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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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Published on: December 11, 2021

Inverse-fluorescence cross-correlation spectroscopy.

Stefan Wennmalm¹, Jerker Widengren

¹Department of Applied Physics, Experimental Biomolecular Physics, Royal Institute of Technology, SE-106 91 Stockholm, Sweden. stewen@kth.se

Analytical Chemistry

|June 10, 2010

Summary

Inverse-fluorescence cross-correlation spectroscopy (iFCCS) extends iFCS for analyzing labeled particles. This method quantifies particle volume, detection volume, or binding interactions, even with only one labeled molecule.

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Area of Science:

Biophysics
Spectroscopy
Biochemistry

Background:

Inverse-fluorescence correlation spectroscopy (iFCS) analyzes unlabeled particles by monitoring their displacement of a fluorescent medium.
Traditional fluorescence correlation spectroscopy (FCS) requires labeling of the analyzed particles or biomolecules.

Purpose of the Study:

To introduce inverse-fluorescence cross-correlation spectroscopy (iFCCS) as an extension of iFCS.
To enable analysis of labeled particles and their interactions using cross-correlation with a fluorescent medium signal.
To provide methods for estimating particle volume, detection volume, or binding interactions.

Main Methods:

Cross-correlation of fluorescence signals from labeled particles/biomolecules with the surrounding fluorescent medium.
Analysis of transient signal dips caused by labeled particle diffusion through the detection volume.
Independent estimation of labeled and unlabeled particle amounts to determine the degree of labeling.

Main Results:

iFCCS allows direct estimation of labeled particle volume or detection volume.
The method can detect binding interactions between labeled small molecules and unlabeled particles.
Accurate estimation of the degree of labeling is achievable by independently quantifying labeled and unlabeled particles.

Conclusions:

iFCCS offers a versatile extension to iFCS, broadening the scope of fluorescence correlation spectroscopy.
The technique facilitates the study of labeled molecules and their interactions without requiring dual labeling.
iFCCS provides a robust method for determining particle characteristics and binding affinities.