SCOPE: A Spatial Co-registration and Omics Pipeline for Comparative Mass Spectrometry Imaging Data Analysis

Spatially resolved omics technologies have transformed our ability to map molecular organization within tissues. However, the absence of anatomically consistent spatial units across biological replicates fundamentally limits cross-sample comparison and rigorous population-level inference at high spatial fidelity. Current approaches rely either on segmentation-dependent region definitions, which risk obscuring fine-scale molecular heterogeneity, or on analyses confined to individual specimens without cross-replicate spatial correspondence. Here, we introduce SCOPE, a spatial co-registration and omics pipeline that integrates deformable image co-registration with a shared spatial reference, HexBin, to establish anatomically homologous spatial units across samples. By enabling segmentation-free comparison while preserving high spatial fidelity, SCOPE maintains intrinsic molecular heterogeneity and supports statistically robust cross-replicate comparison. Application of SCOPE to an ex vivo bovine lens model of oxidative stress reveals region- and microdomain-specific remodeling of glutathione metabolism that is obscured by whole-tissue averaging or coarse segmentation. High-spatial-fidelity mapping identifies anterior-posterior asymmetries and localized vulnerabilities within lens fiber cell populations and enables relative quantitative evaluation of the thiol-based antioxidant therapeutic N-acetylcysteine amide. SCOPE advances spatial omics beyond segmentation-dependent and single-section descriptive analyses toward reproducible cross-sample quantitative inference, providing a scalable foundation for high-spatial-fidelity analysis across biological tissues.