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Micro-manipulation system with a two-fingered micro-hand and its potential application in bioscience
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Journal of Biotechnology 133 (2008) 219–224
Micro-manipulation system with a two-fingered micro-hand and its potential application in bioscience Kenji Inoue a,∗ , Tamio Tanikawa b , Tatsuo Arai a a
b
Osaka University, Japan National Institute of Advanced Industrial Science and Technology, Japan
Received 15 January 2007; received in revised form 25 July 2007; accepted 9 August 2007
Abstract A micro-manipulation system using a two-fingered micro-hand, an auto-focusing optical microscope, and user interfaces was developed. This micro-hand has 6 degrees of freedom (DOF): 3 DOF for each of the two fingers. These fingers work just like the thumb and forefinger. Thus, this hand can grasp, move, rotate, and release micro-objects, such as biological cells. A human operator can operate this hand using a joystick or a keyboard, while seeing the microscope image displayed on a monitor. The present paper describes two applications of this system to the field of bioscience. The first application involves extraction of cytoplasm from a cell using two, two-fingered micro-hands. One hand holds the cell firmly, while the other hand makes a hole in the cell and tears it. Then, the hand holding the cell squeezes the cytoplasm from the cell. The second application involves measurement of the mechanical properties of living cells using the micro-finger and a micro-force sensor based on the Atomic Force Microscope (AFM) principle. The AFM cantilever is placed within the microscopic field. The micro-finger holds a cell and presses it against the cantilever tip. By measuring the pressing force and the deformation of the cell, the cell’s force–deformation curve is obtained. © 2007 Elsevier B.V. All rights reserved. Keywords: Micro-manipulation; Two-fingered micro-hand; Cloning; Cell stiffness; Atomic Force Microscope
1. Introduction Recent advancements in biotechnology demand more accurate, faster, and more dexterous micro-manipulation, observation, and measurement of biological objects (Ikuta, 1988; Arai et al., 1997; Sun and Nelson, 2001). Controlling the position and orientation of a single cell under an optical microscope with 100 nm scale accuracy is required of micro-tools and micromanipulators; such dexterous manipulation permits detailed analyses and precise operations on cells. Hence, these techniques will be able to bring about further progress in bioscience and cell engineering.
∗ Corresponding author at: Department of Systems Innovation, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan. Tel.: +81 6 6850 6366; fax: +81 6 6850 6366. E-mail address: [email protected] (K. Inoue).
0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.08.027
For this purpose, we developed a micro-manipulation system using a two-fingered micro-hand, an auto-focusing optical microscope, and user interfaces. The micro-hand has 6 degrees of freedom (DOF): 3 DOF for each of the two fingers. These fingers work just like the thumb and forefinger or chopsticks. Thus, this hand can grasp, move, rotate, and release micro-scale objects. A human operator can control the motion of this hand with a joystick or a keyboard, while seeing the microscope image displayed on a monitor. The present paper describes two applications of this system to the field of bioscience. The first application involves extraction of cytoplasm from a cell using two, twofingered micro-hands. One hand holds the cell firmly. The other hand makes a hole in the cell and tears it. Then, the hand holding the cell squeezes the cytoplasm from the cell. The second application involves measurement of the mechanical properties of living cells using the micro-finger and a micro-force sensor that is based on the Atomic Force Microscope (AFM) principle. The micro-finger holds a cell and presses it against the AFM cantilever tip. By measuring the pressing force and the
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deformation of the cell, the cell’s force–deformation curve can be obtained. 2. Two-fingered micro-hand for manipulation of micro-/nano-scale objects 2.1. Design of the micro-manipulation system for biological objects Recently, observation and measurement techniques for micro-/nano-scale objects have been developed, and the importance of manipulation technology for such objects is increasing. In the biotechnology field, the demand for manipulation of biological objects of micro-/nano-scale has become especially important for such applications as DNA injection or the cloning of single cells. Fig. 1 shows conventional micro-manipulation. The micromanipulator has 3 DOF in the X, Y, and Z directions. Generally, two manipulators are used, one at the left side and the other at the right side of the microscope. Cell operations are very difficult for beginners, and more than 6 months of training are required to attain proficiency. Rotation of the cell is particularly difficult using the conventional system. Thus, a manipulator that is capable of multi-degree motion with a high accuracy is needed. Generally, the serial mechanism, which consists of translational motion actuators in series, such as in the conventional system shown in Fig. 1, cannot perform both high accuracy positioning and multi-degree motion. A parallel mechanism consists of multiple actuators that are parallel to the end plate. The end plate is controlled by the cooperative motion of these actuators, and multi-degree motion can be obtained. The mechanism has high stiffness because the end plate is supported by the actuators in parallel, as a result, high accuracy positioning of the end plate can be performed. 2.2. Micro-manipulator with 3 DOF and thin plate flexure hinges Normally, high accuracy positioning stages use flexure hinge mechanisms. On the other hand, the manufacturing cost of the
Fig. 1. Conventional micro-manipulation system.
Fig. 2. Flexure joints made of thin plate. (a) Revolute joint. (b) Prismatic joint.
parallel mechanism with a multi-directional motion capability is very high because the links must be cut from several directions to make the hinge shape. In order to produce mechanical parts at low cost and for mass production, press machines are used. The press machine process primarily involves punching a hole and bending the plate. Low cost can be easily obtained if only the press process is used to make the joint mechanism. The 3 DOF parallel mechanism can be formed by using the revolute joint and the prismatic joint only. Fig. 2 shows a revolute joint and a prismatic joint made only from a thin plate. The revolute joint is obtained by punching a hole in the thin plate, as shown in Fig. 2(a). In the case of the prismatic joint, holes are punched as shown Fig. 2(b), and then the thin plate is bent. The 3 DOF parallel mechanism can be produced by arranging these joints. In order to simplify construction, a link structure, using three revolute joints and one prismatic joint, was chosen, as shown in Fig. 3. The link structure is the same as the 3 DOF parallel mechanism “Delta structure” introduced by Clavel (1990). By pushing the three links from the base individually, as shown by the arrows in Fig. 3, the end plate can be controlled with 3 DOF. 2.3. Two-fingered micro-hand When single cells are micro-manipulated, rotation is very difficult. Here, the chopsticks motion is considered. The chopsticks motion can easily perform rotation by cooperative control of each chopstick. If a two-finger mechanism is designed based
Fig. 3. 3 DOF parallel mechanism with punched and bent thin plate.
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Fig. 4. Configuration of the two-fingered micro-hand mechanism. (a) Whole view of the two-fingered micro hand. (b) Prototype of the two-fingered micro hand.
on the chopsticks motion strategy, one could easily perform dexterous manipulation, including rotation. Using the flexure joint mechanism made from the thin plate, a two-fingered micro-hand was designed for micro-manipulation. Two modules with a parallel mechanism are put in series, and one finger is placed on the upper module body, which is linked to the end plate of the lower module. Another finger is placed on the end plate of the upper module, as shown in Fig. 4(a). Using this configuration, the hand can be controlled in a manner similar to human chopstick motion (Tanikawa and Arai, 1999). Fig. 4(b) shows a prototype of the two-fingered micro-hand with a flexure joint mechanism made from a thin plate. After calibration, the micro-finger has a repeatability of at least 0.1 m and 1 m positioning accuracy, with a nanometer resolution. The workspace of the micro-finger is 200 m × 200 m × 32 m.
the aspiration and the membrane is torn by rubbing both pipettes together, as shown in Fig. 5(b). Finally, the nucleus is extracted along with some of the cell’s cytoplasm by squeezing the cell with both pipettes, as shown in Fig. 5(c). In this conventional process, cell rotation is often necessary, since the cell being manipulated is not always fixed. Fig. 6 demonstrates the operation of the two-fingered microhands for extraction of a nucleus from an egg cell of a rat. Two, opposing, two-fingered micro-hands are used under a microscope. The micro-hands are controlled with the joysticks. The operator can observe the process in the microscope image. First, the cell is held by the left micro-hand as shown in Fig. 6(a). The cell can then be easily rotated for positioning the tearing point,
3. Extraction of cell cytoplasm using the two-fingered micro-hand Recently, various physical processes, such as DNA injection and cloning, have been performed on single cells. As biological research develops, operations on single cells will become more common. In cloning, the extraction of a nucleus from an egg cell is a very difficult procedure. Fig. 5 shows an overview of the process of nucleus extraction from an egg cell. As shown in Fig. 1, two tools (a holding pipette and an injection pipette) are used. The egg cell is held by the holding pipette, and the injection pipette is inserted into the cell membrane, as shown in Fig. 5(a). Then, the cell is released from the holding pipette by stopping
Fig. 5. Extraction of a nucleus from an egg cell. (a) Insertion. (b) Tearing. (c) Extraction.
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Fig. 6. Extraction of a nucleus from an egg cell using two-fingered micro-hands. (a) Holding. (b) Injection. (c) Tearing. (d) Extraction.
since the micro-hand is capable of rotating an object through multi-directional motion of the finger, just like chopsticks. The right fingers are then inserted with the fingers held close together, as shown in Fig. 6(b). Then, the fingers are opened within the membrane for tearing, as shown in Fig. 6(c). Finally, the left fingers are further closed to squash the cell, and the nucleus is extracted with a part of the cell’s cytoplasm, as shown in Fig. 6(d). During this process, rotation is necessary only during holding. This process appears to be easier than the conventional process, as the cell can be always fixed by the left micro-hand.
4. Measurement of living cells’ mechanical properties 4.1. Purpose and system configuration Analyses of the mechanical properties, such as stiffness, viscosity, and strength, of living cells or other biological materials are expected to contribute to the advancement of medical technology for diagnosis, treatment, and prevention of disease. Various methods and techniques have been applied to the measurement of the mechanical properties of cells, including aspirating a part of the cell with a micropipette (Sato et al., 1987); using an AFM (Miyazaki and Hayashi, 1999; Bottomley et al., 1996; Tao et al., 1992; Radmacher et al., 1996; Goldmann and Ezzell, 1996; Putman et al., 1994); using a scanning acoustic microscope (Bereiter-Hahn et al., 1995); aspirating an entire cell with a micropipette (Hochmuth et al., 1994); stretching an entire cell with a pair of micropipettes (Glerum et al., 1990); and stretching an entire cell using a pair of microplates (Thoumine and Ott, 1997). The use of micropipettes is straightforward and easy, but its associated resolution is low. AFM has a high resolution, but most conventional AFMs are expensive and suitable
only for the observation of sample surface profiles. Since the cantilevers touch the samples in limited directions, it is difficult to take measurements at arbitrary points on the samples. We used our two-fingered micro-hand to measure living cells’ mechanical properties. Fig. 7 shows the system configuration, which consists of a micro-manipulation system and a micro-force sensor based on the AFM principle. The micro-manipulation system consists of the micro-hand, an autofocusing optical microscope, a computer for micro-hand control using a joystick, and a computer with a monitor for image processing. One of the micro-hand’s fingers is used; the finger has a hollow glass tube as its end-effector, which is 1 mm in diameter and 30 mm long, with a tip radius of 2 m. The glass fibers inside the hollow glass tube enhance the capillary effect. A CCD camera captures the microscope image, which is displayed on the monitor. The operator can control the position and velocity of the finger tips using the joystick. We developed a compact and inexpensive AFM system specifically for measuring the mechanical properties of cells. The AFM system consists of a laser diode module, a cantilever placed within the microscopic field, and a photo detector module. The wavelength of the laser diode is 670 nm. The length of the cantilever is 100 m, the radius of curvature of the tip is less than 20 nm, and the spring constant, k, of the cantilever is 0.57 N/m. When the cantilever tip is pressed against the sample, the deflection, d, of the cantilever tip is measured by an optical lever. Because the spring constant, k, of the cantilever is known, the force, f, acting on the cantilever tip can be determined by: f = kd
(1)
The relationship between the deflection, d, and the photo detector output is calibrated beforehand.
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Fig. 8. Method for measuring cell stiffness.
The micro-finger allows for selection and dexterous manipulation of the target cell with a high accuracy, and the AFM force sensor allows high accuracy micro-force sensing. Thus, this combination permits point-by-point analysis of one cell. Using the optical microscope, one can observe the contact among the cell, the cantilever, and the micro-finger tip from above. Since the AFM system is compact, it can be easily installed into general optical microscopes, and the cost is reduced to one-tenth of that of general AFMs. Fig. 7. System for measuring the mechanical properties of living cells.
4.3. Measuring cell stiffness 4.2. Procedure for measuring cell stiffness The stiffness of cells in liquid can be measured using the following procedure: (1) Since the finger is a hollow tube, simply by bringing the finger tip close to a target cell, capillary suction attracts and holds the cell firmly at the finger tip. (2) The finger can then move the cell to the AFM cantilever and press it against the cantilever tip. Thus, a force is applied to the cell, and the cell is deformed (Fig. 8). (3) This pressing force, f, is calculated from the measured deflection, ds , of the cantilever tip. The deformation, d, of the cell is obtained from the difference between the deflection, ds , and the moving distance, df , of the micro-finger tip. d = df − ds
The stiffness of a normal human leukocyte and a yeast cell was measured and compared using the measurement system that we developed. Peripheral blood was collected from one of the authors and centrifuged, following which a sample, primarily from the buffy coat, was aspirated. Spherical leukocytes, 5–8 m-diameter, were identified. The stiffness of an approximately 5 m-diameter leukocyte was measured in normal saline. Fig. 9 shows the microscope images of the experiment, and Fig. 10(a) shows the force–deformation curve that was obtained. Since the leukocyte was very soft, the curve was nonlinear, indicating that the stiffness of the human leukocyte was nonlinear. The stiffness of an approximately 5 m-diameter yeast cell was
(2)
(4) While measuring force, f, and deformation, d, the finger gradually presses harder against the cell, thus applying a larger force. As a result, the relationship between the force, f, and the deformation, d, (the force–deformation curve) of the target cell can be obtained. The stiffness of the cell is then the gradient of the force–deformation curve.
Fig. 9. Measuring the stiffness of a human leukocyte.
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Fig. 10. Force–deformation curves of a human leukocyte and a yeast cell. (a) Human leukocyte. (b) Yeast cell.
also measured. An adhesive yeast cell in a solution of dry yeast in water was chosen, and its stiffness was measured. Fig. 10(b) shows the force–deformation curve of the yeast cell. Similar to the human leukocyte, the stiffness of the yeast cell was nonlinear, but the human leukocyte was slightly softer than the yeast cell. 5. Conclusions A micro-manipulation system using a two-fingered microhand, an auto-focusing optical microscope, and user interface was developed. This micro-hand can grasp, move, rotate, and release micro-objects, such as biological cells. Two bioscience applications of this system were described. The first application involved the extraction of a cell’s cytoplasm using two, two-fingered micro-hands. While one hand holds the cell firmly, the other punctures the cell and tears it. Then, the hand holding the cell squeezes the cytoplasm from the cell. The second application involved the measurement of the mechanical properties of living cells using the micro-finger and a micro-force sensor based on the AFM principle. An AFM cantilever was placed within the microscopic field and the micro-finger was used to hold the cell, pressing it against the cantilever tip. By measuring the force and the deformation of the cell, the force–deformation curve of the cell could be obtained. Future bioscience techniques will require more accurate, faster, and more skillful micro-manipulation, observation, and measurement of biological objects. Since the two-fingered micro-hand is easy to manipulate and has a high positioning accuracy, our system can be used to make detailed analyses and precise operations on cells possible. We plan to apply our system to practical tasks in the fields of bioscience and cell engineering. Acknowledgement This work was supported by MEXT under a Grant-in-Aid for Scientific Research on Priority Areas (Project No. 17076010).
References Arai, F., Morishima, K., Kasugai, T., Fukuda, T., 1997. Bio-micro-manipulation (new direction for operation improvement). In: Proceedings of IROS 1997. Bereiter-Hahn, J., Karl, I., Luers, H., Voth, M., 1995. Mechanical basis of cell shape: investigations with the scanning acoustic microscope. Biomech. Cell Biol. 73, 337–348. Bottomley, L.A., Coury, J.E., First, P.N., 1996. Scanning probe microscopy. Anal. Chem. 68, 185R–230R. Clavel, R., 1990. Device for the movement and positioning of an element in space, US Patent No. 4,976,582, 11 December. Glerum, J.J., Van Mastrigt, R., Van Koeveringe, A.J., 1990. Mechanical properties of mammalian single smooth muscle cells. 3. Passive properties of pig detrusor and human a terme uterus cells. J. Muscle Res. Cell Motil. 11, 453–462. Goldmann, W.H., Ezzell, R.M., 1996. Viscoelasticity in wild-type and vinculindeficient (5.51) mouse F9 embryonic carcinoma cells examined by atomic force microscopy and rheology. Exp. Cell Res. 226, 234–237. Hochmuth, R.M., Ting-Beall, H.P., Beaty, B.B., Needham, D., Tran-Son-Tay, R., 1994. Viscosity of passive human neutrophils undergoing small deformations. Biophys. J. 64, 1596–1601. Ikuta, K., 1988. The application of micro/miniature mechatronics to medical robotics. In: Proceedings of IROS 1988, pp. 9–14. Miyazaki, H., Hayashi, K., 1999. Atomic force measurement of the mechanical properties of intact endothelial cells in fresh arteries. Med. Biol. Eng. Comp. 37, 530–536. Putman, C.A., van der Werf, K.O., de Grooth, B.G., van Hulst, N.F., Greve, J., 1994. Viscoelasticity of living cells allows high resolution imaging by tapping mode atomic force microscopy. Biophys. J. 67, 1749–1753. Radmacher, M., Fritz, M., Racher, C., Cleveland, J.P., Hansma, P.K., 1996. Measuring the viscoelastic properties of human platelets with the Atomic Force Microscope. Biophys. J. 70, 556–567. Sato, M., Levesque, M.J., Nerem, R.M., 1987. An application of the micropipette technique to the measurement of the mechanical properties of cultured bovine aortic endothelial cells. Trans. ASME, J. Biotechnol. Eng. 109, 27–34. Sun, Y.U., Nelson, B.J., 2001. Microrobotic cell injection. In: Proceedings of IEEE ICRA 2001, pp. 620–625. Tanikawa, T., Arai, T., 1999. Development of a micro-manipulation system having a two-fingered micro-hand. IEEE Trans. Robotics Automation 15 (1), 152–162. Tao, N.J., Lindsay, S.M., Lees, S., 1992. Measuring the microelastic properties of biological material. Biophys. J. 63, 1165–1169. Thoumine, O., Ott, A., 1997. Time scale dependent viscoelastic and contractile regimes in fibroblasts probed by microplate manipulation. J. Cell Sci. 100, 2109–2116.
Journal of Biotechnology 133 (2008) 219–224
Micro-manipulation system with a two-fingered micro-hand and its potential application in bioscience Kenji Inoue a,∗ , Tamio Tanikawa b , Tatsuo Arai a a
b
Osaka University, Japan National Institute of Advanced Industrial Science and Technology, Japan
Received 15 January 2007; received in revised form 25 July 2007; accepted 9 August 2007
Abstract A micro-manipulation system using a two-fingered micro-hand, an auto-focusing optical microscope, and user interfaces was developed. This micro-hand has 6 degrees of freedom (DOF): 3 DOF for each of the two fingers. These fingers work just like the thumb and forefinger. Thus, this hand can grasp, move, rotate, and release micro-objects, such as biological cells. A human operator can operate this hand using a joystick or a keyboard, while seeing the microscope image displayed on a monitor. The present paper describes two applications of this system to the field of bioscience. The first application involves extraction of cytoplasm from a cell using two, two-fingered micro-hands. One hand holds the cell firmly, while the other hand makes a hole in the cell and tears it. Then, the hand holding the cell squeezes the cytoplasm from the cell. The second application involves measurement of the mechanical properties of living cells using the micro-finger and a micro-force sensor based on the Atomic Force Microscope (AFM) principle. The AFM cantilever is placed within the microscopic field. The micro-finger holds a cell and presses it against the cantilever tip. By measuring the pressing force and the deformation of the cell, the cell’s force–deformation curve is obtained. © 2007 Elsevier B.V. All rights reserved. Keywords: Micro-manipulation; Two-fingered micro-hand; Cloning; Cell stiffness; Atomic Force Microscope
1. Introduction Recent advancements in biotechnology demand more accurate, faster, and more dexterous micro-manipulation, observation, and measurement of biological objects (Ikuta, 1988; Arai et al., 1997; Sun and Nelson, 2001). Controlling the position and orientation of a single cell under an optical microscope with 100 nm scale accuracy is required of micro-tools and micromanipulators; such dexterous manipulation permits detailed analyses and precise operations on cells. Hence, these techniques will be able to bring about further progress in bioscience and cell engineering.
∗ Corresponding author at: Department of Systems Innovation, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan. Tel.: +81 6 6850 6366; fax: +81 6 6850 6366. E-mail address: [email protected] (K. Inoue).
0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.08.027
For this purpose, we developed a micro-manipulation system using a two-fingered micro-hand, an auto-focusing optical microscope, and user interfaces. The micro-hand has 6 degrees of freedom (DOF): 3 DOF for each of the two fingers. These fingers work just like the thumb and forefinger or chopsticks. Thus, this hand can grasp, move, rotate, and release micro-scale objects. A human operator can control the motion of this hand with a joystick or a keyboard, while seeing the microscope image displayed on a monitor. The present paper describes two applications of this system to the field of bioscience. The first application involves extraction of cytoplasm from a cell using two, twofingered micro-hands. One hand holds the cell firmly. The other hand makes a hole in the cell and tears it. Then, the hand holding the cell squeezes the cytoplasm from the cell. The second application involves measurement of the mechanical properties of living cells using the micro-finger and a micro-force sensor that is based on the Atomic Force Microscope (AFM) principle. The micro-finger holds a cell and presses it against the AFM cantilever tip. By measuring the pressing force and the
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deformation of the cell, the cell’s force–deformation curve can be obtained. 2. Two-fingered micro-hand for manipulation of micro-/nano-scale objects 2.1. Design of the micro-manipulation system for biological objects Recently, observation and measurement techniques for micro-/nano-scale objects have been developed, and the importance of manipulation technology for such objects is increasing. In the biotechnology field, the demand for manipulation of biological objects of micro-/nano-scale has become especially important for such applications as DNA injection or the cloning of single cells. Fig. 1 shows conventional micro-manipulation. The micromanipulator has 3 DOF in the X, Y, and Z directions. Generally, two manipulators are used, one at the left side and the other at the right side of the microscope. Cell operations are very difficult for beginners, and more than 6 months of training are required to attain proficiency. Rotation of the cell is particularly difficult using the conventional system. Thus, a manipulator that is capable of multi-degree motion with a high accuracy is needed. Generally, the serial mechanism, which consists of translational motion actuators in series, such as in the conventional system shown in Fig. 1, cannot perform both high accuracy positioning and multi-degree motion. A parallel mechanism consists of multiple actuators that are parallel to the end plate. The end plate is controlled by the cooperative motion of these actuators, and multi-degree motion can be obtained. The mechanism has high stiffness because the end plate is supported by the actuators in parallel, as a result, high accuracy positioning of the end plate can be performed. 2.2. Micro-manipulator with 3 DOF and thin plate flexure hinges Normally, high accuracy positioning stages use flexure hinge mechanisms. On the other hand, the manufacturing cost of the
Fig. 1. Conventional micro-manipulation system.
Fig. 2. Flexure joints made of thin plate. (a) Revolute joint. (b) Prismatic joint.
parallel mechanism with a multi-directional motion capability is very high because the links must be cut from several directions to make the hinge shape. In order to produce mechanical parts at low cost and for mass production, press machines are used. The press machine process primarily involves punching a hole and bending the plate. Low cost can be easily obtained if only the press process is used to make the joint mechanism. The 3 DOF parallel mechanism can be formed by using the revolute joint and the prismatic joint only. Fig. 2 shows a revolute joint and a prismatic joint made only from a thin plate. The revolute joint is obtained by punching a hole in the thin plate, as shown in Fig. 2(a). In the case of the prismatic joint, holes are punched as shown Fig. 2(b), and then the thin plate is bent. The 3 DOF parallel mechanism can be produced by arranging these joints. In order to simplify construction, a link structure, using three revolute joints and one prismatic joint, was chosen, as shown in Fig. 3. The link structure is the same as the 3 DOF parallel mechanism “Delta structure” introduced by Clavel (1990). By pushing the three links from the base individually, as shown by the arrows in Fig. 3, the end plate can be controlled with 3 DOF. 2.3. Two-fingered micro-hand When single cells are micro-manipulated, rotation is very difficult. Here, the chopsticks motion is considered. The chopsticks motion can easily perform rotation by cooperative control of each chopstick. If a two-finger mechanism is designed based
Fig. 3. 3 DOF parallel mechanism with punched and bent thin plate.
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Fig. 4. Configuration of the two-fingered micro-hand mechanism. (a) Whole view of the two-fingered micro hand. (b) Prototype of the two-fingered micro hand.
on the chopsticks motion strategy, one could easily perform dexterous manipulation, including rotation. Using the flexure joint mechanism made from the thin plate, a two-fingered micro-hand was designed for micro-manipulation. Two modules with a parallel mechanism are put in series, and one finger is placed on the upper module body, which is linked to the end plate of the lower module. Another finger is placed on the end plate of the upper module, as shown in Fig. 4(a). Using this configuration, the hand can be controlled in a manner similar to human chopstick motion (Tanikawa and Arai, 1999). Fig. 4(b) shows a prototype of the two-fingered micro-hand with a flexure joint mechanism made from a thin plate. After calibration, the micro-finger has a repeatability of at least 0.1 m and 1 m positioning accuracy, with a nanometer resolution. The workspace of the micro-finger is 200 m × 200 m × 32 m.
the aspiration and the membrane is torn by rubbing both pipettes together, as shown in Fig. 5(b). Finally, the nucleus is extracted along with some of the cell’s cytoplasm by squeezing the cell with both pipettes, as shown in Fig. 5(c). In this conventional process, cell rotation is often necessary, since the cell being manipulated is not always fixed. Fig. 6 demonstrates the operation of the two-fingered microhands for extraction of a nucleus from an egg cell of a rat. Two, opposing, two-fingered micro-hands are used under a microscope. The micro-hands are controlled with the joysticks. The operator can observe the process in the microscope image. First, the cell is held by the left micro-hand as shown in Fig. 6(a). The cell can then be easily rotated for positioning the tearing point,
3. Extraction of cell cytoplasm using the two-fingered micro-hand Recently, various physical processes, such as DNA injection and cloning, have been performed on single cells. As biological research develops, operations on single cells will become more common. In cloning, the extraction of a nucleus from an egg cell is a very difficult procedure. Fig. 5 shows an overview of the process of nucleus extraction from an egg cell. As shown in Fig. 1, two tools (a holding pipette and an injection pipette) are used. The egg cell is held by the holding pipette, and the injection pipette is inserted into the cell membrane, as shown in Fig. 5(a). Then, the cell is released from the holding pipette by stopping
Fig. 5. Extraction of a nucleus from an egg cell. (a) Insertion. (b) Tearing. (c) Extraction.
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Fig. 6. Extraction of a nucleus from an egg cell using two-fingered micro-hands. (a) Holding. (b) Injection. (c) Tearing. (d) Extraction.
since the micro-hand is capable of rotating an object through multi-directional motion of the finger, just like chopsticks. The right fingers are then inserted with the fingers held close together, as shown in Fig. 6(b). Then, the fingers are opened within the membrane for tearing, as shown in Fig. 6(c). Finally, the left fingers are further closed to squash the cell, and the nucleus is extracted with a part of the cell’s cytoplasm, as shown in Fig. 6(d). During this process, rotation is necessary only during holding. This process appears to be easier than the conventional process, as the cell can be always fixed by the left micro-hand.
4. Measurement of living cells’ mechanical properties 4.1. Purpose and system configuration Analyses of the mechanical properties, such as stiffness, viscosity, and strength, of living cells or other biological materials are expected to contribute to the advancement of medical technology for diagnosis, treatment, and prevention of disease. Various methods and techniques have been applied to the measurement of the mechanical properties of cells, including aspirating a part of the cell with a micropipette (Sato et al., 1987); using an AFM (Miyazaki and Hayashi, 1999; Bottomley et al., 1996; Tao et al., 1992; Radmacher et al., 1996; Goldmann and Ezzell, 1996; Putman et al., 1994); using a scanning acoustic microscope (Bereiter-Hahn et al., 1995); aspirating an entire cell with a micropipette (Hochmuth et al., 1994); stretching an entire cell with a pair of micropipettes (Glerum et al., 1990); and stretching an entire cell using a pair of microplates (Thoumine and Ott, 1997). The use of micropipettes is straightforward and easy, but its associated resolution is low. AFM has a high resolution, but most conventional AFMs are expensive and suitable
only for the observation of sample surface profiles. Since the cantilevers touch the samples in limited directions, it is difficult to take measurements at arbitrary points on the samples. We used our two-fingered micro-hand to measure living cells’ mechanical properties. Fig. 7 shows the system configuration, which consists of a micro-manipulation system and a micro-force sensor based on the AFM principle. The micro-manipulation system consists of the micro-hand, an autofocusing optical microscope, a computer for micro-hand control using a joystick, and a computer with a monitor for image processing. One of the micro-hand’s fingers is used; the finger has a hollow glass tube as its end-effector, which is 1 mm in diameter and 30 mm long, with a tip radius of 2 m. The glass fibers inside the hollow glass tube enhance the capillary effect. A CCD camera captures the microscope image, which is displayed on the monitor. The operator can control the position and velocity of the finger tips using the joystick. We developed a compact and inexpensive AFM system specifically for measuring the mechanical properties of cells. The AFM system consists of a laser diode module, a cantilever placed within the microscopic field, and a photo detector module. The wavelength of the laser diode is 670 nm. The length of the cantilever is 100 m, the radius of curvature of the tip is less than 20 nm, and the spring constant, k, of the cantilever is 0.57 N/m. When the cantilever tip is pressed against the sample, the deflection, d, of the cantilever tip is measured by an optical lever. Because the spring constant, k, of the cantilever is known, the force, f, acting on the cantilever tip can be determined by: f = kd
(1)
The relationship between the deflection, d, and the photo detector output is calibrated beforehand.
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Fig. 8. Method for measuring cell stiffness.
The micro-finger allows for selection and dexterous manipulation of the target cell with a high accuracy, and the AFM force sensor allows high accuracy micro-force sensing. Thus, this combination permits point-by-point analysis of one cell. Using the optical microscope, one can observe the contact among the cell, the cantilever, and the micro-finger tip from above. Since the AFM system is compact, it can be easily installed into general optical microscopes, and the cost is reduced to one-tenth of that of general AFMs. Fig. 7. System for measuring the mechanical properties of living cells.
4.3. Measuring cell stiffness 4.2. Procedure for measuring cell stiffness The stiffness of cells in liquid can be measured using the following procedure: (1) Since the finger is a hollow tube, simply by bringing the finger tip close to a target cell, capillary suction attracts and holds the cell firmly at the finger tip. (2) The finger can then move the cell to the AFM cantilever and press it against the cantilever tip. Thus, a force is applied to the cell, and the cell is deformed (Fig. 8). (3) This pressing force, f, is calculated from the measured deflection, ds , of the cantilever tip. The deformation, d, of the cell is obtained from the difference between the deflection, ds , and the moving distance, df , of the micro-finger tip. d = df − ds
The stiffness of a normal human leukocyte and a yeast cell was measured and compared using the measurement system that we developed. Peripheral blood was collected from one of the authors and centrifuged, following which a sample, primarily from the buffy coat, was aspirated. Spherical leukocytes, 5–8 m-diameter, were identified. The stiffness of an approximately 5 m-diameter leukocyte was measured in normal saline. Fig. 9 shows the microscope images of the experiment, and Fig. 10(a) shows the force–deformation curve that was obtained. Since the leukocyte was very soft, the curve was nonlinear, indicating that the stiffness of the human leukocyte was nonlinear. The stiffness of an approximately 5 m-diameter yeast cell was
(2)
(4) While measuring force, f, and deformation, d, the finger gradually presses harder against the cell, thus applying a larger force. As a result, the relationship between the force, f, and the deformation, d, (the force–deformation curve) of the target cell can be obtained. The stiffness of the cell is then the gradient of the force–deformation curve.
Fig. 9. Measuring the stiffness of a human leukocyte.
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Fig. 10. Force–deformation curves of a human leukocyte and a yeast cell. (a) Human leukocyte. (b) Yeast cell.
also measured. An adhesive yeast cell in a solution of dry yeast in water was chosen, and its stiffness was measured. Fig. 10(b) shows the force–deformation curve of the yeast cell. Similar to the human leukocyte, the stiffness of the yeast cell was nonlinear, but the human leukocyte was slightly softer than the yeast cell. 5. Conclusions A micro-manipulation system using a two-fingered microhand, an auto-focusing optical microscope, and user interface was developed. This micro-hand can grasp, move, rotate, and release micro-objects, such as biological cells. Two bioscience applications of this system were described. The first application involved the extraction of a cell’s cytoplasm using two, two-fingered micro-hands. While one hand holds the cell firmly, the other punctures the cell and tears it. Then, the hand holding the cell squeezes the cytoplasm from the cell. The second application involved the measurement of the mechanical properties of living cells using the micro-finger and a micro-force sensor based on the AFM principle. An AFM cantilever was placed within the microscopic field and the micro-finger was used to hold the cell, pressing it against the cantilever tip. By measuring the force and the deformation of the cell, the force–deformation curve of the cell could be obtained. Future bioscience techniques will require more accurate, faster, and more skillful micro-manipulation, observation, and measurement of biological objects. Since the two-fingered micro-hand is easy to manipulate and has a high positioning accuracy, our system can be used to make detailed analyses and precise operations on cells possible. We plan to apply our system to practical tasks in the fields of bioscience and cell engineering. Acknowledgement This work was supported by MEXT under a Grant-in-Aid for Scientific Research on Priority Areas (Project No. 17076010).
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