Transcript poster - Davidson College

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.
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2.
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Mammen, M., Helmerson, K., Kishore, R., Choi, S. (1996). Optically controlled collisions of biological objects to evaluate potent polyvalent inhibitors of virus-cell
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4.
Minoura, I., Kamiya, R. (1995). Strikingly Different Propulsive Forces Generated by Different Dynein-Deficient Mutants in Viscous Media. Cell Motil. Cytoskel. 31,
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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.
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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