Abstract
Publisher Summary This chapter discusses the applications of optical traps in vivo. A number of groups have combined sophisticated optical-trapping setups with bead assays to study the function of single molecules in vitro in detail. The basic optical trap is quite simple: a single-mode laser with Gaussian beam profile (TM 00 ) is passed through a beam expander (so that the beam fills the back aperture of the microscope objective to use the entire lens, which generates the largest gradient in the focused beam) and then steered with a combination of mirrors and lenses into the microscope objective. This objective focuses the light in the sample plane of the microscope to a diffraction limited spot, forming the trap. In vitro optical traps are used to study polymers, such as microtubules or DNA, and to measure properties of single molecules, such as titin, or molecular motors, such as kinesin. To calibrate the applied force involves initial in vitro calibration, followed by corrections to compensate for the differences between working in buffer and in the relatively uncontrolled cytoplasm.
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