Automatic Coregistration of High-Resolution MALDI-MSI and Raman Imaging Applied to Cardiac Tissue of Fabry Disease Mouse Models

Understanding early molecular changes in biological tissues is crucial for diagnosing pathological and genetic diseases and for elucidating their underlying mechanisms. However, localized molecular alterations of low-molecular-weight compounds (<2000 Da) are not inferred from conventional staining or genetic methods. Here, we established a multimodal imaging approach that integrates Raman spectroscopy and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-MALDI-MSI): two complementary, label-free techniques enabling molecular profiling of a broad spectrum of biomolecules from one single tissue section. This method was applied to detect globotriaosylceramide (Gb3) accumulation in the heart tissue of murine models of Fabry disease, including mice deficient in α-galactosidase A (GLA) activity (GLA knockout) and transgenic mice with a GLA knockout and an upregulation of Gb3 synthase. With AP-MALDI-MSI, we were able to discern the heterogeneous expression of Gb3 lipoforms with down to 5 μm pixel size and reveal the significantly increased Gb3 content in mice containing a GLA knockout combined with human Gb3 synthase overexpression compared to GLA knockout and wild-type samples. By employing Raman microscopy with a pixel size of 2 μm, we were able to contextualize the physiological alterations in cardiac tissue by identifying components associated with nuclei, tissue, collagen, and lipids for the same three genotypes. An automated coregistration algorithm aligned Raman and AP-MALDI-MSI data from the same tissue section with a (5.1 ± 1.6) μm precision, enabling overlay at 5 and 2 μm resolutions. The method resolved heterogeneous Gb3 distributions and distinct lipid species in cardiac mouse tissue.