Molecular Imaging Technologies in Analytical Chemistry: A Critical Review of Mass Spectrometry Imaging, Raman Imaging, Imaging Mass Cytometry, and Emerging Spatial Analytical Platforms.

Molecular imaging technologies have transformed analytical chemistry by enabling molecular identification and spatial localization within intact biological specimens. Advances in mass spectrometry imaging (MSI), Raman imaging, imaging mass cytometry (IMC), and emerging spatial analytical platforms have expanded the investigation of tissue heterogeneity and molecular organization. However, quantitative reliability, reproducibility, standardization, computational complexity, and clinical translation remain significant challenges. This review critically evaluates the analytical performance of current molecular imaging technologies, emphasizing spatial resolution, sensitivity, molecular coverage, quantitative capability, multiplexing, reproducibility, computational integration, artificial intelligence (AI)-assisted analysis, and clinical readiness. No single modality simultaneously provides optimal spatial resolution, molecular coverage, quantitative accuracy, throughput, and reproducibility. MSI enables comprehensive molecular profiling, Raman imaging provides label-free chemical specificity, IMC supports highly multiplexed protein analysis, and emerging spatial omics platforms offer systems-level biological insights. Nevertheless, matrix effects, workflow variability, computational demands, limited standardization, and insufficient multicenter validation restrict broader analytical and clinical implementation. AI-assisted analysis and multimodal data integration offer opportunities to improve performance but require transparent validation and harmonized workflows. Future progress will depend on standardized analytical protocols, quantitative validation, explainable AI, multimodal integration, automated quality control, and international harmonization to establish molecular imaging as robust, reproducible, and clinically translatable analytical platforms.