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Author Notes:

Correspondence: Keith M. Berland; Email: kberlan@emory.edu

Author Contributions: Conceived and designed the experiments: NA and KB.

Performed the experiments: NA.

Analyzed the data: NA.

Contributed reagents/materials/analysis tools: NA and KB.

Wrote the paper: NA and KB.

Derived theory: KB.

Designed software used in analysis: NA.

Disclosures: The authors have declared hat no competing interests exist.

Subject:

Research Funding:

This work was supported by National Science Foundation grants MCB0817966 and DMR0907435.

τFCS: Multi-Method Global Analysis Enhances Resolution and Sensitivity in Fluorescence Fluctuation Measurements

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Journal Title:

PLoS ONE

Volume:

Volume 9, Number 2

Publisher:

, Pages e90456-e90456

Type of Work:

Article | Final Publisher PDF

Abstract:

Fluorescence fluctuation methods have become invaluable research tools for characterizing the molecular-level physical and chemical properties of complex systems, such as molecular concentrations, dynamics, and the stoichiometry of molecular interactions. However, information recovery via curve fitting analysis of fluctuation data is complicated by limited resolution and challenges associated with identifying accurate fit models. We introduce a new approach to fluorescence fluctuation spectroscopy that couples multi-modal fluorescence measurements with multi-modal global curve fitting analysis. This approach yields dramatically enhanced resolution and fitting model discrimination capabilities in fluctuation measurements. The resolution enhancement allows the concentration of a secondary species to be accurately measured even when it constitutes only a few percent of the molecules within a sample mixture, an important new capability that will allow accurate measurements of molecular concentrations and interaction stoichiometry of minor sample species that can be functionally important but difficult to measure experimentally. We demonstrate this capability using τFCS, a new fluctuation method which uses simultaneous global analysis of fluorescence correlation spectroscopy and fluorescence lifetime data, and show that τFCS can accurately recover the concentrations, diffusion coefficients, lifetimes, and molecular brightness values for a two component mixture over a wide range of relative concentrations.

Copyright information:

© 2014 Anthony, Berland

This is an Open Access work distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).
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