Fluorescence Brightness, Photostability and Energy Transfer Enhancement of Immobilized Single Molecules in Zero-Mode Waveguides Nanoapertures

TL;DR

This study investigates how zero-mode waveguides (ZMWs) affect the fluorescence properties of single molecules, revealing enhanced brightness and energy transfer without compromising photostability, thus advancing single-molecule biophysics techniques.

Contribution

The paper provides a comprehensive analysis of ZMW influence on fluorophore photophysics, including brightness, photostability, and FRET, with new quantitative insights into energy transfer enhancement.

Findings

Fluorescence brightness and photon counts increase significantly in ZMWs.

Photostability remains comparable to glass references despite plasmonic effects.

FRET rate constant is enhanced by 50% in ZMWs.

Abstract

Zero-mode waveguide (ZMW) nanoapertures are widely used to monitor single molecules beyond the range accessible to normal microscopes. However, several aspects of the ZMW influence on the photophysics of fluorophores remain inadequately documented and sometimes controversial. Here, we thoroughly investigate the ZMW influence on the fluorescence of single immobilized Cy3B and Alexa 647 molecules, detailing the interplays between brightness, lifetime, photobleaching time, total number of emitted photons and F\"orster resonance energy transfer (FRET). Despite the plasmonic-enhanced excitation intensity in the ZMW, we find that the photostability is preserved with similar photobleaching times as on the glass reference. Both the fluorescence brightness and the total numbers of photons detected before photobleaching are increased, with an impressive gain near five times found for Alexa 647 dyes. Finally, the single-molecule data importantly allow a loophole-free characterization of the ZMW influence on the FRET process. We show that the FRET rate constant is enhanced by 50%, demonstrating that nanophotonics can mediate the energy transfer. These results deepen our understanding of the fluorescence enhancement in ZMWs and are of immediate relevance for single-molecule biophysical applications.