/* * =================================================================================== * Latest release data: 12.01.2019 (v1.0) * * (c) Tarik Exner, Carlo Beretta * * For any problems, contact Tarik.Exner@med.uni-heidelberg.de * * This macro has been published under doi:yyy * If you use this code for your analysis, please cite as xxx * * Detects lipid droplets (LDs) in widefield microscopy images and summed Z-stacks. * * The script uses a separation procedure containing a DoG filter for the enhancement of * LD contrast towards their surrounding structures. LDs are defined as the overlap of * two geometrical characteristics: A central fluorescence maximum and a circular edge. * * * For a full documentation of functions, see: * * * * * =================================================================================== */ // 1. Close all the open images before starting the macro function CloseAllWindows() { wait(1000); while(nImages > 0) { selectImage(nImages); close(); } } // 2. Check plugin installation function CheckPluginInstallation() { // Check ImageJ version requires("1.48"); currentVersionScript = "LDquanti v1.0\n"; List.setCommands; if (List.get("Watershed Segmentation") == "") { print("Before to start to use this macro you need to install the Classic Watershed Segmentation plugin!"); wait(3000); print("1. Dowload the Classic Watershed Segmentation plugin at http://bigwww.epfl.ch/sage/soft/watershed/"); exec("open", "http://bigwww.epfl.ch/sage/soft/watershed/"); print("2. Drag and drop the Classic Watershed plugin in the Fiji.app plugin directory"); print("3. After restarting ImageJ/Fiji you should be able to run this macro!"); print("4. Please contact Tarik.Exner@med.uni-heidelberg.de in case of any further issue"); exit(); } else { print("Running " + currentVersionScript); print("Classic Watershed plugin is installed!"); } // Checks if the MorphoLibJ is installed if (List.get("Morphological Filters") == "" || List.get("Morphological Filters (3D)") == "" || List.get("Directional Filtering") == "") { Dialog.create("Plugin not found"); Dialog.addMessage("The MorphoLibJ suite is not installed.\n \nSelect the Help button for instructions."); Dialog.addHelp("" + "Go to Fiji => Help => Update => Manage update sites and enable the IJPB-plugins (http://sites.imagej.net/IJPB-plugins/) "); Dialog.show(); exit(); } else { print("MorphoLibJ is installed!"); print("All necessary plugins are installed!"); wait(2000); print("\\Clear"); } } // 3. Close B&C window function CloseBCWindow() { if (isOpen("B&C")) { selectWindow("B&C"); run("Close"); } } // 4. Close the ROI Manager function CloseROIsManager() { if (isOpen("ROI Manager")) { selectWindow("ROI Manager"); run("Close"); } } // 5. Open the ROI Manager function OpenROIsManager() { if (!isOpen("ROI Manager")) { run("ROI Manager..."); eval("script","f = WindowManager.getFrame('ROI Manager'); f.setLocation(0,0); f.setSize(10,10);"); } } // 6. Close result window function CloseResultsWindow() { if (isOpen("Results")) { selectWindow("Results"); run("Close"); } } // 7. General settings function Setting() { // Set the Measurments parameters run("Set Measurements...", "area redirect=None decimal=3"); run("Input/Output...", "jpeg=100 gif=-1 file=.csv use use_file copy_column copy_row save_column save_row"); // Set binary background to 0 run("Options...", "iterations=1 count=1 white"); } // 8. Close Log window function CloseLogWindow(dirOutRoot) { if (isOpen("Log")) { selectWindow("Log"); saveAs("txt", dirOutRoot + "Warnings_LOG"); run("Close"); } else { print("Log window has not been found!"); run("Close"); } } // 9. Input dialog box user setting function InputUserSettingParameters(previewMode) { width=512; height=512; experimentTitle = "Insert Title"; ampliCycle = 5; varBlurring = 1; blurAfterAmpliNumber = 1; findMaximum = 4000; ALT = 5; lowerSize = 10; upperSize = 1000000; lowerCircularity = 0.10; upperCircularity = 1.00; volumeDet = false; blurAfterAmpli = true; wsBlurring = 2; savingSetting = true; loadPrevious = false; previewMode = false; Dialog.create("Analysis settings..."); Dialog.addString("Title:", experimentTitle); Dialog.addMessage("_____________________________________________________________________________"); Dialog.addMessage("Separation procedure:"); Dialog.addMessage(""); Dialog.addNumber("Separation Cycles:", ampliCycle, 0, 10, ""); Dialog.addToSameRow(); Dialog.addNumber("Blur During Separation Cycles:", varBlurring, 0, 10, ""); Dialog.addNumber("Blur-Sigma After Separation", blurAfterAmpliNumber, 0, 10, ""); Dialog.addToSameRow(); Dialog.addCheckbox("Blur After Separation", blurAfterAmpli); Dialog.addMessage("_____________________________________________________________________________"); Dialog.addMessage("LD Number Detection (Maxima):"); Dialog.addMessage(""); Dialog.addNumber("Find Maximum Noise Tolerance:", findMaximum, 0, 10, ""); Dialog.addMessage("_____________________________________________________________________________"); Dialog.addMessage("LD Number Detection (Edges):"); Dialog.addMessage(""); Dialog.addNumber("Auto Local Threshold Radius:", ALT, 0, 10, ""); Dialog.addNumber("Minimum LD Size (in pixel^2):", lowerSize, 0, 10, ""); Dialog.addToSameRow(); Dialog.addNumber("Maximum LD Size (in pixel^2):", upperSize, 0, 10, ""); Dialog.addNumber("Minimum LD Circularity:", lowerCircularity, 0, 10, ""); Dialog.addToSameRow(); Dialog.addNumber("Maximum LD Circularity:", upperCircularity, 0, 10, ""); Dialog.addMessage("_____________________________________________________________________________"); Dialog.addMessage("Volume Estimation:"); Dialog.addMessage(""); Dialog.addCheckbox("Estimate LD Volume", volumeDet); Dialog.addNumber("Classic Watershed Blurring:", wsBlurring, 0, 10, ""); Dialog.addMessage("_____________________________________________________________________________"); Dialog.addMessage(""); Dialog.addCheckbox("Save settings", savingSetting); Dialog.addMessage(""); Dialog.addCheckbox("Enter Preview Mode", previewMode); Dialog.addMessage(" The preview mode will guide you through the crucial steps to evaluate your settings."); Dialog.addMessage(""); Dialog.addCheckbox("Load previous settings", loadPrevious); Dialog.show(); experimentTitle = Dialog.getString(); ampliCycle = Dialog.getNumber(); varBlurring = Dialog.getNumber(); blurAfterAmpliNumber = Dialog.getNumber(); findMaximum = Dialog.getNumber(); ALT = Dialog.getNumber(); lowerSize = Dialog.getNumber(); upperSize = Dialog.getNumber(); lowerCircularity = Dialog.getNumber(); upperCircularity = Dialog.getNumber(); blurAfterAmpli = Dialog.getCheckbox(); volumeDet = Dialog.getCheckbox(); wsBlurring = Dialog.getNumber(); savingSetting = Dialog.getCheckbox(); previewMode = Dialog.getCheckbox(); loadPrevious = Dialog.getCheckbox(); if(loadPrevious == true) { previousSettings = LoadPreviousSettings(); ampliCycle = previousSettings[0]; varBlurring = previousSettings[1]; blurAfterAmpliNumber = previousSettings[3]; findMaximum = previousSettings[4]; ALT = previousSettings[5]; lowerSize = previousSettings[6]; upperSize = previousSettings[7]; lowerCircularity = previousSettings[8]; upperCircularity = previousSettings[9]; blurAfterAmpli = previousSettings[2]; volumeDet = previousSettings[11]; wsBlurring = previousSettings[10]; } inputDialogSetting = newArray(experimentTitle, ampliCycle, varBlurring, blurAfterAmpliNumber, findMaximum, ALT, lowerSize, upperSize, lowerCircularity, upperCircularity, blurAfterAmpli, volumeDet, wsBlurring, savingSetting, previewMode); return inputDialogSetting; } // 10. Save chosen user setting function SaveUserSettingParameters(savingSetting, experimentTitle, ampliCycle, blurAfterAmpli, blurAfterAmpliNumber, varBlurring, findMaximum, ALT, lowerSize, upperSize, lowerCircularity, upperCircularity, volumeDet, wsBlurring, dirOutRoot) { if(savingSetting == true) { print("Experiment title:\t" + experimentTitle); print("Separation Cycles:\t" + ampliCycle); print("Blur During Separation Cycles:\t" + varBlurring); if (blurAfterAmpli == true) { print("Blur-Sigma After Separation (sigma):\t" + blurAfterAmpli + "\t(" + blurAfterAmpliNumber + ")"); } else { print("Blur-Sigma After Separation (sigma):\t" + "0"); } print("Find Maximum Noise Tolerance:\t" + findMaximum); print("Auto Local Threshold Radius:\t" + ALT); print("Minimum LD Size (in pixel^2):\t" + lowerSize); print("Maximum LD Size (in pixel^2):\t" + upperSize); print("Minimum LD Circularity:\t" + lowerCircularity); print("Maximum LD Circularity:\t" + upperCircularity); if (volumeDet == true) { print("Classic Watershed Blurring:\t" + wsBlurring); } else { print("No volume analysis is performed"); } saveAs("txt", dirOutRoot + "Experimental_settings"); if (isOpen("Log")) { selectWindow("Log"); run("Close"); } } } // 11. Track analysis time for documentation function MacroStartUpTime() { monthNames = newArray("Jan","Feb","Mar","Apr","May","Jun","Jul","Aug","Sep","Oct","Nov","Dec"); dayNames = newArray("Sun", "Mon","Tue","Wed","Thu","Fri","Sat"); getDateAndTime(year, month, dayOfWeek, dayOfMonth, hour, minute, second, msec); timeString = "Date: " + dayNames[dayOfWeek] + " "; if (dayOfMonth<10) {timeString = timeString + "0";} timeString = timeString + dayOfMonth + "-" + monthNames[month] + "-" + year + "\nTime: "; if (hour<10) {timeString = timeString + "0";} timeString = timeString + hour +":"; if (minute<10) {timeString = timeString + "0";} timeString = timeString + minute + ":"; if (second<10) {timeString = timeString + "0";} timeString = timeString + second; return timeString; } // 12. Screen location function ScreenLocation() { imageWidth = getWidth(); imageHeight = getHeight(); imageReshapeWidth = screenWidth *0.5; imageReshapeHeight = imageWidth *10; imageShape = newArray(imageWidth, imageHeight, imageReshapeWidth, imageReshapeHeight); return imageShape; } // 13. Input image calibration and bit depth function InputImageProperties(title, unit, pixelWidth, pixelHeight) { // Select input image selectImage(title); imageDepth = bitDepth(); getDimensions(width, height, channels, slices, frames); // Checks if the input file is a multichannels, Z-stacks or a time series if(channels > 1 || slices > 1 || frames > 1) { selectWindow("Log"); run("Close"); exit("Multichannels/z-stack/time series images are not supported."); } // Checks for image bitdepth if(imageDepth != 8 && imageDepth != 16) { selectWindow("Log"); run("Close"); exit ("8-bit or 16-bit images are required."); } // Check bit deapth and reset min and max intensity value if (imageDepth == 16) { run("Apply LUT"); setMinAndMax(0, 65535); } getPixelSize(unit, pixelWidth, pixelHeight); if (unit != "pixels" || lengthOf(unit) != 1) { userPixelSize = pixelWidth; invertedUserPixelSize = 1/userPixelSize; run("Properties...", "channels=1 slices=1 frames=1 unit=um pixel_width=["+userPixelSize+"] pixel_height=["+userPixelSize+"] voxel_depth=["+userPixelSize+"]"); run("Set Scale...", "distance=["+invertedUserPixelSize+"] known=1 pixel=1 unit=micron global"); } if (unit == "pixels" || lengthOf(unit) == 1) { Dialog.create("Image Properties"); Dialog.addMessage("Uncalibrated input image"); Dialog.addNumber("Enter the pixel size: ", 0.10638); Dialog.show(); Dialog.setLocation(255, 255); userPixelSize = Dialog.getNumber(); invertedUserPixelSize = 1/userPixelSize; run("Properties...", "channels=1 slices=1 frames=1 unit=um pixel_width=["+userPixelSize+"] pixel_height=["+userPixelSize+"] voxel_depth=["+userPixelSize+"]"); run("Set Scale...", "distance=["+invertedUserPixelSize+"] known=1 pixel=1 unit=micron global"); } imageProperties = newArray(invertedUserPixelSize, imageDepth); return imageProperties; } // 14.Polygon selection function PolygonSelection(title, imageReshapeWidth, imageReshapeHeight, imageWidth, imageHeight) { selectImage(title); setBatchMode("show"); setLocation(0, 0, imageReshapeWidth, imageReshapeHeight); // Draw a polygon selection around the ROI setTool("polygon"); waitForUser("Select the Region of Interest. Then press Ok!"); // Update the progress bar showProgress(0); // Check if selection exist type = selectionType(); // If selection does not exist use the whole image if (type == -1) { makeRectangle(0, 0, imageWidth, imageHeight); Roi.setName("ROI_drawn"); roiManager("Add"); } // Otherwise save the ROI in the ROI manager if ((type >= 0 && type <= 4) || type == 9) { Roi.setName("ROI_drawn"); roiManager("Add"); } // Do not show the images setBatchMode("hide"); } // 15. Amplification cycles for deblurring function AmplificationCircles(imageDepth, ampliCycle, title, saveTitle, varBlurring) { if (imageDepth == 16) { for (j=0; j 0) { done = true; return b; } if (b < -1) { // End functions CloseAllWindows(); // 1 CloseLogWindow(dirOutRoot); // 8 CloseBCWindow(); // 3 CloseROIsManager(); // 4 CloseThresholdWindows(); // 21 // Display the image and clear the memory setBatchMode(false); call("java.lang.System.gc"); exit("No LDs have been found! Please run the macro with a different setting."); } } } // 18. Print summary function (Modified from ImageJ/Fiji Macro Documentation) function PrintSummary(textSummary) { titleSummaryWindow = "ParticlesAreaVolume"; titleSummaryOutput = "["+titleSummaryWindow+"]"; outputSummaryText = titleSummaryOutput; if (!isOpen(titleSummaryWindow)) { // Create the results window run("Text Window...", "name="+titleSummaryOutput+" width=90 height=20 menu"); // Print the header and output the first line of text print(outputSummaryText, "% Id\t" + "% Area (um^2)\t" + "% Volume (um^3)\t" + "\n"); print(outputSummaryText, textSummary +"\n"); eval("script","f = WindowManager.getFrame('ParticlesAreaVolume'); f.setLocation(0,0); f.setSize(10,10);"); } else { print(outputSummaryText, textSummary +"\n"); } } // 19. Check for warning function Warning(analysisPath, saveTitle, warningException, warningConsole) { // Prints the console window or exception window if it opens (indicates problems) for further bug-management if (isOpen("Exception")) { selectWindow("Exception"); saveAs("txt", analysisPath + "Exception_Log_" + saveTitle); run("Close"); warningException = true; } if (isOpen("Console")) { selectWindow("Console"); saveAs("txt", analysisPath + "Console_Log_" + saveTitle); run("Close"); warningConsole = true; }´ outPutWarning = newArray(warningException, warningConsole); return outPutWarning; } // 20. Print warnings function PrintWarning(warningException, warningConsole, analysisPath, title) { if (warningException == true || warningConsole == true) { print("> Warning: Log file can not be found " + analysisPath); } else { print("> Processed file: " + title); } } // 21. Close thresholod window function CloseThresholdWindows() { if (isOpen("Threshold")) { selectWindow("Threshold"); run("Close"); } } // 22. Load previous settings function LoadPreviousSettings() { experiment = getDirectory("Choose the previous experiment directory to load the setting"); if(File.exists(experiment + "experimental_settings.txt")) { parameters = File.openAsString(experiment + "experimental_settings.txt"); singleParameters = split(parameters,"\n"); ampliCycle = parseInt(replace(singleParameters[1],"Separation Cycles:\t","")); varBlurring = parseInt(replace(singleParameters[2], "Blur During Separation Cycles:\t", "")); blurAfterAmpli_raw = replace(singleParameters[3], "Blur-Sigma After Separation \\(sigma\\):\t",""); bracketPosition = indexOf(blurAfterAmpli_raw, "("); blurAfterAmpli = parseInt(substring(blurAfterAmpli_raw, bracketPosition-2,bracketPosition-1)); blurAfterAmpliNumber = parseInt(substring(blurAfterAmpli_raw, bracketPosition+1, bracketPosition+2)); findMaximum = parseInt(replace(singleParameters[4], "Find Maximum Noise Tolerance:\t", "")); ALT = parseInt(replace(singleParameters[5], "Auto Local Threshold Radius:\t", "")); lowerSize = parseInt(replace(singleParameters[6], "Minimum LD Size \\(in pixel\\^2\\):\t", "")); upperSize = parseInt(replace(singleParameters[7], "Maximum LD Size \\(in pixel\\^2\\):\t", "")); lowerCircularity = parseFloat(replace(singleParameters[8], "Minimum LD Circularity:\t", "")); upperCircularity = parseFloat(replace(singleParameters[9], "Maximum LD Circularity:\t","")); wsBlurring = parseInt(replace(singleParameters[10], "Classic Watershed Blurring:\t","")); //print("Previous settings loaded from " + experiment); if(isNaN(wsBlurring) == true && File.exists(experiment + "experimental_settings.txt") == true) { addVolumeAnalysis = getBoolean("Do you want to analyze the LD volume?"); if(addVolumeAnalysis == true) { volumeDet = true; Dialog.create("Analysis settings"); Dialog.addNumber("Classic Watershed Blurring:", 2, 0, 7, ""); Dialog.show(); wsBlurring = Dialog.getNumber(); } else { volumeDet = 0; wsBlurring = 0; } } else { addVolumeAnalysis = getBoolean("Do you want to analyze the LD volume?"); if(addVolumeAnalysis == true) { volumeDet = 1; } else { volumeDet = 0; wsBlurring = 0; } } } else { exit("No settings found in the selected folder"); } previousSettings = newArray(ampliCycle, varBlurring, blurAfterAmpli, blurAfterAmpliNumber, findMaximum, ALT, lowerSize, upperSize, lowerCircularity, upperCircularity, wsBlurring, volumeDet); return previousSettings; } // 23. PreviewDialog: Segmentation function PreviewDialogSegmentation() { Dialog.create("Analysis settings..."); Dialog.addMessage("_____________________________________________________________________________"); Dialog.addMessage("Separation procedure:"); Dialog.addMessage(""); Dialog.addNumber("Separation Cycles:", ampliCycle, 0, 10, ""); Dialog.addToSameRow(); Dialog.addNumber("Blur During Separation Cycles:", varBlurring, 0, 10, ""); Dialog.addNumber("Blur-Sigma After Separation", blurAfterAmpliNumber, 0, 10, ""); Dialog.addToSameRow(); Dialog.addCheckbox("Blur After Separation", blurAfterAmpli); Dialog.show(); ampliCycle = Dialog.getNumber(); varBlurring = Dialog.getNumber(); blurAfterAmpliNumber = Dialog.getNumber(); blurAfterAmpli = Dialog.getCheckbox(); previewSegmentationSetting = newArray(ampliCycle, varBlurring, blurAfterAmpli, blurAfterAmpliNumber); return previewSegmentationSetting; } // 24. PreviewDialog: FindMaximum function PreviewDialogFindMaximum() { Dialog.create("Analysis settings..."); Dialog.addMessage("_____________________________________________________________________________") Dialog.addNumber("Find Maximum Noise Tolerance:", findMaximum, 0, 10, ""); Dialog.addMessage(""); Dialog.show(); findMaximum = Dialog.getNumber(); return findMaximum; } // 25. PreviewDialog: Edge Detection function PreviewDialogEdges() { Dialog.create("Analysis settings..."); Dialog.addMessage("_____________________________________________________________________________"); Dialog.addMessage("LD Number Detection (Edges):"); Dialog.addMessage(""); Dialog.addNumber("Auto Local Threshold Radius:", ALT, 0, 10, ""); Dialog.addNumber("Minimum LD Size (in pixel^2):", lowerSize, 0, 10, ""); Dialog.addToSameRow(); Dialog.addNumber("Maximum LD Size (in pixel^2):", upperSize, 0, 10, ""); Dialog.addNumber("Minimum LD Circularity:", lowerCircularity, 0, 10, ""); Dialog.addToSameRow(); Dialog.addNumber("Maximum LD Circularity:", upperCircularity, 0, 10, ""); Dialog.show(); ALT = Dialog.getNumber(); lowerSize = Dialog.getNumber(); upperSize = Dialog.getNumber(); lowerCircularity = Dialog.getNumber(); upperCircularity = Dialog.getNumber(); previewEdgesSetting = newArray(ALT, lowerSize, upperSize, lowerCircularity, upperCircularity); return previewEdgesSetting; } // 26. PreviewDialog: Classic Watershed function PreviewDialogClassicWatershed() { Dialog.create("Analysis settings..."); Dialog.addMessage("_____________________________________________________________________________"); Dialog.addMessage("Volume Estimation:"); Dialog.addNumber("Classic Watershed Blurring:", wsBlurring, 0, 10, ""); Dialog.show(); wsBlurring = Dialog.getNumber; return wsBlurring; } // 27. print preview Settings to the analysis folder without messing up the log window function PrintPreviewSettings(savingSetting, experimentTitle, ampliCycle, blurAfterAmpli, blurAfterAmpliNumber, varBlurring, findMaximum, ALT, lowerSize, upperSize, lowerCircularity, upperCircularity, volumeDet, wsBlurring, analysisPath) { titleWindow = "Preview Mode Settings"; titleOutput = "["+titleWindow+"]"; if (!isOpen(titleWindow)) { // Create the results window run("Text Window...", "name="+titleOutput+" width=90 height=20 menu"); eval("script","f = WindowManager.getFrame('Preview Mode Settings'); f.setLocation(0,0); f.setSize(10,10);"); print(titleOutput, "Experiment title:\t" + experimentTitle + "\n"); print(titleOutput, "Separation Cycles:\t" + ampliCycle + "\n"); print(titleOutput, "Blur During Separation Cycles:\t" + varBlurring + "\n"); if (blurAfterAmpli == true) { print(titleOutput, "Blur-Sigma After Separation (sigma):\t" + blurAfterAmpli + "\t(" + blurAfterAmpliNumber + ")" + "\n"); } else { print(titleOutput, "Blur-Sigma After Separation (sigma):\t" + "0" + "\n"); } print(titleOutput, "Find Maximum Noise Tolerance:\t" + findMaximum + "\n"); print(titleOutput, "Auto Local Threshold Radius:\t" + ALT + "\n"); print(titleOutput, "Minimum LD Size (in pixel^2):\t" + lowerSize + "\n"); print(titleOutput, "Maximum LD Size (in pixel^2):\t" + upperSize + "\n"); print(titleOutput, "Minimum LD Circularity:\t" + lowerCircularity + "\n"); print(titleOutput, "Maximum LD Circularity:\t" + upperCircularity + "\n"); if (volumeDet == true) { print(titleOutput, "Classic Watershed Blurring:\t" + wsBlurring + "\n"); } else { print(titleOutput, "No volume analysis is performed" + "\n"); } selectWindow("Preview Mode Settings"); saveAs("Text", analysisPath + "experimental_settings.txt"); run("Close"); } } // 28. merge the volume text windows function MergeSummaryWindows() { titleMergedSummary = "Single_LD_Volumes"; titleMergedSummaryOutput = "["+titleMergedSummary+"]"; outputMergedSummaryText = titleMergedSummaryOutput; run("Text Window...", "name="+titleMergedSummary+" width=90 height=20 menu"); print(outputMergedSummaryText, "% Id\t" + "% Area (um^2)\t" + "% Volume (um^3)\t" ); eval("script","f = WindowManager.getFrame('Single_LD_Volumes'); f.setLocation(0,0); f.setSize(10,10);"); for (aa=0; aa Renaming input image: " + title); } return title; } // ############################################# MACRO ################################################### // Use exidecimal code to create the macro logo macro "LD finding Action Tool - 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 [6]" { // Start up functions CloseAllWindows(); // 1 CheckPluginInstallation(); // 2 CloseBCWindow(); // 3 CloseROIsManager(); // 4 OpenROIsManager(); // 5 CloseResultsWindow(); // 6 Setting(); // 7 // Choose the input root directory and get the file list dirIn = getDirectory("Choose the INPUT root directory"); list = getFileList(dirIn); // General output root path dirOutRoot = dirIn; // Tiff/tif file list files = newArray(); saveTitleArray = newArray(); for(a=0; a Processing input file: 0" + i + "_" + title); if(i >= 1 && previewMode == true) { Dialog.create("Preview Mode"); options = newArray("Yes", "No"); Dialog.addRadioButtonGroup("Do you want to keep the preview mode on?", options, 2, 1, ""); Dialog.show(); decision = Dialog.getRadioButton(); if(decision == "Yes") { previewMode = true; } else { previewMode = false; } } // 12. Set screen location and image size imageShape = ScreenLocation(); // 29. Replace empty characters in image file name title = ReplaceEmptyCharc(title); // Remove input file estention .tiff/tif dotIndex = indexOf(title, "."); saveTitle = substring(title, 0, dotIndex); saveTitleArray = Array.concat(saveTitleArray, saveTitle); // 13. Function: check input image bit depth and pixel size // Pixel size unit getPixelSize(unit, pixelWidth, pixelHeight); imageProperties = InputImageProperties(title, unit, pixelWidth, pixelHeight); invertedUserPixelSize = imageProperties[0]; imageDepth = imageProperties[1]; // Duplicate the input image to keep the original input file selectImage(title); run("Duplicate...", "title=" + saveTitle + "_original"); title10 = getTitle(); //setLocation(0, 0, imageShape[2], imageShape[3]); // Create the output directory analysisPath = dirOutRoot + saveTitle + "_Analysis" + File.separator; File.makeDirectory(analysisPath); // 14. Function: polygon selection imageWidth = imageShape[0]; imageHeight = imageShape[1]; imageReshapeWidth = imageShape[2]; imageReshapeHeight = imageShape[3]; PolygonSelection(title, imageReshapeWidth, imageReshapeHeight, imageWidth, imageHeight); ////////////////////////////////////////////////////////////////////// ///////////////// Amplification/Segmentation Cycles ////////////////// ////////////////////////////////////////////////////////////////////// // Update the progress bar showProgress(0.2); while (amplificationResult == 1) { // Process the input image selectImage(title); //setLocation(0, 0, imageShape[2], imageShape[3]); makeRectangle(0, 0, imageShape[0], imageShape[1]); // 15. Amplification/Segmentation Cycles title = AmplificationCircles(imageDepth, ampliCycle, title, saveTitle, varBlurring); if (previewMode == 1) { // Display the output image selectImage(title); setBatchMode("show"); setLocation(0,0,imageReshapeWidth,imageReshapeHeight); waitForUser("Filtered image for LDs separation\nZoom in to check the LDs detection"); amplificationResult = getBoolean("Do you want to repeat the image filtering?"); if (amplificationResult == 1) { // Close the output of amplifcation selectImage(title); close(title); // Close the Log window selectWindow("Log"); run("Close"); // Create amplification input image selectImage(title10); run("Duplicate...", "title=" + title); title = getTitle(); // 9. Input dialog box setting previewSegmentationSetting = PreviewDialogSegmentation(); ampliCycle = previewSegmentationSetting[0]; varBlurring = previewSegmentationSetting[1]; blurAfterAmpli = previewSegmentationSetting[2]; blurAfterAmpliNumber = previewSegmentationSetting[3]; // 11. Update start up macro recording time function timeString = MacroStartUpTime(); print(timeString, "\n"); print("Processing input file: 0" + i + "_" + title); //amplificationResult = true; previewMode = true; } if (amplificationResult == false) { amplificationResult = false; } // Do not display the images setBatchMode("hide"); } else { amplificationResult = false; } } while (findMaximumResult == true) { if (previewMode == true) { selectImage(title); roiManager("Select", FindSpecificROI("ROI_drawn")); run("Find Maxima...", "noise=["+findMaximum+"] output=[Point Selection]"); Roi.setName("total_maxima_found_preliminary"); roiManager("Add"); roiManager("show none"); selectImage(title10); roiManager("Select", FindSpecificROI("total_maxima_found_preliminary")); setBatchMode("show"); setLocation(0,0,imageReshapeWidth,imageReshapeHeight); waitForUser("Overlay of local maxima on raw image.\nZoom in to check if the LDs are properly labeled."); findMaximumResult = getBoolean("Do you want to adjust the Noise Tolerance?"); if (findMaximumResult == true) { roiManager("show none"); roiManager("Select", FindSpecificROI("total_maxima_found_preliminary")); roiManager("Delete"); findMaximum = PreviewDialogFindMaximum(); //findMaximumResult = true; previewMode = true; setBatchMode("hide"); } else { findMaximumResult = false; roiManager("Select", FindSpecificROI("total_maxima_found_preliminary")); roiManager("Delete"); setBatchMode("hide"); } } else { findMaximumResult = false; } } while (edgeDetectionResult == true) { if (previewMode == true) { selectWindow(title); makePoint(0,0); setBatchMode("hide"); run("Duplicate...", "title=" + "LD edges"); rename("LD edges"); selectImage("LD edges"); run("Find Edges"); run("8-bit"); run("Auto Local Threshold", "method=Phansalkar radius=["+ALT+"] parameter_1=0 parameter_2=0 white"); run("Invert"); run("Fill Holes"); run("Watershed"); roiManager("select", FindSpecificROI("ROI_drawn")); run("Analyze Particles...", "size=["+lowerSize+"] - ["+upperSize+"] pixel circularity=["+lowerCircularity+"] - ["+upperCircularity+"] show=Masks"); selectWindow(title10); setBatchMode("show"); setLocation(0,0,imageReshapeWidth,imageReshapeHeight); selectImage("Mask of LD edges"); setBatchMode("show"); setLocation(screenWidth/2,0,imageReshapeWidth,imageReshapeHeight); waitForUser("LDs edges. Check if the detection is sufficient.\n\nNote that the particles are also important for the volume analysis."); edgeDetectionResult = getBoolean("Do you want to repeat the edge detection?"); if (edgeDetectionResult == true) { selectImage("Mask of LD edges"); close(); if(isOpen("LD edges")) { selectImage("LD edges"); close(); } previewEdgesSetting = PreviewDialogEdges(); ALT = previewEdgesSetting[0]; lowerSize = previewEdgesSetting[1]; upperSize = previewEdgesSetting[2]; lowerCircularity = previewEdgesSetting[3]; upperCircularity = previewEdgesSetting[4]; previewMode = true; } else { setBatchMode("hide"); if (isOpen("Mask of LD edges")) { selectImage("Mask of LD edges"); close(); } if (isOpen("LD edges")) { selectImage("LD edges"); close(); } } } else { edgeDetectionResult = false; } } // Hide the duplicated original image selectImage(title10); setBatchMode("hide"); // Duplicate the input image for edges detection selectImage(title); run("Duplicate...", "title=" + saveTitle + "_LD_edges"); title6 = getTitle(); ////////////////////////////////////////////////////////////////////// ///////////////////// Volume Determination /////////////////////////// ////////////////////////////////////////////////////////////////////// if (volumeDet == true) { while (classicWatershedResult == true) { // Update the progress bar showProgress(0.3); selectImage(title6); run("Duplicate...", "title=" + saveTitle + "_volumes"); title16 = getTitle(); // Select the ROI using the ROI name roiManager("Select", FindSpecificROI("ROI_drawn")); if(blurAfterAmpli == true) { run("Gaussian Blur...", "sigma=["+blurAfterAmpliNumber+"]"); } // Find maxima run("Find Maxima...", "noise=["+findMaximum+"] output=[Point Selection]"); Roi.setName("total_maxima_found"); roiManager("Add"); selectImage(title16); run("8-bit"); run("Duplicate...", "title=" + saveTitle + "_temp_edge"); title18 = getTitle(); // *** Morphological reconstruction using the maxima defined above => lipid droplets get an area run("Find Edges"); run("Auto Local Threshold", "method=Phansalkar radius=["+ALT+"] parameter_1=0 parameter_2=0 white"); run("Invert"); run("Fill Holes"); run("Analyze Particles...", "size=["+lowerSize+"] - ["+upperSize+"] pixel circularity=0.00-1.00 show=Masks clear"); eval("script","f = WindowManager.getFrame('Summary'); f.setLocation(0,0); f.setSize(10,10);"); selectImage("Mask of " + title18); title18Mask = getTitle(); roiManager("Select", FindSpecificROI("total_maxima_found")); run("Interactive Morphological Reconstruction", "type=[By Dilation] connectivity=8"); rename(saveTitle + "_reconstructed"); run("Invert"); title20 = getTitle(); // Close image no longer used selectImage(title18Mask); close(title18Mask); selectImage(title18); close(title18); // *** selectImage(title16); makeRectangle(0, 0, 0, 0); run("Gaussian Blur...", "sigma=["+wsBlurring+"]"); imageCalculator("Subtract create 32-bit", title16, title20); rename(saveTitle + "_watershedding"); title19 = getTitle(); setMinAndMax(0, 255); run("8-bit"); setMinAndMax(0,255); run("Invert"); // Close image not longer used selectImage(title16); close(title16); selectImage(title20); close(title20); selectImage(title19); nBins = 256; getHistogram(values, counts, nBins); row = 0; for (x=0; x 1) { run("Find Maxima...", "noise=1 output=[Point Selection]"); Roi.setName("overlayed_counted_lipid_droplets"); roiManager("Add"); } if(numberFound <= 1) { // End functions CloseAllWindows(); // 1 CloseLogWindow(dirOutRoot); // 8 CloseBCWindow(); // 3 CloseROIsManager(); // 4 CloseThresholdWindows(); // 21 // Display the image and clear the memory setBatchMode(false); call("java.lang.System.gc"); exit("LDs detection failed! Please run the macro with a different setting."); } // newImage(saveTitle + "_FM_only", "8-bit white", imageShape[0], imageShape[1], 1); title11 = getTitle(); roiManager("Select", FindSpecificROI("total_maxima_found")); setMinAndMax(255, 255); run("Apply LUT"); run("Analyze Particles...", "size=0-Infinity circularity=0.00-1.00 show=Nothing summarize"); // Measure maxima that overlayed with edge-particles newImage(saveTitle + "_FM_edge_overlay", "8-bit white", imageShape[0], imageShape[1], 1); title12 = getTitle(); roiManager("Select", FindSpecificROI("overlayed_counted_lipid_droplets")); setMinAndMax(255, 255); run("Apply LUT"); run("Analyze Particles...", "size=0-Infinity circularity=0.00-1.00 show=Nothing summarize"); // ROIs are saved, partly individually, in the corresponding analysis folder. roiManager("Select", FindSpecificROI("overlayed_counted_lipid_droplets")); saveAs("Selection", analysisPath + "overlayed_counted_lipid_droplets"); roiManager("Select", FindSpecificROI("total_maxima_found")); saveAs("Selection", analysisPath + "total_maxima_found"); roiManager("deselect"); roiManager("Save", analysisPath + "edge_particles_and_maxima" + ".zip"); // Close the image are not used selectImage(title6); close(title6); selectImage(title7); close(title7); selectImage(title8); close(title8); selectImage(title9); close(title9); selectImage(title11); close(title11); selectImage(title12); close(title12); // Update the progress bar showProgress(0.8); // Make an image with both ROIs as a colored overlap and save the image selectImage(title10); run("Duplicate...", "title=" + saveTitle + "_single_ROI"); title14 = getTitle(); roiManager("Select", FindSpecificROI("total_maxima_found")); run("Properties... ", "stroke=red point=Dot size=Tiny"); run("Add Selection..."); roiManager("Deselect"); roiManager("Select", FindSpecificROI("overlayed_counted_lipid_droplets")); run("Properties... ", "stroke=green point=Dot size=Tiny"); run("Add Selection..."); saveAs("jpeg", analysisPath + saveTitle + "_both_maxima"); title14 = getTitle(); if(previewMode == true) { setBatchMode("show"); setLocation(0,0,imageReshapeWidth,imageReshapeHeight); waitForUser("Please check if the LD detection is accurate. Green dots indicate positive LDs while red dots indicate false positive LDs"); LDDetectionResult = getBoolean("Is the LD detection correct?"); if(LDDetectionResult==false) { startOver = getBoolean("Do you want to repeat the image processing?"); if(startOver == true) { i=i-1; } } if (savingSetting == true) { PrintPreviewSettings(savingSetting, experimentTitle, ampliCycle, blurAfterAmpli, blurAfterAmpliNumber, varBlurring, findMaximum, ALT, lowerSize, upperSize, lowerCircularity, upperCircularity, volumeDet, wsBlurring, analysisPath); } print("\nWarning: Overwriting previous setting. The new setting is applied to all the images starting from: " + title, "\nThe Settings is saved in the respective analysis folder"); } // selectImage(title10); run("Duplicate...", "title=" + saveTitle + "_edge_ROI"); title15 = getTitle(); roiManager("Select", FindSpecificROI("overlayed_counted_lipid_droplets")); roiManager("delete"); roiManager("Select", FindSpecificROI("total_maxima_found")); roiManager("delete"); roiManager("Show All without labels"); run("Flatten"); title15Flat = getTitle(); saveAs("jpeg", analysisPath + saveTitle + "_edges"); title15Flat = getTitle(); // Update the progress bar showProgress(0.9); // Save the input image selectImage(title10); saveAs("jpeg", analysisPath + saveTitle + "_original"); title10 = getTitle(); // Reset the scale if (i == files.length-1) { run("Set Scale...", "distance=0 known=0 pixel=1 unit=pixel"); } close(title10); // Close all the open images one by one selectImage(title14); close(title14); selectImage(title15); close(title15); selectImage(title15Flat); close(title15Flat); selectImage(title); close(title); // Close results window CloseResultsWindow(); // Clear the roiManger roiManager("reset"); // 19. Check for warnings outPutWarning = Warning(analysisPath, saveTitle, warningException, warningConsole); warningException = outPutWarning[0]; warningConsole = outPutWarning[1]; // 20. Print warnings PrintWarning(warningException, warningConsole, analysisPath, title); } } // Update the progress bar showProgress(1); // Save the summary table if (isOpen("Summary")) { selectWindow("Summary"); eval("script","f = WindowManager.getFrame('Summary'); f.setLocation(0,0); f.setSize(500,500);"); selectWindow("Summary"); tableTitle = "Summary"; firstFolderName = File.getName(dirIn); secondFolder = File.getParent(dirIn); secondFolderName = File.getName(secondFolder); thirdFolder = File.getParent(secondFolder); thirdFolderName = File.getName(thirdFolder); forthFolder = File.getParent(thirdFolder); forthFolderName = File.getName(forthFolder); saveAs("results", dirIn + tableTitle + "_" + experimentTitle + "_" + forthFolderName + "_" + thirdFolderName + "_" + secondFolderName + "_" + firstFolderName + ".xls"); } if (isOpen("Log")) { // Enlarges the Log window for better visibility selectWindow("Log"); eval("script","f = WindowManager.getFrame('Log'); f.setLocation(0,500); f.setSize(500,500);"); } // Output processing speed stop = getTime(); seconds = (stop - start) /1000; speed = files.length /seconds; if (files.length == 1) { print("\nProcessed " + files.length + " file in " + seconds + " seconds!"); } if (files.length != 1) { print("\nProcessed " + files.length + " files in " + seconds + " seconds!"); } if (volumeDet == true) { MergeSummaryWindows(); } // End functions print("\n%%% Congratulation your files have been successfully processed %%%\n"); CloseBCWindow(); // 3 CloseROIsManager(); // 4 CloseThresholdWindows(); // 21 wait(3000); CloseLogWindow(dirOutRoot); // 8 // Display the images and clear the memory setBatchMode(false); call("java.lang.System.gc"); // Display status showStatus("Finished!"); wait(3000); }