Green fluorescent protein in inertially injected aqueous nanodroplets

We inertially inject and study the contents of optically trappable aqueous nanodroplets (hydrosomes) emulsified in a perfluorinated matrix. A new piezoelectric actuated device for production of single hydrosomes on demand is introduced. Hydrosomes containing enhanced green fluorescent protein (EGFP) were injected, optically trapped, and held at the focus of an excitation laser in a confocal microscope, and single-molecule photobleaching events were observed. The rotational diffusion time of EGFP in trapped hydrosomes was measured using time-resolved fluorescence anisotropy. In free solution, the mean rotational diffusion time was determined to be 13.8 A? 0.1 ns at 3 I?M and 14.0 A? 0.2 ns at 10 I?M. In hydrosomes, the mean rotational diffusion time was similar and determined to be 12.6 A? 1.0 ns at 3 I?M and 15.5 A? 1.6 ns at 10 I?M.