Intramolecular fluorescence correlation spectroscopy in a feedback tracking microscope

TL;DR

This paper develops a theoretical framework for intramolecular fluorescence correlation spectroscopy using feedback tracking microscopy, demonstrating enhanced sensitivity to molecular dynamics and applying it to genomic DNA analysis.

Contribution

It introduces a general expression for fluorescence autocorrelation considering intramolecular dynamics and showcases experimental validation with DNA molecules.

Findings

Tracking enhances sensitivity to diffusion and molecular size.

The derived autocorrelation function applies to linear intramolecular dynamics.

Experimental results on DNA demonstrate the method's effectiveness.

Abstract

We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system, and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity and long time-scale decays in comparison to traditional fluorescence correlation spectroscopy. We demonstrate our approach by using a three-dimensional tracking microscope to study genomic $\lambda$-phage DNA molecules with various fluorescence label configurations.