Cross-Modality Alignment of Spatial Transcriptomics, Multiplexed Imaging, and Histology with PHARAOH.

Accurate cross-modality alignment between spatial transcriptomics (ST), multiplexed imaging, and histology is essential for spatially contextualized molecular analysis. In imaging-based ST platforms, molecular measurements are defined in the coordinate system of nuclear-stained images rather than in histological space, requiring precise registration for accurate integration with tissue morphology. Existing approaches either rely on labor-intensive manual landmark placement or lack the precision required for cellular-resolution alignment. Here we present PHARAOH, a hierarchical framework for cross-modality alignment of ST, multiplexed imaging, and histology. By combining global structural alignment with automated nuclear landmark anchoring, PHARAOH enables accurate coordinate transfer between molecular imaging and histological space with minimal user input. Across diverse tissue types, imaging platforms, and staining conditions, PHARAOH consistently achieved fast, robust, and spatially precise alignment, including across adjacent tissue sections. The framework further generalizes to multiplexed immunofluorescence imaging, providing a unified and scalable strategy for integrating spatial molecular measurements with histological architecture.