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Final output files

The IMC Segmentation Pipeline produces a number of output files during pre-processing, ilastik pixel classification, segmentation and measurement. They are listed below:

analysis
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├── cpinp 
    ├── acquisition_metadata.csv 
    ├── full_channelmeta.csv
    ├── probab_channelmeta_manual.csv
├── cpout      
    ├── images
        ├── {XYZ}_s0_a1_ac_full.tiff
        ├── {XYZ}_s0_a1_ac_full.csv
        ├── ...
    ├── masks
        ├── {XYZ}_s0_a1_ac_ilastik_s2_Probabilities_mask.tiff
        ├── ...
    ├── probabilities
        ├── {XYZ}_s0_a1_ac_ilastik_s2_Probabilities_s1.tiff
        ├── ...
    ├── cell.csv
    ├── Experiment.csv
    ├── Image.csv
    ├── Object relationships.csv
    ├── panel.csv
    ├── var_cell.csv
    ├── var_Image.csv
├── crops 
    ├── {XYZ}_s0_a1_ac_ilastik_x{X}_y{Y}_w{W}_h{H}.tiff
    ├── ...
├── histocat
    ├── {XYZ}_s0_a1_ac
        ├── {channel_label}_{channel_name}.tiff
        ├── ...
    ├── ...
├── ilastik
    ├── {XYZ}_s0_a1_ac_ilastik.tiff
    ├── {XYZ}_s0_a1_ac_ilastik.csv
    ├── {XYZ}_s0_a1_ac_ilastik_s2.h5
    ├── {XYZ}_s0_a1_ac_ilastik_s2_Probabilities.tiff
    ├── ...
├── ometiff
    ├── {XYZ}
        ├── {XYZ}_s0_a1_ac.ome.tiff
        ├── {XYZ}_s0_a1_ac.ome.csv
        ├── ...
        ├── {XYZ}_s0_p1_pano.png
        ├── ...
        ├── {XYZ}_s0_slide.png
        ├── {XYZ}_schema.xml

Here XYZ indicates the sample name.

The main output folder

The cpout folder contains all relevant output files:

  • cpout/images: contains the hot pixel filtered full stacks for analysis as well as .csv files indicating the channel order.
  • cpout/masks: contains single-channel segmentation masks in 16-bit .tiff format. Segmentation masks are single-channel images that match the input images in size, with non-zero grayscale values indicating the IDs of segmented objects.
  • cpout/probabilities: contains 3 channel images in 16-bit .tiff format representing the downscaled pixel probabilities after Ilastik pixel classification.
  • cpout/cell.csv: contains features (columns) for each cell (rows).
  • cpout/Experiment.csv: contains metadata related to the CellProfiler version used.
  • cpout/Image.csv: contains image-level measurements (e.g. channel intensities) and acquisition metadata.
  • cpout/Object relationships.csv: contains neighbor information in form of an edge list between cells.
  • cpout/panel.csv: a copy of the panel file used for the experiment.
  • cpout/var_cell.csv: contains feature metadata for all single-cell features.
  • cpout/var_Image.csv: contains feature metadata for all image features.

The CellProfiler input folder

The cpinp folder contains metadata files for CellProfiler input:

  • cpinp/acquisition_metadata.csv: containing acquisition metadata.
  • cpinp/full_channelmeta.csv: containing full stack channel metadata.
  • cpinp/probab_channelmeta_manual.csv: containing probability stack channel metadata.

Ilastik folder

The following folders contain files for Ilastik pixel classification:

  • analysis/ilastik: contains the Ilastik stacks ({XYZ}_s0_a1_ac_ilastik.tiff), matched .csv files indicating the correct channel order ({XYZ}_s0_a1_ac_ilastik.csv), the upscaled ilastik stacks in .h5 format (XYZ}_s0_a1_ac_ilastik_s2.h5) and upscaled pixel probabilities ({XYZ}_s0_a1_ac_ilastik_s2_Probabilities.tiff).
  • analysis/crops: this folder contains the image crops of the Ilastik stack in .h5 format for Ilastik training.

Image data folders

The following folders contain data in different formats for use with other software or histoCAT.

  • analysis/ometiff: contains individual folders (one per sample) of which each contains multiple .ome.tiff files (one per acquisition).
  • analysis/histocat: contains individual folders (one per acquisition) of which each contains multiple single-channel .tiff files for upload to histoCAT.