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The Design and Construction of Optical Tweezers to Measure Piconewton Scale Biological Forces Chlamydomonas reinhardtii Chlamydomonas reinhardtii R.P. McCord, J.N. Yukich, and K.K. Bernd, Davidson College, Davidson, North Carolina Abstract Experimental technique Optical tweezers have many applications in measuring biological forces due to their ability to exert piconewton scale forces and to manipulate biological material with minimal damage. This research involves the construction and calibration of a dual-beam optical tweezers laser trap apparatus and the use of this trap to measure the swimming force exerted by the unicellular flagellated algae Chlamydomonas reinhardtii. The data presented will demonstrate the efficacy of this technique of force measurement by comparing the force exerted by wild type Chlamydomonas cells to that exerted by oda1 cells, a mutant strain lacking the entire dynein outer arm of the flagella. This work will both contribute to knowledge of optical tweezers and their applications to investigations of living biological systems. The swimming force of Chlamydomonas reinhardtii cells is measured as follows: -The laser power is set to 1.0 W and the polarizer is adjusted so that all of the light travels through one path creating a maximum power trap spot at the microscope. - A single swimming Chlamydomonas cell is trapped in a sample on a microscope slide. - Using the polarizer, the laser power, and thus the trap force, is decreased until the swimming cell can just escape the trap - This escape power is recorded as directly related to the swimming force exerted by the Chlamydomonas flagella. Origin of Trapping Force Background Preliminary data Lens Arrangement to Obtain Tight Focus Escape Velocity vs. Power y = 5.8671x + 0.6981 Distribution of escape powers Oda1 2 R = 0.9475 Wild Type 200 L1 L2 L3 Objective Lens Fraction escaped Escape Velocity (um/s) 180 160 140 120 100 80 60 40 0.5 0.4 0.3 0.2 0.1 0 0-5 6 to 11 16- 21- 26- 31- 36- 41- 46- 51- >5 10 to 20 25 30 35 40 45 50 55 5 15 20 0 f1 + f2 Figure from: Svoboda and Block, 1994. A dielectric particle is trapped by optical tweezers due to opposing scattering and gradient forces. - The scattering force occurs in the direction of the laser beam and is caused by the collision of photons with the trapped object - The gradient force (shown in diagram) is caused by the refraction of the laser light ray through the object. The force is equal and opposite to the change in momentum of the beam. As shown, an object will be pushed toward the brightest part of the beam and held in place at a tight focus. f3 0 16 cm 5 10 15 20 25 30 Power (mW) Power range (mW) Image created from Physlet at http://webphysics.davidson.edu/Course_Material/Py230L/optics/lenses.htm Physlet by Dr. Wolfgang Christian and Mike Lee The best trap is obtained when the maximum light gradient is obtained. This corresponds to a very tight focus, which is obtained with four lenses. - L1 and L2 form a telescope to expand the laser beam to fill the back of the microscope. - L3 focuses the beam 16 cm away from the back of the objective lens. - The objective lens has a high numerical aperture which creates a tight focus of the beam. This graph shows a preliminary calibration of the optical trap. Dead Chlamydomonas cells were dragged with the piezoelectric stage through the viscous fluid. The velocity at which the viscous drag force caused the cell to escape from the trap is directly proportional to the optical force at that corresponding optical power. The data follow a linear trend. Strain Sample Size Average Power (mW) wild type 93 43.85 oda1 86 8.65 This graph shows the distribution of escape powers recorded for mutant oda1cells and wild type Chlamydomonas cells. The table below the graph gives the sample size and average escape power for each strain. Preliminary Conclusions - The force exerted on a Chlamydomonas cell by the optical trap is linearly related to the laser power. Apparatus - As expected for a living system, the swimming force exerted by individual Chlamydomonas cells is highly variable. - Despite this variation, the average measured swimming forces and distribution of swimming forces demonstrate a clear difference between the dynein deficient oda1 mutant strain and the wild type strain. The mutant oda1 cells, which lack an important component of their flagella, exert a smaller swimming force than do wild type cells. Future Work -This optical method of force measurement will be used to examine whether flagella regenerated by Chlamydomonas after acid-induced deflagellation or flagella resorbtion are functionally equivalent to the original flagella in force production. -This force measurement may be used to investigate the phenomenon of chemotaxis or phototaxis and the swimming forces exerted by cells attracted to such stimulants. -The dual beam optical trap may be used for other Chlamydomonas investigations, such as a measurement of the adhesive forces between the cells during mating agglutination. Active Layer References Laser Trap Setup This photograph of the optical tweezers setup shows the 1.0 W Nd:YAG laser in the foreground. The lenses and mirrors that direct and modify the beam are seen behind this laser, followed by the microscope for sample manipulation and the camera and television screen which are used to view the trapped samples. This schematic diagram of the apparatus shows the splitting of the laser beam with the combination of a polarizer and two beam-splitting cubes. This creates two independently manipulable traps. The diagram also shows the path of the beam through the lenses and microscope. 1. Ashkin, A. (1997). Optical trapping and manipulation of neutral particles using lasers. Proc Natl Acad Sci U S A. 94, 4853-60. 2. Konig, K., Svaasand, L., Liu, Y., Sonek, G., Patrizio, P., Tadir, Y., Berns, M.W., Tromberg, B.J. (1996). Determination of motility forces of human spermatozoa using an 800 nm optical trap. Cellular and Molecular Biology (Noisy-le-grand). 42, 501-9. 3. Mammen, M., Helmerson, K., Kishore, R., Choi, S. (1996). Optically controlled collisions of biological objects to evaluate potent polyvalent inhibitors of virus-cell adhesion. Chemistry and Biology 3, 757-763. 4. Minoura, I., Kamiya, R. (1995). Strikingly Different Propulsive Forces Generated by Different Dynein-Deficient Mutants in Viscous Media. Cell Motil. Cytoskel. 31, 130-139. 5. Smith, S.P., Bhalotra, S.R., Brody, A.L., Brown, B.L., Boyda, E.K., Prentiss, M. (1999). Inexpensive Optical Tweezers for Undergraduate Laboratories. Am. J. Phys. 67, 26-34. 6. Svoboda K., and Block S. (1994) Biological applications of optical forces. Ann. Rev. Biophys. Biomol. Struct. 23, 247-285. Acknowledgements This work has been supported by: Davidson College, The National Institute of Standards and Technology, and The Duke Foundation