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

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.