Mass Spectrometry Imaging of Arabidopsis thaliana Leaves at the Single-Cell Level by Infrared Laser Ablation Atmospheric Pressure Photoionization (LAAPPI)

Abstract

In this study, we show that infrared laser ablation atmospheric pressure photoionization mass spectrometry (LAAPPI-MS) imaging with 70 μm lateral resolution allows for the analysis of Arabidopsis thaliana (A. thaliana) leaf substructures ranging from single-cell trichomes and the interveinal leaf lamina to primary, secondary, and tertiary veins. The method also showed its potential for depth profiling analysis for the first time by mapping analytes at the different depths of the leaf and spatially resolving the topmost trichomes and cuticular wax layer from the underlying tissues. Negative ion LAAPPI-MS detected many different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids, whose distributions varied significantly between the different substructures of A. thaliana leaves. The results show that LAAPPI-MS provides a highly promising new tool to study the role of metabolites in plants.

Deuterium- and Alkyne-Based Bioorthogonal Raman Probes for In Situ Quantitative Metabolic Imaging of Lipids within Plants

“…Performing in situ monitoring of individual metabolites has historically been challenging due to the complicated endogenous cellular milieu with high concentrations of complex compounds. ,, This is further complicated by the differences between plant and animal cells since most probes developed for animal cells cannot be immediately applied for plant-based studies. General imaging techniques such as mass spectrometry imaging have been used to overcome these limitations within metabolite research for biomedical applications due to their high sensitivity and multiplex capabilities. , Despite limitations in spatial resolution and tissue damage, they have shown tremendous promise for fast and direct imaging of intracellular molecules without the development of specific probes …”

“…11,13 Despite limitations in spatial resolution and tissue damage, they have shown tremendous promise for fast and direct imaging of intracellular molecules without the development of specific probes. 16 Conversely, bulky bioorthogonal probes, such as those with fluorescent tags or click chemistry reactivities, represent highly specific probes with strong potential for multiplex imaging applications. However, large tags often interfere with their endogenous metabolism and localization and potentially their cellular dynamics, hampering their applications in studying metabolic processes.…”

“…Mass spectrometry (MS) imaging techniques have also been developed and utilized for in situ metabolite analysis that can be performed under ambient conditions. − Notably, matrix-assisted laser/desorption ionization and desorption electrospray ionization are commonly employed for MS imaging due to their rapid analysis, high sensitivity, and lateral resolution. , Infrared laser ablation has also seen use in plants due to its ability for spot-to-spot sampling with matrix-free measurements that allow for ionization and desorption of analytes directly from the sample surface. , Due to the accuracy and speed of MS measurements, a range of compounds can be detected unambiguously and simultaneously within a large spatial range. , However, challenges including the limited resolution due to the laser spot size (average ∼20 μm), sample damage, and limited penetration beneath the epidermal layer have hampered widespread adoption of MS for intracellular metabolite tracking within plants. Despite these limitations, the use of MS imaging has been used to quantify a variety of metabolites within different plant tissues including sugars, lipids, and secondary metabolites, and considerable research efforts have been made to further develop general application of MS for single-cell resolution imaging. − …”

Advances in Plant Metabolomics and Its Applications in Stress and Single-Cell Biology

“…Infrared laser ablation atmospheric pressure photoionization MS (LAAPPI-MS) imaging was employed to conduct metabolite profiling at various layers of Arabidopsis leaf tissues, ranging from trichomes to veins. This demonstrated that the distributions of different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids varied significantly between the different substructures in the leaves [212].…”

Chemical Imaging Reveals Diverse Functions of Tricarboxylic Acid Metabolites in Root Growth and Development

“…1c, Supplementary Information Figs. 1-7) [5][6][7] . Between 20 to 30 small molecules were localized per each developmental zone (Supplementary Figs.…”