3DOrganoidAnalysis_

• This repository contains a robust methodology for identifying and characterizing organoids using the Cellpose algorithm.
• The script allows for precise 3D identification of individual organoids based on the overlay of GFP and Tomato markers

Download 3DOrganoidAnalysis_

1. Go to the GitHub repository
2. Click on <Code>>Download ZIP
3. The repo will be found at Downloads directory.

Running 3DOrganoidAnalysis_ in headless mode through ImageJ/Windows Windows Terminal (ALL parameters)

ImageJ-win64.exe --ij2 --headless --run "/absolute_path/to/groovyscript/3DOrganoidAnalysisAll_.groovy" "inputFilesOrganoid='/home/anaacayuela/organoid_labels',inputFilesRaw='/home/anaacayuela/organoid_raw_images',outputDir='/home/anaacayuela/output',tomaChannel=0,gfpChannel=1"

Parameters Explanation:

• inputFilesOrganoid : Directory in which the images (tiff, png... files cp_masks.tiff) to be analyzed are located. e.g.'/home/anaacayuela/organoid_labels'
• inputFilesRaw : Directory in which the raw images (tiff, jpeg... files) to be analyzed are located. e.g.'/home/anaacayuela/organoid_raw_images'
• outputDir : Directory in which the outputs are saved. '/home/anaacayuela/output'
• tomaChannel : Channel in which Tomato marker is located e.g.0
• gfpChannel : Channel in which GFP marker is located e.g.1
  • Channel indexes start from 0. e.g. Channel 1 is equal to 0

Features

• 3D Identification: Utilizes the Cellpose algorithm for accurate 3D segmentation of organoids.
• Fluorescence Analysis: Calculates fluorescence intensity distributions for GFP and Tomato markers for each segmented organoid.
• Volume Analysis both in physical units and pixels.
• Fluorescence Metrics: Computes maximum, minimum, mean, and integrated density of fluorescence intensities for both GFP and Tomato markers.
• Classification:
  • Organoids are classified as GFP-positive (GFP+) or GFP-negative (GFP-), and Tomato-positive (Tomato+) or Tomato-negative (Tomato-) based on fluorescence expression within a predefined eroded area.
  • Further classification of GFP+ organoids based on GFP intensity distribution:
    • Full: GFP+ intensity above the mean GFP intensity plus two standard deviations.
    • Empty: GFP+ intensity below this threshold.
• Similar classification procedure for Tomato+ organoids.