4i Analysis Pipeline
An end-to-end Nextflow pipeline for tiling, processing, segmenting, and analysing multiplex imaging data.
Nextflow Python Multiplex Imaging
Overview
An end-to-end Nextflow pipeline for processing 4i (iterative indirect immunofluorescence imaging) data from raw tiles through to per-cell/organelle measurements. Handles the full stack: tiling, flat-field correction, drift correction across imaging cycles, nuclei and cell segmentation, ROI matching, and configurable single-cell analysis.
Key Features
- Tile stacking with automatic flat-field correction from Harmony FFC profiles
- SIFT-based drift correction via Fiji/ImageJ, aligning all cycles to a reference
- StarDist nuclei segmentation + Cellpose cell segmentation with cytosol derivation
- JSON/YAML analysis config to select from expandable library of analysis functions
- Config validator with Pydantic to catch typos and missing fields before a run starts
- MCP Server to conversationally configure pipeline with static validation
- Outputs per-cell measurements in long-format CSV; optional wide format
Stack
Nextflow DSL2 · Python · StarDist · Cellpose · pyimagej