JP2022066093A - Processing apparatus for small fish and their fertilized eggs - Google Patents

Abstract

To provide a processing system that can perform high-speed processing of small fish, such as zebrafish, and their fertilized eggs without damaging them.SOLUTION: Small fish, such as zebrafish, and their fertilized eggs are preferably individually housed in each well of a common multi-well plate, and appropriate position control is performed by sequentially forming an ascending water flow and a descending water flow formed in a well. Since each well of the common multi-well plate is large enough for a fertilized egg, each fertilized egg can be easily dropped into each well one by one by sequentially dropping the fertilized eggs from a fertilized egg storage container having a simple structure.SELECTED DRAWING: None

Description

The present invention relates to a processing system for small fish such as zebrafish or fertilized eggs thereof.

Zebrafish, which has advantages such as fertility, high-speed growth, and ease of observation, are significantly advantageous in the fields of life science and drug discovery as compared with conventional test animals such as nude mice. However, because zebrafish and its fertilized eggs are small and easily damaged, a system that automatically processes a series of complicated tasks including injection of target materials into fertilized eggs and fry, breeding of fry, observation of adult fish, etc. as precisely as possible. Is expected to be realized at a reasonable cost.

However, it has been found that such an assumed automatic processing system still has many problems. For example, injection of the target material into a large number of fertilized eggs must be performed within a very short time immediately after the onset of division of each fertilized egg. This suggests that the work process needs to be simplified.

Patent Document 1 discloses a multi-well plate in which a large number of wells each accommodating one zebrafish are arranged in a matrix. Each well has a rectangular bottom on which an adult zebrafish can lie down. The water in each well is drained from a drainage hole formed along the long side of the bottom surface of this rectangle. The zebrafish in the well is urged by this drainage stream to lie on the bottom of the well. The sides of the well are slanted to reduce variability in the recumbent position of the zebrafish on the bottom of the rectangle.

Patent Document 2 discloses a fertilized egg holding structure which is a positioning plate for setting a fertilized egg at a predetermined position on the bottom surface of each well of the above-mentioned multi-well plate. This structure overlaid on the multi-well plate has a tapered tube that hangs near the bottom of the well. Since the structure is fixed to the multi-well plate, each tapered tube is positioned on a predetermined position on the bottom surface of each well. Therefore, the fertilized egg can be implanted at a predetermined position in each well through this tapered tube portion.

Japanese Unexamined Patent Publication No. 2014-169951 Japanese Unexamined Patent Publication No. 2020-20591

From various experimental results, it was found that in the processing system of zebrafish and its fertilized egg, simplification and speeding up of the positioning work of the processing tool for zebrafish and its fertilized egg are particularly important for shortening the processing time. Furthermore, it was found that mechanical contact of the processing tool with the zebrafish and its fertilized eggs should be avoided as much as possible in various processing operations including this positioning operation. This is because zebrafish and its fertilized eggs have a thin and vulnerable surface membrane.

An object of the present invention is to provide a processing system capable of performing necessary processing on small fish such as zebrafish and their fertilized eggs at high speed without damaging them as much as possible.

Three aspects of the processing system for small fish and fertilized eggs thereof of the present invention are described below. The first and second aspects relate to the treatment of fertilized eggs with multi-well plates. The third aspect relates to the treatment of small fish using multi-well plates. However, preferably, each well of the multi-well plate is commonly used for both the treatment of fertilized eggs and the treatment of hatched small fish.

According to the first aspect of the present invention, in order to carry out the operation of individually inserting the small fish fertilized eggs into each well of the multi-well plate, first, a large number of small fish fertilized eggs are fertilized eggs having a substantially inverted pyramid shape together with water. It is housed in the storage tube. The fertilized egg storage tube portion has a fertilized egg discharge port slightly larger than the fertilized egg at the lower end, and the fertilized egg and water fall from this discharge port. A large number of fertilized eggs gradually become dense as they descend near the fertilized egg outlet. However, the diameter of the fertilized egg outlet is limited so that a plurality of fertilized eggs do not fall from the fertilized egg outlet at the same time.

As a result of the experiment, it was found that the falling frequency of the fertilized egg that falls from the fertilized egg outlet together with water can be controlled by the pressure applied to the water surface in the fertilized egg storage tube. When this pressure is high, the drainage flow rate from the fertilized egg discharge port increases, and the frequency of fertilized egg discharge increases accordingly. This pressure can be controlled by a process such as controlling the height of the water surface in the fertilized egg accommodating cylinder or adding air pressure to the water surface.

In one example, the fertilized egg discharge frequency is maintained within a predetermined range by feedback-controlling the water surface height to a predetermined level. In another example, air pressure is applied to the water surface at the timing when the fertilized egg is required to fall. This makes it possible to drop the fertilized egg into the well at the required timing.

The fertilized egg storage tube is moved above the multi-well plate relative to the multi-well plate in a direction parallel to the upper surface of the multi-well plate. As a result, the fertilized egg discharge port is sequentially positioned at a predetermined position directly above each well.

Preferably, the fertilized egg outlet is stopped directly above this well until one fertilized egg falls into the well directly below. After the fall of the fertilized egg is detected, the fertilized egg outlet is quickly moved directly above the next well. As a result, the work of individually inserting the fertilized egg into each well can be realized simply and surely. Further, according to this fertilized egg arrangement method, it is possible to better prevent the fertilized egg from being damaged as compared with the conventional method.

According to the preferred first aspect and the second aspect of the present invention, the multi-well plate is tilted in order to set the fertilized egg inserted into the well by dropping or the like in a predetermined seating position on the bottom surface of each well. The fertilized egg positioning process is executed. Since the fertilized egg has a slightly higher density than water, this tilted posture of the multi-well plate causes the fertilized egg to move to the above-mentioned seating position corresponding to the lowest position on the bottom surface of the well. Therefore, the fertilized egg can be seated in the above-mentioned seating position without applying an unfavorable mechanical contact operation to the fertilized egg. As a result, the automatic injection work of the target substance into the fertilized egg and the automatic image inspection of the fertilized egg become reliable and easy.

Preferably, the fertilized egg is placed near a predetermined position on the side wall that partitions the well. This side wall can further regulate the displacement of the fertilized egg from the seating position. In one example, the surface of the bulkhead facing the well is formed approximately perpendicular to the bottom plate of the well. This makes it possible to increase the total number of wells in the multi-well plate without reducing the volume of the wells. Preferably, the well has a substantially square bottom surface. Further, the tilted posture of the multi-well plate is controlled so that the fertilized egg is in the lowest position in the vertical direction at one of the four corners of the bottom surface. As a result, the fertilized egg can be stably held in a predetermined seating position.

Preferably, the fertilized egg ejection port of the fertilized egg accommodating cylinder portion is predetermined with respect to the vertical direction so that the ejected fertilized egg is accelerated toward a predetermined seating position on the bottom surface of the well by the water flow simultaneously discharged. It is installed diagonally only at an angle. As a result, it is possible to shorten the time required for the fertilized egg discharged from the fertilized egg discharge port together with the water flow at a predetermined speed to reach a predetermined seating position on the bottom surface of the well.

According to a preferred second aspect and a third aspect of the invention, each well of the multi-well plate has a nearly rectangular bottom surface on which the grown small fish can lie down. According to the third aspect, the multi-well plate may be a breeding well that breeds small fish but does not perform fertilized egg treatment. This multi-well plate has a well water supply / drainage subwell adjacent to one short side of the rectangular bottom surface of each well. The bottom of the partition between the well and the subwell has a communication hole for water supply and drainage of the well.

Before imaging the small fish or injecting the target material into the small fish, the small fish in the well are laid down on the bottom of the well. In this lying operation, first, a water injection step of injecting water from the sub well into the well through the communication hole is executed. As a result, an ascending water flow is formed in the well, and small fish float in the well. As a result, even if the small fish is in close contact with the bottom or side surface of the well, the small fish will be separated from the bottom surface or side surface of the well by this rising water flow and will float.

Next, a drainage step is performed to drain the water in the wells into the subwells through the communication holes. As a result, a descending water flow toward the communication hole that opens on the short side of the bottom surface of the well is formed in the well. This descending stream flowing along the surface of the small fish in the well changes the posture of the small fish.

According to the experiment, the small fish is in a posture that minimizes the fluid resistance to this descending water flow. Specifically, the large head of the small fish is urged to the position farthest from the communication hole, and conversely, the thin tail of the small fish is urged to the position close to the communication hole. As a result, the positions of the heads of each small fish lying on the bottom of each well can be aligned. This makes it possible to reduce the burden of image processing for each small fish. In addition, various processing operations for each small fish lying down become easy. The water supply / drainage cycle consisting of this water injection step and the subsequent drainage step can be continuously executed a plurality of times as needed.

In a preferred embodiment, the water supply / drainage cycle is used to perform an eggshell separation step to separate the small fish in the well from its eggshell. The rising water flow formed in the well by the water injection step raises the eggshell and small fish in the well, but since the eggshell has a lower density than the small fish, it rises relatively larger than the small fish. After that, the descending water flow formed in the well by the drainage step lowers the eggshell and small fish in the well, but since the eggshell has a lower density than the small fish, it stays in the upper space in the well, and the small fish stays in the upper space. It descends relatively larger than the eggshell. The velocity of the lower precipitation flow in the well is relatively higher in the lower space than in the upper space in the well. In the end, it was found that the eggshell could be well separated above the small fish by this water supply / drainage cycle.

FIG. 1 is a block diagram showing a part of the zebrafish processing system of the embodiment. FIG. 2 is a schematic plan view showing a multi-well plate. FIG. 3 is a schematic cross-sectional view showing wells and sub wells of a multi-well plate. FIG. 4 is a schematic cross-sectional view showing wells and sub wells of a multi-well plate. FIG. 5 is a flowchart showing a zebrafish processing sequence. FIG. 6 is a plan view showing wells and sub wells of the multi-well plate. FIG. 7 is a cross-sectional view of the fertilized egg container. FIG. 8 is a schematic diagram showing a step of sequentially arranging fertilized eggs from the fertilized egg container to each well of the multi-well plate. FIG. 9 is a plan view of a multi-well plate for showing the seated state of the fertilized egg on the bottom corner of each well. FIG. 10 is a schematic cross-sectional view of a multi-well plate for showing a zebra fish floating in a well. FIG. 11 is a schematic cross-sectional view of a multi-well plate for showing a zebrafish that settles in a well. FIG. 12 is a schematic cross-sectional view of a multi-well plate for showing zebrafish and eggshells floating in the wells. FIG. 13 is a schematic cross-sectional view of a multi-well plate for showing zebrafish that selectively settles in the wells.

Suitable examples of the zebrafish treatment method of the present invention are described below. However, the present invention should not be construed as being limited to the following examples, and it is naturally possible to add or change to known technical elements. For example, this embodiment can treat other small fish of the same species instead of zebrafish.

(First Example)
This embodiment describes a common multi-well plate type zebrafish processing system in which both fertilized zebrafish eggs and their hatched fish are treated with a common multi-well plate. Each well of the multi-well plate is essentially infused with one zebrafish fertilized egg. The hatched fish hatched from the fertilized egg in each well is bred as it is in each well. Further, in this embodiment, the target substance is injected into the fertilized egg.

It is also possible to treat the fertilized egg and its hatched fish in a separate multi-well plate. This processing method is called a multi-well plate method. The advantages of the multi-well plate method are that the fertilized egg can be easily positioned and the well density of the multi-well plate for fertilized egg can be improved. The advantage of the common multi-well plate method is that the step of transferring the fry hatched in the fertilized egg multi-well plate to the fry breeding multi-well plate can be omitted.

The common multi-well plate method requires a wide well bottom for zebrafish breeding. However, the misalignment of the fertilized egg is increased by this wide bottom of the well. As a result, the alignment work becomes difficult in the process of injecting a drug into the fertilized egg and the process of photographing the fertilized egg. This problem is well solved by this embodiment.

FIG. 1 is a block diagram showing a basic configuration of the zebrafish processing system of this embodiment. This system consists of an egg sequence block 1, an injection block 2, a growth block 3, an imaging block 4, an operating robot 5, and a management block 6.

The egg arrangement block 1 performs an egg arrangement step in which one fertilized zebrafish egg is placed in each well of the multi-well plate. The injection block 2 executes an injection step of injecting a chemical solution, cells, or the like necessary for each fertilized egg arranged. The growth block 3 has a breeding step of raising fry hatched in each well of the multi-well plate, an eggshell separation step of separating eggshells from zebrafish, and lying down the adult fish in each well on the bottom of each well after breeding. Perform the lying process. In this growing step, work of supplying clean water and food to each well of the multi-well plate, work of discharging contaminated water including feces and residual food from each well, and the like are carried out as necessary. The imaging block 3 executes an imaging step of evaluating a fertilized egg in each well and a zebrafish lying on the bottom surface of each well by a process such as imaging.

The above-mentioned injection step, growth step, and imaging step have been filed and made known by the present inventors, but their description is omitted because they have little relation to the gist of the present invention. The plurality of application documents previously submitted by the present inventors disclose these technical contents, so please refer to them as necessary.

The operating robot 5 moves the multi-well plate between each block based on the reception command and the built-in program. The operation robot 5 further operates the multi-well plate and various tools in each block. The operating robot 5 having one or a plurality of robot arms performs various operations such as gripping, moving, bed fixing, and injection of the multi-well plate.

The management block 6 comprises a computer device that controls the blocks 1-4 and the operating robot 5 based on a predetermined sequence. The management block 6 manages the progress of a series of processes described later by periodically communicating with the built-in microcomputers of the blocks 1-4 and the operating robot 5.

FIG. 2 is a partial schematic plan view of the multi-well plate 7 adopted in this embodiment. FIG. 3 is a partial Y-direction vertical cross-sectional view of the multi-well plate 7. FIG. 4 is a partial X-direction vertical cross-sectional view of the multi-well plate 7.

As shown in FIGS. 3 and 4, the multi-well plate 7 is composed of a rectangular bottom plate 71, a square frame-shaped frame plate 72, and a rectangular lid plate 73, and has a rectangular thick plate shape as a whole. ing. The bottom plate 71 is made of a transparent acrylic flat plate or a glass flat plate. This enables vertical imaging of a zebrafish or fertilized egg lying on the bottom plate 71. The frame plate 72 made of a colored resin molded body is adhered on the bottom plate 71.

As shown in FIG. 2, the frame plate 72 is composed of a rectangular outer wall portion 721, a large number of partition wall portions 722, and a partition wall portion 723. The thin rectangular parallelepiped space in the outer wall portion 721 is partitioned by the partition wall portions 722 and the partition wall portions 723 arranged in a grid pattern, and is divided into a large number of wells 74 and sub wells 75. A large number of partition walls 722 extend in the Y direction, and a large number of partition walls 723 extend in the X direction. However, the partition wall portion 722 and the partition wall portion 723 are shown by a solid line in FIG.

The large number of wells 74 and the large number of subwells 75 are arranged in a matrix. The wells 74 are adjacent to each other with the partition wall portion 723 in the Y direction. Wells 74 and subwells 75 are arranged alternately in the X direction.

The well 74 has a substantially rectangular horizontal cross-sectional shape, and the sub well 75 has a substantially square horizontal cross-sectional shape. The partition wall portion 723 which is the long side of the well 74 extends in the X direction, and the partition wall portion 722 which is the short side of the well 74 extends in the Y direction. Therefore, the subwell 75 is adjacent to the short side of the well 74. The number of wells 74 is equal to the number of subwells 75. Each well 74 has the same shape as each other, and each subwell 75 has the same shape as each other.

As shown in FIGS. 3 and 4, the partition walls 722 and 723 facing the well 74 become thicker toward the bottom plate 71. The upper end opening of the well 74 in contact with the lid plate 73 is wider than the bottom surface 740 of the well 74 formed by the bottom plate 71. As a result, the volume of the well 724 can be increased to improve the living environment of the zebrafish while reducing the misalignment of the zebrafish lying on the bottom plate 71 in the well 724. However, the tapered shapes of the partition wall portions 722 and 723 described above can be omitted.

FIG. 3 shows the vertical cross-sectional shapes of the wells 74 and the sub wells 75 arranged in the X direction. FIG. 4 shows the vertical cross-sectional shape of the wells 74 arranged in the Y direction. The bottom of the partition wall 722 between the subwell 75 and the well 74 on the left side thereof has a communication hole 76 for communicating the well 74 and the subwell 75. In other words, the communication hole 76 for water flow between the well 74 and the sub well 75 adjacent to each other is opened facing the short side of the well bottom surface 740.

The lid plate 73 placed on the frame plate 72 is made of a transparent acrylic resin flat plate. The lid plate 73 has a well opening 731 located at the center of the well 74 and a sub well opening 732 located at the center of the subwell 75. The lid plate 73 can be omitted.

FIG. 5 is a flowchart showing a zebrafish processing routine executed by the management block 6. The egg arrangement step (S100) executed by the egg arrangement block 1, the injection step (S102) executed by the injection block 2, the growth step (S104) executed by the growth block 3, and the lying step performed by the growth block 3. (S106) and the imaging step (S108) executed by the imaging block 4 are comprehensively managed by the management block 6. The management block 6 controls the operation of the operating robot 5 in order to carry out these steps. Preferably, blocks 1-4 operate in parallel and control block 6 supervises the progress of each process.

The egg arrangement process is described with reference to FIGS. 6-9. FIG. 6 is an enlarged plan view showing a pair of wells 74 and sub wells 75 of the multi-well plate 7. The egg arrangement block 1 has an inclined bed (support stand) on which the multi-well plate 7 is placed, and the upper surface of the inclined bed is inclined at a predetermined angle.

First, a predetermined amount of water is injected into each well 74 of the multi-well plate 7 from which the lid plate 73 has been removed. Next, the multi-well plate 7 is placed on the upper surface of the inclined bed. When the multi-well plate 7 is set on the inclined bed, the first corner portion 741 of the bottom surface 740 of the well 74 becomes the lowest position in the vertical direction than the other corner portions 742-744, and the third corner portion 743 of the bottom surface 740. Is the highest position in the vertical direction than the other corners 742-744. These operations are performed by the operating robot 5.

In this egg arrangement step, the fertilized egg container 8 shown in FIG. 7 is used. The operation robot 5 grips the fertilized egg container 8 by its arm, moves in order in the arrangement direction of each well 74 directly above the oblique multi-well plate 7, and inserts an egg into each well 74. Are executed sequentially. As a result, the fertilized eggs 9 in the fertilized egg container 8 are sequentially put into each well 74, and one fertilized egg is arranged in each well 74.

FIG. 7 is a schematic view showing a vertical cross section of the fertilized egg container 8. The fertilized egg container 8 has a tubular portion 81 having a substantially inverted conical shape, a lid portion 82 for closing the upper end opening of the tubular portion 81, and a piezoelectric element 83 fixed to the lower surface of the lid portion 82. The lower end of the tubular portion 81 has a fertilized egg discharge port 84. The lid 82 has an air hole 85.

A large number of zebrafish fertilized eggs 9 are housed in the fertilized egg container 8 together with water. Since these fertilized eggs have a slightly higher specific density than water, they tend to gradually descend to the bottom of the fertilized egg container 8.

The fertilized egg discharge port 84 is formed to be slightly larger in diameter than the fertilized egg. Preferably, the inner diameter of the fertilized egg discharge port 84 is 1.5-1.8 mm. Since the outer diameter of the zebrafish fertilized egg is generally 0.9-1.2 mm, it is prevented that a plurality of zebrafish fertilized eggs fall from the fertilized egg outlet 84 at the same time.

In this embodiment, a piezoelectric extrusion method is adopted in which the fertilized egg is dropped from the discharge port 84 by applying a pulse to the piezoelectric element 83. The piezoelectric element 83 applies sound energy to the water in the container 8 and the fertilized egg 9 toward the fertilized egg discharge port 84. This acoustic energy is sufficient to release almost one fertilized egg 9 from the fertilized egg outlet 84. As a result, almost one fertilized egg is released from the fertilized egg discharge port 84.

However, it is assumed that a plurality of fertilized eggs 9 may be released into one well 74. In this case, various measures are taken such as avoiding the inspection of the zebrafish in the well 74 or eliminating the excess fertilized egg by the dropper attached to the robot arm.

In this embodiment, the acoustic (dynamic) energy of the piezoelectric element 83 is applied to the fertilized egg 9 in the vicinity of the fertilized egg discharge port 84 through the water in the container 8 and the fertilized egg 9, and the fertilized egg 9 is dropped from the fertilized egg discharge port 84. The basic principle of the fertilized egg ejection method is essentially the same as that of the conventional inkjet printing method. However, this energy is regulated to a level that does not cause biological damage to the fertilized egg. It is also possible to adopt another fertilized egg discharging method in order to impart falling energy to the fertilized egg 9 in the vicinity of the fertilized egg discharging port 84. For example, it is possible to apply air pressure to the upper part of the container 8 at a predetermined timing.

It is desirable that this fertilized egg discharge be performed when the discharge port 84 reaches a predetermined position on one well 74. It is desirable that the moving scanning of the fertilized egg container 8 by the operating robot 5 be stopped when the fertilized egg is discharged. After optically confirming this fertilized egg discharge, the container 8 may be moved directly above the next well 74.

As yet another method for discharging fertilized eggs, the height of the water surface in the fertilized egg container 8 can be controlled. For example, the height of the water surface in the fertilized egg container 8 is controlled to a constant level. As a result, water containing the fertilized egg continuously falls from the fertilized egg outlet. Since the falling water flow velocity is substantially proportional to the water level, the water level is adjusted so that an appropriate fertilized egg falling frequency can be obtained. As a result, the fertilized egg 9 is released from the fertilized egg outlet 84 with a substantially constant interval.

Preferably, the moving speed of the fertilized egg container 8 by the operation robot 5 is synchronized with the fertilized egg discharge interval from the discharge port 84. As a result, fertilized eggs can be charged one by one into most of the wells 74 of the multi-well plate 7. When a well 74 in which a fertilized egg is not inserted is generated, the two-dimensional address of this well is stored in advance as a defective well and is removed from the inspection target.

Another feature of this egg arranging process will be described with reference to FIGS. 8 and 9. According to this embodiment, the multi-well plate 7 is tilted and placed on the inclined bed as described above. As a result, each well 74 of the multi-well plate 7 has a posture in which one corner portion 741 is tilted at the lowest position in the vertical direction.

FIG. 8 is a schematic diagram briefly showing the tilted state of the multi-well plate 7 in the egg arrangement step. As a result, the fertilized egg 9 charged into each well 74 from the fertilized egg container 8 slides on the well bottom surface 740 toward the lowest position corner 741, and finally sits on the lowest position corner 741. do. The septum 722 and 723 adjacent to the lowest corner 741 regulate further displacement of the fertilized egg 9. It is also possible to form a shallow recess in the bottom surface 740 where the fertilized egg 9 can sit, located at the lowest corner portion 741.

Further, according to the egg arrangement step of this embodiment, as shown in FIG. 8, the fertilized egg container 8 has a predetermined inclination of the axis 87 of the discharge port 84 with respect to the upper surface of the multi-well plate 7 during the fertilized egg discharge period. It has a posture that tilts in the direction. For example, this tilting direction of the axis 87 is equal to or parallel to the direction in which the fertilized egg 9 released from the discharge port 84 is directed toward the lowest corner portion 741. As a result, the time required for the fertilized egg 9 to reach the corner 741 can be shortened.

The effect of seating the fertilized egg 9 on the corner 741 of the bottom surface 740 of the well 74 will be described below. In microinjection in which the target material is injected into a fertilized egg using a microneedle, the alignment of the microneedle with respect to the fertilized egg is very important. For example, it is preferable to insert the tip of the microneedle near the top of the small fertilized egg 9. However, this injection must be completed in a short period of time immediately after the start of cell division of the fertilized egg. However, it is not easy to accurately align the microneedles for each fertilized egg 9 in order to align the fertilized egg with the multi-well plate 7.

For this reason, in the past, it was common to use a multi-well plate dedicated to holding fertilized eggs and to hold the fertilized eggs in the narrow well dedicated to holding eggs. However, the adoption of such a multi-well plate dedicated to holding fertilized eggs requires that the hatched zebrafish be individually transferred from the wells of the multi-well plate dedicated to holding fertilized eggs to the wells of the multi-well plate dedicated to fry breeding. Is derived.

After all, according to this embodiment, it is possible to realize a reliable injection step into the fertilized egg by using the multi-well plate 7 having a large well capable of raising fry. FIG. 9 is a schematic partial plan view of a multi-well plate 7 for showing a state in which a fertilized egg 9 is seated on a corner portion 741 of a bottom surface 740 of each well 74.

Next, the operating robot 5 transfers the multi-well plate 7 from the egg arrangement block 1 to the injection block 2. The injection block 2 has a bed on which the multi-well plate 7 can be placed horizontally, and the operating robot 5 fixes the multi-well plate 7 on the bed. The bed is formed with a stopper that regulates the displacement of the multi-well plate 7 in the X-direction and the Y-direction, and the operating robot 5 brings the corner portion of the multi-well plate 7 into contact with the stopper. Thereby, the multi-well plate 7 can be positioned with respect to the injection block 2.

The injection block 2 for performing the injection step has one or more microneedles for injecting a target drug solution, cells, or the like into a fertilized egg 9 in each well 74. Since each fertilized egg 9 is stably maintained in the vicinity of the corner portion 741 of the multi-well plate 7, the microneedle of the injection block 2 can be accurately plunged into substantially the crown of each fertilized egg 9. After the completion of this injection step, the operating robot 5 transfers the multi-well plate 7 to the growth block 3. Zebrafish fry hatched from each fertilized egg 9 of the multi-well plate 7 are grown in this growth block 3 for a predetermined time.

In this growing process, clean water is periodically supplied to the sub-well 75, and is supplied from the sub-well 75 to the well 74 through the communication port 76. In this embodiment, the lid plate 73 is put on the well 74 and the sub well 75 in the growing step. This is to improve the hygiene of the wells 74 and subwells 75 and prevent the zebrafish from popping out of the wells 74. Feeding the zebrafish is carried out through the opening 731 of the lid plate 73.

According to this embodiment, the discharge of feces and residual food in the well 74 is carried out by supplying water from the sub well 75 to the well 74 and overflowing the water from the opening 731 on the well 74. The overflow from the well 74 flows along the upper surface of the lid plate 73 and is discharged to the outside. In order to prevent this overflow from flowing into the well 74 and the sub well 75 again from the other openings 731 and 732 of the lid plate 73, it is preferable that a protrusion is provided on the upper peripheral periphery of the opening 731 and the opening 732. .. Detailed explanation of other growth processes is omitted.

According to this embodiment, the growing block 3 further performs a lying step of lying down the zebrafish on the bottom surface 740 of the well 74 at the final stage of this growing step. In this lying step, the anesthetic is mixed with the water supplied from the subwells 75 to the wells 74, whereby each zebrafish becomes stationary in the wells 74.

In this lying step, the water supply step from the sub well 75 to the well 74 and the drainage step from the well 74 to the sub well 75 are sequentially executed. The water supply step of this lying step is almost the same as the water supply step to the well 74 in the zebrafish growing step.

This water supply step in the lying process will be described with reference to FIG. In this water supply step, a water supply head composed of a water supply tank in which a predetermined number of water supply nozzles 11 hang down is used. This water supply tank communicates with each water supply nozzle 11. The management block 6 controls the injection of water into the water supply tank and its discharge. When water is injected into the water supply tank, the water pressure in the water supply tank increases, and water is ejected from the water supply nozzle 11. When water is discharged from the water supply tank, the water pressure in the water supply tank decreases, and the water supply nozzle 11 absorbs water from the outside.

The operating robot 5 sets the water supply head above the multi-well plate 7 immediately before this water supply step. Each water supply nozzle 11 rests directly above the opening 732 on each subwell 75. Next, the operating robot 5 lowers the water supply head. As a result, each water supply nozzle 11 is individually inserted into each opening 732. A ring-shaped stopper 12 is individually attached to the water supply nozzle 11, and the water supply nozzle 11 is lowered until the stopper 12 comes into contact with the lid plate 73. As a result, the lower end of the water supply nozzle 11 reaches the vicinity of the bottom surface of the sub well 75. The management block 6 controls the water supply to the water supply tank, and the water pressure of the water supply tank is controlled by the control block 6.

In the water supply step of the lying process shown in FIG. 10, a predetermined amount of water is discharged from the water supply nozzle 11 into the subwell 75. As a result, the water in the sub well 75 flows into the bottom of the well 74 through the communication hole 76. As a result, the zebrafish 10 in the well 74 rises.

In the drainage step of the lying process shown in FIG. 11, the pressure in the water supply tank is reduced. As a result, the water in the subwell 75 is collected in the water supply tank through the water supply nozzle 11. As a result, the water in the well 74 flows into the sub well 75 through the communication hole 76. As a result, a descending water flow toward the communication hole 76 is formed in the well 74, and this descending water flow changes the attitude of the zebrafish. This attitude change urges the zebrafish 10 so that the fluid resistance of the zebrafish 10 to the descending water flow is minimized.

Experiments have shown that this descending stream causes the zebrafish 10 to have its large head away from the communication hole 76 and the narrow tail of the zebrafish 10 close to the communication hole 76. Finally, the water in the well 74 is almost completely drained and the zebrafish 10 lies on the bottom 740 of the well 74 in a proper lying position.

It is preferable that the lying step including the water supply step and the drainage step is carried out a plurality of times. As a result, it is possible to reduce the positional variation of the zebrafish in the imaging work of the zebrafish 10 and the injection work of the reagent, cells, etc., which will be described later.

Next, the eggshell separation step will be described with reference to FIGS. 12 and 13. This eggshell separation step is appropriately carried out in the growing block 3 during the growing step or immediately before the lying step in the same manner as the lying step. This eggshell separation step is essentially the same as the lying down step.

The need for this eggshell separation step is explained. Most of the zebrafish fry that hatch from the fertilized egg can completely withdraw from the eggshell of the fertilized egg. At this time, the eggshell that stays in the well 74 can be discharged from the opening 731 of the lid plate 73 due to the overflow of water from the well 74. However, the event that eggshell attaches to zebrafish fry rarely occurs. According to this case, since the eggshell cannot be discharged, the shooting of the zebrafish and the image determination are hindered.

According to this eggshell separation step, the eggshell 13 is separated from the zebrafish 10 by the water supply step and the drainage step of the lying down step. FIG. 12 is a schematic diagram showing a water supply step in the eggshell separation step. FIG. 12 is essentially the same as the water supply step of the lying down step shown in FIG.

In this water supply step, water is ejected from the communication hole 76 into the well 74, whereby an ascending water flow acts on the zebrafish 10 and the eggshell 13 attached thereto. Since this rising water stream gives the eggshell 13 a relatively stronger rising force than the zebrafish 10, the eggshell 13 is separated from the zebrafish 10 and the eggshell 13 rises to the upper part of the well 74. On the other hand, the rise of zebrafish 10 is relatively slow.

FIG. 13 is a schematic diagram showing a drainage step carried out immediately after this water supply step. FIG. 13 is essentially the same as the drainage step of the lying down step shown in FIG. The water in the lower part of the well 74 is absorbed into the sub well 75 through the communication hole 76. As a result, the descending water flow acts on the zebrafish 10 and the eggshell 13. This descending stream lowers the zebrafish and eggshell. However, since the zebrafish 10 is heavier than the eggshell 13, the zebrafish 10 preferentially descends.

Preferably, the eggshell 13 can be levitated near the water surface by executing the set of the water supply step and the drainage step a plurality of times. The eggshell 13 is discharged from the opening 731 of the lid plate 73, for example, by the overflow treatment described above.

After the recumbent step and the eggshell separation step are completed, the water supply head is moved upward and the water supply nozzle 11 is pulled upward from the opening 732. Next, the operating robot 5 transfers the multi-well plate 7 to the imaging block 4 in order to execute the imaging process. The imaging block 4 has a bed on which the multi-well plate 7 is placed horizontally. The operating robot 5 places a multi-well plate 7 on which the zebrafish lies on this bed. The lighting device and the imaging camera are located above the bed. The imaging block 4 performs imaging, image processing, and optical inspection of the zebrafish in each well 74. A detailed description of this imaging process will be omitted.

(Second Example)
In this embodiment, the target material is injected into zebrafish fry or adult fish. In this embodiment, the injection routine (S102) shown in the flowchart shown in FIG. 5 is executed after the lying routine (S106). Generally, after this injection routine is performed, the growth routine (S104) is re-executed, followed by the imaging routine (S108). As the target material, a drug solution, a genetic material, a cancer cell, or the like can be adopted. Therefore, in this injection routine, the microneedles of the injection block 2 inject the target drug solution, cells, or the like into the zebrafish in each well 74. After all, the system of the present invention can be utilized for injecting the target material into fertilized eggs and / or small fish. This second embodiment is essentially the same as the first embodiment except that the execution order (sequence) of each partial routine shown in FIG. 5 is different.

(Deformation mode)
The eggshell separation step described above can be carried out in the breeding step instead of the lying step. For example, this eggshell separation step can be performed on fry to which the eggshell adheres.

(Deformation mode)
The pulse energization of the piezoelectric element in the egg arrangement step described above can be executed a plurality of times. The fertilized eggs arranged in the lower end tube of the fertilized egg container 8 having the fertilized egg discharge port 84 move toward the fertilized egg discharge port 84 by a predetermined small distance by one pulse energization. The fertilized egg can be moved by the required distance by energizing multiple pulses. It is also possible to detect the fall of a fertilized egg and stop the pulse energization. Of course, it is possible to appropriately design the pulse energization waveform.

1 Egg arrangement block (fertilized egg arrangement means)
2 Injection block 3 Growth block 4 Imaging block 5 Operation robot (fertilized egg arrangement means, relative movement means, fertilized egg setting means)
6 Management block (water supply control means)
7 Multi-well plate 8 Fertilized egg storage container (fertilized egg setting means)
9 Fertilized egg 10 Zebrafish 11 Water supply nozzle (water supply / drainage means)
74 Well 75 Subwell 76 Communication hole 81 Fertilized egg storage tube 83 Piezoelectric element (water supply adjusting means)
84 Fertilized egg outlet

Claims (11)

It has a large number of wells partitioned by partition walls erected on a transparent bottom plate, and the upper end of each well is an open multi-well plate.
A water supply / drainage means for controlling water supply and drainage to each well,
A fertilized egg arranging means for inserting one small fish fertilized egg into each of the wells,
Equipped with
The fertilized egg arranging means
An almost inverted cone-shaped fertilized egg storage tube that has a fertilized egg outlet slightly larger than the small fish fertilized egg at the lower end and stores a large number of small fish fertilized eggs together with water.
A water supply adjusting means for dropping the fertilized small fish eggs in the fertilized egg accommodating tube one by one at a predetermined timing by controlling the discharge rate of water from the fertilized egg discharge port.
A relative moving means for inserting one small fish fertilized egg into each well by moving the fertilized egg accommodating tube portion above the multi-well plate in parallel with the upper surface of the multi-well plate.
A small fish fertilized egg processing device characterized by being equipped with. It has a fertilized egg setting means for setting each small fish fertilized egg at a predetermined position on the bottom surface in each well.
The fertilized egg setting means is
The small fish fertilized egg processing apparatus according to claim 1, wherein the small fish fertilized egg is arranged at a predetermined position close to a predetermined partition surface of the well by inclining the multi-well plate. The bottom surface of each well has a substantially rectangular shape and has a substantially rectangular shape.
2. The fertilized egg setting means tilts the multi-well plate so that the fertilized small fish egg moves to the predetermined position close to one of the four corners of the bottom surface of the substantially square shape. The described small fish fertilized egg processing device. The fertilized egg arranging means drops the small fish fertilized egg from the fertilized egg accommodating tube portion into each well in each well of the multi-well plate in the inclined state.
2. The discharge port of the fertilized egg accommodating cylinder portion is obliquely provided at a predetermined angle with respect to the vertical direction so as to apply kinetic energy toward the predetermined position to the fertilized egg to be discharged. The described small fish fertilized egg processing device. The well has a substantially rectangular bottom surface on which the grown small fish can lie down.
The multi-well plate is partitioned on the bottom surface by the partition wall and is formed on a large number of subwells individually adjacent to the short side of the bottom surface of each well, and on the bottom of the partition wall between the well and the subwell. It has a communication hole that is penetrated and communicates with the well and the sub-well.
The water supply / drainage means supplies water from the sub-well toward the short side of the bottom surface of the well, and then drains the water in the well to the sub-well, thereby causing the small fish to flow into the well. The small fish fertilized egg processing apparatus according to claim 1, which is laid down on the bottom surface. The water supply / drainage means supplies water from the sub-well to the well and then drains the water in the well to the sub-well so that the small fish lies on the bottom surface of the well and remains in the well. The small fish fertilized egg processing apparatus according to claim 5, wherein the eggshell of the fertilized egg is levitated on the upper part of the well. It has a large number of wells partitioned by partition walls erected on a transparent bottom plate, and the upper end of each well is an open multi-well plate.
A water supply / drainage means for controlling water supply and drainage to each well,
A fertilized egg arranging means for inserting one small fish fertilized egg into each of the wells,
It has a fertilized egg setting means for setting each small fish fertilized egg at a predetermined position on the bottom surface in each well.
The fertilized egg setting means is
A small fish fertilized egg processing apparatus, characterized in that the small fish fertilized egg is arranged at a predetermined position close to a predetermined partition wall surface of the well by inclining the multi-well plate. The bottom surface of each well has a substantially rectangular shape and has a substantially rectangular shape.
7. The fertilized egg setting means tilts the multi-well plate so that the fertilized small fish egg moves to the predetermined position close to one of the four corners of the bottom surface of the substantially square shape. The described small fish fertilized egg processing device. The fertilized egg arranging means drops the small fish fertilized egg from the fertilized egg accommodating tube portion into each well in each well of the multi-well plate in the inclined state.
7. The discharge port of the fertilized egg accommodating cylinder portion is obliquely provided at a predetermined angle with respect to the vertical direction so as to apply kinetic energy toward the predetermined position to the fertilized egg to be discharged. The described small fish fertilized egg processing device. It has a large number of wells partitioned by partition walls erected on a transparent bottom plate, and the upper end of each well is an open multi-well plate.
A water supply / drainage means for controlling water supply and drainage to each well,
Have,
The well has a nearly rectangular bottom surface on which small fish in the well can lie down.
The multi-well plate is partitioned on the bottom surface by the partition wall and is formed on a large number of subwells individually adjacent to the short side of the bottom surface of each well, and on the bottom of the partition wall between the well and the subwell. It has a communication hole that is penetrated and communicates with the well and the sub-well.
The water supply / drainage means supplies water from the sub-well toward the short side of the bottom surface of the well, and then drains the water in the well to the sub-well to allow the small fish to flow to the bottom surface of the well. A small fish processing device characterized by lying down. The water supply / drainage means supplies water from the sub-well to the well and then drains the water in the well to the sub-well so that the small fish lies on the bottom surface of the well and remains in the well. The small fish processing apparatus according to claim 10, wherein the eggshell of the fertilized egg of the small fish is levitated on the upper part of the well.

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