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

• Published in: Nature communications 2019-03, Vol.10 (1), p.1232-1232, Article 1232
• Main Authors: Halabi, Elias A., Pinotsi, Dorothea, Rivera-Fuentes, Pablo
• Format: Article
• Language: English
• Subjects: 13/106; 13/109; 14/34; 14/63; 631/80/2373/2238; 639/638/439/945; 639/638/92/56; 639/638/92/96; Chemical compounds; Fluorescence; Fluorescent Dyes - chemistry; Fluorescent Dyes - radiation effects; Fluorophores; HeLa Cells; Humanities and Social Sciences; Humans; Intravital Microscopy - methods; Isomerism; Light; Localization; Microscopy; Microscopy, Fluorescence - methods; Molecular Probes - chemistry; Molecular Probes - radiation effects; multidisciplinary; Neurons - chemistry; Neurons - cytology; Neurons - metabolism; Organelles; Photobleaching; Phototoxicity; Science; Science (multidisciplinary); Single Molecule Imaging - methods; Spectrometry, Fluorescence; Stochasticity; Switching; Synaptic Vesicles - chemistry; Synaptic Vesicles - metabolism; Time-Lapse Imaging - methods
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-019-09217-7

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. Super-resolution microscopy with spontaneously blinking dyes is dependent on pH and polarity of the medium. Here the authors introduce a photoactivatable fluxional fluorophore for live cell imaging that allows control over the fraction of spontaneously blinking molecules independently of medium properties.