Rotational Fluorescence Recovery after Orientational Photobleaching via surface electromagnetic waves on dielectric stacks

TL;DR

This paper introduces a novel optical technique using dielectric stacks and surface electromagnetic waves to measure protein rotational dynamics at interfaces, overcoming low signal challenges.

Contribution

It presents Fluorescence Recovery after Orientational Photobleaching, a new rotational measurement method leveraging anisotropic photobleaching and surface waves.

Findings

Successfully measured rotational friction coefficient of neutravidin at surface.

Rotational friction at interface is significantly higher than in bulk solutions.

Method provides a high-sensitivity platform for interfacial biomolecular dynamics.

Abstract

Protein rotational kinetics are essential for understanding macromolecular behavior in crowded environments, yet measuring these dynamics at solid-liquid interfaces remains a significant challenge due to low signal strengths. Here, we experimentally demonstrate a label-based optical technique for measuring rotational diffusion kinetics using an all-dielectric multilayer stack that sustains both transverse electric and transverse magnetic polarized surface electromagnetic waves. We introduce the concept of Fluorescence Recovery after Orientational Photobleaching, a rotational analogue to the standard translatory fluorescence recovery after photobleaching technique, which utilizes anisotropic photobleaching via resonant transverse electric excitation followed by real-time monitoring of the orientational relaxation towards isotropy. Our ratiometric analysis of the transverse electric and magnetic polarized fluorescence components allows for a distance-independent estimation of the rotational friction coefficient. Applying this method to covalently bound neutravidin, we observe a rotational friction coefficient (about 5.8E-18 J s) significantly higher than in bulk solutions, highlighting the impact of surface anchoring and molecular crowding. The proposed approach provides a robust, high-sensitivity platform for resolving biomolecular dynamics in complex interfacial environments.