FRET Enhancement in Aluminum Zero‐Mode Waveguides

Zero‐mode waveguides (ZMWs) can confine light into attoliter volumes, which enables single molecule fluorescence experiments at physiological micromolar concentrations. Of 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 concentrations 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 that diffuse into aluminum zero‐mode waveguides. Aluminum ZMWs are important structures thanks to their commercial availability and the large amount of literature that describe their use for single‐molecule fluorescence spectroscopy. We also compared the results between ZMWs milled in gold and aluminum, and found that although 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 observed that the FRET rate scales linearly with the isolated donor decay rate and the local density of optical states. Detailed information about FRET in ZMWs unlocks their application as new devices for enhanced single‐molecule FRET at physiological concentrations.