donorCAfter.setTitle("Donor after bleaching (bl. corr.) - " + new Date().toString()); new ImageConverter(donorCAfter).convertToGray32(); setAfterButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("subtractCBefore")) { if (donorCBefore == null) { accBlWindow.logError("No image is set as donor before bleaching. (bl. corr.)"); return; } else if (donorCBefore.getRoi() == null) { accBlWindow.logError("No ROI is defined for donor before bleaching. (bl. corr.)"); return; } ImageProcessor ipDB = donorCBefore.getProcessor(); int width = donorCBefore.getWidth(); int height = donorCBefore.getHeight(); double sum = 0; int count = 0; for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (donorCBefore.getRoi().contains(i, j)) { sum += ipDB.getPixelValue(i,j); count++; } } } float backgroundAvgDB = (float)(sum/count); float value = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { value = ipDB.getPixelValue(x,y); value = value - backgroundAvgDB; ipDB.putPixelValue(x, y, value); } } donorCBefore.updateAndDraw(); donorCBefore.killRoi(); accBlWindow.log("Subtracted background ("+backgroundAvgDB+") of donor before bleaching. (bl. corr.)"); subtractBeforeButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("subtractCAfter")) { if (donorCAfter == null) { accBlWindow.logError("No image is set as donor after bleaching. (bl. corr.)"); return; } else if (donorCAfter.getRoi() == null) { accBlWindow.logError("No ROI is defined for donor after bleaching. (bl. corr.)"); return; } ImageProcessor ipDA = donorCAfter.getProcessor(); int width = donorCAfter.getWidth(); int height = donorCAfter.getHeight(); double sum = 0; int count = 0; for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (donorCAfter.getRoi().contains(i, j)) { sum += ipDA.getPixelValue(i,j); count++; } } } float backgroundAvgDA = (float)(sum/count); float value = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { value = ipDA.getPixelValue(x,y); value = value - backgroundAvgDA; ipDA.putPixelValue(x, y, value); } } donorCAfter.updateAndDraw(); donorCAfter.killRoi(); accBlWindow.log("Subtracted background ("+backgroundAvgDA+") of donor after bleaching. (bl. corr.)"); subtractAfterButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setCBeforeThreshold")) { if (donorCBefore == null) { accBlWindow.logError("No image is set as donor before bleaching. (bl. corr.)"); return; } IJ.selectWindow(donorCBefore.getTitle()); IJ.run("Threshold..."); setBeforeThresholdButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setCAfterThreshold")) { if (donorCAfter == null) { accBlWindow.logError("No image is set as donor after bleaching. (bl. corr.)"); return; } IJ.selectWindow(donorCAfter.getTitle()); IJ.run("Threshold..."); setAfterThresholdButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("calculate")) { if (donorCBefore == null) { accBlWindow.logError("No image is set as donor before bleaching. (bl. corr.)"); return; } else if (donorCAfter == null) { accBlWindow.logError("No image is set as donor after bleaching. (bl. corr.)"); return; } else { DecimalFormat df = new DecimalFormat("#.###"); ImageProcessor ipDB = donorCBefore.getProcessor(); ImageProcessor ipDA = donorCAfter.getProcessor(); float[][] corrImgPoints = null; int width = ipDB.getWidth(); int height = ipDB.getHeight(); if(showBlCImagesCB.isSelected()) { corrImgPoints = new float[width][height]; } double sumc = 0; double countc = 0; for (int i = 0; i < width; i++) { for (int j = 0; j < height; j++) { if (ipDA.getPixelValue(i, j) > 0 && ipDB.getPixelValue(i, j) > 0) { double current = ipDB.getPixelValue(i, j) / ipDA.getPixelValue(i, j); sumc += current; countc++; if(showBlCImagesCB.isSelected()) { corrImgPoints[i][j] = (float)current; } } } } float avg = (float)(sumc / countc); mode1ResultLabel.setText(df.format(avg).toString()); float[] ipDBP = (float[])ipDB.getPixels(); float[] ipDAP = (float[])ipDA.getPixels(); double avgBefore = 0; double avgAfter = 0; countc = 0; sumc = 0; for (int i = 0; i < ipDBP.length; i++) { if (ipDBP[i] > 0) { sumc += ipDBP[i]; countc++; } } avgBefore = sumc / countc; countc = 0; sumc = 0; for (int i = 0; i < ipDAP.length; i++) { if (ipDAP[i] > 0) { sumc += ipDAP[i]; countc++; } } avgAfter = sumc / countc; mode2ResultLabel.setText(df.format((float)(avgBefore/avgAfter)).toString()); calculateButton.setBackground(accBlWindow.greenColor); donorCBefore.changes = false; donorCAfter.changes = false; if(showBlCImagesCB.isSelected()) { ImagePlus corrImg = new ImagePlus("Donor bleaching correction image", new FloatProcessor(corrImgPoints)); corrImg.show(); } } } else if (e.getActionCommand().equals("setfactor")) { if (quotientsButton.isSelected()) { if (mode1ResultLabel.getText().equals("")) { accBlWindow.logError("The correction factor has to be calculated before setting it. (bl. corr.)"); return; } accBlWindow.setBleachingCorrection(mode1ResultLabel.getText()); setButton.setBackground(accBlWindow.greenColor); accBlWindow.calculateDBCorrButton.setBackground(accBlWindow.greenColor); } else { if (mode2ResultLabel.getText().equals("")) { accBlWindow.logError("The correction factor has to be calculated before setting it. (bl. corr.)"); return; } accBlWindow.setBleachingCorrection(mode2ResultLabel.getText()); setButton.setBackground(accBlWindow.greenColor); accBlWindow.calculateDBCorrButton.setBackground(accBlWindow.greenColor); } } else if (e.getActionCommand().equals("registerCImages")) { if (donorCBefore == null) { accBlWindow.logError("No image is set as donor before bleaching. (bl. corr.)"); return; } else if (donorCAfter == null) { accBlWindow.logError("No image is set as donor after bleaching. (bl. corr.)"); return; } else { FHT fht1 = new FHT(donorCBefore.getProcessor().duplicate()); fht1.transform(); FHT fht2 = new FHT(donorCAfter.getProcessor().duplicate()); fht2.transform(); FHT res = fht1.conjugateMultiply(fht2); res.inverseTransform(); ImagePlus image = new ImagePlus("Result of registration", res); ImageProcessor ip = image.getProcessor(); int width = ip.getWidth(); int height = ip.getHeight(); int maximum = 0; int maxx = -1; int maxy = -1; for (int i = 0; i < width; i++) { for (int j = 0; j < height; j++) { if (ip.getPixel(i, j) > maximum) { maximum = ip.getPixel(i, j); maxx = i; maxy = j; } } } int shiftX = 0; int shiftY = 0; if (maxx != 0 || maxy != 0){ ShiftDialog sd = new ShiftDialog(accBlWindow); if (maxy > height/2) { accBlWindow.log("Shifting donor after image up " + (height-maxy) + " pixel" + ((height-maxy)>1?"s":"") + ". (bl. corr.)"); sd.shiftUp(donorCAfter, height-maxy); } else if (maxy != 0) { accBlWindow.log("Shifting donor after image down " + maxy + " pixel" + (maxy>1?"s":"") + ". (bl. corr.)"); sd.shiftDown(donorCAfter, maxy); } if (maxx > width/2) { accBlWindow.log("Shifting donor after image to the left " + (width-maxx) + " pixel" + ((width-maxx)>1?"s":"") + ". (bl. corr.)"); sd.shiftLeft(donorCAfter, width-maxx); } else if (maxx != 0) { accBlWindow.log("Shifting donor after image to the right " + maxx + " pixel" + (maxx>1?"s":"") + ". (bl. corr.)"); sd.shiftRight(donorCAfter, maxx); } actionPerformed(new ActionEvent(registerButton, 1, "registerCImages")); } else { accBlWindow.log("Registration finished. Maximum: x=" + maxx + " y=" + maxy + " (bl. corr.)"); registerButton.setBackground(accBlWindow.greenColor); } } } } catch (Throwable t) { accBlWindow.logException(t.toString(), t); } } } class AcceptorCTCorrDialog extends JDialog implements ActionListener{ private AccPbFRET_Plugin accBlWindow; private ImagePlus donorCBefore, acceptorCBefore; private JPanel panel; private JButton setDonorBeforeButton, setAcceptorBeforeButton; private JButton setDonorBeforeThresholdButton, setAcceptorBeforeThresholdButton, calculateButton, setButton; private JButton subtractDonorBeforeButton, subtractAcceptorBeforeButton; private JButton resetButton; private ButtonGroup buttonGroup; private JRadioButton averagesButton, quotientsButton; private JLabel mode1ResultLabel, mode2ResultLabel; private JCheckBox showCTCImagesCB; public AcceptorCTCorrDialog(AccPbFRET_Plugin accBlWindow) { setTitle("Acceptor cross-talk correction factor"); this.accBlWindow = accBlWindow; setDefaultCloseOperation(JFrame.DISPOSE_ON_CLOSE); setModal(false); createDialogGui(); Dimension screen = Toolkit.getDefaultToolkit().getScreenSize(); setSize(300, 380); setLocation((screen.width - getWidth())/2, (screen.height - getHeight())/2); } public void createDialogGui() { GridBagLayout gridbaglayout = new GridBagLayout(); GridBagConstraints gc = new GridBagConstraints(); panel = new JPanel(); panel.setLayout(gridbaglayout); gc.insets = new Insets(2,2,6,2); gc.fill = GridBagConstraints.BOTH; gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 0; JLabel infoLabel = new JLabel("<html><center>This factor is calculated based on images of donor and acceptor channels of an acceptor only labeled sample, before photobleaching.</center></html>"); panel.add(infoLabel, gc); gc.insets = new Insets(2,2,2,2); gc.gridwidth = 3; gc.gridx = 0; gc.gridy = 1; setDonorBeforeButton = new JButton("Set donor before bleaching (acc. only)"); setDonorBeforeButton.addActionListener(this); setDonorBeforeButton.setActionCommand("setDonorCBefore"); panel.add(setDonorBeforeButton, gc); gc.gridx = 0; gc.gridy = 2; setAcceptorBeforeButton = new JButton("Set acceptor before bleaching (acc. only)"); setAcceptorBeforeButton.addActionListener(this); setAcceptorBeforeButton.setActionCommand("setAcceptorCBefore"); panel.add(setAcceptorBeforeButton, gc); gc.gridx = 0; gc.gridy = 3; subtractDonorBeforeButton = new JButton("Subtract background of donor before"); subtractDonorBeforeButton.addActionListener(this); subtractDonorBeforeButton.setActionCommand("subtractDonorCBefore"); panel.add(subtractDonorBeforeButton, gc); gc.gridx = 0; gc.gridy = 4; subtractAcceptorBeforeButton = new JButton("Subtract background of acceptor before"); subtractAcceptorBeforeButton.addActionListener(this); subtractAcceptorBeforeButton.setActionCommand("subtractAcceptorCBefore"); panel.add(subtractAcceptorBeforeButton, gc); gc.gridx = 0; gc.gridy = 5; setDonorBeforeThresholdButton = new JButton("Set donor before threshold"); setDonorBeforeThresholdButton.addActionListener(this); setDonorBeforeThresholdButton.setActionCommand("setDonorCBeforeThreshold"); panel.add(setDonorBeforeThresholdButton, gc); gc.gridx = 0; gc.gridy = 6; setAcceptorBeforeThresholdButton = new JButton("Set acceptor before threshold"); setAcceptorBeforeThresholdButton.addActionListener(this); setAcceptorBeforeThresholdButton.setActionCommand("setAcceptorCBeforeThreshold"); panel.add(setAcceptorBeforeThresholdButton, gc); gc.gridx = 0; gc.gridy = 7; gc.gridheight = 2; JPanel radioPanel = new JPanel(); GridBagLayout gbl = new GridBagLayout(); GridBagConstraints gcr = new GridBagConstraints(); radioPanel.setLayout(gbl); gcr.insets = new Insets(0,4,4,4); gcr.fill = GridBagConstraints.BOTH; JLabel modeLabel = new JLabel("Mode:"); JLabel resultLabel = new JLabel("Result:"); mode1ResultLabel = new JLabel("", JLabel.CENTER); mode2ResultLabel = new JLabel("", JLabel.CENTER); quotientsButton = new JRadioButton("point-by-point"); quotientsButton.setToolTipText("The factor is the averaged ratio of corresponding pixel values in the donor before and acceptor before photobleaching images."); averagesButton = new JRadioButton("average pixels"); averagesButton.setToolTipText("The factor is the ratio of the gated pixel averages in the donor before and acceptor before photobleaching images."); quotientsButton.setSelected(true); buttonGroup = new ButtonGroup(); buttonGroup.add(quotientsButton); buttonGroup.add(averagesButton); gcr.gridx = 0; gcr.gridy = 0; radioPanel.add(modeLabel, gcr); gcr.gridx = 1; gcr.gridy = 0; radioPanel.add(quotientsButton, gcr); gcr.gridx = 2; gcr.gridy = 0; radioPanel.add(averagesButton, gcr); gcr.gridx = 0; gcr.gridy = 1; radioPanel.add(resultLabel, gcr); gcr.gridx = 1; gcr.gridy = 1; radioPanel.add(mode1ResultLabel, gcr); gcr.gridx = 2; gcr.gridy = 1; radioPanel.add(mode2ResultLabel, gcr); panel.add(radioPanel, gc); gc.gridx = 0; gc.gridy = 9; gc.gridheight = 1; showCTCImagesCB = new JCheckBox("show correction image (for manual calc.)"); panel.add(showCTCImagesCB, gc); gc.gridwidth = 1; gc.gridheight = 1; gc.gridx = 0; gc.gridy = 10; calculateButton = new JButton("Calculate"); calculateButton.addActionListener(this); calculateButton.setActionCommand("calculate"); panel.add(calculateButton, gc); gc.gridx = 1; gc.gridy = 10; setButton = new JButton("Set selected"); setButton.addActionListener(this); setButton.setActionCommand("setfactor"); panel.add(setButton, gc); gc.gridx = 2; gc.gridy = 10; resetButton = new JButton("Reset"); resetButton.addActionListener(this); resetButton.setActionCommand("reset"); panel.add(resetButton, gc); getContentPane().add(panel); } public void actionPerformed(ActionEvent e) { try { if (e.getActionCommand().equals("reset")) { donorCBefore = null; acceptorCBefore = null; setDonorBeforeButton.setBackground(accBlWindow.originalButtonColor); setAcceptorBeforeButton.setBackground(accBlWindow.originalButtonColor); setDonorBeforeThresholdButton.setBackground(accBlWindow.originalButtonColor); setAcceptorBeforeThresholdButton.setBackground(accBlWindow.originalButtonColor); calculateButton.setBackground(accBlWindow.originalButtonColor); setButton.setBackground(accBlWindow.originalButtonColor); subtractDonorBeforeButton.setBackground(accBlWindow.originalButtonColor); subtractAcceptorBeforeButton.setBackground(accBlWindow.originalButtonColor); mode1ResultLabel.setText(""); mode2ResultLabel.setText(""); } else if (e.getActionCommand().equals("setDonorCBefore")) { donorCBefore = WindowManager.getCurrentImage(); if (donorCBefore == null) { accBlWindow.logError("No image is selected. (ct. corr.)"); return; } if (donorCBefore.getNChannels() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+donorCBefore.getNChannels()+"). Please split it into parts. (ct. corr.)"); donorCBefore = null; return; } else if (donorCBefore.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+donorCBefore.getNSlices()+"). Please split it into parts. (ct. corr.)"); donorCBefore = null; return; } if (donorCBefore != null && acceptorCBefore != null && donorCBefore.equals(acceptorCBefore)) { accBlWindow.logError("The two images must not be the same. Please select and set an other image. (ct. corr.)"); donorCBefore.setTitle(""); donorCBefore = null; return; } donorCBefore.setTitle("Donor before bleaching (ct. corr.) - " + new Date().toString()); new ImageConverter(donorCBefore).convertToGray32(); setDonorBeforeButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setAcceptorCBefore")) { acceptorCBefore = WindowManager.getCurrentImage(); if (acceptorCBefore == null) { accBlWindow.logError("No image is selected. (ct. corr.)"); return; } if (acceptorCBefore.getNChannels() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+acceptorCBefore.getNChannels()+"). Please split it into parts. (ct. corr.)"); acceptorCBefore = null; return; } else if (acceptorCBefore.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+acceptorCBefore.getNSlices()+"). Please split it into parts. (ct. corr.)"); acceptorCBefore = null; return; } if (donorCBefore != null && acceptorCBefore != null && donorCBefore.equals(acceptorCBefore)) { accBlWindow.logError("The two images must not be the same. Please select and set an other image. (ct. corr.)"); acceptorCBefore.setTitle(""); acceptorCBefore = null; return; } acceptorCBefore.setTitle("Acceptor before bleaching (ct. corr.) - " + new Date().toString()); new ImageConverter(acceptorCBefore).convertToGray32(); setAcceptorBeforeButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("subtractDonorCBefore")) { if (donorCBefore == null) { accBlWindow.logError("No image is set as donor before bleaching. (ct. corr.)"); return; } else if (donorCBefore.getRoi() == null) { accBlWindow.logError("No ROI is defined for donor before bleaching. (ct. corr.)"); return; } ImageProcessor ipDB = donorCBefore.getProcessor(); int width = donorCBefore.getWidth(); int height = donorCBefore.getHeight(); double sum = 0; int count = 0; for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (donorCBefore.getRoi().contains(i, j)) { sum += ipDB.getPixelValue(i,j); count++; } } } float backgroundAvgDB = (float)(sum/count); float value = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { value = ipDB.getPixelValue(x,y); value = value - backgroundAvgDB; ipDB.putPixelValue(x, y, value); } } donorCBefore.updateAndDraw(); donorCBefore.killRoi(); accBlWindow.log("Subtracted background ("+backgroundAvgDB+") of donor before bleaching. (ct. corr.)"); subtractDonorBeforeButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("subtractAcceptorCBefore")) { if (acceptorCBefore == null) { accBlWindow.logError("No image is set as acceptor before bleaching. (ct. corr.)"); return; } else if (acceptorCBefore.getRoi() == null) { accBlWindow.logError("No ROI is defined for acceptor before bleaching. (ct. corr.)"); return; } ImageProcessor ipAB = acceptorCBefore.getProcessor(); int width = acceptorCBefore.getWidth(); int height = acceptorCBefore.getHeight(); double sum = 0; int count = 0; for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (acceptorCBefore.getRoi().contains(i, j)) { sum += ipAB.getPixelValue(i,j); count++; } } } float backgroundAvgDA = (float)(sum/count); float value = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { value = ipAB.getPixelValue(x,y); value = value - backgroundAvgDA; ipAB.putPixelValue(x, y, value); } } acceptorCBefore.updateAndDraw(); acceptorCBefore.killRoi(); accBlWindow.log("Subtracted background ("+backgroundAvgDA+") of acceptor before bleaching. (ct. corr.)"); subtractAcceptorBeforeButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setDonorCBeforeThreshold")) { if (donorCBefore == null) { accBlWindow.logError("No image is set as donor before bleaching. (ct. corr.)"); return; } IJ.selectWindow(donorCBefore.getTitle()); IJ.run("Threshold..."); setDonorBeforeThresholdButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setAcceptorCBeforeThreshold")) { if (acceptorCBefore == null) { accBlWindow.logError("No image is set as acceptor before bleaching. (ct. corr.)"); return; } IJ.selectWindow(acceptorCBefore.getTitle()); IJ.run("Threshold..."); setAcceptorBeforeThresholdButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("calculate")) { if (donorCBefore == null) { accBlWindow.logError("No image is set as donor before bleaching. (ct. corr.)"); return; } else if (acceptorCBefore == null) { accBlWindow.logError("No image is set as acceptor before bleaching. (ct. corr.)"); return; } else { DecimalFormat df = new DecimalFormat("#.###"); ImageProcessor ipDB = donorCBefore.getProcessor(); ImageProcessor ipAB = acceptorCBefore.getProcessor(); float[][] corrImgPoints = null; int width = ipDB.getWidth(); int height = ipDB.getHeight(); if(showCTCImagesCB.isSelected()) { corrImgPoints = new float[width][height]; } double sumc = 0; double countc = 0; for (int i = 0; i < width; i++) { for (int j = 0; j < height; j++) { if (ipAB.getPixelValue(i, j) > 0 && ipDB.getPixelValue(i, j) > 0) { double current = ipDB.getPixelValue(i, j) / ipAB.getPixelValue(i, j); sumc += current; countc++; if(showCTCImagesCB.isSelected()) { corrImgPoints[i][j] = (float)current; } } } } float avg = (float)(sumc / countc); mode1ResultLabel.setText(df.format(avg).toString()); float[] ipDBP = (float[])ipDB.getPixels(); float[] ipABP = (float[])ipAB.getPixels(); double avgDonorBefore = 0; double avgAcceptorBefore = 0; countc = 0; sumc = 0; for (int i = 0; i < ipDBP.length; i++) { if (ipDBP[i] > 0) { sumc += ipDBP[i]; countc++; } } avgDonorBefore = sumc / countc; countc = 0; sumc = 0; for (int i = 0; i < ipABP.length; i++) { if (ipABP[i] > 0) { sumc += ipABP[i]; countc++; } } avgAcceptorBefore = sumc / countc; mode2ResultLabel.setText(df.format((float)(avgDonorBefore/avgAcceptorBefore)).toString()); calculateButton.setBackground(accBlWindow.greenColor); donorCBefore.changes = false; acceptorCBefore.changes = false; if(showCTCImagesCB.isSelected()) { ImagePlus corrImg = new ImagePlus("Cross-talk correction image", new FloatProcessor(corrImgPoints)); corrImg.show(); } } } else if (e.getActionCommand().equals("setfactor")) { if (quotientsButton.isSelected()) { if (mode1ResultLabel.getText().equals("")) { accBlWindow.logError("The correction factor has to be calculated before setting it. (ct. corr.)"); return; } accBlWindow.setCrosstalkCorrection(mode1ResultLabel.getText()); accBlWindow.calculateAccCTCorrButton.setBackground(accBlWindow.greenColor); setButton.setBackground(accBlWindow.greenColor); } else { if (mode2ResultLabel.getText().equals("")) { accBlWindow.logError("The correction factor has to be calculated before setting it. (ct. corr.)"); return; } accBlWindow.setCrosstalkCorrection(mode2ResultLabel.getText()); accBlWindow.calculateAccCTCorrButton.setBackground(accBlWindow.greenColor); setButton.setBackground(accBlWindow.greenColor); } } } catch (Throwable t) { accBlWindow.logException(t.toString(), t); } } } class AcceptorPPCorrDialog extends JDialog implements ActionListener{ private AccPbFRET_Plugin accBlWindow; private ImagePlus donorCAfter, acceptorCBefore; private JPanel panel; private JButton setDonorAfterButton, setAcceptorBeforeButton; private JButton setDonorAfterThresholdButton, setAcceptorBeforeThresholdButton, calculateButton, setButton; private JButton subtractDonorAfterButton, subtractAcceptorBeforeButton; private JButton resetButton; private ButtonGroup buttonGroup; private JRadioButton averagesButton, quotientsButton; private JLabel mode1ResultLabel, mode2ResultLabel; private JCheckBox showPPImagesCB; public AcceptorPPCorrDialog(AccPbFRET_Plugin accBlWindow) { setTitle("Acceptor photoproduct correction factor"); this.accBlWindow = accBlWindow; setDefaultCloseOperation(JFrame.DISPOSE_ON_CLOSE); setModal(false); createDialogGui(); Dimension screen = Toolkit.getDefaultToolkit().getScreenSize(); setSize(300, 380); setLocation((screen.width - getWidth())/2, (screen.height - getHeight())/2); } public void createDialogGui() { GridBagLayout gridbaglayout = new GridBagLayout(); GridBagConstraints gc = new GridBagConstraints(); panel = new JPanel(); panel.setLayout(gridbaglayout); gc.insets = new Insets(2,2,6,2); gc.fill = GridBagConstraints.BOTH; gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 0; JLabel infoLabel = new JLabel("<html><center>This factor is calculated based on images of a sample labeled with acceptor only, donor after and acceptor before photobleaching.</center></html>"); panel.add(infoLabel, gc); gc.insets = new Insets(2,2,2,2); gc.gridwidth = 3; gc.gridx = 0; gc.gridy = 1; setDonorAfterButton = new JButton("Set donor after bleaching (acc. only)"); setDonorAfterButton.addActionListener(this); setDonorAfterButton.setActionCommand("setDonorCAfter"); panel.add(setDonorAfterButton, gc); gc.gridx = 0; gc.gridy = 2; setAcceptorBeforeButton = new JButton("Set acceptor before bleaching (acc. only)"); setAcceptorBeforeButton.addActionListener(this); setAcceptorBeforeButton.setActionCommand("setAcceptorCBefore"); panel.add(setAcceptorBeforeButton, gc); gc.gridx = 0; gc.gridy = 3; subtractDonorAfterButton = new JButton("Subtract background of donor after"); subtractDonorAfterButton.addActionListener(this); subtractDonorAfterButton.setActionCommand("subtractDonorCAfter"); panel.add(subtractDonorAfterButton, gc); gc.gridx = 0; gc.gridy = 4; subtractAcceptorBeforeButton = new JButton("Subtract background of acceptor before"); subtractAcceptorBeforeButton.addActionListener(this); subtractAcceptorBeforeButton.setActionCommand("subtractAcceptorCBefore"); panel.add(subtractAcceptorBeforeButton, gc); gc.gridx = 0; gc.gridy = 5; setDonorAfterThresholdButton = new JButton("Set donor after threshold"); setDonorAfterThresholdButton.addActionListener(this); setDonorAfterThresholdButton.setActionCommand("setDonorCAfterThreshold"); panel.add(setDonorAfterThresholdButton, gc); gc.gridx = 0; gc.gridy = 6; setAcceptorBeforeThresholdButton = new JButton("Set acceptor before threshold"); setAcceptorBeforeThresholdButton.addActionListener(this); setAcceptorBeforeThresholdButton.setActionCommand("setAcceptorCBeforeThreshold"); panel.add(setAcceptorBeforeThresholdButton, gc); gc.gridx = 0; gc.gridy = 7; gc.gridheight = 2; JPanel radioPanel = new JPanel(); GridBagLayout gbl = new GridBagLayout(); GridBagConstraints gcr = new GridBagConstraints(); radioPanel.setLayout(gbl); gcr.insets = new Insets(0,4,4,4); gcr.fill = GridBagConstraints.BOTH; JLabel modeLabel = new JLabel("Mode:"); JLabel resultLabel = new JLabel("Result:"); mode1ResultLabel = new JLabel("", JLabel.CENTER); mode2ResultLabel = new JLabel("", JLabel.CENTER); quotientsButton = new JRadioButton("point-by-point"); quotientsButton.setToolTipText("The factor is the averaged ratio of corresponding pixel values in the donor after and acceptor before photobleaching images."); averagesButton = new JRadioButton("average pixels"); averagesButton.setToolTipText("The factor is the ratio of the gated pixel averages in the donor after and acceptor before photobleaching images."); quotientsButton.setSelected(true); buttonGroup = new ButtonGroup(); buttonGroup.add(quotientsButton); buttonGroup.add(averagesButton); gcr.gridx = 0; gcr.gridy = 0; radioPanel.add(modeLabel, gcr); gcr.gridx = 1; gcr.gridy = 0; radioPanel.add(quotientsButton, gcr); gcr.gridx = 2; gcr.gridy = 0; radioPanel.add(averagesButton, gcr); gcr.gridx = 0; gcr.gridy = 1; radioPanel.add(resultLabel, gcr); gcr.gridx = 1; gcr.gridy = 1; radioPanel.add(mode1ResultLabel, gcr); gcr.gridx = 2; gcr.gridy = 1; radioPanel.add(mode2ResultLabel, gcr); panel.add(radioPanel, gc); gc.gridx = 0; gc.gridy = 9; gc.gridheight = 1; showPPImagesCB = new JCheckBox("show correction image (for manual calc.)"); panel.add(showPPImagesCB, gc); gc.gridwidth = 1; gc.gridheight = 1; gc.gridx = 0; gc.gridy = 10; calculateButton = new JButton("Calculate"); calculateButton.addActionListener(this); calculateButton.setActionCommand("calculate"); panel.add(calculateButton, gc); gc.gridx = 1; gc.gridy = 10; setButton = new JButton("Set selected"); setButton.addActionListener(this); setButton.setActionCommand("setfactor"); panel.add(setButton, gc); gc.gridx = 2; gc.gridy = 10; resetButton = new JButton("Reset"); resetButton.addActionListener(this); resetButton.setActionCommand("reset"); panel.add(resetButton, gc); getContentPane().add(panel); } public void actionPerformed(ActionEvent e) { try { if (e.getActionCommand().equals("reset")) { donorCAfter = null; acceptorCBefore = null; setDonorAfterButton.setBackground(accBlWindow.originalButtonColor); setAcceptorBeforeButton.setBackground(accBlWindow.originalButtonColor); setDonorAfterThresholdButton.setBackground(accBlWindow.originalButtonColor); setAcceptorBeforeThresholdButton.setBackground(accBlWindow.originalButtonColor); calculateButton.setBackground(accBlWindow.originalButtonColor); setButton.setBackground(accBlWindow.originalButtonColor); subtractDonorAfterButton.setBackground(accBlWindow.originalButtonColor); subtractAcceptorBeforeButton.setBackground(accBlWindow.originalButtonColor); mode1ResultLabel.setText(""); mode2ResultLabel.setText(""); } else if (e.getActionCommand().equals("setDonorCAfter")) { donorCAfter = WindowManager.getCurrentImage(); if (donorCAfter == null) { accBlWindow.logError("No image is selected. (pp. corr.)"); return; } if (donorCAfter.getNChannels() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+donorCAfter.getNChannels()+"). Please split it into parts. (pp. corr.)"); donorCAfter = null; return; } else if (donorCAfter.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+donorCAfter.getNSlices()+"). Please split it into parts. (pp. corr.)"); donorCAfter = null; return; } if (donorCAfter != null && acceptorCBefore != null && donorCAfter.equals(acceptorCBefore)) { accBlWindow.logError("The two images must not be the same. Please select and set an other image. (pp. corr.)"); donorCAfter.setTitle(""); donorCAfter = null; return; } donorCAfter.setTitle("Donor after bleaching (pp. corr.) - " + new Date().toString()); new ImageConverter(donorCAfter).convertToGray32(); setDonorAfterButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setAcceptorCBefore")) { acceptorCBefore = WindowManager.getCurrentImage(); if (acceptorCBefore == null) { accBlWindow.logError("No image is selected. (pp. corr.)"); return; } if (acceptorCBefore.getNChannels() > 1) { accBlWindow.logError("Current image contains more than 1 channel ("+acceptorCBefore.getNChannels()+"). Please split it into parts. (pp. corr.)"); acceptorCBefore = null; return; } else if (acceptorCBefore.getNSlices() > 1) { accBlWindow.logError("Current image contains more than 1 slice ("+acceptorCBefore.getNSlices()+"). Please split it into parts. (pp. corr.)"); acceptorCBefore = null; return; } if (donorCAfter != null && acceptorCBefore != null && donorCAfter.equals(acceptorCBefore)) { accBlWindow.logError("The two images must not be the same. Please select and set an other image. (pp. corr.)"); acceptorCBefore.setTitle(""); acceptorCBefore = null; return; } acceptorCBefore.setTitle("Acceptor before bleaching (pp. corr.) - " + new Date().toString()); new ImageConverter(acceptorCBefore).convertToGray32(); setAcceptorBeforeButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("subtractDonorCAfter")) { if (donorCAfter == null) { accBlWindow.logError("No image is set as donor after bleaching. (pp. corr.)"); return; } else if (donorCAfter.getRoi() == null) { accBlWindow.logError("No ROI is defined for donor after bleaching. (pp. corr.)"); return; } ImageProcessor ipDA = donorCAfter.getProcessor(); int width = donorCAfter.getWidth(); int height = donorCAfter.getHeight(); double sum = 0; int count = 0; for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (donorCAfter.getRoi().contains(i, j)) { sum += ipDA.getPixelValue(i,j); count++; } } } float backgroundAvgDB = (float)(sum/count); float value = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { value = ipDA.getPixelValue(x,y); value = value - backgroundAvgDB; ipDA.putPixelValue(x, y, value); } } donorCAfter.updateAndDraw(); donorCAfter.killRoi(); accBlWindow.log("Subtracted background ("+backgroundAvgDB+") of donor after bleaching. (pp. corr.)"); subtractDonorAfterButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("subtractAcceptorCBefore")) { if (acceptorCBefore == null) { accBlWindow.logError("No image is set as acceptor before bleaching. (pp. corr.)"); return; } else if (acceptorCBefore.getRoi() == null) { accBlWindow.logError("No ROI is defined for acceptor before bleaching. (pp. corr.)"); return; } ImageProcessor ipAB = acceptorCBefore.getProcessor(); int width = acceptorCBefore.getWidth(); int height = acceptorCBefore.getHeight(); double sum = 0; int count = 0; for (int i=0; i<width; i++) { for (int j=0; j<height; j++) { if (acceptorCBefore.getRoi().contains(i, j)) { sum += ipAB.getPixelValue(i,j); count++; } } } float backgroundAvgDA = (float)(sum/count); float value = 0; for (int x=0; x < width; x++) { for (int y=0; y < height; y++) { value = ipAB.getPixelValue(x,y); value = value - backgroundAvgDA; ipAB.putPixelValue(x, y, value); } } acceptorCBefore.updateAndDraw(); acceptorCBefore.killRoi(); accBlWindow.log("Subtracted background ("+backgroundAvgDA+") of acceptor before bleaching. (pp. corr.)"); subtractAcceptorBeforeButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setDonorCAfterThreshold")) { if (donorCAfter == null) { accBlWindow.logError("No image is set as donor after bleaching. (pp. corr.)"); return; } IJ.selectWindow(donorCAfter.getTitle()); IJ.run("Threshold..."); setDonorAfterThresholdButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("setAcceptorCBeforeThreshold")) { if (acceptorCBefore == null) { accBlWindow.logError("No image is set as acceptor before bleaching. (pp. corr.)"); return; } IJ.selectWindow(acceptorCBefore.getTitle()); IJ.run("Threshold..."); setAcceptorBeforeThresholdButton.setBackground(accBlWindow.greenColor); } else if (e.getActionCommand().equals("calculate")) { if (donorCAfter == null) { accBlWindow.logError("No image is set as donor after bleaching. (pp. corr.)"); return; } else if (acceptorCBefore == null) { accBlWindow.logError("No image is set as acceptor before bleaching. (pp. corr.)"); return; } else { DecimalFormat df = new DecimalFormat("#.###"); ImageProcessor ipDA = donorCAfter.getProcessor(); ImageProcessor ipAB = acceptorCBefore.getProcessor(); float[][] corrImgPoints = null; int width = ipDA.getWidth(); int height = ipDA.getHeight(); if(showPPImagesCB.isSelected()) { corrImgPoints = new float[width][height]; } double sumc = 0; double countc = 0; for (int i = 0; i < width; i++) { for (int j = 0; j < height; j++) { if (ipAB.getPixelValue(i, j) > 0 && ipDA.getPixelValue(i, j) > 0) { double current = ipDA.getPixelValue(i, j) / ipAB.getPixelValue(i, j); sumc += current; countc++; if(showPPImagesCB.isSelected()) { corrImgPoints[i][j] = (float)current; } } } } float avg = (float)(sumc / countc); mode1ResultLabel.setText(df.format(avg).toString()); float[] ipDAP = (float[])ipDA.getPixels(); float[] ipABP = (float[])ipAB.getPixels(); double avgDonorAfter = 0; double avgAcceptorBefore = 0; countc = 0; sumc = 0; for (int i = 0; i < ipDAP.length; i++) { if (ipDAP[i] > 0) { sumc += ipDAP[i]; countc++; } } avgDonorAfter = sumc / countc; countc = 0; sumc = 0; for (int i = 0; i < ipABP.length; i++) { if (ipABP[i] > 0) { sumc += ipABP[i]; countc++; } } avgAcceptorBefore = sumc / countc; mode2ResultLabel.setText(df.format((float)(avgDonorAfter/avgAcceptorBefore)).toString()); calculateButton.setBackground(accBlWindow.greenColor); donorCAfter.changes = false; acceptorCBefore.changes = false; if(showPPImagesCB.isSelected()) { ImagePlus corrImg = new ImagePlus("Photoproduct correction image", new FloatProcessor(corrImgPoints)); corrImg.show(); } } } else if (e.getActionCommand().equals("setfactor")) { if (quotientsButton.isSelected()) { if (mode1ResultLabel.getText().equals("")) { accBlWindow.logError("The correction factor has to be calculated before setting it. (pp. corr.)"); return; } accBlWindow.setPhotoproductCorrection(mode1ResultLabel.getText()); setButton.setBackground(accBlWindow.greenColor); accBlWindow.calculateAccPPCorrButton.setBackground(accBlWindow.greenColor); } else { if (mode2ResultLabel.getText().equals("")) { accBlWindow.logError("The correction factor has to be calculated before setting it. (pp. corr.)"); return; } accBlWindow.setPhotoproductCorrection(mode2ResultLabel.getText()); setButton.setBackground(accBlWindow.greenColor); accBlWindow.calculateAccPPCorrButton.setBackground(accBlWindow.greenColor); } } } catch (Throwable t) { accBlWindow.logException(t.toString(), t); } } } class HelpWindow extends JFrame { private AccPbFRET_Plugin accBlWindow; private JPanel panel; public HelpWindow(AccPbFRET_Plugin accBlWindow) { setTitle("AccPbFRET Help"); this.accBlWindow = accBlWindow; setDefaultCloseOperation(JFrame.DISPOSE_ON_CLOSE); createGui(); Dimension screen = Toolkit.getDefaultToolkit().getScreenSize(); setSize(600, 800); setLocation((screen.width - getWidth())/2, (screen.height - getHeight())/2); } public void createGui() { GridBagLayout gridbaglayout = new GridBagLayout(); GridBagConstraints gc = new GridBagConstraints(); panel = new JPanel(); panel.setLayout(gridbaglayout); setFont(new Font("Helvetica", Font.PLAIN, 12)); gc.insets = new Insets(4,4,4,4); gc.fill = GridBagConstraints.HORIZONTAL; gc.gridwidth = GridBagConstraints.REMAINDER; gc.gridx = 0; gc.gridy = 0; gc.weightx = 2; JLabel label1 = new JLabel("<html><center><b>AccPbFRET Help</b></center></html>"); label1.setFont(new Font("Helvetica", Font.BOLD, 14)); panel.add(label1, gc); gc.gridy = GridBagConstraints.RELATIVE; JLabel label1b = new JLabel("<html><b><br><u>Menu structure</u></b></html>"); panel.add(label1b, gc); JLabel label1c = new JLabel("<html>The \"File\" and \"Image\" menus include ImageJ commands that are likely to be frequently used in<br>the analysis process.<br><br>Also, messages generated during image processing can be saved or cleared from the \"File\" menu.<br>Furthermore, analysis data can be reset from the \"File\" menu and the plugin can be switched to<br>semi-automatic mode here as well.<br><br>In the \"Corrections\" menu, the checkboxes of desired correction algorithms need to be checked.<br><br>Donor bleaching is almost inevitable during the acquisition of sequential donor images and using<br>this factor (or at least checking its value) is highly recommended. When this correction is chosen,<br>the default value of 1% unwanted donor bleaching is assigned (yielding a factor of 1.01).<br><br>Acceptor crosstalk can be a problem depending on the filter sets and laser of choice. When this<br>correction is chosen, the default value of 1% unwanted acceptor bleed-through to the donor<br>channel is assigned (yielding a factor of 0.01).<br><br>Acceptor photoproduct(s) can be fluorescent and cross-talk into the donor channel. When<br>correction for this possibility is chosen, the default value of 1% unwanted acceptor photoprodocut<br>cross-talk to the donor channel is assigned (yielding a factor of 0.01).<br><br>Partial acceptor photobleaching causes the underestimation of FRET. The correction for this<br>assumes a linear relation between the number of available (unbleached) acceptor molecules<br>and the efficiency of FRET. The default value 0 for this correction factor means a total (100%)<br>photodestruction of the acceptor.<br></html>"); panel.add(label1c, gc); JLabel label2 = new JLabel("<html><b><br><u>Step 1: Opening and setting images</u></b></html>"); panel.add(label2, gc); JLabel label3 = new JLabel("<html>Image files can be opened with the \"Open\" button, or with the \"Open image\" item in the \"File\"<br>menu. After opening, images can be set as donor before or after, or acceptor before or after<br>using the \"Set image\" buttons. If multichannel images are used (either having before and after<br>images, or donor and acceptor channel images, or both) in consecutive layers of stacked image<br>files, the opened image has to be split (item available from the \"Image\" menu) before setting. If<br>the \"Use LSM\" checkbox is checked, the LSM image files (up to AIM v. 4.x, files with *.lsm<br>extension) containing donor and acceptor channels in a time series encompassing both before<br>and after photobleaching images are split and set automatically after opening with the \"Open &<br>Set LSM\" button. Every previously opened image window will be closed after pressing this button.<br></html>"); panel.add(label3, gc); JLabel label4 = new JLabel("<html><b><br><u>Step 2: Registration of donor images</u></b></html>"); panel.add(label4, gc); JLabel label5 = new JLabel("<html>To register the donor images, press the \"Register\" button. If the checkbox is checked, acceptor<br>image pairs will automatically be resgistered using the same shift. This is important for<br>deterimining the correction factor for incomplete bleaching of the acceptor.</html>"); panel.add(label5, gc); JLabel label6 = new JLabel("<html><b><br><u>Step 3: Subtraction of background of images</u></b></html>"); panel.add(label6, gc); JLabel label7 = new JLabel("<html>To subtract background (the average of pixels in a selected ROI), the \"Subtract\" button has to<br>be pressed for each relevant image. The \"Copy\" button copies the ROI of the first image to the<br>others. This should be done after marking the ROI and before applying the subtraction. To avoid<br>incidental reusing of the ROI in further operations (such as Gaussian blurring), the ROI is<br>automatically deleted after applying the background correction.</html>"); panel.add(label7, gc); JLabel label8 = new JLabel("<html><b><br><u>Step 4: Gaussian blurring of images</u></b></html>"); panel.add(label8, gc); JLabel label9 = new JLabel("<html>Images can be blurred with the given radius by pressing the corresponding \"Blur\" button.<br>Blurring (together with thresholding) can be reverted using the \"Reset\" buttons in Step 5.</html>"); panel.add(label9, gc); JLabel label10 = new JLabel("<html><b><br><u>Step 5: Setting thresholds for the images</u></b></html>"); panel.add(label10, gc); JLabel label11 = new JLabel("<html>Thresholds can be applied to the images by pressing the corresponding \"Threshold\" button.<br>After setting the threshold, the \"Apply\" button has to be pressed on bottom menu of the<br>\"Threshold\" window. After this, select \"Set background pixels to NaN\" and press \"Ok\".<br>Closing \"Threshold\" window will apply the thresholding LUT to the active image and therefore<br>should be avoided.<br>The \"Reset\" buttons reset both blur and threshold settings of the corresponding image.</html>"); panel.add(label11, gc); JLabel label12 = new JLabel("<html><b><br><u>Correction 1: Calculation and setting of donor bleaching correction factor</u></b></html>"); panel.add(label12, gc); JLabel label13 = new JLabel("<html>After pressing the \"Calculate\" button, a new window pops up, where the calculation of this factor<br>can be done with setting donor before and after photobleaching images of a sample labeled with<br>donor only, and taking similar steps as in the main window of<br>the program.</html>"); panel.add(label13, gc); JLabel label14 = new JLabel("<html><b><br><u>Correction 2: Calculation and setting of acceptor cross-talk correction factor</u></b></html>"); panel.add(label14, gc); JLabel label15 = new JLabel("<html>After pressing the \"Calculate\" button, a new window pops up, where the calculation of this factor<br>can be done with setting donor and acceptor images of a sample labeled with acceptor only<br>(before photobleaching), and taking similar steps as in the main window of the program.</html>"); panel.add(label15, gc); JLabel label16 = new JLabel("<html><b><br><u>Correction 3: Calculation and setting of acceptor photoproduct correction factor</u></b></html>"); panel.add(label16, gc); JLabel label17 = new JLabel("<html>After pressing the \"Calculate\" button, a new window pops up, where the calculation of this factor<br>can be done with setting donor (after photobleaching) and acceptor (before photobleaching)<br>images of a sample labeled with acceptor only, and taking similar steps as in the main window of<br>the program.</html>"); panel.add(label17, gc); JLabel label18 = new JLabel("<html><b><br><u>Correction 4: Calculation of partial acceptor photobleaching correction factor</u></b></html>"); panel.add(label18, gc); JLabel label19 = new JLabel("<html>To calculate this factor, press the \"Calculate\" button. The acceptor before and after photobleaching<br>images have to be set before the calculation. Usually this will already have been done in step 1,<br>where the measurement files (or stacked file) is opened.</html>"); panel.add(label19, gc); JLabel label20 = new JLabel("<html><b><br><u>Step 6: Creation of the transfer (FRET efficiency) image</u></b></html>"); panel.add(label20, gc); JLabel label21 = new JLabel("<html>After pressing the \"Create\" button, the transfer image will be calculated and displayed. If the<br>\"Results\" window is not open it will be opened too. If the \"Use also acceptor before image as<br>mask\" option is checked, the FRET image will be created by AND-ing this mask to the threshold<br>masks of the donor before and after images. At this point, the acceptor before image can be<br>cleared (at step 1), and any other relevant image can be loaded and applied as an additional<br>mask after processing and thresholding this image as desired (going through steps 3c, 4c and<br>5c).</html>"); panel.add(label21, gc); JLabel label22 = new JLabel("<html><b><br><u>Step 7: Making measurements</u></b></html>"); panel.add(label22, gc); JLabel label23 = new JLabel("<html>After the creation of the transfer image, ROIs can be selected on it, and measurements can be<br>made by pressing the \"Measure\" button. FRET histograms can be most easily viewed and<br>exported by clicking the \"Histogram\" item in the \"Image\" menu.</html>"); panel.add(label23, gc); JLabel label24 = new JLabel("<html><br></html>"); panel.add(label24, gc); JScrollPane logScrollPane = new JScrollPane(panel, JScrollPane.VERTICAL_SCROLLBAR_ALWAYS, JScrollPane.HORIZONTAL_SCROLLBAR_AS_NEEDED); getContentPane().add(logScrollPane); } }