WO2009084374A1 - チップ駆動装置及びカンチレバーチップ - Google Patents

チップ駆動装置及びカンチレバーチップ Download PDF

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Publication number
WO2009084374A1
WO2009084374A1 PCT/JP2008/072199 JP2008072199W WO2009084374A1 WO 2009084374 A1 WO2009084374 A1 WO 2009084374A1 JP 2008072199 W JP2008072199 W JP 2008072199W WO 2009084374 A1 WO2009084374 A1 WO 2009084374A1
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WO
WIPO (PCT)
Prior art keywords
tip
chip
cells
adapter
tip portion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2008/072199
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English (en)
French (fr)
Japanese (ja)
Inventor
Kiyohiko Tateyama
Yasuo Sasaki
Yuka Imaoka
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Olympus Corp
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Olympus Corp
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Filing date
Publication date
Application filed by Olympus Corp filed Critical Olympus Corp
Publication of WO2009084374A1 publication Critical patent/WO2009084374A1/ja
Priority to US12/813,077 priority Critical patent/US20100248342A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/54—Supports specially adapted for pipettes and burettes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/32—Micromanipulators structurally combined with microscopes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries

Definitions

  • the present invention is a chip driving device capable of moving the tip portion toward the object while holding the tip portion formed in the object direction at a predetermined angle with respect to the flexible support portion at a predetermined angle.
  • the present invention also relates to a cantilever tip that can be attached to such a tip drive device via a predetermined member.
  • WO 04/092 369 discloses a microinjection method and apparatus for introducing a substance to be introduced such as a gene into cells.
  • the introduced substance is electrically adsorbed to the tip of the microneedle which is the tip portion, and the microneedle is made to penetrate into cells.
  • the substance to be introduced is detached from the tip of the fine needle and introduced into the cell.
  • the penetration of the microneedles into cells is carried out by finely moving the microneedles using a piezoelectric element that expands and contracts coaxially with the microneedles.
  • a technique is a system in which a gene is held at the tip of a fine needle, and it is possible to introduce a gene into cells with low invasiveness, and a high survival rate can be obtained.
  • the penetration volume of the microneedle provided at the tip of the cantilever tip into the cell is small, and the cell membrane has fluidity. Therefore, even if the cantilever tip is moved to a position where the tip of the fine needle is considered to have penetrated into the cell, the cell membrane may flow over the surface of the needle portion and may not be able to penetrate the cell membrane. For this reason, there is a disadvantage that the chip driving is not stable and a good introduction rate can not be obtained.
  • the present invention has been made in view of the above-described problems, and a substance is introduced into cells with high efficiency or electrical stimulation is applied to cells while maintaining low survival rates and high survival rates.
  • An object of the present invention is to provide a tip drive device and a cantilever tip that can be observed efficiently.
  • the tip portion is moved in the direction of the object while maintaining the tip portion formed in the direction of the object at a predetermined angle with respect to the flexible support portion.
  • Possible chip drive devices The tip portion includes a tip end of the support portion and a tip driving device having a contact side which contacts an object with a predetermined pressure in a cross section along the extension direction.
  • a flexible support portion and a tip portion formed at a predetermined angle with respect to the support portion are provided, and the tip portion can be moved in a predetermined direction.
  • a cantilever tip that can be attached to a flexible tip drive via a predetermined member,
  • the tip portion includes a tip of the support portion, and a cantilever tip having a contact side which is at least partially in contact with the object at a predetermined pressure in a cross section along the extension direction.
  • FIG. 1 is a whole block diagram showing a chip driving apparatus according to a first embodiment of the present invention.
  • FIG. 2 is a diagram showing the configuration of the characterizing portion of the chip driving device according to the first embodiment.
  • FIG. 3 is a view showing the configuration of the needle.
  • FIG. 4 is a side view of a cantilever tip having a sharpened tip portion used in a general tip drive device.
  • FIG. 5 is a perspective view showing the configuration of a tip unit used in the tip drive device according to the first embodiment.
  • FIG. 6 is a side view showing the configuration of the tip unit.
  • FIG. 7 is an enlarged perspective view of the tip portion in the first embodiment.
  • FIG. 8 is a diagram for explaining the interference due to the angle of the needle.
  • FIG. 8 is a diagram for explaining the interference due to the angle of the needle.
  • FIG. 9 is a view for explaining the movable range of the needle.
  • FIG. 10 is a block diagram showing the electrical configuration of the chip driving device according to the first embodiment.
  • FIG. 11 is a diagram showing a flowchart for explaining a chip driving method using the chip driving device according to the first embodiment.
  • FIG. 12 is a diagram showing the state of chip introduction by the conventional sharp chip portion.
  • FIG. 13 is a diagram showing the state of chip introduction by the chip unit in the first embodiment.
  • FIG. 14 is a view showing a microscope image immediately after introducing a gene expressing GFP fluorescent protein into Hela S3 cells by the chip driving device according to the first example.
  • FIG. 15 is a diagram showing a phase-contrast observation microscope image 24 hours after the gene for expressing the GFP fluorescent protein is introduced into the HelaS3 cell by the chip driving device according to the first embodiment.
  • FIG. 16 is a diagram showing a microscope image 24 hours after the gene for expressing GFP fluorescent protein is introduced into Hela S3 cells by the chip driving device according to the first example.
  • FIG. 17 is a perspective view showing the shape of a tip portion in a tip driving apparatus according to a second embodiment of the present invention.
  • FIG. 18 is a side view showing the shape of the tip portion in the second embodiment.
  • FIG. 19 is a diagram for explaining a method of manufacturing the tip portion in the second embodiment.
  • FIG. 20 is a view showing the state of chip introduction by the chip portion in the second embodiment.
  • the chip driving device 10 according to the first embodiment of the present invention is used by being attached to an inverted microscope 12 for observing cells, as shown in FIG.
  • the inverted microscope 12 includes an illumination device 14, a microscope XY stage 16, a microscope XY stage handle 18, an objective lens (not shown), and an eyepiece lens 20.
  • the illumination device 14 illuminates the cells on the dish 22 containing the cells.
  • the microscope XY stage 16 moves the dish 22 in the X and Y directions.
  • the microscope XY stage handle 18 drives the microscope XY stage 16.
  • the objective lens and eyepiece lens 20 are an optical system for observing the light reflected or transmitted from the cells on the dish 22 or the fluorescence generated from the cells. At least the bottom surface of the dish 22 is formed of a transparent material such as glass so that the cells can be observed.
  • the inverted microscope 12 operated manually was demonstrated here, it may be a motorized inverted microscope which drive-controls the microscope XY stage 16 by computer. Furthermore, it may be an inverted microscope which has a CCD camera or the like and displays an observation image on a monitor.
  • the illumination device 14 includes a transmissive illumination light source 24, a condenser lens 26, and an epi-illumination light source 28.
  • the transmission illumination light source 24 irradiates the cells on the dish 22 with illumination light from the side opposite to the eyepiece lens 20.
  • the condenser lens 26 condenses the illumination light emitted from the transmissive illumination light source 24 on the cells.
  • the epi-illumination light source 28 irradiates the cells on the dish 22 with illumination light from the same direction as the eyepiece lens 20.
  • the chip driving device 10 is configured of an apparatus body 30, a microscope adapter 32, and an operation module 34.
  • the microscope adapter 32 is a mounting portion of the device main body 30 to the condenser lens 26.
  • FIG. 1 shows a state in which the device body 30 is mounted on the right side of the condenser lens 26 with respect to the front side of the inverted microscope 12, which is the side on which the eyepiece 20 is provided.
  • the operation module 34 is connected to the device body 30 via a cable (not shown), and can be installed at an arbitrary position.
  • the device body 30 includes an adapter holding portion 36, a Z drive portion 38, and a needle tip XY adjustment knob 40.
  • the tip portion 42 to be driven is provided on the needle 44.
  • a needle 44 with such a tip 42 is attached to the adapter 46.
  • An adapter 46 mounted with such a needle 44 is attached to the adapter holding portion 36.
  • the Z drive unit 38 moves the tip unit 42 in the Z direction by moving the adapter holding unit 36 in the Z direction.
  • the needle tip XY adjustment knob 40 adjusts the XY position of the tip unit 42 by moving the adapter holding unit 36 in the X direction and the Y direction.
  • the adapter holding unit 36 is not shown in a linear movement mechanism (not shown) of the Z drive unit 38, and an XY driving mechanism (not shown).
  • Z-axis drive mounting portion 48 for mounting via drive).
  • the adapter holding portion 36 has a mounting member for detachably mounting the adapter 46 on the side opposite to the Z-axis drive portion mounting portion 48 in the longitudinal direction.
  • this mounting member for example, if the adapter 46 is made of metal or provided with a metal portion at the corresponding portion, it is the magnet 50.
  • the right side of the dashed dotted line of the adapter holding portion 36 is a portion accommodated in the apparatus main body 30. That is, the magnet 50 is provided at a position outside the apparatus main body 30.
  • a fitting portion 52 to be fitted to a hole or a groove provided in the adapter 46 is disposed for positioning of the adapter 46.
  • the fitting portion 52 protrudes toward the front side of the inverted microscope 12 so that the adapter 46 can be attached by being inserted from the front side.
  • the magnet 50 and the fitting portion 52 may be provided on the back surface side of the adapter holding portion 36 so that the adapter 46 can be attached even when the device body 30 is attached to the left side of the condenser lens 26.
  • the adapter holding portion 36 may be configured to be replaceable according to the mounting position of the device body 30.
  • the needle 44 attached to the adapter 46 is composed of a cantilever tip 54 and a shaft 56 for holding the cantilever tip 54, as shown in FIG.
  • the tip portion 42 is formed on the cantilever tip 54.
  • the cantilever tip 54 is bonded to the tip of the shaft 56.
  • the cantilever tip 54 is manufactured by a silicon process, and includes a silicon base portion 58, a flexible lever portion 60, and the tip portion 42.
  • the silicon base portion 58 is a portion for bonding with the other portion, that is, the shaft 56.
  • the lever portion 60 extends from the silicon base portion 58 and has, for example, a thickness of 2.7 ⁇ m, a length of 240 ⁇ m, and an elastic constant of about 2 N / m.
  • the tip portion 42 is formed at the free end of the lever portion 60 at an angle of approximately 90 degrees with respect to the longitudinal direction of the lever portion 60.
  • the tip portion in a general chip driving device, as shown in FIG. 4, a tip portion 62 whose tip is sharpened is used.
  • the tip portion in the tip drive device 10 according to the present embodiment, as shown in FIGS. 5 and 6, the tip portion is formed as the tip portion 42 which is flattened substantially parallel to the lever portion 60. That is, as shown in FIG. 7, in the cross section along the extension direction including the tip of the lever portion 60, the tip portion 42 includes a first region including a contact side that contacts cells with a predetermined pressure (tip And a second region (side surface) 66 connected to the lever portion 60.
  • the needle 44 incorporating the tip portion 42 as described above is inserted and fixed in a hole (not shown) formed in the adapter 46 and then the adapter 46 attached with the needle 44 is It is mounted on the device body 30.
  • the needle 44 which is a component (consumable) having a high degree of replacement, and it is possible to use the tip drive device 10 repeatedly without the risk of contamination.
  • the elongated needle 44 is directly attached to the apparatus main body 30, the workability is poor, and there is a possibility that the tip portion 42 may be hit somewhere in the inverted microscope 12 such as the microscope XY stage 16 at the attachment operation. is there.
  • the risk of such breakage is reduced. be able to.
  • the adapter 46 is configured to hold the shaft 56 of the needle 44 obliquely downward at a predetermined angle when the adapter 46 is attached to the apparatus main body 30.
  • the cantilever tip 54 is bonded to the shaft 56 at a predetermined angle.
  • the tip portion 42 is provided so as to extend in a direction intersecting the longitudinal direction of the lever portion 60. Therefore, when the adapter 46 is attached to the apparatus main body 30, the tip portion 42 of the tip portion 42 is held so that the tip thereof is directed substantially vertically downward at the free end of the lever portion 60.
  • the fixed angle at which the adapter 46 holds the shaft 56 is determined as follows. That is, as shown by reference numeral 68 in FIG. 8, when the shaft 56 is raised too much, it interferes with the condenser lens 26. If the length of the needle 44 is, for example, about 50 mm, raising the shaft 56 more than 60 degrees interferes with the condenser lens 26. Conversely, if the shaft 56 is laid too much, it will interfere with the side wall of the dish 22, as shown by the reference numeral 70 in FIG. In general, in a 35 mm glass bottom dish frequently used in cell culture, the side wall of the dish 22 may be interfered when lying down more than 30 degrees. Therefore, in the present embodiment, the fixed angle at which the adapter 46 holds the shaft 56 is set to 45 degrees, which is in the middle of 30 degrees to 60 degrees.
  • a movable range 72 as shown by an alternate long and short dash line in FIG. 9 is obtained.
  • the glass surface (about ⁇ 14 mm) of the 35 mm glass bottom dish can be worked without interfering with the side walls of the condenser lens 26 and the dish 22.
  • the fixed angle at which the adapter 46 holds the shaft 56 is determined to provide the needle 44 with a sufficient movable range 72 in consideration of interference with the condenser lens 26 and the dish 22 used. Then, in the adapter 46, a hole (not shown) for inserting and fixing the needle 44 is formed with an angle that holds the shaft 56 at this fixed angle.
  • the operation module 34 of the chip driving device 10 has a Z adjustment handle 74, a speed setting dial 76, a fine adjustment (upper) button 78, a fine adjustment (lower) button 80, and a movement amount setting dial 82, and a Z value set button 84.
  • the Z adjustment handle 74 and the speed setting dial 76 are used for coarse movement (in mm) of the adapter holder 36 in the Z direction.
  • the speed setting dial 76 switches and sets the driving amount in accordance with the rotation operation of the Z adjustment handle 74 in three stages of large, medium, and small.
  • the fine adjustment buttons 78 and 80 and the movement amount setting dial 82 are used for fine Z direction movement (in ⁇ m units) of the adapter holder 36.
  • the fine adjustment (upper) button 78 or the fine adjustment (lower) button 80 is operated, the adapter holding unit 36 is finely driven in the Z direction using the Z drive unit 38 according to the button.
  • the movement amount setting dial 82 switches and sets a minute drive amount in accordance with one ON operation of the fine adjustment buttons 78 and 80 in three steps of large, medium, and small.
  • the Z value set button 84 is a button for giving an instruction to store an arbitrary position in the Z direction. Even if the Z adjustment handle 74 or the fine adjustment buttons 78, 80 are operated, the adapter holding portion 36 is lowered below the position stored by the Z value set button 84 (in the direction of the sample in the dish 22) Will not.
  • the Z value set button 84 is provided with a latch mechanism (not shown), and when the operator performs a pressing operation, that is, an ON operation, the pressed state, that is, the ON state is maintained until the pressing operation is performed again.
  • first mode the operation of the Z adjustment handle 74 and the fine adjustment buttons 78 and 80 when the Z value set button 84 is in the OFF state
  • second mode the operation of the adjustment buttons 78 and 80 when the Z value set button 84 is in the ON state
  • the device body 30 is a position for detecting the position of the adapter holding portion 36 in addition to the Z driving portion 38.
  • a detection unit 86 is provided.
  • the position detection unit 86 the position of the adapter holding unit 36 may be directly detected optically or may be detected indirectly by detecting the amount of drive of the Z drive unit 38. It is good. Further, the position detection unit 86 may be provided separately from the apparatus main body 30.
  • the operation module 34 includes an input unit 88, a storage unit 90, a determination unit 92, an indicator light 94, a control unit 96, and a power supply 98.
  • the input unit 88 includes a movement instruction unit 88A, a speed setting unit 88B, a movement amount setting unit 88C, and a Z value setting unit 88D.
  • the movement instruction unit 88A outputs a movement instruction signal in response to the ON operation of the Z adjustment handle 74 and the fine adjustment buttons 78 and 80.
  • the speed setting unit 88B outputs a speed setting signal indicating the moving speed set by the speed setting dial 76.
  • the movement amount setting unit 88C outputs a movement amount setting signal indicating the movement amount set by the movement amount setting dial 82.
  • the Z value setter 88D outputs a Z value set signal in response to the ON operation of the Z value set button 84.
  • Each signal output from the input unit 88 is input to the control unit 96.
  • the storage unit 90 stores, as a Z value, the position of the adapter holding unit 36 detected by the position detection unit 86 when the Z value set button 84 is turned on.
  • the determination unit 92 compares the position of the adapter holding unit 36 detected by the position detection unit 86 with the Z value stored in the storage unit 90, and determines whether the adapter holding unit 36 has reached the position of the Z value It is determined whether or not.
  • the indicator light 94 lights up in response to the Z value set signal from the Z value setting unit 88D. The operator can confirm the storage of the Z value by turning on the indicator light 94.
  • the control unit 96 controls the entire chip drive device 10. Then, the power supply 98 supplies power to operate each part of the chip drive device 10.
  • the attachment side of the apparatus body 30 is selected, and the apparatus body 30 is attached to the condenser lens 26 via the microscope adapter 32 (step S10).
  • the needle 44 is inserted into and attached to the adapter 46 removed from the apparatus body 30 (step S12). Then, the adapter 46 to which the needle 44 is attached is attached to the adapter holding portion 36 of the apparatus main body 30 from the front side of the inverted microscope 12 (step S14).
  • step S16 chip positioning is performed (step S16). That is, the position of the tip portion 42 formed at the tip of the needle 44 is visually set to the center position (field center position) of an objective lens (not shown). This is performed by operating the needle tip XY adjustment knob 40 of the apparatus main body 30 and the Z adjustment handle 74 of the operation module 34 while observing with the eyepiece lens 20. In addition, this operation is performed without placing the dish 22 on the microscope XY stage 16. In the Z direction, the speed setting dial 76 of the operation module 34 is set large or small, and the lever portion 60 of the cantilever tip 54 can be visually confirmed in the field of view by operating the Z adjustment handle 74. The lowering operation of the tip unit 42 is performed.
  • the sample is set, that is, the dish 22 is placed on the microscope XY stage 16 (step S18).
  • the procedure is as follows. That is, first, the Z adjustment handle 74 of the operation module 34 is operated to retract the tip portion 42 at the tip of the needle 44 to a safe area (the upper side in the Z direction). Further, the support 100 (see FIG. 1) of the inverted microscope 12 is turned backward. Thus, the entire device body 30 moves. After thus securing the space for the sample set, the dish 22 (sample) is placed on the microscope XY stage 16. And then, the pillar 100 of the inverted microscope 12 is put back.
  • the above-mentioned dish 22 (sample) is set in a dispersed state of the substance to be introduced in order to introduce the substance into the cells cultured in the culture solution in the dish 22.
  • step S20 cells to be introduced are selected (step S20).
  • the microscope XY stage handle 18 is operated to place cells to be observed in the dish 22 under microscope observation.
  • the Z drive unit 38 is operated to bring the tip unit 42 of the needle 44 closer to the cell from above the cell. That is, first, the lowering operation of the tip portion 42 in the Z direction is performed until the lever portion 60 of the cantilever tip 54 can be visually confirmed in the visual field while observing with the eyepiece lens 20. This is performed by operating the Z adjustment handle 74 by setting the speed setting dial 76 of the operation module 34 to a small size.
  • the descent operation in the Z direction is performed using the lever portion 60 that can be roughly identified even if the focus is not larger than the tip portion 42. Then, if the lever portion 60 is lowered to the visual field so that the lever portion 60 can be visually confirmed, next, while observing with the eyepiece lens 20, the microscope XY stage 16 is adjusted in the XY direction by visual observation. The position considered to be the tip part 42 is set right above. As described above, cells to be introduced are selected (determined).
  • the subsequent operation differs depending on whether the Z value is set in the storage unit 90 of the operation module 34 or not.
  • the chip is introduced in the first mode (without the Z value) (step S24). That is, while operating the Z adjustment handle 74 or the fine adjustment buttons 78 and 80 of the operation module 34, observation is performed with the eyepiece lens 20 to confirm “distortion of cells” or “deflection of the lever portion 60”. Determine the optimal position of the direction. At this time, the operation of the Z adjustment handle 74 is performed while appropriately switching the sensitivity of the speed setting dial 76 in the large, middle, and small sizes. Further, the fine adjustment buttons 78 and 80 are operated while switching the sensitivities of the movement amount setting dial 82 large, medium, and small.
  • the tip portion 42 is lowered and brought close to the bottom surface of the dish 22, and the tip of the tip portion 42 comes in contact with the cells in the dish 22 while being lowered.
  • the tip of the tip section 42 makes holes or flaws in the cell, that is, the cell membrane and the nucleus.
  • the substance dispersed in the dish 22 flows through the holes or the wound thus formed, whereby the substance flows into the cells.
  • the physical stimulation by deforming the cells in the tip portion 42 also causes the channel bound to the stretch receptor or the like to open. In this way, the introduction of the substance takes place.
  • the control unit 96 of the operation module 34 determines that the Z value set button 84 is pressed. In response to the determination, the control unit 96 causes the storage unit 90 to store the current position of the adapter holding unit 36 detected by the position detection unit 86 in step S24 as a Z value indicating the optimum position (step S26). At this time, the indicator 94 is turned on.
  • the cell 102 is scratched by point contact + sliding. If the tip end of the tip portion 62 is sharp as described above, there is a high possibility that the tip end of the tip portion 62 is broken when sliding as shown by the arrow 106.
  • the tip portion 42 having a flattened tip since the tip portion 42 having a flattened tip is used, the tip end of the tip portion 42 may not be sharp and may be damaged at the time of sliding. Is low.
  • the contact area between the tip portion 42 and the cell 102 increases, so that the cell 102 can have a large scratch 104 to a certain extent, and the efficiency of substance introduction can be increased.
  • damage to the cell 102 can be reduced.
  • the introduction amount is controlled in two regions Therefore, it is easy to optimize the chip introduction amount, which contributes to the stabilization of the introduction amount.
  • the area ratio of the first region 64 to the second region 66 of the chip portion 42 may be determined so that the chip introduction amount becomes optimum.
  • step S28 the operator operates the Z adjustment handle 74 of the operation module 34 to raise the needle 44, thereby retracting the tip portion 42 (step S28).
  • the speed setting dial 76 of the operation module 34 is set to middle or small, and the tip portion 42 is moved upward by operating the Z adjustment handle 74 in the first mode (without setting the Z value). .
  • the cell membrane After raising the tip 42 and withdrawing the tip 42 from the cell 102, the cell membrane recovers by self-repair and the substance is taken into the cell when a certain time elapses.
  • step S28 the operator turns off the power switch (not shown) of the apparatus main body 30 if it is not necessary to introduce a substance into the next sample cell (step S30). It will end by operating.
  • Step S30 the process returns to the above-mentioned Step S20, and the introduction of the substance is repeatedly performed on an arbitrary sample cell. That is, the operator operates the microscope XY stage handle 18 while observing with the eyepiece lens 20 to operate the microscope XY stage 16 and set the tip unit 42 right above the cell 102 to be introduced. That is, the cell 102 to be introduced is selected (step S20).
  • the chip introducing operation in the second mode is performed (step S32).
  • the tip unit is not concerned with excessive operations by the Z adjustment handle 74 and the fine adjustment buttons 78 and 80. It is possible to lower it to the optimum position only by performing an operation to lower 42 sufficiently. That is, the determination unit 92 of the operation module 34 compares the position of the adapter holding unit 36 detected by the position detection unit 86 with the Z value set in the storage unit 90 to obtain the adapter holding unit 36 (chip unit 42). Determines whether the position of the Z value has been reached. Then, if it is determined that the determination unit 92 has reached that point, the control unit 96 of the operation module 34 further controls the Z drive unit 38 even if the Z adjustment handle 74 and the fine adjustment buttons 78 and 80 are operated. Control not to descend.
  • the adapter holding unit 36 (chip unit 42) may be automatically lowered to that position. That is, the steering wheel operation in the second mode may be automated.
  • the motorized inverted microscope drives and controls the microscope XY stage 16 by a computer, is equipped with a CCD camera and the like, and displays an observation image on a monitor.
  • the tip drive device 10 according to the present embodiment is applied to such a motorized inverted microscope instead of the manually operated inverted microscope 12, cells in need of introduction of a substance are selected on the image in advance. It may be made to move to that position automatically. That is, the adjustment of the microscope XY stage 16 in the XY directions may be automated.
  • the substance to be introduced into the cell may be a gene, a dye, a fluorescent reagent such as a quantum dot, an ion, a peptide, a protein, a polysaccharide, or the like as long as it can be dispersed in the dish 22.
  • the cross-sectional shape of the flat surface (first region 64) in contact with the cell 102 is not limited to the triangle as shown in FIGS. 5 and 7, and it may be a quadrangle or an n-gon (polygon) It may be That is, the first area 64 may be a surface other than a point or a pipe.
  • the introduced gene is a gene that expresses the GFP fluorescent protein, and the correctness of the introduction can be confirmed by fluorescence observation.
  • FIG. 14 shows a microscopic observation image of cells to which introduction was attempted immediately after gene introduction. We are trying to select and introduce multiple cells in the observation image.
  • FIG.15 and FIG.16 is the microscope observation image which confirmed the success or failure of introduction
  • FIG. 15 is a phase contrast observation image, showing the state of cells after 24 hours.
  • FIG. 16 shows the cells observed by fluorescence observation. In cells into which the introduction was successful, the gene was expressed and strong fluorescence intensity was obtained. From this result, it can be confirmed that the gene has been introduced into the cell very efficiently.
  • the tip portion 42 having the As a result the amount of introduced chips is stabilized, so that the substance to be introduced can be introduced into the cells with certainty and high introduction efficiency while the invasiveness is low and the survival rate is high as in the prior art.
  • the chip portion 42 in the chip driving device according to the second embodiment of the present invention makes the area of the second region 66 smaller than in the first embodiment, as shown in FIGS.
  • the first area 64 is a quadrangle in FIG. 17, it may be a surface of an n-gon (polygon) having a triangle or more, as in the first embodiment.
  • Such a tip portion 42 in the present embodiment can be manufactured, for example, by cutting a commercially available cantilever tip on which a sharp tip portion 62 is formed as shown in FIG. That is, a commercially available cantilever chip having a sharp tip portion 62 can be manufactured by cutting from two directions as shown by a dashed dotted line and a dashed line in FIG.
  • the chip portion 42 in the first embodiment manufactured by the silicon process may be manufactured by cutting from one direction as indicated by a dashed dotted line in FIG.
  • the area ratio of the first region 64 to the second region 66 of the chip portion 42 may be determined so that the chip introduction amount becomes optimal. Therefore, the commercially available cantilever tip on which the sharp tip portion 62 is formed is cut in two directions so as to achieve such a ratio. Alternatively, the cantilever tip 54 on which the tip portion 42 having the first and second regions 64 and 66 as in the first embodiment is formed is cut from one direction.
  • the tip portion 42 in the present embodiment has the tip of the tip portion 42 slid as shown by the arrow 106 as shown in FIG.
  • the cells can be injured and materials can be introduced.
  • the area of the second region 66 of the tip portion 42 is smaller than that of the first embodiment, the amount of introduction proportional to the volume of the tip portion 42 decreases, but the load applied to the cell 102 is the above. It is smaller than the first embodiment.
  • the chip introduction amount is stabilized, so that the invasiveness is still low and the survival rate is high as in the conventional case.
  • the introduced substance can be introduced into cells reliably and with high introduction efficiency.
  • the chip unit 42 in the first embodiment is suitable for chip introduction to a cell (small cell or thin cell) weaker than the target cell.
  • first and second embodiments an example is described in which only one tip drive device 10 is attached to the inverted microscope 12 and used, but a plurality of tip drive devices 10 may be used at the same time. Can be mounted on both sides of the condenser lens 26 and used.
  • the said electrical stimulation is not limited to using the some chip part 42, but provides an electrical potential difference between the one chip part 42 and the predetermined
  • prescribed electrode for example, glass bottom etc. with ITO etc.
  • the tip portion 42 preferably has conductivity.
  • cells can be electrically stimulated to efficiently observe living cells while maintaining a high survival rate with low invasiveness.
  • the microscope adapter 32 extended from the device body 30 of the chip driving device 10 is attached to the condenser lens 26.
  • the present invention is not limited to this. It may be attached to

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PCT/JP2008/072199 2007-12-27 2008-12-05 チップ駆動装置及びカンチレバーチップ Ceased WO2009084374A1 (ja)

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Citations (3)

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Publication number Priority date Publication date Assignee Title
JPH06343478A (ja) * 1993-06-08 1994-12-20 Hitachi Ltd マイクロインジェクション方法及び装置
JP2003325161A (ja) * 2002-03-06 2003-11-18 National Institute Of Advanced Industrial & Technology 細胞操作装置及び方法
WO2004092369A1 (ja) * 2003-04-11 2004-10-28 Riken マイクロインジェクション方法および装置

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DE3718066A1 (de) * 1987-05-29 1988-12-08 Zeiss Carl Fa Verfahren zur mikroinjektion in zellen bzw. zum absaugen aus einzelnen zellen oder ganzer zellen aus zellkulturen
WO1999046361A1 (fr) * 1998-03-12 1999-09-16 Center For Advanced Science And Technology Incubation, Ltd. Procedes permettant de percer une cellule en un site specifique
JP4200665B2 (ja) * 2001-05-08 2008-12-24 株式会社日立製作所 加工装置
JP4370397B2 (ja) * 2003-07-18 2009-11-25 独立行政法人産業技術総合研究所 物質細胞挿入の力学解析方法
JP2007166981A (ja) * 2005-12-22 2007-07-05 Fujitsu Ltd 注入装置及び方法
JP2007319037A (ja) * 2006-05-30 2007-12-13 Olympus Corp 細胞操作装置および細胞操作方法
US20080213899A1 (en) * 2006-10-12 2008-09-04 University Of Connecticut Rotationally Oscillating Injector

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06343478A (ja) * 1993-06-08 1994-12-20 Hitachi Ltd マイクロインジェクション方法及び装置
JP2003325161A (ja) * 2002-03-06 2003-11-18 National Institute Of Advanced Industrial & Technology 細胞操作装置及び方法
WO2004092369A1 (ja) * 2003-04-11 2004-10-28 Riken マイクロインジェクション方法および装置

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