Optimized Sodium-Enhanced MALDI-2 Enables Multimodal Imaging of Triacylglycerols.

Triacylglycerols (TAGs) are essential neutral lipids with critical roles in cellular function and disease. However, spatial characterization of these chemical species remains difficult because neutral molecules often exhibit low ionization efficiency with common mass spectrometry-based imaging strategies, such as matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Here, we assess how ammonium formate (AmF) or sodium carbonate buffer solution (CBS) tissue washes, combined with varying primary laser power, affect TAG sensitivity with and without MALDI-2 postionization. We found that Na+ doping using Na+ CBS washes combined with MALDI-2 at 31% total laser power was optimal for both signal intensity and molecular coverage. Additionally, Na+ CBS-washed tissues enabled MALDI-2 to be performed using lower laser powers, making this method compatible with sequential microscopy experiments as part of multimodal workflows. Applying this strategy to diseased brain tissue enabled spatial mapping of TAGs linked to Alzheimer's disease. This approach balances sensitivity and tissue preservation, providing a robust platform for multimodal spatial lipidomic studies across diverse tissues and disease contexts.