Enhanced MALDI-2 Sensitivity with Reflecting Post-Ionization Laser for High-Resolution MS Imaging Combined with Real-Time Microscope Imaging

Laser-induced matrix-assisted laser desorption/ionization post-ionization (MALDI-2) could improve the MALDI sensitivity of biological metabolites by over 1 order of magnitude. Herein, we demonstrate that MALDI-2 sensitivity can be further enhanced with reflecting post-ionization laser that multiplies the intersection times between laser and MALDI plume. This method, which we named MALDI-2+, typically brought over 2 times sensitivity improvement from conventional MALDI-2. Advancing in sensitivity thereby prompted us to pursue higher mass spectrometry imaging (MSI) spatial resolution. A dedicated T-shaped ion guide was designed to allow perpendicular incidence of ablation laser in reflection geometry MALDI. Although 8-10 μm pixel was used in MALDI imaging due to the limited precision of the motorized stage, the laser spot diameter could be down to 2.5 μm for potentially higher spatial resolution. In addition, this ion source enabled real-time and high-quality microscope imaging from backward of the sample plate. Beneficially, we were able to monitor the actual laser spot condition in real time as well as obtain high-resolution microscopic sample images that inherently register with MSI images. All of these benefits have been demonstrated by analyzing standard samples and imaging of cells. We believe that the enhancement in sensitivity, spatial resolution, and microscope capacity of our design could facilitate spatial omics studies.