Fluorescence resonance energy transfer (FRET) from individual, dye-labeled RNA molecules confined in freely-diffusing attoliter-volume aqueous droplets is carefully compared to FRET from unconfined RNA in solution. The use of freely-diffusing droplets is a remarkably simple and highthroughput technique that facilitates a substantial increase in signal-to-noise for single-molecularpair FRET measurements. We show that there can be dramatic differences between FRET in solution and in droplets, which we attribute primarily to an altered pH in the confining environment. We also demonstrate that a sufficient concentration of a non-ionic surfactant mitigates this effect and restores FRET to its neutral-pH solution value. At low surfactant levels, even accounting for pH, we observe differences between the distribution of FRET values in solution and in droplets which remain unexplained. Our results will facilitate the use of nanoemulsion droplets as attoliter volume reactors for use in biophysical and biochemical assays, and also in applications such as protein crystallization or nanoparticle synthesis, where careful attention to the pH of the confined phase is required. V C 2015 AIP Publishing LLC. [http://dx.doi.org/10.1063/1.4921202]Single-molecular-pair fluorescence resonance energy transfer (spFRET) is widely used in molecular biophysics to understand folding, binding, and structural changes in proteins 1 and RNA. 2 In the simplest and most frequently used application of spFRET, fluorescent photons are detected as a molecule diffuses through a femtoliter-volume confocal detection region. The number of photons detected depends on the brightness of the molecule and length of time spent (dwell time) in the detection volume, typically <1 ms. To some extent, the dwell time, and therefore the signal, can be increased by adding sucrose or glycerol to increase the viscosity of the solution. An increase in the detection volume also increases the dwell time, but the gain in signal is offset by a concurrent increase in background that limits this option.Alternatively, the dwell time of a biomolecule can be dramatically increased, and the background minimized, by confinement in a nanocontainer that is larger than the biomolecule but smaller than the detection volume. Two common candidates for molecular confinement are droplets 3 and liposomes; 4 their relative merits have been discussed elsewhere. 5 It has long been assumed that confinement does not perturb FRET measurements; here we test that assumption for molecules confined in water-in-perfluorinated-liquid nanodroplets.The Stokes-Einstein diffusivity for a spherical particle is D ¼ k B T/6pgr, where r is the hydrodynamic radius of the particle, k B is the Boltzmann constant, T is the temperature, and g is the dynamic viscosity. The dwell time s / w 2 /D where w is the waist of the confocal detection volume (260 nm), so that s scales with the radius of the particle and the viscosity of the medium. The droplets used in this study had a log-normal size distribution, with hri ¼ ...