Fast single-molecule FRET spectroscopy: theory and experiment

Area of Science:

• Biophysics
• Chemical Physics
• Spectroscopy

Background:

• Single-molecule spectroscopy offers unique insights into macromolecular dynamics beyond ensemble methods.
• Studying fast molecular dynamics is constrained by photon count rate, linked to illumination intensity.
• Increasing illumination intensity is often problematic due to photophysical and photochemical issues.

Purpose of the Study:

• To enhance the dynamic range of single-molecule fluorescence spectroscopy.
• To improve the analysis of fast molecular dynamics using existing photon count rates.
• To present a maximum likelihood method for analyzing photon trajectory data.

Main Methods:

• Utilizing all detected photons in trajectory analysis.
• Applying a maximum likelihood method to determine molecular dynamics model parameters.
• Analyzing photon colors and inter-photon times to reconstruct dynamics.

Main Results:

• The maximum likelihood method allows for improved parameter extraction from photon trajectories.
• This method has been successfully applied to analyze fast two-state protein folding.
• Transition path times can be accurately measured using this approach.

Conclusions:

• The maximum likelihood method significantly improves the dynamic range of single-molecule fluorescence spectroscopy.
• This technique enables the study of faster molecular dynamics than previously possible.
• Integrating fluorescence lifetimes offers further dimensions for analysis, such as in 2D FRET efficiency-lifetime histograms.