A Photoactivatable Plasma Membrane Probe Based on a Self‐Triggered Photooxidation Cascade for Live Cell Super‐Resolution Microscopy

Overview & Synopsis

Super‐resolution imaging based on the localization of single emitters requires a spatio‐temporal control of the ON and OFF states. To this end, photoactivatable fluorophores are adapted as they can be turned on upon light irradiation. Here, we present a concept called self‐triggered photooxidation cascade (STPC) based on the photooxidation of a plasma membrane‐targeted leuco‐rhodamine (LRhod‐PM), a non‐fluorescent reduced form of a rhodamine probe. Upon visible light irradiation the small number of oxidized rhodamines, Rhod‐PM, acts as a photosensitizer to generate singlet oxygen capable of oxidizing the OFF state LRhod‐PM thereby switching it to its ON state. We showed that this phenomenon is kinetically favored by a high local concentration and propagates quickly when the probe is embedded in membrane bilayers. In addition, we showed that the close proximity of the dyes favors the photobleaching. At the single‐molecule level, the concomitant activation/bleaching phenomena allow reaching a single‐molecule blinking regime enabling single‐molecule localization microscopy for super‐resolution of live cellular membranes and their thin processes including filopodia and tuneling nanotubes.