//Macro_Opera_RGB_merger macroName = "Opera_RGB_merger"; macroShortDescription = "This macro creates RGB images from Opera microscope .flex images (16-bit)."; macroDescription = "This macro merges up to 4 channels to RBG images from Opera automated microscope." + "\nOptionally background substraction and auto-contrast can be adjusted for each channel" + "\nand in the manual mode the macro allows interactively to process each image manually."; macroRelease = "third release 26-06-2014 by Martin Stöter (stoeter(at)mpi-cbg.de)"; macroHelpURL = "http://idisk-srv1.mpi-cbg.de/knime/FijiUpdate/TDS%20macros/" + macroName + ".htm"; macroHtml = "" +"" + macroName + "/n" + macroRelease + "
" +"Check for help on this web page:
" +"" + macroHelpURL + "
" +"...get this URL from Log window!
" +""; //print macro name and current time to Log window getDateAndTime(year, month, dayOfWeek, dayOfMonth, hour, minute, second, msec); month++; print("\\Clear"); print(macroName,"\nStart:",year+"-"+month+"-"+dayOfMonth+", "+hour+"-"+minute+"-"+second); print(macroHelpURL); //start macro Dialog.create("Fiji macro: " + macroName); Dialog.addMessage("Fiji macro: " + macroName + " (Fiji-Tools by TDS@MPI-CBG)\n \n" + macroShortDescription + "\nClick 'OK' to go on, 'Cancel' to quit or 'Help' for online description."); Dialog.addHelp(macroHtml); Dialog.show; //choose folders inputPath = getDirectory("Choose image folder... "); outputPath = getDirectory("Choose result image folder... or create a folder"); printPaths = "inputPath = \"" + inputPath + "\";\noutputPath = \"" + outputPath + "\";"; print(printPaths); tempLogFileNumber = 1; while (File.exists(outputPath + "Log_temp_" + tempLogFileNumber +".txt")) tempLogFileNumber ++; //find last tempLog file to prevent overwriting of log //set array variables for RGB merge availableRGBcolors = newArray("Red", "Green", "Blue", "Grays"); //possible color selections in pull-down colorsForRGBmerge = newArray("*None*", "*None*", "*None*", "*None*"); //array of color selection for channel 1-4 availableChannels = newArray("*None*", "Channel_0", "Channel_1", "Channel_2", "Channel_3"); //array of color selection for channel 1-4 changeColor = false; channelColorVector = newArray(4); channelsForRGBmerge = newArray(4); availableRGBfileFormats = newArray("jpeg","png"); //possible output RGB file formats //choose colors for each channel Dialog.create("RGB merging"); Dialog.addNumber("Number of fields per well for RGB (0=all):", 0); //set number of field for RBG images Dialog.addNumber("Start at well index (first well = 0):", 0); Dialog.addCheckbox("Adjust image contrast manually?", true); Dialog.addCheckbox("Do background substraction?", false); Dialog.addCheckbox("Set batch mode (no images shown)?", false); Dialog.addString("Add prefix to all RGB image names:", ""); Dialog.addString("Add suffix to all RGB image names:", "_RGB"); Dialog.addChoice("Save RGB images :", availableRGBfileFormats); Dialog.addCheckbox("Show all files in log window?", false); Dialog.show(); numberOfSelectedChannels = 0; numberOfFields = Dialog.getNumber(); startWellIndex = Dialog.getNumber(); manualMode = Dialog.getCheckbox(); bkgCorrection = Dialog.getCheckbox(); batchMode = Dialog.getCheckbox(); rgbFilePrefix = Dialog.getString(); rgbFileSuffix = Dialog.getString(); rgbFileFormat = Dialog.getChoice(); fileListToLog = Dialog.getCheckbox(); seriesString = ""; for (i = 1; i <= numberOfFields; i++) seriesString = seriesString + " series_" + i; // " series_1 series_2 series_3 series_4" saveRGB = false; makeMontage = false; saveMontage = false; montageScale = 0.5; percentileArray = newArray(0,1); if (manualMode) run("Color Balance..."); //run("Arrange Channels..."); //run("Channels Tool..."); //set variables for auto contrast and background corrections autoContrastValue = newArray(0.35, 0.35, 0.35, 0.35); autoContrast = false; bkgValue = newArray(200, 200, 200, 200); if (bkgCorrection) { Dialog.create("Background substraction parameter (0 = not applied)"); Dialog.addMessage("Background substraction\nRolling ball radius (0 = not applied)"); for (i = 1; i <= 4; i++) Dialog.addNumber(availableChannels[i], bkgValue[i-1]); Dialog.show(); for (i = 0; i < 4; i++) bkgValue[i] = Dialog.getNumber(); } //initialize => default settings run("Set Measurements...", "area mean standard min centroid center shape integrated median display redirect=None decimal=3"); run("Input/Output...", "jpeg=95 gif=-1 file=.txt copy_column copy_row save_column save_row"); run("Close All"); setBatchMode((batchMode && !manualMode)); ////////////////// M A C R O : R G B - M E R G E A R R A Y S C A N I M A G E S /////////////////////////////// //get file list and well list fileList = getFileList(inputPath); Array.sort(fileList); //also done in function getAllWellsFunction l = fileList.length; if (fileListToLog) for (i = 0; i < l; i++) { print(fileList[i]); } wellList = getAllWellsArrayScanFunction(fileList, false, fileListToLog); exampleFileName = getImageFileExampleArrayScanFunction(fileList); //now load well-field by well-field and merge to RGB for (currentWell = startWellIndex; currentWell < wellList.length; currentWell++) { showProgress(currentWell, wellList.length); fileName = substring(exampleFileName,0,lengthOf(exampleFileName)-14) + wellList[currentWell] + substring(exampleFileName,lengthOf(exampleFileName)-8,lengthOf(exampleFileName)); //to log window //print("well:", wellList[currentWell], ", field:", currentField, ", channel:", currentChannel, " file:", fileName); print("well:", wellList[currentWell], ", file:", fileName); //if (imageFormat == "Opera (.flex)") run("TIFF Virtual Stack...", "open=" + inputPath + fileName); if (File.exists(inputPath + fileName)) { IJ.redirectErrorMessages(); if (bkgCorrection) setBatchMode(true); if (seriesString == "") {// white spaces dont work!!! run("Bio-Formats", "open=" + inputPath + fileName + " autoscale color_mode=Composite concatenate_series open_files open_all_series view=Hyperstack stack_order=XYCZT"); } else { run("Bio-Formats", "open=" + inputPath + fileName + " autoscale color_mode=Composite concatenate_series open_files view=Hyperstack stack_order=XYCZT" + seriesString); } imageTitle = getTitle(); getDimensions(width, height, channels, slices, frames); print(width, height, channels, slices, frames); fileTitle = getTitle(); //print(fileTitle); if (bkgCorrection) { labelArray = newArray(frames * channels); for (i = 1; i <= frames * channels; i++) { setSlice(i); labelArray[i-1] = getMetadata("Label"); //print(i,labelArray[i-1]); } run("Split Channels"); mergeString = ""; for (currentChannel = 1; currentChannel <= channels; currentChannel++) { selectWindow("C" + currentChannel + "-" + fileTitle); if (bkgValue[currentChannel] > 0) run("Subtract Background...", "rolling=" + bkgValue[currentChannel]); mergeString = mergeString + "c" + currentChannel + "=[C" + currentChannel + "-" + fileTitle + "] "; } //run("Merge Channels...", "c1=[C1-002002000.flex - Well B-2; Field #1] c2=[C2-002002000.flex - Well B-2; Field #1] c3=[C3-002002000.flex - Well B-2; Field #1] create"); run("Merge Channels...", mergeString + "create"); for (i = 1; i <= frames * channels; i++) { setSlice(i); setMetadata("Label",labelArray[i-1]); } setBatchMode(false); } //if (autoContrast && autoContrastValue[currentChannel] > 0) run("Enhance Contrast", "saturated=" + autoContrastValue[currentChannel]); goToNextWell = false; sliceCounter = 1; do { if (sliceCounter < channels * frames) setSlice(sliceCounter); if (manualMode && !goToNextWell) { waitForUser("Adjust contrast manually, then 'OK'"); Dialog.create("Manual mode! Well: " + wellList[currentWell]); //enable use inveractivity Dialog.addCheckbox("Stay in manual mode?", manualMode); Dialog.addCheckbox("Make Montage?", makeMontage); Dialog.addCheckbox("Save Montage?", saveMontage); Dialog.addNumber("Montage scale? (0=dont save)", montageScale); Dialog.addString("Add prefix to all RGB image names:", rgbFilePrefix); Dialog.addString("Add suffix to all RGB image names:", rgbFileSuffix); Dialog.addCheckbox("Save current RGB image", saveRGB); Dialog.addNumber("Save number of fields automatically as RGB (-1= none,0=all):", -1); Dialog.addCheckbox("Change auto-contrast values?", false); Dialog.addCheckbox("Change color of channels?", false); Dialog.addCheckbox("Go to next field?", false); Dialog.addCheckbox("Go to next well?", false); Dialog.show(); manualMode = Dialog.getCheckbox(); makeMontage = Dialog.getCheckbox(); saveMontage = Dialog.getCheckbox(); montageScale = Dialog.getNumber(); rgbFilePrefix = Dialog.getString(); rgbFileSuffix = Dialog.getString(); saveRGB = Dialog.getCheckbox(); numberOfFieldsToBeSavedAsRGB = Dialog.getNumber(); changeAutoContrastDialog = Dialog.getCheckbox(); changeColorDialog = Dialog.getCheckbox(); goToNextField = Dialog.getCheckbox(); goToNextWell = Dialog.getCheckbox(); } else { goToNextWell = true; // for automaten mode (manual mode == false) } sliceLabel = getInfo("slice.label"); //set auto-contrast values if (changeAutoContrastDialog) { Dialog.create("Set auto-contrast (0 = not applied)"); Dialog.addMessage("Set auto-contrast\nEnhance contast value (0-1, 0 = not applied)"); for (i = 1; i <= channels; i++) Dialog.addNumber(availableChannels[i], autoContrastValue[i-1]); Dialog.addCheckbox("Apply auto-contrast values?", autoContrast); Dialog.show(); for (i = 0; i < channels; i++) autoContrastValue[i] = Dialog.getNumber(); autoContrast = Dialog.getCheckbox(); changeAutoContrastDialog = false; } //assign different color to channels if (changeColorDialog) { Dialog.create("Specifiy colors"); Dialog.addMessage("Assign colors to channels"); for (i = 1; i <= channels; i++) Dialog.addChoice(availableChannels[i+1], availableRGBcolors, availableRGBcolors[i-1]); Dialog.show(); for (i = 0; i < channels; i++) channelColorVector[i] = Dialog.getChoice(); changeColor = true; changeColorDialog = false; } print(changeColor); if (changeColor) { for (i = 1; i <= channels; i++) { setSlice(i); //set channels 1,2,3... print(i,channelColorVector[i-1]); run(channelColorVector[i-1]); } setSlice(sliceCounter); } if (makeMontage) { //run("Stack to RGB", "frames keep"); //rgbStack = getTitle(); montageColumns = floor(sqrt(frames - 1) + 1); montageRows = floor((frames - 1)/montageColumns + 1); run("Make Montage...", "columns=" + montageColumns + " rows=" + montageRows + " scale=" + montageScale + " first=1 last=" + frames + " increment=1 border=0 font=12 label"); if (autoContrast) { for (currentChannel = 1; currentChannel <= channels; currentChannel++) { setSlice(currentChannel); if ((autoContrastValue[currentChannel - 1] > 0) && (autoContrastValue[currentChannel - 1] <= 1)) run("Enhance Contrast", "saturated=" + autoContrastValue[currentChannel - 1]); } } fileNameMontage = rgbFilePrefix + substring(fileName,0,lengthOf(fileName)-5) + substring(sliceLabel,7,indexOf(sliceLabel,";")) + "_montage" + rgbFileSuffix + "." + rgbFileFormat; rename(fileNameMontage); if (saveMontage) { saveAs(rgbFileFormat, outputPath + fileNameMontage); print("Saved file:", fileNameMontage); } //if (isOpen(rgbStack)) close(rgbStack); if (manualMode) waitForUser("This is the well montage -> 'OK'"); if (isOpen(fileNameMontage)) close(fileNameMontage); } fileNameRGB = rgbFilePrefix + substring(fileName,0,lengthOf(fileName)-5) + substring(sliceLabel,7,lengthOf(sliceLabel)) + rgbFileSuffix + "." + rgbFileFormat; if (saveRGB) { saveAs(rgbFileFormat, outputPath + fileNameRGB); print("Saved file:", fileNameRGB); rename(imageTitle); } if (goToNextField || !manualMode) sliceCounter = sliceCounter + channels; if (makeMontage && !manualMode && !saveRGB) goToNextWell = true; //go to next well if only montage should be saved in automatic mode if ((sliceCounter > frames) && !manualMode) goToNextWell = true; //go to next well if all RGB fields are saved and in automatic mode } while (!goToNextWell); if (isOpen(imageTitle)) close(imageTitle); selectWindow("Log"); //save temp log saveAs("Text", outputPath + "Log_temp_" + tempLogFileNumber + ".txt"); } } //print current time to Log window and save log getDateAndTime(year, month, dayOfWeek, dayOfMonth, hour, minute, second, msec); month++; print("Macro executed successfully.\nEnd:",year+"-"+month+"-"+dayOfMonth+", "+hour+"-"+minute+"-"+second); selectWindow("Log"); saveAs("Text", outputPath + "Log_"+year+"-"+month+"-"+dayOfMonth+", "+hour+"-"+minute+"-"+second+".txt"); if (File.exists(outputPath + "Log_temp_" + tempLogFileNumber +".txt")) File.delete(outputPath + "Log_temp_" + tempLogFileNumber + ".txt"); //delete current tempLog file ///////////////////////////////////////////////////////////////////////////////////////////// //////// F U N C T I O N S ///////////// ///////////////////////////////////////////////////////////////////////////////////////////// function getAllWellsArrayScanFunction(fileList, closeWindow, fileListToLog) { //function get all unique wells from a file list //the file list needs to be a list of files exported from ArrayScan/HCSView (.tif or .TIF) //the function goes through the sorted list and finds the well-text in file name (MFGTMP_131004100001_C02f00d1.TIF position 12 to 9 counted form end of file name => C02) //unique well-text and number of found images per well (fields x channels) are put to a list/array //message pops up information about number of wells found and their number of images, that will be closed then second parameter = true Array.sort(fileList); wellList = newArray(fileList.length); wellImageCountList = newArray(fileList.length); wellIndex = -1; //= no entry in wellList (length(wellList)=0) wellImageCount = 0; currentFile = 0; while (currentFile < fileList.length) { //for all files found if (endsWith(fileList[currentFile],".flex")){ //exclude metadata files if (wellIndex == -1) { //for first image found set first well wellIndex++; //= go to first well wellList[wellIndex] = substring(fileList[currentFile],lengthOf(fileList[currentFile])-14,lengthOf(fileList[currentFile])-8); //= write first well to list } else { if (wellList[wellIndex] != substring(fileList[currentFile],lengthOf(fileList[currentFile])-14,lengthOf(fileList[currentFile])-8)) { //if new well wellImageCountList[wellIndex] = wellImageCount; //= write number of fields into wellCountList wellImageCount = 0; //= reset field count in well wellIndex++; //= go to next well wellList[wellIndex] = substring(fileList[currentFile],lengthOf(fileList[currentFile])-14,lengthOf(fileList[currentFile])-8); //= write next well to list } } wellImageCount++; } //for debugging: if (fileListToLog) print(fileList[currentFile], currentFile, wellList[wellIndex], wellIndex, wellImageCount); currentFile++; } wellImageCountList[wellIndex] = wellImageCount; //= write number of fields from last well into wellCountList //trim array lists and show in window wellList = Array.slice(wellList,0,wellIndex+1); wellImageCountList = Array.slice(wellImageCountList,0,wellIndex+1); //show result of well list Array.show("Wells & images found (indexes)",wellList,wellImageCountList); waitForUser("Number of well found: " + (wellIndex+1) + "\n " + " \n" +"Check if number of wells and number of images in well" + "\n" + "are as expected! Otherwise press 'ESC' and check image list!"); //tidy up and close windows if (closeWindow) { windowList = getList("window.titles"); for (i = 0; i < windowList.length; i++) { if (windowList[i] == "Arrays") { selectWindow("Arrays"); run("Close"); } } } //end of function: return well list return wellList; } ///////////////////////////////////////////////////////////////////////////////////////////////// function getImageFileExampleArrayScanFunction(fileList) { //function get an image file name as an example from a file list //the file list needs to have image file name with these extensoins: .tif or .TIF //message pops up if no image is found and macro is aborted currentFile = 0; do { if (endsWith(fileList[currentFile],".flex")){ //exclude metadata files //end of function: first image name return fileList[currentFile]; } currentFile++; } while (currentFile < fileList.length); //for all files found //show file list and abort macro Array.show(fileList); exit("No image files found!?" + "\n " + " \n" +"Check image list!"); } //////////////////////////////////////// E N D O F M A C R O ////////////////////////////