JP6664909B2 - Imaging optical device - Google Patents

Description

  The present invention relates to an imaging optical device that images a movable object such as a cell or a microorganism that exists in a medium such as a liquid.

  Conventionally, cells, microorganisms, and the like have been cultured in a flat container such as a petri dish. A culture solution for culturing cells, microorganisms, and the like is observed with a microscope from the bottom side of the container or from the upper side of the container, and the state of division and growth thereof is visualized (for example, see Patent Document 1). The quality of growth of cells, microorganisms, and the like has been determined by imaging cells, microorganisms, and the like in a stationary state with a camera and visually recognizing the image with the naked eye.

JP-A-2004-348104

  Cells cultured by stirring in a large-capacity culture vessel are always in a state of floating movement in the culture solution, and can freely move three-dimensionally in the culture vessel. There is also a technique of collecting and observing cells in culture from a culture vessel, but this technique only observes a part of cells in the culture solution, and the range of cells that can be observed is limited. It has been difficult to accurately judge the overall culture state of the existing cells.

  Moreover, in the cell culture equipment, it is necessary to install as many culture vessels as possible in order to culture a large amount of cells. For this reason, the culture vessels are arranged adjacent to each other so that no gap is created. However, when the observation device is installed in a culture vessel, a gap is generated between adjacent culture vessels and the number of culture vessels is reduced, the original purpose of culturing a large number of cells can be achieved. It becomes difficult.

  The present invention has been made in view of the above points, and an object of the present invention is to enable non-invasive, high-speed and continuous observation of cells, microorganisms, and the like remotely and in a plurality of culture vessels. It is an object of the present invention to provide an imaging optical device capable of observing cultured cells and microorganisms.

An embodiment of the imaging optical device according to the present invention includes:
A light-emitting unit that emits light, and a light-emitting imaging unit that has an imaging unit that captures an image of the inside of a container irradiated with the light emitted from the light-emitting unit,
A reflector that reflects light, and a reflector that is disposed so as to face the light-emitting imaging unit with the container interposed therebetween,
The container has a surrounding wall that surrounds the medium to be housed housed in the container, and has a surrounding wall through which light emitted from the light emitting unit can pass,
The light emitted from the light emitting unit passes through the surrounding wall toward the reflector, is reflected by the reflector, and then illuminates the storage medium.

  The cells can be observed remotely and non-invasively at high speed and continuously, and the cells cultured in each of the plurality of culture vessels can be observed.

FIG. 1 is a perspective view schematically showing an imaging optical device 100. FIG. 2 is a plan view schematically showing the imaging optical device 100. FIG. 2A is a perspective view schematically illustrating the light-emitting imaging device 120, and FIG. 2B is a front view illustrating the light-emitting unit 130 and the CCD camera 150 of the light-emitting imaging device 120. FIG. 3 is a perspective view illustrating a configuration of a reflector 200. FIG. 3 is a perspective view showing a state where the imaging optical device 100 is attached to a container 300. FIG. 3 is a conceptual diagram illustrating an optical path of light emitted from a light-emitting imaging device. It is a schematic diagram when light enters into interface S1 from container 300 (plastic). FIG. 3 is a conceptual diagram illustrating an optical path of light emitted from a light-emitting imaging device. It is a figure showing other modes of a side of a container. FIG. 9 is a perspective view schematically showing a housing 910. FIG. 9 is a plan view schematically showing a housing 910. FIG. 3 is a schematic diagram showing total reflection generated at an interface S1 due to unevenness formed on an inner peripheral surface 202 of a reflector 200.

  Hereinafter, embodiments will be described with reference to the drawings.

According to a first embodiment of the present invention,
A light emitting unit that emits light (for example, a light emitting unit 130 to be described later) and an imaging unit that captures the inside of a container (for example, a container 300 to be described later) irradiated with the light emitted from the light emitting unit (for example, to be described later) Light-emission imaging means (e.g., a light-emission imaging device 120 described later) having
A reflector (eg, a reflector 200 described later) that is a reflector that reflects light and that is arranged to face the light-emission imaging unit with the container interposed therebetween.
The container is a surrounding wall that surrounds a medium to be housed (for example, a culture solution to be described later) housed in the container, and is a surrounding wall through which light emitted from the light emitting unit can pass (for example, a container 300 to be described later). Cylindrical side surface 304, etc.)
The light emitted from the light emitting unit passes through the surrounding wall toward the reflector (for example, light A described later), and is reflected by the reflector, and then illuminates the medium to be accommodated (for example, , Light B described below).

  The imaging optical device includes a light emitting imaging unit and a reflector.

  The light-emission imaging unit has both a light-emitting unit and an imaging unit. The light emitting section emits light having a predetermined intensity at a predetermined wavelength. The light emitted from the light emitting unit is irradiated on the container to illuminate the container. The imaging unit captures an image of the inside of the illuminated container.

  Since the light-emitting unit and the imaging unit are located on the same side of the container as the light-emitting imaging means, it is not necessary to secure a space for installing the light-emitting unit and a space for installing the imaging unit separately and at different positions. Therefore, even when a plurality of containers are installed, the light emission imaging means can be provided so that no gap is generated between adjacent containers. In this way, a large amount of cells, microorganisms, and the like can be cultured by arranging a plurality of containers densely without gaps while saving space and effectively utilizing the space.

  It is preferable that the light-emitting imaging unit is one in which the light-emitting unit and the imaging unit are integrally formed. In particular, it is preferable that the light emitting unit and the imaging unit are arranged concentrically. For example, it is more preferable that a light emitting unit is arranged around the imaging unit around the imaging unit. By arranging the light-emitting imaging unit in a predetermined direction, the light-emitting unit emits light in a predetermined direction, and the imaging unit can receive light from a predetermined direction.

  The reflector reflects light. The reflector is disposed so as to face the light-emitting imaging means with the container interposed therebetween. The container is located between the reflector and the luminescence imaging means, and is sandwiched between the reflector and the luminescence imaging means.

  The container has a surrounding wall. The surrounding wall surrounds the storage medium stored in the container. The surrounding wall is formed so that light emitted from the light emitting unit can pass therethrough. Since the light emitted from the light emitting section can pass through the surrounding wall, the medium to be accommodated contained in the container is illuminated by the light emitted from the light emitting section.

  First, the light emitted from the light emitting unit passes through the surrounding wall and travels toward the reflector. Thereafter, the light is reflected by the reflector and illuminates the medium to be accommodated. The light reflected by the reflector illuminates the medium to be contained.

  As described above, the accommodated medium is illuminated not only with the light emitted from the light emitting unit, but also with the light reflected by the reflector, so that the accommodated medium can function as a background and be imaged brightly.

  Since the image is emitted by irradiating the light with the light emission imaging unit, it is possible to observe the culture state of the cells and microorganisms while maintaining an environment in which cells and microorganisms can be cultured without human intervention. Further, since the medium to be housed functions as a background and is imaged brightly, it can be imaged at high speed, for example, even at a high shutter speed, and can clearly image cells, microorganisms, and the like moving in the container.

  The imaging optical device is effectively used for imaging a movable object such as a cell or a microorganism (hereinafter, simply referred to as a “cell or the like”) existing in a medium such as a liquid, and particularly, an embryonic stem cell (ES cell). And an artificial pluripotent stem cell (iPS cell), an embryoid body (embryoid body) of a stem cell such as a somatic stem cell, or a cell aggregate.

A second embodiment of the present invention is the first embodiment of the present invention,
The reflector is arranged along the surrounding wall.

  Since the reflector is arranged along the surrounding wall, the light emitted from the surrounding wall can be accurately directed to the reflector, and the light reflected by the reflector can be accurately returned to the surrounding wall. The light emitted from the light emitting section can be efficiently reflected by the reflector without wasting, and the medium to be accommodated can be illuminated by the reflected light.

  Furthermore, a reflector can be arranged so as not to interfere with an adjacent container, and a large number of cells can be cultured by disposing many containers.

A third embodiment of the present invention is the first embodiment of the present invention,
The refractive index of the surrounding wall is larger than the refractive index of air.

  Since the refractive index of the surrounding wall is larger than the refractive index of air, light emitted from the light emitting unit can be reflected by the surrounding wall. By reducing the amount of light emitted from the light emitting unit to leak to the outside of the surrounding wall (outside of the container), and returning the light reflected by the surrounding wall to the medium to be stored, the medium to be stored is illuminated more brightly, and The medium can function as a bright background.

A fourth embodiment of the present invention is the first embodiment of the present invention,
The surrounding wall is
An inner wall surface (for example, an inner peripheral surface 202 described later) that forms an interface between the accommodation medium and the surrounding wall,
An outer wall surface (for example, an outer peripheral surface 204 described later) that forms an interface between the air outside the container and the surrounding wall,
The outer wall surface reflects light incident from the storage medium and returns the light to the storage medium.

  The surrounding wall has an inner wall surface and an outer wall surface. The inner wall forms an interface between the contained medium and the surrounding wall. The outer wall forms an interface between the air outside the container and the surrounding wall. The outer wall surface reflects light incident from the storage medium and returns the light to the storage medium. By returning the light reflected by the outer wall surface of the surrounding wall to the storage medium side, the storage medium can be illuminated more brightly.

A fifth embodiment of the present invention is the first embodiment of the present invention,
The reflector has irregularities formed on a reflection surface.

  Since unevenness is formed on the reflecting surface of the reflector, diffuse reflection occurs on the reflecting surface. By causing diffuse reflection, even when illumination with high directivity is used, it is possible to generate a large amount of light that is totally reflected by setting the incident angle larger than the critical angle. By passing a large amount of light that repeats total reflection through the storage medium, the storage medium serving as the background can be illuminated more brightly, and the contrast can be further increased by increasing the chance of illuminating the subject (cells). The critical angle is determined by the ratio of the refractive index of air existing outside the container (about 1) to the refractive index of the container (about 1.5 to 1.6).

According to a sixth embodiment of the present invention,
The light-emitting unit (for example, a light-emitting unit 130 described later) and the imaging unit (for example, a CCD camera 150 described later) include a container (for example, a container described later) that stores a medium to be stored (for example, a culture solution described later). 300) and a reflector (for example, a reflector 200 described later) is interposed therebetween.
Light emitted from the light emitting unit (for example, light A described later) and light emitted from the light emitting unit and reflected by the reflector after passing through the container and returned to the container (for example, light described later) B etc.), and illuminating the medium to be accommodated contained in the container by:
An imaging step of imaging the inside of the container by an imaging unit.

  Since the light emitting unit and the imaging unit are located on the same side of the container, there is no need to secure a space for installing the light emitting unit and a space for installing the imaging unit separately and at different positions. Therefore, even when a plurality of containers are installed, the light emitting unit and the imaging unit can be provided so that no gap is generated between adjacent containers. Thus, a large amount of cells and the like can be cultured by arranging a plurality of containers densely without gaps while saving space and effectively utilizing the space.

  The accommodated medium is illuminated not only with the light emitted from the light emitting unit, but also with the light reflected by the reflector, so that the accommodated medium can function as a background and be imaged brightly.

  Since light is emitted from the light emitting unit and the image is taken by the imaging unit, it is possible to observe the culture state of the cells and the like while maintaining an environment where cells and the like can be cultured without human intervention. Furthermore, since the accommodation medium functions as a background and is imaged brightly, high-speed imaging can be performed, for example, even at a high shutter speed, and cells and the like moving in the container can be clearly imaged.

  More specifically, there are the following embodiments. In the following, a cell or the like to be imaged will be described as a “cell”, and a medium to be accommodated will be described as a “culture solution”.

Another embodiment of the present invention provides:
An area camera (for example, a CCD camera 150 and the like described below) closely or closely contacted with a transparent culture vessel (for example, a container 300 and the like described below) made of resin or glass,
A lens having a focal length for observing the culture vessel through transmission (for example, an objective lens 156 described later),
(For example, an imaging optical device 100 described later).

  Further, a light guiding fiber (for example, an optical fiber 132 described later) is arranged on an outer peripheral portion of a lens (for example, an objective lens 156 described later) of an area camera (for example, a CCD camera 150 described later). The light-guiding fiber guides illumination light for illuminating the culture vessel, and emits illumination light from an emission surface (for example, an emission surface 136 described below) to function as a lighting device. By integrating the illumination device and the area camera, the overall size can be reduced, and the space at the outer peripheral portion of the objective lens 156 can be effectively used.

  In addition, a light guide fiber (for example, an optical fiber 132 described later) is connected to a light source device (for example, a common light source 400 described later), and the light emitted from the light source device is cultured via the light guide fiber. It leads to a container and illuminates the inside of the culture container. By using a light-guiding fiber (for example, an optical fiber 132 to be described later), a high-intensity light source device can be installed at a position separated from the culture vessel. Since the light source device is installed at a position separated from the culture container, the heat generated from the light source device is not easily transmitted to the culture container, and the influence of the heat on the culture environment can be prevented.

  Furthermore, when a multi-branch fiber is used as the light guiding fiber, each of the culture vessels can be illuminated by branching the light emitted from a single light source device and supplying it to a plurality of culture vessels. Since the number of light source devices can be reduced, space saving and energy saving can be achieved.

  Furthermore, the illumination light emitted from the emission surface (for example, the emission surface 136 described below) of the light guide fiber travels inside the culture vessel while spreading within a range of the emission angle (about 70 degrees). Part of the illumination light emitted from the emission surface is applied to a subject (for example, cells) existing inside the culture vessel. The remaining illumination light that has not been irradiated on the subject reaches the opposite side of the culture container facing the area camera (for example, a CCD camera 150 described later) with the culture container therebetween.

  Furthermore, a reflector (made of metal, mirror, film, etc.) for reflecting light is installed on the opposite side of the culture vessel facing the area camera with the culture vessel in between. The reflector is formed according to the shape of the culture vessel. Further, magnesium oxide, barium sulfate, or the like having a reflective effect may be applied to the outer peripheral surface on the opposite side of the culture container facing the area camera with the culture container interposed therebetween. The illumination light can be returned to the culture container by reflecting the illumination light with a reflector or the like. Note that a reflection medium may be provided or applied to the inner peripheral surface of the culture container as long as it does not affect the culture environment of the cells and the like.

  In addition, when the reflection angle of the illumination light returned to the culture container becomes a critical angle or more due to the shape of the culture container (cylindrical), the illumination light is scattered and reflected in the culture container a plurality of times, and Light is trapped in the culture vessel. By scattering and reflecting the illumination light multiple times, the culture solution is illuminated at multiple locations, enhancing the effect of the cell contour and background (contrast between the culture solution and the cells), and floating and moving at high speed. The shutter can be imaged at high speed with the shuttered cells. Since images can be taken at a high speed with the shutter released, an image can be obtained as a still image of cells moving at a high speed.

  Furthermore, by taking images with the shutter released at a high speed, continuous imaging is facilitated, and the number of times of imaging within the imaging depth of focus is increased, making it possible to observe a large number of cells contained in a large-capacity culture container. Can be more.

<<<< Imaging optical device 100 >>>>
FIG. 1 is a perspective view showing the configuration of the imaging optical device 100. FIG. 2 is a plan view showing the configuration of the imaging optical device 100. As shown in FIGS. 1 and 2, the imaging optical device 100 includes a housing 110, a light-emitting imaging device 120, and a reflector 200. In FIG. 2, the housing 110, the light-emitting imaging device 120, and the reflector 200 are separated from each other in order to clarify the relationship. Further, in FIG. 2, the container 300 is shown by an imaginary line, and the stirring blade 310 is omitted (see FIG. 5).

  FIG. 5 is a perspective view showing a state in which the imaging optical device 100 is attached to the container 300. The container 300 has a circular bottom surface 302 and a cylindrical side surface 304 (surrounding wall). As will be described later, light enters, reflects, and exits on the cylindrical side surface 304. As described above, in the present embodiment, the incidence, reflection, and refraction of light on a curved surface such as the cylindrical side surface 304 are used. The side surface 304 of the container 300 may be made of a material that can transmit and reflect light, and may be made of a transparent material or a translucent material.

  The container 300 contains a culture solution containing various cells such as IiPS cells. A stirring blade 310 is rotatably provided in the container 300. The stirring blade 310 is rotationally driven by a stirrer (not shown). By rotating the stirring blade 310, the culture solution contained in the container 300 is stirred. For example, the stirring blade 310 can be rotated at 40 to 60 rpm (rotation per minute). As long as the culture solution is stirred by the stirring blade 310, the cells and the like always float and move in accordance with the flow of the culture solution.

<< Case 110 >>
As shown in FIG. 1, the housing 110 has a cylindrical band 112 having a substantially cylindrical shape. The cylindrical band portion 112 has a cylindrical inner peripheral surface 114 and a cylindrical outer peripheral surface 116. The housing 110 is detachably attached to the container 300 so as to wind the side surface 304 of the container 300 (see FIG. 5).

  By winding the housing 110 around the side surface 304, the housing 110 can be attached to the container 300 in close contact, and even when vibration or impact occurs, the housing 110 can be stably held on the container 300, By keeping the relative position between the housing 110 and the container 300 constant, cells and the like can be clearly imaged by the light-emitting imaging device 120.

  The cylindrical band portion 112 has a structure that can be expanded and contracted in the longitudinal direction (circumferential direction) by an elastic body such as rubber or a spring, and the housing 110 can be attached to the container 300 in close contact. Alternatively, the housing 110 can be attached to the container 300 by using a fixing member such as a screw or a bolt so as to be tightly attached to the container 300.

<< Light Emission Imaging Device 120 (Light Emission Imaging Unit) >>
The light-emitting imaging device 120 is attached to the cylindrical band 112 of the housing 110. The cylindrical band portion 112 is provided with a holding portion 118 (see FIG. 2). The light-emitting imaging device 120 is fixed to the cylindrical band portion 112 via the holding portion 118 by a locking member (not shown) such as a bolt or a screw.

  The light-emitting imaging device 120 includes a light-emitting unit 130 including an optical fiber 132 and a CCD camera 150. The CCD camera 150 functions as an area camera.

  FIG. 3A is a perspective view schematically showing the light-emitting imaging device 120, and FIG. 3B is a front view showing the light-emitting unit 130 and the CCD camera 150 of the light-emitting imaging device 120.

<CCD camera 150 (imaging unit)>
The CCD camera 150 includes an optical unit 152 and an electric unit 160. The optical system section 152 has a substantially cylindrical shape, and mainly includes various optical elements such as an objective lens 156 and a filter (not shown). The optical system section 152 has an opening 154, and light reflected by the imaging target enters the opening 154. The light path of the light incident on the opening 154 is adjusted by various optical elements such as the objective lens 156. As the CCD camera 150, a monochrome area camera or the like can be used.

  The electrical system section 160 has a substantially rectangular parallelepiped shape, and includes a CCD image sensor section (not shown), a signal processing circuit section (not shown), and the like. The light whose optical path has been adjusted by the optical system 152 enters the CCD image sensor to form an image. The electric system unit 160 converts an image formed by the CCD image sensor unit into an electric signal, performs various kinds of signal processing such as digitization, and outputs the image signal as an imaging signal.

<Light emitting unit 130 (light emitting unit)>
The light emitting unit 130 includes a plurality of optical fibers 132. Each of the plurality of optical fibers 132 has an input surface 134 and an output surface 136. As shown in FIG. 2, all the incident surfaces 134 of the plurality of optical fibers 132 are connected to the common light source 400. The common light source 400 is formed of a light emitting body such as an LED (light emitting diode). The light emitted from the common light source 400 is branched into each of the plurality of optical fibers 132, propagates, and is emitted from the emission surface 136 (FIGS. 3A and 3B). As described above, light can be emitted from the plurality of optical fibers 132 using the single common light source 400.

  Since the light emitted from the common light source 400 is propagated by the plurality of optical fibers 132, the common light source 400 can be arranged at a position separated from the container 300. For this reason, it is possible to prevent the heat generated from the common light source 400 from being transmitted to the culture solution or the cells contained in the container 300, and to reduce the influence of the heat generated from the common light source 400 to keep the culture environment constant. it can.

  As shown in FIG. 3A, each end of the plurality of optical fibers 132 is arranged on the outer peripheral surface of the optical system 152 of the light emitting unit 130 along the longitudinal direction. Further, as shown in FIG. 3B, the emission surfaces 136 of the plurality of optical fibers 132 are arranged at equal intervals so as to go around the outer peripheral surface of the opening 154 of the optical system 152.

  A substantially cylindrical holder 170 is attached to the outside of the plurality of optical fibers 132 (see FIG. 3B). The plurality of optical fibers 132 are held between the outer peripheral surface of the optical system 152 and the inner peripheral surface of the holder 170. That is, the plurality of optical fibers 132 are arranged so as to cover the optical system section 152, and the holder 170 is covered so as to cover the optical system section 152 and the plurality of optical fibers 132. By holding the plurality of optical fibers 132 by the holding body 170, the direction of light emitted from the emission surface 136 can be kept constant.

  As shown in FIG. 6, the CCD camera 150 moves the opening 154 (objective lens 156) of the optical system unit 152 and the emission surface 136 of each of the plurality of optical fibers 132 toward the side surface 304 of the container 300. It is attached. For example, the CCD camera 150 is mounted such that the axial direction of the optical system 152 is substantially perpendicular to the housing 110.

<< Reflector 200 >>
FIG. 4 is a perspective view showing the configuration of the reflector 200. The reflector 200 has an elongated shape, and has a shape curved along the longitudinal direction, for example, a substantially semi-cylindrical shape. As shown in FIGS. 1 and 5, the height (transverse direction) of the reflector 200 is the same as the height (width) of the housing 110 or is higher than the height (width) of the housing 110. short. The reflector 200 has an inner peripheral surface 202 (inner wall surface) and an outer peripheral surface 204 (outer wall surface).

  As shown in FIGS. 1 and 2, the reflector 200 is provided along the cylindrical band 112. Specifically, the outer peripheral surface 204 of the reflector 200 is attached so as to be in close contact with the inner peripheral surface 114 of the cylindrical band 112. For example, the reflector 200 can be fixed to the cylindrical band 112 using an adhesive or the like. When the reflector 200 is attached to the cylindrical inner peripheral surface 114 of the cylindrical band portion 112 and the casing 110 is attached to the container 300, the inner peripheral surface 202 of the reflector 200 is attached closely to the side surface 304 of the container 300. be able to.

  The reflector 200 is formed of a predetermined metal, for example, a thin plate of aluminum, copper, or the like. The metal only needs to have a high reflectance. By forming the reflector 200 by bending a thin metal plate, the reflector 200 can be provided along the cylindrical band portion 112. By forming the reflector 200 with a thin metal plate, light can be reflected on the inner peripheral surface 202 of the reflector 200.

  Further, fine irregularities are formed on the inner peripheral surface 202 of the reflector 200. The unevenness allows not only the light incident on the inner peripheral surface 202 of the reflector 200 to be simply reflected but also diffusely reflected (reflected while being scattered). By appropriately changing the surface roughness, for example, the shape and size of the unevenness, the tendency and degree of spread of diffuse reflection can be determined.

  Further, the base of the reflector 200 may be formed of not only a thin metal plate but also a flexible material such as plastic. In this case, by forming a metal thin film or the like on the inner peripheral surface 202 of the reflector 200 by coating, vapor deposition, sputtering, or the like, light can be reflected similarly to a thin metal plate. The film may be formed using a material having high reflectance. For example, magnesium oxide, barium sulfate, or the like having a reflective effect can be used.

  Furthermore, the reflector 200 may be formed using a flexible film-shaped material as a base material. Since it is in the form of a film, it is easily deformed, so that the adhesion between the inner peripheral surface of the reflector 200 and the side surface 304 of the container 300 is improved, and the light reflected by the reflector 200 can be accurately returned to the container 300.

  Furthermore, diffuse reflection can be achieved by forming irregularities on the surface of the base material before forming the metal thin film.

  In the above-described example, the case where the reflector 200 has a substantially semi-cylindrical shape has been described. However, the shape is not limited thereto, and the reflector may be provided at a portion other than a portion where light necessary for the light-emitting imaging device 120 passes. it can. In addition, although the reflector 200 is configured separately from the container 300, the reflector 200 may be configured so that a part of the container 300 is the reflector 200. Further, the material forming the reflector 200 is not limited to metal, but may be any material that can reflect light, and may be a resin sheet or the like having a high reflectance.

<<< Lighting of culture solution in container 300 >>
FIG. 6 is a conceptual diagram illustrating an optical path of light emitted from the light-emitting imaging device 120. In FIG. 6, the housing 110 and the stirring blade 310 are omitted to clearly show the optical path. The container 300 is made of plastic, and a culture solution is accommodated inside the container 300. Air exists outside the container 300. FIG. 6 mainly considers light emitted from the common light source 400 and light reflected by the reflector 200.

  Light emitted from the common light source 400 and traveling through the optical fiber 132 is emitted from the emission surface 136 of the optical fiber 132. The light A emitted from the emission surface 136 travels while spreading at a light distribution angle (emission angle) θD. For example, the light distribution angle θD is about 70 degrees.

  The light A emitted from the emission surface 136 passes through the side surface 304 of the container 300, enters the culture solution contained in the container 300, and illuminates the culture solution (light of the arrow A). After that, the light A that has passed through the culture solution passes through the side surface 304 and reaches the reflector 200. The light that has reached the reflector 200 is reflected by the reflector 200. The light B reflected by the reflector 200 passes through the side surface 304 and enters the culture solution. The light that has entered the culture solution illuminates the culture solution (light of arrow B).

  As described above, not only the light A emitted from the common light source 400 passes through the culture solution, but also the light B reflected by the reflector 200 passes through the culture solution. In this manner, by passing the light B reflected by the reflector 200 through the culture solution, the light can enter the culture solution a plurality of times, and the culture solution can be brightly illuminated.

<<< Imaging by Light Emission Imaging Device 120 >>>
<Cell imaging>
An objective lens 156 is provided in the optical system section 152 of the light-emitting imaging device 120. For example, a telecentric lens can be used as the objective lens 156. By appropriately setting the magnification of the objective lens 156, an area within the imaging depth of focus can be determined as the imaging area M (see FIG. 6). The CCD camera 150 focuses on the cells located in the imaging region M by the objective lens 156 to clearly image the cells.

  The imaging region M is located between the stirring blade 310 and the light-emitting imaging device 120 and can be located closer to the light-emitting imaging device 120 than the stirring blade 310. More preferably, the imaging region M can be located at a position closer to the luminescence imaging device 120 than to the center between the stirring blade 310 and the luminescence imaging device 120. Specifically, the position of the imaging region M can be set to a position facing the stirring blade 310 about 1 mm from the side surface 304 of the container 300.

  By setting the position of the imaging region M at a position out of focus with the stirring blade 310, it is difficult to image the stirring blade 310, and the cells can be clearly imaged. The position of the imaging region M may be any position where the stirring blade 310 is hardly imaged and cells can be clearly imaged, and can be determined by appropriately determining the position of the light-emitting imaging device 120 and the magnification of the objective lens 156.

  In FIG. 5, in order to clearly show the configuration of the housing 110, the light-emitting imaging device 120, and the reflector 200 in a state where the housing 110 is attached to the container 300, the stirring blade 310 is shifted upward from the light-emitting imaging device 120. I showed it. However, even when the stirring blade 310 is positioned below the position in FIG. 5 and is positioned in front of the imaging region M of the light-emitting imaging device 120, as described above, the position of the imaging region M is This is a position where the wing 310 is out of focus, and the image of the agitating wing 310 is difficult to be imaged, so that cells and the like located in the imaging region M can be clearly imaged.

<Imaging of culture solution>
The container 300 contains a culture solution containing cells. As described above, the culture solution is illuminated by the light A emitted from the emission surface 136 and the light B reflected by the reflector 200. The CCD camera 150 images the culture solution illuminated with the light A and the light B as a background. That is, although the objective lens 156 does not focus on most of the culture solution other than the imaging region M, the light illuminating the culture solution enters the CCD camera 150 via the objective lens 156, and the brightly illuminated culture solution Acts as a background.

<Imaging of cells and culture medium>
The CCD camera 150 focuses on the cells located in the imaging region M by the objective lens 156, and images the cells. Further, light illuminating the culture solution enters the CCD camera 150 via the objective lens 156. In this way, the CCD camera 150 can use the light illuminating the culture solution as a background and take an image in comparison with the cell as the target. By increasing the amount of light that passes through the culture, the culture can be brightly illuminated and act as a background, increasing the contrast with the focused cells and clearly imaging the target cells. it can.

  When the reflector 200 is not provided, the light is not returned to the culture solution by the reflector 200 after passing through the culture solution once. For this reason, the light passing through the culture solution cannot be increased, the background cannot be brightened, the contrast with the cells cannot be increased, and it becomes difficult to clearly image the cells.

  The imaging optical device 100 does not illuminate the culture medium only with the light A emitted from the common light source 400, but also illuminates the culture medium with the light B reflected by the reflector 200. Can work.

  The light emitting imaging device 120 is disposed so as to face the reflector 200 with the container 300 interposed therebetween. In the light-emitting imaging device 120, the light-emitting unit 130 and the CCD camera 150 are integrally formed. If the luminescence imaging device 120 is installed in the container 300, both the light emitting unit 130 and the CCD camera 150 can be installed in the container 300 at the same time. By integrating the light emitting unit 130 and the CCD camera 150, it is not necessary to secure a space for installing the light emitting unit 130 and a space for installing the CCD camera 150 separately and at different positions. Therefore, the imaging optical devices 100 adjacent to each other are densely arranged to save space, so that space can be effectively used.

<< Influence of rotation of stirring blade 310 >>
The culture solution is stirred by the rotation of the stirring blade 310. By agitating the culture solution, the cells in the growth process always move in the container 300. The position of the imaging region M is a position where the focus is not focused on the stirring blade 310, and the stirring blade 310 is hardly imaged. Therefore, of the moving cells, only the cells located in the imaging region M can be clearly imaged. Even in a state where the culture solution is stirred by rotating the stirring blade 310, the cells can be imaged by the imaging optical device 100. Therefore, cells in the culture process can be imaged without interrupting the culture process or collecting the cells.

  The cells are constantly moving in the container 300 due to the stirring of the culture solution, and the shutter speed is determined according to the moving speed of the cells. As described above, the culture solution contained in the container 300 is brightly illuminated by the light A emitted from the common light source 400 and the light B reflected by the reflector 200. Therefore, even when the shutter speed is increased, cells can be clearly imaged.

  Further, even when the output itself of the common light source 400 is reduced, the culture solution can be sufficiently illuminated by the light A and the light B. By reducing the output of the common light source 400, the influence of heat generated from the common light source 400 can be reduced and the culture environment can be kept constant.

  As described above, the shutter speed is determined according to the moving speed of the cells in the container 300 (for example, the rotation speed of the stirring blade 310), and the intensity of the common light source 400 can be optimized according to the determined shutter speed. In this way, cells can be imaged with optimal intensity of illumination, and cells can be clearly imaged.

<< Diffuse reflection due to unevenness of reflector 200 >>
As described above, fine irregularities are formed on the inner peripheral surface 202 of the reflector 200, and diffuse reflection occurs. Due to the diffuse reflection, light B1 to B4 (see FIG. 6) that gradually diffuses from the inner peripheral surface 202 is generated, and the light B1 to B4 illuminates the culture solution over a wide range. By illuminating the culture solution over a wide range, the brightness of the culture solution as the background can be made uniform regardless of the location.

  Since irregularities are formed on the inner peripheral surface 202 of the reflector 200, diffuse reflection occurs due to the irregularities. By causing diffuse reflection, even when illumination with high directivity is used, much light that is totally reflected at the interface S1 of the side surface 304 of the container 300 can be generated. By causing light having an incident angle larger than the critical angle to enter the interface S1, total reflection can be caused at the interface S1.

  For example, as shown in FIG. 12, the light X1 is incident on the inner peripheral surface 202 of the reflector 200. Irregularities are formed on the inner peripheral surface 202, and the inclination of the irregularities differs depending on the location of the inner peripheral surface 202. For this reason, depending on the inclination of the unevenness at the position where the light X1 is incident, the light is reflected in a direction (light X2) that is more inclined than when the unevenness is not formed on the inner peripheral surface 202 (light X2 '). Furthermore, when the light X2 advances and is reflected by the inner peripheral surface 202 of the reflector 200 and returns to the culture solution (light X3), the incident angle θI may be larger than the critical angle and enter the interface S1 of the container 300. . In this case, total reflection (θR = θI) occurs (light X4). The critical angle is determined by the ratio of the refractive index of air existing outside the container 300 (about 1) to the refractive index of the container 300 (about 1.5 to 1.6).

  As described above, by forming the unevenness on the inner peripheral surface 202 of the reflector 200, a large amount of light totally reflected at the interface S1 can be generated. By passing light that repeats total reflection through the culture solution, the culture solution serving as the background can be illuminated more brightly, and the contrast can be further increased by increasing the chance of illuminating the cells.

<<< Interface in Container 300 >>>
7A and 7B are diagrams illustrating a case where light enters the interface S1 from the side surface 304 of the container 300. FIG. 7A and 7B, the container 300 is made of plastic, and a culture solution is contained inside the container 300. Air exists outside the container 300. The interface between the air and the side surface 304 of the container 300 is called an interface S1, and the interface between the side surface 304 of the container 300 and the culture solution is called an interface S2. FIG. 7A is a schematic diagram illustrating an example where the incident angle θI at the interface S1 is smaller than the critical angle. FIG. 7B is a schematic diagram showing an example where the incident angle θI at the interface S1 is larger than the critical angle.

  Refractive index of air <refractive index of culture solution <refractive index of plastic. For example, air has a refractive index of about 1, culture medium has a refractive index of about 1.4, and plastic has a refractive index of about 1.5.

  In FIG. 7A, the incident angle θI is smaller than the critical angle. Therefore, the incident light I is separated into the reflected light R and the refracted light F at the interface S1. The reflected light R is reflected at the interface S1 and returns to the side surface 304 of the container 300. After passing through the interface S2, the reflected light returns to the culture solution and illuminates the culture solution. On the other hand, the refracted light F exits from the interface S1 to the air.

  In FIG. 7B, the angle of incidence θI is greater than the critical angle and total internal reflection occurs. That is, the incident light I is totally reflected at the interface S1 to become the reflected light R, and there is no light emitted from the interface S1 to the air. The reflected light R reflected at the interface S1 returns to the side surface 304 of the container 300, passes through the interface S2, returns to the culture solution, and illuminates the culture solution.

  In the above-described example, the case where the container 300 is made of resin such as plastic is shown, but other types of resin or glass may be used. Further, the refractive index of the container 300 can be set to 1.5 to 1.6.

  In the example described above, the refractive index of air is about 1, the refractive index of the culture solution is about 1.4, and the refractive index of plastic is about 1.5, and it is mainly incident, reflected, and refracted at the interface S1. Was considered. Depending on the refractive index of the culture solution and the magnitude of the refractive index of the container 300, the amount of light reflected at the interface S2 and returning to the culture solution may increase. The light reflected at the interface S2 returns to the culture solution to illuminate the culture solution.

<< Continuous formation of reflected light >>
FIG. 8 is a conceptual diagram illustrating an optical path of light emitted from the light-emitting imaging device 120. The same components as those in FIG. 6 are denoted by the same reference numerals. In FIG. 8, as in FIG. 6, the housing 110 and the stirring blade 310 are omitted to clearly show the optical path. The container 300 is made of plastic, and a culture solution is accommodated inside the container 300. Air exists outside the container 300. In FIG. 8, an optical path is shown at a position separated from the side surface 304 to clearly show the state of reflection.

  The interface between the air and the side surface 304 of the container 300 is the interface S1, and the interface between the side surface 304 of the container 300 and the culture solution is the interface S2. Refractive index of air <refractive index of culture solution <refractive index of container 300 (plastic). In FIG. 8, light emitted from the common light source 400 and light reflected at the interface S1 (see FIG. 7) are considered.

  As described with reference to FIG. 7, when light enters the interface S1 from the side surface 304, if the incident angle is smaller than the critical angle, a part of the incident light is reflected and becomes reflected light. When light enters the interface S1 from the side surface 304 and the incident angle is larger than the critical angle, total reflection occurs, and all of the incident light becomes reflected light. In either case, there is a difference in intensity, but reflected light is generated. In the following, attention is paid to light that is reflected at the interface S1 and returns to the culture solution.

  As in FIG. 6, the light emitted from the common light source 400 travels through the optical fiber 132 and is emitted from the emission surface 136 of the optical fiber 132 (point P1). The light A emitted from the emission surface 136 reaches the reflector 200 (light of the arrow A). The light that has reached the reflector 200 is reflected by the reflector 200 (point P2). The light B reflected by the reflector 200 passes through the side surface 304 and enters the culture solution (light of arrow B).

  As described above, fine irregularities are formed on the inner peripheral surface 202 of the reflector 200, and light B <b> 1 to B <b> 4 (see FIG. 8) diffused by the irregularities on the inner peripheral surface 202 is generated. Hereinafter, the light B1 among the lights B1 to B4 will be described. The light B1 enters the culture solution and illuminates the culture solution.

  The light B1 that has passed through the culture solution reaches an interface S1 (position P3) between the air and the side surface 304 of the container 300. As described with reference to FIG. 7, at the interface S1, reflected light R reflected on the container 300 side is generated. The reflected light R (light C) reflected on the culture solution side passes through the culture solution and illuminates the culture solution.

  The light C that has passed through the culture solution reaches an interface S1 (position P4) between the air and the side surface 304 of the container 300. Also at the interface S1 at the position P4, reflected light R reflected on the container 300 side is generated. The reflected light R (light D) reflected on the culture solution passes through the culture solution and illuminates the culture solution.

  As described above, the light emitted from the light emitting unit 130 reaches the reflector 200 after passing through the culture solution. The light that has reached the reflector 200 is reflected by the reflector 200 and returns to the culture solution. The light returned to the culture solution repeats reflection at the interface S1 one after another (positions P3 → P4 →...), And passes through the culture solution one after another as indicated by B1 → C → D →. By increasing the light passing through the culture, the culture can be illuminated more brightly as a background. For this reason, the difference between the background and the cells can be further increased so that the cells can be imaged more clearly.

  As described with reference to FIG. 7, the light incident from the interface S2 can also finally enter the culture solution after being reflected. Such light can increase the amount of light passing through the culture solution.

<< Case 910 >>
The housing 110 shown in FIGS. 1 and 5 has a band shape without a bottom surface. As shown in FIG. 1, the housing 110 has a so-called band shape or belt shape.

  On the other hand, as shown in FIGS. 10 and 11, a housing 910 having a bottom surface may be used. 10 and 11, the same reference numerals are given to the same components as those in FIGS. 1 and 5.

  The housing 910 has a disk-shaped bottom portion 920 and a cylindrical side portion 912 provided along the outer periphery of the bottom portion 920, and has a shape in which an upper end surface is open. Since the container 300 can be placed on the bottom portion 920, the container 300 can be easily attached to the housing 910, and the container 300 can be stably held. The bottom surface 302 of the container 300 is placed on the bottom surface 920 of the housing 910, and the container 300 is mounted on the housing 910.

  The side surface portion 912 has a cylindrical inner peripheral surface 114 and a cylindrical outer peripheral surface 116 similarly to the cylindrical band portion 112. As in the case 110, the light-emitting imaging device 120 is attached to the side surface portion 912.

  Since the upper end surface of the housing 910 is open, attachment and detachment of the container 300 can be facilitated, and work efficiency can be improved.

  The reflector 200 is provided along the side surface part 912. Specifically, the reflector 200 is attached so that the outer peripheral surface 204 thereof is in close contact with the cylindrical inner peripheral surface 114 of the side surface portion 912. For example, the reflector 200 can be fixed to the side surface portion 912 using an adhesive or the like. By providing the reflector 200 on the side surface portion 912, the light A emitted from the common light source 400 and the light B reflected by the reflector 200 can be used as a culture solution even when the housing 910 is used, as in FIG. To brightly illuminate the entire culture solution.

  Also in the case where the housing 910 is used, as in FIG. 8, the light passing through the culture solution can be increased one after another, and the difference between the background and the cells can be further increased to make the cells clearer. Images can be taken.

  In FIG. 11, similarly to FIG. 5, in order to clearly show the configuration of the housing 910, the light-emitting imaging device 120, and the reflector 200 in a state where the stirring blade 310 is attached to the container 300, the stirring blade 310 is moved from the light-emitting imaging device 120. Are also shifted upward. However, even when the stirring blade 310 is located below the position in FIG. 11 and is located in front of the imaging region M of the light-emitting imaging device 120, the position of the imaging region M is focused on the stirring blade 310. It is a position that does not match, and it is difficult to image the stirring blade 310, and it is possible to clearly image cells and the like located in the imaging region M.

<< Example of other containers >>
9A to 9D are plan views showing examples of other containers. 9A to 9D, the stirring blade 310 is omitted to clearly show the shape of the container.

  The side surface 304 of the container 300 described above has a cylindrical shape, and the entire side surface 304 has a curved shape. In this case, light can be returned to the side surface 304 by using the semi-cylindrical reflector 200 corresponding to the shape of the side surface 304 as the side surface 304. The container of the present embodiment is not limited to a container having a cylindrical side surface, and may have a shape that surrounds or circulates a culture solution. With such a shape, the inside of the container can be illuminated by sequentially reflecting light on the inner side surface of the side surface.

  The side surface of the container 500 shown in FIG. 9A has a flat portion 504a and a curved portion 504b. The side surface of the container 600 shown in FIG. 9B has a flat portion 604a and a bent portion 604b. The side surface of the container 700 shown in FIG. 9C has a curved portion 704a and a bent portion 704b. The side surface of the container 800 shown in FIG. 9D has a flat portion 804a, a bent portion 804b, and a curved portion 804c.

  As described above, the side surfaces of the containers 300, 500, 600, 700, and 800 may be formed by flat portions, curved portions, and bent portions, and may surround the accommodated culture solution so as to circulate therearound.

<< other >>
In the example described above, a case where a monochrome area camera is used as the CCD camera 150 has been described, but a color-compatible area camera may be used. It is also assumed that the culture solution changes color or becomes turbid as the cells grow during the culture process. In such a case, by using a color-compatible area camera, not only changes in cells and the like but also changes in the culture solution can be imaged to accurately determine the culture process.

  In the field of regenerative medicine, it is urgently necessary to stably culture cells and the like in large quantities. In particular, there is a need to shorten the time required for application to medical treatment, reduce costs, and stably culture cells at high speed and in large quantities. For this reason, it is necessary to culture cells in a culture container having a large capacity without manual operation.

  In addition, the concentration of cells in the culture solution is low, and in the case of a method for collecting cells, the number of cells that can be collected is increased by stopping stirring to bring the cells into a sediment state. In some cases, it is necessary to remove the culture vessel from the culture apparatus and collect it. For this reason, cells must be observed in an environment different from the culture environment. Furthermore, the cells collected in this manner cannot be returned to the original culture solution in consideration of the effect on other cells, and the cells must be discarded after observation.

  Furthermore, the culture of cells requires a sterile environment. In order for humans to be able to enter and leave the cultivation equipment, costs for maintaining a sterile environment are also required. Therefore, when culturing a large volume of cells, it is necessary to maintain a sterile state by automating the culture apparatus and observing the cell state remotely (outside the clean room).

  As described above, in the field of regenerative medicine, there is a demand for an observation device that can remotely and non-invasively observe cells cultured in a large-capacity cell culture device at high speed and continuously. The optical device is also suitable for observing such cells and the like.

REFERENCE SIGNS LIST 100 Imaging optical device 120 Emission imaging device 200 Reflector 300, 500, 600, 700, 800 Container

Claims (6)

A light-emitting unit having an emission surface for emitting light; and an imaging unit for imaging an inside of a container irradiated with the light emitted from the light-emitting unit, the imaging unit having an objective lens on which imaging light is incident. Light emission imaging means;
A reflector that reflects light, and a reflector that is disposed so as to face the light-emitting imaging unit with the container interposed therebetween,
The container has a surrounding wall that surrounds the medium to be housed housed in the container, and has a surrounding wall through which light emitted from the light emitting unit can pass,
With the objective lens of the imaging unit as the center, the emission surface of the light emitting unit is arranged around the objective lens,
An imaging optical device, wherein light emitted from the light emitting unit passes through the surrounding wall toward the reflector, is reflected by the reflector, and illuminates the medium to be accommodated.   The container has a cylindrical surrounding wall,
  The imaging optical device according to claim 1, wherein the emission surface of the light emitting unit and the objective lens of the imaging unit are mounted toward the cylindrical surrounding wall.   The imaging optical device according to claim 1, wherein the reflector is arranged along the surrounding wall.   The imaging optical device according to claim 1, wherein a refractive index of the surrounding wall is larger than a refractive index of air. The surrounding wall is
An inner wall surface forming an interface between the contained medium and the surrounding wall,
An outer wall forming an interface between the air outside the container and the surrounding wall,
The imaging optical device according to claim 1, wherein the outer wall surface reflects light incident from the storage medium and returns the light to the storage medium. The imaging optical device according to claim 1, wherein the reflector has a reflection surface that causes diffuse reflection by unevenness.

Images