JP4677555B2 - Specimen operation control apparatus and method - Google Patents

Description

  The present invention relates to a specimen operation control apparatus and method, and more particularly to injecting an injection into each of a large number of specimens suspended in a specimen suspension in a specimen suspension tank.

  Conventionally, as a technique for manipulating the position and posture of a living cell suspended in a specimen suspension, such as an egg, a method using a manipulator, a method using an alternating electric field, a method using a laser beam, a method using a vibration, and the like are known. It has been.

  The method using a manipulator changes the posture of an object by using a method such as hooking a holding pipette on the object, and the method using an alternating electric field allows cells as dielectrics to be applied by applying an alternating electric field. Control is performed (see Patent Document 1), and a method using a laser beam is a method in which a sample is manipulated without contact with the laser beam.

In addition to this, an example of a method using vibration is to vibrate a rod-shaped vibrator such as a glass pipette and generate at least one standing wave in the vibrator, thereby capturing the sample at the position of the standing wave node. Such a method has been proposed (see Patent Document 2).
JP 2001-239500 A International Publication WO01 / 072951

  However, according to the method using vibration disclosed in Patent Document 2, the position where the sample can be operated is limited by the vibration mode, so that it is actually difficult to operate the sample at the tip of the pipette.

  The present invention has been made in consideration of the above points, and a specimen in which a large number of specimens suspended in a specimen suspension are guided one by one to the pipette tip position, and an injection can be injected. An operation control apparatus and method are proposed.

In order to solve such a problem, in the present invention, the specimen suspension liquid tank 2 holding the specimen suspension liquid 3 for suspending the specimen 4 and the specimen 4A before processing from the specimen injection area of the specimen suspension liquid tank 2 to the injection operation area 2A. The flow forming means (31A, 31B, 35, 36, 37, 38) for introducing and processing the flow of the processed specimen 4B from the injection operation area 2B to the specimen storage area 2C, and a predetermined amount in the injection operation area 2B Vibrating means 6 and 51 for forming a local flow for guiding the pre-treatment specimen 4A at the position, specimen adsorption means 9 and 51 for adsorbing and fixing the pre-treatment specimen 4A guided to the injection operation region 2B, and specimen adsorption means the infusate provided with a infusate injection means 11 for injecting process in a fixed pre-processed specimen 4A in 9,51, flow forming means (31A, 31B, 35, 36, 37, 38) is a specimen suspension 3 The sample suspension 3 to flow through the injection operation region 3B to include a rotor (31A, 31B) for circulating the sample suspension tank 2.

According to the present invention , the sample suspension is circulated in the sample suspension tank by the rotor so as to flow from the sample introduction region through the injection operation region to the sample storage region, and in the injection operation region, the vibrating means, By performing the injection process using the adsorbing unit and the injection agent injection unit, it is possible to realize a sample operation control apparatus that can automatically process the input pre-process samples one by one.

  Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings.

  In FIG. 1, reference numeral 1 denotes a specimen operation control apparatus as a whole, and a large number of specimens 4 to be injected are floated in a specimen suspension 3 held in a specimen suspension tank 2 having a flat dish shape. The injection processing unit 5 injects the specimen 4 one by one.

  The injection processing unit 5 generates a local flow in the specimen suspension 3 to vibrate the specimen 4 to a predetermined drawing position, and a specimen that is fixed in a state where the specimen 4 guided to the drawing position can be punctured. The adsorbing unit 5B and an injecting agent injecting unit 5C for injecting an injecting agent by puncturing the fixed specimen.

  In the case of this embodiment, the vibration means 5A is constituted by a rod-shaped vibrator 7 having no hollow portion whose tip is vibrated in the direction along the bottom plate 2A of the sample suspension tank 2 by the vibration drive unit 6. Yes.

  The specimen adsorbing means 5B is constituted by a specimen fixing pipette 9 that generates a negative pressure in the pipette by the adsorption pump 8.

  Further, the injecting agent injection means 5C is constituted by an injecting pipette 11 that sends out the injecting agent by an injecting operation unit 10 including an injecting agent injection pump and a forward / reverse drive mechanism.

  In the case of this embodiment, the specimen operation control device 1 automatically fertilizes a large number of eggs floating as the specimen 4 in the specimen suspension tank 2.

  The state of the region in the sample suspension liquid tank 2 including the tip portions of the sample fixing pipette 9 and the injection pipette 11 in the sample suspension liquid tank 2 is provided at a position below the bottom plate 2A through the bottom plate 2A of the sample suspension liquid tank 2. The image is captured by the image capturing unit 12.

  The imaging unit 12 magnifies the projection light LA from the specimen suspension tank 2 coming through the bottom plate 2A by the microscope lens 12A and enters the camera unit 12B.

  The camera unit 12B is composed of a CCD element device or a CMOS element device, and the pixel data D1 is supplied to the capture board 14 of the control unit main body 13.

  The capture board 14 develops the pixel data D1 on the XY coordinates, and thereby the state at each time point in the sample suspension liquid tank 2 captured by the camera unit 12B through the microscope lens 12A is converted into image data D2 including XY coordinate position information. Convert and send.

  The control unit main body 13 has a personal computer configuration, and a central processing unit (CPU) 15 uses a program and parameters stored in advance in a ROM configuration program memory 17 via a bus 16 and uses a RAM configuration operation memory 18. By performing the execution calculation, the image data D2 sent from the capture board 14 is calculated.

The calculation processing result is accumulated in the operation memory 18, and a drive signal S 1 is given to the adsorption pump 8 of the sample fixing pipette 9 through the input / output interface 19 and is also driven to the injection operation unit 10 of the injection pipette 11. A signal S2 is given, and a drive signal S3 is given to the vibration drive unit 6 of the vibrator 7.

  In this way, the image data D2 stored in the operation memory 18 is given to the display 21 by the CPU 15, thereby displaying the state of the injection operation area 2B.

  As shown in FIG. 2, the sample suspension liquid tank 2 has a sample input region 2A for holding a large number of pre-processing samples 4A made up of the samples 4 before the injection processing at the rear center position, and from the sample input region 2A. The pre-treatment specimen 4A flows in the forward direction through the introduction path indicated by the arrow a1, and thereby the pre-treatment specimen 4A is sent one by one to the injection operation area 2B in the substantially central portion of the specimen suspension tank 2.

  The processed specimen 4B, which is the specimen 4 that has been injected in the injection operation area 2B, flows forward from the injection operation area 2B through the lead-out path indicated by the arrow a2, and is provided at a substantially front center position of the sample suspension liquid tank 2. Is sent to the specimen storage area 2C.

A sample fixing pipette 9 is arranged at the left position of the injection operation area 2B so as to extend from the outside of the left side plate portion 2L of the sample suspension tank 2 to the injection operation area 2B through a substantially central position in the front-rear direction. The vibrator 7 is arranged so as to be close to the specimen fixing pipette 9 .

  On the other hand, at the right side position of the injection operation area 2B, it extends from the outside of the right side plate portion 2R of the sample suspension tank 2 to the injection operation area 2B through the substantially central position in the front-rear direction of the sample suspension tank 2. An injecting pipette 11 is arranged.

  The tips of the sample fixing pipette 9 and the injection pipette 11 are arranged so as to face each other with the flow path of the sample 4 in between in the injection operation region 2B. When the injection pipette 11 is driven forward to the left by the injection operation unit 10 in the state where the sample is adsorbed, the tip of the injection pipette 11 can puncture the sample 4 adsorbed by the sample fixing pipette 9. It is made like that.

  In the rear right side of the specimen suspension tank 2, a flow formation is performed to form a flow of the specimen suspension 3 that feeds a large number of pre-treatment specimens 4A introduced into the specimen introduction area 2A in the direction of the introduction path a1. A rotor 31A is provided.

  The flow-forming rotor 31A is a water wheel that rotates counterclockwise, whereby the sample suspension 3 in the center of the sample suspension tank 2 is circulated to the sample input region 2A through the suspension liquid circulation path indicated by the arrow b1. Let

  In the portion from the sample injection region 2A to the injection operation region 2B, a partially circular flow channel wall 35 disposed along the periphery of the flow forming rotor 31A, and the sample facing the flow channel wall 35 A flow path wall 36 is formed which forms a tapered funnel-shaped introduction path from the charging area 2A toward the injection operation area 2B.

  Thus, the pre-treatment specimen 4A flows from the specimen introduction area 2A to the injection operation area 2B by the specimen suspension 3 circulated through the suspension liquid circulation path b1 by the flow forming rotor 31A, and the introduction path a1 thereof. Are tapered by the flow path walls 35 and 36, a large number of pre-treatment specimens 4A are arranged in an elongated shape and are sent one by one to the injection operation region 2B.

On the other hand, the flow forming rotor 31B is also provided on the front right side of the sample suspension tank 2 so as to face the flow forming rotor 31A, and the arrow is shown when the flow forming rotor 31B rotates counterclockwise. A floating liquid circulation path indicated by b2 is formed. At the same time, flow path walls 37 and 38 similar to the flow path walls 35 and 36 are formed, whereby the sample suspension 3 in the center of the sample suspension tank 2 is led out between the flow path walls 37 and 38. By flowing in the forward direction, the processed specimen 4B processed in the injection operation area 2B is guided to the specimen storage area 2C.

  In the case of this embodiment, a protective net member 40 is formed at a position between the flow forming rotors 31A and 31B in the injection operation region 2B, and thereby the specimen floats from the injection operation region 2B in the direction of the floating liquid circulation path b1. The specimen 4 in the injection operation area 2B is protected from flowing out in the direction of the liquid 3.

In addition to this, a protective mesh member 41 is formed between the outlet of the floating liquid circulation path b1 and the sample introduction region 2A, and the protective mesh member is formed between the floating liquid circulation path b2 and the sample storage region 2C. 42 is formed to partition the sample 4 so that the sample 4 does not flow out of the sample input region 2A and the sample storage region 2C.

Water wheel constituting the flow-forming rotors 31A and 31B, as shown in FIG. 1, a rotating magnet 45A and 45B provided on the lower position of the bottom plate 2A of the specimen suspension tank 2, by a driving motor 46A and 46B Rotation / stop control is performed by being driven using the drive signals S3 and S4 supplied from the CPU 15 via the input / output interface 19 of the control unit body 13 by the configured rotation drive means.

  In the injection operation region 2B, a vibrator 7 is disposed so that the tip thereof is along the tip of the sample fixing pipette 9, and the vibrator 7 is vibrated by the vibration driving part 6 to thereby inject the A local flow is generated in the specimen suspension 3 in the operation region 2B, whereby the specimen 4A before processing is processed to be adsorbed and fixed to the tip surface of the specimen fixing pipette 9 in a predetermined posture (orientation).

In this embodiment, the vibrator 7 is configured to disposed the tip portion 7A on the bottom plate 2A of the specimen suspension tank 2, the distal end portion 7A as shown by the arrow d in FIG. 2, the bottom plate 2A It is vibrated at a frequency of 10 to 30 [Hz] along the direction.

  As shown in FIG. 3, the vibration drive unit 6 has a pair of vertical plate members 6B1 and 6B2 attached to both front and rear end faces of the piezoelectric element 6A, and the right end surfaces of the vertical plate members 6B1 and 6B2 are leaf spring members. It is attached to 6C.

  The front vertical plate member 6B2 is fixed to the base 6D, whereas the rear end face of the vibrator 7 is fixed to the vertical surface of the rear vertical plate member 6B1 by the fixing member 6E.

Thus given the sinusoidal voltage as the drive signal S3 to the piezoelectric element 6 A, in response thereto, as shown by an arrow k, when expansion and contraction in the horizontal direction, the vertical plate member oscillator 7 is fixed 6B1 Rotates horizontally around the leaf spring member 6C, and in response to this, the tip 7A of the vibrator 7 has a small width (for example, 20 to 28 [ reciprocally oscillate in the horizontal direction within the range of [μm].

  When the tip 7A of the vibrator 7 vibrates in this way, as shown in FIG. 4, the specimen suspension 3 in the injection operation region 2B is applied to both sides of the tip 7A as indicated by arrows f1 and f2. A relatively large local flow that flows toward the side surface of the tip 7A and reverses occurs.

Further, the front end surface of the distal end portion 7A, as indicated by arrow g1 and g2, pull stream comprising a local flow such as from the region side of the obliquely rearward and obliquely front side stream flowing toward to the front end surface occurs.

  Further, in the vicinity of the tip portion 7A, as indicated by arrows j1 and j2, a spiral flow (both sides of the tip surface) that returns to the original position while being bent toward both sides toward the outside of the tip at both front and rear positions on the tip surface. Swirls in the opposite direction).

  Further, as indicated by arrows h1 and h2, a leading flow of local flow is generated on the distal end surface of the distal end portion 7A so as to flow toward the front and rear end portions of the distal end surface from the front region facing the distal end surface. .

In practice, as shown in FIG. 5, pretreatment sample 4A enters the injection operation area 2B came flowing along the bottom plate 2 A in a state sunk in the sample suspension in 3, no pull-stream g1 to h1 and spiral flow j1 while being captured is guided to the front end rear end portion position of the vibration Doko 7 by the and rotates in that position by the flow of these local analyte suspension 3.

  Similarly, as shown in FIG. 6, the pretreatment specimen 4A is guided to the position near the front end of the front end of the vibrator 7 by the front drawing flows g2 to h2 and the spiral flow j2, and starts rotating.

Thus, the position where the specimen 4A before processing is drawn is a position sufficient to suck the specimen fixing pipette 9 at its tip when the specimen fixing pipette 9 performs the suction operation (according to the experiment, it is within a radius of 500 [μm]). Therefore, the drawn pre-treatment sample 4A is fixed by being adsorbed to the tip of the sample fixing pipette 9 .

  In contrast, when the pre-treatment sample 4A rides on the flow of the reverse flow f1, as shown in FIG. 7, it is sent back to the position near the tip 7A of the vibrator 7 in the reverse direction, and then the drawn flows g1 to h1. It will be drawn in to the tip position of tip part 7A by drawing operation by.

  In the above configuration, the CPU 15 of the control unit main body 13 executes the sample injection processing procedure RT0 as shown in FIGS. 8A and 8B.

First, when a large number of pre-treatment specimens 4A are placed in the specimen input area 2A of the specimen suspension tank 2 by the user in step SP1, the CPU 15 drives the flow forming rotors 31A and 31B in the next step SP2, thereby floating the specimen. Circulation of the specimen suspension 3 is started so as to pass through the liquid circulation paths b1 and b2.

  At this time, the pre-treatment specimen 4A is sent in the direction of the injection operation region 2B through the introduction path a1 while being rearranged by the funnel shape.

Eventually, one of the pre-treatment specimens 4A enters the injection operation area 2B, and when the image data is captured by the imaging unit 12 and the image data D2 is taken into the operation memory 18, the CPU 15 enters the injection operation area 2B in step SP3. After confirming that one of the pre-processing specimens 4A has entered, the rotation of the flow forming rotors 31A and 31B is stopped.

  At this time, the CPU 15 moves to the next step SP4 to give the drive signal S3 to the vibration drive unit 6 to vibrate the vibrator 7, thereby pulling in the specimen 4A before processing by the local flow generated in the injection operation region 2B. At the same time, the rotation of the drawn pre-processing specimen 4A is started.

  Subsequently, the CPU 15 proceeds to step SP5 and performs image analysis on the image data D2 fetched into the operation memory 18, thereby determining whether or not the posture of the pre-processing specimen 4A is ready to be injected by the injection pipette 11. Check.

  If the confirmation can be made, the CPU 15 proceeds to the next step SP6 and applies the drive signal S1 to the adsorption pump 8 through the input / output interface 19 to cause the specimen fixing pipette 9 to perform the suction operation.

  At this time, the pre-processing sample 4A is kept in its posture because the pre-processing sample 4A is drawn into a range in which the sample fixing pipette 9 can be adsorbed to the tip (FIGS. 5 and 6). Adsorb.

By this time CPU15 is to provide a drive signal S2 to the injection operation unit 10 via the input output interface 18, to puncture action against pretreatment sample 4A being adsorb the analyte fixed pipette 9 injection pipette 11 After that, the injection is injected through the injection pipette 11.

  In the case of this embodiment, since the ovum is used as the specimen 4 and the sperm is used as the injection agent, the puncture of the injection pipette 11 needs to select a position avoiding the polar body of the ovum. In order to realize this, the pre-treatment specimen 4A (that is, the egg) is rotated.

  When this process is completed, the CPU 15 in step SP7 receives the processed sample 4B adsorbed on the tip of the sample fixing pipette 9 (that is, injected with an injecting agent) by the drive signal S1 of the adsorption pump 8 via the input / output interface 19. Release the adsorption to the egg.

  At this time, the processed specimen 4B floats in the injection operation area 2B, but the CPU 15 rotates the flow forming rotors 31A and 31B in the next step SP8, thereby causing the processed specimen 4B to flow. It is sent out from the injection operation area 2B to the specimen storage area 2C along the flow generated in the outlet path a2 by the flow of the suspended liquid circulation path b2 formed by the rotor 31B.

Subsequently, the CPU 15 determines whether or not the pre-processing sample 4A does not enter the injection operation area 2B in the next step SP9.

  Here, when the flow forming rotors 31A and 31B are driven so as to send the processed specimen 4B from the injection operation area 2B to the specimen storage area 2C in step SP8, the processed specimen 4B is sent out through the derivation path a2. At the same time, if the pre-treatment sample 4A is in the introduction path a1, the next one pre-treatment sample 4A enters the injection operation region 2B.

At this time, the CPU 15 obtains a negative result in step SP9, thereby returning to the above-described step SP3 and repeating the injection processing for the new pre-processing sample 4A.

Eventually, when the CPU 15 obtains a positive result in step SP9, this means that the injection process has been completed for all of the unprocessed specimens 4A inserted into the specimen insertion area 2A, and at this time the CPU 15 determines that the operator by taking out the processed sample 4B are stored in the sample reservoir region 2C from the sample suspension tank 2, and terminates the injection procedure RT0 to the analyte in step SP11.

  According to the above configuration, for a large number of pre-treatment specimens 4A inserted into the specimen introduction area 2A of the specimen suspension tank 2, the pre-treatment specimens 4A are sent one by one to the injection operation area 2B and are supplied to the specimen fixing pipette 9. Adsorption is performed in a predetermined posture and injection processing is performed by the injection pipette 11, whereby all the pre-processing samples 4 </ b> A can be automatically processed as processed samples 4 </ b> B.

  FIG. 9 shows a second embodiment. In the above-described embodiment, the function of the specimen adsorbing means 5B of the specimen 4A before processing by the specimen fixing pipette 9 and the function of the vibrating means 5A by the vibrator 7 are shown. Are operated by the vibration / adsorption pipette 51 (by the drive signals S1 and S3).

  The vibration / adsorption pipette 51 has a configuration in which the tip 51A is bent by 45 ° along the bottom plate 2A of the specimen suspension tank 2, and the vibration / fixed drive unit 50 causes the tip 51A to move back and forth as indicated by an arrow n. It vibrates in the direction, thereby functioning as the vibration means 5A.

  Such a vibration operation is executed with the same configuration as described above with reference to FIG. 3, thereby causing the local flow described with reference to FIGS. 4 to 7 by the tip 51 </ b> A.

  In addition, the vibration / fixed drive unit 50 operates as the sample adsorbing means 5B by causing the vibration / adsorption pipette 51 to perform the aspiration operation when the pre-processing sample 4A is drawn into a position facing the distal end surface of the distal end portion 51A. In this state, the pretreatment sample 5A adsorbed on the tip 51A is injected by the function of the injection pipette 11 as the injection agent injection means 5C.

  Also with the vibration / suction pipette 51 having the configuration shown in FIG. 9, the same effects as those of the above-described embodiment can be obtained.

  In the embodiment of FIG. 2, the vibrator 7 may be omitted, and the injection pipette 11 may be vibrated to cause local flow in the injection operation region 2B.

  The present invention can be used when an injection such as sperm is injected into a specimen such as an egg.

1 is a schematic system diagram illustrating an overall configuration of a sample motion control apparatus according to an embodiment of the present invention. It is a top view which shows the detailed structure of the sample suspension liquid tank of FIG. It is a top view which shows the detailed structure of a vibration drive part. It is a rough-line perspective view with which it uses for description of the local flow which arises in the injection | pouring operation area | region 2B with a vibrator | oscillator. It is a rough-line perspective view with which it uses for description of the drawing-in operation of the sample by a vibrator. It is a rough-line perspective view with which it uses for description of the drawing-in operation of the sample by a vibrator. It is a rough-line perspective view with which it uses for description of the transfer operation | movement of the sample before a process which remove | deviated drawing operation to the drawing position of a vibrator. It is a flowchart which shows the injection process procedure to a test substance. It is a flowchart which shows the injection process procedure to a test substance. It is a perspective view which shows 2nd Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Specimen operation control apparatus, 2 ... Specimen suspension liquid tank, 2A ... Specimen input area, 2B ... Injection | pouring operation area, 2C ... Specimen storage area, 2X ... Bottom plate, 3 ... Specimen suspension liquid, 4 ...... Sample, 4A ... Pre-processed sample, 4B ... Processed sample, 5 ... Injection processing unit, 5A ... Vibration means, 5B ... Sample adsorption means, 5C ... Injection injection means, 6 ... Vibration Drive unit, 7 ... vibrator, 8 ... adsorption pump, 9 ... specimen fixing pipette, 10 ... injection operation unit, 11 ... injection pipette, 12 ... imaging unit, 12A ... microscope lens, 12B ...... Camera unit, 13 ... Control unit body, 14 ... Capture board, 15 ... Central processing unit (CPU), 16 ... Bus, 17 ... Program memory, 18 ... Operation memory, 19 ... Input output Interface, 21 ... Display, 31A, 3 B ...... flow forming rotor, 35,36,37,38 ...... channel wall, 40, 41, 42 ...... protective net member, 50 ...... vibration fixed drive unit, 51 ...... vibration and suction pipettes.

Claims (6)

A specimen suspension tank for holding a specimen suspension for suspending the specimen;
A flow forming means for introducing a pre-treatment sample from the sample input region of the sample suspension liquid tank into the injection operation region and forming a flow for deriving the processed sample from the injection operation region to the sample storage region;
Vibration means for forming a local flow for guiding the pre-treatment specimen at a predetermined position in the injection operation region;
Specimen adsorbing means for adsorbing and fixing the pre-treatment specimen guided to the injection operation area;
An injectant injection means for injecting an injectant into the pre-treatment specimen fixed to the specimen adsorption means ,
The specimen operation control apparatus , wherein the flow forming means includes a rotor for circulating the specimen suspension in the specimen suspension tank so that the specimen suspension flows through the injection operation region . 2. The specimen motion control apparatus according to claim 1, further comprising: a rotational drive unit that rotationally drives the rotor from the outside of the specimen suspension tank. The specimen motion control apparatus according to claim 1, wherein the tip surface of the vibrating pipette member constitutes the specimen adsorption means, and the peripheral surface of the tip portion of the pipette member constitutes the vibration means. A vibrator constituting the vibrating means is disposed in the vicinity of the tip of the pipette constituting the specimen adsorbing means, and the local flow is formed at the tip of the pipette by the vibration of the vibrator. The sample motion control apparatus according to claim 1. The specimen movement control apparatus according to claim 1, wherein the injection means reciprocates with respect to a distal end surface of the specimen adsorption means. The specimen suspension tank holds the specimen suspension that floats the specimen,
The flow forming means introduces the pre-treatment sample from the sample input region of the sample suspension tank to the injection operation region, and forms a flow for deriving the processed sample from the injection operation region to the sample storage region,
By the vibrating means, a local flow that guides the pre-treatment specimen at a predetermined position in the injection operation region is formed,
The specimen adsorption means adsorbs and fixes the pre-treatment specimen guided to the injection operation area,
Injecting agent is injected into the pre-treatment specimen fixed to the specimen adsorbing means by means of injecting means.
The specimen operation control method, wherein the flow forming means includes a rotor for circulating the specimen suspension in the specimen suspension tank so that the specimen suspension flows through the injection operation region .

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