Version Compatibility

Reference examples tested with: matplotlib 3.8+, numpy 1.26+, pandas 2.2+, scipy 1.12+

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

• Python: pip show <package> then help(module.function) to check signatures

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

Quality Metrics

"Assess quality of my IMC acquisition" → Evaluate IMC data quality through signal-to-noise ratios, channel correlations, tissue integrity scores, and acquisition-specific QC metrics.

• Python: numpy/scipy for SNR calculation and channel correlation analysis

Signal-to-Noise Ratio

python
import numpy as np
from scipy import ndimage
from skimage import io

def calculate_snr(image, mask=None):
    '''Calculate signal-to-noise ratio for an image channel.'''
    if mask is None:
        mask = image > np.percentile(image, 10)

    signal = np.mean(image[mask])
    noise = np.std(image[~mask])

    if noise == 0:
        return np.inf

    snr = signal / noise
    return snr

def calculate_snr_all_channels(image_stack, channel_names, tissue_mask=None):
    '''Calculate SNR for all channels in stack.'''
    results = {}
    for i, name in enumerate(channel_names):
        snr = calculate_snr(image_stack[i], tissue_mask)
        results[name] = snr
    return results

image_stack = io.imread('imc_image.tiff')
channel_names = ['CD45', 'CD3', 'CD68', 'panCK', 'DNA']
snr_values = calculate_snr_all_channels(image_stack, channel_names)

for ch, snr in snr_values.items():
    status = 'PASS' if snr > 3 else 'WARN' if snr > 1.5 else 'FAIL'
    print(f'{ch}: SNR = {snr:.2f} [{status}]')

Channel Correlation

python
def calculate_channel_correlation(image_stack, channel_names):
    '''Calculate pairwise correlation between channels.'''
    n_channels = image_stack.shape[0]
    flat_data = image_stack.reshape(n_channels, -1)

    corr_matrix = np.corrcoef(flat_data)

    import pandas as pd
    corr_df = pd.DataFrame(corr_matrix, index=channel_names, columns=channel_names)
    return corr_df

def flag_unexpected_correlations(corr_df, expected_pairs=None, threshold=0.7):
    '''Flag unexpected high correlations (possible spillover).'''
    issues = []

    if expected_pairs is None:
        expected_pairs = []

    for i, ch1 in enumerate(corr_df.columns):
        for j, ch2 in enumerate(corr_df.columns):
            if i >= j:
                continue

            corr = corr_df.loc[ch1, ch2]
            pair = (ch1, ch2)
            is_expected = pair in expected_pairs or (ch2, ch1) in expected_pairs

            if corr > threshold and not is_expected:
                issues.append({'channel_1': ch1, 'channel_2': ch2, 'correlation': corr, 'expected': is_expected})

    return pd.DataFrame(issues)

corr_matrix = calculate_channel_correlation(image_stack, channel_names)
print('Channel correlations:')
print(corr_matrix.round(2))

expected = [('CD3', 'CD45')]
issues = flag_unexpected_correlations(corr_matrix, expected)
if len(issues) > 0:
    print('\nUnexpected high correlations:')
    print(issues)

Tissue Integrity

python
def assess_tissue_integrity(dna_channel, min_coverage=0.3):
    '''Assess tissue coverage and integrity from DNA channel.'''
    threshold = np.percentile(dna_channel, 50)
    tissue_mask = dna_channel > threshold

    total_pixels = dna_channel.size
    tissue_pixels = np.sum(tissue_mask)
    coverage = tissue_pixels / total_pixels

    labeled, n_fragments = ndimage.label(tissue_mask)
    fragment_sizes = ndimage.sum(tissue_mask, labeled, range(1, n_fragments + 1))

    largest_fragment = np.max(fragment_sizes) if len(fragment_sizes) > 0 else 0
    fragmentation = 1 - (largest_fragment / tissue_pixels) if tissue_pixels > 0 else 1

    return {
        'coverage': coverage,
        'n_fragments': n_fragments,
        'fragmentation': fragmentation,
        'intact': coverage > min_coverage and fragmentation < 0.5
    }

dna_channel = image_stack[channel_names.index('DNA')]
integrity = assess_tissue_integrity(dna_channel)

print(f"Tissue coverage: {integrity['coverage']:.1%}")
print(f"Fragments: {integrity['n_fragments']}")
print(f"Fragmentation: {integrity['fragmentation']:.2f}")
print(f"Status: {'PASS' if integrity['intact'] else 'FAIL'}")

Acquisition QC

python
def check_acquisition_artifacts(image_stack, channel_names):
    '''Check for common acquisition artifacts.'''
    results = []

    for i, name in enumerate(channel_names):
        channel = image_stack[i]

        saturated = np.sum(channel >= channel.max() * 0.99) / channel.size
        if saturated > 0.01:
            results.append({'channel': name, 'issue': 'saturation', 'severity': saturated})

        hot_pixels = np.sum(channel > np.percentile(channel, 99.9) * 2) / channel.size
        if hot_pixels > 0.001:
            results.append({'channel': name, 'issue': 'hot_pixels', 'severity': hot_pixels})

        dead_regions = np.sum(channel == 0) / channel.size
        if dead_regions > 0.05:
            results.append({'channel': name, 'issue': 'dead_regions', 'severity': dead_regions})

        row_means = np.mean(channel, axis=1)
        row_cv = np.std(row_means) / np.mean(row_means)
        if row_cv > 0.3:
            results.append({'channel': name, 'issue': 'striping', 'severity': row_cv})

    return pd.DataFrame(results)

artifacts = check_acquisition_artifacts(image_stack, channel_names)
if len(artifacts) > 0:
    print('Artifacts detected:')
    print(artifacts)
else:
    print('No major artifacts detected')

Dynamic Range

python
def assess_dynamic_range(channel, percentiles=(1, 99)):
    '''Assess if channel uses full dynamic range.'''
    low, high = np.percentile(channel, percentiles)
    channel_range = high - low
    max_possible = channel.max()

    utilized = channel_range / max_possible if max_possible > 0 else 0

    return {
        'range_low': low,
        'range_high': high,
        'range_utilized': utilized,
        'adequate': utilized > 0.1
    }

for i, name in enumerate(channel_names):
    dr = assess_dynamic_range(image_stack[i])
    status = 'OK' if dr['adequate'] else 'LOW'
    print(f"{name}: {dr['range_utilized']:.1%} range used [{status}]")

Segmentation Quality Metrics

python
def segmentation_qc(segmentation_mask, image_stack, channel_names):
    '''QC metrics for cell segmentation.'''
    from skimage.measure import regionprops

    props = regionprops(segmentation_mask)
    n_cells = len(props)

    if n_cells == 0:
        return {'error': 'No cells found'}

    areas = [p.area for p in props]
    eccentricities = [p.eccentricity for p in props]

    area_cv = np.std(areas) / np.mean(areas)
    very_small = np.sum(np.array(areas) < np.percentile(areas, 5)) / n_cells
    very_large = np.sum(np.array(areas) > np.percentile(areas, 95)) / n_cells
    elongated = np.sum(np.array(eccentricities) > 0.9) / n_cells

    return {
        'n_cells': n_cells,
        'mean_area': np.mean(areas),
        'area_cv': area_cv,
        'pct_very_small': very_small,
        'pct_very_large': very_large,
        'pct_elongated': elongated,
        'quality': 'GOOD' if area_cv < 0.5 and elongated < 0.1 else 'REVIEW'
    }

seg_mask = io.imread('cell_segmentation.tiff')
seg_qc = segmentation_qc(seg_mask, image_stack, channel_names)
print(f"Cells: {seg_qc['n_cells']}")
print(f"Mean area: {seg_qc['mean_area']:.1f} pixels")
print(f"Quality: {seg_qc['quality']}")

Batch QC Summary

Goal: Generate a consolidated quality report across all acquisitions in a batch to identify samples requiring re-acquisition or exclusion.

Approach: For each image, compute SNR, tissue integrity, segmentation metrics, and artifact counts, then aggregate into a summary table with pass/fail calls based on combined threshold criteria.

python
def batch_qc_report(image_files, seg_files, channel_names, output_file):
    '''Generate QC report for batch of images.'''
    all_results = []

    for img_file, seg_file in zip(image_files, seg_files):
        image_stack = io.imread(img_file)
        seg_mask = io.imread(seg_file)

        result = {'sample': Path(img_file).stem}

        snr_values = calculate_snr_all_channels(image_stack, channel_names)
        result['mean_snr'] = np.mean(list(snr_values.values()))
        result['min_snr'] = min(snr_values.values())

        dna_idx = channel_names.index('DNA') if 'DNA' in channel_names else 0
        integrity = assess_tissue_integrity(image_stack[dna_idx])
        result['tissue_coverage'] = integrity['coverage']

        seg_qc = segmentation_qc(seg_mask, image_stack, channel_names)
        result['n_cells'] = seg_qc.get('n_cells', 0)

        artifacts = check_acquisition_artifacts(image_stack, channel_names)
        result['n_artifacts'] = len(artifacts)

        result['pass_qc'] = (result['min_snr'] > 1.5 and result['tissue_coverage'] > 0.3 and result['n_artifacts'] == 0)

        all_results.append(result)

    results_df = pd.DataFrame(all_results)
    results_df.to_csv(output_file, index=False)

    print(f"QC Summary: {results_df['pass_qc'].sum()}/{len(results_df)} samples passed")
    return results_df

Visualization

python
import matplotlib.pyplot as plt

def plot_qc_summary(image_stack, channel_names, output_file):
    '''Generate QC summary visualization.'''
    n_channels = len(channel_names)

    fig, axes = plt.subplots(2, n_channels, figsize=(3*n_channels, 6))

    for i, name in enumerate(channel_names):
        channel = image_stack[i]

        axes[0, i].imshow(channel, cmap='viridis')
        axes[0, i].set_title(name)
        axes[0, i].axis('off')

        axes[1, i].hist(channel.flatten(), bins=100, log=True)
        axes[1, i].set_xlabel('Intensity')
        axes[1, i].set_ylabel('Count')

    plt.tight_layout()
    plt.savefig(output_file, dpi=150)
    plt.close()

plot_qc_summary(image_stack, channel_names, 'qc_summary.png')

Related Skills

• data-preprocessing - Clean data before QC
• cell-segmentation - Segmentation affects QC metrics
• interactive-annotation - Manual review of QC failures
• phenotyping - Analysis after QC passes