Green fluorescent proteins as optically controllable elements in bioelectronics

Cinelli, Riccardo A. G.; Pellegrini, Vittorio; Ferrari, Aldo; Faraci, Paolo; Nifosı̀, Riccardo; Tyagi, Mudit; Giacca, Mauro; Beltram, Fabio

doi:10.1063/1.1419047

Cited by 65 publications

(72 citation statements)

References 21 publications

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“…3). No apparent recovery of the fluorescence was detected neither by keeping the molecules up to Х10 hours in the dark, nor by irradiating the gels with IR radiation at shorter wavelengths than the excitation, as found for other mutants (Cinelli et al, 2001). We call bleaching time (T bleaching ) the time at which the transition occurs .…”

Section: Fluorescence Kineticsmentioning

confidence: 97%

Single molecule spectroscopic characterization of GFP‐mut2 mutant for two‐photon microscopy applications

Cannone¹,

Caccia²,

Bologna³

et al. 2004

Microscopy Res & Technique

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Green Fluorescent Protein (GFP) mutants are extensively used in optical microscopy studies of in vivo biological processes in cells. Nonetheless, blinking and bleaching of the GFP chromophore at the single molecule level greatly limits its usefulness. We have worked out what we think are the best experimental conditions for the use of the GFP mutant, GFP-mut2, as a single molecule marker in two-photon excitation measurements. We have measured molecular brightness, excited state lifetime, blinking and photo-bleaching times versus the two-photon excitation intensity on proteins embedded in silica gel matrices versus the excitation wavelength in the range 700-1,000 nm. Our results indicate that GFPmut2 can be employed as a long-lived reporter of biological processes.

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Section: Fluorescence Kineticsmentioning

confidence: 97%

Single molecule spectroscopic characterization of GFP‐mut2 mutant for two‐photon microscopy applications

Cannone¹,

Caccia²,

Bologna³

et al. 2004

Microscopy Res & Technique

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“…In addition, many GFPs exhibit another, long lived, dark state that can be depopulated by illumination with light around 400 nm [46,97,107] or by two-photon excitation between 780 and 870 nm [108]. This switching was attributed to spontaneous protonation of the chromophore.…”

Section: Single Vfp Intensity Trajectoriesmentioning

confidence: 99%

“…Illumination with light of short wavelengths into the absorbance band of the protonated chromophore favours the deprotonation of the chromophore in the excited state. Deprotonation results in the reconstitution of the red-shifted absorption and an efficiently emitting chromophore so that seemingly bleached chromophores were reactivated and regained their fluorescence [46,97,107,108]. The light-induced switching of fluorescent proteins between different emitting or non-emitting states bears great potential for cellular time / s Fig.…”

Section: Single Vfp Intensity Trajectoriesmentioning

confidence: 99%

Single-molecule spectroscopy of fluorescent proteins

Blum

Subramaniam

2008

Anal Bioanal Chem

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The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the details of the photophysics of these reporters is essential for accurate interpretation of the biological and biochemical processes illuminated by fluorescent proteins. Some aspects of the complex photophysics of fluorescent proteins can only be observed and understood at the singlemolecule level, which removes averaging inherent to ensemble studies. In this paper we review how singlemolecule emission detection has helped understanding of the complex photophysics of fluorescent proteins.

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“…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.…”

mentioning

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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Green fluorescent proteins as optically controllable elements in bioelectronics

Cited by 65 publications

References 21 publications

Single molecule spectroscopic characterization of GFP‐mut2 mutant for two‐photon microscopy applications

Single molecule spectroscopic characterization of GFP‐mut2 mutant for two‐photon microscopy applications

Single-molecule spectroscopy of fluorescent proteins

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

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