The Fluorescence Dynamics of Single Molecules of Green Fluorescent Protein

Peterman, Erwin J.G.; Brasselet, Sophie; Moerner, W. E.

doi:10.1021/jp991968o

Cited by 143 publications

(180 citation statements)

References 37 publications

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168

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“…In the notation of their scheme YFP -) A, YFPH ) I and YFPHrb ) N. Our data suggest however that direct decay of N* to A on photoactivation does not occur, but leads to I* and then I as a dark state intermediate. Although Dickson et al (6) suggested A and I states were related by protonation state, as we propose, this conclusion was questioned by Peterman et al (9) who found the blinking rates on the millisecond to seconds time scale were insensitive to pH over the range of 6 to 10. However the latter study focused on GFP variants with lower pK values and laser powers were used up to 5 kW cm -2 , as a result the on-times were much shorter than those reported by Dickson et al (6) and approached the temporal resolution limit of their imaging device.…”

Section: Discussioncontrasting

confidence: 66%

“…The YFP class, in particular, exhibit the phenomenon of emission blinking on the seconds time scale, as well as a long-lived low fluorescence state that can be activated by near UV irradiation. Previous studies of GFP and related proteins have identified at least 3 ground-state species and have suggested interconversions between them that give rise to blinking and switching behavior (2,3,(6)(7)(8)(9). However the ground-state kinetics have not been fully rationalized in a manner that is consistent with the observed pK values.…”

Section: Discussionmentioning

confidence: 89%

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Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

et al. 2005

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Yellow fluorescent protein (YFP 10C) is widely used as a probe in biology, but its complex photochemistry gives rise to unusual behavior that requires fuller definition. Here we characterize the kinetics of protonation and reversible bleaching over time scales of picoseconds to hours. Stopped-flow and pressure-jump techniques showed that protonation of the fluorescent YFP -anion state is two-step with a slow transition that accounts for blinking of 527 nm emission at the single molecule level on the seconds time scale. Femtosecond spectroscopy revealed that the protonated excited-state (YFPH*) decayed predominantly by a radiationless mechanism, but emission at 460 nm was detected within the first picosecond. Limited excited-state proton transfer leads to 527 nm emission characteristic of the YFP -* anion. Prolonged continuous wave illumination at the peak of YFP -absorbance (514 nm) yields, irreversibly, a weakly fluorescent product that absorbs at 390 nm. This "photobleaching" process also gives a different species (YFPHrb) that absorbs at 350/430 nm and spontaneously regenerates YFP -in the dark on the time scale of hours but can be photoactivated by UV light to regenerate YFP -within seconds, via a ground-state protonated intermediate. Using a pulsed laser for photobleaching resulted in decarboxylation of YFP as indicated by the mass spectrum. These observations are accounted for in a unifying kinetic scheme.Green fluorescent protein (GFP) 1 and its color variants have found wide use in biology to probe protein dynamics in vitro and within cells (1). They have also attracted the attention of spectroscopists owing to their complex photochemistry (2-5). In particular, those in the YFP class (i.e. containing a T203Y or T203F mutation to shift the emission to 527 nm) have been shown to undergo fluctuations of emission intensity over a wide range of times scales and reversible photobleaching (6-8). Fluctuations in emission were observed by wide field microscopy at the single molecule level by immobilizing individual YFP molecules in acrylamide or agarose gels and using TIRF excitation (6, 9). Molecules were found to blink on the seconds time scale at low laser powers (<500 W cm -2 ). Increasing laser power reduced the lifetime of the emitting state, but not in direct proportion to the laser power. Initial studies discussed the possibility that the fluctuations were related to the protonation of the phenolate moiety of the fluorophore derived from Y66 (6), but later studies found little pH dependence of the onand off-times when monitored at a laser power of 5000 W cm -2 (9).Members of the YFP class also showed a photochromic switching behavior. Molecules that were bleached by 488 nm irradiation, could be induced to fluoresce by illumination with near UV or blue light (6-8). This reversible photobleaching reaction of YFP was observed in FRET measurements (10). Illumination of YFP at 514 nm resulted in bleaching of the YFP acceptor and dequenching of a donor CFP molecule. Once the YFP was bleached, illumina...

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Section: Discussioncontrasting

confidence: 66%

Section: Discussionmentioning

confidence: 89%

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

et al. 2005

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“…169 It is also possible to observe additional unique behavior such as fluorescence intermittency (i.e., blinking) that cannot be observed for bulk samples. 170 Ensemble studies benefit significantly from the clarification of fundamental photophysical behavior that is exposed on the single particle level, which has catalyzed single molecule studies for many fluorescent systems including dyes, 171-174 polymer chains, 175,176 proteins, [177][178][179] semiconductor quantum dots, 170,180,181 and single-walled carbon nanotubes. 72,77,182 The observation of fluorescence from individual SWNTs was first reported in 2003 and, importantly, confirmed spectral assignments of (n,m) made on the ensemble level.…”

Section: Principles Of Single Molecule Spectroscopymentioning

confidence: 99%

Photophysics of Individual Single-Walled Carbon Nanotubes

2008

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Single-walled carbon nanotubes (SWNTs) are cylindrical graphitic molecules that have remained at the forefront of nanomaterials research since 1991, largely due to their exceptional and unusual mechanical, electrical, and optical properties. The motivation for understanding how nanotubes interact with light (i.e., SWNT photophysics) is both fundamental and applied. Individual nanotubes may someday be used as superior near-infrared fluorophores, biological tags and sensors, and components for ultrahigh-speed optical communications systems. Establishing an understanding of basic nanotube photophysics is intrinsically significant and should enable the rapid development of such innovations. Unlike conventional molecules, carbon nanotubes are synthesized as heterogeneous samples, composed of molecules with different diameters, chiralities, and lengths. Because a nanotube can be either metallic or semiconducting depending on its particular molecular structure, SWNT samples are also mixtures of conductors and semiconductors. Early progress in understanding the optical characteristics of SWNTs was limited because nanotubes aggregate when synthesized, causing a mixing of the energy states of different nanotube structures. Recently, significant improvements in sample preparation have made it possible to isolate individual nanotubes, enabling many advances in characterizing their optical properties. In this Account, single-molecule confocal microscopy and spectroscopy were implemented to study the fluorescence from individual nanotubes. Single-molecule measurements naturally circumvent the difficulties associated with SWNT sample inhomogeneities. Intrinsic SWNT photoluminescence has a simple narrow Lorentzian line shape and a polarization dependence, as expected for a one-dimensional system. Although the local environment heavily influences the optical transition wavelength and intensity, single nanotubes are exceptionally photostable. In fact, they have the unique characteristic that their single molecule fluorescence intensity remains constant over time; SWNTs do not “blink” or photobleach under ambient conditions. In addition, transient absorption spectroscopy was used to examine the relaxation dynamics of photoexcited nanotubes and to elucidate the nature of the SWNT excited state. For metallic SWNTs, very fast initial recovery times (300–500 fs) corresponded to excited-state relaxation. For semiconducting SWNTs, an additional slower decay component was observed (50–100 ps) that corresponded to electron–hole recombination. As the excitation intensity was increased, multiple electron–hole pairs were generated in the SWNT; however, these e−h pairs annihilated each other completely in under 3 ps. Studying the dynamics of this annihilation process revealed the lifetimes for one, two, and three e−h pairs, which further confirmed that the photoexcitation of SWNTs produces not free electrons but rather one-dimensional bound electron–hole pairs (i.e., excitons). In summary, nanotube photophysics is a rapidly developing area o...

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“…The bleaching rate was then plotted as a function of the excitation rate, as shown in Figure 5. The excitation rate can be calculated from the excitation intensity and the absorption cross-section 6 ; for these calculations, the measured cross-section of the dye in toluene is used. The bleaching quantum yield was determined from the slope of the best-fit line relating the bleaching rate to the excitation rate.…”

Section: Bulk Photophysical Propertiesmentioning

confidence: 99%

“…Over the last 14 years, scientists around the world have been performing optical spectroscopy and microscopy on single reporter molecules in liquid, glass, protein, or crystal environments [1][2][3][4][5][6][7][8] . In these single-molecule experiments, a complex condensed phase system is probed with an object typically only 1-2 nm in size, and the changes in emitted fluorescence allow the nanoscale heterogeneity of the system to be explored with high sensitivity.…”

Section: Introductionmentioning

confidence: 99%

Novel Fluorophores for Single-Molecule Imaging

Willets

Ostroverkhova

et al. 2003

J. Am. Chem. Soc.

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A new class of fluorophores has been identified that can be imaged at the single-molecule level and offer additional beneficial properties such as a significant ground state dipole moment, moderate hyperpolarizability, and sensitivity to local rigidity. These molecules contain an amine donor and a dicyanodihydrofuran (DCDHF) acceptor linked by a conjugated unit (benzene, thiophene, alkene, styrene, etc.) and were originally designed to deliver both high polarizability anisotropy and dipole moment as nonlinear optical chromophores for photorefractive applications. Surprisingly, we have found that these molecules are also well-suited for single-molecule fluorescence imaging in polymers and other reasonably rigid environments. We report the bulk (ensemble) and single-molecule photophysical properties measured for six dyes in this new class of single-molecule reporters, with absorption maxima ranging from 486 to 614 nm.

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The Fluorescence Dynamics of Single Molecules of Green Fluorescent Protein

Cited by 143 publications

References 37 publications

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

Protonation, Photobleaching, and Photoactivation of Yellow Fluorescent Protein (YFP 10C): A Unifying Mechanism

Photophysics of Individual Single-Walled Carbon Nanotubes

Novel Fluorophores for Single-Molecule Imaging

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