Single-molecule Force Studies
Wesley P. Wong, P.I.
Single-molecule techniques enable complex biomolecular interactions to be studied in a detailed way. In particular, molecular transitions can be observed directly rather than inferred from ensemble averages, and systems can be studied out of equilibrium and under force. Such investigations require the use of high-precision ultrasensitive force probes with nanometer length and piconewton force resolution.
We have developed a unique dual-mode optical tweezers system designed to explore both forward and reverse biomolecular transitions (e.g bond rupture &formation, protein unfolding & refolding). Our system incorporates a high-resolution 3D particle tracking technique based on reflection-interference imaging, as well as feedback systems which result in longtime stability of 1-2 nm. The system is described in more detail below.
In the two complimentary modes of operation, a functionalized glass probe bead is held by an optical trap near a reactive substrate, allowing single molecular tethers to bridge the two surfaces. The nanoscale motion of the probe bead reports molecular transitions such as bond rupture/formation and protein unfolding/refolding. The optical trap acts as a tunable spring, allowing precise and controllable application of force to the probe bead, and thus the molecular tether.
3D high-resolution, feedback-stabilized optical tweezers
1:00 AM
Our optical tweezers system incorporates high-resolution 3D particle tracking with active feedback for longterm stability, to enable the measurement of both forward and reverse molecular transitions, and near-equilibrium phenomena.
Horizontal Mode
... for “far-from-equilibrium” studies
(e.g. bond rupture, protein unfolding)
Vertical Mode
... for “near-equilibrium” studies
(e.g. bond formation, protein refolding)
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• High-speed 1D tracking
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-Real-time video tracking and edge-detection
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-~2 nm resolution@ 2500 samples/s
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• High-resolution 3D tracking
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-Reflection Interference Contrast Microscopy
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-~0.2 nm (z), ~1 nm (x-y) resolution @ 200 samples/s
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-Absolute distance measurement ideal for small (nm) distances
3D tracking example: bead positions for short spectrin tether with planar slice (inset)
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• Active feedback for high-repeatability
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-Feedback loops for touch control, force application, and bead alignment
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• Active feedback for high-stability
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-Continuous autofocus system corrects for slow drift
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-long-term stability: 1-2 nm in trap height, 10 fN in force
relevent group publications
V. Heinrich*, W.P. Wong*, K. Halvorsen, and E. Evans, “Imaging biomolecular interactions by fast three-dimensional tracking of laser-confined carrier particles,” Langmuir 24, 1194-1203 (2008). [pdf]
K. Halvorsen, “Probing weak single-molecule interactions: development and demonstration of a new instrument,” Ph. D Thesis (advisor: E. Evans), Department of Biomedical Engineering, Boston University, USA (2007). [pdf]
W.P. Wong, “Exploring single-molecule interactions through 3D optical trapping and tracking: from thermal noise to protein refolding,” Ph. D Thesis (advisors: D.R. Nelson and E. Evans), Department of Physics, Harvard University, USA (2006). [pdf]
W.P. Wong, V. Heinrich, and E. Evans, “Exploring reaction pathways of single-molecule interactions through the manipulation and tracking of a potential-confined microsphere in three dimensions,” Mat. Res. Soc. Symp. Proc. 790, P5.1.1-P5.1.12 (2004). [pdf]
*contributed equally