Precision Biomarker Discovery Powered by Microscopy Image Fusion-assisted High Spatial Resolution Ambient Ionization Mass Spectrometry Imaging

Mass spectrometry imaging (MSI) using the ambient ionization technique enables a direct chemical investigation of biological samples with minimal sample pretreatment. However, detailed morphological information of the sample is often lost due to its limited spatial resolution. In this study, predictive high-resolution molecular imaging was produced by the fusion of ambient ionization MSI with optical microscopy of routine hematoxylin and eosin (H&E) staining. Specifically, desorption electrospray ionization (DESI) and nanospray desorption electrospray ionization (nanoDESI) mass spectrometry were employed to visualize lipid and protein species on mice tissue sections. The resulting molecular distributions obtained by ambient ionization MSI-microscopy fusion were verified with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MSI and immunohistochemistry (IHC) staining. Label-free molecular imaging with 5-μm spatial resolution can be acquired using DESI and nanoDESI, whereas the typical spatial resolution of ambient ionization MSI was ∼100 μm. In this regard, sharpened molecular histology of tissue sections was achieved, providing complementary references to the pathology. Such a multi-modal integration enables the discovery of potential tumor biomarkers. After image fusion, more than a dozen potential biomarkers on a metastatic mouse lung tissue section and Luminal B breast tumor tissue section were identified.