Version Compatibility

Reference examples tested with: Cellpose 3.0+, 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.

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

Complete steinbock Workflow

Step 1: Setup and Preprocessing

# Initialize steinbock project
steinbock preprocess imc \
    --mcd data/*.mcd \
    --panel panel.csv \
    --output raw/

# Hot pixel filtering
steinbock preprocess imc hotpixel \
    --input raw/ \
    --output img/ \
    --threshold 50

# Create nuclear and membrane channels
steinbock preprocess mosaic \
    --input img/ \
    --channels panel.csv \
    --output mosaics/

Step 2: Cell Segmentation

# Using Cellpose
steinbock segment cellpose \
    --input img/ \
    --panel panel.csv \
    --channel DNA1 DNA2 \
    --output masks/ \
    --diameter 20

# Alternative: Using Mesmer
steinbock segment mesmer \
    --input img/ \
    --panel panel.csv \
    --nuclear DNA1 DNA2 \
    --membrane CD45 \
    --output masks/

Step 3: Single-cell Quantification

# Extract intensities
steinbock measure intensities \
    --input img/ \
    --masks masks/ \
    --panel panel.csv \
    --output intensities/

# Measure cell properties (area, etc.)
steinbock measure regionprops \
    --masks masks/ \
    --output regionprops/

# Extract neighbor relationships
steinbock measure neighbors \
    --masks masks/ \
    --output neighbors/ \
    --distance 15

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'] = [idx.split('_')[0] for idx in intensities.index]
adata.obs['cell_id'] = intensities.index

# Add spatial coordinates
adata.obsm['spatial'] = regionprops[['centroid_y', 'centroid_x']].values

# === 3. PREPROCESSING ===
# Arcsinh transform (cofactor 5 for IMC)
adata.X = np.arcsinh(adata.X / 5)

# 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
sq.gr.spatial_neighbors(adata, coord_type='generic', delaunay=True)

# 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
fig, ax = plt.subplots(figsize=(10, 10))
sq.pl.spatial_scatter(adata[adata.obs['image_id'] == 'image1'],
                      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 ===
# Compare conditions
adata.obs['condition'] = adata.obs['image_id'].map({
    'image1': 'Control', 'image2': 'Control',
    'image3': 'Treatment', 'image4': 'Treatment'
})

# Cell type proportions
proportions = adata.obs.groupby(['image_id', 'condition', 'cell_type']).size().unstack(fill_value=0)
proportions = proportions.div(proportions.sum(axis=1), axis=0)

# Save results
adata.write('imc_analysis.h5ad')
proportions.to_csv('cell_type_proportions.csv')
print('Analysis complete!')

R Alternative (imcRtools)

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

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

# Transform
assay(spe, 'exprs') <- asinh(counts(spe) / 5)

# Cluster
spe <- runDR(spe, features = rownames(spe), exprs_values = 'exprs', dr = 'UMAP')
spe <- cluster(spe, features = rownames(spe), exprs_values = 'exprs',
               xdim = 10, ydim = 10, maxK = 20)

# Spatial analysis
spe <- buildSpatialGraph(spe, img_id = 'image_id', type = 'expansion', threshold = 20)
spe <- aggregateNeighbors(spe, colPairName = 'neighborhood', by = 'cluster_id')

# Spatial context
cn <- detectCommunity(spe, colPairName = 'neighborhood',
                       size_threshold = 10, group_by = 'image_id')

# Plot
plotSpatial(spe, img_id = 'image1', 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 interactions with tumor
tumor_cells = adata[adata.obs['cell_type'] == 'Tumor'].obs_names
sq.gr.ligrec(adata, cluster_key='cell_type', source_groups=['Tumor'],
             target_groups=['T cells', 'Macrophages'])

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/interactive-annotation - Manual cell labeling
• imaging-mass-cytometry/quality-metrics - QC metrics
• single-cell/clustering - Clustering methods
• spatial-transcriptomics/spatial-statistics - Related spatial methods