Global analysis in fluorescence correlation spectroscopy and fluorescence lifetime microscopy
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Updated: May 15, 2026

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
Published on: December 11, 2021
1Department of Physics, Emory University, Atlanta, Georgia, USA.
Abstract:
Fluorescence correlation spectroscopy (FCS) and related fluctuation spectroscopy and microscopy methods have become important research tools that enable detailed investigations of the chemical and physical properties of molecules and molecular systems in a variety of complex environments. Information recovery via curve fitting of fluctuation data can present complicating challenges due to limited resolution and/or problems with fitting model verification. We discuss a new approach to data analysis called τFCS that couples multiple modes of signal acquisition, here specifically FCS and fluorescence lifetimes, with global analysis. We demonstrate enhanced resolution using τFCS, including the capability to recover the concentration of both molecular species in a two-component mixture even when the species have identical diffusion coefficients and molecular brightness values, provided their fluorescent lifetimes are distinct. We also demonstrate how τFCS provides useful tools for model discrimination in FCS curve fitting.
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