''' HYPERSTACK GENERATOR in Fiji Read all tif images from an experiment inFolder that contains tif images for the different wells, channel, slices and timepoints The script will automatically reads the field from the filename (using index in the string - FOLLOWING ACQUIFER-IM04 NAMING CONVENTION ex: -A001--...) to generate the well's stack NB : issue if all the images in the folder dont have the same size TO DO : - Add projections to a stack - Make plate montage for each time point and/or Z-slice with slider that update the full plate display Requirements - Stack Focuser https://imagej.nih.gov/ij/plugins/stack-focuser.html ''' #@PrefService prefs import os, ij from ij import IJ, VirtualStack from ij.plugin import HyperStackConverter,ZProjector from ij.WindowManager import getCurrentImage, getCurrentWindow from fiji.util.gui import GenericDialogPlus from java.awt.event import ActionListener from loci.formats import ChannelSeparator def dimensionsOf(path): fr = None try: fr = ChannelSeparator() fr.setGroupFiles(False) fr.setId(path) return fr.getSizeX(), fr.getSizeY() except: # Print the error, if any print sys.exc_info() finally: fr.close() class ListenerClass_All(ActionListener): # extends action listener '''Class associated to the Select all button''' def actionPerformed(this,event): '''Called when 'Select All' is clicked''' global Win for checkbox in Win.getCheckboxes()[-96:]: checkbox.setState(True) class ListenerClass_None(ActionListener): # extends action listener '''Class associated to the Select none button''' def actionPerformed(this,event): '''Called when 'Select None' is clicked''' global Win for checkbox in Win.getCheckboxes()[-96:]: checkbox.setState(False) ListenerAll = ListenerClass_All() # create an instance of the EventListener class ListenerNone = ListenerClass_None() # create an instance of the EventListener class # Recover input from persistence Path0 = prefs.get("ImageDirectory","MyPath") pickChannel0 = prefs.getInt("pickChannel",False) Channel0 = prefs.get("Channels","") pickSlice0 = prefs.getInt("pickSlice",False) Slice0 = prefs.get("Slices","") pickTime0 = prefs.getInt("pickTime",False) Time0 = prefs.get("Times","") ShowStack0 = prefs.getInt("ShowStack",True) SaveStack0 = prefs.getInt("SaveStack",False) ProjMethod0 = prefs.get("ProjMethod","None") ShowProj0 = prefs.getInt("ShowProj",False) SaveProj0 = prefs.getInt("SaveProj",False) # Input GUI window Win = GenericDialogPlus("Hyperstack generator - Parameters") # Title of the window Win.addDirectoryField("Image directory",Path0) Win.addStringField("Image file extension",".tif") Win.addCheckbox("Use specific channel(s)",pickChannel0) Win.addToSameRow() Win.addStringField("Channel(s) separated by , (from 1 to 6) : ",Channel0) Win.addCheckbox("Use specific Z-slice(s)",pickSlice0) Win.addToSameRow() Win.addStringField("Z-slice(s) separated by , : ",Slice0) Win.addCheckbox("Use specific timepoint(s)",pickTime0) Win.addToSameRow() Win.addStringField("Timepoint(s) separated by , : ",Time0) Win.addCheckbox("Show stack ?",ShowStack0) Win.addCheckbox("Save stack ?",SaveStack0) Win.addChoice("Make Z-projection ?",['None','avg','sd','max','min','sum','median','Extended Focus'],ProjMethod0) Win.addToSameRow() Win.addCheckbox("Show projection ?",ShowProj0) Win.addToSameRow() Win.addCheckbox("Save projection ?",SaveProj0) #Disclaimer = "This is my link" # this will pop up a small window with some documentation Disclaimer = "https://github.com/acquifer/ImageInLife/wiki/Stacks" # this will directly open the doc page Win.addHelp(Disclaimer) # Generate tick box with plate layout to select well Wells = [] for letter in ['A','B','C','D','E','F','G','H']: for column in range(1,13): Wells.append(letter+'%(column)03d' %{"column":column}) # force max 3 digits and leading 0 Win.addCheckboxGroup(8,12,Wells,[True]*96) Win.addButton('Select all' ,ListenerAll) # add our event listener to the button. Button clicked -> event -> call actionPerformed(MyEvent) Win.addButton('Select none',ListenerNone) Win.showDialog() ## Recover input if (Win.wasOKed()): inFolder = Win.getNextString() image_extension = Win.getNextString() Bool = [checkbox.getState() for checkbox in Win.getCheckboxes()] i = 0 pickChannel = Bool[i] ChoiceChannel = Win.getNextString() i+=1 pickSlice = Bool[i] ChoiceSlice = Win.getNextString() i+=1 pickTime = Bool[i] ChoiceTime = Win.getNextString() i+=1 ShowStack = Bool[i] i+=1 SaveStack = Bool[i] i+=1 ProjMethod = Win.getNextChoice() ShowProj = Bool[i] i+=1 SaveProj = Bool[i] i+=1 BoolWell = Bool[i:] Selection = {well:choice for well,choice in zip(Wells,BoolWell)} # create a dictionnary associating a well to a boolean . Chosen or not #print Selection #print len(Wells) # OK 96 # Save input into memory/persistence prefs.put("ImageDirectory",inFolder) prefs.put("pickChannel",pickChannel) prefs.put("Channels",ChoiceChannel) prefs.put("pickSlice",pickSlice) prefs.put("Slices",ChoiceSlice) prefs.put("pickTime",pickTime) prefs.put("Times",ChoiceTime) prefs.put("ShowStack",ShowStack) prefs.put("SaveStack",SaveStack) prefs.put("ProjMethod",ProjMethod) prefs.put("ShowProj",ShowProj) prefs.put("SaveProj",SaveProj) # Stop execution if no well selected if BoolWell == [False]*96: IJ.error("No well selected") ''' message = "No well selected" IJ.log(message) raise Exception(message) ''' # reformat after the input has been saved in memory if ChoiceChannel != "": # prevent bug when field empty and we convert to int #ChoiceChannel = list(map(int,ChoiceChannel.split(","))) ChoiceChannel = eval(ChoiceChannel) if type(ChoiceChannel)==int: # in case it is a single integer turn it to a list ChoiceChannel = [ChoiceChannel] if ChoiceSlice != "": ChoiceSlice = eval(ChoiceSlice) if type(ChoiceSlice)==int: # in case it is a single integer turn it to a list ChoiceSlice = [ChoiceSlice] if ChoiceTime != "": ChoiceTime = eval(ChoiceTime) if type(ChoiceTime)==int: # in case it is a single integer turn it to a list ChoiceTime = [ChoiceTime] # Find image files for the given channel #inFolder = str(inFolder) # convert filename object to string object WellList = [] # full list of well to know how many iteration for stacking TimeList = [] ChannelList = [] SliceList = [] MainList = [] # contains tuple with (Filename, Well, TimePoint, Channel, Z-Slice) for fname in os.listdir(inFolder): # extract metadata from image filename if fname.endswith(image_extension): # first extract the well field Well = fname[1:5] # if well is selected in the gui, then extract the other metadata if Selection[Well]: try : # int conversion throw an error if the argument string is empty, hence the try Time = int(fname[15:18]) Channel = int(fname[22:23]) Slice = int(fname[27:30]) except : IJ.error("Could not get experimental parameters from filename. Check filename pattern (IM04 convention)") #raise Exception("Could not get experimental parameters from filename. Check filename pattern (IM04 convention)") WellList.append(Well) ## Channel if not pickChannel: # we take all channel ChannelList.append(Channel) elif pickChannel and (Channel in ChoiceChannel): # We take only a set of timepoints ChannelList.append(Channel) else: # This particular channel was not selected continue ## Time if not pickTime: # we take all time points TimeList.append(Time) elif pickTime and (Time in ChoiceTime): # We take only a set of timepoints TimeList.append(Time) else: # Timepoints that were not selected continue ## Z-Slice if not pickSlice: # we take all time points SliceList.append(Slice) elif pickSlice and (Slice in ChoiceSlice): # We take only a set of timepoints SliceList.append(Slice) else: # Timepoints that were not selected continue # Append to main list only if it managed to pass the previous if (thanks to the continue statement) MainList.append((fname,Well,Time,Channel,Slice)) else : # the well was not selected in the GUI, go for the next image filename continue if len(MainList) < 1: IJ.error("No image files found in %s satisfying the channel/slice/time selection. Check if extension match and if filenames match IM04 naming convention" % inFolder) #raise Exception("No image files found in %s satisfying the channel/slice/time selection. Check if extension match and if filenames match IM04 naming convention" % inFolder) # Sort list to prepare iteration WellList = list(set(WellList)) # turn to a set object to have unique item once only WellList.sort() # sort should be after set since set may change order TimeList = list(set(TimeList)) TimeList.sort() ChannelList = list(set(ChannelList)) ChannelList.sort() SliceList = list(set(SliceList)) SliceList.sort() # Verification print 'Well : ', WellList print 'Time : ', TimeList print 'ChannelIdx : ', ChannelList print 'Z-Slice : ' , SliceList MainList.sort(key = lambda field:(field[1], field[2], field[4], -field[3]) ) # sort by Well, Time, Z-slice then Channel (- to have C06 ie BF first) since the hyperstack is created following the default "xyczt" order ''' for i in MainList : print i ''' # Initialise HyperStackConverter and Projector object HyperStacker = HyperStackConverter() Proj = ZProjector() ## Loop over wells to do stack and projection for well in WellList: # - TO DO: Add another level in MainList : one per well # Gather images for a given well, and put them into a sublist IJ.log('Process well : '+well+'...') SubList = [item for item in MainList if item[1]==well] # Sublist containing (fname1,Well,Time1,ChannelX,Slice),(fname2,Well,Time2,ChannelX,Slice) for one given well ex: only A001 #print 'StackSize : ',len(SubList) # Get image dimensions of the first image in the list to initialise VStack width, height = dimensionsOf( os.path.join(inFolder,SubList[0][0]) ) # Initialise Virtual Stack for this well VStack = VirtualStack(width, height, None, inFolder) # Append slices in the VStack for item in SubList: filename = item[0] VStack.addSlice(filename) # conversion to ImageProcessor object to append to stack # Once we have finished appending slices, we reconvert back to an ImagePlus object to be able to show and save it ImpStack = ij.ImagePlus(well, VStack) # well is the title of this hyperstack - Reminder : 1 hyperstack/well #print ImpStack #ImpStack.show() # Good ## Make a hyperstack out of the ImagePlus virtual stack if ImpStack.getStackSize()==1: HyperStack = ImpStack # issue when a single slice in the Hyperstack else: HyperStack = HyperStacker.toHyperStack(ImpStack,len(ChannelList), len(SliceList), len(TimeList),"xyczt","grayscale") # convert PROPERLY ORDERED Stack to Hyperstack ## Display resulting stack if ShowStack : HyperStack.show() ## Save resulting stack if SaveStack : outFolder = os.path.join(inFolder,'HyperStack') if not os.path.exists(outFolder): os.makedirs(outFolder) # create folder if it does not exist (for the first image to save basicly) outPath = os.path.join(outFolder,well+'.tif') IJ.save(HyperStack,outPath) ## PROJECTION if ProjMethod != 'None': # Report message = 'Do projection' #print message IJ.log(message) # Do projection if ProjMethod == 'Extended Focus': IJ.run(HyperStack, "Stack Focuser ", "enter=3") # return void but open a new image window Projected = getCurrentImage().duplicate() # duplicate otherwise lost once the window is closed if not ShowProj: getCurrentWindow().close() else: # other classical projection method Projected = Proj.run(HyperStack,ProjMethod) # Display projection (automatic with the stack focuser) if ShowProj: Projected.show() # Save projection if SaveProj: outFolder = os.path.join(inFolder,'Projected') if not os.path.exists(outFolder): os.makedirs(outFolder) # create folder if it does not exist (for the first image to save basicly) outPath = os.path.join(outFolder,well+'.tif') IJ.save(Projected,outPath)