Automatic colocalization 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 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 Gb3 accumulation in heart tissue of murine models of Fabry disease, including mice deficient in α-galactosidase A (GLA) activity (GLA knock-out) and transgenic mice with a GLA knock-out and an upregulation of globotriaosylceramides (Gb3) synthase. With AP-MALDI MSI we were able to discern the heterogenous expression of Gb3 lipoforms with down to 5 µm pixel size and reveal the significantly increased Gb3 content in mice containing a GLA knock-out combined with human Gb3 synthase overexpression compared to GLA knock-out 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 co-localization algorithm aligned Raman and AP-MALDI-MSI data from the same tissue section with (5.1 ± 1.6) µm precision, enabling overlay at 5 µm and 2 µm resolution. The method resolved heterogeneous Gb3 distributions and distinct lipid species in cardiac mouse tissue.