Bio Imaging Mass Cytometry Data Preprocessing

Version Compatibility

Reference examples tested with: anndata 0.10+, numpy 1.26+, pandas 2.2+, scanpy 1.10+, scipy 1.12+, 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
• 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.

IMC Data Preprocessing

"Preprocess my imaging mass cytometry data" → Load MCD files, apply hot pixel removal, channel cropping, and signal normalization to prepare multiplexed images for segmentation and analysis.

• CLI: steinbock preprocess for automated IMC preprocessing pipeline

Load MCD Files with steinbock

# steinbock CLI workflow (Docker-based)
# Convert MCD to TIFF
steinbock preprocess imc \
    --mcd raw/*.mcd \
    --panel panel.csv \
    -o img

# Output: img/*.tiff (one per acquisition)

Panel File Format

# panel.csv
channel,name,keep,ilastik
1,DNA1,1,1
2,CD45,1,1
3,CD3,1,0
4,CD8,1,0
5,CD4,1,0

Python-Based Loading

import readimc
import numpy as np
from pathlib import Path

# Read MCD file
mcd_file = Path('acquisition.mcd')
with readimc.MCDFile(mcd_file) as mcd:
    # List acquisitions
    for acquisition in mcd.acquisitions:
        print(f'Acquisition: {acquisition.id}')
        print(f'  Channels: {len(acquisition.channel_metals)}')
        print(f'  Size: {acquisition.width} x {acquisition.height}')

    # Load specific acquisition
    acq = mcd.acquisitions[0]
    img = mcd.read_acquisition(acq)  # Returns (C, H, W) array

    # Channel names
    channel_names = acq.channel_names

Hot Pixel Removal

from scipy import ndimage
import numpy as np

def remove_hot_pixels(img, threshold=50):
    '''Remove hot pixels using median filtering comparison'''
    filtered = ndimage.median_filter(img, size=3)
    diff = np.abs(img - filtered)
    hot_pixels = diff > threshold

    # Replace hot pixels with median
    result = img.copy()
    result[hot_pixels] = filtered[hot_pixels]

    return result

# Apply to each channel
img_clean = np.stack([remove_hot_pixels(img[c]) for c in range(img.shape[0])])

Spillover Correction

Goal: Remove channel crosstalk caused by isotope impurities in IMC data so that each channel reflects only its intended metal target.

Approach: Invert the measured spillover matrix (channels x channels) and multiply each pixel's channel vector by the inverse, clipping negative values to zero.

import numpy as np
import pandas as pd

def apply_spillover_correction(img, spillover_matrix):
    '''Apply spillover correction to IMC image

    spillover_matrix: (n_channels, n_channels) DataFrame or array
                      rows = measured, cols = emitting
    '''
    n_channels, height, width = img.shape

    # Reshape to (pixels, channels)
    pixels = img.reshape(n_channels, -1).T

    # Invert spillover matrix
    sm = np.array(spillover_matrix)
    sm_inv = np.linalg.inv(sm)

    # Apply correction
    corrected = pixels @ sm_inv.T
    corrected = np.clip(corrected, 0, None)  # No negative values

    # Reshape back to image
    return corrected.T.reshape(n_channels, height, width)

# Load spillover matrix (from CATALYST or manual measurement)
spillover = pd.read_csv('spillover_matrix.csv', index_col=0)
img_corrected = apply_spillover_correction(img_clean, spillover)

Estimate Spillover from Single-Stain Controls

def estimate_spillover(single_stains, channel_names):
    '''Estimate spillover matrix from single-stain controls'''
    n_channels = len(channel_names)
    spillover = np.eye(n_channels)

    for i, (primary_channel, control_img) in enumerate(single_stains.items()):
        primary_idx = channel_names.index(primary_channel)
        primary_signal = control_img[primary_idx].flatten()
        mask = primary_signal > np.percentile(primary_signal, 95)

        for j, ch in enumerate(channel_names):
            if i != j:
                secondary_signal = control_img[j].flatten()[mask]
                spillover[j, primary_idx] = np.median(secondary_signal / primary_signal[mask])

    return pd.DataFrame(spillover, index=channel_names, columns=channel_names)

Image Normalization

def percentile_normalize(img, low=1, high=99):
    '''Normalize to percentiles (per channel)'''
    normalized = np.zeros_like(img, dtype=np.float32)

    for c in range(img.shape[0]):
        channel = img[c]
        p_low = np.percentile(channel, low)
        p_high = np.percentile(channel, high)

        normalized[c] = np.clip((channel - p_low) / (p_high - p_low), 0, 1)

    return normalized

def arcsinh_transform(img, cofactor=5):
    '''Arcsinh transformation (similar to flow cytometry)'''
    return np.arcsinh(img / cofactor)

# Apply transformations
img_norm = percentile_normalize(img_clean)
img_asinh = arcsinh_transform(img_clean)

steinbock Preprocessing Pipeline

# Complete preprocessing with steinbock

# 1. Extract images from MCD
steinbock preprocess imc --mcd raw/*.mcd -o img

# 2. Apply hot pixel removal
steinbock preprocess filter --img img -o img_filtered

# 3. Generate probability maps (for segmentation)
# Requires trained Ilastik classifier
steinbock classify ilastik \
    --img img_filtered \
    --ilastik-project pixel_classifier.ilp \
    -o probabilities

Visualize with napari

import napari
import tifffile

# Load image
img = tifffile.imread('acquisition.tiff')
channel_names = ['DNA1', 'CD45', 'CD3', 'CD8', 'CD4']

# Create viewer
viewer = napari.Viewer()

# Add channels
for i, name in enumerate(channel_names):
    viewer.add_image(img[i], name=name, colormap='gray', blending='additive')

napari.run()

Create AnnData Object

import anndata as ad
import pandas as pd

# After segmentation, create AnnData from single-cell data
def create_anndata(intensities, cell_info, channel_names):
    '''Create AnnData from segmented single-cell data'''

    # Intensities: cells x channels
    adata = ad.AnnData(X=intensities)

    # Channel names
    adata.var_names = channel_names

    # Cell metadata
    adata.obs = cell_info  # DataFrame with area, centroid_x, centroid_y, etc.

    return adata

# Example usage
adata = create_anndata(
    intensities=cell_intensities,  # (n_cells, n_channels)
    cell_info=cell_metadata,       # DataFrame
    channel_names=channel_names
)

adata.write('imc_data.h5ad')

Batch Processing

from pathlib import Path
import tifffile

def process_batch(input_dir, output_dir):
    '''Process all images in directory'''
    input_dir = Path(input_dir)
    output_dir = Path(output_dir)
    output_dir.mkdir(exist_ok=True)

    for img_path in input_dir.glob('*.tiff'):
        img = tifffile.imread(img_path)

        # Preprocessing
        img = np.stack([remove_hot_pixels(img[c]) for c in range(img.shape[0])])
        img = percentile_normalize(img)

        # Save
        output_path = output_dir / img_path.name
        tifffile.imwrite(output_path, img.astype(np.float32))

        print(f'Processed: {img_path.name}')

process_batch('raw_images', 'processed_images')

Related Skills

• cell-segmentation - Segment preprocessed images
• spatial-transcriptomics/spatial-data-io - Similar data loading concepts