Authors: Francesco Michelotti, Elisabetta Sepe, Agostino Occhicone, Norbert Danz, Alberto Sinibaldi

Citations: N/A

Published: 2026-04-02T10:17:26Z

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.