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The commonly used analytical tools for metabolomics cannot directly probe metabolic activities or distinguish metabolite differences between cells and suborgans in multicellular organisms. These issues can be addressed byin-vivoisotope labeling and mass spectrometry imaging (MSI), respectively, but the combination of the two, a newly emerging technology we call MSIi, has been rarely applied to plant systems. In this study, we explored MSIiofArabidopsis thalianawith D₂O labeling to study and visualize D-labeling in three classes of lipids: arabidopsides, chloroplast lipids, and epicuticular wax. Similar to other stress responses, D₂O-induced stress increased arabidopsides in an hour, but it was relatively minor for matured plants and reverted to the normal level in a few hours. The D-labeling isotopologue patterns of arabidopsides matched with those of galactolipid precursors, supporting the currently accepted biosynthesis mechanism. Matrix-assisted laser desorption/ionization (MALDI)-MSI was used to visualize the spatiotemporal distribution of deuterated chloroplast lipids, pheophytina, MGDGs, and DGDGs, after growing day-after-sowing (DAS) 28 plants in D₂O condition for 3–12 days. There was a gradual change of deuteration amount along the leaf tissues and with a longer labeling time, which was attributed to slow respiration leading to low D₂O concentration in the tissues. Finally, deuterium incorporation in epicuticular wax was visualized on the surfaces of the stem and flower. The conversion efficiency of newly synthesized C30 aldehyde to C29 ketone was very low in the lower stem but very high at the top of the stem near the flower or on the flower carpel. This study successfully demonstrated that MSIican unveil spatiotemporal metabolic activities in various tissues ofA. thaliana. 

Applying solutions of matrix or derivatization agent via microdroplets is a common sample preparation technique for matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) experiments. Mobilized nebulizer sprayers are commonly used to create a homogeneous matrix or reagent layer across large surfaces. Electrospray devices have also been used to produce microdroplets for the same purpose but are rarely used for large tissues due to their immobility. Herein, we present a movable electrospray device that can be used for large tissue sample preparation by a simple modification to an automatic commercial nebulizer device. As demonstrated for on-tissue chemical derivatization (OTCD) with Girard's reagent T using a mimetic tissue model, the sprayer has the additional benefit of being able to investigate reaction acceleration in OTCD when comparing electrostatically charged spray to electrostatically neutral spray. Finally, MALDI-MSI of fatty alde-hydes is successfully demonstrated in rat brain tissues using this device for both OTCD and matrix application. 

Abstract Mass spectrometry imaging (MSI) of volatile metabolites is challenging, especially in matrix‐assisted laser desorption/ionization (MALDI). Most MALDI ion sources operate in vacuum, which leads to the vaporization of volatile metabolites during analysis. In addition, tissue samples are often dried during sample preparation, leading to the loss of volatile metabolites even for other MSI techniques. On‐tissue chemical derivatization can dramatically reduce the volatility of analytes. Herein, a derivatization method is proposed utilizing N,N,N‐trimethyl‐2‐(piperazin‐1‐yl)ethan‐1‐aminium iodide to chemically modify short‐chain fatty acids in chicken cecum, ileum, and jejunum tissue sections before sample preparation for MSI visualization. 

Abstract Derivatization reactions are commonly used in mass spectrometry to improve analyte signals, specifically by enhancing the ionization efficiency of those compounds. Vicinal diols are one group of biologically important compounds that have been commonly derivatized using boronic acid. In this study, a boronic acid with a tertiary amine was adapted for the derivatization of vicinal diol metabolites in B73 maize tissue cross‐sections for mass spectrometry imaging analysis. Using this method, dozens of vicinal diol metabolites were derivatized, effectively improving the signal of those metabolites. Many of these metabolites were tentatively assigned using high‐resolution accurate mass measurements. In addition, reaction interference and cross‐reactivity with various other functional groups were systematically studied to verify data interpretation.