IBEX-PyReg

pyreg.py registers multiplexed immunofluorescence cycle stacks using DAPI as the reference channel.

The pipeline performs two sequential steps per non-reference cycle:

1. Coarse XY translation via phase cross-correlation.
2. Deformable refinement via ANTsPyx SyN registration.

Outputs are saved to a reg_output/ subfolder inside each input directory.

Requirements

• Python packages: numpy, tifffile, antspyx, scikit-image, scipy

Usage

python pyreg.py <input_dir> [<input_dir> ...]

• input_dir: one or more directories containing cycle TIFF stacks

Examples:

python pyreg.py "D:\\202504 Ibex\\APP30"
python pyreg.py "D:\\202504 Ibex\\APP30" "D:\\202504 Ibex\\APP32"
python pyreg.py data --crop_size 1024 --threads 8
python pyreg.py data --axes ZCYX --dapi_channel 0

Options

• --crop_size N center crop size for phase-correlation pre-alignment (default: 2048)
• --threads N ITK/ANTs thread count (default: 4)
• --axes MODE input axis mode: auto, ZCYX, or CZYX (default: auto)
• --dapi_channel N channel index used as DAPI reference (default: 0)

Notes

• The first TIFF stack in each directory is treated as the fixed reference cycle.
• Input stacks must be 4D and represent either ZCYX or CZYX layouts.
• In auto axis mode, ambiguous small-dimension stacks may still require explicit --axes.
• Output TIFF is written in ImageJ-compatible TZCYX layout.