Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy

Andresen, Martin A.; Stiel, André C.; Fölling, Jonas; Wenzel, Dirk; Schönle, Andreas; Egner, Alexander; Eggeling, Christian; Hell, Stefan W.; Jakobs, Stefan

doi:10.1038/nbt.1493

Cited by 280 publications

(320 citation statements)

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“…Water A267 is in turn hydrogen bonded to the carboxylate of Glu144 at a distance of 3.06 Å 22 . This may suggest that a situation exists in Dronpa, which is similar to GFP 23 , which stabilizes the protonated form of the phenolic oxygen via electrostatic repulsion from the carboxylate group.…”

Section: Resultsmentioning

confidence: 99%

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

et al. 2013

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The reversible photoswitching between the 'on' and 'off' states of the fluorescent protein Dronpa involves photoisomerization as well as protein side-chain rearrangements, but the process of interconversion remains poorly characterized. Here we use time-resolved infrared measurements to monitor the sequence of these structural changes, but also of proton transfer events, which are crucial to the development of spectroscopic contrast. Light-induced deprotonation of the chromophore phenolic oxygen in the off state is a thermal ground-state process, which follows ultrafast (9 ps) trans-cis photoisomerization, and so does not involve excited-state proton transfer. Steady-state infrared difference measurements exclude protonation of the imidazolinone nitrogen in both the on and off states. Pump-probe infrared measurements of the on state reveal a weakening of the hydrogen bonding between Arg66 and the chromophore C ¼ O, which could be central to initiating structural rearrangement of Arg66 and His193 coinciding with the low quantum yield cis-trans photoisomerization

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Section: Resultsmentioning

confidence: 99%

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

et al. 2013

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“…Dreiklang may also provide new avenues to multicolor applications in combination with other RSFPs or conventional fluorescent proteins. In this case, contrast may be obtained either by switching 19 , or by different colors or by dissimilar fluorescence lifetimes. In addition to applications in life sciences, the decoupling of switching from fluorescence excitation should also offer new options in subdiffraction reading and writing in protein layers.…”

Section: Discussionmentioning

confidence: 99%

“…However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued.…”

Section: A R T I C L E Smentioning

confidence: 99%

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

et al. 2011

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VOLUME 29 NUMBER 10 OCTOBER 2011 nature biotechnology A r t i c l e sFluorescent proteins (FPs) 1 whose fluorescence can be reversibly or irreversibly switched by optical irradiation have opened new opportunities for the imaging of cells. They have facilitated in vivo protein-tracking schemes 2,3 , applications based on singlemolecule observations 4,5 and fluorescence microscopy with subdiffraction resolution [6][7][8][9][10] .Still, photoswitchable proteins have not displayed their full potential, because proteins that are just photoactivatable 11-13 can be switched only once, which implies that repeated measurements with the same molecule are impossible. On the other hand, photochromic or reversibly switchable fluorescent proteins (RSFPs) can be repeatedly photoswitched between a fluorescent and a nonfluorescent state by irradiation with light of two different wavelengths. However, in all previously characterized RSFPs, the wavelength used for generating the fluorescence emission is identical to one of the wavelengths used for switching the fluorescence on or off. The result is a complex interlocking of switching and fluorescence readout [14][15][16][17][18][19][20][21][22] , impeding or even precluding many applications, including fluorescence nanoscopy (super-resolution microscopy). Hence, the identification of an RSFP in which the generation of fluorescence is disentangled from switching has long been pursued. RESULTS Generation of the RSFP DreiklangNumerous GFP variants exhibit some degree of (generally undesirable) reversible photoswitching 4,23,24 . We found that the fluorescence of the yellow fluorescent protein Citrine 25,26 , a derivative of GFP, can be reversibly modulated to a small extent by alternate irradiation with light of 365 nm (on switching) and 405 nm (off switching), whereas fluorescence is excited at 515 nm. However, the achievable contrast was low, especially at pH values >6, rendering the reversible switching of Citrine unusable (Supplementary Fig. 1).To further develop this unusual switching behavior, we performed extensive random mutagenesis as well as directed PCR-mediated mutagenesis on a plasmid encoding Citrine. We transformed Escherichia coli with the plasmid, and screened with an automated home-built fluorescence microscope for bacterial colonies expressing fluorescent proteins whose fluorescence was excited with green light (515 nm) and which could be reversibly photoswitched from a fluorescent state to a long-lived nonfluorescent state by irradiation with near-UV (405 nm) light and back to a fluorescent state by UV (365 nm) light (Fig. 1a). In several consecutive screening rounds ~70,000 individual clones were analyzed. Finally, we identified a mutant differing from Citrine at four positions (Citrine-V61L, F64I, Y145H, N146D) ( Supplementary Fig. 2), which can be effectively switched and excited to fluoresce. We named this switchable fluorescent protein Dreiklang, the German word for a three-note chord in music.At thermal equilibrium, Dreiklang adopts the brightly fluorescent ...

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“…§ Literature values in ref. 26. is relatively stable, remaining 20.0 for rsEGFP2 after 800 switching cycles and 9.3 for Dronpa after 100 switching cycles.…”

Section: Development Of Skylan-nsmentioning

confidence: 91%

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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Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy

Cited by 280 publications

References 30 publications

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

Ground-state proton transfer in the photoswitching reactions of the fluorescent protein Dronpa

A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching

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

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