JP4621942B2 - Microinjection method using liquid expansion pressure by laser heating - Google Patents

Description

  The present invention relates to a liquid discharge method, and more particularly to a liquid discharge method that can be applied to a microinjection method.

  In order to introduce target substances such as nucleic acids and proteins into cells, a microinjection method using a microcapillary (also called a micropipette) is used. In the conventional microinjection method, a liquid containing a target substance is held inside a microcapillary whose tip is needle-shaped, the tip of the capillary is inserted into a cell, and the capillary is made using air pressure or hydraulic pressure. The target substance is injected into the cell by discharging the liquid held inside the cell into the cell.

  When the outer diameter of the capillary tip is 1 μm or less, a liquid containing the target substance can be injected into the cell without damaging the cell, and the capillary inner tip is injected into the cell. The amount can be set to a size that can be precisely controlled.

  However, when the liquid held inside the capillary is discharged using air pressure or hydraulic pressure, a sufficient discharge pressure cannot be obtained. Therefore, if the outer diameter of the capillary tip is 1 μm or less, the tip of the capillary is There is a risk of clogging. For this reason, when the liquid retained inside the capillary is discharged using air pressure or hydraulic pressure, the liquid is injected into the cell without damaging the cell, and the amount of liquid injected into the cell is precisely determined. It is difficult to realize control.

Under such circumstances, as shown in FIG. 7, a nucleic acid is introduced from a liquid inlet 32 into a glass microcapillary 30 having a liquid outlet 31 at one end and a liquid inlet 32 at the other end. Liquid sample L containing liquid, liquid alloy Galinstan G and silicon oil S are sequentially introduced, the liquid inlet 32 is sealed with a glass cap 40 filled with epoxy resin E, and hot air is blown from the outside of the microcapillary 30. A method of generating a thermal expansion pressure of Galinstan G and discharging the liquid sample L from the liquid discharge port 31 has been developed (Non-patent Document 1). According to this method, by using the thermal expansion pressure generated by the thermal expansion of the Galinstan G, it is possible to obtain a larger liquid discharge pressure than when using air pressure or hydraulic pressure, and the outer diameter of the tip portion is 1 μm or less. The liquid sample L can be discharged using the microcapillary 30 that is.
Knoblauch et al., “Nature Biotechnology”, 1999, Vol. 17, pp.906-909

  However, when the Galinstan G is heated by blowing hot air from the outside of the microcapillary 30, it is difficult to control the temperature of the Galinstan G, and the range in which the temperature of the Galinstan G can be controlled is narrow.

  In addition, since the microcapillary 30 is thermally expanded together with the Galinstan G by blowing hot air, it is necessary to use a special liquid metal called Galinstan having a high coefficient of thermal expansion.

  Furthermore, since the microcapillary 30 may vibrate by blowing hot air, it is difficult to perform precise work in a narrow space on the microscope stage.

  Furthermore, a part of Galistan G may fall on the tip of the microcapillary 30 and the tip may be clogged.

  Furthermore, since the operation | work which seals the liquid inlet 32 with the glass cap 40 with which the epoxy resin E was filled is complicated and requires time, work efficiency falls.

  Therefore, the present invention heats the liquid held inside the capillary without blowing hot air from the outside of the capillary, and uses the thermal expansion pressure of the liquid to obtain the target liquid held inside the capillary. It is an object of the present invention to provide a liquid discharge method that can be discharged, and a capillary holder and a laser irradiation apparatus that can be used for the liquid discharge method.

  To achieve the above object, the present invention provides a first liquid layer containing a first liquid layer and a second liquid layer containing a laser light absorber inside a capillary having a liquid discharge port at one end. Is held so as to be located on the liquid discharge port side, forming a sealed portion that seals the second liquid layer inside the capillary, irradiating the second liquid layer with laser light, A liquid discharge method is provided, wherein the liquid in the first liquid layer is discharged from a liquid discharge port.

  Further, the present invention provides a capillary holding portion, an optical fiber mounting portion, and a laser beam emitted from an optical fiber mounted on the optical fiber mounting portion in a target region on an outer wall surface of the capillary held by the capillary holding portion. There is provided a capillary holder comprising an optical path for guiding the laser beam so as to be irradiated.

  Furthermore, the present invention controls the capillary holder of the present invention, an optical fiber mounted on the optical fiber mounting portion of the capillary holder, a laser light source connected to the optical fiber, and an output of the laser light emitted from the laser light source. Provided is a laser beam irradiation apparatus comprising a controller.

  In the liquid ejection method of the present invention, when the second liquid layer is irradiated with laser light, the laser light absorbent contained in the second liquid layer absorbs the laser light and generates heat, and the second liquid layer is heated. And thermally expands. Since the second liquid layer is sandwiched between the first liquid layer and the sealed portion and is held in a sealed state inside the capillary, thermal expansion pressure is generated by the thermal expansion of the second liquid layer To do. Since the movement of the second liquid layer in the direction of the sealing portion is prevented by the sealing portion, the thermal expansion pressure of the second liquid layer is converted into the discharge pressure of the first liquid layer, and the liquid of the first liquid layer Is discharged from the liquid discharge port of the capillary.

  In the liquid ejection method of the present invention, the laser beam is transmitted through the capillary and irradiated onto the second liquid layer, but the capillary does not thermally expand due to the transmission of the laser beam, so the thermal expansion of the second liquid layer The pressure is quickly and efficiently converted into the discharge pressure of the first liquid layer, and an amount of liquid approximately equal to the volume expansion amount of the second liquid layer is discharged with good response. Therefore, it is not necessary to use a special liquid metal such as galinstan as the liquid constituting the second liquid layer.

  In the liquid ejection method of the present invention, the temperature rise of the second liquid layer can be controlled by adjusting the output of the laser beam, and by controlling the temperature rise of the second liquid layer, It is possible to control the discharge amount of the liquid constituting the liquid layer.

  In the liquid ejection method of the present invention, when irradiating laser light, the capillary does not vibrate unlike when hot air is blown, and a precise operation can be performed even in a narrow space on the microscope stage.

  In the liquid ejection method of the present invention, when the liquid in the first liquid layer held in the capillary is ejected, the amount of liquid in the first liquid layer decreases, but the second liquid layer is adjusted by adjusting the output of the laser beam. By controlling the temperature rise, a sufficiently large discharge pressure of the first liquid layer can be obtained, so that the liquid of the first liquid layer is discharged even if the liquid amount of the first liquid layer decreases. be able to. Therefore, the liquid of the first liquid layer held in the capillary can be repeatedly discharged.

  In a preferred aspect of the liquid ejection method of the present invention, a liquid constituting the first liquid layer, a liquid constituting the second liquid layer, and an inside of the capillary from an opening of the capillary at the other end, and After sequentially introducing a curable liquid resin, the liquid resin is cured to form the sealed portion. According to this aspect, the sealing part firmly fixed to the capillary can be easily formed.

  In the above aspect, the liquid resin is preferably made of an energy ray curable resin. In this case, the liquid resin can be cured by the energy ray irradiation without thermally expanding the second liquid layer.

  In a preferred aspect of the liquid ejection method of the present invention, the first liquid layer contains a target substance, and a laser beam is applied to the second liquid layer while the liquid ejection port of the capillary is inserted into a cell. Then, the liquid in the first liquid layer is discharged into the cell from the liquid discharge port of the capillary. According to this aspect, the target substance can be injected into the cell. By adjusting the output of the laser beam to control the temperature rise of the second liquid layer, a sufficiently large discharge pressure of the first liquid layer can be obtained, so that the tip of the capillary inserted into the cell The target substance can be injected into cells without clogging the tip of the capillary even if the outer diameter of the tube is 1 μm or less. By setting the outer diameter of the tip of the capillary inserted into the cell to 1 μm or less, the target substance can be injected into the cell without damaging the cell, and the inner diameter of the tip of the capillary is injected into the cell. The amount of liquid to be controlled can be set to a size that can be precisely controlled.

  The liquid ejection method of the present invention can be carried out using the capillary holder or the laser beam irradiation apparatus of the present invention.

  According to the present invention, the liquid held inside the capillary is heated without blowing hot air from the outside of the capillary, and the target liquid held inside the capillary is heated using the thermal expansion pressure of the liquid. Provided are a liquid discharge method capable of discharging, a capillary holder and a laser irradiation apparatus which can be used for the liquid discharge method.

Hereinafter, an embodiment of a liquid ejection method of the present invention will be described with reference to the drawings.
In this embodiment, the capillary 1 shown in FIG.
As shown in FIG. 1A, the capillary 1 has a liquid discharge port 11 at one end and a liquid introduction port 12 communicating with the liquid discharge port 11 at the other end. As shown in FIG. 1 (a), the tip of the capillary 1 has a needle shape so that the tip of the capillary 1 can be inserted into a cell. The outer diameter of the tip of the capillary 1 is usually 0.1 to 1 μm, preferably 0.1 to 0.5 μm, so that damage to the cells can be suppressed when piercing the cells.

  The inner diameter of the capillary 1 is adjusted so that the liquid introduced from the liquid inlet 12 into the capillary 1 moves toward the liquid outlet 11 by capillary action and is held in the capillary 1. The inner diameter of the liquid inlet 12 of the capillary 1 is usually 400 to 800 μm, preferably 500 to 700 μm, and the inner diameter of the liquid outlet 11 is usually 0.06 to 0.6 μm, preferably 0.06 to 0.3 μm. .

  The capillary 1 is made of a material that can transmit laser light. Examples of the material that can transmit laser light include glass and quartz. One of these materials can be used alone, or two or more of them can be used in combination.

  As the capillary 1, a microcapillary or a micropipette generally used in a microinjection method in which a substance such as a nucleic acid is injected into a cell can be used. When the capillary 1 is made of glass, the capillary 1 can be produced from a glass tube using a known method such as one-step drawing or two-step drawing.

  In this embodiment, first, a hydrophilic liquid containing a target substance is introduced into the capillary 1 from the liquid inlet 12 of the capillary 1. The liquid moves toward the liquid discharge port 11 by capillary action, and is held inside the capillary 1 in a state where the first liquid layer 2 is formed as shown in FIG.

  The hydrophilic liquid is not particularly limited as long as it is a liquid having the property of mixing with water (aqueous). Examples of the hydrophilic liquid include water, DMSO, polyethylene glycol and the like, and one of these can be used alone or two or more can be used in combination. It is preferable to use water as the hydrophilic liquid because it can contain the target substance in a stable state and does not adversely affect the cells when injected into the cells.

  The target substance is a substance to be injected into the cell, and can be appropriately selected according to the purpose of the injection into the cell. Examples of the target substance include nucleic acids, peptides, proteins, sugars, drugs and the like, and one of these can be used alone or in combination of two or more. The target substance may be contained in the first liquid layer 2 in any state such as dissolution or dispersion.

  The nucleic acid may be any of DNA, RNA, analogs or derivatives thereof (for example, peptide nucleic acid (PNA), phosphorothioate DNA, etc.). Further, the nucleic acid may be either single-stranded or double-stranded, and may be either linear or circular.

  The first liquid layer 2 may contain a buffer, an isotonic agent, a pH adjuster, a dye, and the like in addition to the target substance. The first liquid layer 2 preferably contains a dye from the viewpoint that the liquid discharge amount can be visually controlled. Examples of the dye include fluorescein; rhodamine; phycoerythrin; Cy dyes such as Cy2, Cy3, Cy3.5, Cy5, and Cy7; Alexa-488, Alexa-532, Alexa-546, Alexa-633, Alexa-680 Alexa dyes such as BODIPY FL and BODIPY dyes such as BODIPY TR, and the like, and one of these can be used alone or two or more can be used in combination.

  In the present embodiment, a liquid that contains a laser light absorbent and can form an interface with the first liquid layer 2 is then introduced from the liquid inlet 12 of the capillary 1 into the capillary 1. The liquid moves toward the liquid discharge port 11 by capillary action, forms an interface with the first liquid layer 2, and the capillary is formed with the second liquid layer 3 formed as shown in FIG. 1 is held inside. When introducing the liquid from the liquid inlet 12 of the capillary 1 into the capillary 1, care should be taken not to mix air between the first liquid layer 2 and the second liquid layer 3. If the thermal expansion pressure of the second liquid layer 3 is absorbed by the air layer, it becomes difficult to control the conversion of the thermal expansion pressure of the second liquid 3 into the discharge pressure of the first liquid layer 2. is there.

  Since the first liquid layer 2 is composed of a hydrophilic liquid, a hydrophobic liquid can be used as a liquid that can form an interface with the first liquid layer 2. The hydrophobic liquid is not particularly limited as long as it is a liquid that does not mix with water (water-insoluble). Examples of the hydrophobic liquid include higher fatty acids or esters thereof (for example, oleic acid, methyl oleate, isobutyl oleate, octyl oleate, 2-ethylhexyl oleate, decyl oleate, lauryl oleate, oleyl oleate). Linoleic acid, methyl linoleate, ethyl linoleate, linolenic acid, methyl linolenate, palmitic acid, isopropyl palmitate, 2-ethylhexyl palmitate, stearic acid, butyl stearate, 2-ethylhexyl stearate, isotridecyl stearate, lauric acid Acid, methyl laurate, cetyl 2-ethylhexanoate, octyldodecyl erucate, methyl caprate, coconut oil fatty acid methyl, beef tallow fatty acid methyl, palm oil fatty acid methyl, etc.), aliphatic or aromatic polybasic acid ester Dialkyl esters of aliphatic dicarboxylic acids such as dioleyl adipate, diisobutyl adipate, diisodecyl adipate; aromatics such as didecyl phthalate, ditridecyl phthalate, di-2-ethylhexyl phthalate, cyclohexyl 2-ethylhexyl phthalate, etc. Dialkyl esters of aromatic dicarboxylic acids; polyalkyl esters of aromatic polycarboxylic acids such as tri-2-ethylhexyl trimellitic acid, triethyl trimellitic acid, tri-n-butyl trimellitate, triisodecyl trimellitic acid), glycerin fatty acid esters (eg, oleic acid) Monoglyceride, oleic acid diglyceride, sorbitan monooleate, sorbitan monolaurate, capric acid monoglyceride, capric acid diglyceride, diacetyl capric acid glycerin Oily liquids such as Lido, diacetyl palm oil fatty acid glycerides), epoxidized fatty acid esters (eg, epoxidized fatty acid butyl, epoxidized fatty acid octyl, etc.), and one of these is used alone or two or more Can be used in combination. Among the oily liquids, it is preferable to use a liquid that is liquid at room temperature, has a boiling point of 100 ° C. or higher, is non-toxic, odorless, and hardly vaporized. Examples of such oily liquid include oleic acid, Examples include methyl oleate, linoleic acid, methyl linoleate, and linolenic acid. As the hydrophobic liquid, a liquid metal may be used, but a non-metallic liquid is preferably used. As the hydrophobic liquid, mineral oil, liquid paraffin, and the like can be used in addition to the oily liquid.

The laser light absorber is not particularly limited as long as it is a substance that can generate heat by absorbing laser light. Examples of the laser light absorber include carbon blacks such as acetylene black, channel black, and furnace black; black iron oxides such as Fe ₃ O ₄ and FeO · Fe ₂ O ₃ ; titanium black obtained by reducing titanium dioxide, etc. The black type compound of these is mentioned, Among these, 1 type can be used individually or in combination of 2 or more types. These black compounds can generate heat by absorbing laser light having a wavelength of 150 to 11000 nm. The laser light absorber may be contained in the second liquid layer 3 in any state such as dissolution or dispersion.

  In this embodiment, a curable liquid resin is then introduced into the capillary 1 from the liquid inlet 12 of the capillary 1. The liquid resin moves in the direction of the liquid discharge port 11 by capillary action, forms an interface with the second liquid layer 3, and forms the liquid resin layer 4 as shown in FIG. Retained inside. When introducing the liquid resin into the capillary 1 from the liquid inlet 12 of the capillary 1, care should be taken not to mix air between the second liquid layer 3 and the liquid resin layer 4. If the thermal expansion pressure of the second liquid layer 3 is absorbed by the air layer, it becomes difficult to control the conversion of the thermal expansion pressure of the second liquid 3 into the discharge pressure of the first liquid layer 2. is there.

  As the curable liquid resin, for example, a liquid resin made of an active energy ray curable resin can be used. Examples of the active energy rays include ultraviolet rays, electron beams, radiations, and the like, and the active energy ray curable resins include, for example, prepolymers and / or monomers having a property of being polymerized by irradiation with active energy. Correspondingly, there may be mentioned resin compositions containing other monofunctional or polyfunctional monomers, various polymers, photopolymerization initiators, sensitizers, antistatic agents and the like. Examples of the prepolymer having the property of being polymerized by irradiation with active energy include polyvalent (meth) polyesters such as polyester poly (meth) acrylate, urethane poly (meth) acrylate, epoxy poly (meth) acrylate, and polyol poly (meth) acrylate. Examples of the monomer having a property of being polymerized by irradiation of active energy include mono (meth) acrylates such as mono (meth) acrylates of monoalcohols and mono (meth) acrylates of polyols. Can be mentioned. Examples of the photoinitiator include acetophenones, benzophenones, Michler's ketone, benzyl, benzoin, benzoin ether, benzyl ketals, thioxanthones, and the like. Examples of the sensitizer include amines, diethylaminoethyl methacrylate, and the like. Can be mentioned.

  As the curable liquid resin, a liquid resin made of a thermosetting resin may be used. However, when the thermosetting resin is cured, the heat applied to the thermosetting resin is transmitted to the second liquid layer 3 and an expansion pressure of the second liquid layer 3 may be generated. It is preferable to use a liquid resin made of a curable resin.

  As the curable liquid resin, it is preferable to use a liquid resin having a small volume change (volume reduction) generated during curing. This is because if the volume reduction generated during the curing is large, bubbles may be generated in the liquid of the first liquid layer 2 or the second liquid layer 3.

  In this embodiment, the liquid resin layer 4 is then cured to form the sealed portion 5 as shown in FIG.

  The sealing part 5 is firmly fixed to the capillary 1 and the movement of the second liquid layer 3 in the direction of the sealing part 5 is prevented even if the second liquid layer 3 is thermally expanded. Yes.

  As shown in FIG. 1 (d), one liquid surface of the second liquid layer 3 is in contact with the first liquid layer 2, and the contact between the second liquid layer 3 and the outside air through the liquid discharge port 11 is as follows. It is prevented. In addition, as shown in FIG. 1D, the other liquid surface of the second liquid layer 3 is in contact with the sealing portion 5, and contact between the second liquid layer 3 and the outside air through the liquid inlet 12 is prevented. Has been. Therefore, the second liquid layer 3 is sandwiched between the first liquid layer 2 and the sealed portion 5 and is held in a sealed state inside the capillary 1.

  When a liquid is introduced into the inside of the capillary 1 from the liquid inlet 12 of the capillary 1, as shown in FIG. 2, it is possible to use a syringe 200 to which a chip 100 that can be inserted into the capillary 1 from the liquid inlet 12 is attached. it can. As the chip 100 that can be inserted into the capillary 1 from the liquid inlet 12, for example, a plastic chip whose tip is stretched and thinned can be used. Moreover, a commercial item (for example, microloader (made by Eppendorf)) can also be used.

  In the present embodiment, next, as shown in FIG. 3, the second liquid layer 3 held inside the capillary 1 is irradiated with laser light with the liquid discharge port 11 of the capillary 1 inserted into the cell C. To do.

  The insertion of the liquid discharge port 11 of the capillary 1 into the cell C can be performed on a microscope stage while observing with a microscope, for example.

  The wavelength of the laser light applied to the second liquid layer 3 is a wavelength that can be absorbed by the laser light absorbent contained in the second liquid layer 3, and is usually 150 to 11000 nm.

When the second liquid layer 3 is irradiated with laser light, the laser light absorbent contained in the second liquid layer 3 absorbs the laser light and generates heat, and the second liquid layer 3 is heated and heated. Inflate. Since the second liquid layer 3 is sandwiched between the first liquid layer 2 and the sealed portion 5 and is held in a sealed state inside the capillary 1, the second liquid layer 3 is thermally expanded. Thermal expansion pressure is generated. Since the movement of the second liquid layer 3 in the direction of the sealing portion 5 is prevented by the sealing portion 5, the thermal expansion pressure of the second liquid layer 3 is converted into the discharge pressure of the first liquid layer 2, and the first The liquid in the liquid layer 2 is discharged into the cell C from the liquid discharge port 11 of the capillary 1. Thus, the target substance is injected into the cell C. The amount of liquid discharged into one cell C is usually 0.01 to 100 fL (fL = 1 μm ³ ), preferably 0.01 to 10 fL.

  The laser light passes through the capillary 1 and is applied to the second liquid layer 3. However, since the capillary 1 does not thermally expand due to the transmission of the laser light, the thermal expansion pressure of the second liquid layer 3 is the first. The liquid layer 2 is quickly and efficiently converted to the discharge pressure of the liquid layer 2, and an amount of liquid substantially equal to the volume expansion amount of the second liquid layer 3 is discharged with good response.

The thermal expansion pressure (MPa / ° C.) generated when the temperature of the second liquid layer 3 rises by 1 ° C. is expressed by the following formula: thermal expansion pressure (MPa / ° C.) = Thermal expansion coefficient (m ³ / ° C.) / Compression Calculated by the rate (m ³ / MPa). The thermal expansion pressure generated when the temperature of the second liquid layer 3 rises by 1 ° C. is preferably 0.1 to 10 MPa / ° C., more preferably 1 to 10 MPa / ° C. When the thermal expansion pressure generated when the temperature of the second liquid layer 3 rises by 1 ° C. is in the above range, a sufficiently large discharge pressure of the first liquid layer 2 can be obtained. When oleic acid is used as the liquid constituting the second liquid layer 3, the thermal expansion pressure generated when the temperature rises by 1 ° C. is about 1 MPa / ° C.

  The temperature rise of the second liquid layer 3 can be controlled by adjusting the output of the laser beam, and by controlling the temperature rise of the second liquid layer 3, the liquid discharge amount (the injection amount of the target substance) ) Can be controlled. By generating a high-power laser beam from the laser and irradiating the laser beam on the second liquid layer 3, the temperature of the second liquid layer 3 can be quickly raised.

  When the first liquid layer 2 contains a fluorescent dye, the liquid discharge amount can be visually controlled by observing with a fluorescent microscope.

  When irradiating the laser beam, the capillary 1 is not vibrated unlike when hot air is blown, and a precise operation can be performed even in a narrow space on the microscope stage.

  The biological species from which the cell C is derived is not particularly limited, and may be any animal, plant, microorganism, or the like. The type of cell C is not particularly limited, and may be any of somatic cells, germ cells, stem cells, or cultured cells thereof.

  When the target substance is injected into one cell C, the amount of the liquid in the first liquid layer 2 decreases. However, by adjusting the output of the laser light and controlling the temperature rise in the second liquid layer 3, it is sufficient Therefore, even when the liquid amount of the first liquid layer 2 is reduced, the liquid of the first liquid layer 2 can be discharged. Therefore, the capillary 1 shown in FIG. 1 (e) can be repeatedly used for injection of the target substance into the plurality of cells C.

  When irradiating the second liquid layer 3 with laser light, the laser light irradiation device 6 shown in FIGS. 4 and 5 can be used.

  As shown in FIGS. 4 and 5, the laser beam irradiation device 6 includes a capillary holder 7, an optical fiber 8 having one end attached to the capillary holder 7, a laser 9 connected to the other end of the optical fiber 8, And a controller 10 for controlling the output of the emitted laser light.

  As shown in FIGS. 4, 5 and 6, the capillary holder 7 includes a main body 71, a capillary holding member 72, a plate spring 73 attached to the main body 71 and the capillary holding member 72 by screws 74 and 75, and a main body. A gripping portion 76 attached to the portion 71.

  As shown in FIGS. 4, 5, and 6, the main body 71 includes an optical fiber mounting unit 711, an optical path 712 for guiding laser light emitted from the optical fiber 8 mounted on the optical fiber mounting unit 711, and an optical fiber mounting unit 711. The lens 713 which expands the irradiation range of the laser beam emitted from the mounted optical fiber 8 and the insertion hole 714 into which the part on the liquid inlet 12 side of the capillary 1 can be inserted are provided.

  As shown in FIGS. 4 and 6, the capillary holding member 72 is urged in the direction of the optical path 712 by the urging force of the leaf spring 73, and the capillary holding member 72 is not held in the capillary holder 7. 72 abuts on the main body 71. When holding the capillary 1 in the capillary holder 7, a force in the direction opposite to the urging force of the leaf spring 73 is applied to the capillary holding member 72, and the capillary holding member 72 that is in contact with the main body portion 71 is pulled away from the main body portion 71. 1 is inserted into the insertion hole 714 of the main body 71, and the force applied to the capillary holding member 72 (force opposite to the urging force of the leaf spring 73) is released. Then, as shown in FIG. 5, the capillary 1 is held in a state of being sandwiched between the main body 71 and the capillary holding member 72 by the biasing force of the leaf spring 73. As shown in FIG. 5, the capillary 1 is held by the capillary holder 7 so that the portion on the liquid discharge port 11 side protrudes from the capillary holder 7.

  Operations such as inserting the liquid discharge port 11 of the capillary 1 held in the capillary holder 7 into the cell 5 can be performed with the gripping portion 76 of the capillary holder 7.

  When the laser light whose output is controlled by the controller 10 is emitted from the laser 9, the laser light is sent to the capillary holder 7 through the optical fiber 8. The laser beam sent to the capillary holder 7 passes through the lens 713 and is guided by the optical path 712 to be a target region on the outer wall surface of the capillary 1 held between the main body 71 and the capillary holding member 72. (That is, the outer wall surface of the wall portion in contact with the second liquid layer 3) is irradiated. When the laser beam sent to the capillary holder 7 passes through the lens 713, the irradiation range of the laser beam is expanded, and the laser beam is irradiated over the entire target region, so that the second liquid held inside the capillary 1 The layer 3 can be heated efficiently.

  As shown in FIG. 5, the laser light guided by the optical path 712 is not irradiated to the first liquid layer 2 in the capillary 1, so the target substance contained in the first liquid layer 2 Can be prevented. As shown in FIGS. 4 and 5, the optical path 712 is surrounded by a wall portion, and the laser light guided by the optical path 712 is prevented from leaking from the capillary holder 7. Can prevent adverse effects.

Examples of the laser medium of the laser 9 include gases such as CO ₂ , excimer, and rare gases; solids such as YAG (yttrium / aluminum / garnet single crystal), ruby, and semiconductor, and YAG is preferable. The wavelength of the laser light emitted by the YAG laser is 1064 nm near infrared, and the laser light in this wavelength region has a high light collecting property and can be transmitted by a quartz optical fiber. The YAG laser can generate a high repetition pulse, a Q switch pulse, and continuous oscillation with an output of several hundred W to 1 kW.

  The embodiment described above is described for facilitating understanding of the present invention, and is not described for limiting the present invention. Therefore, each element disclosed in the above embodiment is intended to include all design changes and equivalents belonging to the technical scope of the present invention.

  The liquid discharge method of the present invention is useful when discharging a small amount (usually 0.01 to 100 fL, preferably 0.01 to 10 fL) of liquid, and for various purposes other than the injection of a target substance into cells. Can be used.

  In the present embodiment, the second liquid layer 3 is composed of a single layer, but the second liquid layer 3 may be composed of a plurality of layers. When the second liquid layer 3 is composed of a plurality of layers, the laser light absorbent may be contained in any layer, but the laser light absorbent is contained in a layer composed of a hydrophobic liquid (particularly an oily liquid). It is preferably contained. When the laser light absorber is contained in a layer composed of a hydrophobic liquid (particularly an oily liquid), a sufficiently large thermal expansion pressure can be obtained. When the second liquid layer 3 is composed of a plurality of layers, the two layers in contact with each other can be composed of two types of liquids (for example, a hydrophilic liquid and a hydrophobic liquid) that can form an interface.

  In the present embodiment, since the discharge target liquid is a hydrophilic liquid, the hydrophilic liquid is used as the liquid constituting the first liquid layer 2, but when the discharge target liquid is a hydrophobic liquid, A hydrophobic liquid is used as the liquid constituting the first liquid layer 2.

  In the present embodiment, the sealing portion 5 is formed by curing a curable liquid resin. However, the sealing portion 5 may be formed by attaching a sealing member such as a cap to the liquid inlet 12 of the capillary 1. . However, in this case, it is necessary to firmly attach the sealing member to the liquid inlet 12 so that the second liquid layer 3 is not detached even when the thermal expansion pressure of the second liquid layer 3 is applied. The sealing member can be firmly bonded to the liquid inlet 12 with an adhesive, for example.

[Example 1] Injection into a germ cell group of higher plant torenia (1) Injection by a conventional method A glass tube (inner diameter: about 600 μm) is drawn in two stages, and a liquid discharge port (inner diameter: about 0.7 μm) is provided at one end. And a capillary having a liquid inlet (inner diameter: about 600 μm) at the other end. The tip of this capillary (the part on the liquid discharge port side) has a needle shape, and its outer diameter is about 1.2 μm. After sequentially introducing an aqueous solution (liquid sample) containing 7kbp plasmid and fluorescent dye Alexa and silicon oil from the liquid inlet into the capillary, the part on the liquid inlet side of the capillary is a holder (NARISHIGE, product name : HI-7), and the capillary was fixed to the holder with a screw type presser foot. The holder is hollow, and one end of a Teflon (registered trademark) tube is connected to the opposite side of the capillary inlet, and a syringe (NARISHIGE, product name: IM-26) is connected to the other end of the Teflon (registered trademark) tube. -2) is connected. When the syringe is pushed in, the pressure generated thereby is transmitted as a pressure medium using silicone oil filling the inside of the Teflon (registered trademark) tube, the holder and the capillary, and the liquid is discharged from the tip of the capillary. We tried to inject the higher plant Torenia into the reproductive subgroup by the hydraulic injection method using such a syringe. At this time, red fluorescence of the fluorescent dye Alexa was observed with green excitation light of a fluorescence microscope, and the liquid discharge amount was controlled by visual observation.

  FIG. 8 (a) shows the state of injection into the central cell next to the egg cell. The central cell was a relatively large cell, but it was still deformed and damaged. Further, it is difficult to control the liquid discharge amount, and an excessive amount is often injected. Moreover, the liquid sample also leaked to the culture medium. The small bright spot is the autofluorescence of the chloroplast.

  In order to perform precise injection, it was necessary to make the outer diameter of the tip of the capillary smaller, but if the outer diameter of the tip of the capillary was made smaller, it was impossible to discharge the liquid sample.

(2) Injection by the method of the present invention One-stage drawing of a glass tube (inner diameter: about 600 μm) has a liquid discharge port (inner diameter: about 0.06 μm) at one end and a liquid introduction port (inner diameter: about 0.06 μm) at the other end. A capillary having 600 μm) was produced. The tip of this capillary (the part on the liquid discharge port side) has a needle shape, and its outer diameter is about 0.1 μm. From the liquid inlet to the inside of the capillary, an aqueous solution (liquid sample) containing a 7 kbp plasmid and the fluorescent dye Alexa, oleic acid containing carbon black, and an ultraviolet curable resin (TESK Co., Ltd., A-1428F) After sequentially introducing the liquid resin, the ultraviolet light was irradiated to cure the liquid resin. When introducing various liquids into the capillary, care was taken not to mix air.

  As shown in FIG. 5, a capillary is attached to the laser irradiation apparatus shown in FIG. 4, and the YAG laser is applied to oleic acid held inside the capillary in a state where the capillary liquid outlet is inserted into the germ cells of higher plant torenia. The emitted laser beam (1064 nm) was irradiated, and the liquid sample was discharged from the liquid discharge port. At this time, red fluorescence of the fluorescent dye Alexa was observed with green excitation light of a fluorescence microscope, and the liquid discharge amount was controlled by visual observation.

  As a result, as shown in FIG. 8B, a liquid sample can be discharged even when the outer diameter of the tip of the capillary is about 0.1 μm, and more precise injection than the conventional method was possible. . Moreover, as shown in FIG.8 (b), the injection to an egg cell was also able to be performed easily. The right figure in FIG. 8B shows a fluorescent image obtained by observing the fluorescent dye Alexa, and the left figure shows a bright field image of the same field.

[Example 2] Injection of Torenia's polar nucleus (central cell nucleus) and auxiliary cells Using the method of the present invention in the same manner as in Example 1, the liquid sample was mixed with the Torenia polar nucleus (central cell nucleus) and auxiliary cells. The cells were injected.

  FIG. 9A shows the state of injection into the polar core of Torenia, and FIG. 9B shows the state of injection into the helper cell. In addition, the upper figure of Fig.9 (a) and (b) is a bright field image, and the lower figure is a fluorescence image of the same visual field as the upper figure.

  As shown in FIGS. 9 (a) and 9 (b), the liquid sample could be injected without damage to the target nuclei and very weak auxiliaries that were easily ruptured.

[Example 3] Comparison of injection efficiency A two-stage drawing of a glass tube (inner diameter: about 600 μm) has a liquid discharge port (inner diameter: about 0.7 μm) at one end and a liquid introduction port (inner diameter: about 600 μm) at the other end. ) Was prepared. The tip of the capillary A (the part on the liquid discharge port side) has a needle shape, and its outer diameter is about 1.2 μm.

  In addition, a capillary tube B having a liquid discharge port (inner diameter: about 0.18 μm) at one end and a liquid introduction port (inner diameter: about 600 μm) at the other end is produced by one-step drawing of a glass tube (inner diameter: about 600 μm). did. The tip of the capillary B (part on the liquid discharge port side) has a needle shape, and its outer diameter is about 0.3 μm.

  A liquid sample was injected into a plant culture cell (BY-2 cell) by a hydraulic injection method using capillaries A and B. Using the same capillary, injection was performed until the tip of the capillary was clogged, and the number of injections was examined.

  On the other hand, a liquid sample was injected into plant cultured cells (BY-2 cells) by the method of the present invention using capillary B. Using the same capillary, injection was performed until the tip of the capillary was clogged, and the number of injections was examined.

  As a result, the average number of injections in the hydraulic injection method using capillary A was about 3 times, and the average number of injections in the hydraulic injection method using capillary B was less than one. In contrast, the average number of injections in the method of the present invention using capillary B was 20 or more.

  According to the method of the present invention, not only precise injection can be performed, but also one capillary can be used repeatedly to perform injection efficiently.

(A) is an empty capillary, (b) is a capillary holding a first liquid layer, (c) is a capillary holding a first liquid layer and a second liquid layer, and (d) is a first capillary. (A) is a partial cross-sectional view showing a capillary in which a first liquid layer and a second liquid layer are held and a sealed portion is formed. It is. FIG. 3 is a partial cross-sectional view showing a state in which a liquid is introduced from the liquid inlet of the capillary into the capillary using a syringe attached with a chip that can be inserted into the capillary from the liquid inlet of the capillary. FIG. 4 is a partial cross-sectional view showing a state in which laser light is irradiated to a second liquid layer held inside a capillary in a state where a liquid discharge port of the capillary is inserted into a cell. It is a partial cross section figure which shows the laser beam irradiation apparatus before capillary mounting. It is a partial cross section figure which shows the laser beam irradiation apparatus after capillary mounting. (A) is a plan view of the capillary holder, (b) is a side view of the capillary holder, and (c) is a front view of the capillary holder. It is a partial cross section figure of the capillary used with the conventional microinjection method. (A) is a figure which shows a mode that it injected into the Torenia germinal subgroup by the conventional hydraulic injection method, (b) is a figure which shows a mode that it injected into the Torenia germinal group follicle by the method of this invention. (A) is a figure which shows a mode that it injected into the torenia polar nucleus by the method of this invention, (b) is a figure which shows a mode that it injected into the torenia auxiliary cell by the method of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Capillary 11 ... Liquid discharge port 12 ... Liquid introduction port 2 ... 1st liquid layer 3 ... 2nd liquid layer 4 ... Liquid resin 5 ... Sealing part 6 ... Laser beam irradiation device 7 ... Capillary holder 8 ... Optical fiber 9 ... Laser light source 10 ... Controller C ... Cell

Claims (6)

A first liquid layer and a second liquid layer containing a laser light absorber are held inside a capillary having a liquid discharge port at one end so that the first liquid layer is located on the liquid discharge port side. Let
Forming a sealing portion for sealing the second liquid layer inside the capillary;
Irradiating the second liquid layer with laser light;
A liquid discharge method, wherein the liquid of the first liquid layer is discharged from a liquid discharge port of the capillary.   After sequentially introducing the liquid constituting the first liquid layer, the liquid constituting the second liquid layer, and the curable liquid resin into the capillary from the opening at the other end of the capillary, The method according to claim 1, wherein the liquid resin is cured to form the sealed portion.   The method according to claim 2, wherein the liquid resin comprises an energy ray curable resin. The first liquid layer contains a target substance;
In a state where the liquid discharge port of the capillary is inserted into the cell, the second liquid layer is irradiated with laser light,
The method according to claim 1, wherein the liquid in the first liquid layer is discharged into the cell from a liquid discharge port of the capillary. A capillary holder;
An optical fiber mounting section;
An optical path for guiding the laser light so that laser light emitted from the optical fiber attached to the optical fiber attachment portion is irradiated to a target region of the outer wall surface of the capillary held by the capillary holding portion; Capillary holder characterized by comprising. A capillary holder according to claim 5;
An optical fiber mounted on the optical fiber mounting portion of the capillary holder;
A laser light source connected to the optical fiber;
And a controller for controlling the output of laser light emitted from the laser light source.