Hole-Burning Spectroscopy

Völker, S.

doi:10.1146/annurev.pc.40.100189.002435

Cited by 225 publications

(111 citation statements)

References 69 publications

(180 reference statements)

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“…27 and references therein; ref. 28). By burning into the I-form, not only does the 0-0 hole appear, but, simultaneously, its vibronic band at Ϸ495 nm decreases, while the bands between Ϸ460-480 nm increase (see dashed blue curve in Fig.…”

Section: Resultsmentioning

confidence: 99%

Photophysics and optical switching in green fluorescent protein mutants

Creemers

Lock²,

Subramaniam

et al. 2000

Proceedings of the National Academy of Sciences

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We demonstrate by using low-temperature high-resolution spectroscopy that red-shifted mutants of green fluorescent protein are photo-interconverted among three conformations and are, therefore, not photostable ''one-color'' systems as previously believed. From our experiments we have further derived the energy-level schemes governing the interconversion among the three forms. These results have significant implications for the molecular and cell biological applications of the green fluorescent protein family; for example, in fluorescence resonant energy transfer experiments, a change in "color" on irradiation may not necessarily be due to energy transfer but can also arise from a photo-induced conversion between conformers of the excited species.T he Aequorea victoria green fluorescent protein (GFP) has become a favored marker in molecular and cell biology because of its strong intrinsic visible fluorescence and the feasibility of fusing it to other proteins without affecting their normal functions (1-5). For example, mutants of GFP with different absorption and fluorescence spectra (4-8) are presently used in fluorescence lifetime imaging microscopy and fluorescence resonance energy transfer experiments to study protein-protein interactions, signaling, and trafficking in cellular systems (refs. 3-5 and references therein; refs. 9-12). In all of these studies it is assumed that the color changes observed in the GFP emission are caused by dynamic processes involving the proteins to which GFP is attached and do not arise in the GFP itself. That is, GFP-mutants are generally assumed to be in one conformation (i.e., to emit light of ''one color'') and remain in that conformation under laser illumination (i.e., to be photostable), a supposition that we here prove to be incorrect.The recent determination of the crystal structure of wild-type (wt) GFP and its mutants (13-15) has facilitated a structurebased, rational design of further mutants (3,5,16) in which the amino acids exchanged are either directly involved in the cyclization of the chromophore (serine 65, tyrosine 66, and glycine 67) or are located in their vicinity (4, 6-8, 17, 18). Frequently, the purpose of these mutations is to obtain one-color GFPmutants with a single conformation, in contrast to wt-GFP, which exhibits absorption bands attributed to more than one conformation (3,4,(19)(20)(21)(22)(23).The photophysics of wt-GFP presents a complex problem, and that of its mutants has not been studied in detail. The roomtemperature spectra are broad and rather unstructured (3-7, 18, 19). To determine the energy-level schemes of GFPs it is necessary to go to low temperature, where the spectrum becomes more structured. This has the additional merit that many of the thermally induced conversions are blocked at low temperature and, therefore, discrimination among individual species is facilitated. Energy-level schemes derived from low-temperature experiments put constraints on the interpretation of roomtemperature results. A recent study by high-resolution spec...

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Section: Resultsmentioning

confidence: 99%

Photophysics and optical switching in green fluorescent protein mutants

Creemers

Lock²,

Subramaniam

et al. 2000

Proceedings of the National Academy of Sciences

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“…1,2 Room temperature time-domain measurements, in contrast, while substantially complicated by the broad, relatively featureless, spectral properties of higher temperature systems, offer the essential advantage of allowing for the characterization of samples not only under biological conditions in vitro but even in vivo in living bacterial cells. [3][4][5] To build a complete picture of any physical system, one must of course be able to compare the results obtained using different methods.…”

Section: Introductionmentioning

confidence: 99%

Comments on the optical lineshape function: Application to transient hole-burned spectra of bacterial reaction centers

Reppert

Kell

Pruitt

et al. 2015

The Journal of Chemical Physics

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The vibrational spectral density is an important physical parameter needed to describe both linear and non-linear spectra of multi-chromophore systems such as photosynthetic complexes. Low-temperature techniques such as hole burning (HB) and fluorescence line narrowing are commonly used to extract the spectral density for a given electronic transition from experimental data. We report here that the lineshape function formula reported by Hayes et al. [J. Phys. Chem. 98, 7337 (1994)] in the mean-phonon approximation and frequently applied to analyzing HB data contains inconsistencies in notation, leading to essentially incorrect expressions in cases of moderate and strong electron-phonon (el-ph) coupling strengths. A corrected lineshape function L(ω) is given that retains the computational and intuitive advantages of the expression of Hayes et al. [J. Phys. Chem. 98, 7337 (1994)]. Although the corrected lineshape function could be used in modeling studies of various optical spectra, we suggest that it is better to calculate the lineshape function numerically, without introducing the mean-phonon approximation. New theoretical fits of the P870 and P960 absorption bands and frequency-dependent resonant HB spectra of Rb. sphaeroides and Rps. viridis reaction centers are provided as examples to demonstrate the importance of correct lineshape expressions. Comparison with the previously determined el-ph coupling parameters [Johnson et al., J. Phys. Chem. 94, 5849 (1990); Lyle et al., ibid. 97, 6924 (1993); Reddy et al., ibid. 97, 6934 (1993)] is also provided. The new fits lead to modified el-ph coupling strengths and different frequencies of the special pair marker mode, ωsp, for Rb. sphaeroides that could be used in the future for more advanced calculations of absorption and HB spectra obtained for various bacterial reaction centers.

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“…To address the wide range of ultrafast processes, generally advanced spectroscopic methods are invoked. Initially spectral hole burning [3] was developed to probe the underlying dynamics of inhomogeneously broadened bands in solids. With femtosecond photon echo spectroscopy [4] the inhomogeneous linewidth could be eliminated to study ultrafast dephasing times and solvation dynamics.…”

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confidence: 99%

Single-Molecule Pump-Probe Detection Resolves Ultrafast Pathways in Individual and Coupled Quantum Systems

et al. 2005

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We report the first experimental study of individual molecules with femtosecond time resolution using a novel ultrafast single-molecule pump-probe method. A wide range of relaxation times from below 100 up to 400 fs is found, revealing energy redistribution over different vibrational modes and phonon coupling to the nanoenvironment. Addressing quantum-coupled molecules we find longer decay times, pointing towards inhibited intramolecular decay due to delocalized excitation. Interestingly, each individual system shows discrete jumps in femtosecond response, reflecting sudden breakup of the coupled superradiant state. DOI: 10.1103/PhysRevLett.94.078302 PACS numbers: 82.37.-j, 34.30.+h, 71.35.-y, 82.53.-k Ultrafast processes play a crucial role in the functioning of both natural and synthetic molecular assemblies [1]. For example, energy transfer between chromophores in photosynthetic complexes and conjugated polymers typically occurs on a 0.5 to 2.0 ps time scale [2], while intramolecular dynamics is even faster. The energy transfer is mediated by delocalized electronic excitation, in direct competition with disorder in the system, ultrafast decay through intramolecular vibrations, and coupling to the environment. To address the wide range of ultrafast processes, generally advanced spectroscopic methods are invoked. Initially spectral hole burning [3] was developed to probe the underlying dynamics of inhomogeneously broadened bands in solids. With femtosecond photon echo spectroscopy [4] the inhomogeneous linewidth could be eliminated to study ultrafast dephasing times and solvation dynamics. Currently mainly four-wave mixing and pump-probe spectroscopies [5] are applied to address both dephasing and population dynamics, predominantly of vibrational wave packets. Unfortunately, all these approaches are restricted to processes that can be optically synchronized and therefore yield only the spatial average over some conformational subset.In contrast to this, single-molecule studies [6] commonly reveal how changes in the local environment lead to large variations, in both space and time [7][8][9]. However, single-molecule detection relies essentially on background-free detection of Stokes shifted fluorescence. The limited photon yield is a bottleneck in dynamic studies, restricting real-time observations typically to the microsecond regime [8]. Only at cryogenic conditions are cross sections large enough to allow resonant detection with a faster response [10]. As a result the femtosecond regime has so far remained untouched in the singlemolecule field operating at ambient conditions.Here, we bridge the gap between ''ultrafast'' and ''single-molecule'' detection and present a novel fluorescence-based pump-probe method that gives direct access to ultrafast phenomena in individual quantum systems. The first results on both single and coupled molecules are presented, where from molecule to molecule a wide range of ultrafast decay times is observed.Optical observation of single quantum systems is based on detection of...

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Hole-Burning Spectroscopy

Cited by 225 publications

References 69 publications

Photophysics and optical switching in green fluorescent protein mutants

Photophysics and optical switching in green fluorescent protein mutants

Comments on the optical lineshape function: Application to transient hole-burned spectra of bacterial reaction centers

Single-Molecule Pump-Probe Detection Resolves Ultrafast Pathways in Individual and Coupled Quantum Systems

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