  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.
