Quantitative ambient mass spectrometry imaging in plants: A perspective on challenges and future applications

Abstract

Mass spectrometry imaging (MSI) is a powerful approach to understanding plant chemistry in a native context because it retains key spatial information that is otherwise averaged out, permitting chemical compounds to be mapped to specific tissue structures. Identifying the spatial localization of compounds in plant tissues has provided insights into the synthesis and functional role of a wide range of endogenous molecules. The power and utility of MSI is being further expanded through the development of quantitative methodologies, which enable relative and absolute quantification of target analytes. Here, we briefly summarize applications of MSI in plant studies. We then turn our discussion to the challenges and developments in quantitative MSI, with a particular focus on ambient liquid extraction-based methods. Quantitative MSI is an emerging discipline in plant studies and holds great promise for revealing new information about the molecular composition of plant tissues and the pathways that regulate plant physiology.

Keywords: Mass spectrometry imaging; Plant metabolites; Quantitative analysis.

Figure 1:. Commonly used techniques for ambient liquid extraction-based mass spectrometry imaging.

The main spray-based technique is (a) DESI-MSI, whereas liquid bridge-based methods include (b) nanoDESI-MSI, (c) single probe MSI, and (d) LMJ-SSP MSI. (e) A schematic overview of the MS imaging process, where the desorption spot is moved in a raster scan across the tissue surface to produce a pixelated map. Every pixel contains a full MS scan, and MS images of each individual peak may be produced, showing the variation in intensity across the tissue.

Figure 2:. Overview of liquid desorption mass spectrometry imaging in plants.

(a) Plant parts typically imaged by MSI include fruits/nuts, flowers, leaves, stems, and roots. Tissues may be prepared for (b) direct MS measurement by cryosectioning or mounting of intact tissue to a slide, with or without sample pre-treatment. (c) Indirect MS measurements are typically made of tissue imprints, taken either by pressing or stamping. (d–g) Examples of direct and indirect imaging of plant tissue by DESI-MSI. (d) Direct imaging of maize root cryosections reveals varying distribution patterns of primary and specialized metabolites along the developmental gradient. Adapted from (T. Zhang et al., 2023) [11] and licensed under CC BY 4.0. (e) Direct surface imaging of very long chain fatty acids and flavonoids in untreated leaves and petals of Hypericum perforatum. Reprinted from (B. Li et al., 2013) [32] with permission from Elsevier. (f) Direct surface imaging of phytohormones in a wounded Arabidopsis leaf, after cuticle removal with petroleum ether. Adapted from (C. Zhang et al., 2021) [31] with permission from the Royal Society of Chemistry and licensed by CC BY-NC 3.0. (g) Indirect imaging of gingko leaf imprints on PTFE, where the sensitivity of DESI-MSI is compared to DESI-PPI-MSI. Reprinted (adapted) with permission from (L. Wu et al., 2022) [34]. Copyright 2022 American Chemical Society.

Figure 3:. Common strategies for application of an internal standard (IS).

(a) Homogenously coated vincamine (IS), a plant-derived medicinal compound, shows strong signal variation between two matrices (white and gray matter of the brain) by DESI-MSI. In these circumstances, internal standard normalization can help to account for matrix effects in a way that total ion current (TIC) normalization cannot. Adapted from (L. Gao et al., 2022) [23] with permission from Elsevier. Internal standard can be (b) doped into the perfusion or spray solvent, (c) applied as a thin film over the entire tissue, or (d) dropped onto sample area. Droplets of internal standard pre-mixed with standard may be used in the construction of a calibration curve.

Figure 4:. Common methods for preparation of matrix-matched calibration curves.

(a) An on-tissue dilution series spots standard solutions onto the surface of a tissue or homogenate. (b) A tissue mimetic calibration curve spikes standards into homogenized tissue, which is then either molded, cryosectioned, and mounted or aliquoted directly onto a slide. (c) Tissue extinction coefficients (TEC) require calculation of regional response factors. These are determined by coating the tissue and adjacent slide with the analyte of interest. The TEC for each region is determined from the ratio of the average intensity of the analyte within the ROI to the average on the slide. The TEC is used to normalize the signal from an uncoated sample before comparing to the calibration curve prepared without a tissue matrix. (d) A calibration curve is constructed for absolute quantification by plotting the intensity or a response factor against concentration. Although (a–c) shows four representative solutions, a calibration curve should be constructed with a blank and six standard solutions.