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

Single-molecule imaging promises the observation of individual molecules at work in living cells ^1,2 . In plants, however, the tracking of single molecules is generally limited to mere hundred milliseconds ^3–5 , making it virtually impossible to observe live dynamic cellular events with molecular resolution. Here, we introduce photochromic reversion which uses the reversion of EOS fluorescent protein’s dark state upon blue light illumination ⁶ , thereby stabilizing the fluorescent state of single molecules and extending single-molecule tracking in single particle tracking photoactivated localization microscopy (spt-PALM) experiments. Utilizing photochromic reversion, we tracked single molecules over micrometre distances for seconds. We captured transient spatial arrest events of plasma membrane proteins indicative of the observation of dynamic cellular events under physiological conditions. Finally, we implemented an analysis pipeline leveraging machine learning-based diffusional fingerprinting to automatically detect and quantify spatial arrestment, allowing precise kinetic measurements of molecular events at the nanoscale. We envision that photochromic reversion will constitute a pivotal instrument to decipher fundamental principles underlying membrane dynamics and function in plants.