Researchers at the National Institute of Standards and Technology in Gaithersburg, Md., have developed a technique to contain single protein molecules within optically trappable aqueous nanodroplets. The method is minimally destructive to the protein, and it offers an alternative to surface attachment or lipid encapsulation. The scientists developed a piezoelectric actuated micropipette that inertially injected individual nanodroplets containing enhanced GFP into an immiscible perfluorotriamylamine matrix. Each droplet produced had a diameter of ≥700 nm. The drops could be optically trapped by optical tweezers because the refractive index of the matrix is lower than that of water.To determine how well the technique worked, the investigators needed to study the fluorescence of the GFP in the droplets. Thus, they optically trapped the droplets in the detection volume of a confocal microscope. A 1064-nm laser from IPG Photonics Corp. of Oxford, Mass., formed the optical trap. To show that single GFP molecules were confined, they excited the fluorescent protein with 488-nm CW laser emission. A PerkinElmer avalanche photodiode detected the fluorescence. The researchers observed fluorescence followed by a single photobleaching event, showing that just one molecule was present.They then studied how confinement affected the GFP dynamics to make sure that the molecule was freely diffusing and was not sticking to the water-surfactant-oil interface. To this end, they measured the rotational diffusion time of the molecule using time-resolved fluorescence anisotropy. The setup for these measurements included a mode-locked Coherent Ti:sapphire laser that was frequency-doubled to 461-nm, two photon-counting avalanche diodes from Micro Photon Devices in Bolzano, Italy, and single-photon-counting electronics from Becker & Hickl in Berlin. Measurements showed that GFP in solution had a mean rotational diffusion time of 13.8 ±0.1 ns at 3 μM and 14.0 ±0.2 ns at 10 μM. GFP in the nanodrops had a similar mean rotational diffusion time of 12.6 ±1.0 ns at 3 μM and 15.5 ± 1.6 ns at 10 μM. From these numbers, the researchers concluded that the rotational motion inside the nanodroplets is consistent with rotation in free solution and, therefore, the protein does not aggregate at the interface. The work was published online March 27 by Langmuir.