Manipulating single molecules in solution
The Anti-Brownian ELectrokinetic trap (ABEL trap) is a new device for trapping and manipulating single molecules in solution. It couples fluorescence microscopy to real-time electrokinetic feedback to control the motion of individual molecules in solution at room temperature. By actively canceling the molecule's Brownian motion, the length of time it spends in the observation volume, and thus the length of time it can be studied, is greatly extended. The ABEL trap has been used to trap and study fluorescent quantum dots, molecules of DNA, fluorescently labeled lipid vesicles, single virus particles, single proteins, and even single dye molecules.
Early work with W. E. Moerner, Stanford
An early trap design uses a CCD camera connected to a computer to track the Brownian motion of a single molecule. The molecule's position is determined via real-time image fitting. The computer applies to the solution a time-varying feedback voltage so that the electrophoretic and electroosmotic drifts combine to cancel the Brownian motion.
|This is a picture of a single 200 nm diameter fluorescent polystyrene nanosphere in the ABEL trap.|
By analyzing the statistics of the feedback voltages it is possible to learn about the transport coefficients (diffusion coefficient and electrokinetic mobility) of a particle in the trap. After watching a particle in the trap, we can "undo" the effect of the feedback to reconstruct a pseudo-free trajectory statistically similar to the one it would have followed had it not been trapped.
|The picture on the left shows the actual trajectories of 13 particles of Tobacco Mosaic Virus (TMV) held in the trap (lower right), and the statistically reconstructed pseudo-free trajectories for each of those particles.|
The most recent trap design uses a field-programmable gate array (FPGA) to scan a confocal laser spot. Fluorescence photons are collected by an avalanche photodiode (APD) with single-photon sensitivity, and the position of the laser at their time of detection is used to estimate the particle's position. This design vastly decreases the trap latency relative to CCD-based detection, enabling trapping of smaller, faster-moving molecules. The FPGA tracks the molecule's position in real-time using a recursive Bayesian estimator, and applies appropriate feedback voltages to cancel the observed motion.
- Alex Fields poster presentation at 2009 Biophysical Society Meeting, March 1, 2009
- Adam Cohen's PhD thesis, Trapping and Manipulating Single Molecules in Solution (formatted for single-sided printing), (formatted for double-sided printing), Stanford University, defended Aug. 2006
- Adam Cohen and W. E. Moerner, Controlling Brownian motion of single protein molecules and single fluorophores in aqueous buffer, Optics Express, 16, 6941-6956 (2008)
- Adam Cohen and W. E. Moerner: Suppressing Brownian motion of individual biomolecules in solution, PNAS, 103, 4362-4365, 14 March 2006. Journal link and Supporting Material and Movies.
- Adam Cohen and W. E. Moerner: An all-glass microfluidic cell for the ABEL trap: fabrication and modeling, Proc. SPIE vol. 5930, 2005
- Adam Cohen and W. E. Moerner: The Anti-Brownian Electrophoretic Trap (ABEL Trap): Fabrication and Software, Proc. SPIE vol. 5699, 2005
- Adam Cohen and W. E. Moerner: Method for Trapping and Manipulating Nanoscale Objects in Solution , Appl. Phys. Lett. 86, 093109, Feb. 28 2005
Supporting info and press:
- Robert Brown's original paper on Brownian motion. Fascinating reading!
- Editor's Choice: Canceling brownian motion, Science, v. 307, p. 1694 (18 March 2005)
- Research Highlights: Stop for brownian motion, Nature, v. 434, p. 156 (10 March 2005)
- ScienceNow: Building a better molecule trap, 18 Feb. 2005
- News story on German public radio, 2 June 2006, and transcript
- Analytical Currents: Manipulating nanoscale objects in solution, Analytical Chemistry, v. 77, p. 158A (1 May 2005)
- Movie (680 k) of a single particle of Tobacco Mosaic Virus being manipulated in the ABEL trap. The virus particle is 15 nm wide and 300 nm long and is covalently labeled with Cy3. Note that midway through the movie the trapped particle interchanges with a different particle.
- Movie (3.1 M) of a single fluorescent CdSe quantum dot held in the ABEL trap.
- Movie (845 k) of a single molecule of the chaperonin GroEL held in the ABEL trap. The molecule starts with ~6 fluorescent molecules of Cy3, which photobleach one by one until the molecule becomes too dim to track and is lost from the trap. This experiment was performed in a solution of 50% glycerol to slow down the diffusion of the GroEL enough for the tracking and feedback to keep up with its motion.
- Movie (8.5 M) of single molecules of double-stranded l-DNA being manipulated in the ABEL trap. The red "+" indicates the target position which is being controlled by the computer mouse.
- Movie (4.0 M) of some particles in the trap subject to an arbitrary waveform (make sure sound is on!)