Dataset Information

Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery.

ABSTRACT: Excitation of few-atom Ag cluster fluorescence produces significant steady-state dark state populations that can be dynamically optically depopulated with long wavelength coillumination. Modulating this secondary illumination dynamically repopulates the ground state, thereby directly modulating nanodot fluorescence without modulating background. Both fast and slow modulation enable unmodulated background to be quantitatively removed in fluorescence correlation spectroscopy (FCS) through simple correlation-based averaging. Such modulated dual-laser FCS enables recovery of pure Ag nanodot fluorescence correlations even in the presence of strong, spectrally overlapping background emission. Fluorescence recovery is linear with Fourier amplitude of the modulated fluorescence, providing a complementary approach to background-free quantitation of modulatable emitter concentration in high background environments. Using the expanding range of modulatable fluorophores, such methodologies should facilitate biologically relevant studies in both complex autofluorescent environments and multiplexed assays.

SUBMITTER: Hsiang JC

PROVIDER: S-EPMC5218585 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Publications

Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery.

Hsiang Jung-Cheng JC Fleischer Blake C BC Dickson Robert M RM

The journal of physical chemistry letters 20160620 13

Excitation of few-atom Ag cluster fluorescence produces significant steady-state dark state populations that can be dynamically optically depopulated with long wavelength coillumination. Modulating this secondary illumination dynamically repopulates the ground state, thereby directly modulating nanodot fluorescence without modulating background. Both fast and slow modulation enable unmodulated background to be quantitatively removed in fluorescence correlation spectroscopy (FCS) through simple c ...[more]

PMID: 27299945

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Project description:Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.

Dataset Information

Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery.

Publications

Dark State-Modulated Fluorescence Correlation Spectroscopy for Quantitative Signal Recovery.

Similar Datasets

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