Photoregulated fluxional fluorophores for live-cell super-resolution microscopy with no apparent photobleaching

Abstract

Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.

Unraveling the Nanoscopic Organization and Function of Central Mammalian Presynapses With Super-Resolution Microscopy

“…By harnessing the resolving power of super-resolution optical microscopy, our understanding of synapse structure and function has taken big leaps in recent years. With the incessant quest for further refinements in terms of better hardware, increasingly powerful processing algorithms (e.g., Xu et al, 2017 ), or deep learning strategies ( Ouyang et al, 2018 ; Wang et al, 2019 ; Jin et al, 2020 ), fluorophores with improved or tailored photophysical properties (e.g., Minoshima and Kikuchi, 2017 ; Thiel and Rivera-Fuentes, 2018 ; Halabi et al, 2019 ; Kozma and Kele, 2019 ; Velde et al, 2019 ; Xu et al, 2020 ), and, quite likely, yet new and revolutionary technical approaches altogether, the limits of what can be resolved today are bound to keep shrinking. In parallel, the evolution of strategies for high-throughput SRM are allowing faster imaging of increasing numbers of simultaneous targets over larger sample areas ( Guo et al, 2019 ; Mahecic et al, 2019 ).…”

Recent Progress in Small Spirocyclic, Xanthene-Based Fluorescent Probes

“…The group of Rivera-Fuentes, tried to overcome the problem of photobleaching, a phenomenon that is observed after prolonged excitation, by photo-regulation of fluxional fluorophores [ 267 ]. Fluxional molecules can undergo rapid degenerate rearrangements in the electronic ground state and exhibit stochastic switching behavior [ 268 , 269 , 270 ].…”

“… Fluxional fluorophores for less photobleaching: ( a ) Mechanism of photoactivation and fluxionality of PFF-1 ; ( b ) Bright-field image of a neuronal projection and synapse and an image reconstructed from all frames collected during a 16 s acquisition within the green ticked area, as well as the diffusion coefficients of two vesicles (V1 and V2); ( c ) Bright-field image and fast diffusion of an acidic vesicle (V3) between hotspots (H1 and H2) in 20 s of a single neuronal projection. Adapted with permission of [ 267 ]. Scale bars = 2 µm ( b ) and 1 µm ( c ).…”

Between Life and Death: Reducing Phototoxicity in Super-Resolution Microscopy

“…How does one approach a problem as versatile as measur-ing phototoxicity? An intuitive and common way of assessing photodamage is by measuring photobleaching (40)(41)(42)(43). However, phototoxicity and photobleaching are two separate processes; while toxic ROS are produced during photobleaching, they can also be generated independently of this process (14,44).…”