  Zero-mode waveguides (ZMW) nanoapertures milled in metal films were proposed
to improve the FRET efficiency and enable single molecule FRET detection beyond
the 10 nm barrier, overcoming the restrictions of diffraction-limited detection
in a homogeneous medium. However, the earlier ZMW demonstrations were limited
to the Atto 550 - Atto 647N fluorophore pair, asking the question whether the
FRET enhancement observation was an artefact related to this specific set of
fluorescent dyes. Here, we use Alexa Fluor 546 and Alexa Fluor 647 to
investigate single molecule FRET at large donor-acceptor separations exceeding
10 nm inside ZMWs. These Alexa fluorescent dyes feature a markedly different
chemical structure, surface charge and hydrophobicity as compared to their Atto
counterparts. Our single molecule data on Alexa 546 - Alexa 647 demonstrate
enhanced FRET efficiencies at large separations exceeding 10 nm, extending the
spatial range available for FRET and confirming the earlier conclusions. By
showing that the FRET enhancement inside a ZMW does not depend on the set of
fluorescent dyes, this report is an important step to establish the relevance
of ZMWs to extend the sensitivity and detection range of FRET, while preserving
its ability to work on regular fluorescent dye pairs.
