Probing Photophysical Processes in Individual Multichromophoric Dendrimers by Single-Molecule Spectroscopy

Hofkens, Johan; Maus, Michael; Gensch, Thomas; Vosch, Tom; Cotlet, Mircea; Köhn, F.; Herrmann, Andreas; Müllen, Kläus; Schryver, Frans De

doi:10.1021/ja0012570

Cited by 233 publications

(362 citation statements)

References 60 publications

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“…Four different intensity levels (plus background) can be identified in the trace. The stepwise changes in fluorescence intensity as well as the intensity jumps between discrete levels are signatures of a multichromophoric system (20)(21)(22)(23). As observed in Fig.…”

Section: Results and Analysismentioning

confidence: 69%

“…The unequal reduction of intensity cannot be explained by solely orientational effects and suggests some fluorescence quenching within the complex. Weak quenching of the fluorescence has been observed in other multichromophoric systems and is attributed to coupling between closely interacting chromophores (20)(21)(22)(23). Fig.…”

Section: Results and Analysismentioning

confidence: 91%

“…To explain the occurrence of one-step blinking in DsRed, one would have to postulate that the four chromophores simultaneously jump to an ''off'' state, or that the chromophores are coupled in some fashion, as suggested by Lounis et al (8), so that the resultant emission and on-off blinking proceed from one emitter in the complex. Intensity jumps to the background have also been observed in strongly coupled multichromophoric systems and are understood in terms of photogenerated traps, which quench the fluorescence of all of the other subunits (20)(21)(22)(23).…”

Section: Results and Analysismentioning

confidence: 99%

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The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

García-Parajo

Koopman

Dijk

et al. 2001

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

105

101

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Recent studies on the newly cloned red fluorescence protein DsRed from the Discosoma genus have shown its tremendous advantages: bright red fluorescence and high resistance against photobleaching. However, it has also become clear that the protein forms closely packed tetramers, and there is indication for incomplete protein maturation with unknown proportion of immature green species. We have applied single-molecule methodology to elucidate the nature of the fluorescence emission in the DsRed. Real-time fluorescence trajectories have been acquired with polarization sensitive detection. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Photodissociation of one of the chromophores weakly quenches the emission of adjacent ones. Dual color excitation (at 488 and 568 nm) single-molecule microscopy has been performed to reveal the number and distribution of red vs. green species within each tetramer. We find that 86% of the DsRed contain at least one green species with a red-to-green ratio of 1.2-1.5. On the basis of our findings, oligomer suppression would not only be advantageous for protein fusion but will also increase the fluorescence emission of individual monomers.T he cloning of the red fluorescent protein (drFP583, commercially available as DsRed) from the Indo pacific reef coral Discosoma sp (1) has triggered intense biological interest as a potential expression marker and fusion partner that would be complementary to the Aequorea victoria green fluorescent protein (avGFP). The main advantage of these naturally fluorescent proteins is that they provide strong visible fluorescence and can be genetically fused to other proteins. Since the cloning of the avGFP (2, 3), different mutants have been produced to extend the palette of available colors. However, no GFP mutant has been produced with emission maxima longer than 529 nm (3). The newly cloned DsRed has bright red fluorescence with emission maxima at 583 nm, one of the longest wavelength emissions reported so far in a wild-type species [Fradkov et al. (4) have reported a wild type highly homologous to the drFP583 with 593-nm emission and a hybrid with 616-nm emission]. Potentially, DsRed is an ideal partner of GFP in fluorescence resonance energy transfer (FRET) experiments, particularly in cell biological applications in which cellular autofluorescence poses a problem (5).In the last few months, extensive research has been done in the biochemical and photophysical investigation of DsRed. The absorption spectrum of the mature protein exhibits a strong band with a peak at 558 nm and two minor shoulders at 526 and 490 nm (6). An extinction coefficient of 75,000 mol Ϫ1 ͞cm Ϫ1 and a fluorescence quantum yield of 0.7 at 558-nm excitation wavelength have been reported (7), much higher than initially published by Matz et al. (1). The protein proves to be stable under harsh pH conditions and is extremely resistant to photobleaching (7, 8). However, t...

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Section: Results and Analysismentioning

confidence: 69%

Section: Results and Analysismentioning

confidence: 91%

Section: Results and Analysismentioning

confidence: 99%

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The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

García-Parajo

Koopman

Dijk

et al. 2001

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

105

101

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“…Photon antibunching [2,3], stochastic fluorescence off times due to excursions of the molecule to the triplet manifold [4], and quantum jumps [5] have been reported. Recently, SMS has yielded novel insights into the electronic interactions in multichromophoric assemblies such as light-harvesting pigments [6], conjugated polymers [7], donor-acceptor substituted biopolymers [8], and dendrimers carrying a variable number of chromophores [9]. The optical and electronic properties of such systems are strongly influenced by dipole-dipole interactions between the chromophores, giving rise to excitonic splittings and/ or incoherent Förster type energy transfer.…”

Section: Photon Antibunching and Collective Effects In The Fluorescenmentioning

confidence: 99%

“…We also note that during the off times the fluorescence intensity reaches the background level before and after B1. The complete vanishing of the fluorescence during the off times when both chromophores are still intact indicates a collective behavior of the two chromophores, and accordingly the term collective off times is in use for this effect [9]. The collective behavior is assigned to singlet-triplet annihilation (STA), where the singlet excitation of one chromophore is efficiently quenched when the other chromophore is in the triplet state.…”

Section: P H Y S I C a L R E V I E W L E T T E R Smentioning

confidence: 99%

Photon Antibunching and Collective Effects in the Fluorescence of Single Bichromophoric Molecules

et al. 2003

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A Stochastic Theory of Single Molecule Spectroscopy

Jung

Barkai

Silbey

2002

Advances in Chemical Physics

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A theory is formulated for time dependent fluctuations of the spectrum of a single molecule in a dynamic environment. In particular, we investigate the photon counting statistics of a single molecule undergoing a spectral diffusion process. Based on the stochastic optical Bloch equation, fluctuations are characterized by Mandel's Q parameter yielding the variance of number of emitted photons and the second order intensity correlation function, g (2) (t). Using a semi-classical approach and linear response theory, we show that the Q parameter can be described by a three-time dipole correlation function. This approach generalizes the Wiener-Khintchine formula that gives the average number of fluorescent photons in terms of a one-time dipole correlation function. We classify the time ordering properties of the three-time dipole correlation function, and show that it can be represented by three different pulse shape functions similar to those used in the context of nonlinear spectroscopy. An exact solution is found for a single molecule whose absorption frequency undergoes a two state random telegraph process (i.e., the Kubo-Anderson sudden jump process.) Simple expressions are obtained from the exact solution in the slow and fast modulation regimes based on appropriate approximations for each case. In the slow modulation regime Q can be large even in the long time limit, while in the fast modulation regime it becomes small.

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Probing Photophysical Processes in Individual Multichromophoric Dendrimers by Single-Molecule Spectroscopy

Cited by 233 publications

References 60 publications

The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

Photon Antibunching and Collective Effects in the Fluorescence of Single Bichromophoric Molecules

A Stochastic Theory of Single Molecule Spectroscopy

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