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Fluorescence energy transfer enhancement in aluminum nanoapertures

Zero-mode waveguides (ZMWs) are confining light into attoliter volumes, enabling single molecule fluorescence experiments at physiological micromolar concentrations. Among the fluorescence spectroscopy techniques that can be enhanced by ZMWs, Förster resonance energy transfer (FRET) is one of the most widely used in life sciences. Combining zero-mode waveguides with FRET provides new opportunities to investigate biochemical structures or follow interaction dynamics at micromolar concentration with single molecule resolution. However, prior to any quantitative FRET analysis on biological samples, it is crucial to establish first the influence of the ZMW on the FRET process. Here, we quantify the FRET rates and efficiencies between individual donor-acceptor fluorophore pairs diffusing in aluminum zero-mode waveguides. Aluminum ZMWs are important structures thanks to their commercial availability and the large literature describing their use for single molecule fluorescence spectroscopy. We also compare the results between ZMWs milled in gold and aluminum, and find that while gold has a stronger influence on the decay rates, the lower losses of aluminum in the green spectral region provide larger fluorescence brightness enhancement factors. For both aluminum and gold ZMWs, we observe that the FRET rate scales linearly with the isolated donor decay rate and the local density of optical states (LDOS). Detailed information about FRET in ZMWs unlocks their application as new devices for enhanced single molecule FRET at physiological concentrations.