Developing transmission mode for infrared matrix‐assisted laser desorption electrospray ionization mass spectrometry imaging

Rationale The development and characterization of the novel NextGen infrared matrix‐assisted laser desorption electrospray ionization (IR‐MALDESI) source catalyzed new advancements in IR‐MALDESI instrumentation, including the development of a new analysis geometry. Methods A vertically oriented transmission mode (tm)‐IR‐MALDESI setup was developed and optimized on thawed mouse tissue. In addition, glycerol was introduced as an alternative energy‐absorbing matrix for tm‐IR‐MALDESI because the new geometry does not currently allow for the formation of an ice matrix. The tm geom was evaluated against the optimized standard geometry for the NextGen source in reflection mode (rm). Results It was found that tm‐IR‐MALDESI produces comparable results to rm‐IR‐MALDESI after optimization. The attempt to incorporate glycerol as an alternative matrix provided little improvement to tm‐IR‐MALDESI ion abundances. Conclusions This work has successfully demonstrated the adaptation of the NextGen IR‐MALDESI source through the feasibility of tm‐IR‐MALDESI mass spectrometry imaging on mammalian tissue, expanding future biological applications of the method.