Innovative Application of a Multifunctional Sucrose–Gelatin Hydrogel Matrix in Desorption Electrospray Ionization-Mass Spectrometry Imaging

Desorption electrospray ionization-mass spectrometry imaging (DESI-MSI) is among the most powerful techniques for visualizing the spatial distribution of small organic molecules, particularly lipids, on tissue surfaces. Conventional DESI-MSI analysis typically involves sectioning fresh-frozen tissues or, less commonly, embedding samples in matrices specifically formulated to preserve the tissue integrity for multifunctional analyses. In this study, we present an optimized sucrose-gelatin hydrogel matrix compatible with DESI-MSI, using mouse brain tissue as a model system. The method involves low-temperature embedding of frozen specimens into the hydrogel matrix, followed by snap-freezing at −160 °C. This matrix formulation ensures minimal background interference and prevents metabolite delocalization, thereby preserving the native molecular composition of the tissue. Notably, sucrose-derived adduct ions restricted to the embedding medium serve as stable internal reference signals in both positive and negative ionization modes. These signals enable continuous lock-mass correction throughout acquisition, offering a new solution to the unresolved challenge for accurate mass-based measurements in DESI-MSI without an infusion of exogenous calibration standards. Complementary DESI-MS/MS analyses further facilitate confident lipid identification and resolve structural ambiguities. Moreover, the sucrose–gelatin embedding medium provides excellent preservation of tissue morphology and antigenicity, supporting subsequent histological and immunohistochemical analyses. Overall, this sucrose-based hydrogel embedding protocol offers a robust, reproducible, and multimodal platform for molecular tissue imaging by DESI-MSI, especially in delicate biological specimens with broad translational potential across preclinical and clinical research domains.