Anal. Chem. 1998, 70, 3513-3515
Correspondence
The “Optical Funnel”. A Technique for Measuring a Microorganism’s Power Naoki Mishima, Takashi Kaneta, and Totaro Imasaka*
Department of Chemical Science and Technology, Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
A novel and potentially useful optical technique called an “optical funnel” has been developed. The technique utilizes a system in which a microorganism is suspended by equilibrium between the medium flow and the radiation pressure applied in the opposite direction. At equilibrium, i.e., when the force of the laser pressure is equal to the force of the moving stream, the microorganism is captured. At this point, it has the ability to escape on its own power. Since the force applied to the microorganism is calculated on the basis of a ray-optics model, it is possible to measure the magnitude of the microorganism’s power. A Trachelomonas volvocina cell was determined to have a power of ∼1 pN. This technique is superior to the laser trapping technique, which is currently used in these determinations, since it is possible to capture numerous organisms in a single experiment, thus allowing the calculation of the average power for an organism with minimal effort, in terms of repetitive samplings and measurements. In biological and medical areas, the manipulation of microspheres, such as living cells and bacteria, remains a challenging technique. Ashkin developed a new optical technique for the manipulation of a microsphere using radiation pressure, induced by a laser,1 and this technique is currently used in certain areas of biological research, such as in the manipulation of actively swimming bacteria2 and sperm cells3,4 as well as in measurements of the mechanical compliance of bacterial flagella.5 However, this approach is successful only to a limited extent; for example, only a single cell was monitored in a single experiment. Recently, we proposed a combination of laser radiation pressure and capillary flow in the development of a new technique, the “optical funnel”, which is similar to optical chromatography, a well-known separa* To whom correspondence should be addressed (tel., 81-92-642-3563; fax, 81-92-632-5209; e-mail,
[email protected]). (1) Ashkin, A. Phys. Rev. Lett. 1970, 24, 156-159. (2) Ashkin, A.; Dziedzic, J. M.; Yamane, T. Nature 1987, 330, 769-771. (3) Ko ¨nig, K.; Svaasand, L.; Liu, Y.; Sonek, G.; Patrizio, P.; Tadir, Y.; Berns, M. W.; Tromberg, B. J. Cell. Mol. Biol. 1996, 42, 501-509. (4) Tadir, Y.; Wright, W. H.; Vafa, O.; Ord, T.; Asch, R. H.; Berns, M. W. Fertil. Steril. 1989, 52, 870-873. (5) Block, S. M.; Blair, D. F.; Berg, H. C. Nature 1989, 338, 514-518. S0003-2700(98)00199-1 CCC: $15.00 Published on Web 06/20/1998
© 1998 American Chemical Society
tion technique.6-8 The optical funnel has several advantages in the measurement of a microorganism’s power. The optical funnel is potentially useful for measuring the powers of many microorganisms in a single experiment. Therefore, it is suitable for determining the average value of their activities, which are individually different from each other, thus improving the accuracy in the evaluation. In addition, the optical damage of the microorganism is minimum in the optical funnel, which is also important in order to improve the accuracy in the measurement of the microorganism’s power. To the contrary, in laser trapping, the actively swimming microorganism is manually trapped by tightly focusing the laser beam, and the minimum laser power necessary to hold the microorganism is measured. Consequently, the microorganism is exposed to a larger extent of laser radiation for a longer time period. In the optical funnel, the light density is smaller and the exposure time is shorter since the microorganism escapes from a weaker laser radiation field immediately. Thus, the optical funnel is expected to give a more accurate value for a microorganism’s power. In this report, we describe some experiments which demonstrate the utility of this technique in measuring an microorganism’s power. Figure 1 shows the experimental apparatus for the optical funnel. An argon ion laser, emitting at 514.5 nm, is used as a light source. The laser beam is focused into a quartz cell using a lens with a 50-mm focal length. A sample containing the microorganism to be examined is introduced from the opposite side of the channel. The motion of the microorganism is observed by a videoscope consisting of a charge-coupled device (CCD camera). Microorganisms which are introduced into the cell are pushed by the radiation pressure generated by the laser light and simultaneously pulled by the force induced by a medium flow. The former is exerted on a microorganism that has a larger refractive index than the surrounding medium. This force originates from a momentum change caused by reflection and refraction at the surface of the microorganism. Consequently, both a scattering force and a gradient force are generated. The scattering force accelerates the microorganism in the direction (6) Imasaka, T.; Kawabata, Y.; Kaneta, T.; Ishidzu, Y. Anal. Chem. 1995, 67, 1763-1765. (7) Kaneta, T.; Ishidzu, Y.; Mishima, N.; Imasaka, T. Anal. Chem. 1997, 69, 2701-2710. (8) Hatano, T.; Kaneta, T.; Imasaka, T. Anal. Chem. 1997, 69, 2711-2715.
Analytical Chemistry, Vol. 70, No. 16, August 15, 1998 3513
Table 1. Determination of the Q Value for Trachelomonas volvocina size (µm)
laser power (W)
za (µm)
Q
12.5 12.5 12.0 12.0
0.35 0.29 0.35 0.29
2.05-2.10 1.61-1.66 1.82-1.87 1.55-1.58
0.12-0.13 0.09-0.10 0.10-0.11 0.09-0.09
a
Distance between the beam waist and the trapped position.
Figure 1. Experimental apparatus for the optical funnel.
Figure 2. Picture taken for determination of dimensionless factor Q. A microorganism is trapped at a position at which the laser radiation force is equilibrated with the force induced by the medium flow. A large laser power is used to kill the microorganism for determination of Q. No aggregation of bacteria was observed in this study, and this value is calculated by assuming the microorganism to be a single cell. The velocity of the medium flow is determined by intercepting the laser beam and measuring the velocity of the particle flowing at the center line.
of the incident beam, and the gradient force attracts the microorganism to the center line of the beam. The medium flow pushes the microorganism downstream, i.e., in a direction opposite to that of the laser beam. The microorganism is trapped at the position where the scattering force is identical to the force induced by the medium flow. These two forces are expressed as follows:7
}
(1)
force induced by a medium flow ) 6πRην
(2)
scattering force )
{
n1P 2R2 Q 2 c ω + (λ2z2/π2ω 2) 0 0
where n1, P, c, R, ω0, λ, z, Q, η, and ν are respectively the refractive index of the surrounding medium, the laser power, the velocity of light, the radius of the microorganism, the size of the beam waist, the wavelength of the laser, the coordinate of propagation direction, a dimensionless factor which represents the efficiency in conversion of the momentum of light on the surface of the microorganism and depends on the refractive indexes of the microorganism and the medium, the viscosity of the surrounding 3514 Analytical Chemistry, Vol. 70, No. 16, August 15, 1998
Figure 3. Pictures taken for a microorganism (a) before, (b) on, and (c) after trapping. The arrow is used to show the motion of the microorganism. (a) Microorganism at position A is moving in the direction indicated by the arrow. (b) The microorganism is trapped and then escapes at position B, at which a radiation force is equilibrated with the force induced by the medium flow. (c) The escaped microorganism is moving at position C. The distance from the beam waist to this position is 526 µm.
medium, and the relative velocity of the microorganism against the medium flow. Therefore, it is necessary to know the dimensionless factor, Q, in order to calculate the microorganism’s power. Figure 2 is a picture used for the determination of Q. A laser beam is introduced from the left side, and the sample is flowing from the right. The laser power was adjusted to 290 mW and the flow velocity to 160 µm/s. The results for the determination of Q are summarized in Table 1 and are obtained assuming that eq 1 equals eq 2. Trachelomonas volvocina, a unicellular microorganism, was used as a sample. This bacterium is a flagellate, with a spherical shape and a size of 10-30 µm. The sample was obtained from a pond at Kyushu University. From the results in Table 1, the value of Q was determined to be ∼0.10 for Trachelomonas volvocina. This value was used in the next step for the calculation of the microorganism’s power. It should be
noted that this value may contain an error arising from the nonspherical shape of the bacteria. For example, Bonder et al. reported the powers of sea urchin and human sperm cells to be 10-60 pN.9 On the other hand, Ko¨nig et al. calculated the powers of sperm cells on the basis of the ellipsoidal model and obtained the value of 18 pN for epididymal spermatozoa and 45 pN for ejaculated and frozen-thawed sperm.10 However, the shape of the bacteria observed in this study was almost spherical, and so the error caused by its nonspherical shape should be minimal. This calculation was carried out on the basis of the following concept and approach. First, a microorganism is captured with the optical funnel. At this trapping position, if the microorganism has a power larger than the gradient force, it will be able to escape from the trap. Otherwise, the microorganism will be fixed and will eventually die. By measuring the trapped position where a microorganism can escape, we calculate the gradient force by using the parameter, Q, obtained earlier. In this scenario, the microorganism’s power is larger than the gradient force. The photographs taken before, on, and after the escape of the microorganism from the trap are shown in Figure 3. Initially, a microorganism is moving in the direction indicated by the arrow (position A). The microorganism is then trapped and is suspended at the equilibrium position and in the process of escaping (position B). It then moves to position C. The distance from the beam waist to this position was determined to be 526 µm. It is possible to immediately calculate the gradient force at this position by using the following equation:
() (
)
n 1 R3 r r2 gradient force ) - 4 PQ exp - 2 2 c ω3 ω ω
(3)
where ω and r are the beam radius and the distance from the center line of the beam, respectively. The beam radius was 13.3 µm at the position where the microorganism escaped. The gradient force at 526 µm is calculated to be 0.96 pN, which is
obtained by substituting the value Q ) 0.10, which was previously determined. This value should be equal to or slightly smaller than the power of the microorganism. Using this straightforward technique, it is possible to examine a variety of single-celled organisms in this manner. The value obtained in this experiment is not the most accurate, because an argon ion laser was employed as the light source. The living cell is damaged by the visible light, since visible light is absorbed slightly by the living cell. Therefore, it is probable that the value obtained herein is less than the actual power possessed by the organism in nature. It would be possible to more accurately measure these values with an infrared laser, which barely damages the living cell.2,11 The above experimental data verify that the optical funnel is suitable for measurement of a microorganism’s power. In the future, the optical funnel technique could be used in measurements of a variety of microorganisms, e.g., sperm cells, which could be practically important for solving the issue of pregnancy and population in advanced nations. ACKNOWLEDGMENT This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan.
Received for review February 23, 1998. Accepted May 7, 1998. AC980199M (9) Bonder, E. M.; Colon, J. M.; Dziezic, J. M.; Ashkin, A. J. Cell Biol. 1990, 111, Abstract 2349. (10) Ko ¨nig, K.; Svaasand, L.; Liu, Y.; Sonek, G.; Patrizio, P.; Tadir, Y.; Berns, M. W.; Tromberg, B. J. Cell. Mol. Biol. 1996, 42, 501-509. (11) Wright, W. H.; Sonek, G. J.; Tadir, Y.; Berns, M. W. IEEE J. Quantum Elect. 1990, 26, 2148-2157.
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