Topographic imaging with automatic z-axis correction of Brassica oleracea var. viridis leaves by IR-MALDESI mass spectrometry imaging.

Mass spectrometry (MS) is a versatile technique for elucidating the chemical composition of biological samples. Beyond analysis of crude extracts, MS can be further applied to spatially resolve compounds across the area of a sample with a technique called mass spectrometry imaging (MSI). The infrared matrix-assisted laser desorption ionization (IR-MALDESI) platform combines elements of matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) to enable MSI of mammalian tissue using endogenous water in the sample as a matrix. For laser-based techniques such as IR-MALDESI, changes in topography across the sample surface cause inconsistent ablation as the sample surface moves above and below the focal plane of the laser. The localization of chemical species in plants reveals crucial information about metabolic processes as reported by Nemes and Vertes (Anal. Chem. 79 (21), 8098-8106, 2007) and biosynthetic pathways by Zou et al. (Trends in Plant Science, 2024) and can even inform selective breeding of crops as discussed by Sakurai (Breed Sci 72 (1), 56-65, 2022); however, leaf topography raises a unique challenge. Features such as veins and trichomes exhibit unique topography, but flattening risks delocalization of analytes and activation of unwanted signaling pathways, and transferring metabolites to a membrane for indirect analysis may incur delocalization and limit metabolomic coverage. To overcome these challenges, a chromatic confocal sensor probe (CA probe) was incorporated for IR-MALDESI-MSI of sections of a collard (Brassica oleracea var. viridis) leaf. The CA probe measures the height at all points of the sample, and automatic z-axis corrections (AzC) are generated from height differences to continuously raise and lower the stage. These stage height corrections keep the sample surface in focus of the laser for the duration of analysis. This method has been applied to relatively homogenous samples, but has not yet been characterized on heterogeneous leaf tissue with considerable topography. Herein, data quality is compared between MSI analyses with and without AzC applied, focusing on the localization of analytes known to be concentrated in different layers of collard leaves.