Examination of Lipid Distributions in Hydrogel-Expanded Mouse Brain Tissue Using Imaging Mass Spectrometry

Imaging is an essential tool in biological research, and imaging mass spectrometry uniquely provides a label-free approach with high molecular specificity. However, imaging mass spectrometry is limited in spatial resolution, which in turn limits the biological structures and processes that can be studied at small dimensions. Custom lens setups and altered optical paths have been used to shrink the diameter of the incident laser beam probe in matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry to achieve high spatial resolutions (< 5 μm). However, these research-grade instruments are complex and expensive, making high spatial resolution imaging experiments unrealistic for the broader community. An alternative method for improving spatial resolution is through physical magnification of the substrate, which has been well established in the subfield of expansion microscopy (ExM). ExM leverages superabsorbent hydrogels for isotropic expansion of tissues and retention of analytes containing fluorescent tags. While typical ExM involves covalently anchoring the analyte of interest to the hydrogel network, lipid retention without anchoring has been recently demonstrated for imaging mass spectrometry. Herein, we demonstrate expansion imaging mass spectrometry (ExIMS) of expanded, whole brain tissue and examine lipid distributions in both positive and negative ion mode across multiple brain structures. A linear expansion factor of 4.5-fold is achieved and used to obtain high spatial resolution images of mouse brain cerebellum. Approximately 95% of lipids in both positive and negative ion mode are retained in expanded tissue compared to unexpanded tissue. Additionally, the majority of lipid distributions across the brain are maintained post-expansion. Alterations to the hydrogel formulation ( e.g. , crosslinker density) can significantly affect the ability of ExIMS to maintain accurate lipid distributions in expanded tissue.