JP3809526B2 - Small animal behavior measurement and control device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention measures or controls the behavior (learning, chemotaxis, avoidance, search, etc.) of minute animals (protozoa, metazoans, etc.) in a space having a three-dimensional microstructure such as soil and animal and plant tissues. Relating to the device. This device can be used in fields such as biology, agriculture, and environmental engineering because it enables easy and highly reproducible behavioral analysis of minute animals by artificially creating a behavioral environment close to nature.
[0002]
[Prior art]
Since the habitat of micro-animals is mainly in soil or liquid, the agar medium (Non-patent Document 1), culture medium (Non-patent Document 2), and soil (non-patent document 2) are used as action spaces for measuring the behavior of micro animals. Patent Document 3), a fine particle filled container (Non-Patent Document 4), and the like are used. A method of measuring movement speed, action radius, action trajectory, response time to stimulus, etc. from observation of a micro animal in the inside or surface layer with a microscope or the number of heads appearing outside through a medium is widely used. I came. In addition, studies have been made such as adding solutions having various chemical compositions in the behavior space to control and measure the behavior of micro animals.
[0003]
[Non-Patent Document 1]
PLINE, M. and DUSENBERY, DB 1987.Responses of plant-parasitic nematode Meloidogyne incognita to carbon dioxide determined by video camera-computer tracking.Journal of Chemical Ecology 13: 873-888.
[0004]
[Non-Patent Document 2]
KENNEDY, MJ 1979. The responses of miracidia and cercariae of Bunodera mediovitellata (Trematoda: Allocreadiidae) to light and gravity.Canadian Journal of Zoology 57: 603-609.
[0005]
[Non-Patent Document 3]
WALLACE, HR 1958. Movement of eelworms-1. The influence of pore size and moisture content of the soil on the migration of larvae of the beet eelworm, Heterodera schachtii Schmidt. Annals of Applied Biology 46: 74-85.
[0006]
[Non-Patent Document 4]
ROBINSON, AF 1994. Movement of five nematode species through sand subjected to natural temperature gradient fluctuations. Journal of Nematology 26: 46-58.
[0007]
[Problems to be solved by the invention]
First, an agar medium method conventionally used for measuring the behavior locus, behavior radius, movement speed, and response time to a stimulus of a nematode will be described as an example. While this method has the advantage that the behavior can be observed easily and in detail, it has the problem that it does not necessarily reflect the natural behavior of nematodes that inhabit the soil or animal and plant tissues. In other words, soil and animal and plant tissues are three-dimensional microstructures consisting of obstacles and voids, and the movement of the solution occurs due to capillarity inside. It is thought that it is acting by using a factor. However, it is difficult to say that the natural behavior of the nematode is measured in that the agar medium does not have the above-described three-dimensional microstructure and does not move by a capillary phenomenon. Furthermore, when conducting responses to various chemical compositions and controlling behavior by them, the experiment is performed by dropping the solution on the agar medium, but the behavior of the nematodes in the immersion state is limited in a flat space. Since it is an open system, there is a problem that it is difficult to control the working concentration due to infiltration or transpiration.
[0008]
On the other hand, as a measurement method in a state close to the actual habitat environment, there is a method using a container filled with soil or fine particles used when simply measuring the mobility of nematodes. Although this method has the advantage of being able to measure the mobility in a natural state, the physical conditions in the container (filled state, presence / absence of bubbles, etc.) cannot be observed uniformly. However, there is a problem that the container becomes a black box because it is difficult.
[0009]
The present invention has a three-dimensional microstructure consisting of obstacles and voids, and their form, size, physical conditions, etc., in order to solve the problems of the technology as described above. By providing an artificial behavioral environment that is controlled so as to be close to the environment such as soil and animal and plant tissues, the behavior of micro-animals inside a three-dimensional microstructure can be easily brought close to the natural state. An apparatus capable of measuring and controlling is provided.
[0010]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventor formed a three-dimensional microstructure having a size and shape controlled on a flat plate on the flat plate with the aid of fine precision processing technology, and the fine The idea was to create an action space with a three-dimensional microstructure consisting of obstacles and voids, such as in soil and animal and plant tissues, by attaching a flat lid from the top to the structural surface. By introducing the micro-animal into the space formed in this way, the micro-animal is confined in the fine structure, and the physicochemical conditions are precisely controlled by utilizing the closed space, and the natural state is obtained with a microscope or the like. The present invention has been completed by finding that it is possible to measure and control actions within a three-dimensional microstructure in a close form in a simple and detailed manner.
[0011]
That is, the present invention includes the following inventions (1) to (7).
(1) a first flat plate having a plurality of fine concave structures connected to each other formed on the surface, a second flat plate in contact with the concave structure surface of the first flat plate and covering the opening of the concave structure; A micro-animal behavior measurement and control device that can measure or control the behavior of a micro-animal introduced into a concave structure.
(2) The minute animal behavior measurement control device according to (1), further including a container that can store the first flat plate and the second flat plate.
(3) The micro-animal behavior measurement control device according to (1) or (2), wherein the micro-animal to be introduced is a metazoan or a protozoan.
(4) The minute animal behavior measurement control device according to any one of (1) to (3), wherein a fine electrode is formed on the second flat plate.
(5) The micro-animal behavior measurement control device according to any one of (1) to (4), wherein the depth and the maximum width of the concave structure are 0.02 mm or more.
(6) The minute animal behavior measurement control device according to any one of (1) to (5), wherein a through hole is provided in the first flat plate or the second flat plate.
(7) The minute animal behavior measurement control device according to any one of (1) to (6), wherein the first flat plate and the second flat plate are transparent.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
The micro-animal behavior measurement control device according to the present invention includes a first flat plate having a plurality of fine concave structures connected to each other on a surface thereof, a concave structure surface of the first flat plate, and an opening of the concave structure. And a second flat plate covering the part. Moreover, this apparatus may have the container which accommodates a 1st flat plate and a 2nd flat plate.
[0013]
By covering the opening of the concave structure with the second flat plate, a fine structure space composed of an obstacle and a gap can be formed. In the apparatus of the present invention, the size and shape of the obstacles and voids in the microstructure space, the nature of the void surface, the type of the medium that fills the voids, and other physicochemical conditions can be set freely and precisely. It is possible to control the behavior of micro animals through the control of environmental conditions.
[0014]
In addition, by providing a through hole in the first flat plate or the second flat plate, it is possible to introduce and circulate micro animals, their essential nutrients, other chemical substances, etc. in the microstructure space. By storing the flat plate in a sealable container, it is possible to precisely control the environment of the fine structure space.
[0015]
Any material can be used for the first flat plate as long as it has plasticity, but it has characteristics such as high affinity with the original plate for forming the concave structure, low chemical reactivity, and high transparency. Is preferred. Examples of such materials include polydimethylsiloxane and polydimethylsilane.
[0016]
The shape and dimensions of the first flat plate are not particularly limited as long as a fine structure space in which a micro animal can act can be formed.
[0017]
The concave structure can be formed by using an original plate for microfabricated resin flat plate molding. The original plate for microfabricated resin flat plate molding can be any 3D micromachining method that guarantees micron-order machining accuracy, but it is 0.01mm in consideration of the size of the microanimal. A method capable of forming a microstructure having the above size is preferable. Examples of the manufacturing method include a silicon crystal anisotropic etching method and a focused ion beam method when a silicon substrate is used, and a metal etching method and an electroforming method when a metal substrate is used.
[0018]
The method for molding the first flat plate is not particularly defined, but the original plate is adhered and fixed to a flat plate such as glass, mica, or metal by an adhesive, magnetic force, or capillary phenomenon, and the original plate is covered. Thus, it is preferable that the first flat plate material is dropped and cured.
[0019]
The first flat plate has a plurality of fine concave structures that are connected to each other. The shape of the concave structure may be any shape as long as it can be connected to each other to form a suitable obstacle and space for the micro animal. The size of the concave structure is preferably 0.02 mm or more in depth and maximum width, but may be smaller than this depending on the size of the micro animal and the behavioral form. For example, since plant parasitic nematodes usually have a body length of about 0.4 mm and a body width of about 0.02 mm, the dimensions of the concave structure may be as shown in FIG. 1 with a depth of 0.05 mm. Each of the plurality of concave structures may have a different shape and size, but all preferably have the same shape and size. Although the concave structure may be randomly arranged on the first flat plate, it is preferable that the concave structure is regularly arranged at equal intervals in the vertical and horizontal directions. The range in which the concave structure is formed is not particularly limited, but is preferably near the center of the first flat plate as shown in FIG.
[0020]
The material of the second flat plate is preferably transparent and high in flatness for observation with a microscope or the like, and preferably low in chemical reactivity. Specifically, glass, quartz, acrylic resin and the like can be exemplified.
[0021]
Microelectrodes may be formed on the second flat plate, which makes it possible to electrically detect a minute animal and to control behavior electrically. When a microelectrode is constructed, it is necessary to form a pattern of chromium, platinum, indium tin oxide, or the like on the second flat plate, and therefore, it is necessary to use a material that can withstand these pattern forming steps. In addition, when it is necessary to provide electrical insulation to the surface of the microelectrode, it is preferable to cover a part or the entire surface of the electrode portion with a transparent insulating resin.
[0022]
The shape and dimensions of the second flat plate are not particularly limited as long as they can cover the opening of the concave structure portion of the first flat plate.
[0023]
Any container may be used as long as it can accommodate the first flat plate and the second flat plate. In order to observe the material with a microscope or the like, the material that is in close contact with the first flat plate is preferably transparent and has high flatness, and preferably has low chemical reactivity. Examples of such materials include glass, quartz, and acrylic resin.
[0024]
The minute animal to be measured and controlled is not particularly limited, and may be either a metazoan or a protozoan. Examples of protozoa include ciliates, fleshworms, and flagellates. Examples of metazoans include nematodes, flukes, and protozoa. The most preferred micro animals to be measured and controlled are nematodes.
[0025]
Hereinafter, an embodiment of the apparatus of the present invention will be described with reference to the drawings.
[0026]
FIG. 1 is a dimensional view of a concave structure formed on a first flat plate, FIG. 2 is a dimensional view of a first flat plate formed with a concave structure, and FIG. 3 is a view of a container for housing the first flat plate and the second flat plate. FIG. 4 is a dimensional diagram of the second flat plate, FIG. 5 is an assembly diagram of the minute animal behavior measuring device, and FIG. 6 is a diagram showing an example of use of the minute animal behavior measuring device.
[0027]
In FIG. 1, the black portion represents a portion that is uniformly recessed at a depth of 0.05 mm from the resin flat plate surface. In FIG. 2, the hatched square portion represents a region where the above-mentioned dents are connected in a state where they are aligned vertically and horizontally. The square outside the hatched square represents a uniformly recessed portion. In FIG. 3, the upper drawing shows a top view of a circular container in which a circular space for storing and fixing the first flat plate is formed, and the lower drawing shows a side view thereof. In FIG. 4, the upper drawing is a top view of the second flat plate, and the lower drawing is a side view thereof. FIG. 5 shows a method of combining the parts shown in FIGS. 2 to 4, wherein 51 is a second flat plate, 52 is a first flat plate, and 53 is a container. After the first flat plate is accommodated in the container, the second flat plate is brought into close contact with the first flat plate, and fitted and fixed from above so as to abut against the upper end of the container. In FIG. 6, a measurement method by microscopic observation using the micro-animal behavior measuring apparatus of FIG. 5 is illustrated, in which 61 is a micro-animal introduction liquid feeding pipe, 62 is a solution injection pipe, 63 is a solution delivery pipe, and 64 is A minute animal behavior measuring device, 65 is an inverted microscope, and 66 is an objective lens.
[0028]
Hereinafter, a method for measuring the behavior of a minute animal will be described by taking as an example the case of using a nematode as the minute animal and measuring the moving speed of the nematode by microscopic observation. First, the first flat plate is sunk with the concave structure surface facing upward in a state where distilled water is put in a portion of the container that accommodates the first flat plate. Next, the second flat plate is fitted to the upper end of the container so that the first flat plate is sandwiched between the container and the second flat plate and firmly adhered. Thereby, it becomes a structure where a micro animal does not leak into the container from the three-dimensional microstructure. The nematode settles by its own weight by injecting the nematode suspended in distilled water with a micro animal introduction liquid pipe, a solution injection pipe, and a solution delivery pipe into the micro animal introduction pipe. To reach the microstructured surface. The nematode's behavior is fine because the upper surface is a free space on the fine structure surface that touches the through hole of the micro-animal introduction tube, solution injection tube, and solution delivery tube provided on the second flat plate. However, the nematode behaves along the concave part of the microstructure when it deviates from the through-hole part. As shown in FIG. 6, the behavior of the nematode on the fine structure surface can be observed through the objective lens on the sample stage of the inverted microscope. In order to control the behavior of nematodes in this state, for example, food substances (plants and pulverized products of animal tissues), light irradiation, electromagnetic stimulation, vibration, dissolved gas concentration control in solution, solution flow, etc. Can also be given. For example, when measuring the movement speed of a nematode as an action measurement, a microscope image is stored in a recording device, and the movement distance is calculated by counting the number of concave structures that the nematode has passed within a certain period of time. However, it can be easily measured by dividing by time. Alternatively, the movement speed of the nematode can be measured in real time by automatically tracking the action locus of the nematode by image processing.
[0029]
【Example】
Measurement was carried out by storing the concave structure shown in FIG. 1 on the first flat plate shown in FIG. 2 and storing it in a quartz container with the configuration shown in FIG. The first flat plate was made of polydimethylsilane, and a transparent first flat plate having a thickness of 1 mm and a diameter of 20 mm was prepared from an original plate manufactured by silicon crystal anisotropic etching. A sample in which an infectious larva of the insect parasitic nematode Steinernema carpocapsae was suspended in distilled water in a petri dish was used as a measurement target. While confirming the presence of the worm body with a stereomicroscope, the worm body is aspirated with 5 × 10 ^-3 ml of distilled water using a micropipette and discharged into the micro-animal feeding tube shown in FIG. Introduced. The introduced nematodes were actively activated by light stimulation from the outside, and the movement in the concave structure was recorded by a CCD camera attached to an inverted microscope. In order to analyze the movement trajectory of the nematode, by performing image processing on the recorded moving image, only the image of the worm body is extracted from all the images, and the result is superimposed as shown in FIG. It became clear. The moving speed was measured to be about 0.5 mm / sec for the fastest moving nematode by counting the number of concave structures moved in 10 seconds.
[0030]
【The invention's effect】
As described above, by using the present invention, the behavior (learning, chemotaxis, avoidance, etc.) of minute animals (protozoa, metazoans, etc.) in a space having a three-dimensional microstructure such as soil and animal and plant tissues Search, etc.) can be measured and controlled. Since this device enables behavioral analysis of minute animals in a state close to nature with high reproducibility, it can be used as a measurement device for biological environmental indicators in fields such as biology, agriculture, and environmental engineering. .
[Brief description of the drawings]
FIG. 1 is a dimensional diagram (depth 0.05 mm) of a concave structure formed on a first flat plate.
FIG. 2 is a dimensional diagram of a first flat plate having a concave structure (plate thickness: 1 mm).
FIG. 3 is a dimensional diagram of a container that houses a first flat plate and a second flat plate.
FIG. 4 is a dimension diagram of a second flat plate.
FIG. 5 is an assembly diagram of a minute animal behavior measuring device.
FIG. 6 is a diagram showing an example of use of a minute animal behavior measuring device.
FIG. 7 is a diagram illustrating a result of analyzing a moving locus image of a nematode in a minute animal behavior measuring apparatus.
[Explanation of symbols]
51 Second Flat Plate 52 First Flat Plate 53 Container 61 Liquid Feeding Pipe for Micro Animal Introduction 62 Solution Injection Pipe 63 Solution Delivery Pipe 64 Micro Animal Behavior Measuring Device 65 Inverted Microscope 66 Objective Lens

Claims (7)

A first flat plate having a plurality of fine concave structures connected to each other formed on the surface, and a second flat plate in contact with the concave structure surface of the first flat plate and covering the opening of the concave structure. A micro-animal behavior measurement control device capable of measuring or controlling the behavior of a micro-animal introduced into a concave structure. The micro animal behavior measurement control device according to claim 1, further comprising a container that can store the first flat plate and the second flat plate. The microanimal behavior measurement control device according to claim 1 or 2, wherein the microanimal to be introduced is a metazoan or a protozoan. The minute animal behavior measurement control device according to any one of claims 1 to 3, wherein a fine electrode is formed on the second flat plate. The minute animal behavior measurement control device according to any one of claims 1 to 4, wherein a depth and a maximum width of the concave structure are 0.02 mm or more. The micro animal behavior measurement control device according to any one of claims 1 to 5, wherein a through hole is provided in the first flat plate or the second flat plate. The minute animal behavior measurement control device according to any one of claims 1 to 6, wherein the first flat plate and the second flat plate are transparent.

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