4i / Cyclic IF

Cyclic immunofluorescence images the same plate several times, stripping and re-staining between rounds. Instead of the three or four markers a single round allows, you measure dozens per cell — provided the rounds can be registered accurately enough to say that a nucleus in round 3 is the same nucleus as in round 1.

That registration problem is what this part of OMERO-Screen solves.

The idea

Each round is analysed as an ordinary plate: segmented, measured, stored. The 4i tools then run afterwards, computing a geometric alignment between rounds, matching cells across them, and merging the per-round features into one single-cell table.

Nothing about the per-round analysis is special. If you can run a plate, you can run a 4i experiment.

The workflow

Step	Tool	What it does
1	omero-screen	Analyse each round as a normal plate
2	align_plates.py	Register the rounds against a reference
3	create_masks.py	Fill in cell masks missing from secondary rounds
4	aggregate_plates.py	Merge per-cell features across rounds
5	alignment_qc.py	Check the registration actually worked

The first plate passed to align_plates.py is the reference. Every other round is registered onto it, and the aggregated dataset carries its plate ID.

Before you start

Warning

These are bin/ utilities and still use argparse, unlike the four main commands. They keep argparse conventions — including abbreviated options — and are run with python bin/<name>.py rather than as installed commands.

Important

--sample-alignments must be set consistently across steps 2, 3 and 4. Mixing per-sample and per-well alignments produces silently wrong mappings — no error, just cells matched to the wrong partners.

Analysing the rounds

Run the pipeline on each round separately, exactly as for a single-round experiment:

uv run omero-screen 4126 --stitch
uv run omero-screen 4127 --stitch
uv run omero-screen 4128 --stitch

Each round gets its own segmentation, features and QC. At this point nothing knows the rounds are related — that begins with alignment.

Protocols

Note

Wet-lab protocols for the stripping and re-staining rounds are being prepared and will be published here.