Publication date: 25th July 2016
Lens-based microscopy was unable to discern fluorescently labeled features closer than 200 nm for decades, until the recent breaking of the diffraction resolution barrier by sequentially switching the fluorescence capability of adjacent features on and off quickly made nanoscale imaging routine. Reported nanoscopy variants switch these features either in a target manner with intense laser beams, or molecule by molecule followed by computation in a stochastic fashion. Here, we show that emergent RESOLFT fluorescence nanoscopy enables fast and continuous imaging of living cells and tissues in super resolved detail by producing raw data images using only ultralow levels of light. This advance has been facilitated by the generation of fluorescent proteins (rsFP) that can be reversibly photoswitched numerous times. Distributions of functional rsFP-fusion proteins in living bacteria and mammalian cells are imaged at < 40 nm resolution. Using a fast-switching rsFP variant, we increased the imaging speed 50-fold over our first reported RESOLFT schemes, which in turn enabled us to record spontaneous and stimulated changes of dendritic actin filaments and spine morphology occurring on time scales from seconds to hours. Furthermore, lifetime based experiments enable precise localization of synapses by simultaneous recording of proteins located in the pre and post synaptic side. Our powerful RESOLFT technique represents a new paradigm for non invasive induction and monitoring of ultrastructural dynamics of synaptic plasticity at the nanoscale.
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