Title: Single Molecule Detection in Optically Trapped Nanocontainers

 

 

Authors  A. M. Crawford, J.E. Reiner, R. Kishore, K. Helmerson

Presenter Alice Crawford

Address  Bldg. 216  stop 842 rm B249  lab F109

Division  Atomic Physics

Telephone 301-975-6867

Fax 301-975-8272

Email alice.crawford@nist.gov

Sigma Xi member:  no

 

Category:  Biotechnology

 

Mentor: Kristian Helmerson

 

We demonstrate a new technique for the study of single molecules.  We confine single molecules inside of a nanocontainer, which can be held in an optical trap.

This technique possesses many advantages over other techniques for studying single molecules.

 By combining the optical trap with a laser excited confocal detection setup, the molecule of interest may be interrogated with high efficiency for an extended period of time. In contrast conventional correlation spectroscopy (FCS) of single molecules can only interrogate molecules for a few milliseconds. Also unlike molecules tethered to a surface, the molecule in the nanocontainer is essentially free, unhindered by surface interactions, and far from any surface that may contribute to background fluorescence.  Currently the nanocontainers we are working with are surfactant stabilized microscopic water droplets in a fluorocarbon medium, which we call hydrosomes.  The particular fluorocarbon we are using has a lower index of refraction than water, which makes the hydrosomes amenable to trapping by a single focused beam laser trap (optical tweezers).  We have been able to detect fluorescence from single dye molecules of several different types (sulforhodamine B, Cy3, TMR, and Cy5). Evidence of the single molecule detection is seen from the photobleaching step of the dye molecule held in the hydrosome.  We have also observed fluorescence resonance energy transfer (FRET) between dyes attached to a DNA complex. We are currently studying the dynamics of single molecules inside of a hydrosome using FRET.