τFCS: multi-method global analysis enhances resolution and sensitivity in fluorescence fluctuation measurements.

Area of Science:

• Biophysics
• Analytical Chemistry
• Spectroscopy

Background:

• Fluorescence fluctuation methods are crucial for studying molecular properties like concentration and dynamics.
• Current methods face limitations in resolution and model identification for complex systems.
• Accurate analysis of molecular interactions and minor species remains challenging.

Purpose of the Study:

• To develop a novel fluorescence fluctuation spectroscopy approach with enhanced resolution and fitting capabilities.
• To enable accurate quantification of minor molecular species in complex mixtures.
• To improve the measurement of molecular concentrations and interaction stoichiometry.

Main Methods:

• Coupling multi-modal fluorescence measurements with multi-modal global curve fitting analysis.
• Introducing τFCS: a method combining fluorescence correlation spectroscopy (FCS) and fluorescence lifetime data.
• Simultaneous global analysis of FCS and fluorescence lifetime data.

Main Results:

• Achieved dramatically enhanced resolution and fitting model discrimination in fluctuation measurements.
• Demonstrated accurate measurement of secondary species concentration, even at a few percent.
• Successfully recovered concentrations, diffusion coefficients, lifetimes, and molecular brightness for a two-component mixture.

Conclusions:

• The new τFCS method significantly advances fluorescence fluctuation spectroscopy.
• Enables accurate characterization of minor but functionally important molecular species.
• Provides a powerful tool for detailed analysis of molecular concentrations and interactions in complex systems.