In-source freezing of Trifolium repens leaf tissue to increase spatial resolution and quench stress responses during IR-MALDESI mass spectrometry imaging

Mass spectrometry imaging (MSI) is a robust analytical tool for resolving the spatial localization of diverse metabolites in a tissue sample simultaneously. Infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) is particularly advantageous for analyzing leaf tissue, since it bypasses the cuticle and uses endogenous water in the sample as a matrix, avoiding delocalization and reducing costs associated with sample preparation. While direct analysis of fresh tissue offers substantial benefits, it also presents a less-discussed challenge: plants initiate a cascade of metabolic responses to various forms of stress (e.g., mechanical wounding, heat stress), so freshly-harvested tissue can undergo major metabolic changes before and during analysis. Herein, a method is introduced which retains the benefits of IR-MALDESI MSI but increases spatial resolution and quenches metabolic processes by quickly freezing leaf tissue within the source enclosure immediately after harvest. Leaf samples were mounted on a glass slide with carboxymethylcellulose gel immediately after harvest and moved into the source enclosure, humidity was purged, and the sample was frozen using the Peltier-cooled stage. Camera images demonstrated minimal ice deposition on the sample surface, and evaluation of the phenylpropanoid pathway suggested that freezing the sample inhibited post-harvest stress responses during and prior to analysis. Frozen tissue analysis significantly decreased ablation spot diameter from 65.4 to 43.9 µm, enabling approximately a 1.5-fold increase in spatial resolution with a conventional 2.97-µm IR laser.