MALDI matrix sublimation versus spray coating in the FluoMALDI imaging pipeline

This study advances the FluoMALDI pipeline for tissue autofluorescence applications by integrating slide-scanning fluorescence microscopy (SSFM) with matrix-assisted laser desorption/ionization (MALDI) imaging to target autofluorescent tissue structures.

Mouse brain, liver, and kidney tissues were used to systematically compare the two matrix deposition methods of sublimation and spray coating in the FluoMALDI pipeline. Half of each tissue section was left uncoated as control by covering it during matrix application. Six MALDI matrices including 9-aminoacridine, norharmane, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxyacetophenone, 2,5-dihydroxybenzoic acid, or sinapinic acid were applied in equal amounts. Autofluorescence enhancements were evaluated using three fluorescence channels in the green, red, and far-red range of the spectrum, revealing matrix- and tissue-dependent fluorescence enhancement profiles. Norharmane matrix resulted in the most consistent tissue autofluorescence enhancements across deposition methods, with the highest enhancements in the green channel when applied by spray coating, and in the red and far-red channels when applied by sublimation coating. Matrix crystal size did not significantly impact the observed fluorescence enhancement effects for all six tested matrices. The two ionization modes of MALDI-1 and MALDI-2 imaging were also compared on brain, liver, and kidney tissue sections to assess lipid detection efficiency and spatial distribution in the FluoMALDI imaging pipeline. While the spatial distributions of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) species were consistent across MALDI ionization modes and matrix deposition methods, lipid detection efficiency varied depending on the matrix type, matrix crystal size, tissue type, and MALDI ionization mode. Sublimation, which produced fine crystals without solvents, generally resulted in higher phospholipid detection efficiency than spray coating for lipids including PE(36:2) and PC(36:2) in both MALDI-1 and MALDI-2 imaging.

These findings highlight that the matrix choice and matrix deposition method applied significantly affect tissue autofluorescence enhancement, spatial resolution, and lipid detection efficiency in MALDI-1 and MALDI-2 imaging modes, offering valuable insights for selecting optimal matrix deposition for FluoMALDI imaging based on a given study's research goals.