Anal. Chem. 2001, 73, 5791-5795
An “Optical Channel”: A Technique for the Evaluation of Biological Cell Elasticity Takashi Kaneta, Jun Makihara, and Totaro Imasaka*
Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan
The development of an optical channel, a new analytical technique for evaluating the elasticity of biological cells, is described in this study. Two types of erythrocyte cells, i.e., young and old cells, were examined via their introduction into a flowing medium, to which a laser beam was focused in the opposite direction. An erythrocyte cell is trapped in a laser beam by a gradient force, moves in the downstream direction, and is then elongated at the beam waist. The change in shape was measured directly using a microscope equipped with a charge-coupled-device camera. It is probable the main driving force for the cell deformation is a shear stress generated by a medium flow, since an estimate of the gradient force suggests that it is too small to change the shape of an erythrocyte. The average values of the elongation of young and old cells were 2.4 ( 0.6 and 2.1 ( 0.5, respectively. These values are in reasonably good agreement with values obtained using a rheoscope method. The deformation was measured without any physical contact to the solid surface, and therefore, damage to cells such as these are minimal. Recently, living cells have attracted a great deal of interest from analytical chemists. Numerous reports concerning the separation of biological cells,1-3 their manipulation,4 and use of a cell as a detector for biological substances5 have appeared recently. To clarify the function of biological cells, it would be desirable to develop a new analytical technique that can be directly applied to individual cells. The elasticity of the erythrocyte cell is a wellknown factor and is used in the diagnosis of many diseases, such as sickle cell disease, hereditary spherocytosis, and immune haemolytic anemia.6,7 Elasticity, which is dependent on the age of a cell, is determined by the proteins located in the cell * To whom correspondence should be addressed: (tel) 81-92-642-3563; (fax) 81-92-632-5209; (e-mail)
[email protected]. (1) Armstrong, D. W.; Schulte, G.; Schneiderheinze, J. M.; Westenberg, D. J. Anal. Chem. 1999, 71, 5465-5469. (2) Armstrong, D. W.; Schneiderheinze, J. M. Anal. Chem. 2000, 72, 44744476. (3) Armstrong, D. W.; Schneiderheinze, J. M.; Kullman, J. P.; He, L. FEMS Microbiol. Lett. 2001, 194, 33-37. (4) Ashkin, A.; Dziedzic, J. M.; Yamane, T. Science 1987, 235, 1517-1520. (5) Jardemark K.; Orwar O.; Jacobson I.; Moscho A.; Zare R. N. Anal. Chem. 1997, 69, 3427-3434. (6) Sutera, S. P.; Gardner, R. A.; Boylan, C. W.; Carroll, G. L.; Chang, K. C.; Marvel, J. S.; Kilo, C.; Gonen, B.; Williamson, J. R. Blood 1985, 65, 275282. (7) Bosch, F. H.; Were, J. M.; Schipper, L.; Roerdinkholder-Stoelwinder, B.; Huls, T.; Willekens, F. L. A.; Wichers, G.; Halie, M. R. Eur. J. Haematol. 1994, 52, 35-41. 10.1021/ac010441g CCC: $20.00 Published on Web 11/17/2001
© 2001 American Chemical Society
membrane, the viscosity of the cytoplasm, and the ratio of the surface area to the volume. Elasticity is typically evaluated from differences in cell density. However, the density is not directly related to the membrane properties and cannot be used as a reliable marker for the evaluation of the age of a cell. In our previous paper, we reported on the power of a living cell using a method referred to as an optical funnel.8 This analytical technique, which involves the use of laser radiation force, was applied to the determination of the motility force of a sperm cell.9 The basic operating principle is similar to the reported technique referred to as optical chromatography,10-14 which was developed for the separation of microspheres and microorganisms. The optical funnel technique, which collects the cell and measures the power of the microorganism, has a distinct advantage over other techniques; the cell function can be measured without any physical contact with a solid surface, and as a result, damage to the cell is much less than with other techniques, such as laser trapping. In this study, we report on a new analytical technique for the evaluation of the elasticity of a biological cell and demonstrate several advantages of this method. In this approach, the erythrocyte cell, suspended in an aqueous solution, is introduced into a capillary by a siphon method. The cell is forced to move toward the center line of the laser beam, which is introduced and focused from the opposite direction. The erythrocyte cell flows through a focal point, at which the erythrocyte cell is seriously deformed by the shear stress of the flow, analogous to passing through a channel induced by a laser radiation force. The shape of the cell is monitored by a microscope equipped with a charge-coupleddevice (CCD) camera, and therefore, the elasticity can directly be visualized. It should be noted that this elasticity is measured without any physical contact with the solid surface, and as a result, damage to the cell is substantially reduced. A similar study has already been conducted, e.g., using a laser trapping technique. However, in this approach, degradation of the cell occurs at high laser intensities; i.e., the flexibility of the cell is lost.15,16 However, the intensity of the light that irradiates the cell is much smaller (8) Mishima, N.; Kaneta, T.; Imasaka T. Anal. Chem. 1998, 70, 3513-3515. (9) Kaneta, T.; Mishima, N.; Imasaka T. Anal. Chem. 2000, 72, 2414-2417. (10) Imasaka, T.; Kawabata, Y.; Kaneta T.; Ishidzu, Y. Anal. Chem. 1995, 67, 1763-1765. (11) Kaneta, T.; Ishidzu, Y.; Mishima, N.; Imasaka, T. Anal. Chem. 1997, 69, 2701-2710. (12) Hatano, T.; Kaneta, T.; Imasaka T. Anal. Chem. 1997, 69, 2711-2715. (13) Makihara, J.; Kaneta, T.; Imasaka, T. Talanta 1999, 48, 551-557. (14) Miki, S.; Kaneta, T.; Imasaka, T. Anal. Chim. Acta 2000, 404, 1-6. (15) Ashkin, A.; Dziedzic, J. M.; Yamane, T. Nature 1987, 330, 769-771.
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Figure 1. Experimental setup for the optical channel.
in the present approach, since the laser beam is more loosely focused. Therefore, the extent of degradation is much less than that for the laser trapping technique. EXPERIMENTAL SECTION Analytical Instrument. Figure 1 shows the experimental setup used in the present study. A Nd:YVO4 laser (Jenoptik, JOL-D05, 1064 nm) was used as a light source for the generation of radiation force. A near-infrared laser was used to reduce optical damage to the erythrocyte. An argon ion laser beam (NEC, GLS3070A, 488 nm, 10 mW) was aligned in a collinear fashion to the Nd:YVO4 laser for use as a guide beam. The laser beams were focused by a lens with a focal length of 30 mm into a quartz cell which was connected to inlet and outlet fused-silica capillaries (GL Science, 200-µm i.d., 375-µm o.d.). The motion of the erythrocyte was observed using a microscope equipped with a CCD camera (Nikon, Eclipse E600, objective, 4× or 20×) and connected to a videotape recorder (Sony, model SVO 260). The position of the beam waist was determined using a 10-µm polystyrene bead as follows; the bead was first trapped as in the case of optical chromatography,13 and the laser power was then gradually decreased. As a result, the bead approaches the beam waist, subsequently leaving the focal point. The position of the beam waist was determined as a position at which the acceleration of the bead began. The flow rate was estimated by measuring the velocity of the bead without the introduction of the laser beam. The laser power was measured behind the focusing lens, which was positioned in front of the quartz cell, by means of a power meter (Molectron, OR, SY5100). The lengths of the capillaries used for the inlet and outlet of the solution were each 40 cm. The end of the capillary, used for injection of the sample, was immersed in a solution containing the erythrocytes, and the end of the other capillary was placed in a buffer solution. The sample solution was introduced by gravity using a siphon method. Sample Preparation. Human blood, anticoagulated by EDTA, was centrifuged for 3 min at 1000g. Plasma, which is located in the upper phase, was then removed with a Pasteur pipet. The residue was washed three times with copious amounts of 10 mM (16) Bronkhorst, P. J. H.; Streekstra, G. J.; Grimbergen, J.; Nijhof, E. J.; Sixma, J. J.; Brakenhoff, G. J., Biophys. J. 1995, 69, 1666-1673.
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HEPES buffer (pH 7.4), which contained 140 mM NaCl and 3 mM glucose. The buffer was used to retain a high elasticity of the erythrocyte cell. The solution was diluted to 200-300 cells mL-1 using the buffer solution. Young and old erythrocyte cells were separated by centrifugation. A washed suspension of erythrocytes was centrifuged for 15 min at 10000g, and small fractions of the upper and lower regions of the solution were used as samples representing young and old cells, respectively. Each sample was washed twice by centrifugation with the buffer solution. To decrease elasticity, the erythrocytes were treated with glutaraldehyde. Glutaraldehyde serves to cross-link the amino groups on the surface of the cell membranes, making them less permeable. These samples were used to estimate the optical radiation force applied to an erythrocyte. In this case, a buffer solution containing 0.1% glutaraldehyde was added into the sample solution containing the erythrocytes. After standing for 30 min, the cells were washed twice by centrifugation with the buffer solution and were then diluted. All the sample preparations were carried out at room temperature (25 °C). RESULTS AND DISCUSSION Preliminary Experiment. The erythrocytes that had been treated with glutaraldehyde were introduced into a capillary and were retained at a position at which the optical radiation force is identical to the force induced by the medium flow. This experiment was conducted to estimate the optical radiation force applied to the erythrocyte. No elasticity was observed for an erythrocyte cell that had been treated with 0.2% glutaraldehyde. However, this sample was found not to be suitable for the measurement of radiation force, since the cell rotated spirally and could not be retained on the laser beam axis. The spiral rotation suggests the change in the shape of the cell from a disk to twisted type. Such deformation might be caused in the cell treatment process: when glutaraldehyde is added to the sample solution, the erythrocyte cell might be inhomogeneously stressed by the shear force induced by mixing the solution using a stirrer, resulting in permanent asymmetric distortion in the shape of the cell. Because of this, the concentration of glutaraldehyde was reduced to 0.1%, at which condition erythrocyte had a disk shape and was retained on the laser beam axis, although it continued to rotate. Such a
Figure 2. Photographs of deformed erythrocyte cells: (a) a young cell; laser power, 1.9 W: (b) a young cell; laser power, 1.1 W: (c) an old cell; laser power, 1.1 W.
rotation may arise from an asymmetrical resistance force, which is induced as a result of the asymmetrical shape of the erythrocyte cell. When several erythrocytes are trapped simultaneously, the light-shielding effect, which is induced by the forward erythrocyte, cannot be neglected. Furthermore, some of the erythrocytes were aggregated, since the cells were forced to be retained at the same position. Such an aggregation is unfavorable for the evaluation of cell elasticity. To overcome this problem, the sample solution was diluted appropriately for the measurement of individual cells. Physical Parameters. To measure the flow rate of the solution, the erythrocyte was first trapped and the laser was then switched off. The flow rate was then measured by monitoring the velocity of the erythrocyte. The resistance force induced by a medium flow calculated for an oblate spheroid, FD, is expressed by17
FD ) 8πLηug(W/L)
(1)
where η is the viscosity of the flowing medium, u the flow rate, and g(W/L) is a function of L and W, which are the lengths of the long and short axes of the erythrocyte, respectively. By replacing W/L by E, the function, g(W/L), in eq 1 can be calculated by
g(E) )
(1 - E 2) [(3 - 2E2) cot-1E/x1 - E2] - E
(2)
Assuming that the erythrocyte is an oblate spheroid, 8.5 µm in length and 2.4 µm in thickness, the resistance force induced by the medium flow is calculated by eqs 1 and 2. The scattering force, calculated from the position of the erythrocyte that had been treated with glutaraldehyde, was the same as that of a polystyrene bead having a radius of 2.7 µm. The area projected by the laser light was 2.8-fold larger than the value for a 2.7-µm polystyrene bead. This indicates that the refractive index of the erythrocyte is much smaller than that of the polystyrene bead. The value of the refractive index for an internal solution of an erythrocyte was determined to be 1.41, using a freeze-thaw method, which is in (17) Happel, J.; Brenner, H. Low Reynolds number hydrodynamics with special applications to particulate media, 2nd revised ed.; Noordhoff International Publishing: Lynden, The Netherlands, 1973; pp 145-149.
Table 1. Elongation Parameters (L/W) of Erythrocytes L/W
a
samplea
young cells
old cells
donor 1 (n ) 48) donor 2 (n ) 52) total (n ) 100)
2.9 ( 0.6 2.3 ( 0.4 2.4 ( 0.6
2.2 ( 0.5 2.0 ( 0.4 2.1 ( 0.5
n, number of samples.
contrast with a refractive index of 1.57 obtained for a polystyrene bead. However, the scattering force calculated for a sphere having a refractive index of 1.41 is 4.5-fold smaller than that for a sphere having a refractive index of 1.57.11 Thus, the refractive index of the erythrocyte cell must be larger than 1.41. This inconsistent result might be caused by the presence of a cell membrane, which is composed of lipids and proteins. A cell membrane would increase the apparent refractive index of the erythrocyte cell, although it is difficult to evaluate the refractive index of the cell membrane quantitatively. Evaluation of Elasticity. A fresh erythrocyte cell was introduced into a stream, and the change in shape was visually observed. The erythrocyte was forced to move to the center line of the laser beam axis by the gradient force. The cell was deformed and rotated over a time period of ∼1 s. It passed through the beam waist, since the deformation of the cell led to a reduction in surface area and front cross section, which decreases the optical radiation force applied to the cell. All experiments were performed under the following conditions: an erythrocyte was allowed to flow through the beam waist slowly by adjusting the flow rate of the medium at a constant laser power and, after this, was rapidly accelerated. As shown in Figure 2, the erythrocyte is substantially elongated at a high laser power. The elongation parameters (L/ W) for young and old cells are listed in Table 1. The elasticity of a young cell is apparently larger than that of an old cell. These results are consistent with reported values (2.5 ( 0.1 for young cells, 2.0 ( 0.4 for old cells) obtained by means of a rheoscope method,6 although the standard deviations are slightly larger in the present experiment. This unfavorable result is likely the result of incomplete separation of young and old cells by centrifugation, since it was difficult to retrieve only 10% of the top and bottom fractions6 due to the short (2 cm) test tube used in this Analytical Chemistry, Vol. 73, No. 24, December 15, 2001
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Figure 3. Cell positions measured from the beam waist for (a) young. (b) old, and (c) glutaraldehyde-treated erythrocytes. The circles and bars indicate average values and standard deviations calculated from the data obtained in 10 experiments, respectively.
experiment. It should be noted that the standard deviation is affected not only by the distribution of the elasticity in both young and old cells but also by the errors in the measurement of the length of the erythrocyte cell and in the stabilities of the laser power and the flow rate. In the case of a rheoscope method, the standard deviation is also affected by the distribution of the elasticity and the errors related to the instrument as well. Unfortunately, it is difficult to discuss the difference in the standard deviation of these methods further since the samples used in the experiments are not exactly identical to each other. Recently, laser-assisted optical rotational cell analysis (LORCA), which is based on ektacytometry using a laser diffraction technique,18,19 is employed for the evaluation of the biological cell elasticity. The L/W values obtained by LORCA are 2.1-3.1 for young cells and 1.4-2.17 for old cells, although these values depend on the applied shear stress.19 The values shown in Table 1 (2.9 and 2.2 for donor 1, 2.3 and 2.0 for donor 2) are in reasonable agreement with the values obtained using LORCA. Figure 3 shows the retained position of the erythrocyte cells, as measured from the beam waist. The order for elasticity is young > old > glutaraldehyde-treated cells. This result indicates that the retention distance (the position measured from the beam waist) decreases with increasing elasticity of the erythrocytes. The elongation of the cells may arise both from the resistance force induced by the medium flow and from the radiation force, since the surface area and the front cross section of the cell decrease. The applied radiation force is small for an elongated cell, and as a result, the position of the erythrocyte shifts to the focal point, at which the radiation force is larger. Thus, the present approach, which is similar to optical chromatography, can be used for the evaluation of cell elasticity, although the deformation of the cell cannot be directly visualized. There are two approaches in which a laser radiation force can be used for the visual observation of erythrocyte deformation, i.e., laser trapping and an optical channel. In laser trapping, an erythrocyte is slightly deformed at a laser power of 4-40 mW,15 although a quantitative value for the extent of the deformation has not yet been reported. In the present approach, the laser (18) Hardeman, M. R.; Goehart, P. T.; Dobbe, J. G. G.; Lettinga, K. P. Clin. Hemorheol. 1994, 14, 605-618. (19) Hardeman, M. R.; Ince, C. Clin. Hemorheol. Microcirc. 1999, 21, 277-284.
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power that the erythrocyte cell receives is several tens of milliwatts, which is essentially the same as that used in laser trapping. However, the laser beam is more loosely focused in an optical channel, and as a result, the gradient force applied to the erythrocyte cell is much smaller than that involved in laser trapping. Thus, it is difficult to conclude that the driving force for the deformation can be attributed only to the gradient force induced by the laser beam in the present study. When the flow rate of the medium was adjusted to 300 µm s-1, the shear stress was estimated to be ∼1 dyn cm-2. This value is comparable to the value (1-10 dyn cm-2) obtained by a flow channel technique using a rectangular solid capillary.20 This fact suggests that the deformation of a cell arises from the shear stress induced by the medium flow. The advantage of the present method is that the elasticity can be measured in a one-dimensional flowing stream without any physical contact with the solid surface of the analytical device. In fact, a flow channel technique in which an erythrocyte is passed through a narrow solid channel would be expected to destroy biological cells at the sharp edge of the capillaries. A rheoscope was used for the evaluation of cell elasticity, but it provides data only about the two-dimensional elongation of the biological cell. This situation (two-dimensional elongation) is not identical to the deformation of an erythrocyte flowing in a narrow (one-dimensional) blood capillary. Thus, the optical channel technique would be expected to give more useful data for the evaluation of erythrocyte elasticity. CONCLUSION In the optical channel technique, the elasticity of the individual cell is visually measured in a flowing medium, in which a onedimensional shear strength is applied to the biological cell. Moreover, this approach requires no physical contact with a solid surface of the device. These characteristics are very similar to those of a narrow blood capillary. This is in striking contrast to other techniques such as a flow channel technique, rheoscope, micropipet, and laser diffractometry. In addition, the optical channel technique requires a weakly focused near-infrared laser beam, and the time during which the laser beam is applied to the erythrocyte cell is only a few seconds. Therefore, optical damage may be much less than that which occurs during the laser trapping technique. In fact, the shape of the deformed erythrocyte was easily recovered after it passed through the focal point. This result suggests that the present approach allows soft and repetitive measurements of the cell for evaluating its elasticity. Recently, several studies have been reported on the separation of biological cells and the subsequent measurement of the constituents in a single cell. A problem that arises in the study of biological cells is the difference in the properties of individual cells, e.g., size, functions, elasticity, and concentrations of the constituents. Although not demonstrated herein, the optical channel technique would permit the separation of biological cells based on their elasticities. This method could be further combined with other techniques for the chemical analysis of the constituents of a single cell. Thus, the optical channel technique may have potential for use in correlating cell function with the chemical (20) Chien, S.; Sung, L. A.; Lee, M. M. L.; Skalak, R. Biorheology 1992, 29, 467478.
species in the cell. Therefore, this method represents a powerful technique, especially in the field of cell biology.
tion of Food Co., Ltd. Fukuoka, Japan, for supplying blood samples.
ACKNOWLEDGMENT This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture. The authors thank the Institute of Rheological Func-
Received for review April 16, 2001. Accepted September 10, 2001. AC010441G
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