Illuminating Single Molecules in Condensed Matter

Moerner, W. E.; Orrit, Michel

doi:10.1126/science.283.5408.1670

Cited by 1,082 publications

(869 citation statements)

References 77 publications

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“…[1][2][3][4][5][6][7][8][9][10][11][12][13][14] Single-molecule fluorescence experiments require an optically transparent host containing exactly one highly fluorescent molecule of interest in the probed volume. Such studies have provided insight into a variety of complex condensed-phase phenomena, revealing processes and dynamics that are obscured by typical ensemble measurements.…”

Section: Introductionmentioning

confidence: 99%

Nonlinear Optical Chromophores as Nanoscale Emitters for Single-Molecule Spectroscopy

et al. 2005

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Fluorescence imaging of single molecules at room temperature is a powerful technique for studying complex condensed phase systems and revealing structure and dynamics hidden by ensemble measurements. Successful single-molecule spectroscopic experiments rely upon strong emitters that can be detected at the level of individual copies above the relevant background signals. This Account discusses a class of nonlinear optical chromophores that not only are well-suited for single-molecule imaging but also offer additional beneficial properties such as a significant ground-state dipole moment, moderate hyperpolarizability, and sensitivity to local environment. An overview of the photophysical properties of several members of this class of molecules as well as a mechanism to help understand the environmental sensitivity is presented. Some preliminary applications of the chromophores as single-molecule reporters in cellular and polymer systems are discussed, along with detection of the emitters by two-photon fluorescence.

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Section: Introductionmentioning

confidence: 99%

Nonlinear Optical Chromophores as Nanoscale Emitters for Single-Molecule Spectroscopy

et al. 2005

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“…1 New information has emerged about dynamic heterogeneities in supercooled liquids, 2,3 subwavelength arrangement of cell components, 4 and dynamics of proteins and enzymes. 5,6 Currently, single-molecule spectroscopy mostly relies on fluorescence, as it provides fast, high-contrast, and lowbackground detection of single molecules.…”

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Resonant Plasmonic Enhancement of Single-Molecule Fluorescence by Individual Gold Nanorods

et al. 2014

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Enhancing the fluorescence of a weak emitter is important to further extend the reach of single-molecule fluorescence imaging to many unexplored systems. Here we study fluorescence enhancement by isolated gold nanorods and explore the role of the surface plasmon resonance (SPR) on the observed enhancements. Gold nanorods can be cheaply synthesized in large volumes, yet we find similar fluorescence enhancements as literature reports on lithographically fabricated nanoparticle assemblies. The fluorescence of a weak emitter, crystal violet, can be enhanced more than 1000-fold by a single nanorod with its SPR at 629 nm excited at 633 nm. This strong enhancement results from both an excitation rate enhancement of ∼130 and an effective emission enhancement of ∼9. The fluorescence enhancement, however, decreases sharply when the SPR wavelength moves away from the excitation laser wavelength or when the SPR has only a partial overlap with the emission spectrum of the fluorophore. The reported measurements of fluorescence enhancement by 11 nanorods with varying SPR wavelengths are consistent with numerical simulations.

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“…14,25 By removing the averaging inherent in ensemble measurements, SMS yields a measure of the distribution of molecular properties which is of importance in systems that display static or time-dependent heterogeneity. Concurrently, developments in fluorescence microscopy have made the observation of single molecules of biological interest possible under a wide range of experimental conditions.…”

Section: Introductionmentioning

confidence: 99%

Photophysics of DsRed, a Red Fluorescent Protein, from the Ensemble to the Single-Molecule Level

et al. 2001

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DsRed, a tetrameric fluorescent protein cloned from the Discosoma genus of coral, has shown promise as a longer-wavelength substitute for green fluorescent protein (GFP) mutants for in vivo protein labeling. Bulk and single-molecule studies of the recombinant protein revealed that the DsRed chromophore shows high stability against photobleaching as compared to GFP mutants. Stark modulation spectra confirm that the electronic structure of the DsRed chromophore is similar to that of GFP. However, the tetrameric nature of DsRed leads to intersubunit energy transfer, as evidenced by the molecule's unusually low fluorescence anisotropy when immobilized (0.23 ( 0.02). This value is approximately consistent with an estimate of the energy transfer rates based on preliminary crystallographic information. The fluorescence emission bleaches at a rate linear in the applied excitation intensity, implying that the cessation of emission during pumping at 532 nm is light-driven and, consistent with the tetrameric structure, several photobleaching "steps" were observed for individual complexes. Because more photons are emitted before bleaching, this study suggests that DsRed may be superior to some GFP-based labeling technologies as long as tetramerization is not an issue in physiological studies. IntroductionSingle-molecule spectroscopy (SMS) continues to play a unique role in the elucidation of the dynamics of an increasingly broad range of complex systems. 14,25 By removing the averaging inherent in ensemble measurements, SMS yields a measure of the distribution of molecular properties which is of importance in systems that display static or time-dependent heterogeneity. Concurrently, developments in fluorescence microscopy have made the observation of single molecules of biological interest possible under a wide range of experimental conditions. 26 This versatility can be exploited to examine the molecular complexity of many biological systems.One critical challenge to the application of SMS techniques to biomolecules, especially in vivo, is the introduction of a fluorophore that acts as a reporter of activity, local environment, or spatial location. While certain important proteins display useful amounts of visible fluorescence arising from native cofactors, 12 most require the attachment of an extrinsic fluorophore to serve as the probe. The genetic fusion of naturally fluorescent proteins to a protein of interest ensures a consistent 1:1 stoichiometry in the cell, without the need for external chemical reactions. While the green fluorescent protein (GFP) and its mutants have proved suitable for many applications, 22,27 the relatively high quantum yield of photobleaching (∼10× that for a rhodamine dye 16 ) leaves room for improvement in applications that require a large number of emitted photons, such as in single-molecule studies. Also, no mutant of GFP has been demonstrated to be a strong emitter at long (>550 nm) wavelengths, where the interference from endogenous fluorophores is substantially less severe.Considering th...

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Illuminating Single Molecules in Condensed Matter

Cited by 1,082 publications

References 77 publications

Nonlinear Optical Chromophores as Nanoscale Emitters for Single-Molecule Spectroscopy

Nonlinear Optical Chromophores as Nanoscale Emitters for Single-Molecule Spectroscopy

Resonant Plasmonic Enhancement of Single-Molecule Fluorescence by Individual Gold Nanorods

Photophysics of DsRed, a Red Fluorescent Protein, from the Ensemble to the Single-Molecule Level

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