A significant limiting factor in achieving high spatial resolution for matrix-assisted laser desorption ionizationmass spectrometry (MALDI-MS) imaging is the size of the laser spot at the sample surface. Here, we present modifications to the beam-delivery optics of a commercial MALDI-linear ion trap-Orbitrap instrument, incorporating an external Nd:YAG laser, beam-shaping optics, and an aspheric focusing lens, to reduce the minimum laser spot size from ~50 μm for the commercial configuration down to ~9 μm for the modified configuration. This improved system was applied for MALDI-MS imaging of cross sections of juvenile maize leaves at 5-μm spatial resolution using an oversampling method. A variety of different metabolites including amino acids, glycerolipids, and defense-related compounds were imaged at a spatial resolution well below the size of a single cell. Such images provide unprecedented insights into the metabolism associated with the different tissue types of the maize leaf, which is known to asymmetrically distribute the reactions of C4 photosynthesis among the mesophyll and bundle sheath cell types. The metabolite ion images correlate with the optical images that reveal the structures of the different tissues, and previously known and newly revealed asymmetric metabolic features are observed. KeywordsMass spectrometry imaging, Metabolite, Maize ABSTRACTA significant limiting factor in achieving high spatial resolution for MALDI-MS imaging is the size of the laser spot at the sample surface. Here we present modifications to the beamdelivery optics of a commercial MALDI-linear ion trap-Orbitrap instrument, incorporating an external Nd:YAG laser, beam-shaping optics, and an aspheric focusing lens, to reduce the minimum laser spot size from ~50 µm for the commercial configuration down to ~5 µm for the modified configuration. At the laser energy needed for efficient desorption and ionization of endogenous metabolites from tissue, the analytically practical spot size was slightly larger, at ~9µm. This improved system was applied for MALDI-MS imaging of cross-sections of juvenile maize leaves at 5 µm spatial resolution using an oversampling method. A variety of different metabolites including amino acids, glycerolipids, and defense-related compounds were imaged at a spatial resolution well below the size of a single cell. Such images provide unprecedented insights into the metabolism associated with the different tissue types of the maize leaf, which is known to asymmetrically distribute the reactions of C4 photosynthesis among the mesophyll and bundle sheath cell types. The metabolite ion images correlate with the optical images that reveal the structures of the different tissues, and previously known and newly revealed asymmetric metabolic features are observed.

Advances in mass spectrometry (MS) have made comprehensive lipidomics analysis of complex tissues relatively commonplace. These compositional analyses, although able to resolve hundreds of molecular species of lipids in single extracts, lose the original cellular context from which these lipids are derived. Recently, high-resolution MS of individual lipid droplets from seed tissues indicated organelle-to-organelle variation in lipid composition, suggesting that heterogeneity of lipid distributions at the cellular level may be prevalent. Here, we employed matrix-assisted laser desorption/ ionization-MS imaging (MALDI-MSI) approaches to visualize lipid species directly in seed tissues of upland cotton (Gossypium hirsutum). MS imaging of cryosections of mature cotton embryos revealed a distinct, heterogeneous distribution of molecular species of triacylglycerols and phosphatidylcholines, the major storage and membrane lipid classes in cotton embryos. Other lipids were imaged, including phosphatidylethanolamines, phosphatidic acids, sterols, and gossypol, indicating the broad range of metabolites and applications for this chemical visualization approach. We conclude that comprehensive lipidomics images generated by MALDI-MSI report accurate, relative amounts of lipid species in plant tissues and reveal previously unseen differences in spatial distributions providing for a new level of understanding in cellular biochemistry.