Lattice light-sheet microscopy allows for super-resolution imaging of receptors in leaf tissue.

Abstract

Plant leaf tissues are difficult to image via fluorescence microscopy due to the presence of chlorophyll and other pigments, which provide large background fluorescence. Lattice light-sheet microscopy offers the advantage of using Bessel beams to illuminate a thin focal region of interest for microscopy, allowing for the excitation of fluorescent molecules within this region without surrounding chlorophyll-like objects outside of the region of interest. Here, we apply STORM super-resolution techniques to observe receptor-like kinases in Arabidopsis thaliana leaf cells. By applying this technique with lattice light-sheet microscopy, we can localize immune-response proteins at sub-100-nm length scales and reconstruct three-dimensional locations of proteins within individual leaf cells. Using this technique, we observed the effect of the ATP and flg22 elicitors, where we observed a significant degree of internalization of cognate receptors P2K1 and FLS2. We were also able to similarly observe differences in colocalization due to stimulation with these elicitors, whereby we observe proteins on the membrane becoming less colocalized as a result of stimulation, suggesting an immune-response mechanism involving receptor internalization via distinct pathways. These data show lattice light-sheet microscopy's capabilities for imaging tissue with problematic background fluorescence that otherwise makes super-resolution fluorescence microscopy difficult.