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Fluorescence resonance energy transfer (FRET) from individual, dye-labeledRNA molecules confined in freely-diffusing attoliter-volume aqueous droplets iscarefully compared to FRET from unconfined RNA in solution. The use offreely-diffusing droplets is a remarkably simple and high-throughput techniquethat facilitates a substantial increase in signal-to-noise forsingle-molecular-pair FRET measurements. We show that there can be dramaticdifferences between FRET in solution and in droplets, which we attributeprimarily to an altered pH in the confining environment. We also demonstratethat a sufficient concentration of a non-ionic surfactant mitigates this effectand restores FRET to its neutral-pH solution value. At low surfactant levels,even accounting for pH, we observe differences between the distribution of FRETvalues in solution and in droplets which remain unexplained. Our results willfacilitate the use of nanoemulsion droplets as attoliter volume reactors foruse in biophysical and biochemical assays, and also in applications such asprotein crystallization or nanoparticle synthesis, where careful attention tothe pH of the confined phase is required.

Single-Molecule-Sensitive FRET in Freely-Diffusing Attoliter Droplets