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.