Summary: The present paper describes matrix‐free laser desorption/ionisation mass spectrometric imaging (LDI‐MSI) of highly localized UV‐absorbing secondary metabolites in plant tissues at single‐cell resolution. The scope and limitations of the method are discussed with regard to plants of the genus Hypericum. Naphthodianthrones such as hypericin and pseudohypericin are traceable in dark glands on Hypericum leaves, placenta, stamens and styli; biflavonoids are also traceable in the pollen of this important phytomedical plant. The highest spatial resolution achieved, 10 μm, was much higher than that achieved by commonly used matrix‐assisted laser desorption/ionization (MALDI) imaging protocols. The data from imaging experiments were supported by independent LDI‐TOF/MS analysis of cryo‐sectioned, laser‐microdissected and freshly cut plant material. The results confirmed the suitability of combining laser microdissection (LMD) and LDI‐TOF/MS or LDI‐MSI to analyse localized plant secondary metabolites. Furthermore, Arabidopsis thaliana was analysed to demonstrate the feasibility of LDI‐MSI for other commonly occurring compounds such as flavonoids. The organ‐specific distribution of kaempferol, quercetin and isorhamnetin, and their glycosides, was imaged at the cellular level.

We have developed a method to visualize matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI IMS) data aligned with optically determinable tissue structures in three dimensions. Details of the methodology are exemplified using the 3-D reconstruction of myelin basic protein (MBP) in the corpus callosum of a mouse brain. In this procedure, optical images obtained from serial coronal sections are first aligned to each other to reconstruct a surface of the corpus callosum from segmented contours of the aligned images. The MALDI IMS data are then coregistered to the optical images and superimposed into the surface to create the final 3-D visualization. Correlating proteomic data with anatomical structures provides a more comprehensive understanding of healthy and pathological brain functions, and holds promise to be utilized in more complex anatomical arrangements.

Significance: The ongoing decline of banana yields caused by pathogens and the use of toxic chemicals to manage them has attracted considerable attention because of the importance of bananas as a major staple food for more than 400 million people. We demonstrate that secondary metabolites (phenylphenalenones) of Musa are the reason for differences in cultivar resistance, and detected the phenylphenalenone anigorufone in greater concentrations in lesions in roots of a nematode-resistant cultivar than in those of a susceptible one. An in vitro bioassay identified anigorufone as the most active nematostatic and nematocidal compound. We discovered that large lipid–anigorufone complex droplets are formed in the bodies of Radopholus similis exposed to anigorufone, resulting in the nematode being killed.