JP2017046620A - Optical imaging apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an optical imaging apparatus capable of remotely observing cells at high speed and continuously in a non-invasive manner and observing cells cultured in each of a plurality of culture vessels.SOLUTION: The present invention provides an optical imaging apparatus that comprises: light-emitting imaging means including a light-emitting part for emitting light and an imaging part for imaging the inside of a container on which light emitted from the light-emitting part is irradiated; and a reflector for reflecting light, the reflector being disposed opposite to the light-emitting imaging means across a container. The container has a surrounding wall, which surrounds an accommodated medium in the container, through which light emitted from the light-emitting part can pass. The light emitted from the light-emitting part passes through the surrounding wall and travels toward the reflector and is then reflected by the reflector, followed by illuminating the accommodated medium.SELECTED DRAWING: Figure 6

Description

  The present invention relates to an imaging optical device that captures an image of a movable object such as a cell or a microorganism present 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 was observed with a microscope from the bottom side of the container or from the upper side of the container to visualize the state of division growth (for example, see Patent Document 1). Whether the cells and microorganisms are growing or not has been judged by capturing still cells or microorganisms with a camera and visually observing the images with the naked eye.

JP 2004-348104 A

  Cells cultured by stirring in a large-capacity culture vessel are in a state where they can always float and move in the culture solution and can freely move three-dimensionally in the culture vessel. Although there is a technique for collecting and observing cultured cells from a culture vessel, this technique only observes some of the cells in the culture solution, and there is a limit to the range of cells that can be observed. It was difficult to accurately determine the overall culture state of the cells.

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

  The present invention has been made in view of the above-mentioned points. The object of the present invention is to remotely and non-invasively observe cells and microorganisms at high speed and continuously, and in each of a plurality of culture containers. An object of the present invention is 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 imaging means having a light emitting unit that emits light, and an imaging unit that images the inside of a container irradiated with light emitted from the light emitting unit;
A reflector that reflects light and is disposed opposite to the light emitting imaging means across the container; and
The container has an enclosure wall that encloses a medium to be accommodated contained in the container and through which light emitted from the light emitting unit can pass,
The light emitted from the light emitting unit passes through the surrounding wall, travels toward the reflector, is reflected by the reflector, and then illuminates the medium to be accommodated.

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

1 is a perspective view illustrating an outline of an imaging optical device 100. FIG. 1 is a plan view illustrating an outline of an imaging optical device 100. FIG. FIG. 2 is a perspective view (A) showing an outline of the light emitting imaging device 120 and a front view (B) showing the light emitting unit 130 and the CCD camera 150 of the light emitting imaging device 120. 4 is a perspective view showing a configuration of a reflector 200. FIG. 4 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 the light emitting imaging device 120. FIG. It is the schematic when light injects into the interface S1 from the container 300 (plastic). 3 is a conceptual diagram illustrating an optical path of light emitted from the light emitting imaging device 120. FIG. It is a figure which shows the other aspect of the side surface of a container. 10 is a perspective view illustrating an outline of a housing 910. FIG. FIG. 11 is a plan view showing an outline of a housing 910. It is the schematic which shows the total reflection which arises in the interface S1 by the unevenness | corrugation formed in the internal peripheral surface 202 of the reflector 200. FIG.

  Embodiments will be described below with reference to the drawings.

According to a first embodiment of the invention,
A light emitting unit that emits light (for example, a light emitting unit 130 described later) and an imaging unit that captures the inside of a container (for example, a container 300 described later) irradiated with light emitted from the light emitting unit (for example, described later) A light emitting imaging means (for example, a light emitting imaging device 120 described later),
A reflector that reflects light and is disposed opposite to the light emitting imaging unit with the container interposed therebetween (for example, a reflector 200 described later),
The container is an surrounding wall (for example, a container 300 described later) through which light emitted from the light emitting unit can pass and surrounds a medium (for example, a culture solution described later) stored in the container. A 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 later).

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

  The light emission imaging means has both a light emission part and an imaging part. The light emitting unit emits light having a predetermined intensity at a predetermined wavelength. The light emitted from the light emitting unit is applied to the container to illuminate the container. The imaging unit images 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 unit, it is not necessary to secure the space for installing the light emitting unit and the space for installing the imaging unit separately and at different positions. Therefore, even when a plurality of containers are installed, the light emitting imaging means can be provided so that no gap is generated between adjacent containers. As described above, a large amount of cells, microorganisms, and the like can be cultured by saving space and effectively utilizing the space and arranging a plurality of containers closely without gaps.

  It is preferable that the light-emitting imaging unit has a light-emitting part and an imaging part 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 with the imaging unit as a center. By disposing 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 the predetermined direction.

  The reflector reflects light. The reflector is disposed opposite to the light emitting imaging means with the container interposed therebetween. The container is located between the reflector and the light emission imaging means, and is sandwiched between the reflector and the light emission imaging means.

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

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

  In this way, the medium to be accommodated is illuminated not only by the light emitted from the light emitting unit, but also by the light reflected by the reflector, so that the medium to be accommodated can function as a background to capture a bright image.

  Since imaging is performed by irradiating light with the luminescence imaging means, it becomes possible to observe the culture state of cells, microorganisms, etc. while maintaining an environment where cells, microorganisms, etc. can be cultured without human intervention. Furthermore, since the medium to be accommodated functions as a background and performs bright imaging, imaging can be performed at high speed, for example, imaging can be performed even at a high shutter speed, and cells, microorganisms, and the like moving in the container can be clearly imaged.

  This imaging optical apparatus is effectively used for imaging a movable object of cells, microorganisms (hereinafter simply referred to as “cells”) present in a medium such as a liquid, and in particular, embryonic stem cells (ES cells). It is preferably used for imaging an embryoid body or cell aggregate of stem cells such as artificial pluripotent stem cells (iPS cells) and somatic stem cells.

According to a second embodiment of the present invention, in the first embodiment of the present invention,
The reflector is disposed 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. Thus, the light emitted from the light emitting portion can be efficiently reflected by the reflector without being wasted, and the contained medium can be illuminated by the reflected light.

  Furthermore, reflectors can be arranged so as not to interfere with adjacent containers, and a large number of cells can be cultured by arranging many containers.

According to a third embodiment of the present invention, in 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, the light emitted from the light emitting part can be reflected also by the surrounding wall. By reducing the amount of light emitted from the light emitting part to the outside of the surrounding wall (outside of the container) and returning the light reflected by the surrounding wall to the receiving medium, the receiving medium is illuminated more brightly and stored The medium can function as a bright background.

According to a fourth embodiment of the present invention, in the first embodiment of the present invention,
The Go wall is
An inner wall surface (for example, an inner peripheral surface 202 described later) that forms an interface between the contained 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 accommodation medium and returns it to the accommodation medium.

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

According to a fifth embodiment of the present invention, in the first embodiment of the present invention,
The reflector has irregularities formed on the reflection surface.

  Since unevenness is formed on the reflection surface of the reflector, diffuse reflection occurs on the reflection surface. By causing diffuse reflection, even when highly directional illumination is used, a large amount of light that is totally reflected can be generated by making the incident angle larger than the critical angle. By allowing a lot of light that repeats total reflection to pass through the medium to be stored, the background medium to be stored 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 between the refractive index of air existing outside the container (about 1) and the refractive index of the container (about 1.5 to 1.6).

According to a sixth embodiment of the invention,
A light emitting unit (for example, a light emitting unit 130 described later) and an imaging unit (for example, a CCD camera 150 described later) are containers (for example, a container described later) in which a medium to be stored (for example, a culture solution described later) is stored. 300) and the like (for example, a reflector 200 to be described later) is disposed opposite to each other.
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 (for example, light described later) B), etc., and an illumination process for illuminating the accommodated medium accommodated in the container,
An imaging step of imaging the inside of the container with an imaging unit.

  Since the light emitting unit and the imaging unit are located on the same side of the container, it is not necessary to secure the space for installing the light emitting unit and the 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. As described above, a large amount of cells and the like can be cultured by saving space and effectively utilizing the space and arranging a plurality of containers closely without gaps.

  Since the medium to be accommodated is illuminated not only by the light emitted from the light emitting unit but also by the light reflected by the reflector, the medium to be accommodated can function as a background to capture a bright image.

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

  More specifically, there are the following embodiments. In the following description, the cells to be imaged are described as “cells”, and the contained medium is described as “culture medium”.

Other embodiments of the invention include:
An area camera (for example, a CCD camera 150 to be described later) that is in close contact with or close to a transparent culture container (for example, a container 300 to be described later) made of resin or glass,
A lens (for example, an objective lens 156 described later) having a focal length for observing the culture vessel through transmission;
An area sensor camera (for example, an imaging optical device 100 described later).

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

  Further, a light guide fiber (for example, an optical fiber 132 to be described later) is connected to a light source device (for example, a common light source 400 to be described later), and light emitted from the light source device is cultured through the light guide fiber. Guide to the vessel and illuminate the inside of the culture vessel. By using a light guiding fiber (for example, an optical fiber 132 described later), a high-luminance 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 vessel, the heat generated from the light source device is hardly transmitted to the culture vessel, and the influence of the heat on the culture environment can be prevented.

  Furthermore, when a multi-branch fiber is used as the light guide 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 exit surface of the light guide fiber (for example, the exit surface 136 described later) travels while spreading inside the culture vessel within the range of the exit angle (about 70 degrees). Part of the illumination light emitted from the emission surface is applied to a subject (for example, a cell) existing inside the culture vessel. The remaining illumination light that has not been irradiated onto the subject reaches the opposite side of the culture vessel facing an area camera (for example, a CCD camera 150 described later) across the culture vessel.

  Furthermore, a reflecting plate (made of metal, mirror, film, etc.) for reflecting light is installed on the opposite side of the culture container facing the area camera with the culture container interposed therebetween. The reflector is formed in accordance with the shape of the culture vessel. Moreover, you may apply | coat magnesium oxide, barium sulfate, etc. which have a reflective effect to the outer peripheral surface on the opposite side of the culture container which faces an area camera on both sides of a culture container. The illumination light can be returned to the culture vessel by reflecting the illumination light with a reflector or the like. In addition, as long as the culture environment of cells and the like is not affected, a reflection medium may be installed or applied to the inner peripheral surface of the culture container.

  In addition, when the reflection angle of the illumination light returned to the culture vessel is equal to or greater than the critical angle depending on the shape of the culture vessel (cylindrical shape), the illumination light is scattered and reflected multiple times in the culture vessel. Light is confined in the culture vessel. By scattering and reflecting the illumination light multiple times, the culture solution is illuminated at multiple locations, improving the effect of the cell outline and background (contrast between the culture solution and the cells), and floating at high speed You can take a picture of the cells in the camera at high speed. Since the image can be taken with the shutter released at high speed, an image can be acquired as a still image of cells that move at high speed.

  In addition, it is easy to shoot continuously by shooting with a shutter at high speed, and the opportunity to observe a large number of cells contained in a large-capacity culture vessel by increasing the number of times of imaging within the imaging depth of focus. Can be more.

<<< Imaging Optical Device 100 >>>
FIG. 1 is a perspective view illustrating a 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, these are shown separately from each other in order to clarify the relationship among the housing 110, the light emitting imaging device 120, and the reflector 200. In FIG. 2, the container 300 is indicated by an imaginary line, and the stirring blade 310 is omitted (see FIG. 5).

  FIG. 5 is a perspective view illustrating 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 (enclosure wall). As will be described later, light enters, reflects, or exits from a 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 only needs to be made of a material that can transmit and reflect light, and can 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). The culture solution accommodated in the container 300 is stirred by rotationally driving the stirring blade 310. 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 according to the flow of the culture solution.

<< Case 110 >>
As shown in FIG. 1, the housing 110 has a cylindrical belt portion 112 having a substantially cylindrical shape. The cylindrical belt portion 112 has a cylindrical inner peripheral surface 114 and a cylindrical outer peripheral surface 116. The casing 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 casing 110 around the side surface 304, the casing 110 can be attached in close contact with the container 300, and even when vibration or impact occurs, the casing 110 can be stably held in the container 300, By keeping the relative position of 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 belt 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 casing 110 can be attached in close contact with the container 300. Alternatively, the housing 110 can be attached in close contact with the container 300 using a fixing member such as a screw or a bolt.

<< Light-Emitting Imager 120 (Light-Emitting Imager) >>
The light emitting imaging device 120 is attached to the cylindrical belt portion 112 of the housing 110. The cylindrical belt portion 112 is provided with a holding portion 118 (see FIG. 2). The light emitting imaging device 120 is fixed to the cylindrical belt portion 112 via a 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 constituted by an optical fiber 132 and a CCD camera 150. The CCD camera 150 functions as an area camera.

  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. FIG.

<CCD camera 150 (imaging unit)>
The CCD camera 150 includes an optical system unit 152 and an electrical system unit 160. The optical system unit 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 152 has an opening 154, and light reflected by the imaging target is incident on 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 unit 160 has a substantially rectangular parallelepiped shape, and includes a CCD image sensor unit (not shown), a signal processing circuit unit (not shown), and the like. The light whose optical path is adjusted by the optical system unit 152 is incident on the CCD image sensor unit to form an image. The electric system unit 160 converts the image formed by the CCD image sensor unit into an electric signal, performs various signal processing such as digitization, and outputs the 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 entrance surface 134 and an exit 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 made of a light emitter such as an LED (light emitting diode). The light emitted from the common light source 400 is branched and propagated to each of the plurality of optical fibers 132 and is emitted from the emission surface 136 (FIGS. 3A and 3B). Thus, light can be emitted from the plurality of optical fibers 132 using a 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 disposed 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 and cells contained in the container 300, and to reduce the influence of the heat generated from the common light source 400 and to keep the culture environment constant. it can.

  As shown in FIG. 3A, the end portions of the plurality of optical fibers 132 are arranged along the longitudinal direction on the outer peripheral surface of the optical system portion 152 of the light emitting portion 130. 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 circulate around the outer peripheral surface of the opening 154 of the optical system unit 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 unit 152 and the inner peripheral surface of the holding body 170. That is, the plurality of optical fibers 132 are arranged so as to cover the optical system unit 152, and the holding body 170 is covered so as to cover the optical system unit 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 is configured such that 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 are directed toward the side surface 304 of the container 300. It is attached. For example, the CCD camera 150 is attached so that the axial direction of the optical system unit 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 a shape that curves along the longitudinal direction, for example, a substantially semi-cylindrical shape. As shown in FIGS. 1 and 5, the height (width 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 belt portion 112. Specifically, the outer peripheral surface 204 of the reflector 200 is attached so as to be in close contact with the cylindrical inner peripheral surface 114 of the cylindrical belt portion 112. For example, the reflector 200 can be fixed to the cylindrical belt portion 112 using an adhesive or the like. When the reflector 200 is attached to the cylindrical inner peripheral surface 114 of the cylindrical belt portion 112 and the casing 110 is attached to the container 300, the inner peripheral surface 202 of the reflector 200 is attached in close contact with 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 such as aluminum or copper. The metal should just have a high reflectance. By forming the reflector 200 by curving a thin metal plate, the reflector 200 can be provided along the cylindrical belt portion 112. By forming the reflector 200 with a thin metal plate, light can be reflected by the inner peripheral surface 202 of the reflector 200.

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

  In addition to forming the reflector 200 with a thin metal plate, the base material of the reflector 200 may be formed of a flexible material such as plastic. In this case, by forming a metal thin film on the inner peripheral surface 202 of the reflector 200 by coating, vapor deposition, sputtering, or the like, light can be reflected in the same manner as a metal thin plate. A film may be formed using a material having high reflectance. For example, magnesium oxide, barium sulfate, or the like that exhibits a reflection effect can be used.

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

  Furthermore, diffuse reflection can be performed by forming irregularities on the surface of the substrate in advance of forming the metal thin film.

  In the above-described example, the case where the reflector 200 has a substantially semi-cylindrical shape is shown. However, the shape is not limited to this shape, and the reflector may be provided in a place other than a portion where light necessary for the light emitting imaging device 120 passes. it can. Moreover, although the reflector 200 is configured separately from the container 300, a part of the container 300 may be configured as the reflector 200. Furthermore, the material constituting the reflector 200 is not limited to a metal, but may be any material that can reflect light, such as a resin sheet having a high reflectance.

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

  The 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 (exit 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 arrow A). Thereafter, the light A that has passed through the culture solution passes through the side surface 304 and reaches the reflector 200. The light that reaches 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).

  Thus, 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 way, the light B reflected by the reflector 200 also allows the culture solution to pass through, so that the light can enter the culture solution a plurality of times and the culture solution can be illuminated brightly.

<< Imaging by Light-Emitting Imaging Device 120 >>
<Cell imaging>
An objective lens 156 is provided in the optical system unit 152 of the light emitting imaging device 120. For example, a telecentric lens can be used as the objective lens 156. By appropriately determining the magnification of the objective lens 156, an area within the imaging focal depth 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 and clearly images the cells.

  The imaging region M is a position between the stirring blade 310 and the light emitting imaging device 120, and can be a position closer to the light emitting imaging device 120 than the stirring blade 310. More preferably, the imaging region M can be positioned closer to the light emitting imaging device 120 than the center of the stirring blade 310 and the light emitting 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 making the position of the imaging region M 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 as long as the stirring blade 310 is hard to be imaged and the cells can be clearly imaged, and the position of the light emitting imaging device 120 and the magnification of the objective lens 156 can be determined as appropriate.

  In FIG. 5, in order to clearly show the configuration of the casing 110, the light emitting imaging device 120, and the reflector 200 in a state of being attached to the container 300, the stirring blade 310 is shifted upward from the light emitting imaging device 120. Shown. However, as described above, the position of the imaging region M is agitated even when the stirring blade 310 is positioned below the position of FIG. 5 and is positioned in front of the imaging region M of the light emitting imaging device 120. The agitating blade 310 is hard to be imaged, and the 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 with 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 by the light A and the light B as a background. That is, the objective lens 156 is not focused on most of the culture solution other than the imaging region M, but the light that illuminates the culture solution enters the CCD camera 150 via the objective lens 156, and the brightly lit culture solution is Serves as a background.

<Imaging of cells and culture solution>
The CCD camera 150 focuses on the cell located in the imaging region M by the objective lens 156 and images the cell. Further, light that illuminates the culture solution enters the CCD camera 150 via the objective lens 156. As described above, the CCD camera 150 functions as a background using the light illuminating the culture solution, and can capture an image in comparison with the target cell. By increasing the amount of light passing through the culture solution, the culture solution can be illuminated brightly to function as a background, and the contrast with the focused cells can be increased to clearly image the target cells. it can.

  In the case where the reflector 200 is not provided, the light is not returned to the culture solution by the reflector 200 after once passing through the culture solution. For this reason, the light which passes a culture solution cannot be increased, a background cannot be brightened, contrast with a cell cannot be made high, and it becomes difficult to image a cell clearly.

  The imaging optical apparatus 100 does not illuminate the culture solution only with the light A emitted from the common light source 400, but also illuminates the culture solution with the light B reflected by the reflector 200, so that the culture solution is used as a bright background. Can function.

  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, a light emitting unit 130 and a CCD camera 150 are integrally formed. If the light emitting 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. Since the light emitting unit 130 and the CCD camera 150 are integrated, it is not necessary to secure the space for installing the light emitting unit 130 and the space for installing the CCD camera 150 separately and at different positions. Therefore, it is possible to effectively use the space by arranging the imaging optical devices 100 adjacent to each other closely to save space.

<< Effect of rotation of stirring blade 310 >>
The culture solution is stirred by the rotation of the stirring blade 310. When the culture solution is stirred, the cells in the growth process always move in the container 300. The position of the imaging region M is a position that does not focus on the stirring blade 310, and the stirring blade 310 is difficult to be imaged. Therefore, only the cells located in the imaging region M among the moving cells can be clearly imaged. Even in a state where the stirring blade 310 is rotated to stir the culture solution, cells can be imaged by the imaging optical device 100. For this reason, it is possible to image cells in the culture process without interrupting the culture process or collecting the cells.

  The cells are constantly moving in the container 300 by stirring 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. For this reason, even when the shutter speed is increased, cells can be clearly imaged.

  Even when the output 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.

  Thus, the shutter speed is determined according to the moving speed of the cells in the container 300 (for example, the rotational speed of the stirring blade 310), and the intensity of the common light source 400 can be optimized in accordance with the determined shutter speed. In this way, the cells can be imaged clearly by imaging the cells with illumination of optimum intensity.

<< Diffusion reflection by 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) gradually diffusing from the inner peripheral surface 202 is generated, and the lights B1 to B4 illuminate 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 unevenness is formed on the inner peripheral surface 202 of the reflector 200, diffuse reflection occurs due to the unevenness. By causing diffuse reflection, a large amount of light that is totally reflected at the interface S1 of the side surface 304 of the container 300 can be generated even when highly directional illumination is used. By making light having an incident angle larger than the critical angle incident on the interface S1, total reflection can be generated at the interface S1.

  For example, as shown in FIG. 12, the light X <b> 1 enters the inner peripheral surface 202 of the reflector 200. Concavities and convexities are formed on the inner peripheral surface 202, and the inclination of the concavities and convexities varies 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 inclined relative to the case where the unevenness is not formed on the inner peripheral surface 202 (light X2 ') (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) and the refractive index of the container 300 (about 1.5 to 1.6).

  Thus, by forming irregularities on the inner peripheral surface 202 of the reflector 200, it is possible to generate much light that is totally reflected at the interface S1. By allowing the light that repeats total reflection to pass through the culture solution, the background culture solution can be illuminated more brightly, and the contrast can be further increased by increasing the chances 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 accommodated inside the container 300. Air is present outside the container 300. The interface between the air and the side surface 304 of the container 300 is referred to as an interface S1, and the interface between the side surface 304 of the container 300 and the culture medium is referred to as an interface S2. FIG. 7A is a schematic diagram illustrating an example in which the incident angle θI at the interface S1 is smaller than the critical angle. FIG. 7B is a schematic diagram illustrating an example in which the incident angle θI at the interface S1 is larger than the critical angle.

  The refractive index of air <the refractive index of the culture solution <the refractive index of the plastic. For example, the refractive index of air is about 1, the refractive index of culture broth is about 1.4, and the refractive index of plastic is about 1.5.

  In FIG. 7A, the incident angle θI is smaller than the critical angle. For this reason, the incident light I is separated into reflected light R and refracted light F at the interface S1. The reflected light R is reflected at the interface S1, returns to the side surface 304 of the container 300, passes through the interface S2, and then returns to the culture solution to illuminate the culture solution. On the other hand, the refracted light F is emitted from the interface S1 to the air.

  In FIG. 7B, the incident angle θI is larger than the critical angle, and total reflection occurs. That is, the incident light I is totally reflected at the interface S1 to become reflected light R, and there is no light emitted from the interface S1 to the air. The reflected light R reflected by 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 a resin such as plastic is shown, but other types of resin or glass may be used. Moreover, the refractive index of the container 300 can be 1.5-1.6.

  In the above-described example, 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. Mainly incident, reflected, and refracted at the interface S1. Was considered. Depending on the refractive index of the culture solution and the size of the refractive index of the container 300, the 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 showing an optical path of light emitted from the light emitting imaging device 120. The same components as those in FIG. Also in FIG. 8, as in FIG. 6, the casing 110 and the stirring blade 310 are omitted in order to clearly show the optical path. The container 300 is made of plastic, and a culture solution is accommodated inside the container 300. Air is present outside the container 300. In FIG. 8, the optical path is shown at a position separated from the side surface 304 in order 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 medium is the interface S2. The refractive index of air <the refractive index of the culture solution <the refractive index of the container 300 (plastic). In FIG. 8, the light emitted from the common light source 400 and the light reflected at the interface S1 (see FIG. 7) are considered.

  As described with reference to FIG. 7, when light is incident on 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. Further, when light enters the interface S1 from the side surface 304, if the incident angle is larger than the critical angle, total reflection occurs, and all of the incident light becomes reflected light. In either case, reflected light is generated with a difference in intensity. In the following, attention is focused on the light reflected at the interface S1 and returning 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 indicated by arrow A). The light reaching 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 the interface S1 (position P3) between the air and the side surface 304 of the container 300. As described with reference to FIG. 7, the reflected light R reflected toward the container 300 is generated at the interface S1. The reflected light R (light C) reflected to 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 the 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, the reflected light R reflected to the container 300 side is generated. The reflected light R (light D) reflected toward the culture solution passes through the culture solution and illuminates the culture solution.

  Thus, the light emitted from the light emitting unit 130 reaches the reflector 200 after passing through the culture solution. The light reaching the reflector 200 is reflected by the reflector 200 and returns to the culture solution. The light that has 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 B1 → C → D →. By increasing the light passing through the culture solution, the culture solution can be illuminated more brightly as a background. For this reason, the difference in brightness between the background and the cells can be further increased, and 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 also increase the light passing through the culture solution.

<< Case 910 >>
The housing 110 illustrated in FIGS. 1 and 5 does not have a bottom surface and has a strip shape. 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 configurations common to FIGS. 1 and 5.

  The housing 910 has a disk-shaped bottom surface portion 920 and a cylindrical side surface portion 912 provided along the outer periphery of the bottom surface portion 920, and has a shape with an open upper end surface. Since the container 300 can be placed on the bottom surface 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 portion 920 of the housing 910, and the container 300 is placed on the housing 910.

  Similar to the cylindrical belt portion 112, the side surface portion 912 has a cylindrical inner peripheral surface 114 and a cylindrical outer peripheral surface 116. Similar to 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, the container 300 can be easily attached and detached, and work efficiency can be increased.

  The reflector 200 is provided along the side surface portion 912. Specifically, the outer peripheral surface 204 of the reflector 200 is attached so as to be 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 culture medium can be obtained by using the light A emitted from the common light source 400 and the light B reflected by the reflector 200 in the same manner as in FIG. The whole culture solution can be illuminated brightly.

  In addition, when 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 light and dark between the background and the cells can be further increased to make the cells clearer. An image can be taken.

  11 also shows the stirring blade 310 from the light-emitting imaging device 120 in order to clearly show the configuration of the housing 910, the light-emitting imaging device 120, and the reflector 200 in a state of being attached to the container 300, as in FIG. Also shown shifted upwards. However, even when the stirring blade 310 is positioned below the position of FIG. 11 and positioned in front of the imaging region M of the light emitting imaging device 120, the position of the imaging region M is in focus with the stirring blade 310. The stirring blades 310 are difficult to image, and the cells and the like located in the imaging region M can be clearly imaged.

<< Examples of other containers >>
9A to 9D are plan views showing examples of other containers. In FIG. 9A to FIG. 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 semicylindrical reflector 200 corresponding to the shape of the side surface 304 for the side surface 304. The container according to the present embodiment is not limited to one having a cylindrical side surface, and may have a shape that surrounds or circulates the culture solution. By adopting such a shape, light can be reflected one after another on the inner side surface of the side surface to illuminate the inside of the container.

  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 includes 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 a flat portion, a curved portion, or a bent portion and may surround the accommodated culture medium so as to circulate.

<< Other >>
In the above-described example, a monochrome area camera is used as the CCD camera 150. However, a color-compatible area camera may be used. It may be assumed that the culture medium changes color or becomes cloudy as the cells grow during the culturing process. In such a case, by using a color-compatible area camera, it is possible to accurately determine the culture process by imaging not only changes in cells and the like but also changes in the culture medium.

  In the field of regenerative medicine, there is an urgent need to stably culture a large amount of cells and the like. In particular, there is a demand for shortening the time until it can be applied to medical treatment, reducing costs, and culturing cells stably at high speed and in large quantities. For this reason, it is necessary to culture | cultivate a cell in a culture container with a large capacity | capacitance without manual operation.

  In addition, the concentration of cells in the culture solution is low, and in the case of a method of collecting cells, the number of cells that can be collected is increased by stopping the stirring and making it settle. 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 view of the influence on other cells, and the cells must be discarded after observation.

  Furthermore, a sterile environment is required for cell culture. In order to enable humans to enter and exit the culture facility, maintenance costs for a sterile environment are also required. Therefore, when culturing a large volume of cells, it is necessary to maintain a sterile condition 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 apparatus that can remotely and non-invasively and rapidly observe cells cultured in a large-capacity cell culture apparatus. The optical device is also suitable for observing such cells.

DESCRIPTION OF SYMBOLS 100 Imaging optical device 120 Light emission imaging device 200 Reflector 300, 500, 600, 700, 800 Container

Claims (6)

A light emitting imaging means having a light emitting unit that emits light, and an imaging unit that images the inside of a container irradiated with light emitted from the light emitting unit;
A reflector that reflects light and is disposed opposite to the light emitting imaging means across the container; and
The container has an enclosure wall that encloses a medium to be accommodated contained in the container and through which light emitted from the light emitting unit can pass,
The imaging optical device that illuminates the accommodation medium after the light emitted from the light emitting unit passes through the surrounding wall, travels toward the reflector, and is reflected by the reflector.   The imaging optical device according to claim 1, wherein the reflector is disposed 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 Go wall is
An inner wall surface forming an interface between the medium to be contained and the surrounding wall;
An outer wall surface forming an interface between the outside air of the container and the surrounding wall,
The imaging optical device according to claim 1, wherein the outer wall surface reflects light incident from the accommodated medium and returns the reflected light to the accommodated medium.   The imaging optical device according to claim 1, wherein the reflector has an uneven surface formed on a reflecting surface. The light emitting unit and the imaging unit are arranged to face the reflector across the container in which the medium to be stored is stored,
Illuminating the contained medium contained in the container with light emitted from the light emitting unit and light emitted from the light emitting unit and reflected by the reflector after passing through the container and returned to the container Lighting process to
An imaging process including imaging an inside of the container by an imaging unit.

Images