JP6153191B2 - Observation tool and observation device - Google Patents

Description

  The present invention relates to an observation tool and an observation apparatus including the same.

  In observing living cells, tissues, and the like with a microscope, the activity of the living cells and the like changes immediately with changes in culture conditions such as temperature. For this reason, it is important to perform observation under stable conditions. Conventionally, in order to achieve the above-described conditions, a complicated device in which a temperature maintaining device, a gas supply device, a culture solution introduction / extraction device, and the like are combined with a microscope is required (for example, Patent Documents 1 and 2).

  Moreover, when using the said apparatus, the complicated operation of installing the said culture dish in the said apparatus and also installing the said apparatus was required. And, when a drug treatment or the like is performed on the living cells or the like during the microscopic observation, it is necessary to take a complicated operation of taking out the culture dish from the device and installing it again in the device after the treatment of the drug or the like. there were.

  Furthermore, in the upright microscope, in addition to the complexity of operation when using the apparatus, there is a problem that the culture medium exchange in the culture dish is unstable, and it is difficult to stabilize the culture conditions. As a result, the microscopic observation of the living cells and the like is mainly performed using an inverted microscope, and it is difficult to use an upright microscope including a multiphoton microscope capable of performing more detailed observation.

  For this reason, there is a need for an observation means that allows the culture sample to be exchanged more easily, makes the culture conditions constant, and can be used in both an upright microscope and an inverted microscope.

Japanese Patent No. 4562165 Special table 2003-528637

  Accordingly, the present invention provides an observation tool in which the culture sample to be observed can be easily exchanged, the culture conditions of the culture sample can be easily fixed, and can be used in both an upright microscope and an inverted microscope. Objective.

In order to solve the problems of the invention, the observation tool of the present invention comprises:
An observation tool for observing a culture sample under a microscope,
Including pedestal and tip,
The pedestal has a chip placement area for placing the chip,
The chip includes a culture chamber, a culture solution introduction channel, and a culture solution outlet channel,
Each upper part of the culture chamber, the culture solution introduction channel and the culture solution introduction channel is open,
The culture medium introduction channel and the culture solution outlet channel communicate with the culture chamber,
The culture chamber can be observed from the outside,
A through hole is formed at a position corresponding to the culture chamber of the chip in the chip placement region of the pedestal,
The pedestal includes a chip fixing means for removably fixing the chip to the chip arrangement region.
It is characterized by that.

The observation apparatus of the present invention is
An observation device for observing a culture sample under a microscope,
Including microscope and observation tool,
The observation tool is arranged under the microscope,
The observation tool is the observation tool of the present invention.

  According to the observation tool of the present invention, it is possible to observe simply by fixing a chip on which a culture sample to be observed (for example, cultured cells or cultured tissue) is placed on a pedestal, and the chip can be removed from the pedestal. Therefore, the culture sample to be observed can be easily exchanged. Further, according to the observation tool of the present invention, since the control of the introduction / extraction of the culture solution is easy, the culture sample can be easily observed under certain conditions. Furthermore, according to the observation tool of the present invention, since the culture sample can be observed from both the upper and lower directions of the observation tool, it can be used in both an upright microscope and an inverted microscope. Therefore, the present invention is a very useful tool for microscopic observation such as live imaging in the life science field.

FIG. 1 is a schematic plan view and a schematic cross-sectional view showing an example of the observation tool of the present invention. FIG. 2 is a schematic plan view and a schematic cross-sectional view showing another example of the observation tool of the present invention. FIG. 3 is a schematic plan view and a schematic cross-sectional view showing another example of the observation tool of the present invention. FIG. 4 is a schematic perspective view showing an example of the observation apparatus of the present invention.

In the observation tool of the present invention,
In addition, including fiber material,
It is preferable that the fiber material is disposed in at least one of the culture solution introduction channel and the culture solution extraction channel.

In the observation tool of the present invention,
It is preferable that the chip fixing means is a recess formed in a region including the through hole in the chip arrangement region, and the inner shape of the recess and the outer shape of the chip are substantially the same.

In the observation tool of the present invention,
Furthermore, it is preferable to include a culture solution introduction tube fixing means for fixing the culture solution introduction tube and a culture solution extraction tube fixing means for fixing the culture solution extraction tube.

In the observation apparatus of the present invention,
Furthermore, a culture medium circulating means for circulating the culture liquid is included,
In the chip, it is preferable that the culture solution is introduced from the culture solution introduction channel into the culture chamber by the culture solution circulation means, and the culture solution is led out from the culture chamber to the culture solution outlet channel.

  Hereinafter, the observation tool and the observation apparatus of the present invention will be described in detail with reference to the drawings. However, the present invention is not limited to the following examples. In addition, in the following FIGS. 1-4, the same code | symbol is attached | subjected to the same part. In the drawings, for convenience of explanation, the structure of each part may be simplified as appropriate, and the dimensional ratio of each part may be schematically shown, unlike the actual case.

  In the following description, “upper” indicates the direction on the side where the tip is fixed with respect to the pedestal in the observation tool of the present invention, and “lower” indicates the side where the tip is fixed with respect to the pedestal. Show the opposite direction. “Depth” indicates a distance from the upper surface side of the chip.

[Observation tools]
The observation tool of the present invention is
An observation tool for observing a culture sample under a microscope,
Including pedestal and tip,
The pedestal has a chip placement area for placing the chip,
The chip includes a culture chamber, a culture solution introduction channel, and a culture solution outlet channel,
Each upper part of the culture chamber, the culture solution introduction channel and the culture solution introduction channel is open,
The culture medium introduction channel and the culture solution outlet channel communicate with the culture chamber,
The culture chamber can be observed from the outside,
A through hole is formed at a position corresponding to the culture chamber of the chip in the chip placement region of the pedestal,
The pedestal includes a chip fixing means for removably fixing the chip to the chip arrangement region.
It is characterized by that.

<Chip>
The shape of the chip is not particularly limited, and may be any shape such as a polygonal shape such as a quadrangle, a pentagon, a hexagon, a circle, an ellipse, or a streamline. The height of the chip is not particularly limited, and can be appropriately set according to, for example, the culture sample to be used, the observation apparatus, and the like.

  The material of the chip is not particularly limited, and examples thereof include glass, plastic, metal, ceramics, and the like. The plastic is not particularly limited, and examples thereof include polystyrene, acrylic, and polycarbonate. The metal is not particularly limited, and examples thereof include aluminum, stainless steel, copper, and brass. The chip may be formed from a single material or from a plurality of materials. In the latter case, for example, a case where two or more different materials are used in the culture chamber, the culture solution introduction flow channel, the culture solution discharge flow channel, and other chip regions can be mentioned. When using the two or more different materials, the culture chamber is preferably a transparent material. The transparent material is not particularly limited, and for example, the above-described examples of glass and plastic can be used.

  The shape of the culture chamber is not particularly limited, and may be any shape such as a polygonal shape such as a quadrangle, a pentagon, and a hexagon, a circle, an ellipse, and a streamline shape. From the viewpoint of properties, a circular shape or a streamline shape is preferable. The depth of the culture chamber is not particularly limited. For example, the depth of the culture chamber and the culture solution introduction channel or the culture solution in the communication portion with the culture solution introduction channel or the culture solution extraction channel It is preferable that the depths of the outlet channels are substantially the same depth.

  The culture chamber may be further treated with a protein that becomes a scaffold for cells or the like. The protein or the like is not particularly limited, and examples thereof include collagen, fibronectin, polyerlysine, and polyethyleneimine. For example, when a culture sample to be described later is directly cultured in the culture chamber, the treatment is preferably performed.

  The culture sample is not particularly limited, and examples thereof include tissues, cultured cells, cells collected from living bodies, primary cultured cells, and the like. When culturing the culture sample in the culture chamber, the culture sample may be directly cultured on the culture chamber, or the culture sample cultured on a culture instrument may be cultured in a state including the culture instrument. It may be placed in a room and cultured.

  The shapes of the culture medium introduction channel and the culture solution outlet channel are not particularly limited. The culture medium introduction flow path and the culture liquid discharge flow path may have the same shape or different shapes. The depths of the culture medium introduction channel and the culture solution outlet channel are not particularly limited, and may be, for example, the same depth as the culture chamber or a different depth. Further, the depth of the culture solution introduction channel and the culture solution outlet channel may be constant or different in the channel. By forming the culture medium introduction channel and the culture medium outlet channel, the chip can stably exchange the culture medium in the culture chamber and can stabilize the culture conditions.

  The arrangement of the culture solution introduction channel and the culture solution extraction channel is not particularly limited as long as the culture solution introduction channel, the culture solution extraction channel, and the culture chamber communicate with each other.

  The culture chamber, the culture solution introduction channel, and the culture solution outlet channel can be formed, for example, by machining the substrate of the material chip by cutting or the like. The culture chamber, the culture solution introduction channel, and the culture solution outlet channel are open at the top.

  In the chip, it is preferable that a fiber material is further disposed in the culture solution introduction channel and the culture solution outlet channel. The arrangement of the fiber material is not particularly limited. For example, the fiber material may be arranged in only one of the culture solution introduction channel and the culture solution extraction channel, or both, but preferably both. The arrangement location of the fiber material in the flow channel is not particularly limited, and may be arranged in a part of the flow channel or may be arranged in the whole, but is preferably arranged in the whole. By arranging the fiber material in the flow path, the introduction / extraction of the culture liquid in the culture liquid introduction flow path and the culture liquid discharge flow path can be further stabilized, and the culture conditions in the culture chamber can be stabilized more stably. Can be

  The material of the fiber material is not particularly limited, and examples thereof include glass wool, absorbent cotton, gauze, etc., preferably glass wool.

<Pedestal>
The shape of the pedestal is not particularly limited, and may be any shape such as a polygonal shape such as a quadrangle, pentagon, or hexagon, a circle, an ellipse, or a streamline. The material of the pedestal is not particularly limited, and examples thereof include glass, plastic, metal, ceramics, and the like. Examples of the plastic include polystyrene, acrylic, and polycarbonate. Examples of the metal include aluminum, stainless steel, copper, and brass.

  The shape of the chip arrangement region is not particularly limited, and can be appropriately designed according to the shape of the chip, for example.

  The shape of the through hole is not particularly limited as long as the culture chamber can be observed from below. The arrangement of the through holes is not particularly limited as long as it is formed at a position corresponding to the culture chamber of the chip in the chip arrangement region. By having such a through hole, the pedestal can be observed not only from the upper side but also from the lower side, and can be observed not only from an upright microscope but also from an inverted microscope.

  The chip fixing means is not particularly limited, and examples thereof include a method using a spring, a fastener, rubber, or the like, or a method of forming and fixing a concave portion or a wall portion. The spring is not particularly limited, and examples thereof include a leaf spring and a coil spring. The fastener is not particularly limited, and examples thereof include a screw and a magnet. When the fastener is used, for example, it can be pressed from above or from the side with a plate or the like and fixed with a screw, a magnet, or the like.

  When the chip fixing means is a recess or a wall, the depth of the recess and the height of the wall are not particularly limited, and may be the same as, higher, or lower than the height of the chip, for example. Also good.

  The arrangement of the chip fixing means is not particularly limited. For example, the chip fixing means may be arranged at the boundary of the chip arrangement area, or the chip fixing means may be arranged inside the chip arrangement area. Alternatively, the chip fixing means may be arranged outside the chip arrangement area.

  The pedestal may further include a culture solution introduction tube fixing unit and a culture solution outlet tube fixing unit. The culture medium introduction pipe fixing means and the culture liquid outlet pipe fixing means are not particularly limited, and examples thereof include a spring, a fitting, and rubber. The spring is not particularly limited, and examples thereof include a leaf spring and a coil spring. When the spring is used, for example, it can be fixed by pressing a culture solution introduction tube and a culture solution discharge tube described later. The fitting is not particularly limited, and includes, for example, a hole having an inner diameter substantially the same as the outer diameter of the culture solution introduction tube and the culture solution discharge tube, and the culture solution introduction tube and the culture solution discharge tube; Those formed so as to be fitted with each other are mentioned. When the rubber is used, for example, a culture solution introduction tube and a culture solution discharge tube described later can be fixed by passing the rubber.

  The arrangement of the culture medium introduction pipe fixing means and the culture liquid outlet pipe fixing means is not particularly limited, and the tip of the inlet of the culture liquid introduction pipe is connected to the culture liquid from the upper opening of the culture liquid introduction channel. The tip of the outlet of the culture medium outlet tube can be inserted into the inside of the culture medium outlet channel from the upper opening of the culture medium outlet channel. As long as it can be placed in

<Observation tools>
In the observation tool of the present invention, when the culture solution in the culture chamber, the culture solution introduction channel, and the culture solution extraction channel is circulated, a culture solution introduction tube and a culture solution extraction tube may be further installed. The culture solution introduction tube and the culture solution discharge tube are optional components, and may or may not be present. The culture solution introduction tube and the culture solution outlet tube are not particularly limited, and known ones such as a needle and a gastric sonde can be used. When installing the culture solution introduction tube and the culture solution discharge tube, for example, the tip of the introduction port of the culture solution introduction tube from the upper opening of the culture solution introduction channel to the inside of the culture solution introduction channel The tip of the outlet of the culture solution outlet tube is inserted from the upper opening of the culture solution outlet channel into the culture solution outlet channel.

  The observation tool of the present invention can be used simply by fixing the tip to the pedestal, and since the tip can be removed from the pedestal, the culture sample to be observed can be easily replaced. .

  Next, an example of the observation tool of the present invention will be described.

  In FIG. 1, an example of a structure of the observation tool of this invention is shown. FIG. 1A is a schematic plan view of the observation tool 100, and FIG. 1B is a schematic cross-sectional view of a cross section viewed in the A-A 'direction. As shown in FIG. 1A, the observation tool 100 includes a chip 11 and a pedestal 21 as main components, and the chip 11 includes a culture chamber 12, a culture medium introduction flow path 13, and a culture discharge flow path 14. The pedestal 21 includes a chip arrangement region 22, a through hole 23, and a chip fixing means 24. The chip 11 is fixed to the pedestal 21 via a chip fixing means 24 formed as a recess having a substantially identical outer shape and inner shape of the chip 11 in a region including the through hole 23 in the chip arrangement region 22. . In FIG. 1, the chip 11 is fixed to the pedestal 21, but the chip 11 can be detached from the pedestal 21. When introducing the culture solution, although not shown, the tip of the introduction port of the culture solution introduction tube is inserted into the culture solution introduction channel 13 from the upper opening of the culture solution introduction channel 13 and arranged. The tip of the outlet of the culture solution outlet tube is inserted from the upper opening of the culture solution outlet channel 14 into the culture solution outlet channel 14. In this case, in FIG. 1, the culture solution is introduced from the culture solution introduction tube into the culture chamber 12 through the culture solution introduction channel 13 and from the culture chamber 12 through the culture solution outlet channel 14. It is led out to the liquid lead-out pipe.

  The observation tool of the present invention may further include a fiber material. The structure of the observation tool containing the said fiber material is shown in FIG. 2, the same parts as those in FIG. 1 are denoted by the same reference numerals. FIG. 2A is a schematic plan view of the observation tool 110, and FIG. 2B is a schematic cross-sectional view of a cross section viewed in the B-B ′ direction. As shown in FIGS. 2 (a) and 2 (b), the observation tool 110 includes a fiber material 15 indicated by hatching in the entire flow path of the culture solution introduction flow path 13 and the culture discharge flow path 14. Except for this point, the observation tool shown in FIG. 2 has the same configuration as the observation tool 100 shown in FIG. In the observation tool of the present invention, by arranging the fiber material 15 in the flow path, the culture solution in the culture chamber 12 can be exchanged more stably when the culture solution is introduced and extracted.

  The observation tool of the present invention may further include a culture solution introduction tube fixing unit and a culture solution outlet tube fixing unit. FIG. 3 shows the configuration of an observation tool including the nutrient solution introduction tube fixing means and the culture solution outlet tube fixing means. 3, the same parts as those in FIG. 1 are denoted by the same reference numerals. FIG. 3A is a schematic plan view of the observation tool 120, and FIG. 3B is a schematic cross-sectional view of a cross section viewed in the C-C 'direction. As shown in FIG. 3, the observation tool 120 includes a pedestal 21 including a culture solution introduction tube fixing unit 25 and a culture solution outlet tube fixing unit 26. Except for this point, the observation instrument shown in FIG. 3 has the same configuration as the observation instrument 100 shown in FIG. As shown in FIG. 3, in the observation tool 120 of this example, the culture solution introduction tube 27 and the culture solution discharge tube 28 are fixed by the culture solution introduction tube fixing means 25 and the culture solution discharge fixing means 26, respectively. Yes.

  The fiber material 15 can also be used in combination with the culture solution introduction tube fixing means 25 and the culture solution outlet tube fixing means 26.

[Observation device]
Next, the observation apparatus of the present invention will be described.

The observation apparatus of the present invention is
An observation device for observing a culture sample under a microscope,
Including microscope and observation tool,
The observation tool is arranged under the microscope,
The observation tool is the observation tool of the present invention. The observation apparatus of the present invention is characterized by including the observation tool of the present invention, and other configurations are not limited at all.

  The culture sample is not particularly limited, and is as described above, for example.

  The microscope is not particularly limited, and examples thereof include an upright microscope and an inverted microscope.

  The observation tool is, for example, as described above.

  The arrangement method of the observation tool on the microscope is not particularly limited. For example, the observation tool may be arranged on the observation table of the microscope, or the observation tool may be arranged on the observation table having the arrangement position of the observation tool. The observation tool may be arranged instead of the observation table.

  It is preferable that the observation apparatus further includes a culture medium circulation unit. The culture medium circulation means is not particularly limited, and examples thereof include a pump and natural dripping. The pump is not particularly limited, and examples thereof include a peristaltic pump, a rotary pump, a diaphragm pump, a water pump, and a vacuum pump.

  The arrangement of the culture fluid circulation means is not particularly limited, and the culture fluid circulation means introduces the culture fluid into the culture chamber from the culture fluid introduction channel of the observation tool, and extracts the culture fluid from the culture chamber. What is necessary is just to arrange | position so that the said culture solution can be guide | induced to a flow path. The observation apparatus of the present invention may further include other members between the culture medium circulating means, the culture medium introduction flow path, and the culture liquid discharge flow path. The other members are not particularly limited, and examples thereof include the culture solution introduction tube, the culture solution discharge tube, the tube, the culture solution filtering unit, the culture solution sterilizing unit, and the culture solution temperature adjusting unit.

  When performing observation using the observation apparatus of the present invention, a cover may be provided at the opening of the culture chamber. The cover is not particularly limited, and examples thereof include a cover glass and a silicon rubber sheet. When the microscope is an inverted microscope, the cover is preferably provided, and when the microscope is an upright microscope, the cover is preferably absent.

  Next, an example of the observation apparatus of the present invention will be described.

  FIG. 4 shows an example of the configuration of the observation apparatus of the present invention using an upright microscope as a microscope. FIG. 4 is a schematic perspective view of the observation apparatus 400. In FIG. 4, the same parts as those in FIGS. As shown in FIG. 4, the observation apparatus 400 includes an observation tool 130, an upright microscope 200, and a pump 300 as main components, and the observation tool 130 is directly arranged on the upright microscope 200. Further, the observation tool 130 includes the fiber material 15, the culture solution introduction tube fixing unit 25, and the culture solution outlet tube fixing unit 26 on the chip 11. As shown in the figure, the pump 300 is connected to the culture solution introduction tube 27 and the culture solution discharge tube 28 via tubes 301 or 302, respectively. The culture solution introduction tube 27 and the culture solution discharge tube 28 are respectively connected to the culture solution. The introduction tube fixing means 25 and the culture solution outlet tube fixing device 26 are fixed so that the introduction port and the discharge port are inserted into the culture solution introduction channel 13 and the culture solution extraction channel 14 of the chip 11, respectively. ing. In FIG. 4, the culture solution introduced from the pump 300 is introduced from the tube 301 and the culture solution introduction pipe 27 into the culture chamber 12 through the culture solution introduction channel 13, and from the culture chamber 12 to the culture solution outlet channel. 14. It is led out to the pump 300 through the culture solution outlet tube 28 and the tube 302. In FIG. 4, the lens of the upright microscope 200 is positioned above the culture chamber 12, but the lens is used while immersed in the culture solution in the culture chamber 12 during observation.

  In this example, the case of using an upright microscope has been described, but the observation apparatus of the present invention is not limited to this, and an inverted microscope may be used. When the inverted microscope is used, the explanation of FIG. 4 can be used except that the opening of the culture chamber is covered with the cover glass and the culture chamber is observed from below.

  As mentioned above, although this invention was demonstrated with reference to drawings, this invention is not limited to the said embodiment. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

  As described above, according to the observation tool of the present invention, it is possible to observe only by fixing the chip on which the observation target is placed on the pedestal, and the chip can be detached from the pedestal. Can be easily exchanged. In addition, according to the observation tool of the present invention, since the introduction / extraction of the culture solution is easy, the culture sample can be easily observed under certain conditions. Furthermore, according to the observation tool of the present invention, since the culture sample can be observed from both the upper and lower directions of the observation tool, it can be used in both an upright microscope and an inverted microscope. Therefore, the present invention is a very useful tool for microscopic observation such as live imaging in the life science field.

DESCRIPTION OF SYMBOLS 11 Chip 12 Culture chamber 13 Culture solution introduction flow path 14 Culture solution extraction flow path 15 Fiber material 21 Base 22 Chip arrangement area 23 Through hole 24 Chip fixing means 25 Culture solution introduction pipe fixing means 26 Culture solution outlet pipe fixing means 27 Introducing tube 28 Culture solution outlet tube 100, 110, 120, 130 Observation tool 200 Erecting microscope 300 Pump 301, 302 Tube 400 Observation device

Claims (6)

An observation tool for observing a culture sample under a microscope,
Including pedestal and tip,
The pedestal has a chip placement area for placing the chip,
The chip includes a culture chamber, a culture solution introduction channel, and a culture solution outlet channel,
Each upper part of the culture chamber, the culture solution introduction channel and the culture solution introduction channel is open,
The culture medium introduction channel and the culture solution outlet channel communicate with the culture chamber,
The culture chamber can be observed from the outside,
A through hole is formed at a position corresponding to the culture chamber of the chip in the chip placement region of the pedestal,
The pedestal includes a chip fixing means for removably fixing the chip to the chip arrangement region.
An observation tool characterized by that. In addition, including fiber material,
The observation tool according to claim 1, wherein the fiber material is disposed in at least one of the culture solution introduction channel and the culture solution extraction channel. 2. The chip fixing means is a recess formed in a region including the through hole in the chip arrangement region, and the inner shape of the recess and the outer shape of the chip are substantially the same. Or the observation tool of 2. The observation tool according to any one of claims 1 to 3, further comprising a culture solution introduction tube fixing unit for fixing the culture solution introduction tube and a culture solution extraction tube fixing unit for fixing the culture solution extraction tube. An observation device for observing a culture sample under a microscope,
Including microscope and observation tool,
The observation tool is arranged under the microscope,
An observation device, wherein the observation tool is the observation tool according to any one of claims 1 to 4. Furthermore, a culture medium circulating means for circulating the culture liquid is included,
In the chip, the culture solution is introduced from the culture solution introduction channel into the culture chamber by the culture solution circulation means, and the culture solution is led out from the subculture chamber to the culture solution outlet channel. The observation apparatus according to claim 5.

Images