Ilastik tracking- Fiji Ilastik HDF5 plugin vs BigDataviewer

Hiya,

I want to track cells and within those cells quantify the number of spots.
Channel 1: DNA for cell segmentation and tracking
Channel2 : spots to be counted/analyseed in CellProfiler

I am using multiple softwares for different purposes / trials

  1. Ilastik Pixel Classifier first to generate segmentation, and then the Ilastik tracking module.
  2. fiji to analyse the tracks with MaMuT/TrackMate and hopefully correct some divisions/daughters mis-assignement
  3. cell profiler to use the corrected probability masks and tracks (from MaMuut) in channel 1 but count spots in channel 2.
  1. Data Preparation - H5 format
    it seems I have and hdf5 conversion problem.
    For TrackMate/MaMut, I need to use the BigDataViewer to export into hdf5 and xml (otherwise I can’t export the tracks in the Ilastik Fiji/Mamut plugin version). So I exported my channels separately using the bigdataviewer
    But I trained the pixel classifier and tracking in IIlastik using h5 data subset from Fiji ilastik plugin

  2. pixel classification - tracking in Ilastik
    I trained the classifier on a subset of my movies that were exported into h5 using the fiji-ilastik export module.
    I then went on to the Ilastik Tracking module which worked nicely.
    I wanted to export in a format compatible for MaMut but it did not work as I did not have the xml file (that needs to be generateed with BigDataViewer - hence point 1).
    So I went back to data I havent analysed yet, converted data to h5 using the bigDataViewer and wanted to batch process them using the Ilastik Pixel classifier, but I got an error message

Is this because the classifier was trained on an h5 image that came from a different source (Ilastik Fiji plugin vs BigData Viewer)?

  1. export of probabilities map /tracks to Cell Profiler
    After checking the tracks of channel 1 with trackmate (and correct them??), I would like to use CP to analyse the 2nd channel.

    Would that be the correct export setup (then used for batch files)?
    Would it be doable to export tracks and object IDs

On a side note: I have just been playing with Ilastik for the past day and am really pleased with how well it all works!!
Cheers

Debbi

Hi @Debora_KellerOlivier,

your pipeline sounds very interesting :slight_smile:

currently, the advice that I have to give there is to start off with the data exported from big-data viewer to make sure the dimensionality is correct, and not mix the ilastik-fiji export plugin with that.

this is a sad bug, that is still present in the current ilastik version: https://github.com/ilastik/ilastik/issues/1854
As a workaround for this you could use the ilastik headless mode where you control ilastik via the command line to apply a pre-trained project to new data. Would be happy to help setting that up.

Concerning the spot counting I would hope that CellProfiler is fit for the task, but I don’t know, let’s hope someone knowledgeable stumbles over this thread.

Cheers
Dominik

ouh headless sounds fun! I’d be happy to try as it seems to put a lot of memory in cache if I load my files directly into the GUI (300Mb-1Gb).
How can I run it in headless mode? I guess some python command line (sorry I am a biologist…)

I’ll try the BDV and see what I can get! I am seeing “The Man” (aka JYves) tomorrow so will surely get some useful advice there too.
Cheers
Debbi
Debbi