High-spatial resolution mass spectrometry imaging (MSI) is crucial for the mapping of chemical distributions at the cellular and subcellular level. In this work, we improved our previous laser optical system for matrix-assisted laser desorption ionization (MALDI)-MSI, from ~9 μm practical laser spot size to a practical laser spot size of ~4 μm, thereby allowing for 5 μm resolution imaging without oversampling. This is accomplished through a combination of spatial filtering, beam expansion, and reduction of the final focal length. Most importantly, the new laser optics system allows for simple modification of the spot size solely through the interchanging of the beam expander component. Using 10×, 5×, and no beam expander, we could routinely change between ~4, ~7, and ~45 μm laser spot size, in less than 5 min. We applied this multi-resolution MALDI-MSI system to a single maize root tissue section with three different spatial resolutions of 5, 10, and 50 μm and compared the differences in imaging quality and signal sensitivity. We also demonstrated the difference in depth of focus between the optical systems with 10× and 5× beam expanders. Graphical Abstract ᅟ.

Germination is a highly complex process by which seeds begin to develop and establish themselves as viable organisms. In this study, we utilize a combination of gas chromatography-mass spectrometry, liquid chromatography-fluorescence, and mass spectrometry imaging approaches to profile and visualize the metabolic distributions of germinating seeds from two different inbreds of maize (Zea mays) seeds, B73 and Mo17. Gas chromatography and liquid chromatography analyses demonstrate that the two inbreds are highly differentiated in their metabolite profiles throughout the course of germination, especially with regard to amino acids, sugar alcohols, and small organic acids. Crude dissection of the seed followed by gas chromatography-mass spectrometry analysis of polar metabolites also revealed that many compounds were highly sequestered among the various seed tissue types. To further localize compounds, matrix-assisted laser desorption/ionization mass spectrometry imaging was utilized to visualize compounds in fine detail in their native environments over the course of germination. Most notably, the fatty acyl chain-dependent differential localization of phospholipids and triacylglycerols was observed within the embryo and radicle, showing correlation with the heterogeneous distribution of fatty acids. Other interesting observations include unusual localization of ceramides on the endosperm/scutellum boundary and subcellular localization of ferulate in the aleurone.Plants use seeds as the propagule to ensure reproduction to the next generation, and over the past 10,000 years, human civilizations have established agricultural practices to ensure a seed-based food supply (Larson et al., 2014). Therefore, deciphering the processes that enable seeds to perform their biological functions is of importance in understanding how plants are propagated and also is of practical importance to improve agriculture. Seeds are designed to survive long periods of dormancy in a relatively dry state, and the process of germination is initiated by the imbibition of water. During this germination process, many metabolic changes occur, most of which are associated with the catabolism of seed storage products (proteins, polysaccharides, and lipids) into metabolically usable, simpler chemical forms that are either used as precursors to assemble the growing seedling or are oxidized via energy-producing biochemical pathways to thermodynamically support growth (Bewley, 1997(Bewley, , 2001.The specific metabolic processes that support seed germination are somewhat dependent on the taxonomic clade of the plant, which determines seed tissue/ organ organization and the nature of the seed storage compounds. Specifically, the seeds of the Poaceae family of monocots are characterized by a starch-and protein-filled endosperm, and their catabolism by digestive enzymes produced in the outermost layer of the endosperm (i.e. the aleurone) provides the carbonbased and nitrogenous precursors for the growth of the embryo, which is composed of the embryonic ax...