rsEGFP2 enables fast RESOLFT nanoscopy of living cells

Grotjohann, Tim; Testa, Ilaria; Reuß, Matthias; Brakemann, T.; Eggeling, Christian; Hell, Stefan W.; Jakobs, Stefan

doi:10.7554/elife.00248

Cited by 209 publications

(409 citation statements)

References 37 publications

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“…In previous studies (12,14), both NL-SIM and RESOLFT used an ∼10 J/cm 2 dose of deactivation illumination to saturate the depletion of the bright state of either…”

Section: Skylan-nsmentioning

confidence: 99%

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

Zhang

Dong

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent "on" to "off" state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm 2 ) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view. Skylan-NSI n the last two decades, the power of fluorescence microscopy has been enhanced by the addition of superresolution (SR) imaging techniques (1-7). Although every SR technique has been successfully demonstrated to resolve ultrastructures beyond the diffraction limit, many of them encounter practical limitations when imaging nano-scale dynamics in living biological samples, especially over long times and large fields of view. For example, the thousands of raw frames typically acquired for a single-molecule localizationbased SR image greatly restrict the temporal resolution of the technique (1, 2, 8) and make it susceptible to blurring induced by cellular motion. On the other hand, structured illumination microscopy (SIM) is capable of live-cell time-lapse imaging for tens to hundreds of time points, at speeds as fast as 11 frames per second (9) and illumination intensities of only 1-100 W/cm 2 . However, its major shortcoming is that it improves resolution only twofold, to ∼100 nm. Stimulated emission depletion (STED) microscopy (4) and saturated SIM (SSIM) (7, 10, 11) are not subject to this constraint but rather provide diffraction unlimited resolution by exploiting a nonlinear dependence of the fluorescence emission rate upon an illumination intensity. Saturation of the excited electronic state S 1 to ground state S 0 (S 1 →S 0 ) via stimulated emission is used in STED,...

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“…In previous studies (12,14), both NL-SIM and RESOLFT used an ∼10 J/cm 2 dose of deactivation illumination to saturate the depletion of the bright state of either…”

Section: Skylan-nsmentioning

confidence: 99%

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

Zhang

Dong

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…2, with I sat referring to the intensity required to efficiently photoswitch the fluorophore to a dark state. RESOLFT based on photoswitching fluorescent proteins has been limited for many years by the slow photokinetics of most fluorescent proteins, but recently developed fluorescent probes and experimental designs allow faster RESOLFT at low intensities (1 W cm −2 ) [23,24].…”

Section: Resolft Based On Photoswitching Fluorescent Proteinsmentioning

confidence: 99%

“…Progress in this area will go hand in hand with the development of photoswitchable proteins with faster photokinetics [23,24].…”

Section: Temporal Resolution and Live-cell Sted/resolft Imagingmentioning

confidence: 99%

“…STED imaging can be performed on fusion constructs of fluorescent proteins that also span most of the visible spectrum: green (GFP [112]), yellow (YFP [113] and citrine [114]) and far-red emitting proteins (TagRFP657 [115] and the tetrametric E2-Crimson [116]). Several reversible photoswitchable proteins have also been demonstrated suitable for RESOLFT imaging, exploiting their reversible switching behaviour to deplete the periphery of the focal spot (Dronpa and its counterpart Padron derivative [15,22], rsEGFP2 [23] and Dreiklang [117]). …”

Section: Fluorescent Probes For Sted Imagingmentioning

confidence: 99%

“…Fluorescence nanoscopy makes possible the investigation of the nanoscale dynamic organisation of proteins within subsynaptic domains: The fate of synaptic vesicles after exocytosis and fusion with the membrane has been studied by STED microscopy in fixed [199,200] and live neurons using video-rate (20)(21)(22)(23)(24)(25)(26)(27)(28) [27,201] and time-lapse STED microscopy [202,203]. AMPA receptors trafficking in neuronal cells have been investigated by single particle tracking PALM [204].…”

Section: Neurobiologymentioning

confidence: 99%

See 2 more Smart Citations

Fluorescence nanoscopy. Methods and applications

Requejo-Isidro

2013

J Chem Biol

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Fluorescence nanoscopy refers to the experimental techniques and analytical methods used for fluorescence imaging at a resolution higher than conventional, diffractionlimited, microscopy. This review explains the concepts behind fluorescence nanoscopy and focuses on the latest and promising developments in acquisition techniques, labelling strategies to obtain highly detailed super-resolved images and in the quantitative methods to extract meaningful information from them.

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Super‐resolved insights into human immunodeficiency virus biology

et al. 2016

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Edited by Wilhelm JustThe recent development of fluorescence microscopy approaches overcoming the diffraction limit of light microscopy opened possibilities for studying small-scale cellular processes. The spatial resolution achieved by these novel techniques, together with the possibility to perform live-cell and multicolor imaging, make them ideally suited for visualization of native viruses and subviral structures within the complex environment of a host cell or organ, thus providing fundamentally new possibilities for investigating virus-cell interactions. Here, we review the use of super-resolution microscopy approaches to study virus-cell interactions, and discuss recent insights into human immunodeficiency virus biology obtained by exploiting these novel techniques.

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rsEGFP2 enables fast RESOLFT nanoscopy of living cells

Cited by 209 publications

References 37 publications

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

Fluorescence nanoscopy. Methods and applications

Super‐resolved insights into human immunodeficiency virus biology

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