Evaluating the optimal tissue thickness for mass spectrometry imaging using infrared matrix-assisted laser desorption electrospray ionization.

Rationale: Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) utilizes a 2970 nm mid-IR laser to desorb samples with depth resolutions (Z) on the order of micrometers. Conventionally, 5-20 μm thick tissue sections are used to characterize different applications of the IR-MALDESI source, but an optimal thickness has not been systematically investigated.

Methods: Mouse liver was sectioned to various thicknesses and analyzed using IR-MALDESI mass spectrometry imaging (MSI). Height profiles of tissue sections of various cryosectioned thicknesses were acquired to affirm tissue thickness. Tissue sections of each thickness were measured using a Keyence microscope. Paraffin wax was cryosectioned, mounted on microscope slides, and measured using a chromatic confocal sensor system to determine the cryostat sectioning accuracy.

Results: Analyzing sectioned tissues at higher thickness (>10 μm) leads to lower ion abundance, a decrease in signal over long analysis times, and more frequent instrument cleaning. Additionally, increasing tissue thickness above the optimum (7 μm) does not result in a significant increase in lipid annotations.

Conclusions: This work defines an optimal sample thickness for IR-MALDESI-MSI and demonstrates the utility of optimizing tissue thickness for MSI platforms of comparable Z resolution.