Systematic evaluation of fluorescence correlation spectroscopy data analysis on the nanosecond time scale

Area of Science:

• Biophysics
• Physical Chemistry
• Spectroscopy

Background:

• Fluorescence time traces reveal biomolecular dynamics via quenching interactions.
• Fluorescence correlation spectroscopy (FCS) analyzes dynamic processes from pico- to millisecond timescales.
• High viscosity can cause multiple correlation decays in labeled biomolecules due to diffusion, quenching, and photophysics.

Purpose of the Study:

• Compare parameter estimation for FCS data with multiple correlation decays.
• Evaluate dynamical fingerprint analysis against Levenberg-Marquardt fitting.
• Identify conditions favoring dynamical fingerprint analysis.

Main Methods:

• Dynamical fingerprint analysis.
• Non-linear Levenberg-Marquardt fitting procedure.
• Analysis of fluorescence correlation spectroscopy (FCS) data.

Main Results:

• Identified conditions where dynamical fingerprint analysis offers advantages for FCS data.
• Discovered a novel nanosecond-timescale photophysical process causing fluorescence intermittency in ATTO655, absent in MR121.
• Resolved the viscosity dependence of fluorescence quenching for photoinduced electron transfer probes using an optimized fitting procedure.

Conclusions:

• Dynamical fingerprint analysis is a valuable tool for FCS data with multiple correlation decays.
• The newly identified photophysical process in ATTO655 is crucial for accurate biomolecular dynamics analysis.
• Optimized fitting procedures enhance the resolution of fluorescence quenching dynamics and viscosity dependencies.