Probing the Relationship Between Detected Ion Intensity, Laser Fluence, and Beam Profile in Thin Film and Tissue in MALDI MSI

Rory T. Steven, Alan M. Race, Josephine Bunch

Matrix assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) is increasingly widely used to provide information regarding molecular location within tissue samples. The nature of the photon distribution within the irradiated region, the laser beam profile, and fluence, will significantly affect the form and abundance of the detected ions. Previous studies into these phenomena have focused on circular-core optic fibers or Gaussian beam profiles irradiating dried droplet preparations, where peptides were employed as the analyte of interest. Within this work, we use both round and novel square core optic fibers of 100 and 50 μm diameter to deliver the laser photons to the sample. The laser beam profiles were recorded and analyzed to quantify aspects of the photon distributions and their relation to the spectral data obtained with each optic fiber. Beam profiles with a relatively small number of large beam profile features were found to give rise to the lowest threshold fluence. The detected ion intensity versus fluence relationship was investigated, for the first time, in both thin films of α-cyano-4-hydroxycinnamic acid (CHCA) with phosphatidylcholine (PC) 34:1 lipid standard and in CHCA coated murine tissue sections for both the square and round optic fibers in continuous raster imaging mode. The fluence threshold of ion detection was found to occur at between ~14 and ~64 J/m(2) higher in tissue compared with thin film for the same lipid, depending upon the optic fiber employed. The image quality is also observed to depend upon the fluence employed during image acquisition. Graphical Abstract ᅟ.