# Disentangling Timescales of Molecular Kinetics with spFRET using ALEX-FCS

## TL;DR

This paper uses simulations to show how combining spFRET, ALEX, and FCS can accurately measure molecular conformational dynamics and diffusion times.

## Contribution

The study provides a simulation framework to analyze conformational dynamics and diffusion times using spFRET-ALEX-FCS data.

## Key findings

- Simulations distinguish small changes in diffusion coefficients of molecular conformations.
- Burst selection yields accurate conformational lifetimes from 100 us to 100 ms.
- The framework can be expanded for complex systems with multiple conformational states and binding interactions.

## Abstract

Single-pair Förster resonance energy transfer (spFRET) probes the dynamics of molecular structures with (sub-)nanometer accuracy. When combined with fluorescence correlation spectroscopy (FCS), diffusion times and conformation lifetimes can be obtained. Alternating excitation (ALEX) further complements spFRET measurements on freely diffusing molecules, allowing for burst analysis, which can be used to reduce background signal without significant changes to the experimental setup. ALEX is particularly useful for extracting conformational dynamics, but extracting small differences in FRET levels and/or diffusion times can still be difficult for multi-species samples with fast or slow transition rates. Though the combination of spFRET, FCS and ALEX can help to constrain the fits of correlation curves, a rigorous analysis of the range of lifetimes that can be probed with a combination of these methods is lacking. Here, we simulated spFRET-ALEX-FCS experiments of molecules with two conformations that differ both in FRET levels and in diffusion coefficients, representative of fully wrapped and partially unwrapped nucleosomes. We show that we can distinguish small changes in the diffusion coefficient and that burst selection yields accurate lifetimes ranging from 100 us to 100 ms. The simulations provide a framework that can be expanded for more complex systems having a larger number of conformational states, variable stoichiometries from binding interactions and/or other excitation schemes.

The online version contains supplementary material available at 10.1007/s10895-025-04187-0.