Enhancing IR‐MALDESI MSI Spatial Resolution Through Beam Constriction With a Ring‐Actuated Iris

RATIONALE: Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) mass spectrometry imaging (MSI) enables label-free, spatially resolved molecular analysis of biological tissues under ambient conditions. While improving spatial resolution is important for mapping fine tissue structures, mid-infrared (IR) lasers are diffraction limited to laser spot sizes greater than ~5 μm, necessitating creative approaches to enhance spatial detail for subcellular MSI. METHODS: An adjustable, ring-actuated iris combined with a reflective objective was integrated into the IR-MALDESI MSI platform, replacing the conventional beam expander and collimator assembly. Laser beam diameter on target was systematically varied, and ion abundances were measured using high-resolution mass spectrometry. Normalized ion abundances were fitted to a linear model as a function of beam diameter to predict signal at increasing spatial resolutions. RESULTS: Ion abundance decreased linearly with decreasing laser beam diameter across the tested range. Linear regression of normalized ion abundances enabled prediction of signal levels at progressively higher spatial resolutions. The tunable aperture provided stable beam control and reproducible ablation, allowing quantitative assessment of sensitivity trade-offs associated with reduced laser spot size. CONCLUSIONS: Controlled beam restriction using an adjustable iris enables fine tuning of spatial resolution in IR-MALDESI MSI by deliberately discarding a large fraction of high laser power while maintaining measurable ion abundance. Far-field diffraction and tissue ablation thresholds confine material removal beyond simple geometric scaling, and linear modeling of ion abundance versus beam diameter enables quantitative prediction of signal loss at high spatial resolution.