JP2005168341A - Biological specimen-observing apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biological specimen-observing apparatus which can reduce damages by outer environments to culture the biological specimen for a long period and can accurately measure the fluorescent light intensity of the biological specimen in real time. <P>SOLUTION: This biological specimen-observing apparatus which can culture and observe the biological specimen comprises a culture chamber 10 which can receive a culture container for culturing and holding the biological specimen and whose inside can be observed from the outside, an environment-controlling means for controlling the inside of the culture chamber to an environment necessary for the culture of the biological specimen, and an outer element-removing means which can not be controlled with the environment-controlling means when the observation is not performed, and can remove external elements affecting the biological specimen when the observation is performed. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a biological sample observation apparatus used for detecting information by reaction of biological samples such as cultured cells.

In recent years, gene analysis techniques have advanced, gene sequences in many organisms including humans have been clarified, and the causal relationship between analyzed gene products (proteins) and diseases has been gradually elucidated. In the future, in order to comprehensively analyze various proteins and genes statistically, research and development of inspection methods and inspection apparatuses using biological samples such as cells have been intensively advanced.
In such an inspection method and inspection apparatus, since it is necessary to detect predetermined information while culturing a biological sample such as a cell for a long period of time, for example, a container for culturing a biological sample under a microscope has been proposed.

As a prior art of such a container, as shown in FIG. 12, a space S in which the temperature can be freely controlled is formed by providing a transparent heat generating plate 1 that can be automatically controlled to a set temperature by a temperature regulator. In order to adjust the carbon dioxide (CO ₂ ) concentration in the space S, a carbon dioxide supply port 2 and a discharge port 3 are provided, and the humidity in the space S sealed by the seal packing 4 is set to a desired value. There is a transparent constant-temperature culture container for microscopic observation in which the culture conditions (temperature, CO ₂ concentration and humidity) of various cells can be set while observing under a microscope by installing the evaporating dish 5 for keeping in the container. In the figure, reference numeral 6 is a microscope objective lens, 7 is a flow rate adjusting valve, 8 is water, and 9 is a microscope stage. (For example, see Patent Document 1)

According to the above-mentioned transparent thermostat container for microscopic observation of the prior art, while performing observation of a culture state of various cells and recording such as photography in the research field of living organisms, reproduction or biotechnology, It is possible to freely control the temperature, carbon dioxide concentration and humidity in the space S to set a desired culture state (environmental condition) as appropriate, and to observe and record the change with time continuously and conveniently. Is done.
JP-A-10-28576

By the way, unlike a gene or the like, fluorescence detection in a living state (detection of GFP (Green Fluorescent Protein) expression in a cell) or the like is frequently used as a measurement technique. For this reason, management of environmental conditions for culturing cells is an important item for obtaining accurate measurement results.
However, plastic or glass dishes are generally used as culture vessels that can be mounted under a microscope, and therefore, environmental conditions such as temperature, carbon dioxide concentration, and humidity cannot be controlled. Microscopic observation of time often kills the cells. For this reason, as shown in the above-described prior art, a container capable of managing environmental conditions is required.

Some types of cells are very vulnerable to changes in the external environment, and may be easily killed by, for example, a rapid temperature increase operation or an uneven temperature distribution. Although it depends on the type of cell, it is generally necessary to maintain a constant temperature of 37 ° ± 0.5 ° C. and a carbon dioxide concentration of 3 to 8%, but other environmental management items cannot be ignored. Furthermore, in the long-term culture, it is also an important management item for the culture environment conditions to make it possible to exchange the cell culture solution in a semi-batch without causing contamination. In addition, since the environment and culture solution for culturing the cells are optimal as conditions for the growth of bacteria and molds, contamination must be avoided from this viewpoint.
In addition, certain cells are very susceptible to ambient light, and if they are exposed to constant external light for long periods of time during culture, such cells can become weak or even die. In addition to being unable to withstand long-term culture, accurate measurement results cannot be obtained.

  However, the transparent constant temperature culture vessel for microscopic observation described in Patent Document 1 can be set and maintained with desired environmental conditions. When compared with the non-observation time in which only the culture is continued without observing with a microscope, the same condition (environment) is maintained unless the container is moved. For this reason, especially when observing cells while cultivating them for a long period of time, the external environment that cannot be dealt with only by setting and maintaining the environmental conditions described above, such as external light, impact and vibration, or dust and germs. There is concern that it will be adversely affected by the elements (external elements). In addition, when changing the culture environment by moving the container during non-observation, it is necessary to pay close attention so that the culture solution is swelled by shaking at the time of movement so that the cells being cultured are not adversely affected.

  The present invention has been made in view of the above problems, and enables a biological sample such as a living cell to be cultured for a long period of time while reducing damage caused by the external environment. For example, the fluorescence intensity of a biological sample such as a living cell is provided. It is an object of the present invention to provide a biological sample observation apparatus capable of accurately measuring the above in real time.

The present invention employs the following means in order to solve the above problems.
The biological sample observation device of the present invention is a biological sample observation device capable of culturing a biological sample and observing the biological sample in which the culture is performed, and culturing the biological sample A culture chamber in which a culture vessel for holding the biological sample is stored and in which the inside can be observed from outside, and the culture chamber is controlled to an environment necessary for the culture of the biological sample And environmental control means for removing external elements that cannot be controlled by the environmental control means and that can adversely affect the biological sample during the observation when the observation is not performed. Possible external element removing means.

  According to such a biological sample observation apparatus, a culture chamber capable of culturing a biological sample and storing a culture container holding the biological sample inside and observing the inside from the outside, and the biological sample in the culture chamber Environmental control means to control the environment necessary for the culture of the stubborn, and when the observation is not performed, external elements that cannot be controlled by the environmental control means and can adversely affect the biological sample at the time of observation are removed. And the external element removing means that can be used, so that the culture chamber is cultivated by the environmental control means when observing the biological sample, during observation to continue culturing, or during non-observation where only culturing of the biological sample is continued. Controlled to the necessary environment. During non-observation where only the culture of the biological sample is continued, external elements that cannot be controlled by the environmental control means can be removed by the external element removing means. In the non-observation period that occupies, it is possible to reduce or eliminate the damage that the biological sample receives from external elements.

  In the biological sample observation apparatus, the external element removing unit is preferably configured so that an observer can remove the external element when the observation is not performed. Thus, when not observing, the observer can manually remove external elements from the culture chamber.

  In the biological sample observation device, the external element is light, and the external element removal unit preferably includes a light-blocking unit that removes light irradiated to the inside of the culture chamber. Damage to a biological sample from external light can be minimized by a manually operated shading means.

  In the biological sample observation device, the external element is an impact or vibration, and the external element removing unit preferably includes a vibration isolating unit, whereby the biological sample is damaged by the impact or vibration. Can be minimized by the manually operated vibration isolating means.

  In the above biological sample observation device, the external element is dust or various germs, and the external element removing means includes an intruder prevention means for preventing an intruder from entering the inside of the culture chamber. In this way, damage to biological samples from dust or other germs can be minimized by manually operating intruder prevention means.

  In the biological sample observation device, the external element removing unit is provided so as to be openable and closable with respect to the culture chamber. By closing the external element removing unit, the external element of the biological sample is removed. It may be configured to be able to remove the influence by manual operation, or the external element removing means is provided as a structure capable of covering a predetermined part of the culture chamber, and the culture By covering a predetermined part of the chamber, it may be configured such that the influence of the external element on the biological sample can be removed by manual operation.

  In the biological sample observation device, the external element removing unit is configured to detect that the observation is not performed and automatically remove the external element when the observation is not performed. Thus, the external element removing means automatically operates during non-observation, so that the external element can be reliably and easily removed from the culture chamber.

  In the biological sample observation device, the culture container is supported so as to be movable from a position where the external element can be removed by the external element removal means to a predetermined position where the observation of the biological sample is possible. It is preferable to further include means, whereby the inside of the culture container can be divided into an observation region and a non-observation region.

  In the biological sample observation device, the external element is light, and the external element removal unit preferably includes a light-blocking unit that removes light irradiated to the inside of the culture chamber. Damage to a biological sample from external light can be minimized by automatic light shielding means.

  In the biological sample observation device, the external element is an impact or vibration, and the external element removing unit preferably includes a vibration isolating unit, whereby the biological sample is damaged by the impact or vibration. Can be minimized by automatic vibration isolation.

  In the above biological sample observation device, the external element is dust or various germs, and the external element removing means includes an intruder prevention means for preventing an intruder from entering the inside of the culture chamber. In this way, the damage that the biological sample receives from dust or other germs can be minimized by automatic intruder prevention means.

  In the biological sample observation device, the external element removing unit is provided so as to be openable and closable with respect to the culture chamber. By closing the external element removing unit, the external element of the biological sample is removed. It may be configured to be able to automatically remove the influence, or the external element removing means is provided as a structure capable of covering a predetermined part of the culture chamber, and the culture It may be configured such that the influence of the external element on the biological sample can be automatically removed by covering a predetermined portion of the chamber.

In the biological sample observation device, the biological sample observation device may be fixedly provided with a stage unit in the observation device that performs the observation, or the biological sample observation device performs the observation. It may be configured to be detachable with respect to the stage portion in the observation machine.
In the biological sample observation apparatus in this case, the observation device is preferably a microscope.

  In the biological sample observation apparatus, it is preferable that the environment control unit includes a humidity adjustment unit capable of adjusting the humidity in the culture chamber, whereby an optimum humidity environment can be provided in the culture chamber.

  In the biological sample observation apparatus, it is preferable that the environment control unit includes a temperature adjustment unit capable of adjusting the temperature in the culture chamber, thereby providing an optimal temperature environment in the culture chamber.

  In the biological sample observation apparatus, it is preferable that the environment control means includes a gas convection means capable of convection of the atmosphere in the culture chamber, whereby the atmosphere in the culture chamber can be made substantially uniform over the entire area. it can.

In the biological sample observation device, the environment control means preferably further includes a gas concentration adjusting means capable of adjusting a concentration of a predetermined gas necessary for culturing the biological sample in the culture chamber. An optimal gas concentration environment can be provided in the culture chamber.
In the biological sample observation apparatus in this case, it is preferable that the gas contains carbon dioxide having a predetermined concentration.

  In the biological sample observation apparatus, it is preferable that the biological sample observation apparatus further includes a sterilizing light irradiation means capable of irradiating the inside of the culture chamber with a sterilizing light when the culture is not performed. The inside can be sterilized to prevent contamination.

  In the above biological sample observation device, it is preferable that the biological sample contains cells.

According to the biological sample observation device of the present invention, a culture chamber in which a biological sample is cultured and a culture container holding the biological sample can be housed inside to observe the inside from the outside, and the biological sample in the culture chamber Environmental control means to control the environment necessary for the culture of the stubborn, and when the observation is not performed, external elements that cannot be controlled by the environmental control means and can adversely affect the biological sample at the time of observation are removed. The culture chamber is cultivated by the environmental control means when observing the biological sample, during observation to continue culturing, or when not observing only culturing the biological sample. In the non-observation period in which the environment necessary for the control is controlled and only the biological sample is continuously cultured, the external elements that cannot be controlled by the environmental control means can be removed by the external element removing means.
For this reason, it becomes possible to measure accurately and in real time without damaging a biological sample cultured in a container that maintains an optimal culture environment during observation. Furthermore, during non-observation, which accounts for a large proportion of time in long-term culture, minimize the damage that biological samples receive from external elements, and prevent the deterioration and death of biological materials caused by external elements. Therefore, the remarkable effect that the biological sample can be reliably cultured for a longer period of time can be obtained.

Hereinafter, an embodiment of a biological sample observation device according to the present invention will be described based on the drawings.
The biological sample observation device is configured to be able to cultivate various biological samples and to observe the culture state of biological samples in research fields such as living organisms, reproduction or biotechnology. is there. The biological sample here includes microorganisms such as cells, eggs, biological tissues, algae, fungi, and bacteria, and in each embodiment described below, cells are cultured as an example of a biological sample. Will be described.

<First Embodiment>
A configuration example of a biological sample observation apparatus according to a first embodiment of the present invention will be described with reference to FIGS. 1 is a schematic configuration diagram of the biological sample observation apparatus, FIG. 2 is a plan view of the culture chamber showing a state where the culture container is in a measurement (observation) position, and FIG. 3 is a non-measurement (observation) position of the culture container. It is a top view of the culture room which shows the state in a culture position.
The biological sample observation apparatus 100 includes a culture chamber 10 for culturing cells (biological sample), and environmental control means for controlling the inside of the culture chamber 10 to an environment necessary for cell culture, in other words, culturing cells. Environmental control means capable of forming and maintaining an optimum environment for the maintenance, and external element removal means capable of removing external elements that may adversely affect living cells in culture. Composed.

The culture chamber 10 has a box-like configuration in which an upper opening 12 is formed on the upper surface of a housing 11 having a rectangular parallelepiped shape and a lid member described later is provided so as to close the upper opening 12. A base member 11a for fixing the culture chamber 10 at a predetermined position is provided below the bottom surface of the casing 11, and a space for accommodating a transport mechanism described later is formed on the base member 11a. ing. Such a culture chamber 10 is preferably made of a light-shielding material having high anticorrosion properties such as anodized aluminum or stainless steel such as SUS316, and more preferably, from the viewpoint of heat retention. It is recommended to select a low material.
The inside of the housing 11 forming the culture chamber 10 is divided into a measurement area 13 for observing cultured cells and a non-measurement area 14 for culturing cells only. The culture chamber 10 is configured such that cells can be cultured, a culture container holding the cells can be housed inside, and the cells in the culture container can be observed from the outside of the culture chamber 10. Here, as shown in FIGS. 2 and 3, the case where the microplate 15 is used as a culture container will be described, but a dish or a flask can also be used.

As shown in FIGS. 2 and 3, a culture vessel transport mechanism including an X-axis operation stage 16 and a Y-axis operation stage 17 is installed on the bottom surface of the housing 11.
In this case, the X-axis operation stage 16 reciprocates in the left-right direction in FIG. 2 (hereinafter referred to as “X-axis direction”) with respect to the bottom surface of the casing 11 on the fixed side. Further, the Y-axis operation stage 17 reciprocates in the vertical direction (hereinafter referred to as “Y-axis direction”) in FIG. 2 on the above-described X-axis operation stage 16.
In the example shown in the figure, the above-described microplate 15 is placed on the Y-axis operation stage 17 so that it moves along with the stage in the X-axis direction and the Y-axis direction by a desired amount with respect to the bottom surface of the housing 11. It is possible to make it. That is, the transport mechanism including the X-axis operation stage 16 and the Y-axis operation stage 17 has a function of transporting the macro plate 15 to a desired position and positioning it within the housing 11.

The X-axis operation stage 16 and the Y-axis operation stage 17 described above are configured to reciprocate in the X-axis direction or the Y-axis direction, for example, by rotating a ball screw (not shown).
Further, the X-axis operation stage 16 and the Y-axis operation stage 17 may adopt an electric drive mechanism configured to move the operation stage by rotating a ball screw by an electric motor (not shown), or a ball screw. A manual drive mechanism configured to move the stage by manually rotating the handle such as a handle operation (not shown) may be employed. A drive mechanism for the Y-axis operation stage 17, such as a ball screw, is disposed in the illustrated drive unit box 18, and the drive mechanism for the X-axis operation stage 16 is housed in the space of the base member 11a. . The drive mechanism is separated from the internal space of the culture chamber 10 by being housed in the drive unit box 18. The drive mechanism may be provided outside the culture chamber 10.

  In the configuration example shown in FIG. 1, an X-axis scanning control unit 102 that outputs an operation signal of the X-axis operation stage 16 based on a control signal output from a computer 101 that performs various operation controls of the biological sample observation apparatus 100. In addition, an electric driving mechanism including a Y-axis scanning control unit 103 that outputs an operation signal of the Y-axis operation stage 17 is employed.

In the measurement area 13 and the non-measurement area 14 described above, the inside of the culture chamber 10 is divided in the X-axis direction by a top plate 19 and a pair of partition receivers 20 fixed to the housing 11 side. That is, in FIG. 2, the left area of the partition receiver 20 is the measurement area 13, and the right area is the non-measurement area 14.
A partition plate 21 that moves integrally with the stage is fixedly installed at the left end of the X-axis operation stage 16. The partition plate 21 is a plate-like member having a shape that substantially matches the cross-sectional shape of the housing 11. The Y-axis operation stage 17 is stopped at a predetermined position (home position) and the X-axis operation stage 16 is moved to the non-measurement area 14. By moving to the side, the side surface abuts on the partition receiver 21 described above, and the internal space of the housing 11 is divided into two in the left-right (X-axis) direction. Furthermore, since the upper end of the partition plate 21 contacts the lower surface of the top plate 19 described above, the measurement area 13 and the non-measurement area 14 become two spaces that are divided from each other.

The upper opening 12 of the measurement area 13 described above is detachably covered with a detachable glass top cover 22 that covers the opening of the area. The glass upper cover 22 can be removed as necessary when performing various operations such as replacement of an objective lens of a microscope, which will be described later, and internal cleaning of the measurement area 13. The glass upper cover 22 may be fixed by, for example, fixing the housing 11 with a necessary number of screws.
An open / close lid 23 that can be opened and closed by a hinge or the like is attached to the upper opening 12 of the non-measuring area 14 so as to cover the opening of the area. One end of the opening / closing lid 23 is supported by the top plate 19 in a closed state.

The glass upper cover 22 of the measurement area 13 described above is composed of a glass plate (optical glass member) 22a on the entire surface or substantially the entire surface excluding the surrounding frame portion. It may be a limited area. In this case, it is preferable to use an optical glass material having an antireflection film (AR coating) on both sides for the glass plate 22a. The antireflection film may be coated only on one side, but double-sided coating is preferable when it is assumed that both transmission observation and epi-illumination observation are performed.
The open / close lid 23 of the non-measuring area 14 is entirely made of a light-shielding material (usually the same material as the housing 11), and a peephole cover 23a and a UV irradiation hole cover 23b are provided as necessary. It has been. That is, the non-measuring area 14 has a structure that does not allow external ambient light to pass through, and the open / close lid 23 functions as a light shielding unit described later together with the housing 11.

One peephole cover 23a has a configuration in which a window member such as a glass plate or a resin plate having a low transmittance is fitted into an opening (window) of a peephole provided by partially cutting out the opening / closing lid 23, for example. More preferably, when not in use, the window member may be covered with a detachable or open / close lid made of a light-shielding member similar to the open / close lid 23. The peephole cover 23a is for visually confirming the inside of the culture chamber 10, and is disposed, for example, on the upper surface of a water tank 34 described later.
In addition, the UV irradiation hole cover 23b is detachable from a plate made of a light-shielding member similar to the opening / closing lid 23 with respect to, for example, an opening (window) of the UV irradiation hole provided by partially cutting the opening / closing lid 23. Or it fixes so that opening and closing is possible. The UV irradiation hole cover 23b is removed when the non-measurement area 14 is irradiated with ultraviolet rays for sterilization.
Note that the peephole cover 23a and the UV irradiation hole cover 23b can be shared and integrated by considering the position and size thereof.

In the culture chamber 10 described above, in order to bring the internal environment of the casing 11 whose upper opening 12 is closed with the glass upper lid 22 and the opening / closing lid 23 into a desired state suitable for cell culture, in other words, the culture chamber Environmental control means is provided for making 10 an incubator box having the same function as the incubator.
This environmental control means has a function of setting and maintaining temperature, humidity, and CO ₂ concentration, which are environmental conditions in the culture chamber 10, to desired values. Suitable environmental conditions for general cell culture are a temperature of about 37 ± 0.5 ° C., a high humidity of about 100%, and a CO ₂ concentration of about 3 to 8%.

  In order to obtain such a culture environment, a small or tape-shaped heater is attached to the inner surface of the housing 11 as a temperature adjustment means capable of adjusting the temperature in the culture chamber 10 as a component of the environment control means. Yes. This heater is arranged so that the microplate 15 on the Y-axis operation stage 17 can be heated uniformly and indirectly. That is, for example, a planar heater 31 is used as a heater, and the planar heater 31 does not directly heat the microplate 15 but instead arranges the planar heater 31 so as to warm the surrounding air. Thus, the microplate 15 is indirectly heated. Accordingly, in the illustrated example, the tape-like planar heater 31 is attached to the inner peripheral surface of the housing 11 over substantially the entire circumference.

In addition to the planar heater 31 described above, an auxiliary heater 32 is attached to the upper surface of the Y-axis operation stage 17 as necessary. The auxiliary heater 32 performs auxiliary heating so as to compensate for the insufficiency of indirect heating by the planar heater 31 described above, and is provided at a position that does not directly contact the microplate 15 like the planar heater 31. . The heating capacity of the auxiliary heater 32 may be smaller than that of the planar heater 31.
In order to control the energization heating of the planar heater 31 and the auxiliary heater 32 described above, the set temperature control signal input from the computer 101, the detected temperature signal of the temperature sensor 33, and the detected temperature signal of an internal temperature detecting sensor (not shown) are used. An incubator temperature control unit 104 that performs feedback control based on this is provided. The temperature sensor 33 used here is a small sensor installed so as to be in contact with the microplate 15, and thus can directly detect the temperature of the microplate 15 rather than the ambient air temperature. Reference numeral 33 a in the figure is a signal line of the temperature sensor 33.

In the incubator temperature control unit 104 to which detection temperature signals are input from the temperature sensor 33 and the internal temperature detection sensor, the energization of the planar heater 31 is mainly controlled to keep the environmental temperature at a set value. However, for example, when the temperature difference between the temperature sensor 33 and the internal temperature detection sensor is larger than a predetermined value due to non-uniform internal temperature distribution, for example, the detected temperature of the temperature sensor 33 is the internal temperature detection sensor. If the temperature is lower than the detected temperature by a predetermined value or more, it is determined that the heating amount in the culture chamber 10 is insufficient, and the auxiliary heater 32 is energized and heated in addition to the planar heater 31. Since this auxiliary heater 32 is located close to the microplate 15, it is possible to compensate for the temperature shortage even with a relatively small heating capacity.
In this way, the temperature environment in the culture chamber 10 is controlled by the incubator temperature control unit 104, the planar heater 31, and the auxiliary heater 32 so as to maintain a desired temperature.

Next, in the casing 11 constituting the culture chamber 10, in order to set and maintain the humidity in the culture chamber 10 at a desired value, a humidity adjusting means is provided as a component of the environmental control means. . This humidity adjusting means is a water vapor generating means comprising a water tank 34 installed in the culture chamber 10 and a heater 35 for heating the water tank 34. The water tank 34 of the water vapor generating means is provided with a heater 35 of the heating means and provided with a wide (entire surface in the illustrated example) opening on the upper surface. This water vapor generating means is provided in the water tank 34 in the culture chamber 10. Water in the tank (usually sterile water) is heated to about 37 ° C. by heating with a heater 35 provided in contact with the water tank 34 and evaporated to maintain the inside of the culture chamber 10 at a high humidity of about 100%. Can be done.
The heater 35 for heating the water tank may be small or planar (tape-shaped).

In this case, at the time of culture, the heater 35 may be constantly heated to evaporate water continuously. Alternatively, humidity detection means may be provided at an appropriate place in the culture chamber 10 and water may be evaporated by intermittent operation in which the heater is heated when the detected humidity falls below a desired humidity.
The amount of water in the aquarium 34 can be easily replenished by checking the remaining amount from the peephole from which the peephole cover 23a is removed and opening the open / close lid 23 during long-term culture. Sterile water supply means that can be appropriately supplied from a water storage tank or the like provided outside the culture chamber 10 may be provided to enable long-term continuous operation.

Further, the casing 11 forming the culture chamber 10 is provided with a gas concentration adjusting means for adjusting the concentration of the culture gas as a component of the environment control means. The gas concentration adjusting means for maintaining especially the CO ₂ concentration in the culture chamber 10 is set to a desired value, and a CO ₂ supply port 36 that constitutes the CO ₂ supply means. The CO ₂ supply port 36 can be connected to a culture gas supply source (gas supply means) such as a buffer tank (not shown) via a CO ₂ pipe 36a.
In addition, it is preferable that the CO ₂ supply port 36 and the CO ₂ pipe 36a be configured to be detachable through, for example, a connector.

The buffer tank stores a culture gas adjusted to a desired CO ₂ concentration (for example, 5%). Then, the CO ₂ concentration control unit 105 connected to the computer 101 performs CO ₂ opening / closing control according to a detection value of a CO ₂ concentration detection sensor (gas concentration measuring means) (not shown), for example. _{2 The} amount of culture gas supplied into the culture chamber 10 via the supply port 36 can be adjusted to control the internal CO ₂ concentration.
Since the supply of such culture gas is supplied so as to compensate for a small amount of atmosphere flowing out from the gap in the culture chamber 10 (for example, the observation hole 25 of the Y-axis operation stage 17 or the sliding portion of the drive mechanism). The pressure is slightly higher than the outside. For this reason, by continuing supply of culture gas, it can be set as the culture environment where the external air containing dust and various germs cannot flow into the culture room 10 easily.

As described above, since the environmental control means including the heater 31, the water tank 34, the CO ₂ supply port 36, and the like is provided in the culture chamber 10 that functions as an incubator box, temperature control and CO ₂ are performed in the same manner as a commercially available incubator. It is possible to create a high humidity environment where the concentration is controlled.
Moreover, it is preferable that the environmental control means of the culture chamber 10 described above is provided with a fan 37 of gas convection means that is provided at a suitable place in the non-measurement area 14 and convects the internal atmosphere. By operating the fan 37, the atmosphere of the high humidity environment in which the temperature control and the CO ₂ concentration control are performed convects in the non-measurement area 14, thereby forming a uniform culture environment without uneven distribution over the entire area. can do.

The culture chamber 10 described above further includes external element removal means in addition to the environmental control means. This external element removing means removes external elements that adversely affect cells in culture and enables long-term culture, and may be configured so that an observer can perform various operations manually. Alternatively, various operations may be automatically performed by detecting the non-observation time during which only culture is performed.
Here, specific examples of the external element are given as follows: ambient light such as sunlight and room lighting (hereinafter referred to as external light) irradiated as being inserted from outside the culture chamber 10, cells being cultured and culture There are dust, germs and the like that enter the culture chamber 10 due to impact or vibration acting on the liquid and contamination.

  Further, in order to detect the non-observation time described above, the position of the culture vessel such as the microplate 15 or the stage position of the culture vessel transport mechanism constituted by the X axis operation stage 16 and the Y axis operation stage 17 is used. May be determined by detecting with a position sensor (not shown). As such a position sensor, a photo sensor, a limit switch, a proximity switch, a touch sensor, a photoelectric switch, a linear transducer, an encoder, or the like can be used.

Further, in order to detect the above non-observation time, it is also possible to use a light detection device that measures brightness (light quantity) at a predetermined position in the culture chamber 10. In other words, generally, the illumination light for observation required during observation is turned off during non-observation, so that the brightness at a predetermined position decreases due to this influence. For example, such a change in the amount of light is detected. It is also possible to judge that it is not observed.
Further, in order to detect the non-observation time described above, an operation detection device that detects whether or not the observation device is operating may be used. As a specific example of this motion detection device, for example, the operation completion of the X-axis operation stage 16 and the Y-axis operation stage 17 or the operation end of the observation device is used as a trigger, and the operation end of the observation device can be determined from the signal. Conceivable.

In order to detect the above-mentioned time of sad observation, time management means such as a timer for measuring observation time or culture time, a clock for detecting time for observation or culture, and the like may be used.
Here, for example, a microscope is generally used as the above-described observation device, and a method of viewing from the observation portion of the observation device with the naked eye of the observer, or an image taken by an imaging device such as a camera provided in the observation device is monitored. Any of the methods of displaying and viewing on may be adopted.

  Hereinafter, a specific configuration example of the external element removing unit will be described. The external element removing means includes a light shielding means for removing external elements of light, a vibration isolating means for removing external elements of shock or vibration, and a dustproof means for removing external elements of dust or germs. It comprises at least one.

First, a dustproof means for removing external elements of dust or various germs will be described.
The above-described open / close lid 23 of the non-measuring area 14 functions as a dust-proof cover (intrusion prevention means) that prevents intruders such as dust and germs from entering the inside of the culture chamber 10 by closing the lid. It becomes a dustproof means of the automatic element removing means. In this case, in order to more reliably prevent intrusion, for example, a seal member may be attached at an appropriate position where the opening / closing lid 23 and the housing 11 are in contact with each other to improve the adhesion between them.
Further, when cells are cultured in the non-measurement area 14 of the culture chamber 10, an elastic member such as silicon is provided at a portion where the partition plate 21 abuts on the partition receiver 22 in order to divide the measurement area 13 and the non-measurement area 14. By disposing, the impact at the time of contact can be reduced. Furthermore, if a sealing member or the like is attached to a portion where the partition plate 21 is in contact with the partition receiver 22 or a contact portion between the lower surface of the top plate 19 and the upper end of the partition plate 21, the adhesion between the two can be increased.

In this way, the non-measurement area 14 in the culture chamber 10 is a space that is substantially sealed by the casing 11, the partition plate 21, and the opening / closing lid 23. Can be prevented. For this reason, it is possible to prevent the cells from being adversely affected and weakening or dying, and therefore, culture for a long period of time is possible.
Further, if all of the sealed space of the non-measurement area 14 described above is formed of a light-shielding member, it also serves as a light-shielding means that prevents the intrusion of external light, which is one of external factors, and removes the influence of light. Will not be weakened or killed due to adverse effects, and therefore, long-term culture is possible in this respect as well.

Further, since the observation area 13 is also covered with the glass upper cover 22, no intruder enters from here, and therefore, such a glass upper cover 22 cooperates with the partition plate 21 and the like to be double dustproof. It becomes a dustproof means that functions as a cover.
In addition, in order to increase the dustproof property and the light-shielding property at the time of culture, dustproof covers such as a dustproof cover and a dark curtain that cover the entire culture chamber 10 from the outside or cover the entire observation machine in which the culture chamber 10 is installed are covered. The means and the light shielding means may be used in combination. In addition, the external element removing means functioning as the above-mentioned dustproof means and light shielding means is a rotary lid member that closes the upper opening 12 of the culture chamber 10 so as to cover the entire surface, or a slide shutter that closes by sliding. Such a lid member may be used.

Next, a specific configuration of the external element removing unit will be described with respect to the vibration isolating unit for removing the remaining external factors such as shock and vibration.
For example, in the drive mechanism of the X-axis operation stage 16 and the Y-axis operation stage 17, it is appropriate to place a sliding part such as a ball screw, a rotating part such as an electric motor as a driving source, and a part to which an impact due to contact is applied. An anti-vibration member and an impact absorbing member may be provided on the surface. Anti-vibration members and shock absorbing members that can be used here include, for example, urethane, silicon, rubber, springs, elastic members filled with gas, dampers filled with fluid, etc. By doing so, it is possible to prevent or suppress the generation of vibrations and shocks, or to absorb and prevent the generated vibrations and shocks.

Since vibrations and shocks of external factors can be eliminated in this way, cells in culture are not adversely affected and are not weakened or killed. Become.
In this way, external factors such as dust, germs, external light, vibration, and impact can be removed by the external element removing means described above, so that external factors are prevented from acting on the cells being cultured. Therefore, the cells will not be weakened or killed due to these external factors, and stable measurement over a long period of time can be performed to obtain accurate measurement data.

  By the way, as shown in FIG. 1, for example, the casing 11 constituting the culture chamber 10 described above has the base member 11a of the casing 11 fixedly or detachably installed on a stage 70 of a microscope whose bottom is an observation machine. It may be a thing. Note that in the case of fixed installation, the microscope is a dedicated machine for the biological sample observation apparatus 100, and in the case of being detachable, existing microscopes can be widely used. In the figure, reference numeral 71 is a microscope objective lens, and 72 is a revolver.

  The base member 11a and the X-axis operation stage 16 are provided with observation holes 24 and 25, respectively, as shown in FIG. The observation windows 24 and 25 are provided for observing the biological sample in the microplate 15 from below with the objective lens 71 in the observation area of the measurement area 13. Therefore, in the predetermined observation region, the observation hole 25 of the X-axis operation stage 16 is coincident with the observation hole 24 of the base member 11a. Therefore, the objective lens 71 is inserted so as to penetrate both the observation holes 24 and 25, and the biological sample is inserted. Can be observed. Note that at least the installation position of the microplate 15 of the Y-axis operation stage 17 is provided with an observation hole (not shown) or an observable member so that observation with the objective lens 71 of the microscope can be performed from below. For example, it is made of a transparent glass plate or a resin plate.

Further, when the observation holes 24 and 25 are large and the amount of outflow of the atmosphere at the time of measurement increases remarkably as compared with that at the time of non-measurement, for example, sheet materials 24a and 25a such as soft and slippery rubber are pasted. The objective lens 71 may be penetrated by providing a plurality of cuts 24b and 25b that cross each other and cross the center portion, and the substantial opening area may be minimized.
Further, when the observation holes 24 and 25 described above are large and the outflow amount of the atmosphere at the time of measurement is remarkably increased as compared with that at the time of non-measurement, the supply amount of the culture gas is respectively measured at the time of non-measurement and at the time of measurement. It may be changed according to the outflow amount.
In the above-described embodiment, the casing 11 has a rectangular parallelepiped shape. However, for example, a shape in which a part of the rectangular parallelepiped shape is partially cut away in order to avoid interference depending on the arrangement of a transmission illumination post (not shown). It can be changed as appropriate according to the situation.

Here, with respect to the biological sample observation apparatus 100 having the above-described configuration, basic operations (operations) relating to culture and observation of the biological sample will be described based on the flowchart of FIG.
In the basic operation starting in the first step S1, the opening / closing lid 23 of the culture chamber 10 is opened in the next step S2, and the culture chamber 10 is opened. At this time, the X-axis operation stage 16 and the Y-axis operation stage 17 are at home positions in the non-measurement area 14. Further, the UV irradiation hole cover 23b is in a normal closed state, and when a light-shielding lid member is provided on the peep hole cover 23a, the peep hole cover 23a is covered with the lid member and closed.

When proceeding to the next step S3 from this state, the microplate 15 is set at a predetermined culture position set on the Y-axis operation stage 17. At this time, cells of a biological sample that has been prepared for culture are placed in the microplate 15.
In the subsequent step S4, by closing the open / close lid 23, the upper opening 12 of the culture chamber 10 is closed together with the glass upper lid 22 in the closed state. As a result, the inside of the non-measurement area 14 of the culture chamber 10 is protected from external light irradiation and dust and other bacteria intrusion, which are external factors that adversely affect the cells.

After setting to such a culture state, in the next step S5, cell culture is continuously performed for a predetermined time while operating the environment control means to maintain a desired culture environment. The culture environment at this time is maintained so that the temperature, humidity, and CO ₂ concentration are optimum for the cells to be cultured.
After culturing for a predetermined time in this way, in the next step S 6, the X-axis operation stage 16 and the Y-axis operation stage 17 are operated to move the microplate 15 to the observation position in the measurement area 13. It is possible to observe the above.

In the next step S7, changes in the biological sample due to culture are observed and measured, such as taking and outputting a biological sample image with an observation machine.
Thereafter, the X-axis operation stage 16 and the Y-axis operation stage 17 are operated to move to the home position (S8), and the culture state in step S5 described above is restored. Then, after culturing again for a predetermined time, the above-described steps S5 to S8 are repeated as many times as necessary to observe and measure the temporal change of the biological sample due to the culture. When the necessary number of observations and measurements are completed, the Y-axis operation stage on which the microplate 15 is set is returned to the home position in step S8.

Thus, after repeating culture | cultivation and observation of required number of times, culture | cultivation and observation regarding the biological sample in the microplate 15 are complete | finished in the following step S9.
After completion of the culture and observation, in the next step S10, the opening / closing lid 23 is opened and the culture chamber 10 is opened.
In the subsequent step S11, the microplate 15 is taken out from the Y-axis operation stage 17, and in the next step S12, the open / close lid 23 is closed.

In the last step S13, the UV irradiation hole cover 23b is removed as necessary, and sterilization light is irradiated into the inside of the culture chamber 10, in particular, into the non-measurement area 14 to be disinfected. At this time, for example, a handy type UV lamp may be installed in the UV irradiation hole and irradiated with ultraviolet rays.
When the internal disinfection is thus completed, all the basic operations of the biological sample observation apparatus 100 are finished in step S14.

Furthermore, regarding the biological sample observation apparatus 100 having the above-described configuration, the operations (operations) of the environment control unit and the external element removal unit of the above-described embodiment will be described with reference to the flowchart of FIG.
In the first step S21, the operation of the biological sample observation apparatus 100 is started. At this time, the X-axis operation stage 16 and the Y-axis operation stage 17 are present at the home position in the non-measurement area 14.

In the next step S22, a predetermined amount of sterilized water is supplied into the water tank 34 after the opening / closing lid 23 is opened.
In subsequent step S23, the incubator temperature control unit 104 is driven to energize the planar heater 31 and the like so as to maintain a desired culture environment temperature (for example, 37 ± 0.5 ° C.) inside the culture chamber 10. Implement temperature control.
Subsequently, by driving the CO ₂ concentration control unit 105 proceeds to step S24, to start the supply of the adjusted gas (e.g. air or culture gas) to a desired CO ₂ concentration from the CO ₂ supply port 36.

  Next, it progresses to step S25, a UV lamp is turned on, and the ultraviolet rays for sterilization are irradiated toward the inside of the non-measurement area 14 from the UV irradiation hole from which the UV irradiation hole cover 23b is removed. After the ultraviolet irradiation is continued for a predetermined time to sterilize the inside, the process proceeds to the next step S26, and the UV lamp is turned off. After the UV lamp is turned off, the UV irradiation hole cover 23b is attached and the UV irradiation hole is closed.

After the sterilization in the culture chamber 10 is completed in this way, the process proceeds to the next step S27 and the opening / closing lid 23 is opened.
In the next step S28, the microplate 15 is set at a predetermined position on the Y-axis operation stage 17 at the home position. A biological sample (cell) to be cultured is placed in the microplate 15 in a state where the preparation for culture is completed.
In the subsequent step S29, the open / close lid 23 is closed, and the culture for a predetermined time is started while maintaining the desired culture environment.

After culturing for a predetermined time in this way, observation and measurement are performed for changes in the cultured biological sample.
In the next step S30, the objective lens 71 suitable for observation is selected by operating the revolver 72 of the microscope for observing the biological sample in the microplate 15.

In the next step S31, a shutter (not shown) is opened so that the biological sample is irradiated with light or external light from the blocked light source. Then, the process proceeds to the next step S32, the X-axis operation stage 16 and the Y-axis operation stage 17 are operated and moved into the measurement area 13, and positioned at the measurement start position by the selected objective lens 71. At this time, one of the observation targets is selected from the plurality of biological samples, and the biological sample is positioned at a measurement start position where observation with the objective lens 71 is possible.
In step S33, the focus of the objective lens 71 is adjusted to the cell of the biological sample, and then the process proceeds to the next step S34 to capture and output the cell image.

Such cell image capture and output can be performed, for example, on the number of cells contained in the microplate 15, and thus after observation and measurement of a single cell or a predetermined group of cells is completed. Returning to step S32, the X-axis operation stage 16 and the Y-axis operation stage 17 are moved, and another cell is positioned at an observable position. Then, after the objective lens 71 is focused again in step S33, image capture and output regarding the cell are performed (S34).
In the same manner, the same operation is repeated as many times as necessary for selecting and observing the cells placed in the microplate 15, and then the process proceeds to the next step S35, where the shutter is closed and the biological sample is formed. The irradiated light is blocked.

  Thereafter, in step S36, the X-axis operation stage 16 and the Y-axis operation stage 17 are operated to move to the home position, and the culture state after step S29 and step S30 described above is returned as necessary. That is, after culturing again for a necessary time, the steps from Step S31 to Step S35 are repeated again, and the change with time of the cells due to the culture is observed. Such culturing and observation are repeated as many times as necessary to observe changes over time, and the process proceeds to the next step S37 when the final observation is completed.

After opening the opening / closing lid 23 in this step S37, the process proceeds to the next step S38, and the microplate 15 is removed from the Y-axis operation stage 17.
In the subsequent step S39, the remaining amount of sterilized water is removed from the water tank 34, and then the process proceeds to the next step S40 where the open / close lid 23 is closed.
Thereafter, the process proceeds to the next step S41, the UV lamp is turned on, and sterilizing ultraviolet rays are irradiated toward the inside of the non-measurement area 14 from the UV irradiation hole from which the UV irradiation hole cover 23b is removed. After the ultraviolet irradiation is continued for a predetermined time to sterilize the inside, the process proceeds to the next step S42, and the UV lamp is turned off. After the UV lamp is turned off, the UV irradiation hole cover 23b is attached and the UV irradiation hole is closed.
When the sterilization / disinfection in the culture chamber 10 is thus completed, all the operations of the biological sample observation apparatus 100 are completed in step S43.

Next, a modification of the first embodiment described above will be described with reference to FIGS. In addition, the same code | symbol is attached | subjected to the part similar to 1st Embodiment mentioned above, and the detailed description is abbreviate | omitted.
In this modification, instead of the microplate 15 described above, a culture vessel 40 that can cultivate a biological sample by circulating a culture solution is used. The culture container 40 can accommodate a slide glass 41 on which cells C to be cultured are placed, a glass member 40a for observation with an objective lens 71 is provided on the bottom surface, and light from a transmission light source is provided on the top surface side. A glass member (not shown) for transmission is provided. Note that the glass member on the upper surface side is detachable so that operations in the culture container 40 such as taking in and out of the slide glass 41 can be performed.

The culture system for supplying a culture solution to the culture vessel 40 described above includes a culture solution bottle 42, a culture solution pump 43, a CO ₂ supply means 44, a culture solution pipe 45, and a control computer 110. Composed.
The culture solution bottle 42 is a container for storing a culture solution mixed with nutrients necessary for cell growth and 5% CO ₂ . The culture solution is kept at 37 ° C. by controlling a heater (not shown) by the culture solution bottle temperature control unit 111 to prevent a decrease in cell activity due to a temperature change of the culture solution. Further, the CO ₂ supply means 44 connected to the culture solution bottle 42 receives the control of the CO ₂ concentration control unit 112 and supplies CO ₂ to the culture bottle 42, whereby CO ₂ is mixed into the culture solution.

Since the culture solution mixed with CO ₂ is circulated through the culture vessel 40 connected by the culture solution pipe 45 by operating the culture solution pump 43 under the control of the culture solution pump control unit 113, the culture gas And nutrients are supplied to the cultured cells in the culture vessel 40.
Note that each of the culture solution bottle temperature control unit 111, the CO ₂ concentration control unit 112, and the culture solution pump control unit 113 receives a control signal from the computer 110.

  The culture vessel 40 connected to such a culture system may be used and attached to a predetermined position in the culture chamber 10 that functions as an incubator box to culture and observe cells. Further, when the size (area of the plane) of the culture container 40 is different from that of the microplate 15 described above, for example, as shown in FIG. Combined use with the microplate 15 is possible. In this case, it is preferable that the temperature of the culture solution can be detected by the same temperature sensor 33 as that of the microplate 15.

In such a modification, the CO ₂ pipe 36a connected to the CO ₂ supply port 36 may be removed, or only supply may be stopped. Preferably, as shown in FIG. 7, a connector cap 47 is attached and closed to the CO ₂ supply port 36 from which the CO ₂ pipe 36a has been removed to prevent inflow of outside air.
In addition, since the culture solution circulates in the water tank 34, it is not necessary to add sterilized water to generate water vapor. Therefore, it is possible to remove the water tank 34 during culture and observation.
The heating by the surface heater 31 and the auxiliary heater 32 and the convection by the fan 37 are the same as in the above-described embodiment.

As described above, according to the above-described first embodiment and its modification, the carrier 70 holding the cells can be selected by covering the stage 70 of the observation device (microscope) with the culture chamber 10 in the incubator box. Furthermore, by using the culture vessel 40 and the culture system in combination, it is possible to observe cells over a longer period of time using the slide glass 41.
Further, when the feature amount of the cell is not detected, the cell can be cultured for a long period of time by eliminating the influence of phototoxicity by blocking external ambient light. And since the carbon dioxide density | concentration in atmosphere can be equalize | homogenized, a cell can be culture | cultivated for a long term also by this. In addition, the temperature of the microplate 15 is used to control the temperature of the heaters in a coordinated manner, so that damage to the cells due to temperature changes can be reduced.
In addition, by acquiring an image of a cell with a CCD camera or the like (not shown), it is possible to easily record changes in cells in culture over time.

  Accordingly, the culture chamber 10 can be installed on the microscope stage of the observation machine and the biological sample such as cells can be cultured for a long period of time without being moved as it is. This makes it possible to easily detect changes in biological samples over time and to detect changes in biological samples that occur during the culture process in real time, and to accurately determine the behavior of biological samples such as cultured cells that are always activated (stabilized). A biological sample observation apparatus capable of detection can be realized.

<Second Embodiment>
Hereinafter, a second embodiment of the biological sample observation apparatus according to the present invention will be described with reference to FIGS. In addition, the same code | symbol is attached | subjected to the part similar to embodiment mentioned above, and the detailed description is abbreviate | omitted.
In the biological sample observation apparatus 100A of this embodiment, a fixed UV lamp 26 is attached to the opening (UV irradiation hole) of the opening / closing lid 23 'provided in the non-measurement area 14 of the culture chamber 10'. The UV irradiation lamp 26 is disposed in the casing so as to close the opening and to irradiate the inside of the culture chamber 10 ′ with ultraviolet rays. By adopting such a fixed UV lamp 26, it becomes possible to sterilize and sterilize the inside by simply irradiating ultraviolet rays when necessary.

Further, as shown in FIG. 9, in the temperature adjusting means of this embodiment, a planar heater 31 is attached to the outer surface side of the casing 11, and heat from the planar heater 31 is efficiently applied to the inner surface side of the casing 11. A heat radiating plate 38 for radiating heat is attached. And the outer surface side of the housing | casing 11 is set as the heat retention structure which attached the heat insulation material 39 to the outer side of the planar heater 31, and coat | covered it.
With the temperature adjusting means having such a configuration, the heat generated by the planar heater 31 can be efficiently transmitted from the housing 11 via the heat radiating plate 38 to warm the internal atmosphere. As a result, even if an abrupt temperature change occurs in the planar heater 31, the influence on the atmosphere can be minimized, and a soft (gradual) temperature change environment suitable for a biological sample such as a cell is formed. be able to. In addition, since it was set as the heat insulation structure by the heat insulating material 39, the calorie | heat amount which generate | occur | produces in the planar heater 31 and is thermally radiated to external air can be suppressed to the minimum.

Further, in the humidity adjusting means of this embodiment, as shown in FIGS. 8 and 9, a sterilized water pipe 61 for supplying sterilized water from the sterilized water storage container 60 can be connected to the water tank 34 in the non-measurement area 13. Therefore, the sterilizing water supply connector 62 is attached to the housing 11. The sterilized water supply connector 62 is connected to the water tank 34 through a water supply pipe 63 provided in the culture chamber 10 '. In addition, the code | symbol 35a in a figure is an electric wire for supplying with electricity to the heater 35. FIG.
As a method for supplying the sterilized water from the sterilized water storage container 60 to the water tank 34, the sterilized water may be pumped using a pump or the siphon effect may be used. However, when using the siphon effect, it is preferable to provide flow rate adjusting means such as a needle connector in the middle of the sterilizing water pipe 61 or the water supply pipe 63 so that the supply amount of the sterilizing water can be adjusted.

  In this way, if sterilized water can be supplied from the outside of the culture chamber 10 ′, it is possible to avoid a decrease in the humidity of the atmosphere due to depletion of the sterilized water, and thus culture can be easily performed for a longer period of time. Furthermore, by supplying the sterilized water continuously and stably, the liquid depth in the water tank 34 can be kept shallow, and an efficient supply of humidity can be performed.

Further, in the region where the objective lens 71 is inserted through the observation holes 24 and 25 and observed, a sheet 73 made of an elastic member covers the objective lens 71 and the revolver 72 as shown in FIG. It is suspended from the bottom. By providing such a sheet 73, it is possible to suppress the atmosphere in the culture chamber 10 ′ from flowing out without applying an extra load to the objective lens 71. That is, since the elastic member sheet 73 can be covered and covered with the revolver 72, the objective lens 71 is not directly subjected to a load, and the opening that causes the outflow of the atmosphere can be blocked.
Instead of the elastic member sheet 73, a metal or resin tubular member may be attached as a cover.

Further, the gas concentration adjusting means of this embodiment includes a CO ₂ concentration sensor 64 in the culture chamber 10 ′. The CO ₂ concentration CO ₂ concentration detected by the sensor 64 is input to the CO ₂ concentration control unit 105 '. The CO ₂ concentration control unit 105 ′ controls the supply amount (flow rate) supplied from the CO ₂ supply means 44 into the culture chamber 10 ′ in accordance with the input CO ₂ concentration, so that the internal atmosphere is more appropriate. It becomes possible to keep on.

  Further, for the glass top cover 22 'that is the cover on the measurement area 13 side, for example, as shown in FIG. 8, the area of the optical glass member such as the glass plate 22a' is limited to a minimum area that does not affect the observation. Thus, it is possible to prevent ambient light from entering the culture chamber 10 'more than necessary. As a matter of course, it is preferable to cover the entire apparatus with a light shielding member such as a black curtain. When transmission observation is not required, complete light shielding can be performed by placing a light shielding curtain only on the upper surface of the glass plate 22a '.

<Third Embodiment>
Next, a third embodiment of the biological sample observation apparatus according to the present invention will be described with reference to FIGS. In addition, the same code | symbol is attached | subjected to the part similar to embodiment mentioned above, and the detailed description is abbreviate | omitted.
In the biological sample observation device 100B of this embodiment, the casing 11 ″ of the culture chamber 10 ″ is fixed to the Y-axis operation stage 17. The casing 11 ″ of the culture chamber 10 ″ forms one area for measuring and culturing, and the upper surface opening is provided with a glass plate 22a that is an optical glass member having antireflection films on both sides. A glass top cover 22 is detachably installed as a cover. As in the second embodiment described above, the temperature adjusting means in this case includes the planar heater 31 and the heat insulating material 39 attached to the outer surface side of the casing 11 ″, and the planar heater on the inner surface side of the casing 11 ″. Although the structure which attached the heat sink 38 which thermally radiates the heat | fever of 31 efficiently is employ | adopted, as the structure which attached the planar heater 31 to the inner surface side of housing | casing 11 '' similarly to 1st Embodiment. Also good.

In addition, a dark box 74 that can store the Y-axis operation stage 17 is fixedly installed on the stage 70 of the microscope as a light shielding means in place of the above-described opening / closing lid 23. The dark box 74 has a large opening 74a on one side surface (left side of the drawing) in the X-axis direction in which the X-axis operation stage 16 moves. The opening 74a is closed by contacting a partition plate 75 fixed to the end surface of the Y-axis operation stage 17 at a predetermined (origin) position. As a result, the casing 11 ″ of the culture chamber 10 ″ is housed in a box-like closed space, so that the above-described predetermined position is set as the culture position, thereby blocking ambient light except during measurement. It becomes possible.
In this case, the UV lamp may be fixedly installed at an appropriate position of the dark box 74, or an opening serving as an irradiation hole is provided in the dark box 74, and a handy type UV lamp is used as in the first embodiment. Usually, it may be closed with a UV irradiation hole cover.

In addition, a connector 65 is provided on the side surface of the casing 11 ″ so that a culture atmosphere supply pipe 65a can be connected. In this case, by supplying the culture atmosphere whose humidity has been adjusted, the water tank 34 is provided. Without it, the culture chamber 11 ″ can be downsized.
Here, the supply of the culture atmosphere will be described. This culture atmosphere is supplied from a mixing tank 66 installed outside the culture chamber 11 ″. This mixing tank 66 is, for example, 37 ° C. containing 5% CO ₂ . It has the function of creating steam.

In the mixing tank 66, humidity control is performed by incubating sterile water for storing a predetermined temperature (e.g. 37 ° C.), further, in accordance with the CO ₂ concentration of the CO ₂ concentration sensor 64 culture chamber 11 in "detected by CO ₂ flow rate supplied from the CO ₂ supply means 44 is controlled. Therefore, in the mixing tank 66 is mixed with the supplied CO ₂ and humid air will be desired culture atmosphere, the culture atmosphere Is supplied to the culture chamber 10 ″ via the culture atmosphere pipe 65a by an air pump (not shown).

Further, with the configuration of such an embodiment, the transport mechanism (stage drive unit) of the X-axis operation stage 16 and the Y-axis operation stage 17 can be installed outside the culture chamber 10 ″, so that the high humidity and high temperature CO _2. There is no need to install a drive unit in the presence, and therefore, the selection range of the material used for the drive unit is widened, and the cost can be reduced.

  In addition, this invention is not limited to each embodiment mentioned above, In the range which does not deviate from the summary of this invention, it can change suitably.

It is a schematic block diagram which shows 1st Embodiment about the biological sample observation apparatus which concerns on this invention. It is a top view of the culture room shown in FIG. 1, and has shown the state which has a microplate (culture container) in the measurement position of a measurement area. It is a top view of the culture room shown in FIG. 1, and has shown the state which has a microplate (culture container) in the culture position of a non-measurement area. It is a flowchart which shows the basic operation (operation) regarding culture | cultivation and observation of a biological sample. It is a flowchart which shows operation | movement (operation) of an environmental control apparatus and an external element removal apparatus about the biological sample observation apparatus of 1st Embodiment. It is a figure which shows the modification concerning 1st Embodiment, and is a schematic block diagram of a culture system. It is a figure which shows the modification concerning 1st Embodiment, and is a top view of the culture room using the culture system of FIG. It is a schematic block diagram which shows 2nd Embodiment about the biological sample observation apparatus which concerns on this invention. It is a top view of the culture room shown in FIG. 8, and has shown the state which has a microplate (culture container) in the culture position of a non-measurement area. It is a schematic block diagram which shows 3rd Embodiment about the biological sample observation apparatus which concerns on this invention. It is a top view of the culture room shown in FIG. 10, and has shown the state which has a microplate (culture container) in the culture position of a non-measurement area. It is sectional drawing which shows the structural example of the container which culture | cultivates a biological sample under a microscope as a prior art example.

Explanation of symbols

10, 10 ', 10 "Incubation chamber 11, 11" Case 11a Base member 12 Upper opening 13 Measurement area 14 Non-measurement area 15 Microplate (culture vessel)
16 X-axis operation stage 17 Y-axis operation stage 18 Drive unit box 19 Top plate 20 Partition receiver 21 Partition plate 22, 22 'Glass top cover 23, 23' Open / close cover 26 UV irradiation lamp 31 Planar heater 32 Auxiliary heater 33 Temperature sensor 34 Water tank 35 Heater 36 CO ₂ supply port 36a CO ₂ pipe 37 Fan 38 Heat sink 39 Heat insulating material 40 Culture container 41 Slide glass 42 Culture liquid bottle 43 Culture liquid pump 44 CO ₂ supply means 45 Culture liquid pipe 60 Sterile water storage container 64 CO ₂ concentration sensor 66 Mixing tank 70 Stage 71 Objective lens 72 Revolver 73 Sheet 74 Dark box 74a Opening 75 Partition plate 100, 100A, 100B Biological sample observation device 101 Computer 102 X-axis operation control unit 103 Y-axis operation control unit 104 Incubator temperature control Part 105,1 5 'CO ₂ concentration controller 110 computer 111 culture bottle temperature controller 112 CO ₂ concentration control unit 113 broth pump control unit

Claims (24)

A biological sample observation device capable of culturing a biological sample and observing the biological sample in which the culture is performed,
A culture chamber capable of observing the inside from the outside, housing the culture container for holding the biological sample and performing the culture of the biological sample;
Environmental control means for controlling the culture chamber to an environment necessary for the culture of the biological sample;
When the observation is not performed, the environmental control means cannot control the external element and can remove an external element that can adversely affect the biological sample during the observation. Means,
A biological sample observation apparatus comprising:   2. The biological sample according to claim 1, wherein the external element removing unit is configured to allow an observer to remove the external element when the observation is not performed. Observation device.   The biological sample observation apparatus according to claim 2, wherein the external element is light, and the external element removing unit includes a light shielding unit that removes light irradiated to the inside of the culture chamber. .   The biological sample observation apparatus according to claim 2, wherein the external element is an impact or a vibration, and the external element removing unit includes a vibration isolating unit.   3. The external element is dust or various bacteria, and the external element removing means includes an intruder prevention means for preventing an intruder from entering the inside of the culture chamber. The biological sample observation device described.   The external element removing means is provided so as to be openable and closable with respect to the culture chamber. By closing the external element removing means, the influence of the external element on the biological sample can be removed. The biological sample observation apparatus according to claim 2, wherein The external element removing means is provided as a structure capable of covering a predetermined portion of the culture chamber,
The biological sample observation apparatus according to claim 2, wherein the influence of the external element on the biological sample can be removed by covering the predetermined portion of the culture chamber.   The external element removing means is configured to detect that the observation is not performed and automatically remove the external element when the observation is not performed. The biological sample observation apparatus according to claim 1.   The apparatus further comprises support means for movably supporting the culture container from a position where the external element can be removed by the external element removing means to a predetermined position where the observation of the biological sample is possible. The biological sample observation device according to claim 8.   The biological sample observation apparatus according to claim 8, wherein the external element is light, and the external element removing unit includes a light shielding unit that removes light irradiated to the inside of the culture chamber. .   The biological sample observation apparatus according to claim 8, wherein the external element is an impact or a vibration, and the external element removing unit includes a vibration isolating unit.   9. The external element is dust or bacteria, and the external element removal means includes an intruder prevention means for preventing an intruder from entering the inside of the culture chamber. The biological sample observation device described. The external element removing means is provided to be openable and closable with respect to the culture chamber,
The biological sample observation apparatus according to claim 8, wherein the external element removing unit is closed to remove the influence of the external element on the biological sample. The external element removing means is provided as a structure capable of covering a predetermined portion of the culture chamber,
The biological sample observation apparatus according to claim 8, wherein the influence of the external element on the biological sample can be removed by covering the predetermined portion of the culture chamber. The biological sample observation apparatus according to claim 1, wherein the biological sample observation apparatus is fixedly provided with a stage unit in the observation device that performs the observation. The biological sample observation apparatus according to claim 1, wherein the biological sample observation apparatus is configured to be detachable with respect to a stage unit in the observation device that performs the observation. The biological sample observation apparatus according to claim 15, wherein the observation device is a microscope. The biological sample observation apparatus according to claim 1, wherein the environment control unit includes a humidity adjustment unit capable of adjusting a humidity in the culture chamber. The biological sample observation apparatus according to claim 1, wherein the environment control unit includes a temperature adjustment unit capable of adjusting a temperature in the culture chamber. The biological sample observation apparatus according to claim 1, wherein the environment control means includes gas convection means capable of convection of the atmosphere in the culture chamber. 2. The biological sample observation according to claim 1, wherein the environmental control means further includes gas concentration adjusting means capable of adjusting a concentration of a predetermined gas necessary for culturing the biological sample in the culture chamber. apparatus. The biological sample observation apparatus according to claim 21, wherein the gas contains carbon dioxide having a predetermined concentration. The biological sample observation apparatus according to claim 1, further comprising a sterilizing light irradiation unit capable of irradiating the inside of the culture chamber with sterilizing light when the culture is not performed. The biological sample observation apparatus according to any one of claims 1 to 23, wherein the biological sample includes cells.

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