SequentialMALDI and Nano-DESI MS Imaging of Lipidsand Proteins on Stretched Tissue Samples

Abstract Imaging of both lipids and proteoforms in the same tissue section is a powerful approach to obtaining comprehensive molecular maps of biological systems. However, achieving high spatial resolution for both molecular classes remains challenging. Here, we combine tissue expansion with sequential imaging of lipids via matrix-assisted laser desorption/ionization (MALDI) and intact proteins via nanospray desorption electrospray ionization (nano-DESI) on the same tissue section to achieve comprehensive high-resolution molecular maps. Physical stretching of the tissue on Parafilm “M” produced a 4.5-fold effective resolution enhancement in both the X and Y dimensions (a ∼20-fold increase in area) in the mouse cerebellum for both MALDI and nano-DESI measurements. We further leveraged MALDI data to sharpen nano-DESI images using a partial least-squares (PLS) regression framework with a data consistency constraint. Finally, we applied midlevel fusion of MALDI and nano-DESI data to identify lipids and proteins localized to the same anatomical structures. Together, this straightforward workflow enables multimodal imaging of lipids and proteins from the same tissue section with improved spatial resolution and integrated analysis.