Version Compatibility

Reference examples tested with: Cellpose 4.0+ (cpsam model), anndata 0.10+, matplotlib 3.8+, numpy 1.26+, pandas 2.2+, scanpy 1.10+, scvi-tools 1.1+, squidpy 1.3+, steinbock 0.16+

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show <package> then help(module.function) to check signatures
• R: packageVersion('<pkg>') then ?function_name to verify parameters
• CLI: <tool> --version then <tool> --help to confirm flags

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Imaging Mass Cytometry Pipeline

"Process my imaging mass cytometry data from images to spatial analysis" -> Orchestrate image preprocessing (steinbock), cell segmentation (Cellpose), phenotyping (FlowSOM/scanpy), spatial neighborhood analysis (squidpy), and tissue community detection.

This is a workflow skill: it owns the chaining decisions and hand-offs, not the internals of any one step.

The governing principle

Segmentation is the largest irreversible error source, and it is spatial: every per-cell number is a mask-bounded pixel average, so a wrong boundary fabricates cell types before any expression QC can see them. The seam ORDER — and the patient-level unit — is therefore what decides trustworthiness.

1. The comparison frame (panel + segmentation frame + pixel size) is committed once and inherited by every per-cell number. The summed membrane channel encodes a cell-type bias (sum BROADLY-expressed markers, or segmentation under-performs on cell types lacking a strong membrane marker); Mesmer was trained at model_mpp ~0.5 um and rescales the input to it, so passing the wrong pixel size (Mesmer's image_mpp defaults to None = NO rescaling, assuming the input is already at model resolution — the true pixel size must be passed explicitly; steinbock's --pixelsize flag wraps image_mpp and defaults to 1.0) rescales cells to the wrong learned size and degrades every boundary. No downstream step recovers a merged or split cell.
2. Channel spillover is compensated on PIXELS before segmentation; lateral spillover (REDSEA) runs on the per-cell table AFTER segmentation — they are DIFFERENT problems. Metal-isotope crosstalk is a pixel-level NNLS correction whose compensated value must be what gets averaged into the per-cell mean (post-aggregation is wrong). REDSEA corrects real signal leaking across shared cell boundaries at ~1 um even with perfect segmentation and zero channel spillover — it is defined on segmented neighbors, so it must run after segmentation. Running REDSEA pre-segmentation, or channel comp post-aggregation, is a category error.
3. The experimental unit is the PATIENT, not the cell or the ROI. Cells and ROIs from one patient are not independent replicates; a cell-level or per-image test over correlated cells is pseudoreplication (reports p~0 for trivial effects). Aggregate to per-patient proportions/summaries, then a mixed model / scCODA. Arcsinh cofactor is 1 for IMC integer ion counts, NOT the suspension-CyTOF 5 (which over-compresses them). Impossible lineage-exclusive co-expression is a segmentation/spillover ALARM, not a hybrid cell type.

Made-once commitments

Commitment	Consequence inherited downstream
Panel (metal->antibody; membrane-sum channels)	Which channels extract and phenotype; a narrow membrane sum biases segmentation against some cell types
Segmentation frame (nuclear + membrane channels)	Every per-cell number (all are mask-bounded pixel averages); the largest irreversible error source
Pixel size (steinbock --pixelsize / Mesmer image_mpp, ~1.0 um for IMC)	Boundary quality + all spatial distances; the wrong value rescales cells to the wrong learned size
Arcsinh cofactor = 1 (IMC), not 5 (CyTOF)	Clustering/phenotyping distances; cofactor 5 over-compresses integer ion counts

Pipeline Overview

Raw MCD/TIFF Files ──> Image Processing ──> Cell Masks
                                                 │
                                                 ▼
                ┌─────────────────────────────────────────────┐
                │              imc-pipeline                   │
                ├─────────────────────────────────────────────┤
                │  1. Data Preprocessing (spillover, hot px)  │
                │  2. Cell Segmentation (Cellpose/Mesmer)     │
                │  3. Single-cell Quantification              │
                │  4. Clustering & Phenotyping                │
                │  5. Spatial Analysis                        │
                │  6. Visualization                           │
                └─────────────────────────────────────────────┘
                                                 │
                                                 ▼
                    Cell Types + Spatial Neighborhoods

Decisions Threaded Through This Pipeline

Four reframes govern every stage and are detailed in the depended-on skills: IMC pixels are integer ion COUNTS (arcsinh cofactor 1, not the suspension-CyTOF 5), and spillover is spatial so it must be NNLS-compensated before segmentation; segmentation is the largest irreversible error source, so impossible double-positives are a QC alarm, not biology; a spatial interaction is a hypothesis test whose null silently decides whether the result is real or a density artifact; and the experimental unit is the patient, not the cell, so cross-condition tests aggregate to patients before testing.

Complete steinbock Workflow

Step 1: Setup and Preprocessing

# generate the panel template; edit the keep column before extracting
steinbock preprocess imc panel

# extract per-channel TIFFs (keep-filtered, panel-ordered) with hot-pixel removal
# (--hpf is a signed 8-neighbor difference; 50 is a count, tune to dynamic range)
steinbock preprocess imc images --hpf 50

# channel spillover is compensated with NNLS (CATALYST/cytomapper, R) on the pixel images
# BEFORE segmentation when spatial analysis is the endpoint -- see data-preprocessing

Step 2: Cell Segmentation

# Mesmer/DeepCell whole-cell (nuclear-first); membrane channels aggregated via the panel column.
# --pixelsize is steinbock's CLI flag for the acquisition resolution (it wraps Mesmer's image_mpp);
# steinbock defaults it to 1.0 um for IMC, so pass the true value explicitly rather than relying on it.
steinbock segment deepcell --pixelsize 1.0 --minmax -o masks

# Alternative: Cellpose container (Cellpose 4+ default model cpsam; channel order reversed vs native)
steinbock segment cellpose --minmax -o masks

Step 3: Single-cell Quantification

# Extract per-cell MEAN intensities (mean is the default and the right phenotyping aggregator;
# sum confounds cell size with expression)
steinbock measure intensities -o intensities

# Measure cell properties (area, centroid, eccentricity)
steinbock measure regionprops -o regionprops

# Build the spatial neighbor graph (expansion within a max distance; match the graph to the
# biological claim -- contact vs proximity -- in spatial-analysis)
steinbock measure neighbors --type expansion --dmax 15 -o neighbors

Complete Python Workflow

import pandas as pd
import numpy as np
import anndata as ad
import scanpy as sc
import squidpy as sq
from pathlib import Path

# === 1. LOAD DATA ===
data_dir = Path('steinbock_output')

intensities = pd.read_csv(data_dir / 'intensities.csv', index_col=0)
regionprops = pd.read_csv(data_dir / 'regionprops.csv', index_col=0)
neighbors = pd.read_csv(data_dir / 'neighbors.csv')

print(f'Loaded {len(intensities)} cells')

# === 2. CREATE ANNDATA ===
adata = ad.AnnData(X=intensities.values, obs=regionprops, var=pd.DataFrame(index=intensities.columns))
adata.obs['image_id'] = pd.Categorical([idx.rsplit('_', 1)[0] for idx in intensities.index])   # strip only the trailing cell index: rsplit keeps Patient1_ROI002 distinct from Patient1_ROI001. squidpy library_key requires a categorical, not object/string
adata.obs['cell_id'] = intensities.index

# Add spatial coordinates (skimage regionprops_table names them centroid-0 (y) / centroid-1 (x))
adata.obsm['spatial'] = regionprops[['centroid-0', 'centroid-1']].values

# === 3. PREPROCESSING ===
# Arcsinh transform: cofactor 1 for IMC single-cell means (Hunter 2024), NOT the
# suspension-CyTOF cofactor 5, which over-compresses IMC's lower-count means
adata.layers['counts'] = adata.X.copy()
adata.X = np.arcsinh(adata.X / 1)

# Scale for clustering
sc.pp.scale(adata, max_value=10)
adata.raw = adata.copy()

# === 4. DIMENSIONALITY REDUCTION ===
sc.pp.pca(adata, n_comps=20)
sc.pp.neighbors(adata, n_neighbors=15)
sc.tl.umap(adata)

# === 5. CLUSTERING ===
sc.tl.leiden(adata, resolution=0.8)
print(f'Found {adata.obs["leiden"].nunique()} clusters')

# === 6. PHENOTYPING ===
# Marker expression per cluster
sc.tl.rank_genes_groups(adata, 'leiden', method='wilcoxon')
marker_genes = sc.get.rank_genes_groups_df(adata, group=None)

# Annotate clusters based on markers
cluster_annotations = {
    '0': 'T cells',
    '1': 'Macrophages',
    '2': 'Tumor',
    '3': 'B cells',
    '4': 'Stromal'
}
adata.obs['cell_type'] = adata.obs['leiden'].map(cluster_annotations)

# === 7. SPATIAL ANALYSIS ===
# Build spatial graph PER IMAGE (library_key), else Delaunay fabricates edges across ROIs
sq.gr.spatial_neighbors(adata, coord_type='generic', delaunay=True, library_key='image_id')

# Neighborhood enrichment
sq.gr.nhood_enrichment(adata, cluster_key='cell_type')

# Co-occurrence analysis
sq.gr.co_occurrence(adata, cluster_key='cell_type')

# Ripley's statistics
sq.gr.ripley(adata, cluster_key='cell_type', mode='L')

# === 8. VISUALIZATION ===
import matplotlib.pyplot as plt

# UMAP by cell type
fig, axes = plt.subplots(1, 2, figsize=(14, 5))
sc.pl.umap(adata, color='cell_type', ax=axes[0], show=False)
sc.pl.umap(adata, color='leiden', ax=axes[1], show=False)
plt.savefig('umap_celltypes.png', dpi=150, bbox_inches='tight')

# Spatial plot. Pick the image dynamically: image_id is derived from the cell index, so a hardcoded
# literal selects zero cells and spatial_scatter errors on the empty subset.
fig, ax = plt.subplots(figsize=(10, 10))
first_image = adata.obs['image_id'].iloc[0]
sq.pl.spatial_scatter(adata[adata.obs['image_id'] == first_image],
                      color='cell_type', shape=None, size=10, ax=ax)
plt.savefig('spatial_celltypes.png', dpi=150, bbox_inches='tight')

# Neighborhood enrichment heatmap
sq.pl.nhood_enrichment(adata, cluster_key='cell_type')
plt.savefig('neighborhood_enrichment.png', dpi=150, bbox_inches='tight')

# === 9. DIFFERENTIAL ANALYSIS (patient is the unit, NOT the cell) ===
import statsmodels.formula.api as smf

# aggregate to per-image proportions, then test across PATIENTS -- a cell-level or per-image
# test over correlated cells is pseudoreplication and reports p~0 for trivial effects.
# obs must carry patient and condition columns; see differential-analysis for scCODA
# (compositional) and the spatial differential path.
counts = adata.obs.groupby(['patient', 'condition', 'image_id', 'cell_type'], observed=True).size().unstack(fill_value=0)   # observed=True: image_id is categorical; the default expands the full cartesian product into all-zero phantom rows -> NaN proportions
image_prop = counts.div(counts.sum(axis=1), axis=0).reset_index()
target = 'Tumor'   # an actual cell_type column from cluster_annotations above (single-word for the formula)
res = smf.mixedlm(f'{target} ~ condition', image_prop, groups=image_prop['patient']).fit()  # patient random effect
print(res.summary())

adata.write('imc_analysis.h5ad')
print('Analysis complete!')

R Alternative (imcRtools)

library(imcRtools)
library(cytomapper)
library(CATALYST)

# Read steinbock output
spe <- read_steinbock('steinbock_output/')

# Transform (cofactor 1 for IMC single-cell means, not 5)
assay(spe, 'exprs') <- asinh(counts(spe) / 1)

# Cluster (CATALYST runDR takes assay=; cluster() always uses the 'exprs' assay, no assay arg)
spe <- runDR(spe, features = rownames(spe), assay = 'exprs', dr = 'UMAP')
spe <- cluster(spe, features = rownames(spe), xdim = 10, ydim = 10, maxK = 20)

# Spatial analysis. buildSpatialGraph names the colPair '<type>_interaction_graph';
# aggregateNeighbors counts a label via aggregate_by='metadata' + count_by=.
spe <- buildSpatialGraph(spe, img_id = 'sample_id', type = 'expansion', threshold = 20)
spe <- aggregateNeighbors(spe, colPairName = 'expansion_interaction_graph',
                          aggregate_by = 'metadata', count_by = 'cluster_id')

# Spatial context
spe <- detectCommunity(spe, colPairName = 'expansion_interaction_graph',
                       size_threshold = 10, group_by = 'sample_id')

# Plot (img_id is the colData COLUMN used to facet; read_steinbock names it 'sample_id', not 'image_id')
plotSpatial(spe, img_id = 'sample_id', node_color_by = 'cluster_id')

QC Checkpoints

Stage	Check	Action if Failed
Preprocessing	No hot pixel streaks	Lower threshold
Segmentation	>80% cells detected	Adjust diameter
Quantification	All markers extracted	Check panel.csv
Clustering	5-20 clusters	Adjust resolution
Spatial	Neighbors detected	Check distance

Workflow Variants

High-plex Panels (40+ markers)

# Use batch-aware clustering
import scvi

scvi.model.SCVI.setup_anndata(adata, batch_key='image_id')
model = scvi.model.SCVI(adata)
model.train()
adata.obsm['X_scvi'] = model.get_latent_representation()
sc.pp.neighbors(adata, use_rep='X_scvi')

Tumor Microenvironment Analysis

# Spatial cell-cell co-location around tumor (per-image, then aggregate to patient).
# Note: sq.gr.ligrec keys ligand-receptor pairs on gene symbols from OmniPath, so it is
# usually empty on a ~40-marker antibody panel -- prefer neighborhood enrichment for IMC.
sq.gr.nhood_enrichment(adata, cluster_key='cell_type')   # see spatial-analysis for the null caveat

Common Errors

Symptom	Cause	Fix
Impossible double-positive "hybrid" cell types	Spillover not corrected before phenotyping (channel and/or lateral)	NNLS channel compensation on pixels before segmentation; REDSEA on the per-cell table after; treat lineage-exclusive co-expression as a QC failure until proven
Every boundary degraded, cells the wrong size	Wrong pixel size (Mesmer image_mpp defaults None=no rescaling, model trained at ~0.5; steinbock --pixelsize defaults 1.0)	Pass the true acquisition resolution explicitly (~1.0 um for IMC)
Macrophages under-captured; biased comparison	Nuclear-expansion segmentation cross-compared with whole-cell data	Never quantitatively compare expansion-segmented vs whole-cell; report the expansion radius; use constrained (not free) dilation
p~0 for a trivial effect	Pseudoreplication (cells/ROIs treated as replicates)	Aggregate to per-patient summaries; mixed model with patient random effect / scCODA
Markers over-compressed, noise clusters	Arcsinh cofactor 5 used on IMC	Cofactor 1 for IMC integer ion counts
Acquisition batch drives the clusters	Batch confounded with / not modeled against condition	Randomize acquisition order; batch-aware clustering (Harmony/scVI) for clustering ONLY; model batch as a covariate; no rescue if batch==condition

References

• Windhager J, Zanotelli VRT, Schulz D, et al (2023) An end-to-end workflow for multiplexed image processing and analysis. Nature Protocols 18:3565-3613. DOI 10.1038/s41596-023-00881-0. (steinbock.)
• Greenwald NF, Miller G, Moen E, et al (2022) Whole-cell segmentation of tissue images with human-level performance using large-scale data annotation and deep learning. Nature Biotechnology 40:555-565. DOI 10.1038/s41587-021-01094-0. (Mesmer/DeepCell.)
• Bai Y, Zhu B, Rovira-Clave X, et al (2021) Adjacent cell marker lateral spillover compensation and reinforcement for multiplexed images. Frontiers in Immunology 12:652631. DOI 10.3389/fimmu.2021.652631. (REDSEA.)
• Palla G, Spitzer H, Klein M, et al (2022) Squidpy: a scalable framework for spatial omics analysis. Nature Methods 19:171-178. DOI 10.1038/s41592-021-01358-2.
• Hunter B, Nicorescu I, Foster E, et al (2024) OPTIMAL: an OPTimized Imaging Mass cytometry AnaLysis framework for benchmarking segmentation and data exploration. Cytometry Part A 105:36-53. DOI 10.1002/cyto.a.24803. (arcsinh cofactor 1 for IMC.)

Related Skills

• imaging-mass-cytometry/data-preprocessing - Hot pixel, spillover
• imaging-mass-cytometry/cell-segmentation - Cellpose/Mesmer details
• imaging-mass-cytometry/phenotyping - Cluster annotation
• imaging-mass-cytometry/spatial-analysis - Spatial statistics
• imaging-mass-cytometry/differential-analysis - Patient-level cross-condition testing
• imaging-mass-cytometry/interactive-annotation - Manual cell labeling
• imaging-mass-cytometry/quality-metrics - QC metrics
• single-cell/clustering - Clustering methods
• spatial-transcriptomics/spatial-statistics - Related spatial methods