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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
Published on: December 11, 2021
Dual-color time-integrated fluorescence cumulant analysis
Bin Wu1, Yan Chen, Joachim D Müller
1School of Physics and Astronomy, University of Minnesota, Minneapolis, Minnesota, USA. binmu@physics.umn.edu
Biophysical Journal
|July 4, 2006
Summary
We developed dual-color time-integrated fluorescence cumulant analysis (TIFCA) for analyzing fluorescence data. This method simultaneously measures fluorophore brightness, number, and diffusion time, enabling precise differentiation and quantification in complex biological samples.
Area of Science:
- Biophysics
- Spectroscopy
- Fluorescence analysis
Background:
- Fluorescence fluctuation spectroscopy (FFS) is a powerful technique for studying molecular dynamics.
- Analyzing complex FFS data, especially in multi-component systems, presents significant challenges.
- Existing methods often struggle to simultaneously extract multiple parameters like brightness, concentration, and diffusion times.
Purpose of the Study:
- To introduce and validate a novel dual-color time-integrated fluorescence cumulant analysis (TIFCA) method.
- To enable simultaneous extraction of fluorophore brightness, occupation number, and diffusion time.
- To demonstrate the method's capability in differentiating and quantifying fluorophores based on spectral properties.
Main Methods:
- Development of dual-color TIFCA utilizing bivariate cumulants of fluorescence intensity from two detection channels.
- Derivation of analytical expressions for bivariate factorial cumulants of photon counts.
- Calculation of cumulant variance using the moments-of-moments technique.
- Incorporation and experimental verification of a method to account for non-ideal detector effects (dead-time, afterpulsing).
Main Results:
- Simultaneous determination of brightness, occupation number, and diffusion time for fluorophores.
- Accurate characterization of simple dye solutions and dye mixtures.
- Demonstrated robustness of dual-color TIFCA across a wide concentration range of fluorescent proteins within cells.
- Successful resolution of EGFP/EYFP binary mixtures in living cells using a single measurement.
Conclusions:
- Dual-color TIFCA provides a robust and sensitive approach for analyzing fluorescence fluctuation spectroscopy data.
- The method allows for simultaneous multi-parameter extraction and spectral differentiation of fluorophores.
- Dual-color TIFCA shows significant potential for complex biological measurements, including in vivo studies and resolving closely related fluorescent proteins.
