trapping.pdf - Physics 433/833 2014 Optical trapping Optical trapping ABSTRACT Optical trapping is a technique widely used to probe mechanical responses

trapping.pdf - Physics 433/833 2014 Optical trapping...

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Physics 433/833, 2014 Optical trapping Optical trapping ABSTRACT Optical trapping is a technique widely used to probe mechanical responses in modern biophysics experiments. It uses a focused laser beam to trap microscopic particles, which can be various types of refractive objects. Cells can be trapped at the focus; more com- mon current experiments use micron-sized spheres, to which biological molecules can be biochemically attached for manipulation. Single-molecule manipulation experiments using optical tweezers have provided a great deal of insight into many biological systems, including the mechanisms of molecular motors (including myosin, kinesin, and DNA and RNA poly- merases), the elastic response of DNA, and the unfolding pathways of proteins and nucleic acids. I. OBJECTIVES Learn the physics behind how an optical trap works Apply different techniques to determine the stiffness of an optical trap Learn about how thermal fluctuations impact a bead’s motion, and how an optical trap attenuates the bead’s motion at certain frequencies Determine how experimental variables can be used to change the stiffness of an optical trap Learn how power spectral analysis can be used to determine the rotation frequency of E. coli II. BACKGROUND Optical trapping works based on the change in momentum of light arising from its inter- action with a dielectric object. By tightly focussing a laser beam, the resultant high gradient in the electric field of the light results in a strong attraction of objects towards the focal region. Thus, near the focus, such objects can be attracted and held in three dimensions. A. Optical tweezers in biophysics experiments Since their invention by Arthur Ashkin in 1986 [ 1 ], optical tweezers have become increas- ingly widely used as an important tool in biophysics experiments. Shown in Fig. 1 are just a few of the many experiments that have utilized optical tweezers to better understand biolog- ical systems. These include probing the elasticity of DNA (and developing polymeric models 1 © 2012–4, NF and JB
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Physics 433/833, 2014 Optical trapping to describe its mechanical response), characterizing the activity and mechanisms underlying various molecular motors in the cell, determining the unfolding pathways of proteins and RNA and measuring the response of cells to stress. FIG. 1. Some of the types of biophysics experiments made possible with optical tweezers. Clockwise from upper left: stretching single molecules (e.g., DNA); protein unfolding [ 2 ]; characterizing the elasticity of red blood cells [ 3 ]; T-cell activation (Beckman Laser Institute, UC Irvine); and probing the mechanism of cellular molecular motors such as kinesin (Block lab, Stanford University). Optical tweezers have proven to be particularly suitable for probing the response of biological systems to applied forces, given the range of forces they typically exert. The biophysicist’s energy unit is k B T , where k B is Boltzmann’s constant and T is temperature (in degrees Kelvin). This represents the average thermal energy available to a system. In
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