Hydrosomes: femtoliter containers for fluorescence spectroscopy studies

Abstract

We report on improvements and innovations in the use of hydrosomes to encapsulate and study single molecules. Hydrosomes are optically-trappable aqueous nanodroplets. The droplets are suspended in a fluorocarbon medium that is immiscible with water and has an index of refraction lower than water, so hydrosomes are stable and optically trapped by a focused laser beam (optical tweezers). Using optical tweezers, we hold the hydrosomes within a confocal observation volume and interrogate the encapsulated molecule by fluorescence excitation. This method allows for long observation times of a molecule without the need for surface immobilization or liposome encapsulation. We have developed a new way for creating hydrosomes on demand by inertially launching them into the fluorocarbon matrix using a piezo-activated micropipette. Time-resolved fluorescence anisotropy studies are carried out to characterize the effects of the hydrosome interface boundary on biological molecules and to determine whether molecules encapsulated within hydrosomes diffuse freely throughout the available volume. We measured the fluorescence anisotropy decay of 20mer DNA duplexes, and enhanced green fluorescent protein (GFP). We conclude that the molecules rotate freely inside the nanodroplets and do not stick or aggregate at the boundary