Three-Dimensional (3D) Topographic Mass Spectrometry Imaging of <i>Artemisia annua</i> by UV-MALDESI

Matrix-assisted laser desorption electrospray ionization (MALDESI) enables mass spectrometry imaging (MSI) capabilities to reveal the localization of a wide range of biomolecules across an organism. Three-dimensional (3D) MSI of biological tissues is typically accomplished by imaging two-dimensional sections followed by the creation of a 3D image informatically. In contrast to this sectioning-based approach, we employ an ablation-based 3D MSI technique to image <i>Artemisia annua</i>, a medicinal herb that naturally produces the antimalarial drug artemisinin. We incorporated a novel high-energy burst-mode ultraviolet (UV) laser, and a chromatic confocal aberration (CA) probe with automatic <i>z</i>-axis correction (A<i>z</i>C) to measure the depth of ablation (i.e., <i>z</i>-resolution). The combination of these techniques allowed the visualization of the artemisinin metabolic pathway along with other secondary metabolites beneath the tissue surface, as supported by the resulting data. This approach enabled detailed molecular mapping in 3D, providing a comprehensive view of the plant's molecular landscape layer by layer, offering new insights into its biosynthetic pathways in three dimensions.