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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

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Two-dimensional fluorescence lifetime correlation spectroscopy. 1. Principle.

Kunihiko Ishii¹, Tahei Tahara

¹Molecular Spectroscopy Laboratory, RIKEN , 2-1 Hirosawa, Wako , Saitama 351-0198, Japan.

The Journal of Physical Chemistry. B

|August 28, 2013

Summary

This summary is machine-generated.

We developed two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) to analyze complex molecular dynamics. This new method combines fluorescence correlation spectroscopy (FCS) and time-correlated single photon counting (TCPSC) for microsecond resolution insights.

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Area of Science:

Biophysics
Physical Chemistry
Spectroscopy

Background:

Fluorescence correlation spectroscopy (FCS) is crucial for studying microsecond molecular dynamics.
Extracting meaningful data from FCS for complex systems presents challenges.
Existing methods struggle with unambiguous analysis of equilibrium dynamics.

Purpose of the Study:

To develop a novel method for extracting unambiguous information on equilibrium dynamics of complex molecular systems.
To combine FCS and TCPSC for enhanced molecular dynamics analysis.
To introduce two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS).

Main Methods:

Developed 2D FLCS by combining FCS and TCPSC.
Analyzed fluorescence photon pair correlations based on fluorescence lifetime.
Generated 2D maps of excitation-emission delay times.
Applied inverse Laplace transformation to convert 2D maps to species correlations.
Validated the method using kinetic Monte Carlo simulations of TCPSC-FCS experiments.

Main Results:

Successfully extracted unambiguous information about equilibrium dynamics.
Demonstrated microsecond time resolution at the single-molecule level.
2D FLCS visualizes equilibration dynamics of complex molecules.
Kinetic Monte Carlo simulations confirmed the method's validity.

Conclusions:

2D FLCS offers a powerful approach for analyzing complex molecular dynamics.
The method provides high potential for analyzing dynamic system photon data.
This technique enhances the study of microsecond molecular dynamics in equilibrium systems.