Single-cell mass spectrometry imaging: emerging advancements and future insights

Single-cell mass spectrometry imaging (MSI) has emerged as a transformative technology for dissecting complex biological systems with high spatial precision. It enables the insitu analysis of cellular biomolecules within their native tissue architecture while preserving critical microenvironmental context. This approach offers unparalleled insights into cellular heterogeneity, biomolecular interactions, and dynamic microenvironmental influences. Recent advances in instrumentation, including improved ion optics and ion mobility spectrometry, along with innovative sample preparation techniques such as hydrogel-assisted tissue expansion, have markedly enhanced the spatial resolution, molecular sensitivity, and analyte coverage of single-cell MSI. In this review, we provide a systematic overview of single-cell MSI workflows, encompassing sample preparation strategies, ionization and imaging modalities, and computational tools for data analysis. We critically assess their underlying principles, strengths, and limitations, and highlight recent methodological developments aimed at improving molecular coverage and spatial fidelity. Finally, we offer perspectives on the future trajectory of single-cell MSI and its potential to drive new discoveries in spatial omics and systems biology.