Photochromic reversion enables long-term tracking of single molecules in living plants.

Single-molecule imaging enables the observation of individual molecules in living cells (DEste et al., 2024; Kusumi et al., 2014; Lelek et al., 2021; Nguyen et al., 2023). In plants, however, the tracking of single molecules is typically limited to a few hundred milliseconds (Bayle et al., 2021; Gronnier et al., 2017; Hosy et al., 2015), precluding the observation of dynamic cellular processes at molecular resolution. Here, we describe photochromic reversion, an imaging modality that enables long-term single-molecule tracking of genetically encoded translational fusions. Using this approach, we achieve minute-long tracking of individual cell-surface receptors and reveal previously inaccessible dynamic spatial arrest events of single plasma membrane proteins. We further developed and benchmarked computational analysis of spatial arrests (CASTA), a machine learning-based tool that automatically detects and analyses spatial, temporal, and diffusional properties of these events, thereby enabling precise nanoscale kinetic measurements. Together, these advances provide a powerful framework for deciphering the principles governing membrane dynamics and function.