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.