Ilastik_organoid_segmentation

This repository holds two Ilastik projects and respective training data used for the organoid segmentation of the initial organoid screen in the publication "A semi-automated intestinal organoid screening method demonstrates epigenetic control of epithelial differentiation" (Ostrop et al. 2020).

The image pre-processing was carried out with the ImageJ script available on GitHub: https://github.com/jennyostrop/Fiji_organoid_brightfield_processing, archived on Zenodo as https://doi.org/10.5281/zenodo.3951125

For training, 1 well treated with DMSO vehicle control of 4 biological replicates and 5 timepoints were used (total 20 images). Training data was excluded from further analysis.

The raw images were deposited to the Image Data Resource (https://idr.openmicroscopy.org) under accession number idr0092.
Training data corresponds to Well A01 in plate 1-plate 4.

Project 1: Ilastik1_PixelClass_Edges

Pixel classification, created with Ilastik 1.3.2

Input Raw Data: Summary projection of Sobel edge detection for each z-layer (Output 2d from ImageJ script)

Training classes:
Background: lines over background and debris (size 7) and inside of organoids (size 3)
Object: exact lines following the outer border of organoids (size 1)

Project 2: Ilastik2_ObjectClass_Projection

Object classification [Inputs: Raw Data, Pixel Prediction Map], created with Ilastik 1.3.2

Input Raw Data: Minimum intensity projections of each Zstack (Output 1b from ImageJ script)
Input Prediction Maps: Pixel Prediction Maps generated in project Ilastik1_PixelClass_Edges

Threshold and Size Filter: Method - Simple, Input - 0 (Background), Smooth – 1.0, 1.0, Threshold  - 0.55, Size Filter – Min 10, Max 1000000

Object classes:
Mislabelled: Patch of background enclosed by objects, light
Organoid: Typical organoid, medium dark
Sphere_big: Sphere, almost round, light, size of organoids or bigger
Sphere_small: Sphere, almost round, light to medium dark, small
Cluster: Cluster of several organoids/spheres and organoids segmented as single object
Debris: Debris in Matrigel, irregular borders and structure, mostly small, medium dark
AirBubble: Air bubbles in Matrigel at early timepoints, round, dark with light centre
Edges: Edges of well plate (empty class, included for consistency)