JP2006000057A - Culture vessel and biological sample observation system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture vessel and a biological sample observation system using the same, capable of observing weak light and also capable of cutting both labor and cost involved in observing a plurality of biological samples. <P>SOLUTION: The culture vessel 130 for culturing the biological samples CE by accommodating them inside in a closed condition comprises members 140 to be inoculated with the biological samples CE and culture vessel bodies 131 and 150 to which the members 140 are attached in a detachable manner. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a culture container and a biological sample observation system used for culturing a biological sample and simultaneously observing the cells.

  With recent advances in gene analysis technology, gene sequences in many organisms including humans have been clarified, and the causal relationship between diseases and gene products such as analyzed proteins has begun to be gradually elucidated. In the future, in order to comprehensively and statistically analyze various proteins and genes, various examination methods and apparatuses using biological samples, particularly cells, are beginning to be considered.

Usually, the cells are seeded in a plastic or glass dish or flask and cultured in an incubator. The inside of this incubator is, for example, set at a carbon dioxide concentration of 5%, a temperature of 37 ° C., and a humidity of 100%, and is maintained in an environment suitable for cell growth.
Further, in the incubator, the culture solution is changed every 2-3 days in order to feed the cells with nutrients and maintain a pH suitable for the culture.

Several methods are known for observing cells in culture, and one of them is the above-mentioned dish or flask taken out of an incubator and using an inverted microscope such as a phase contrast microscope. A method for performing observation is known.
In the above method, it is necessary to observe the cells as quickly as possible and return the cells to the incubator after the observation. This is to prevent the activity of the cells from being impaired by placing the cells for a long time in a normal environment (an environment different from an environment suitable for culture).
That is, if the cell activity is unstable, it is difficult to perform accurate evaluation. In addition, when removing the cells from the incubator, care is taken to prevent contamination and the like.
As another cell observation method, a method using a transparent constant temperature culture vessel for microscopic observation capable of setting various cell culture conditions is also known (see, for example, Patent Document 1).
JP-A-10-28576

In the above-mentioned Patent Document 1, a pair of transparent heating plates that can be controlled to a predetermined temperature by a temperature controller, a sealed container having a carbon dioxide supply port and a discharge port for adjusting the carbon dioxide concentration, and a sealed container A transparent constant temperature culture vessel for microscopic observation having an evaporating dish for keeping humidity therein is disclosed.
By using this transparent constant temperature culture container for microscopic observation, the temperature, carbon dioxide concentration and humidity inside the container can be controlled, and observation can be performed while culturing cells. In other words, for example, by observing with an objective lens from below the transparent heat generating plate, it was possible to continuously and easily observe and record the time-dependent change in the cell culture state.

However, in the transparent constant temperature culture container for microscopic observation described in Patent Document 1, cells are seeded directly in a sealed container. Therefore, when observing other cells in the same transparent constant temperature culture container, it is necessary to remove the previously observed cells. There is a problem that it takes time and effort to observe cells.
Alternatively, the transparent constant temperature culture container is made disposable, but unlike a slide glass or the like, the expensive transparent constant temperature culture container is made disposable, so there is a problem that the cost of cell observation becomes high.
This is a problem that occurs even if the seeded cells are removed, other cells do not stick to the surface once seeded.

In order to avoid the above-mentioned problem, a method of arranging a slide glass, a microplate or the like in a culture vessel and seeding cells on the slide glass or the like is known.
However, this method has a problem that it is difficult to detect weak fluorescence emitted from cells.
In other words, the distance between the objective lens for observing the cell and the cell is restricted by the thickness of the cell container and the slide glass, and it cannot be observed with a high NA objective lens suitable for detecting faint light. There was a problem.

  The present invention has been made in order to solve the above-described problem, and is capable of observing feeble light, and a culture container capable of reducing labor and cost required for observing a plurality of types of biological samples. An object is to provide a biological sample observation system.

In order to achieve the above object, the present invention provides the following means.
The invention according to claim 1 is a culture container in which a biological sample is housed in a sealed state and cultured, and a seeded member on which the biological sample is seeded, and a culture container main body to which the seeded member is detachably attached And a cell culture container.

According to the present invention, it is possible to observe different types of biological samples by exchanging only the seeded member in which the biological sample is seeded in the culture container. Therefore, the cost required for observation can be reduced as compared with the case where the entire culture container is replaced. Also, compared to removing seeded cells, the labor required for observation can be saved. In addition, since a biological sample can be observed only through the seeded member, for example, observation is performed via a culture container and a slide glass. Compared with the case, weaker light can be observed. That is, since it can be observed closer to the biological sample, it can be observed using a high NA objective lens having a shorter focal length as an observation means, and weaker light can be observed.

Moreover, in the said invention, it is desirable for the said seeding member to be affixed on the said culture container main body using the adhesive member.
According to the present invention, the seeded member can be easily attached to the culture vessel body, and the culture vessel can be easily sealed.
In addition, as an adhesive member, the thing formed from the material which is not toxic with respect to a biological sample is preferable, Furthermore, the thing which can remove | reattach the to-be-seeded member again is preferable.

Furthermore, in the said invention, it is desirable to provide the flow path into which the culture solution which immerses the said biological sample flows in and out in the said culture container main body.
According to the present invention, since the culture solution can be supplied to the biological sample, the biological sample can be cultured in the culture container for a long period of time, and the biological sample can be observed for a long period of time.

  In the above invention, the culture vessel body includes an outer frame member that forms a side wall, a middle frame member that is disposed inside the outer frame member, and a lid member that is fixed to the outer frame member, The outer frame member is provided with a support portion that supports the seeded member, the middle frame member is disposed on a surface facing the seeded member and the lid member, and contacts the seeded member and the lid member. A sealing member is provided, and the seeded member and the lid member are disposed with the middle frame member sandwiched therebetween with the support portion supporting the seeded member, and the lid member is used as the outer frame member. By fixing, it is desirable to form a sealed container with the seeded member, the middle frame member, and the lid member.

According to the present invention, only the seeded member can be replaced by attaching / detaching the lid member to / from the outer frame member. That is, since the seeded member is sandwiched and held by the support portion of the outer frame member and the lid member via the middle frame member, it can be easily replaced by attaching and removing the lid member.
In addition, since the sealing member seals between the seeded member and the middle frame member and between the middle frame member and the lid member, a sealed container is formed by the seeded member, the middle frame member, and the lid member. can do. Therefore, the culture solution for culturing the biological sample can be held in the sealed container, and the biological sample can be cultured for a long period of time and observed over a long period of time.
The lid member is preferably attached to the outer frame member using a member that can be easily attached and detached, such as a screwing member (for example, a screw) or a locking member (for example, a hook).

  In the above invention, the culture vessel body includes an outer frame member forming a side wall, an inner lid member attached to the outer frame member, a lid member attached to the outer frame member via the inner lid member, An inner frame member disposed inside the outer frame member, and the outer frame member is provided with a support portion for supporting the seeded member, and the seeded member and the inner lid are provided on the inner frame member. A seal member disposed on a surface facing the member and in contact with the seeded member and the inner lid member; and with the support member supported by the support portion, the intermediate frame member is sandwiched between By arranging the seeded member and the inner lid member, fixing the inner lid member to the outer frame member, holding the seeded member, and fixing the lid member to the outer frame member, The seeded member, the inner frame member, the inner lid member, and It is desirable to configure the closed container by Kifuta member.

According to the present invention, only the seeded member can be replaced by attaching / detaching the inner lid member to / from the outer frame member. That is, the seeded member is sandwiched and held by the support portion of the outer frame member and the inner lid member via the middle frame member, so that it can be easily replaced by attaching and removing the inner lid member. it can.
In addition, since the sealing member seals the space between the seeded member and the middle frame member and between the middle frame member and the inner lid member, an opening is formed between the seeded member, the middle frame member, and the inner lid member. An open space can be formed. Therefore, a culture solution for culturing a biological sample can be held in the space and ventilation can be performed through the opening, so that the biological sample can be cultured for a long period of time.

Furthermore, by attaching a lid member, a sealed container can be formed by the seeded member, the middle frame member, the inner lid member, and the lid member, thereby preventing contamination of the biological sample during observation or the like. Can do.
The lid member and the inner lid member are preferably attached to the outer frame member by using a member that can be easily attached and detached, such as a screwing member (for example, a screw) or a locking member (for example, a hook).

In the above invention, it is desirable that the middle frame member is provided with a flow path for allowing the culture solution to flow into and out of the sealed container.
According to the present invention, since the culture solution can be supplied to the biological sample in the sealed container, the biological sample can be cultured in the sealed container for a long period of time, and the biological sample can be observed for a long period of time.

In the said invention, it is desirable to form the said flow path so that it may be branched into plurality in the said inner frame member, and may have several opening toward the said airtight container.
According to the present invention, since the culture solution can be uniformly flowed into the sealed container, it is possible to prevent the generation of the region where the culture solution flow is stagnant. By preventing the generation of starch, it is possible to prevent uneven supply of nutrients and the like to the biological sample and to culture the biological sample uniformly.

In the above invention, it is desirable that the flow path forming portion in which the flow path is formed in the middle frame member is formed detachably with respect to the middle frame member.
According to the present invention, since only the flow path forming portion can be replaced, it is possible to reduce labor and cost required for observation of a biological sample.
That is, the components constituting the culture solution tend to remain at the branch of the flow channel in the flow channel forming unit. By replacing the flow path forming part, it is possible to save the trouble of washing the deposits or to make it easy to wash. Further, by replacing only the flow path forming portion, the cost required for replacement can be reduced as compared with the case of replacing the middle frame member.

In the said invention, it is desirable that the sealed container of the said culture container is comprised using the said to-be-seeded member by which the said biological sample was seed | inoculated.
According to the present invention, since the seeding member can be seeded under an environment suitable for seeding the biological sample, the biological sample can be more reliably adhered to the seeding member.

In the said invention, it is desirable to seed | inoculate the said biological sample to the said to-be-seeded member after the structure of the airtight container of the said culture container.
According to the present invention, since it is not necessary to move the seeded member seeded with the biological sample, damage to the biological sample due to the movement can be prevented.

In the said invention, it is desirable to supply the said culture solution containing the said biological sample in the said culture container via the said flow path, and to seed | inoculate the said biological sample to the said to-be-seeded member.
According to the present invention, since the biological sample is supplied into the culture container together with the culture solution, for example, the biological sample can be seeded easily even when the culture container is configured.

In the said invention, after seed | inoculating the said biological sample, it is desirable to supply the said culture solution so that the liquid level may become below the said flow path, and to culture | cultivate the said biological sample.
According to the present invention, the supplied culture solution can be easily cultured without flowing out of the flow path, and can be adhered to the seeded member.

In the above invention, it is desirable that the seeded member includes a slide glass capable of transmitting light necessary for observing the biological sample from the outside.
According to the present invention, a general slide glass on which a biological sample can be seeded can be directly attached as a part of a culture vessel for observation.

  The invention according to claim 13 is a biological sample observation system for acquiring information on a biological sample to be observed, a culture container in which the biological sample is housed in a sealed state and cultured, and the biological sample is observed. An observing means for acquiring information, and a seeding member on which the biological sample is seeded is disposed on a surface of the culture container facing the observation means, and the seeding member is a member of the culture container. And a biological sample observation system that is detachably arranged with respect to the culture vessel.

According to the present invention, it is possible to observe different types of biological samples by exchanging only the seeded member in which the biological sample is seeded in the culture container. Therefore, the cost required for observation can be reduced as compared with the case where the entire culture container is replaced. Further, compared to the case of removing the seeded cells, it is possible to save labor for observation. Also, since the observation means observes the biological sample only through the seeded member, for example, a culture container and a slide glass are used. Compared with the case of observing via, weaker light can be observed. That is, since the distance between the observation means and the biological sample becomes shorter, it is possible to observe using a high NA objective lens having a shorter focal distance as the observation means, and it is possible to observe weaker light.

According to the biological sample observation system of the present invention, since the seeded member on which the biological sample is seeded is detachably arranged with respect to the culture container, the biological sample can be easily exchanged and a plurality of types of biological samples are observed. There is an effect that it is possible to reduce labor and cost required.
In addition, since the seeded member constitutes a part of the sealed container in the culture container, the distance between the observation means and the living cells can be shortened, and an observation means suitable for weak light observation can be used. Play.

[First Embodiment]
Hereinafter, a biological sample observation system according to a first embodiment of the present invention will be described with reference to FIGS.
FIG. 1 is a perspective view showing an outline of a biological sample observation system according to the present embodiment. FIG. 2 is a schematic diagram showing the system configuration of the biological sample observation system.

  The biological sample observation system 10 is schematically configured from a detection unit 20 and a culture unit 70 as shown in FIGS. 1 and 2. The detection unit 20 and the culture unit 70 are desirably arranged close to each other, and more preferably, both units 20 and 70 are disposed in contact with each other.

As shown in FIGS. 1 and 2, the detection unit 20 is schematically configured from a heat retaining box 21 that houses a biological sample and a detection unit 40 that measures a cell (biological sample) CE.
The heat insulation box 21 includes a heater 21H that keeps the inside of the heat insulation box 21 at a predetermined temperature, a stage 22 that holds an incubator box 100 described later, a transmission light source 23 that irradiates the cells CE with light, The heat insulation box 21 includes a fan 24 that makes the temperature uniform, a UV lamp 25 that sterilizes the inside of the heat insulation box 21, a carrier 26 that protects a culture solution circulation pipe 77 and a culture gas supply pipe 97, which will be described later, and an incubator box 100. An open / close door 27 used when taking in and out the main unit and a main power switch 28 for turning on / off the main power of the detection unit 20 are provided.

The stage 22 includes an X-axis operation stage 22 </ b> X and a Y-axis operation stage 22 </ b> Y that move relative to each other in an orthogonal direction, and scanning control is performed by a stage scanning unit 29.
The stage scanning unit 29 includes an X-axis coordinate detection unit 30 that detects an X-axis coordinate value of the X-axis operation stage 22X, an X-axis scan control unit 31 that controls the operation (scanning) of the X-axis operation stage 22X, and a Y-axis The Y-axis coordinate detection unit 32 that detects the Y-axis coordinate value of the operation stage 22Y and the Y-axis scan control unit 33 that controls the operation (scanning) of the Y-axis operation stage 22Y are configured.
The X-axis coordinate detection unit 30 and the Y-axis coordinate detection unit 32 are arranged to output the detected X-coordinate of the X-axis operation stage 22X and Y-coordinate of the Y-axis operation stage 22Y to the computer PC. . The X-axis scanning control unit 31 and the Y-axis scanning control unit 33 are arranged so as to control scanning of the X-axis operation stage 22X and scanning of the Y-axis operation stage 22Y based on instructions from the computer PC.

As a mechanism for driving the X-axis operation stage 22X and the Y-axis operation stage 22Y, for example, a combination of a motor and a ball screw can be used.
As described above, the computer PC controls the scanning of the X-axis operation stage 22X and the Y-axis operation stage 22Y, controls the detection system of the cell CE, and analyzes the image of the captured cell CE, as will be described later. The X-axis operation stage 22X, the Y-axis operation stage 22Y, the detection system, and the analysis system are controlled in conjunction with each other.

Between the transmissive light source 23 and the incubator box 100, a condenser lens 34 that condenses the light emitted from the transmissive light source 23 onto the cells CE is disposed.
Note that the shutter 35 may not be provided between the condenser lens 34 and the incubator box 100, or the shutter 35 may be provided.
The fan 24 is disposed on the wall surface of the heat insulating box 21. By operating this fan 24, the air in the heat insulation box 21 can be convected, and the temperature in the heat insulation box 21 can be easily maintained uniformly.

The UV lamp 25 is connected to a UV lamp switch 36 disposed on the wall surface of the detection unit 20, and a timer 37 that temporally controls the operation of the UV lamp 25 is provided between the UV lamp 25 and the UV lamp switch 36. Has been placed. Further, a sterilization indicator lamp (not shown) for displaying the lighting of the UV lamp 25 is disposed.
For example, when the UV lamp switch 36 is pressed when the cell CE is not measured, the timer 37 starts counting, power is supplied to the UV lamp 25, and UV light (ultraviolet light) is irradiated into the heat insulation box 21. The At the same time, the sterilization indicator lamp is lit. When a predetermined time (for example, 30 minutes) elapses and the timer 37 finishes counting, the timer 37 stops supplying power to the UV lamp 25, and the UV light irradiation ends. The sterilization indicator lamp is also turned off.
Note that the UV lamp 25 is controlled separately from the main power switch 28 and can operate even when the main power is turned off.
Note that the lighting time of the UV lamp 25 may be 30 minutes as described above, or may be less than 30 minutes or longer than 30 minutes as long as the germs in the heat insulating box 21 can be killed. good.

The open / close door 27 is made of an alumite-treated metal such as aluminum or a translucent resin having a high light shielding property. As the structure of the open / close door 27, a structure having a hollow double structure, or a structure in which the inside is the metal and the outside is a resin can be considered. By using resin on the outside of the opening / closing door 27, it is possible to prevent the heat in the heat insulating box 21 from escaping from the opening / closing door 27 to the outside. In addition, by making the inside of the opening / closing door 27 alumite-treated metal, it is possible to prevent the life of the opening / closing door 27 from being impaired by the UV lamp 25.
In addition, when the opening / closing door 27 has a double structure of metal or metal and resin, since the light is completely shielded, it is desirable to provide a viewing window at a position to look into the incubator box 100. It is desirable that a transparent resin or glass is fitted in the viewing window, and a cover that can be opened and closed is disposed on the outside.

  As shown in FIGS. 1 and 2, the detection unit 40 includes a heater 40H that keeps the detection unit 40 at a predetermined temperature, epi-illumination light sources 41A and 41B that irradiate cells CE from the detection unit 40 side, and an epi-illumination light source. An optical path switching unit 42 that switches the optical paths from 41A and 41B, a light amount adjustment mechanism 43 that adjusts the amount of irradiation light, a lens system 44 that collects the irradiation light toward the cell CE, and the wavelength and detection light of the irradiation light A filter unit 45 that controls the wavelength of the light, an autofocus (AF) unit 46 that performs a focusing operation on the cell CE, a revolver 47 that includes a plurality of objective lenses (observation means) 48 having different magnifications and properties, A detector 49 that detects detection light from the cell CE, a light amount monitor 50 that measures the amount of detection light, a fan 51 that makes the temperature in the detection unit 40 uniform, and a cooling that cools the detection unit 40 A fan 52, are provided.

The incident light sources 41 </ b> A and 41 </ b> B are made of, for example, a mercury lamp, are arranged outside the detection unit 40, and are connected to a power source 53 that supplies electric power.
Normally, one incident light source, for example, the incident light source 41A is used, but when the amount of light from the incident light source 41A decreases to a predetermined value or less, illumination light is irradiated from the incident light source 41B, and the power source of the incident light source 41A Is turned off.

The optical path switching unit 42 is formed to guide the illumination light of either the incident light source 41 </ b> A or the incident light source 41 </ b> B to the light amount adjustment mechanism 43. Further, the optical path switching unit 42 is provided with an optical path control unit 54 that is connected to a computer PC described later and controls the optical path switching unit 42 based on an instruction from the computer PC.
A shutter 42S is disposed on the illumination light exit side of the optical path switching unit 42 to perform illumination light transmission / blocking control.

A light amount adjustment mechanism 43 is disposed on the illumination light emission side of the shutter 42S to adjust the amount of illumination light transmitted through the shutter 42S. As the mechanism, for example, a known diaphragm mechanism or the like may be used, or a known mechanism or technique capable of adjusting the other light amount may be used.
The light amount adjustment mechanism 43 is provided with a light amount control unit 55 that is connected to a computer PC described later and controls the light amount adjustment mechanism 43 based on an instruction from the computer PC.
A lens system 44 is disposed on the illumination light exit side of the light amount adjustment mechanism 43. The lens system 44 includes a pair of lenses 44A and 44B and a diaphragm 44C disposed between the lens 44A and the lens 44B.

The filter unit 45 includes an excitation filter 56, a dichroic mirror 57, and an absorption filter 58. The excitation filter 56 is a filter that transmits a wavelength (excitation light) that contributes to fluorescence emission of the cell CE from the illumination light, and is arranged so that the illumination light emitted from the lens system 44 enters the excitation filter 56. ing. The dichroic mirror 57 is an optical element that separates excitation light and fluorescence. The dichroic mirror 57 is arranged to reflect the excitation light transmitted through the excitation filter 56 toward the cell CE and to transmit fluorescence from the cell CE. Yes. The absorption filter 58 is an optical element that separates fluorescence from the cell CE and other unnecessary scattered light, and is arranged so that light transmitted through the dichroic mirror 57 enters.
The filter unit 45 is provided with a filter control unit 46C that controls the wavelengths of excitation light and detection light (fluorescence) emitted from the filter unit 45 based on instructions from the computer PC described later.
The excitation filter 56, the dichroic mirror 57, and the absorption filter 58 may be used one by one or plural.

The AF unit 46 is disposed on the excitation light emission side of the filter unit 45, and is disposed so as to collect the excitation light on the cell CE via the objective lens 48 based on an instruction from the computer PC described later.
The revolver 47 is disposed on the excitation light emission light side of the AF unit 46, and a plurality of objective lenses 48 having different magnifications are disposed. The revolver 47 is provided with an objective lens control unit 59 that selects and controls the objective lens 48 on which the excitation light is incident based on an instruction from the computer PC to be described later.

The objective lens 48 has a structure in which the inside of the incubator box in the heat retaining box 21 can be observed from the detection unit 40 through holes provided in the X-axis operation stage 22X and the Y-axis operation stage 22Y.
In the X-axis operation stage 22X and the Y-axis operation stage 22Y, the size of the hole is expected to be slightly larger in consideration of the range in which the stage operates.
Therefore, even if the atmosphere is kept at a humidity suitable for cell culture in the heat insulation box 21, the atmosphere escapes to the detection unit 40 through the hole, and the temperature suitable for cell culture cannot be maintained, and the cell cannot be maintained. May cause decreased activity.
Therefore, suppression means 99 that suppresses the conduction of the atmosphere having a temperature suitable for cell culture between the heat insulating box 21 and the detection unit 40 may be provided.
The suppression means 99 only needs to be able to suppress the flow of air so as not to disturb the movement of the revolver 47 and the objective lens 48. For example, a sheet made of a flexible material such as a film or a transparent sheet is used as a boundary between the heat insulating box 21 and the detection unit 40. It is possible to use a curtain-like one that is attached around the hole provided in the slab and attached in a suspended state around the revolver.

On the detection light emission side of the filter unit 45, a condenser lens 60 that condenses the detection light on the detector 49 and the light amount monitor 50 is disposed.
A half mirror 61 that reflects part of the detection light toward the detector 49 and transmits the remaining detection light toward the light amount monitor 50 is disposed on the detection light emission side of the condenser lens 60.
The detector 49 is disposed at a position where the detection light reflected from the half mirror 61 is incident. The detector 49 is connected to a detector calculation unit 62 that calculates a detection signal from the detector 49 and outputs the detection signal to a computer PC described later.
The detector 49 may use a line sensor, may use an area sensor, may share the line sensor and the area sensor, and is not particularly limited.
The light quantity monitor 50 is arranged to measure the detection light transmitted through the half mirror 61 and output the measured value to the computer PC.
As described above, the light amount of the detection light may be measured using the light amount monitor 50, or the light amount of the detection light may be measured using an illuminance meter, a power meter, or the like.

The heater 40H controls the temperature of the detection unit 40 so that the temperature is, for example, 30 ° C. to 37 ° C. The fan 51 is arranged so that the air in the detection unit 40 is convected and the temperature in the detection unit 40 is made uniform. Therefore, the temperature in the detection unit 40 is maintained at a temperature close to that of the heat insulation box 21, and the temperature of the heat insulation box 21 is more easily stabilized.
The cooling fan 52 is driven to lower the temperature in the detection unit 40 based on the output of a temperature sensor (not shown) disposed in the detection unit 40. Therefore, for example, an abnormal temperature rise in the detection unit 40 due to heat generated by a motor or the like can be prevented.

FIG. 3 is a perspective view showing the incubator box according to the present embodiment.
As shown in FIG. 3, the incubator box 100 is schematically configured from a housing 101 that houses a chamber (culture vessel) 130 and a cover 102 that forms a sealed container together with the housing 101.
The housing 101 and the cover 102 are subjected to a magnetic shielding process for blocking a magnetic field from the outside and an electrostatic removal process for removing static electricity generated in the incubator box 100.

The housing 101 is formed of a bottom plate 103 and side walls 104, and a region corresponding to the measurement area of the bottom plate 103 is formed of a material having translucency such as glass. The other region of the bottom plate 103 and the side wall 104 are preferably made of a light-shielding material with high anticorrosion properties such as anodized aluminum or stainless steel such as SUS316, and more preferably from the viewpoint of heat retention. A material with low thermal conductivity may be selected.
In addition, an adapter 105 for holding the chamber 130 and a temperature sensor 106 for measuring the temperature of the chamber 130 are arranged on the bottom plate 103. Note that the chamber 130 may be held using the adapter 105 as described above, or the chamber 130 may be held without using the adapter 105.
The output of the temperature sensor 106 is input to the computer PC via the incubator temperature detection unit 106S, and is also input to the temperature display unit 107 disposed on the wall surface of the detection unit 20. The computer PC controls the heater 21H and the like via the incubator temperature control unit 106C shown in FIG. 2 so that the temperature in the incubator box 100 is kept constant.

The cover 102 includes a glass plate 117 that transmits illumination light and a support portion 117A that supports the glass plate 117. On the glass plate 117, antireflection films may be formed on both sides in a region corresponding to the measurement area. By forming the antireflection film on both sides, reflection by the glass plate 117 in transmission observation and epi-illumination observation is prevented.
In addition, the area of the glass plate 117 may be substantially the same as that of the bottom plate 103 of the incubator box 100, or may be a minimum area necessary for performing measurement without any problem.

FIG. 4 is an exploded perspective view showing the culture container according to the present embodiment, and FIG. 5 is a cross-sectional view showing the culture container according to the present embodiment.
As shown in FIGS. 4 and 5, the chamber 130 includes an outer frame portion (outer frame member, culture vessel body) 131 that forms a part of the outer side wall and the bottom surface of the chamber 130, and a slide glass on which cells CE are seeded. (Seeding member) 140, a core (inner frame member) 141 disposed in the outer frame part 131, and a lid (lid member, culture vessel main body) 150 attached to the outer frame part 131. ing.

The outer frame portion 131 includes a frame portion side wall 132 arranged in a rectangular shape and a bottom plate 133 in which a bottom plate opening 134 is formed. The outer frame portion 131 is made of a corrosion-resistant metal (for example, stainless steel).
The frame side wall 132 is formed with a U-shaped groove 135 in which a joint 143 to be described later is disposed, and a screw hole 136 is formed on the upper surface (upward surface in the drawing). . The bottom plate 133 is formed with a bottom plate opening 134 that is slightly smaller than a slide glass 140 described later, and a stepped portion (support portion) 137 that supports the slide glass 140. The step portion 137 is formed to support the slide glass 140 from the lower side in the drawing.
The surface (the upper surface in the drawing) of the slide glass 140 that is inside the chamber 130 is coated with a coating (for example, a PLL (Poly-L-Lysine) coat) that promotes the adhesion of the cells CE.

The core 141 includes a rectangular core frame 142, a joint 143 disposed at a position facing the core frame 142, and O-rings (seal members) 144 disposed on the upper and lower surfaces of the core frame 142. It is configured. The core frame 142 and the joint 143 are made of a resin that is harmless to the cell CE (for example, PEEK (polyether ether ketone) resin).
The joint 143 and the core frame 142 are formed with a passage 145 through which a culture solution for immersing the cells CE flows. Arrangement grooves 146 for arranging O-rings 144 are formed on the upper and lower surfaces of the core frame 142. The O-ring 144 is disposed so that the slide glass 140, the core 141, and the lid 150 form a sealed container 160 by contacting the slide glass 140 and the lid 150, and the culture solution is held in the sealed container 160. Has been.

  The lid 150 is formed of a lid glass 151 that transmits illumination light and a glass support portion 152 that supports the lid glass 151. An antireflection film for light is formed on the surface of the cover glass 151 that contacts the outside air (upper surface in the figure), and a highly hydrophilic film is formed on the surface that contacts the culture medium (lower surface in the figure). Yes. A through hole 153 for a screw 155 that fixes the lid 150 and the outer frame portion 131 is formed in the glass support portion 152.

As shown in FIGS. 1 and 2, the culture unit 70 is generally configured by a sterilization box 71 that stores a culture solution therein and a mixing unit 90 that generates culture gas.
The sterilization box 71 includes a heater 71H that keeps the inside of the sterilization box 71 at a predetermined temperature, a culture solution bottle 72 that stores a culture solution therein, a reserve tank 73 that stores a reserve culture solution, and a used culture. Turn on / off the main power of the waste liquid tank 74 for storing the liquid, the UV lamp 25 for sterilizing the inside of the sterilization box 71, the open / close door 75 used when the culture liquid bottle 72 and the like are taken in and out of the sterilization box 71 A main power switch 76 is provided.

In the culture solution bottle 72, a culture solution circulation pipe 77 that circulates the culture solution to and from the incubator box 100, a supply pipe 78 that supplies a spare culture solution from the reserve tank 73, and a culture solution bottle 72 that has been used. A waste liquid pipe 79 for discharging the culture liquid to the waste liquid tank 74 is disposed.
The culture fluid circulation pipe 77 is provided with a culture fluid pump (culture fluid exchange means) 80 for sending the culture fluid from the culture fluid bottle 72 to the incubator box 100 and circulating the culture fluid. Since the culture medium in the chamber 130 can be exchanged for a new one by the culture medium pump 80, the period during which the cells CE can be cultured can be made longer than that in which the culture medium cannot be exchanged.
A replenishment pump 81 for sending the culture solution from the reserve tank 73 to the culture solution bottle 72 is arranged in the replenishment piping 78, and a waste solution pump for sending the used culture solution from the culture solution bottle 72 to the waste solution tank 74 in the waste solution pipe 79. 82 is arranged.
As described above, the waste liquid tank 74 for storing the used culture medium may be used, or a discharge port for directly discharging the used culture liquid to the outside without using the waste liquid tank 74 may be provided. .

  The culture solution bottle 72 is provided with a culture solution temperature sensor (not shown) for detecting the temperature of the internal culture solution, and the output of the culture solution temperature sensor is input to the computer PC via the culture solution temperature detector 83. Are arranged as follows. The culture medium temperature data input to the computer PC is stored as text data in a memory and can be used for comparison / verification with the detection result of the cell CE.

A culture medium temperature control unit 84 that controls the temperature of the culture medium via the temperature in the sterilization box 71 based on an instruction from the computer PC is disposed in the heater 71H. The temperature of the culture solution supplied from the culture solution bottle 72 is maintained at approximately 37 ° C. by the culture solution temperature control unit 84, thereby preventing a decrease in the activity of the cells CE due to a change in the temperature of the culture solution. In addition, a temperature display unit 85 that displays the culture solution temperature detected by the above-described culture solution temperature sensor is disposed on the wall surface of the culture unit 70.
The culture solution pump 80 is provided with a culture solution pump control unit 86 for controlling the circulation of the culture solution based on an instruction from the computer PC. The replenishment pump 81 and the waste liquid pump 82 are also arranged so that their operations are controlled based on instructions from the computer PC.

The UV lamp 25 is connected to a UV lamp switch 36 disposed on the wall surface of the culture unit 70, and a timer 37 that temporally controls the operation of the UV lamp 25 is provided between the UV lamp 25 and the UV lamp switch 36. Has been placed. Further, a sterilization indicator lamp (not shown) for displaying the lighting of the UV lamp 25 is disposed.
Note that the UV lamp 25 is controlled separately from the main power switch 76, and can operate even when the main power is turned off.

  As shown in FIGS. 1 and 2, the mixing unit 90 includes a heater (not shown) that keeps the inside of the mixing unit 90 at a predetermined temperature, and the carbon dioxide gas concentration in the culture gas supplied to the incubator box 100. A culture gas mixing tank 91 to be adjusted and a CO2 pump 93 for supplying carbon dioxide gas from a CO2 tank 92 disposed outside the culture unit 70 to the culture gas mixing tank 91 are arranged.

In the culture gas mixing tank 91, a CO2 concentration detector 94 for detecting the carbon dioxide gas concentration inside is arranged, and the output of the CO2 concentration detector 94 is arranged to be input to the computer PC. The CO2 pump 93 is provided with a CO2 concentration control unit 95 that controls the amount of carbon dioxide gas supplied to the culture gas mixing tank 91 based on an instruction from the computer PC. A CO2 concentration display unit 96 that displays the carbon dioxide gas concentration in the culture gas mixing tank 91 detected by the CO2 concentration detection unit 94 is disposed on the wall surface of the culture unit 70.
Furthermore, a culture gas supply pipe 97 is disposed between the culture gas mixing tank 91 and the culture solution bottle 72. Therefore, the culture gas is supplied to the culture solution via the culture gas supply pipe 97, and the culture gas can be sufficiently dissolved in the culture solution. Thus, by producing a culture solution in which a culture gas having a carbon dioxide gas concentration of 5% is dissolved in the culture solution bottle 72, a culture solution containing the culture gas and nutrients necessary for the growth of the cells CE is contained. Is supplied to a chamber 130 described later. In addition, the pH of the culture solution can be adjusted by dissolving the culture gas in the culture solution.
The carbon dioxide gas concentration input to the computer PC from the CO2 concentration detector 94 is stored as data in the memory and can be processed in the computer PC.

Next, seeding and culturing of cells CE in the chamber 130 having the above-described configuration will be described with reference to FIGS. 4 and 5.
First, seeding and adhesion of the cells CE on the slide glass 140 are performed.
Specifically, first, the slide glass 140 is put in a dish or the like, and the cells CE are seeded on the slide glass 140. Then, the cells are cultured in an incubator (which may be different from the incubator 100 described above) until the cells CE adhere to the slide glass 140.
When the cell CE is adhered to the slide glass 140, the slide glass 140 is pulled up from the dish, and a PLL coating or the like is applied to sufficiently remove the culture solution attached to the surface other than the surface to which the cell CE is adhered.

Next, the chamber 130 is formed using the slide glass 140 to which the cells CE are adhered.
First, the slide glass 140 is disposed on the stepped portion 137 so that the surface to which the cells CE are adhered is inside the chamber 130, and the core 141 is disposed on the outer frame portion so that the joint 143 is disposed in the groove portion 135. It arranges in 131. Then, the lid 150 is fixed to the outer frame portion 131 with screws 155, and the chamber 130 is formed.
Thereafter, the culture solution circulation pipe 77 or the like is connected to the joint 143, the culture solution is supplied to the chamber 130 through the flow path 145, the cells CE are cultured, and the cells CE are observed at a predetermined timing.

Next, an observation method in the biological sample observation system 10 having the above configuration will be described.
First, scanning method and selection of a detection range in the present embodiment will be described with reference to FIGS. 6 (a), (b), (c), and (d).
FIGS. 6A, 6 </ b> B, 6 </ b> C, and 6 </ b> D are diagrams illustrating examples of scanning method and detection range selection in the present embodiment.

In the example shown in FIG. 6A, by specifying the upper left point a and the lower right point b of the measurement target range M on the displayed image, the measurement target range M (enclosed by a broken line in the figure). Range). Specifically, the measurement target range M may be set by dragging between the points a and b using a device such as a mouse, or the coordinate values of the points a and b are input and instructed. Also good.
The measurement portion by the detector 49 is scanned in the left-right direction within the set measurement target range M as indicated by arrows in the figure. That is, when scanning from the left to the right in the figure, the scanning is performed in parallel with the X-axis direction, and when scanning from the right to the left, the scanning is performed obliquely downward to the left. Among these, imaging of the cell CE is performed when scanning from left to right.

FIG. 6B shows two examples of the measurement target range M set by the method described above. First, two measurement target ranges MA and MB are set by the method described above. The measurement target range MA and the measurement target range MB are set so that they are arranged at a predetermined interval in the X-axis direction in the drawing and all overlap in the Y-axis direction.
The measurement part by the detector 49 in this example is scanned so as to measure the measurement target ranges MA and MB in parallel, as indicated by arrows in the figure. That is, when scanning from left to right in the figure, the measurement target range MA is scanned from the measurement target range MB, and when scanning from right to left, the measurement target range MB is scanned from the measurement target range MA. Is done.

FIG. 6C is an example in which the measurement target ranges set by the above-described method are two examples, and the arrangement of the two measurement target ranges MA and MB is different. Here, the measurement target range MA and the measurement target range MB are set so as to be arranged at a predetermined interval in the X-axis direction in the figure and partially overlapped in the Y-axis direction in the figure.
In this example, as shown by the arrows in the drawing, only the portions overlapping in the Y-axis direction of the measurement target ranges MA and MB are continuously scanned. That is, first, a portion where the measurement target range MA does not overlap is scanned. Next, scanning of overlapping portions of the measurement target ranges MA and MB is continuously performed. Then, the non-overlapping portion of the measurement target range MB is scanned.

FIG. 6D shows two examples of the measurement target range M set by the above-described method, and the arrangement of the two measurement target ranges MA and MB is the same as that in FIG. Is a different example.
In the measurement part by the detector 49 in this example, the measurement object ranges MA and MB are separately scanned as indicated by arrows in the drawing. That is, first, the entire range of the measurement target range MA is scanned, and then the entire range of the measurement target range MB is scanned.

  Among the scanning methods shown in FIGS. 6A, 6B, 6C, and 6D described above, a scanning method in which the total movement distance or scanning time is the shortest is the set parameters and It is automatically selected by the computer PC based on the measurement mode.

In addition, when imaging the range in which cells are cultured, if the configuration allows the imaging to be divided into a plurality of areas (detection ranges) such as setting the measurement target range M in this way, imaging of only the necessary part is performed as necessary. Can be performed.
For example, if the setting of the computer PC is changed so that scanning of the entire range to be scanned and scanning of a predetermined part of the region are alternately performed, it is peculiar to biological samples that occur only for a short period of time. Can capture the phenomenon. As an example, when scanning of the entire range to be scanned is performed every 30 minutes, if scanning of a predetermined measurement target range M in which there are cells to be noticed is performed in between, only about 15 minutes are performed. It is also possible to capture that a unique phenomenon that does not occur has occurred in a cell of interest.
Further, since only the necessary measurement target range M is scanned when necessary, the scanning time can be shortened and the light irradiation time for other cells can be shortened.

Next, the procedure for measuring the cell CE will be described using each flowchart.
First, measurement parameters are set before the measurement of the cell CE. Accordingly, the flow of setting the measurement parameters will be described with reference to FIG.
FIG. 7 is a flowchart for explaining the flow of setting measurement parameters.
First, measurement parameters are set (STEP 1).
Thereafter, default condition setting is performed (STEP 2). The conditions set here are, for example, culture conditions and measurement conditions such as a CO2 concentration of 5% and a temperature of 37 ° C., and these set conditions can be changed to predetermined conditions by the user.

Next, a measurement target is selected (STEP 3). The measurement target is a cell CE container such as the microplate 120 or the slide glass 116.
Next, the measurement mode is selected (STEP 4). Measurement modes include an area imaging mode, a line imaging mode, and an automatic mode. The automatic mode is a mode for automatically selecting a measurement mode with a short measurement time from other measurement modes.

Next, the measurement magnification is selected (STEP 5), and then the detection wavelength is selected (STEP 6). The measurement magnification and the detection wavelength can be selected from two or more options.
Here, as a method for selecting the detection wavelength, a list of fluorescent proteins to be used, for example, GFP, HC-Red, and the like is stored in advance in the computer PC, and is selected from the stored list. The computer PC automatically selects an excitation filter 56 and an absorption filter 58 that are optimal for observation based on the selected fluorescent protein. In this way, predetermined fluorescence can be detected from the cell CE.
Note that changes in the excitation filter 56, the absorption filter 58, the objective lens 48, and the like during measurement are automatically performed in synchronization with the driving of the X-axis operation stage 22X and the Y-axis operation stage 22Y.

Next, a measurement interval is set (STEP 7).
Then, a preview screen is captured (STEP 8), and a preview image is displayed on the monitor (STEP 9). Here, when the user issues an instruction using a preview button or the like for instructing display of the preview image on the monitor, the preview image is displayed on the monitor. Then, the user can confirm the preview image displayed on the monitor.

Next, the measurement range is selected (STEP 10). After selecting the measurement range, the preview image may be displayed on the monitor again to check whether the measurement range is a predetermined range.
Next, a predetermined measurement interval is selected from the set plurality of measurement intervals (STEP 11).
When a measurement start switch (not shown) is pressed (STEP 12), the measurement of the cell CE is started (STEP 13). If the measurement start switch is not pressed, the process waits until the measurement start switch is pressed (STEP 12).

  In STEP 12, when the measurement start switch is not pressed, the setting may be set so as to return to various STEPs so that various settings can be reset.

When the setting of the measurement parameters is completed, the cell CE is measured next. Accordingly, the flow of measurement of the cell CE will be described with reference to FIG.
FIG. 8 is a flowchart for explaining the flow of measurement.
First, when measurement is started, the measurement target range is read (STEP 21). Thereafter, the magnification is read (STEP 22), and the detection wavelength is read (STEP 23).
Next, the measurement mode is read (STEP 24). Here, the optimum stage scanning method is determined based on the read measurement object range, magnification, detection wavelength (fluorescence wavelength), and the like. When the measurement mode is set to the automatic mode, the imaging mode is also determined here.

Next, the operation method of the X-axis operation stage 22X, the Y-axis operation stage 22Y and the like according to the determined stage scanning method is analyzed (STEP 25), and the analyzed operation method data (operation data) is stored in the computer PC. It is stored in a table (STEP 26).
Thereafter, measurement is performed by a different measurement method depending on whether or not the area sensor mode is selected (STEP 27).

First, the case where the area sensor mode is selected will be described.
When the measurement start switch is pressed, the X-axis operation stage 22X and the Y-axis operation stage 22Y are moved to the measurement start position (STEP 30). Here, the measurement start position inputted by the computer PC is read, the X-axis operation stage 22X and the Y-axis operation stage 22Y are moved to the measurement start position, and the cell CE is positioned within the imaging field of the objective lens 48. Moved.
Then, the shutter 35 is set to OPEN (STEP 31), and the objective lens 48 is selected (STEP 32). Here, based on the measurement magnification set by the computer PC, the revolver 47 is driven to select the objective lens 48 having a predetermined magnification.

Next, the filter unit 45 is selected (STEP 33). Here, based on the fluorescent protein set by the computer PC, the filter control unit 46C selects an excitation filter 56, an absorption filter 58, and the like that are optimal for measurement.
The operations up to this point (from STEP 30 to STEP 33) after the measurement start switch is pressed are automatically selected and executed in accordance with the measurement mode.
Thereafter, the focus position is detected (STEP 34), the image is captured and the image data is output to the image memory unit of the computer PC (STEP 35).

If acquisition of a necessary image has not been completed, operations from selection of the objective lens 48 (STEP 32) to image capture and image data output to the image memory unit of the computer PC (STEP 35) are performed again. The process is repeated until the acquisition is completed (STEP 36).
When the necessary image acquisition is completed, the X-axis operation stage 22X or the Y-axis operation stage 22Y is driven by one step (STEP 37). If the position to which the X-axis operation stage 22X or Y-axis operation stage 22Y has moved is within the measurement target range, the operation from the selection of the objective lens 48 (STEP 32) to the one-step stage drive (STEP 37) is repeated again. This repetitive operation is repeated until the position where the stage 22 has moved is outside the measurement target range (STEP 38).

When the movement destination of the X-axis operation stage 22X or the Y-axis operation stage 22Y is outside the measurement target range, the shutter 35 is turned off (STEP 39).
Thereafter, when a predetermined measurement time interval is reached, the operation from the time when the shutter 35 is again set to OPEN (STEP 31) to the time when the shutter 35 is closed (STEP 39) is repeated until the measurement time ends (STEP 40).

Next, a case where the area sensor mode is not selected will be described.
When the measurement start switch is pressed, the X-axis operation stage 22X and the Y-axis operation stage 22Y are moved to the measurement start position (STEP 50). Here, the measurement start position inputted by the computer PC is read, the X-axis operation stage 22X and the Y-axis operation stage 22Y are moved to the measurement start position, and the cell CE is positioned within the imaging field of the objective lens 48. Moved.
Then, the shutter 35 is set to OPEN (STEP 51), and the focus position is detected (STEP 52).

Next, the objective lens 48 is selected (STEP 53). Here, based on the measurement magnification set by the computer PC, the revolver 47 is driven to select the objective lens 48 having a predetermined magnification.
Then, the filter unit 45 is selected (STEP 54). Here, based on the fluorescent protein set by the computer PC, the filter control unit 46C selects an excitation filter 56, an absorption filter 58, and the like that are optimal for measurement. The operations up to this point (from STEP 50 to STEP 54) after the measurement start switch is pressed are automatically selected and executed in accordance with the measurement mode.

Thereafter, the driving of the X-axis operation stage 22X and the Y-axis operation stage 22Y is started (STEP 55), and image capture and image data output to the memory unit of the computer PC are performed (STEP 56).
If necessary image acquisition is not completed, the operations from selection of the objective lens 48 (STEP 53) to image capture and image data output to the memory unit of the computer PC (STEP 56) are repeated. The process is repeated until the image acquisition is completed (STEP 57).

When the acquisition of a necessary image is completed, the shutter 35 is closed (STEP 58).
Thereafter, when a predetermined measurement time interval is reached, the operation from when the shutter 35 is again set to OPEN (STEP 51) until the shutter 35 is closed (STEP 58) is repeated until the measurement time ends (STEP 59).

When the imaging of the cell CE is completed, the captured image is processed next. Therefore, a processing method of the captured image will be described with reference to FIG.
FIG. 9 is a flowchart illustrating an image processing method.
First, the image processing unit of the computer PC extracts a background image from the captured image stored in the memory unit (STEP 71) and removes the background image (background) from the captured image (STEP 72).
Next, the maximum luminance range of the image that can be enhanced is read (STEP 73), and the image is enhanced by multiplying a predetermined coefficient, for example, according to the maximum luminance range (STEP 74). By these processes, the image is enhanced so that each cell CE can be easily recognized in granular form from the image from which the background is removed.

Then, by extracting, for example, a portion having a luminance equal to or higher than a predetermined threshold from the emphasized image, the luminance of each cell CE is recognized as a clear granularity (STEP 75).
Next, geometric features such as the center of gravity and area of the cell CE, chemical features, and optical features such as fluorescence luminance are more accurately recognized and extracted in association with the location information of the cell CE. (STEP 76). By extracting these feature amounts, each cell CE can be identified.
After extracting the feature quantity of the cell CE, the enhancement work (STEP 74) performed for recognizing the cell CE is corrected (STEP 77). By this correction, the influence of the predetermined coefficient used for image enhancement is removed.
Next, the corrected feature amount is output to, for example, a file and stored in the file (STEP 78).

  Therefore, the image processing unit of the computer PC can image the fluorescence amount distribution of the cells CE at each position on the entire surface such as a slide glass or a microplate. In addition, since the image processing unit can accurately track each cell CE, for example, paying attention to only a predetermined number of cells CE, the fluorescence distribution inside the cells CE is locally long while culturing. It is also possible to measure time. Furthermore, while culturing the cell CE, for example, the entire surface of a slide glass, a microplate, or the like is measured at regular intervals, and the fluorescence amount of the cell CE with respect to time can be automatically measured.

Next, data processing performed after data such as the feature amount of the cell CE is extracted from the captured image will be described with reference to FIG.
FIG. 10 is a flowchart for explaining the flow of data processing.
Here, the data (feature value) of the cell CE accumulated in the file is processed by the data processing unit of the computer PC.
First, the data processing unit reads the raw data (features) of the cells CE accumulated in the file (STEP 81) and rearranges the data so as to be arranged in time series for each cell CE (STEP 82). When the data is rearranged, the data processing unit graphs the luminance, that is, the change over time in the expression level for each cell CE (STEP 83).
When the graphing is completed, the data processing unit displays a preview of the graph (STEP 84), and outputs the graphed data to a file (STEP 85).

  By performing this treatment, it is possible to easily observe the temporal change of one cell when the cell CE is cultured for a long time. Accordingly, it is possible to accurately and easily measure a change in the expression level of the cell CE over time during the culture.

Next, adjustment of the irradiation light amount performed at the time of measuring the cell CE will be described with reference to FIG.
FIG. 11 is a flowchart for explaining the flow of light amount adjustment.
First, the irradiation light quantity irradiated to the cell CE is measured (STEP 91). The irradiation light amount may be calculated from the output of the light amount monitor 50, may be measured by providing an illuminometer, or may be calculated from the output of the power meter by providing a power meter.

If the measured irradiation light quantity is within the allowable range, the process returns to the measurement of the irradiation light quantity (STEP 91) again and is repeated until the irradiation light quantity is outside the allowable range (STEP 92).
When the amount of irradiation light falls outside the allowable range, the ND filter (not shown) included in the light amount adjustment mechanism 43 is replaced (STEP 93), and the amount of irradiation light is adjusted to be within the allowable range. Thereafter, the process returns to the measurement of the irradiation light amount (STEP 91), and the adjustment of the irradiation light amount is repeated.

Next, a method for controlling the supply / exchange of the culture solution to the chamber 130 will be described with reference to FIG.
FIG. 12 is a flowchart for explaining a culture medium replenishment / exchange method.
First, the background value of the captured image is analyzed (STEP 101). Autofluorescence of the culture solution is imaged in the background of the captured image, and the brightness of the autofluorescence of the culture solution is analyzed.
Here, since the brightness of the autofluorescence increases as the culture solution becomes older, the replacement time of the culture solution can be detected by measuring the brightness of the autofluorescence.

If the temporal variation of the analyzed background value is within a predetermined specified value, the process returns to the background value analysis (STEP 101) again until the temporal variation of the background value becomes larger than the predetermined predetermined value. Repeated (STEP 102).
When the fluctuation of the background value with time becomes larger than a predetermined specified value, the culture liquid waste pump 82 is driven (STEP 103), and then the culture liquid supply pump 81 is driven (STEP 104).

As described above, the timing of replenishment / exchange of the culture solution may be determined by autofluorescence of the culture solution, may be performed continuously, or automatically at time intervals designated in advance by the user. Replenishment / exchange may be performed, or by selecting a cell CE from a pre-registered table, it may be possible to appropriately designate the replacement time of the culture solution. Further, the amount to be replaced may be set by the user, may be determined by the autofluorescence of the culture solution, or all of the culture solution in the chamber 130 may be replaced at once, or may be registered in advance. By selecting the cell CE from the existing table, the exchange amount of the culture solution may be appropriately designated.
It may be set automatically by converting the weight.
In the present embodiment, the value of the background autofluorescence is detected using the captured image, but this detection may be detected from an image captured where a cell CE does not exist. For example, an optical detector may be provided in the vicinity of the culture solution bottle 72 for detection.
By the measurement procedure as described above, a cell tracking image representing a change in position of each cell over time as shown in FIG. 13 can be acquired.

According to the above configuration, different types of cells CE can be observed by exchanging only the slide glass 140 in which the cells CE are seeded in the chamber 130. Therefore, the cost required for observation can be reduced as compared with the case where the entire chamber 130 is replaced. Moreover, compared with the case where the seeded cells CE are removed, the labor required for observation can be saved.
Since the slide glass 140 is sandwiched and held between the step portion 137 and the lid 150 via the core 141, the slide glass 140 can be easily replaced by attaching and removing the lid 150.

Since the space between the slide glass 140 and the core 141 and the space between the core 141 and the lid 150 are sealed by the O-ring 144, the sealed glass 160, the core 141, and the lid 150 form a sealed container 160. can do. Therefore, the culture solution for culturing the cell CE can be held in the sealed container 160, and the cell CE can be cultured for a long period of time and observed over a long period of time.
Furthermore, since the culture solution can be supplied to the cell CE via the joint 143, the cell CE can be cultured in the chamber 130 for a long period of time, and the cell CE can be observed for a long period of time.

  In addition, since the objective lens 48 observes the cell CE only through the slide glass 140, for example, weaker light can be observed as compared with the case of observation through the chamber container and the slide glass. That is, since the distance between the objective lens 48 and the cell CE becomes shorter, it can be observed using a high NA objective lens having a shorter focal length, and weaker light can be observed.

  In addition, since the chamber 130 is assembled after the cell CE is seeded on the slide glass 140, the cell CE can be seeded on the slide glass 140 in an environment suitable for the seeding of the cell CE. 140 can be more reliably adhered to the surface.

[Second Embodiment]
Next, a second embodiment of the present invention will be described with reference to FIG.
The basic configuration of the biological sample observation system of the present embodiment is the same as that of the first embodiment, but the configuration of the chamber is different from that of the first embodiment. Therefore, in this embodiment, only the periphery of the chamber will be described with reference to FIG. 14, and description of the culture unit and the like will be omitted.
FIG. 14 is a cross-sectional view of the chamber according to the present embodiment.
In addition, about the same component, the same code | symbol is attached | subjected and the description is abbreviate | omitted.

The chamber (culture vessel) 230 includes an outer frame portion (outer frame member, culture vessel main body) 231 that forms part of the outer side wall and bottom surface of the chamber 230, a slide glass 140 on which cells CE are seeded, and an outer frame portion. The lid 150 is attached to the H.231 and an O-ring 244 disposed on the upper surface of the outer frame portion 231.
The outer frame part 231 includes a frame part side wall 232 arranged in a rectangular shape and a bottom plate 233 in which a bottom plate opening 134 is formed. The outer frame portion 231 is formed of a resin that is not toxic to the cell CE (for example, PEEK resin).
A joint 243 is formed on the frame side wall 232, and a screw hole (not shown) is formed on the upper surface thereof. An arrangement groove 246 for arranging the O-ring 244 is formed on the upper surface of the frame side wall 232 on the inner side of the screw hole. A passage 245 through which the culture solution flows is formed through the joint 243 and the frame side wall 232. A bottom plate opening 134 is formed in the bottom plate 233, and a step portion 137 that supports the slide glass 140 is formed.

Next, seeding and culturing of cells CE in the chamber 230 having the above configuration will be described with reference to FIG.
First, the slide glass 140 not seeded with the cells CE is attached to the outer frame portion 231. The slide glass 140 is disposed on the stepped portion 137 so that the surface on which the PLL coat or the like is applied faces the inside of the chamber 230. The slide glass 140 and the stepped portion 137 are adhered to each other by an adhesive that is not toxic to the cells CE, such as an adhesive member (adhesive material) G that can be easily peeled, such as double-sided tape or silicon.

Next, the slide glass 140 and the outer frame portion 231 are sterilized. Then, the joint 243 is closed with a cap (not shown), the cells CE are seeded on the slide glass 140, and a predetermined amount of culture solution is supplied.
In this state, the lid 150 is placed on the outer frame portion 231 and cultured in an incubator (which may be different from the incubator 100 described above) until the cells CE adhere to the slide glass 140.

  When the cell CE adheres to the slide glass 231, the cap of the joint 243 is removed, and the culture solution circulation pipe 77 and the like are connected. Then, using the screw 155, the lid 150 is fixed to the outer frame portion 231 to form the sealed container 160. When the sealed container 160 is formed, a culture solution is supplied to expel air from the sealed container 160, fill with the culture solution, and observe the cells CE at a predetermined timing.

According to said structure, by using the adhesive member (adhesive material) G, the slide glass 140 can be easily affixed on the outer frame part 231, and the airtight container 160 of the chamber 230 can be comprised easily. .
In addition, since the cell CE is seeded after the slide glass 140 is attached to the outer frame portion 231, there is no need to move the slide glass 140 seeded with the cell CE, and damage to the cell CE due to the movement is prevented. be able to.

[Third Embodiment]
Next, a third embodiment of the present invention will be described with reference to FIGS. 15 and 16.
The basic configuration of the biological sample observation system of the present embodiment is the same as that of the first embodiment, but the configuration of the chamber is different from that of the first embodiment. Therefore, in the present embodiment, only the periphery of the chamber will be described using FIGS. 15 and 16, and the description of the culture unit and the like will be omitted.
FIG. 15 is an exploded perspective view of the chamber according to the present embodiment.
In addition, about the same component, the same code | symbol is attached | subjected and the description is abbreviate | omitted.

  As shown in FIG. 15, the chamber (culture container) 330 includes an outer frame portion 131 that forms a part of the outer side wall and the bottom surface of the chamber 330, a slide glass 140 on which cells CE are seeded, and an inner frame portion 131. It is roughly constituted by a core (inner frame member) 341 disposed on the outer frame 131 and a lid 150 attached to the outer frame 131.

FIG. 16A is a perspective view of the core, and FIG. 16B is a cross-sectional view of the core.
As shown in FIGS. 15 and 16A and 16B, the core 341 includes a rectangular core frame 142, a joint 143 disposed at a position facing the core frame 142, and a core frame. 142 and O-rings 144 arranged on the upper and lower surfaces of 142. The core frame 142 and the joint 143 are formed from a resin (for example, PEEK resin) that is harmless to the cell CE.
Of the pair of joints 143, the joint 143 through which the culture solution flows into the chamber 330 and the core frame 142 in which the joint 143 is disposed have a flow path 345 through which the culture solution flows, as shown in FIG. It is formed through. The flow path 345 is formed to branch into a plurality from the outside toward the inside in the core frame 142.
One flow path 145 is formed in the other joint 143 and the core frame 142 as in the first embodiment.

  According to said structure, since the culture solution can be uniformly flowed in in the airtight container 160 by the flow path 345 branched into plurality, generation | occurrence | production of the area | region where a culture solution flow stagnates can be prevented. By preventing the generation of starch, non-uniform supply of nutrients to the cell CE can be prevented, and the cell CE can be uniformly cultured.

[Fourth Embodiment]
Next, a fourth embodiment of the present invention will be described with reference to FIGS.
The basic configuration of the biological sample observation system of the present embodiment is the same as that of the third embodiment, but the configuration of the chamber is different from that of the first embodiment. Therefore, in the present embodiment, only the periphery of the chamber will be described with reference to FIGS. 17 and 18, and description of the culture unit and the like will be omitted.
FIG. 17 is an exploded perspective view of the chamber according to the present embodiment.
In addition, about the same component, the same code | symbol is attached | subjected and the description is abbreviate | omitted.

  As shown in FIG. 17, the chamber (culture container) 430 includes an outer frame portion 131 that forms part of the outer side wall and the bottom surface of the chamber 430, a slide glass 140 on which cells CE are seeded, and an inner frame portion 131. It is schematically configured from a core (inner frame member) 441 disposed on the outer frame 131 and a lid 150 attached to the outer frame 131.

18A is an exploded perspective view of the core, and FIG. 18B is a cross-sectional view of the separation core.
As shown in FIGS. 17 and 18A and 18B, the core 441 includes a rectangular core frame 442, a joint 143 disposed at a position facing the core frame 442, and the core frame. And an O-ring 144 arranged on the upper and lower surfaces of 442.
As shown in FIG. 18A, an insertion port 450 is formed in the core frame 442, and a separation core (flow path forming portion) 451 configured to be detachable from the insertion port 450 is disposed. Has been.
The insertion port 450 is formed with a locking portion (not shown) that meshes with a hook 453 of a separation core 451 described later.

In the separation core 451, one joint 143 of the pair of joints 143 is disposed, and an O-ring 452 that maintains watertightness between the separation core 451 and the core frame 442 is disposed. In addition, the separation core 451 is provided with a pair of hooks 453 for detachably locking the separation core 451 to the core frame 442.
Further, as shown in FIG. 18B, the separation core 451 is formed with a through hole 454 substantially orthogonal to the joint 143, and the flow path 445 is being separated using the through hole 451 as a branch channel. A plurality of branches are formed from the outer side to the inner side of the child 451. At both ends of the through-hole 454, plugs 455 for preventing the culture medium from flowing out are detachably disposed.

According to the above configuration, since only the separation core 451 can be replaced, it is possible to reduce labor and cost required for observation of the cell CE. That is, by replacing the flow path 445 in which the deposit such as the culture solution is easily formed with the separation core 451, it is possible to save the trouble of washing the deposit. Further, by replacing only the separation core 451, the cost required for replacement can be reduced as compared with the case where the entire core 441 is replaced.
By forming the branch portion of the flow path 445 with the through hole 451 and the plug 455, the branch portion can be easily cleaned. That is, by removing the plug 455, the cleaning property of the through hole 451 can be improved.

[Fifth Embodiment]
Next, a fifth embodiment of the present invention will be described with reference to FIG. 19 and FIG.
The basic configuration of the biological sample observation system of the present embodiment is the same as that of the first embodiment, but the configuration of the chamber is different from that of the first embodiment. Therefore, in this embodiment, only the periphery of the chamber will be described with reference to FIGS. 19 and 20, and description of the culture unit and the like will be omitted.
FIG. 19 is an exploded perspective view of the chamber according to the present embodiment, and FIG. 20 is a cross-sectional view of the chamber according to the present embodiment.

As shown in FIGS. 19 and 20, the chamber (culture container) 530 includes an outer frame 131 that forms a part of the outer side wall and the bottom of the chamber 530, a slide glass 140 on which cells CE are seeded, and an outer frame. It is schematically configured by a core 141 disposed in the portion 131, an inner lid (inner lid member) 531 attached to the outer frame portion 131, and a lid 150 attached to the outer frame portion 131.
The inner lid 531 is made of a plate-like member having an opening 532 formed substantially at the center, and at least a pair of hooks 533 for detachably locking the inner lid 531 to the outer frame 131 are disposed. The inner lid 531 is formed with a through hole 534 through which a screw 155 for fixing the lid 150 to the outer frame portion 131 is inserted.
The opening 532 is formed to be slightly smaller than the O-ring 144, and the inner lid 531 is formed so as to contact the O-ring 144.

Next, seeding and culturing of the cells CE in the chamber 530 having the above configuration will be described with reference to FIGS. 19 and 20.
First, the slide glass 140 is placed on the stepped portion 137 so that the PLL-coated surface is inside the chamber 530, and a cap (not shown) is placed on the joint 143 to prevent the culture medium from flowing out. . Then, the core 141 is disposed in the outer frame portion 131 so that the joint 143 is disposed in the groove portion 135, and the inner lid 531 is fixed to the outer frame portion 131 by the hook 533.

Next, the cell CE is seeded on the slide glass 140, and an appropriate amount of the culture solution is supplied into the chamber 530. Thereafter, the lid 150 is placed on the inner lid 531 and cultured in an incubator box until the cells CE adhere to the slide glass 140.
When the cell CE adheres to the slide glass 140, first, the cap of the joint 143 is removed, and the culture solution circulation pipe 77 and the like are connected. Next, the lid 150 is fixed to the outer frame portion 131, the inside of the chamber 530 is sealed, a culture solution is supplied, the air in the chamber 530 is expelled, and the cell CE is observed at a predetermined timing.

According to the above configuration, the slide glass 140 is sandwiched and held between the stepped portion 137 and the inner lid 531 via the core 141, so that the inner lid 531 is attached to and detached from the outer frame portion 131. Only the slide glass 140 can be replaced.
Further, since the space between the slide glass 140 and the core 141 and between the core 141 and the inner lid 531 are sealed by the O-ring 144, the slide glass 140, the core 141, and the inner lid 531 A space opened at the opening 532 can be formed. Therefore, the culture medium for culturing the cell CE can be held in the space, and ventilation can be performed through the opening 532, so that the cell CE can be cultured for a long time.
Further, by attaching the lid 150, the sealed glass 160 can be formed by the slide glass 140, the core 141, the inner lid 531 and the lid 150, thereby preventing contamination of the cell CE during observation or the like. be able to.

[Sixth Embodiment]
Next, a sixth embodiment of the present invention will be described with reference to FIGS.
The basic configuration of the biological sample observation system of the present embodiment is the same as that of the first embodiment, but the configuration of the chamber is different from that of the first embodiment. Therefore, in the present embodiment, only the periphery of the chamber will be described using FIGS. 21 to 24, and the description of the culture unit and the like will be omitted.
FIG. 21 is a perspective view of the chamber according to the present embodiment, and FIG. 22 is a cross-sectional view of the chamber according to the present embodiment.

As shown in FIGS. 21 and 22, the chamber (culture container) 630 includes a frame portion 631 that forms the outer side wall of the chamber 630, a slide glass 140 on which cells CE are seeded, and an upper portion that covers the upper portion of the frame portion 631. A glass plate 632 is schematically configured.
In the frame portion 631, a joint 143 is disposed at a position facing the frame portion 631, and a commutator 633 is disposed on the joint 143 side into which the culture solution flows out of the pair of joints 143. The joint 143 is disposed so as to be offset above the frame portion 631 (upward in the drawing).

The commutator 633 is formed of a rectifying unit 634 in which a plurality of rectangular columnar shapes are arranged in parallel, and a connecting unit 635 that connects the plurality of rectifying units 634 and connects the rectifying unit 634 to the frame portion 631. The connection part 635 may be arrange | positioned so that the upper end of the rectification | straightening part 634 may be connected, and may be arrange | positioned so that the upper and lower ends of the rectification | straightening part 634 may be connected.
Note that the commutator 633 may be configured by arranging a plurality of quadrangular prisms in parallel as described above, or may be arranged by crossing a plurality of quadrangular prisms in a cross shape as shown in FIG. . Further, it may be formed from a quadrangular column, a cylindrical column, a triangular column, or the like.

Next, seeding and culturing of cells CE in the chamber 630 having the above configuration will be described.
FIG. 24A is a diagram for explaining seeding of cells in the chamber in the present embodiment, and FIG. 24B is a diagram for explaining culture of cells in the chamber in the present embodiment.

In the present embodiment, the chamber 630 is formed in advance, and the cells CE are seeded in the formed chamber 630 and further cultured.
First, as shown in FIG. 24A, the tube 636 is inserted into the joint 143, and an injector (for example, a syringe or pipette) 637 holding the cell suspension is connected to the tube 636. Then, as shown in FIG. 24B, the cell suspension is supplied into the chamber 630, the tube is removed from the joint 143, and the cap 638 is attached.

The amount of the culture solution supplied is desirably such that the culture solution level in the chamber 630 is below the flow path 145 of the joint 143.
The cap 638 is provided with a filter 639 that transmits carbon dioxide, and carbon dioxide can be supplied into the chamber 630 even when the cap 638 is attached to the joint 143.
Thereafter, the suspended cells CE are cultured in the incubator box until they adhere to the slide glass 140.

According to said structure, since cell CE is supplied in the chamber 630 with a culture solution, seed | inoculation of the cell CE can be performed easily also in the state by which the chamber was comprised, for example.
Further, since the cap 638 is attached to the joint 143, the cell CE can be easily cultured without adhering to the supplied culture solution from the channel 145, and can be adhered to the slide glass 140.
Since the commutator 633 is disposed in the frame portion 631, by uniformizing the flow of the culture solution flowing into the chamber 630, the supply of nutrients to the cells CE is made uniform, and the cells CE are uniformly cultured. be able to.

The technical scope of the present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.
For example, in the above-described embodiment, the description has been made by adapting to the configuration for detecting the fluorescence intensity. However, the present invention is not limited to the configuration for detecting the fluorescence intensity, and other various indicators such as cell shape change are detected. It can be adapted to the configuration.
Further, the present invention is not limited to the configuration for observing cells, but can be applied to a configuration for observing various biological samples such as bacteria, microorganisms, and eggs.

1 is a perspective view of a biological sample observation system according to a first embodiment of the present invention. It is a schematic diagram showing the system configuration of the biological sample observation system. It is a perspective view which shows an incubator box same as the above. It is a disassembled perspective view which shows a culture container same as the above. It is sectional drawing which shows a culture container same as the above. It is a figure which shows the example of selection of a scanning method and a detection range similarly. 3 is a flowchart showing a flow of setting measurement parameters. It is a flowchart which shows the flow of a measurement similarly. 3 is a flowchart illustrating an image processing method. 4 is a flowchart showing the flow of data processing. It is a flowchart which shows the flow of adjustment of light quantity similarly. 2 is a flowchart showing a method for replenishing / changing a culture solution. It is a figure which shows the cell tracking image showing the time-dependent change of the cell. It is sectional drawing of the chamber which concerns on 2nd Embodiment by this invention. It is a disassembled perspective view of the chamber which concerns on 3rd Embodiment by this invention. It is the perspective view and sectional drawing of a core same as the above. It is a disassembled perspective view of the chamber which concerns on the 4th Embodiment by this invention. It is the exploded perspective view and sectional drawing of a core same as the above. It is a disassembled perspective view of the chamber which concerns on 5th Embodiment by this invention. It is sectional drawing of a chamber same as the above. It is a perspective view of the chamber which concerns on the 6th Embodiment by this invention. It is sectional drawing of a chamber same as the above. It is a figure which shows another Example of a commutator similarly. It is a figure explaining seeding | inoculation and culture | cultivation of the cell to a chamber same as the above.

Explanation of symbols

10 Biological sample observation system 48 Objective lens (observation means)
130, 230, 330, 430, 530, 630 Chamber (culture vessel)
131, 231 Frame (outer frame member, culture vessel body)
137 Step part (support part)
140 slide glass (seeded material)
141, 341, 441 Core (inner frame member)
144 O-ring (seal member)
145, 345 channel 150 lid (lid member, culture vessel body)
160 Sealed container 451 Separation core (flow path forming part)
531 Inner lid (inner lid member)
532 Opening CE cell (biological sample)
G Adhesive member

Claims (14)

A culture vessel for culturing a biological sample in a sealed state inside,
A cell culture container comprising a seeded member on which a biological sample is seeded and a culture container main body to which the seeded member is detachably attached.   The cell culture container according to claim 1, wherein the seeded member is attached to the culture container body using an adhesive member.   The cell culture container according to claim 1 or 2, wherein a flow path for inflow and outflow of a culture solution in which the biological sample is immersed is provided in the culture container body. The culture vessel main body includes an outer frame member that forms a side wall, a middle frame member that is disposed inside the outer frame member, and a lid member that is fixed to the outer frame member,
The outer frame member is provided with a support portion for supporting the seeded member,
The middle frame member is provided with a seal member that is disposed on a surface facing the seeded member and the lid member, and is in contact with the seeded member and the lid member,
In a state where the seeded member is supported by the support portion, the seeded member and the lid member are disposed across the middle frame member, and the lid member is fixed to the outer frame member, The culture container according to claim 1, wherein a sealed container is constituted by the seeded member, the inner frame member, and the lid member. The culture vessel body includes an outer frame member forming a side wall, an inner lid member attached to the outer frame member, a lid member attached to the outer frame member via the inner lid member, and the outer frame member An inner frame member disposed inside,
The outer frame member is provided with a support portion for supporting the seeded member,
The middle frame member is disposed on a surface facing the seeded member and the inner lid member, and a seal member that contacts the seeded member and the inner lid member is provided.
In a state where the seeded member is supported by the support portion, the seeded member and the inner lid member are arranged across the middle frame member, and the inner lid member is fixed to the outer frame member. Holding the seeded member,
The culture container according to claim 1, wherein the lid member is fixed to the outer frame member, whereby a sealed container is configured by the seeded member, the middle frame member, the inner lid member, and the lid member.   The culture container according to claim 4 or 5, wherein the middle frame member is provided with a flow path for allowing the culture solution to flow into and out of the sealed container.   The culture container according to claim 6, wherein the flow path is branched into a plurality in the middle frame member and has a plurality of openings toward the sealed container.   The culture container according to claim 6 or 7, wherein a flow path forming portion in which the flow path in the middle frame member is formed is detachably attached to the middle frame member.   The culture container according to any one of claims 1 to 8, wherein a sealed container of the culture container is configured using the seeded member on which the biological sample has been seeded.   The culture container according to any one of claims 1 to 8, wherein the biological sample is seeded on the seeded member after the closed container of the culture container is configured.   The culture solution containing the biological sample is supplied into the culture vessel through the flow path, and the biological sample is seeded on the seeded member. The culture vessel described.   12. The seed according to any one of claims 3, 6, 7, 8, and 11, wherein after the seeding of the biological sample, the culture solution is supplied so that the liquid level is below the flow path, and the biological sample is cultured. A culture vessel according to any one of the above.   The culture container according to any one of claims 1, 9, and 10, wherein the seeded member includes a slide glass capable of transmitting light necessary for observing the biological sample from the outside. In a biological sample observation system that acquires information on a biological sample to be observed,
A culture vessel in which the biological sample is housed in a sealed state and cultured;
Observation means for acquiring information by observing the biological sample,
The seeded member on which the biological sample is seeded is disposed on the surface of the culture container facing the observation means,
The biological sample observation system in which the seeded member constitutes a part of the culture container and is detachably disposed with respect to the culture container.

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