UniFORM: Towards Universal Immunofluorescence Normalization for Multiplex Tissue Imaging

Multiplexed tissue imaging (MTI) technologies enable high-dimensional spatial analysis of tumor microenvironments but face challenges with technical variability in staining intensities. Existing normalization methods, including z-score, ComBat, and MxNorm, often fail to account for the heterogeneous, right-skewed expression patterns of MTI data, compromising signal alignment and downstream analyses. We present UniFORM, a non-parametric, Python-based pipeline for normalizing both feature- and pixel-level MTI data. UniFORM preserves marker intensity distribution shapes while maintaining positive population proportions without prior assumptions and uses automated normalization. Benchmarking across two distinct MTI platforms and datasets demonstrates that UniFORM outperforms existing methods in mitigating batch effects while maintaining biological signal fidelity. This is evidenced by improved marker distribution alignment, enhanced kBET scores, and improved downstream analyses such as UMAP visualizations and Leiden clustering. UniFORM also introduces a novel guided fine-tuning option for complex and heterogeneous datasets. Although optimized for fluorescence-based platforms, UniFORM provides a scalable and robust solution for MTI data normalization, enabling accurate and biologically meaningful interpretations.