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An integrated laser trap/flow control video microscope for the study of single biomolecules
G J Wuite1, R J Davenport, A Rappaport
1Department of Physics, University of California, Berkeley, California 94720 USA.
Biophysical Journal
|August 2, 2000
Summary
We developed an automated laser trap and flow control microscope for precise mechanical manipulation of single biopolymers. This system accurately measures DNA elasticity and observes molecular processes like RNA polymerase translocation.
Area of Science:
- Biophysics
- Molecular Biology
- Instrumentation
Background:
- Precise mechanical manipulation of single biopolymers is crucial for understanding their function.
- Existing techniques may be limited by laser-induced damage or lack of precise force control.
- High-throughput methods are needed to accelerate biopolymer research.
Purpose of the Study:
- To develop and validate an integrated laser trap and flow control video microscope for mechanical manipulation of single biopolymers.
- To enable precise force application and displacement measurement for biopolymer characterization.
- To provide a versatile platform for studying biological systems, especially those sensitive to laser damage.
Main Methods:
- An automated single-beam optical trap was integrated with a computer-controlled flow system within a microfluidic chamber.
- Micron-scale polystyrene beads, held by optical tweezers and micropipette suction, served as attachment points for macromolecules.
- Forces were applied using the optical trap and controlled fluid flow, with displacements measured by video microscopy or laser deflection.
Main Results:
- The instrument successfully assembled and characterized the elasticity of individual biotinylated DNA molecules between streptavidin-coated beads.
- DNA extension was accurately measured under varying forces applied by both the laser trap and fluid flow.
- The system demonstrated its utility by observing the translocation of transcribing RNA polymerase for extended periods (up to 650 s).
Conclusions:
- The developed integrated laser trap/flow control video microscope offers automated, high-throughput mechanical manipulation of single biopolymers.
- This versatile instrument minimizes laser-induced damage, making it ideal for studying sensitive biological processes.
- The system provides a robust platform for detailed biopolymer mechanics studies and real-time observation of molecular motors.