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Global analysis in fluorescence correlation spectroscopy and fluorescence lifetime microscopy.

Neil Anthony¹, Keith Berland

¹Department of Physics, Emory University, Atlanta, Georgia, USA.

Methods in Enzymology

|January 2, 2013

Summary

τFCS enhances fluorescence correlation spectroscopy (FCS) analysis by combining signal acquisition modes. This novel approach improves resolution, enabling the differentiation of molecular species with identical diffusion and brightness but distinct fluorescence lifetimes.

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Area of Science:

Biophysics
Analytical Chemistry
Spectroscopy

Background:

Fluorescence correlation spectroscopy (FCS) is vital for studying molecular properties in complex systems.
Data analysis challenges in FCS include limited resolution and model verification issues.
Existing methods struggle to differentiate species with similar diffusion and brightness.

Purpose of the Study:

Introduce τFCS, a new data analysis approach for fluctuation spectroscopy.
Demonstrate enhanced resolution and model discrimination capabilities of τFCS.
Showcase τFCS's ability to resolve complex molecular mixtures.

Main Methods:

Coupling multiple signal acquisition modes: FCS and fluorescence lifetimes.
Utilizing global analysis for integrated data interpretation.
Applying τFCS to analyze fluctuation spectroscopy data.

Main Results:

τFCS achieves enhanced resolution in fluctuation spectroscopy data analysis.
Successfully resolved concentrations of two molecular species with identical diffusion and brightness.
Demonstrated successful differentiation based on distinct fluorescence lifetimes.
Provided effective tools for model discrimination in FCS curve fitting.

Conclusions:

τFCS offers a significant advancement in analyzing complex molecular systems.
The method overcomes limitations of traditional FCS data analysis.
τFCS enables precise characterization of molecular mixtures previously indistinguishable.