Truthful visualizations for mass spectrometry imaging enable high-spatial-resolution interactive m/z mapping and exploration.

Mass spectrometry imaging (MSI) produces high-dimensional molecular data, but practical interpretation remains limited by visualizations that incompletely preserve global structure. We present MSI-VISUAL, an open-source framework for interactive MSI analysis that integrates truthful dimensionality reduction visualizations with region-of-interest selection, statistical comparison, and direct mass/charge ratio (m/z) mapping. MSI-VISUAL introduces four visualization strategies: SALO and SPEAR (optimization-based methods designed to improve global structure preservation across distance metrics) and TOP3 and PR3D (lightweight approaches for memory-efficient, rapid visualization of large datasets). Across benchmarks and lipidomics case studies, including mouse brain and kidney pathology examples, the proposed methods outperform commonly used alternatives in our benchmarks, improve detection of subtle tissue differences, and reveal fine molecular-anatomical patterns that support biological insights. These results establish MSI-VISUAL as a scalable framework for discovery-oriented and diagnostic MSI workflows.