The Biological Physics Training Laboratory Contacts Projects Biophysical forces   and Laser tweezing Electrophysiology Biological Pattern   Formation Biological Fluid   Dynamics Biology, Mathematics and Physics Initiative For further information contact Applied Mathematics (520-621-2016)

The University of Arizona Biological Physics Teaching Laboratory Biophysical Forces Optical Tweezing and Kinesin Motility Kinesin is a molecular motor that converts the energy derived from adenosine triphosphate (ATP) hydrolysis into mechanical work and moves along polymeric tubes of the protein tubulin. These tubes, called microtubules, are found in virtually all eucaryotic cells and provide lines of transport and communication between different levels in the cell. Thus kinesin functions as an intracellular transport vehicle, binding to organelles or supply vesicles and carrying them down microtubules to all parts of the cell. While the details of how chemical energy stored in ATP is released as mechanical energy in kinesin stepping remain unknown, it is clear that kinesin hydrolyses one ATP molecule for each 8 nm step forward. In this module, students investigate kinesin velocity, measured in steps taken per second, as a function of ATP concentration. An image captured by differential interference contrast (DIC) microscopy, shows a small glass bead (diameter ~1 um) which is being carried by Kinesin molecular motor(s) along polymeric tubes of the protein tubulin (the thin strands). Although the kinesin motor is far too small to be observed with standard microscopy techniques, the activity of kinesin motors is still possible to observe thanks to the fact that the kinesin tail binds well to materials like glass. Individual, or collections of motors, can be made to carry a small glass bead (diameter ~1 um) which can be easily observed using differential interference contrast (DIC) microscopy (right). Knowledge of the techniques of laser tweezing, microtubule polymerization and the preparation of a kinesin motility assay are required before measurements commence. Data are captured onto VHS tape via CCD camera from DIC microscopy observations. The frame-by-frame position of transported beads is analyzed with computer software. Brownian motion, Stokes flow, and Activated processes Thermal fluctuations are ubiquitous at the cellular level and below, yet it is rare for students in the biological sciences to see quantitative treatments of their effects. Likewise, physicists and mathematicians interested in the biological world often study stochastic phenomena in the abstract, with no true sense of their significance. The goal of this core experimental module is to use the techniques of video microscopy and optical trapping to quantify several phenomena associated with Brownian motion. A small latex bead (diameter ~1 um) is shown at high magnification.Brownian motion affects the bead position over time. A first goal is to learn the principles of optical trapping "laser tweezers", used also in other experiments, and calibrate traps of varying intensity through Stokes drag on latex beads of varying sizes (left). The fluctuations of those beads in the trap will be compared with theoretical calculations using the basic principles of statistical mechanics. Observations of Brownian motion in the absence of a trap will be used to determine Avogadro's number from the universal gas constant, along the lines of the original Einstein work on Perrin's results. Fluids of different viscosities and particles of different sizes will be used to check the This collection of optical components (mirrors, lenses, and beamsplitters) is used to split an infrared laser beam into to components of with orthogonal polarization, to control the position of the two beams, and to recombine them before they are focused by the microscope objective at the sample plane. The result is two steerable optical traps which can capture and manipulate microscopic objects. Stokes-Einstein relation (fluctuation-dissipation theorem). Finally, a double trap has been constructed (right) to examine Kramers' theory, which attempts to characterize a chemical reaction by the rate at which a Brownian particle can move over the energy barrier of a one-dimensional double-well potential. Experimentally, such a potential is readily obtained by using two optical traps positioned closely together. By video-recording the images a glass particle (~500 nm in diameter) jumping from one optical trap into the other, and subsequent Particle tracking (below left) using a dedicated image-processing computer, the transition rate can be determined, and Kramers' results can be experimentally verified. A collection of data points that are samples of the position of a microsphere over time that hops back and forth between two nearby optical traps of approximately equal depth.

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Last modified October 2004

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