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Zero-mode waveguide (ZMW) nanoapertures are widely used to monitor singlemolecules beyond the range accessible to normal microscopes. However, severalaspects of the ZMW influence on the photophysics of fluorophores remaininadequately documented and sometimes controversial. Here, we thoroughlyinvestigate the ZMW influence on the fluorescence of single immobilized Cy3Band Alexa 647 molecules, detailing the interplays between brightness, lifetime,photobleaching time, total number of emitted photons and F\"orster resonanceenergy transfer (FRET). Despite the plasmonic-enhanced excitation intensity inthe ZMW, we find that the photostability is preserved with similarphotobleaching times as on the glass reference. Both the fluorescencebrightness and the total numbers of photons detected before photobleaching areincreased, with an impressive gain near five times found for Alexa 647 dyes.Finally, the single-molecule data importantly allow a loophole-freecharacterization of the ZMW influence on the FRET process. We show that theFRET rate constant is enhanced by 50%, demonstrating that nanophotonics canmediate the energy transfer. These results deepen our understanding of thefluorescence enhancement in ZMWs and are of immediate relevance forsingle-molecule biophysical applications.

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