Optically trapped femtoliter containers for single molecule studies

 

A.M. Jofre*1, J. E. Reiner1, A. M. Crawford1, R. B. Kishore1, Lori. S. Goldner1, M. K. Gilson2, K. Helmerson1

1Physics Laboratory, National Institute of Standards and Technology, 100 Bureau Dr., Gaithersburg, MD 20899

2Center for Advanced Research in Biotechnology, 9600 Gudelsky Dr. Rockville, MD 20850

 

 

We demonstrate a novel technique for creating, manipulating, and combining femtoliter volume chemical containers. Possible uses include creating controlled chemical reactions involving small quantities of reagent, and studying the dynamics of single molecules. The containers, which we call hydrosomes, are stable aqueous droplets in a low index-of-refraction fluorocarbon medium. The index of refraction mismatch between the container and fluorocarbon is such that individual hydrosomes can be optically trapped by single focus laser beams, i.e. optical tweezers. We demonstrate the detection of single dye and red fluorescent protein molecules in a hydrosome, and the observation of single pair fluorescence resonance energy transfer (spFRET) from Cy3-Cy5 molecules attached to a single-stranded 16mer DNA molecule. Furthermore, we demonstrate the controlled fusion of two hydrosomes for studying reactions. Previous work on single molecules usually involved the tethering of the molecule to a surface, in order to interrogate the molecule for an extended period of time. The use of hydrosomes opens up the possibility for studying free molecules, away from any perturbing surface, and under non-equilibrium conditions where the local environment is rapidly changed. Preliminary results from polarization spectroscopy studies indicate that single molecules in optically trapped hydrosomes freely diffuse and rotate; however, if we incorporate a surfactant into the hydrosomes, it appears that the rotational motion of the molecules is constrained. As a comparison with another type of femtoliter container, we also perform polarization measurements of single molecules in optically trapped liposomes.

 

 

Fig. 1. Left: Schematic of the confocal set-up for detection of single molecules encapsulated in hydrosomes held stationary by optical tweezers. Right: Counts measured on single photon counting modules (SPCM) of the fluorescence from single molecules contained in individual hydrosomes. The figures from top to bottom correspond to the detection of one, two and three sulfo-rhodamine B dye molecules, respectively.