Python Script for reading CZI images

Hi all, I am playing around for a while now with all the nice options on how to read CZI images in Fiji using python scripting. What I got is not perfect, but works quite well in most cases.

So feel free to use it and even improve it or share feedback.

You find the script here:

Sebi

6 Likes

Hi there, thanks for the contribution. Sorry, I haven’t had the chance to try your script yet, but I was wondering if you could add a comment on how your script differs and/or improves on Fiji’s capabilities with .czi files. I have been using Fiji to read and analyze .czi files for some time and find it rather satisfactory already, but perhaps your script provides features I don’t (yet) know I need?

Hi,

for most things just using the very powerful BioFormats plugin suffienct and will do the job thanks to work of the BioFormats guys and the the good communication with our DevTeam at ZEISS.

The reason why I created this script has several reasons.

  • CZI image do have a pyramidal structure with different resolution levels. While it is possible to open them using BioFormats by choosing the correct image series, this is cumbersome to automate
  • the script allows you to directly specify the desired pyramid level to read
  • additional is sets the scaling for the pyramid level correctly, which did not work using the BioFormats plugin only (or I do not know how … :-))
  • the need to automate opening CZI in headless mode required to write some kind of script and not using IJ.run method, because IJ.run and BioFormats do not work in headless mode
  • see this post: Strange Issue with using BioFormats from python script in headless mode only
  • this headless things is really crucial to run our Docker containers on APEER (www.apeer.com), where we use Fiji + BoFormats quite often to create image processing modules like this one: Measure 3D Objects

So basically it offers a bit more flexibility and allows automating reading CZIs via BioFormats by exposing all the options of BioFormats (and some additional minor things) and wrapping them inside a python script by using the cool scripting parameters, which makes life in headless mode and inside Docker containers easy.

Feel free to test it and let me know if it works or how it could be improved, since I am pretty sure my script still has some hidden “glitches” …

Sebi (from ZEISS)

4 Likes

Hi, I’ve found this thread after struggling for a while to figure out how to open CZI file via fiji in headless mode. It seems that - just like you described in your post - I can’t simply use open(“file.czi”) in a myMacro.ijm to open it (I get Java Error: Bad Operand).

My goal is to automate the process of:

  1. read czi file
  2. Stack to RGB (in Fiji, the macro command is: run(“Stack to RGB”)
  3. save as Tiff (in Fiji: saveAs(“Tiff”, outputPath/file))

Would your fjij python toolkit library for reading czi help to automate the pipeline above? I am not sure how to use your python scripts with fiji, but if the toolkit would be able to be used for the described task, I’d greatly appreciate if you could suggest a direction. Thanks!

Hi @KSL,

to read a CZI you should either use the BioFormats plugin from the macro or for more flexibility have a look into this script (requires fijipytools.py)

Can confirm, the bioformats importer doesn’t work in Fiji in headless mode, so you will need something else (that script should certainly work, but I haven’t tried personally); to do the rest of your macro in python in Fiji, you basically just need to add a from ij import IJ to the import section and put IJ. in front of each of your commands).

If you aren’t wedded to a Fiji-only solution, that would be a super simple pipeline in CellProfiler- configure the input modules to read your CZI files, one “GrayToColor” module to make your RGB image, and one “SaveImages” module to save it out. But obviously without knowing the rest of your workflow it’s not clear if that would be simpler here.

Hi @bcimini and @KSL,

for me the BioFormats Importer does work quite well in headless mode or do I misunderstand the problem?

Or do you mean how to read CZI in "normal python? Here I always use AICSImageIO or aicspylibczi, which both work very nicely.

@sebi06 @bcimini Thanks for the reply.

@sebi06 For me BioFormats importer does not work in headless mode it seems.

For example, I wrote a simple ijm macro to just open a file:

#@String file

open(file);

or

#@String file

run("Bio-Formats Importer", "open=[" + file + "] color_mode=Default view=Hyperstack stack_order=XYCZT");

When I run either macro

/Applications/Fiji.app/Contents/MacOS/ImageJ-macosx --ij2 --headless --console --run test.ijm ‘file=“test.czi”’

I get the same following error:

java.lang.VerifyError: Bad type on operand stack
Exception Details:
  Location:
    loci/plugins/in/MainDialog.rebuildDialog(Lij/gui/GenericDialog;)V @1926: invokestatic
  Reason:
    Type 'ij/gui/GenericDialog' (current frame, stack[0]) is not assignable to 'java/awt/Container'
  Current Frame:
    bci: @1926
    flags: { }
    locals: { 'loci/plugins/in/MainDialog', 'ij/gui/GenericDialog', 'java/util/List', 'java/util/List', 'java/util/List', 'java/awt/Label', 'java/awt/Label', 'java/awt/Label', 'java/awt/Label', '[Ljava/awt/Component;', 'java/lang/String', 'java/lang/String', 'com/jgoodies/forms/builder/PanelBuilder', 'com/jgoodies/forms/layout/CellConstraints', integer }
    stack: { 'ij/gui/GenericDialog' }
  Bytecode:
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    0x0000570: b600 9757 840e 0219 0c2a b400 632a 190d
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    0x00006e0: 5784 0e02 190c 2ab4 006f 2a19 0d08 150e
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    0x0000790: b1
  Stackmap Table:
    full_frame(@58,{Object[#164],Object[#4],Object[#309],Object[#309],Object[#309],Object[#95],Object[#95],Object[#95],Object[#95],Object[#310],Integer},{})
    same_frame(@117)
    same_frame(@168)
    same_frame(@195)
    chop_frame(@201,1)
    same_frame_extended(@641)

	at loci.plugins.in.ImporterPrompter.promptMain(ImporterPrompter.java:132)
	at loci.plugins.in.ImporterPrompter.statusUpdated(ImporterPrompter.java:79)
	at loci.plugins.in.ImportProcess.notifyListeners(ImportProcess.java:476)
	at loci.plugins.in.ImportProcess.step(ImportProcess.java:772)
	at loci.plugins.in.ImportProcess.execute(ImportProcess.java:140)
	at loci.plugins.in.Importer.showDialogs(Importer.java:140)
	at loci.plugins.in.Importer.run(Importer.java:76)
	at loci.plugins.LociImporter.run(LociImporter.java:78)
	at ij.IJ.runUserPlugIn(IJ.java:235)
	at ij.IJ.runPlugIn(IJ.java:198)
	at ij.Executer.runCommand(Executer.java:150)
	at ij.Executer.run(Executer.java:68)
	at ij.IJ.run(IJ.java:317)
	at ij.IJ.run(IJ.java:328)
	at ij.macro.Functions.doRun(Functions.java:686)
	at ij.macro.Functions.doFunction(Functions.java:98)
	at ij.macro.Interpreter.doStatement(Interpreter.java:278)
	at ij.macro.Interpreter.doStatements(Interpreter.java:264)
	at ij.macro.Interpreter.run(Interpreter.java:160)
	at ij.macro.Interpreter.run(Interpreter.java:93)
	at ij.macro.Interpreter.run(Interpreter.java:104)
	at ij.plugin.Macro_Runner.runMacro(Macro_Runner.java:161)
	at ij.IJ.runMacro(IJ.java:153)
	at ij.IJ.runMacro(IJ.java:142)
	at net.imagej.legacy.IJ1Helper$3.call(IJ1Helper.java:1148)
	at net.imagej.legacy.IJ1Helper$3.call(IJ1Helper.java:1144)
	at net.imagej.legacy.IJ1Helper.runMacroFriendly(IJ1Helper.java:1095)
	at net.imagej.legacy.IJ1Helper.runMacro(IJ1Helper.java:1144)
	at net.imagej.legacy.plugin.IJ1MacroEngine.eval(IJ1MacroEngine.java:145)
	at org.scijava.script.ScriptModule.run(ScriptModule.java:157)
	at org.scijava.module.ModuleRunner.run(ModuleRunner.java:165)
	at org.scijava.module.ModuleRunner.call(ModuleRunner.java:124)
	at org.scijava.module.ModuleRunner.call(ModuleRunner.java:63)
	at org.scijava.thread.DefaultThreadService.lambda$wrap$2(DefaultThreadService.java:225)
	at java.util.concurrent.FutureTask.run(FutureTask.java:266)
	at java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1149)
	at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:624)
	at java.lang.Thread.run(Thread.java:748)

I’ve tried searching google for similar errors related to opening czi in ImageJ via headless mode, but have not been able to find a solution yet.

FYI, I am working with a czi file that is a histology slice of a brain region. I tried opening a different czi file (like cell microscopy images), but I got the same error, so I do not think the error is due to different czi files.

Did you not encounter a similar error when trying to run a macro in headless mode to open a czi file?

I tried this on my local (mac) machine and remote computing cluster (centOS), but both had the same error so I thought czi cannot be read in headlessmode via ImageJ.

@KSL,

hmmm, so have no clue why this does not work for me. Can you share a CZI file, which does not work for you?

Sure thing! Here’s the test.czi file I could not open in headless mode (in GUI Fiji I can open just fine)

@sebi06 , I’ve run into that before, and I’m not the only one, curious to know if you have any counter-example scripts where you’ve tried to invoke the Bioformats importer in headless ImageJ and HAVE had it work! That might help with the tracing and fixing of the issue.

Hi @bcimini ,

i created a little test python script.

# @File(label = "Image File", persist=True) FILENAME
# @OUTPUT String FILENAME
# @UIService uiService
# @LogService log

import os
import sys
from loci.formats import MetadataTools
from loci.formats import ImageReader
from loci.plugins. in import ImporterOptions
from loci.plugins.util import LociPrefs
from loci.plugins import BF
from loci.formats. in import ZeissCZIReader
from loci.formats. in import DynamicMetadataOptions
from ij import IJ, ImagePlus, ImageStack, Prefs
from ij.measure import Calibration
from org.scijava.log import LogLevel


def setCZIReaderOptions(imagefile, czi_options, setflatres=True):

        czireader = ZeissCZIReader()
        czireader.setFlattenedResolutions(setflatres)
        czireader.setMetadataOptions(czi_options)
        czireader.setId(imagefile)

        return czireader


def setReaderOptions(imagefile, stitchtiles=False,
                                    setflatres=True,
                                    setconcat=False,
                                    openallseries=False,
                                    showomexml=False,
                                    attach=False,
                                    autoscale=True):

        czi_options = DynamicMetadataOptions()
        czi_options.setBoolean("zeissczi.autostitch", stitchtiles)
        czi_options.setBoolean("zeissczi.attachments", attach)

        # set the option for the CZIReader
        czireader = setCZIReaderOptions(imagefile, czi_options, setflatres=setflatres)

        # Set the preferences in the ImageJ plugin
        # Note although these preferences are applied, they are not refreshed in the UI
        Prefs.set("bioformats.zeissczi.allow.autostitch", str(stitchtiles).lower())
        Prefs.set("bioformats.zeissczi.include.attachments", str(attach).lower())

        # set the option for the BioFormats import
        reader_options = ImporterOptions()
        reader_options.setOpenAllSeries(openallseries)
        reader_options.setShowOMEXML(showomexml)
        reader_options.setConcatenate(setconcat)
        reader_options.setAutoscale(autoscale)
        reader_options.setStitchTiles(stitchtiles)
        reader_options.setId(imagefile)

        return reader_options, czireader



# get the FILENAME as string
imagefile = FILENAME.toString()
pylevel = 0

# define the reader options
reader_options, czireader = setReaderOptions(imagefile, stitchtiles=True,
                                                        setflatres=False,
                                                        setconcat=True,
                                                        openallseries=True,
                                                        showomexml=False,
                                                        attach=False,
                                                        autoscale=True)

# open the ImgPlus using BioFormats
imps = BF.openImagePlus(reader_options)

# get the series
imp = imps[pylevel]

# close czireader
czireader.close()

# get the stack and some info
imgstack = imp.getImageStack()
slices = imgstack.getSize()
width = imgstack.getWidth()
height = imgstack.getHeight()

log.log(LogLevel.INFO, '---------------------------------------------')
log.log(LogLevel.INFO, 'Filename : ' + imagefile)
log.log(LogLevel.INFO, 'Slices   : ' + str(slices))
log.log(LogLevel.INFO, 'Width    : ' + str(width))
log.log(LogLevel.INFO, 'Height   : ' + str(height))
log.log(LogLevel.INFO, 'Done.')

# exit
os._exit()

# to run it use with your respective (!) paths (on WIN10, Fiji is up-to-date):
# ImageJ-win64.exe --ij2 --headless --console --run "C:\Fiji\scripts\czireader_headless.py" "FILENAME='C:\Testdata_Zeiss\CellDivision_T=3_Z=5_CH=2_X=240_X=170.czi'"

# or if the script should really exit (this trick I was told by Curtis Rueden a long time ago ...) you should 
# us the complete commandline, which one can get using: ImageJ-win64.exe --dry-run --ij2 --headless --console
#
# java -Dpython.cachedir.skip=true -Dplugins.dir=C:\\Fiji -Xmx24446m -Djava.awt.headless=true -Dapple.awt.UIElement=true -Xincgc -XX:PermSize=128m -Djava.class.path=C:\\Fiji/jars/imagej-launcher-5.0.3.jar -Dimagej.dir=C:\\Fiji -Dij.dir=C:\\Users\\m1srh\\DOCUME~1\\Fiji -Dfiji.dir=C:\\Fiji -Dfiji.defaultLibPath=bin/server/jvm.dll -Dfiji.executable=C:\\Fiji\\ImageJ-win64.exe -Dij.executable=C:\\Fiji\\ImageJ-win64.exe -Djava.library.path=C:\\Fiji/lib/win64;C:\\Fiji/mm/win64 -Dscijava.context.strict=false net.imagej.launcher.ClassLauncher -ijjarpath jars -ijjarpath plugins net.imagej.Main --run "C:\Fiji\scripts\czireader_headless.py" "FILENAME='C:\Testdata_Zeiss\CellDivision_T=3_Z=5_CH=2_X=240_X=170.czi'"

If you run this one from the commandline I see the following output:

(PrismaLearn) C:\Fiji>Java HotSpot(TM) 64-Bit Server VM warning: ignoring option PermSize=128m; support was removed in 8.0
Java HotSpot(TM) 64-Bit Server VM warning: Using incremental CMS is deprecated and will likely be removed in a future release
[WARNING] Not overwriting extension 'py':
        proposed = net.haesleinhuepf.clijx.te_oki.TeOkiLanguage [file:/C:/Fiji/plugins/clijx-assistant_-0.4.2.21.jar
        existing = org.scijava.plugins.scripting.jython.JythonScriptLanguage [file:/C:/Fiji/jars/scripting-jython-1.0.0.jar
[WARNING] Not overwriting extension 'ijm':
        proposed = net.clesperanto.macro.interpreter.ClEsperantoMacroLanguage [file:/C:/Fiji/plugins/clijx-assistant_-0.4.2.21.jar
        existing = net.imagej.legacy.plugin.IJ1MacroLanguage [file:/C:/Fiji/jars/imagej-legacy-0.37.4.jar
[INFO] Overriding Ice ; identifier: script:LUTs/_CMOcean/Ice_.ijm; jar: file:/C:/Fiji/jars/scijava-common-2.83.3.jar
[INFO] Overriding BIOP VSI Reader; identifier: script:ActionBar/Biop Bars/BIOP_VSI_Reader.ijm; jar: file:/C:/Fiji/jars/scijava-common-2.83.3.jar
[INFO] Overriding BIOP VSI Reader; identifier: script:ActionBar/Shared Bars/BIOP_VSI_Reader.ijm; jar: file:/C:/Fiji/jars/scijava-common-2.83.3.jar
[INFO] Overriding Common Tools; identifier: script:ActionBar/Shared Bars/Common_Tools.ijm; jar: file:/C:/Fiji/jars/scijava-common-2.83.3.jar
[INFO] Overriding JaCoP Tools; identifier: script:ActionBar/Shared Bars/JaCoP_Tools.ijm; jar: file:/C:/Fiji/jars/scijava-common-2.83.3.jar
[INFO] Overriding BIOP Run Macro...; identifier: command:ch.epfl.biop.macrorunner.B_Run_Macro; jar: file:/C:/Fiji/plugins/BIOP/B_Run_Macro-1.0.0-SNAPSHOT.jar
[INFO] Overriding Get Spine From Circle Rois; identifier: command:Cirlces_Based_Spine; jar: file:/C:/Fiji/plugins/Max_Inscribed_Circles-1.1.0.jar
[INFO] Overriding Distribution Plotter; identifier: script:Distribution_Plotter.ijm; jar: file:/C:/Fiji/jars/scijava-common-2.83.3.jar
[INFO] Overriding Visualise vector field (experimental); identifier: command:net.haesleinhuepf.clijx.piv.visualisation.VisualiseVectorFieldsPlugin; jar: file:/C:/Fiji/plugins/clijx_-0.30.1.21.jar
[INFO] Overriding ROI Color Coder; identifier: script:ROI_Color_Coder.ijm; jar: file:/C:/Fiji/jars/scijava-common-2.83.3.jar
[INFO] ---------------------------------------------
[INFO] Filename : C:\Testdata_Zeiss\CellDivision_T=3_Z=5_CH=2_X=240_X=170.czi
[INFO] Slices   : 30
[INFO] Width    : 240
[INFO] Height   : 170
[INFO] Done.

I hope it helps.

Ok! So it seems like the underlying LIBRARIES work fine in headless mode, just not the ImageJ command “Run Bioformats Headless Importer” (or Exporter, based on your 2016 post in the linked thread from my last post).

@ctrueden or @hinerm or whomever, does it make sense to clarify that in the ImageJ documentation or anywhere else?

Hi @bcimini ,

just to add this, the BioFormats Exporter also works headless for me (despite writing OME-TIFF is reather slow) by using code along those lines:

paramstring = "outfile=[" + savepath + "] windowless=true compression=Uncompressed saveROI=false"
            plugin = LociExporter()
            plugin.arg = paramstring
            exporter = Exporter(plugin, imp)
            exporter.run()