JP2000241712A - Holder for mounting particle moving/fixing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a holder having a structure to allow the exchange of a particle moving/fixing device. SOLUTION: The particle moving/fixing device 2 is mounted at a microscope stage 5 by the holder 3. This holder 3 serves also as a container for being kept filled with a medium 13. Electrodes 17 are formed at a base material 15 constituting the device 2. The electric field generated at the time of voltage impression to the electrodes 17 renders dielectric migration force. The particles 12 in the medium 13 are moved or fixed by the dielectric migration force. The holder 3 has substrate holding parts 45 and 46. The substrate holding parts 45 and 46 hold the base material 15 from both sides Consequently, the holder 3 is attachably and detachably held at the base material 15.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] 1. Field of the Invention [0002] The present invention relates to a holder used when a particle moving / fixing device is mounted on a microscope stage.

[0002]

2. Description of the Related Art In recent years, a technique called microinjection has been known as a kind of micromanipulation technique for manipulating a minute particle-shaped object.

[0003] Microinjection refers to a technique of injecting DNA, RNA, organelles, various proteins, various chemicals, and the like into cells using, for example, an injection needle called a micropipette formed by processing a glass tube. Such a technique has attracted particular attention in recent years as an effective technique for selectively introducing a specific gene or the like into cells. Such a method has the advantage that the introduction efficiency is higher and the cost is lower than that of a similar method such as laser injection.

In the above technique, a liquid is injected into a cell suspended in a medium while the micropipette is piercing the cell, and it is necessary to fix the cell by some means at the time of piercing. In addition, when it is desired to introduce a gene or the like into a specific site in a cell, it is necessary to fix the cell in a state where the site is oriented in a predetermined direction in which the pipette is easily pierced. And conventionally,
The cells were fixed by sucking the cells to the tip using a micropipette for piercing and another pipette.

However, the conventional method of sucking and fixing cells is troublesome and time-consuming, and it has been difficult to expect an improvement in productivity. Therefore, as an alternative, the present inventors have conceived a unit provided with a cell moving / fixing device 81 as shown in FIG. In the figure, a cell moving / fixing device 81 is supported in a mounting holder 83 which also serves as a container for filling a medium.
A cell chamber 86 for accommodating cells 85 one by one is provided at the center of a base material 84 constituting the device 81. The cell chamber 86 serving as a particle container is provided with a base material 84.
Are tapered through-holes that are opened on both side surfaces. A plurality of electrodes 87 are arranged adjacent to the cell chamber 86. The electrode 87 is connected to a pad 89 via a wiring pattern 88, and a lead wire 90 is joined to the pad 89. In addition, the device 81 includes a holder 8
3 is fixed on an observation stage 91 of a microscope.
An objective lens 92 is arranged below the stage 91, and a micropipette 93 is arranged above the stage 91.

Accordingly, each electrode 8 is connected via each lead wire 90.
When a high-frequency voltage is applied to 7, an electric field is generated in the cell chamber 86, and the electric field provides a suitable dielectrophoretic force to the cells 85. Therefore, the cells 85 are transferred to the cell chamber 86.
It is possible to fix the cell 85 after moving it inside and correcting the position. Therefore, the operator can
While observing the cell 85 from the lower side of 1, the micropipette 93 can be operated to pierce the tip of the cell 85 into the cell 85.

[0007]

However, in FIG. 6, the support 81 is provided at a plurality of positions on the inner bottom surface of the holder 83 and the lower surface of the base material 84 is adhered to the upper surface thereof, whereby the device 81 is mounted on the holder 83. Was held within.
Therefore, once the device 81 is adhered to the holder 83, the device 81 cannot be removed and replaced.

In addition, depending on the structure of the holder 83, if the bonding is incomplete, the medium 82 may be outside the holder 83.
Leaks, and the function as a container may be impaired.

The present invention has been made in view of the above problems, and an object of the present invention is to provide a holder for mounting a particle moving / fixing device having a structure in which the particle moving / fixing device can be replaced.

It is another object of the present invention to provide a holder for mounting a particle moving / fixing device, which can reliably fill a medium without leaking the medium.

[0011]

In order to solve the above-mentioned problems, according to the first aspect of the present invention, a medium is formed by a dielectrophoretic force caused by an electric field generated when a voltage is applied to an electrode formed on a substrate. A holder for moving or fixing particles therein, which is used when attaching the particle moving / fixing device to a microscope stage, and also serves as a container for filling the medium, wherein the holder holds the base material on both sides. A holder for mounting a particle moving / fixing device, comprising a base material holding portion for holding the base material in a detachable manner by being sandwiched between the holders.

According to a second aspect of the present invention, in the first aspect, the holder has an annular lower jig fixed on the microscope stage and having a base material holding portion on an inner peripheral edge;
An annular upper jig that has a base material holding portion on the inner peripheral edge and fits on the upper end surface side of the lower jig, and a portion that comes into contact with the base material in the base material holding portion includes an elastic body. It is assumed that the endless sealing member is mounted.

According to a third aspect of the present invention, in the second aspect, the upper jig is fixed to the lower jig using fastening means. The invention according to claim 4 is the invention according to claim 2.
In 3 or 3, it is assumed that at least one of the pair of jigs has a fitting recess for fixing the particle moving / fixing device in a positioned state.

The "action" of the present invention will be described below. According to the first aspect of the present invention, the base material is held by the holder by the base material holding portion of the holder for attaching the particle moving / fixing device holding the base material from both sides.
When the sandwiching is released, the held base material is easily separated from the holder. That is, if this holder is used, the device can be attached to and detached from the holder, so that the device can be replaced. In addition, since this structure eliminates the bonding portion, problems caused by incomplete bonding,
That is, the leakage of the medium from the bonding portion is eliminated.

According to the second aspect of the present invention, when the base material is sandwiched between the base material holding portions, the endless sealing member made of an elastic body comes into contact with the base material. As a result, the gap between the base material and the base material holding portion is sealed, and the flow of the medium through the contact portion is cut off. Therefore, the inside of the holder can be reliably filled without leaking the medium, and the function as a container is secured.

According to the third aspect of the present invention, when the upper jig is fixed to the lower jig, the tightening force of the fastening means acts, so that the seal member at the base material holding portion moves in a predetermined direction. Compressed. As a result, the sealing member is brought into close contact with the base material in an elastically deformed state, whereby the contact portion between the base material and the base material holding portion is reliably sealed.

According to the fourth aspect of the present invention, the base is fitted into the fitting recess, so that the apparatus is fixed in a state where it is positioned on the jig. In addition, with such a fixing structure, there is no need to use a fastening means such as a bolt or an adhesive, so that the device can be fixed easily and without displacement.

[0018]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A cell manipulation system 1 embodying a holder for mounting a particle moving / fixing device according to the present invention will be described below in detail with reference to FIGS.

This cell manipulation system 1
Comprises a cell moving / fixing device 2, a mounting holder 3, an objective lens 4 as a lower observation means, a microscope stage 5, a micropipette 6, a manipulator (not shown), a light source (not shown), and the like. . Further, one cell moving / fixing unit UN1 is constituted by the cell moving / fixing device 2 and the mounting holder 3.

As shown in FIG. 1, the microscope stage 5
Is provided with a through hole 11 at the center. This through hole 11
The object lens 4 is disposed below the object in an upward state. The objective lens 4 can be moved in the vertical direction by operating an operation means (not shown). The light source is located above the through hole 11 and is spaced apart therefrom. The micropipette 6 is arranged above the microscope stage 5. The micropipette 6 is manufactured by thinly stretching a glass tube heated and melted. The tip of the micropipette 6 is a plant cell 12 having a diameter of about several tens μm to several hundred μm.
Is formed in a pointed shape so that it can be pierced. The micropipette 6 is driven three-dimensionally by operating the manipulator. As a driving method of the manipulator, for example, there are a hydraulic type, a pneumatic type, a mechanical type, and the like. The base end of the micropipette 6 is connected to a pressure device such as a syringe (not shown). From the pressurizer, for example, a solution containing DNA is sent under pressure.

As shown in FIG. 1, the mounting holder 3 is mounted on the center of the upper surface of the observation stage 5. When the holder 3 is attached, the upper opening of the through hole 11 is closed. The holder 3 is for attaching the apparatus 2 to the microscope stage 5, and also serves as a container for filling a culture solution 13 as a medium.

As shown in FIG. 2A, cell migration /
The base material 15 constituting the fixing device 2 has a disk shape. In the present embodiment, a base material 15 made of glass (specifically, borosilicate glass), which is a kind of transparent material, is used. The thickness of the borosilicate glass substrate 15 is 0.2 mm
It is set to about 1 mm. At the center of the base material 15, a cell chamber 42 for accommodating each of the plant cells 12, which is a particulate object, is formed. A plurality of cell chambers 42 serving as particle storage units may be provided on the base material 15. This cell chamber 42 is
Is a non-penetrating concave portion that is opened only on the upper side surface. This recess is formed in a circular cross section by isotropic etching. The lower portion of the inner side surface of the etching concave portion is a round surface forming an angle of 0 ° to 90 ° with respect to the thickness direction of the base material 15. The bottom surface of the etching recess is substantially parallel to the surface direction of the base material 15. This detail chamber 42
It is almost bowl-shaped. The depth of the cell chamber 42 composed of the etching concave portion is 50 μm to 150 μm.
The diameter is set to about 150 μm to 300 μm.

As shown in FIGS. 1, 2 and 4B, a plurality of electrodes 17 are arranged adjacent to the cell chamber 42 on the base material 15. In the present embodiment, four electrodes 17 are formed on each of the upper surface side and the lower surface side of the base material 15. In FIG. 4B, U1, U2, U3, and U4 are attached to the four electrodes 17 on the upper surface side of the base material 15 for convenience of describing the positional relationship between the electrodes 17. In the figure, D1, D2, D3, and D4 are attached to four electrodes 17 on the lower surface side of the base material 15. According to this, the electrodes U1-D1, U2-D2, U3-D3,
It can be seen that U4-D4 has a corresponding positional relationship.

Pads 19 are formed on the outer peripheral portions of both sides of the base material 15. Each electrode 17 is electrically connected to these pads 19 via a wiring pattern 18.
The conductor other than the pad 19 is protected by an insulating layer 21 made of an insulating material such as polyimide.

As shown in FIG. 1, the mounting holder 3 includes a pair of jigs 43 and 44 for holding the base material 15 detachably by sandwiching the base material 15 from both sides. . The lower jig 43 and the upper jig 44 are both annular. The lower jig 43 is used while being fixed horizontally on the microscope stage 5. A fitting recess 5 a for fixing the lower jig 43 in a positioned state is formed on the microscope stage 5 and around the through hole 11. The lower jig 43 is formed so as to have a dimension capable of being fitted into and removed from the fitting concave portion 5a. The upper jig 44 is used in a state of being fitted to the upper end surface side of the lower jig 43. Above the inner peripheral edge of the lower jig 43, a base material holding portion 45 is provided over the entire periphery. Under the inner peripheral edge of the upper jig 44, a base material holding portion 46 is provided over the entire periphery thereof. The base material sandwiching portions 45 and 46 are in a positional relationship just opposite to each other. Annular grooves 47 and 48 are formed on the outer peripheral side of the substrate holding portions 45 and 46, respectively.

An annular concave portion 49 is formed over the entire periphery of the portion (here, the lower edge) of the substrate holding portion 45 that comes into contact with the substrate 15. An O-ring 50 as a sealing member is mounted in the annular concave portion 49. The O-ring 50 has an endless shape and an elliptical cross section, and is made of an elastic material such as rubber. As shown by a two-dot chain line in FIG. 2A, the O-ring 50 is formed on the base 15 in the region where the wiring pattern 18 is formed (in other words, each electrode 17 and each pad 1).
9).

Screw insertion holes 51 are provided at a plurality of locations on the outer peripheral portion of the upper jig 44. A female screw hole 52 is provided at a position corresponding to each of the screw insertion holes 51 on the outer peripheral portion of the lower jig 43. A screw 53 serving as a fastening means is screwed into each female screw hole 52 while being inserted through each screw insertion hole 51. Therefore, by fastening each screw 53 in a state where the upper jig 44 is fitted to the lower jig 43, the two jigs 43 and 44 are fixed. Lower jig 43
A fitting recess 54 having an inner diameter substantially equal to the outer dimensions of the base material 15 is formed on the upper surface side of the base material 15. Therefore, the particle moving / fixing device 2 can be fitted into the fitting recess 54. When the particle moving / fixing device 2 is fitted, the device 2 is horizontally supported on the base material holding portion 45 and is fixed to the lower jig 43 in a positioned state.

As shown in FIG. 1, a pair of jigs 43,
Reference numeral 44 includes a plurality of contacts 61 and 62.
The contacts 61 and 62 are formed using a conductive spring material. Contact 6 of lower jig 43
Reference numeral 1 denotes a substantially linear shape, and a substantially C-shaped bent portion 61a is provided on the inner end side.
have. The contact 62 of the upper jig 44 has a shape in which two portions are bent at right angles, and has a substantially C-shaped bent portion 62a on the inner end side. The convex surface of each of the bent portions 61a and 62a faces the pad 19 side. Contact 61, 62
Is pressed against each pad 19 with the sandwiching operation by the jigs 43 and 44. The bent portions 61a and 62a of the contacts 61 and 62 are regions isolated from the region A1 filled with the culture solution 13 via the O-ring 50 when the holder 2 holds the device 2 and the culture solution 13 is filled. A2. Specifically, the region A1 refers to a region on the inner peripheral side of the O-ring 50, and the region A2
Means an area on the outer peripheral side of the O-ring 50. Note that jigs 43 and 44 constituting the holder 3 of the present embodiment.
Is an insert molded product in which the contacts 61 and 62 are inserted into an insulating resin molding material.

The outer ends of the contacts 61 and 62 are connected to the jig 4
The sockets 63 protrude from the outer peripheral surfaces of the base wires 3 and 44, and a female socket 63 provided at the end of the lead wire 20 is detachable. When the socket 63 is mounted, several tens of leads are connected to the contacts 61 and 62 via the lead wires 20.
A high frequency alternating voltage of kHz to several MHz can be applied.

FIG. 4A shows eight electrodes 17 (U1 to U).
4, D1 to D4) are tables for explaining how to energize. To rotate the plant cell 12 clockwise, U
Applying a positive voltage to only one specific electrode 17 in the order of 1 → U2 → U3 → U4 may be performed in a clockwise order. If this operation is performed in the opposite direction such as U4 → U3 → U2 → U1, the plant cell 12 can be rotated to the left. To raise the plant cells 12, a positive voltage is applied to U1 and U2, and U3
And a negative voltage may be applied to U4. If it is desired to lower the plant cells 12, a positive voltage may be applied to U1 to U4. When it is desired to fix the plant cells 12 without moving them, the voltage values applied to U1 to U4 and D1 to D4 may be fixed.

The above operation can be controlled because when a high-frequency voltage is applied to the electrode 17 via the contacts 61 and 62, an electric field is generated in the cell chamber 42, and the electric field is suitable for the plant cells 12. This is because a dielectrophoretic force is provided. In this case, the plant cells 12 which are particulate objects
Is generally different from the dielectric constant of the culture solution 13 which is a medium, and the electric field causes the plant cells 12 to undergo electrostatic polarization. As described above, since the inner surface of the cell chamber 42 is at a predetermined angle with respect to the thickness direction of the base material 15, when a voltage is applied, an imbalance occurs in the strength of the electric field. As a result, a suitable dielectrophoretic force is generated in the cell chamber 42, and the electrostatically polarized plant cells 12 are attracted to the stronger electric field.

Next, a method for manufacturing the cell moving / fixing device 2 will be described with reference to FIG. First, a base material 15 made of borosilicate glass is prepared, and patterning is performed on the base material 15 by a conventionally known method.
7, conductor portions such as the wiring pattern 18 and the pad 19 are formed (see FIG. 3A). As a patterning method, there are a baking printing method, a sputtering method, a CVD method, and the like, in addition to a general plating method. Examples of the conductive metal material used for forming the conductor portion include copper, tin, nickel, lead, iron, chromium, titanium, gold, and platinum. When performing isotropic etching on glass, it is desirable that the metal material be resistant to hydrofluoric acid as an etchant. In view of this, the present embodiment employs a gold / chromium layer composed of two kinds of gold and chromium (that is, a gold layer having a chromium layer as a base).

After the patterning, a photosensitive polyimide resin is uniformly applied to almost all areas on both surfaces of the base material 15 and dried. In this state, exposure and development are performed by disposing a photomask to form an insulating layer 21 for protecting the electrodes 17 and the wiring patterns 18 (see FIG. 3B). The central portion of the base material 15 and the pads 19 are not covered with the insulating layer 21 and are exposed to the outside.

After performing the insulating layer forming step, an etching mask 65 is provided on the base material 15 (see FIG. 3C).
The etching mask 65 has an opening 66 at a central portion on the upper surface side of the base material 15, that is, at a location where the cell chamber 42 is to be formed. The diameter of the opening 66 is the cell chamber 4
2 (several tens of μm).
Note that a material for forming the etching mask 65 is preferably a material that can withstand hydrofluoric acid as an etchant.

Then, by performing isotropic etching using hydrofluoric acid in a state where the mask is provided, a non-penetrating etching concave portion serving as the cell chamber 42 is formed on the upper surface side of the base material 15 (FIG. 3D). reference). Thereafter, by removing the unnecessary etching mask 65, the desired cell moving / fixing device 2 is completed (see FIG. 3E).

Next, the procedure for assembling the cell moving / fixing unit UN1 will be described. First, the cell moving / fixing device 2 is fitted into the fitting recess 54 of the lower jig 43. At this time, the device 2 is horizontally supported on the substrate holding portion 45.
Next, the respective screw insertion holes 51 and the female screw holes 52 are aligned, and the upper jig 44 is fitted to the upper end surface of the lower jig 43. By tightening each screw 53 in this state, the jigs 43 and 44 are fixed. At this time, as a result of the base material 15 being sandwiched from both upper and lower sides by the base material holding portions 45 and 46 having the opposed positional relationship, the apparatus 2 is securely held by the holder 3.

In this case, the O-ring 50 is compressed in its own axial direction by the action of the tightening force of the screw 53. As a result, the O-ring 50 comes into close contact with the upper surface of the device 2 while being elastically deformed. Due to this close contact, a high sealing property is obtained between the region A1 and the region A2. That is, the bent portions 61a, 62a of the pads 19 and the contacts 61, 62
The culture solution 13 in the area A1 does not enter the area A2 having the same.

When the tightening force of the screw 53 acts, each bent portion 61
a and 62a are brought into pressure contact with each other. Due to this pressure contact, the conductive portion on the holder 3 side (that is, the contacts 61 and 62)
The conductive portion (pad 19, wiring pattern 18,
Conduction with the electrode 17). Therefore, each contact 61,
If the socket 63 is attached to the outer end of 62 and energized,
A high-frequency voltage can be applied to each electrode 17.

Next, an example of a specific method of using the present system 1 will be described. The holder 3 is set on the microscope stage 5, and the cell moving / fixing device 2 is fixed on the inner bottom surface of the holder 3. In this state, the inside of the holder 3 is filled with the sterilized culture solution 13. As a result, both the upper surface and the lower surface of the substrate 15 are in an environment where the culture solution 13 is present, and the inside of the cell chamber 42 is reliably filled with the culture solution 13. Thus, the death of the plant cells 12 that are vulnerable to drying can be avoided. The plant cells 12 may be single cells such as protoplasts or pollen, or may be specific organs such as ovules or cell groups such as callus (ie, multicells).

The operator transfers one plant cell 12 into the cell chamber 42 by another micropipette (not shown). Next, the operator applies a high-frequency voltage to the electrode 17 while performing microscopic observation to correct the position of the plant cell 12. In this case, if the site into which the specific gene is to be introduced is, for example, an embryo, the embryo is adjusted so as to face the tip of the micropipette 6.

Next, the operator drives the micropipette 6 while observing the microscope, and slowly advances the tip of the micropipette 6 toward the target plant cell 12. After confirming with a microscope that the tip of the micropipette 6 has pierced the embryo of the plant cell 12, the operator gently presses the head of the syringe to push out the liquid containing DNA to the outside. Then, the liquid is discharged from the tip portion via the micropipette 6. Therefore, DNA can be injected into the embryo of the plant cell 12. After the above-described introduction processing is completed, the operator needs to retract the micropipette 6 and remove the tip from the plant cell 12. As a result, the specific gene can be selectively and efficiently introduced into the plant cell 12. Thereafter, the plant cells 12 into which the gene has been introduced are gently sucked out of the cell chamber 42 again by another micropipette.

Therefore, according to the present embodiment, the following effects can be obtained. (1) The cell manipulation system 1 of the present embodiment
According to the above, by applying a voltage to the electrode 17, a suitable dielectrophoretic force acts on the plant cell 12. As a result, the plant cell 12 can be moved or fixed in the cell chamber 42 without bringing a device such as a fixing micropipette into contact with the plant cell 12 at all. Therefore, different from the conventional method of sucking and fixing the particulate object, various operations can be performed without damaging the soft plant cells 12. In addition, according to this system 1, since no troublesome operation of the fixing micropipette is involved, an improvement in productivity can be expected.

(2) The holder 3 of the present embodiment is characterized in that it has the above-described substrate holding portions 45 and 46. Therefore, the base material 15 is held by the holder 3 by the base material holding portions 45 and 46 sandwiching the base material 15 from both upper and lower sides. On the other hand, when the sandwiching is released, the held base material 15 is easily separated from the holder 3. That is, if the holder 3 is used, the device 2 can be attached to and detached from the holder 3, and the device 2 that cannot be achieved by the bonding structure can be replaced. Also, by adopting this structure,
An adhesive portion between the holder 3 and the device 2 can be eliminated. Therefore, it is possible to solve the problem caused by the incomplete adhesion, that is, the leakage of the culture solution 13 from the adhered portion.

(3) The holder 3 of the present embodiment comprises an annular lower jig 43 and an annular upper jig 44. An O-ring 50 is mounted on the lower edge of the substrate holding portion 46 of the upper jig 44. Accordingly, the base material 15 is
As the O-ring 50 comes into contact with the base material 15 when being sandwiched between the base materials 6, the gap between the base material 15 and the base material holding portion 46 is sealed. As a result, the culture solution 13 through the contact site
Is cut off, and the culture solution 13 in the area A1 does not enter the area A2 including the pad 19 and the bent portions 61a and 62a of the contacts 61 and 62. That is, the culture solution 13
Can be reliably filled inside the holder 3 without leaking, and the function as a container can be secured. Also, by pressing the O-ring 50, both jigs 43 and 4 are pressed.
Since the stress at the time of setting 4 is also reduced, it is possible to prevent the device 2 from tilting.

(4) In the holder 3 of the present embodiment, the O-ring 50 is compressed in its own axial direction by the tightening force of the screw 53 acting upon fixing. As a result, the O-ring 50 comes into close contact with the upper surface of the device 2 while being elastically deformed,
Thereby, the space between the area A1 and the area A2 is reliably sealed. In other words, this configuration contributes to more reliably preventing the culture solution 13 from leaking.

(5) In the holder 3 of the present embodiment, the base material 1
By fitting 5 into fitting recess 54, device 2 can be fixed in a state where it is positioned on lower jig 43.
With such a fixing structure, it is not particularly necessary to use a fastening means such as a bolt or an adhesive, so that the device 2 can be fixed easily and without displacement. Of course, since no adhesive is used, the unit UN1 can be disassembled. That is, the replacement of the device 2 is not hindered.

The embodiment of the present invention may be modified as follows. The O-ring 50 serving as the seal member is not limited to being provided only on the upper surface side of the base material 15 so as to be able to press-contact. FIG. 5 shows another example of the cell micromanipulation system 71. In the device 2 used in the system 71, the cell chamber 73 is a tapered through hole. Therefore, both the upper and lower surfaces of the substrate 15 need to be filled with the culture solution 13. Holder 3A is lower jig 7
2 and an upper jig 44. The central portion of the lower jig 72 needs to have at least light transmittance. An annular concave portion 49 is provided at an upper end edge of the substrate holding portion 45 of the lower jig 72, and an O-ring 50 is mounted thereon. If the holder 3A having the above configuration is used, not only the leakage of the culture solution 13 on the upper surface side of the base material 15,
Leakage of the culture solution 13 on the lower surface side can also be prevented. In addition to this alternative example, an O-ring 50 as a sealing member
May be provided so as to be pressed against only the lower surface side of the base material 15.

The sealing member itself such as the O-ring 50 can be omitted. In this case, for example, jig 4
A structure is conceivable in which the entire body 3, 44 is formed using an elastic body.

The fitting recess 54 may be formed on the side of the upper jig 44, or the fitting recess 54 may not be provided in any of the jigs 43 and 44. The material for forming the jigs 43 and 44 is not limited to an insulating material such as a resin. If the insulation with the contacts 61 and 62 is secured by any means, for example,
The jig 43 is made of a conductive material such as stainless steel.
Making 44 is also acceptable.

When the jigs 43 and 44 are fixed,
A fastening unit other than the screw 53 may be used, and another unit (for example, a locking unit such as a hook) may be used instead of the fastening unit.

The culture solution 1 for plants described in the embodiment.
Not only is No. 3 used as a medium, but also simple distilled water containing almost no nutrient may be used as a medium. Of course,
The medium can be appropriately changed according to the change in the type of the particles. Further, the particles applied to the present invention, plants,
It is not limited to living things such as animals and microorganisms, and may be non-living things, for example.

Next, in addition to the technical ideas described in the claims, the technical ideas grasped by the above-described embodiments are listed below together with their effects. (1) In any one of claims 2 to 4, the base material holding portions of the two jigs are in a facing positional relationship. Therefore, according to the invention described in the technical idea 1,
The sealing member is easily brought into close contact, and higher sealing properties can be obtained.

(2) In any one of the second to fourth aspects and the technical concept 1, the substrate holding portion of each of the two jigs may be located on an outer peripheral side of a region where the electrodes are formed, and the pad may be located between the jigs. Sandwiching the base material in a region on the inner peripheral side of the formed region; According to the invention described in the technical idea 2, the pad-side region can be reliably sealed.

(3) Claims 2 to 4, technical idea 1,
In any one of the two, the two jigs may include an insertion portion through which fastening means can be inserted at a plurality of spaced locations. Therefore, according to the invention described in the technical idea 3, as a result of obtaining a uniform tightening force, the sealing property is improved.

(4) A microscope unit having a stage and a lens, a particle moving / fixing device, and a particle moving / fixing unit having a holder according to any one of claims 1 to 4, and technical ideas 1 to 3. And a cell manipulation system, wherein the unit is used when attaching the device to the stage, serves also as a container for filling the medium, and includes a micropipette for cell manipulation.

[0056]

As described in detail above, according to the first to fourth aspects of the present invention, it is possible to provide a holder for mounting a particle moving / fixing device having a structure capable of replacing the particle moving / fixing device. it can. Moreover, the inside of the medium can be reliably filled without leaking the medium.

According to the second aspect of the present invention, the gap between the device and the holder is sealed by the seal member.
Leakage of the medium can be reliably prevented. According to the third aspect of the present invention, since the contact portion between the base material and the base material holding portion is securely sealed, leakage of the medium is more reliably prevented.

According to the fourth aspect of the present invention, the device can be fixed to the holder simply and without displacement.

[Brief description of the drawings]

FIG. 1 is a schematic cross-sectional view showing a cell manipulation system using a cell moving / fixing unit including a holder according to an embodiment of the present invention.

2A is a plan view of a cell migration / fixation device according to an embodiment, and FIG. 2B is a cross-sectional view of the device.

FIGS. 3A to 3E are cross-sectional views illustrating a procedure for manufacturing the cell migration / fixation device according to the embodiment.

FIG. 4A is a table for explaining a method of energizing each electrode, and FIG. 4B is a schematic diagram for explaining a positional relationship of each electrode.

FIG. 5 is a schematic cross-sectional view showing a cell manipulation system using a cell moving / fixing unit provided with another example of a holder.

FIG. 6 is a schematic sectional view showing a cell manipulation system using a cell migration / fixation unit previously devised by the present inventors.

[Explanation of symbols]

2. Cell migration / fixation device as particle migration / fixation device
3, 3A: holder for attaching the particle moving / fixing device, 5:
Microscope stage, 12 ... plant cells as particles, 13 ...
Culture medium as a medium, 15 ... substrate, 17 (U1, U2,
U3, U4, D1, D2, D3, D4) ... electrodes, 43 ...
Lower jig, 44 ... Upper jig, 45, 46 ... Substrate holding portion, 50
... O-ring as a seal member, 53... Screws as fastening means, 54.

Claims (4)

[Claims] A particle moving / fixing device for moving or fixing particles in a medium by a dielectrophoretic force caused by an electric field generated when a voltage is applied to an electrode formed on a substrate is used when the device is mounted on a microscope stage. And a holder also serving as a container for filling the medium,
The holder for mounting a particle moving / fixing device, wherein the holder includes a substrate holding portion that holds the substrate detachably by sandwiching the substrate from both sides. 2. The holder has an annular lower jig fixed on the microscope stage while having a base material holding portion on an inner peripheral edge, and a base material holding portion on an inner peripheral edge and having a base material holding portion on the lower jig. An endless seal member made of an elastic body is attached to a portion of the base material holding portion that comes into contact with the base material, the end device being a ring-shaped upper jig fitted to the end surface side. The holder for mounting the particle moving / fixing device according to claim 1. 3. The holder according to claim 2, wherein said upper jig is fixed to said lower jig using fastening means. 4. A fitting recess for fixing the particle moving / fixing device in a positioned state is formed in at least one of the pair of jigs. Or a holder for mounting a particle moving / fixing device according to item 3.