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

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

Published on: December 11, 2021

Modulated fluorescence correlation spectroscopy with complete time range information.

Gustav Persson¹, Per Thyberg, Jerker Widengren

¹Experimental Biomolecular Physics, Department of Applied Physics, Royal Institute of Technology, AlbaNova University Center, Stockholm, Sweden.

Biophysical Journal

|September 25, 2007

Summary

Two methods enhance fluorescence correlation spectroscopy (FCS) using modulated excitation. These techniques enable full correlation data extraction and improve dye photophysical property optimization, aiding diverse applications.

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Area of Science:

Biophysics
Physical Chemistry

Background:

Fluorescence Correlation Spectroscopy (FCS) is a powerful technique for studying molecular dynamics.
Standard FCS methods can be limited by photophysical properties like triplet state buildup.
Modulated excitation offers potential improvements for FCS measurements.

Purpose of the Study:

To develop and validate methods for combining FCS with modulated excitation.
To enable the extraction of complete correlation data across all timescales.
To demonstrate the advantages of modulated excitation for optimizing FCS experiments and analyzing complex processes.

Main Methods:

Development of two distinct methods to integrate modulated excitation with FCS.
Experimental verification of the developed methods using standard hardware correlators and time-resolved acquisition.
Application of the methods to study dye photophysics (rhodamine 6G) and protonation kinetics (fluorescein).

Main Results:

Successful extraction of distortion-free correlation data with standard hardware under specific conditions.
A second method requiring time-resolved acquisition accommodates broader experimental conditions.
Modulated excitation demonstrated superior suppression of triplet population buildup compared to continuous wave reduction.
Distinguished protonation kinetics of fluorescein at varying pH by suppressing photoinduced states.

Conclusions:

The developed methods allow for complete correlation timescale analysis in FCS.
Modulated excitation provides an effective strategy to optimize FCS measurements by managing photophysical properties.
This approach enhances the utility of FCS for investigating complex molecular processes and kinetics.