Cell observation system and program

Abstract

To solve a problem that unevenness in a captured image or temporal fluctuation of an image occurs in a cell observation system that radiates light from a side face of a culture vessel.SOLUTION: A cell observation system has: a mounting part which mounts a culture vessel, which has a culture surface on which cells can be cultured and whose side face constituting a peripheral wall of the culture surface is optically transparent; a light irradiation part which irradiates the culture surface with irradiation light from the side face; and an imaging part which images the culture surface, where the system has at least either one characteristic of: a) controlling the variation of the light quantity of irradiation light radiated on the culture surface caused by absorption of medium in the culture vessel; and b) correcting an effect occurred by the variation of the light quantity of irradiation light radiated on the culture surface caused by absorption of medium in the culture vessel.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to a cell observation system. [Background technology]

[0002] When culturing cells, a technique for observing a wide area of ​​the culture surface in a short time is important for cell culture. For example, the ratio of cells occupying the culture surface (cell occupancy rate) can be used as an indicator for the timing of subculture, etc. In addition, when a colony of cells is formed, it is also possible to observe the shape of the colony of cultured cells from the scattering of irradiated light. Patent Document 1 proposes a system that can observe the colony shape by irradiating light from the side of the culture vessel and observing the scattered light from the colony with a camera installed at the bottom of the culture vessel. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2016-077226 A Summary of the Invention [Problem to be solved by the invention]

[0004] However, an observation system that irradiates light from the side of a culture vessel has a problem in that the captured image has unevenness and fluctuates over time. [Means for solving the problem]

[0005] The present disclosure has been made in consideration of the above problems. The present inventors have considered the following reasons as the cause of unevenness in the captured image and fluctuation of the image over time in an observation system that irradiates light from the side of a culture vessel. That is, in an observation system that irradiates light from the side of a culture vessel, the irradiated light passes through the culture vessel and advances while being scattered by cells and the like near the culture surface. Therefore, in the culture vessel, the light amount is lower on the side farther from the light source than on the side closer to the light source. In addition, since culture media generally contain a pH indicator such as phenol red and exhibit a color change depending on the pH, the color change due to the pH of the culture medium also causes fluctuations in the light amount. It was considered that the fluctuations in the light amount of the irradiated light irradiated to the culture surface in the culture vessel as described above cause unevenness in the captured image and fluctuations in the image over time.

[0006] Therefore, we have found that the above problems can be solved by providing a cell observation system that suppresses and corrects unevenness in a captured image and fluctuations over time of the image caused by fluctuations within the surface or over time of the amount of irradiated light irradiated to the culture surface. Furthermore, we have found that it is possible to calculate the characteristics of a medium from the unevenness in a captured image and the fluctuations over time of the image and present them to a user.

[0007] That is, the present disclosure is a mounting portion on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; and An imaging unit that images the culture surface, A) controlling fluctuations in the amount of light irradiated onto the culture surface due to light absorption by the medium in the incubator; or a) correcting the influence caused by fluctuations in the amount of light irradiated onto the culture surface due to light absorption by the medium in the incubator; The present invention has at least one of the following characteristics: A cell observation system is provided.

[0008] Also, a mounting section on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; An imaging unit that images the culture surface, and An information processing unit is provided, The information processing unit includes: an image information acquiring step of acquiring image information of a captured image from the imaging unit; A characteristic calculation step of calculating characteristics of the medium in the incubator from the image information; and A characteristic output step of outputting characteristics of the medium in the incubator is carried out. A cell observation system is provided. Effect of the Invention

[0009] In an observation system that irradiates light from the side of a culture vessel, it is possible to suppress unevenness in the captured image and fluctuations in the image over time. It is possible to calculate the characteristics of the medium from the unevenness in the captured image and fluctuations in the image over time and present them to the user. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram illustrating a first embodiment. [Diagram 2] 1 shows the absorption spectrum of phenol red. [Diagram 3] A preferred example of the spectrum of the irradiated light is shown below. [Figure 4] A preferred example of the spectrum of the irradiated light is shown below. [Diagram 5] FIG. 11 is a schematic diagram illustrating a second embodiment. [Figure 6] 11 is a schematic diagram illustrating a process executed by an information processing unit in the second embodiment. FIG. [Figure 7] 2 is a schematic diagram illustrating an example of a hardware configuration of an information processing unit. FIG. [Figure 8] FIG. 11 is a schematic diagram illustrating a process executed by an information processing unit in the third embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present disclosure provides a mounting section on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; and An imaging unit that images the culture surface, A) controlling fluctuations in the amount of light irradiated onto the culture surface due to light absorption by the medium in the incubator; or a) correcting the influence caused by fluctuations in the amount of light irradiated onto the culture surface due to light absorption by the medium in the incubator; The present invention has at least one of the following characteristics: A cell observation system is provided. Preferred embodiments of the present disclosure will now be described with reference to the drawings.

[0012] First Embodiment The first embodiment of the present disclosure is a mounting portion on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; and An imaging unit that images the culture surface, A) controlling fluctuations in the amount of light irradiated onto the culture surface due to light absorption by the medium in the incubator; or a) correcting the influence caused by fluctuations in the amount of light irradiated onto the culture surface due to light absorption by the medium in the incubator; In a cell observation system having at least one of the following characteristics: The above-mentioned feature (a) is present, The irradiation light has a wavelength λ 2A [nm] and wavelength λ 2B A cell observation system in which the intensity at wavelength λ1 [nm] is 50% or less of the intensity at wavelength λ1 [nm].

[0013] however, In the spectrum of the irradiation light, the intensity is maximum at a wavelength λ1 [nm], When the medium is acidic, the wavelength λ 2A The absorption maximum is at [nm], When the medium is alkaline, the wavelength λ in the absorption spectrum of the medium 2B [nm] has maximum absorption. This embodiment will be described below with reference to FIGS.

[0014] (System Configuration) As shown in the schematic diagram of FIG. 1, the cell culture system 100 according to this embodiment includes a mounting unit 003, a light irradiation unit 004, and an imaging unit 006. The culture vessel 002 is mounted on the mounting unit 003. The culture vessel 002 is a holding vessel for holding a medium for culturing cells, and a culture surface 001 on which cells can adhere and grow is provided at the bottom of the vessel. In FIG. 1, the mounting unit 003 is provided with an opening corresponding to an imaging area so that the imaging unit 006 arranged below the mounting unit 003 can image the cells in the culture vessel 002 through the transparent bottom surface of the culture vessel 002. The light irradiation unit 004 emits irradiation light 005 from the side direction of the culture vessel 002, which is irradiated onto the culture surface 001 or its vicinity through the side surface. The imaging unit 006 images the scattered light of the irradiation light 005 irradiated to the cells adhered to the culture surface 001, and acquires the shape of the colony, etc. In Figure 1, an example is shown in which the imaging unit 006 captures images from below the culture vessel 002, but the imaging unit 006 does not necessarily have to be placed below the culture vessel 002, and may be placed above or at an angle, etc.

[0015] (Culture container) As described above, the culture vessel 002 holds a medium for culturing cells and cells placed in the medium. Note that the cell observation system of the present disclosure does not include the culture vessel 002 as a component, but the cell observation system of the present disclosure can use the culture vessel 002 described below.

[0016] The material of the culture vessel 002 is preferably a transparent resin or glass that is easy to process and observe. Resins that can be used include polystyrene, polycarbonate, acrylic, and the like. Examples of the shape include those with a rectangular or circular bottom. The culture surface 001 is preferably made of a transparent material that is easy to observe and is configured so that cells can attach and grow thereon. In the culture vessel 002, the culture surface 001 may be the bottom surface of the vessel, or may be a separate structure from the vessel disposed on the bottom surface of the vessel. However, from the viewpoint of cost and preparation, it is preferable that the culture surface 001 is made of the same material as the culture vessel 002 and constitutes a part of the culture vessel 002. At this time, it is preferable to perform a surface treatment such as a plasma treatment so that cells can easily adhere only to the culture surface 001 . Any culture vessel as described above can be used without limitation, and commercially available culture dishes, culture flasks, etc. can also be used.

[0017] (Placement section) The mounting section 003 is a platform for placing the culture vessel 002. The mounting section 003 needs to be able to image the cells in the culture vessel 002 through the transparent bottom surface of the culture vessel 002 by the imaging section 006 disposed below the mounting section 003. For this reason, the mounting section 003 can be provided with an opening corresponding to the imaging area. Most of the culture surface 001 can be observed without being hidden by the mounting section 003. It is desirable that the opening has a shape similar to that of the bottom surface of the culture vessel 002 and is slightly larger than the bottom surface so that the culture vessel 002 can be stably mounted and as wide an area as possible can be observed from the imaging section 006. For example, if the culture vessel 002 is a circular petri dish, the opening can be a circle slightly smaller than the petri dish, and if the culture vessel 002 is a square, the opening can be a square slightly smaller than the culture vessel 002. Alternatively, it is also preferable to use a transparent material such as glass for the mounting portion 003, in which case the culture surface 001 can be observed without providing an opening.

[0018] The shape of the mounting portion 003 is preferably one that does not block the irradiating light 005 onto the culture surface 001, but having a masking effect that blocks the irradiating light 005 from being irradiated onto areas other than the culture surface 001 is effective in improving the quality of the acquired image. The mounting portion 003 may have a frame or a clasp for stably mounting the culture vessel 002 thereon.

[0019] (Light irradiation part) From the viewpoint of image quality contrast, it is preferable that the irradiation light 005 from the light irradiation unit 004 is irradiated only to the culture surface 001 with as little stray light as possible. In addition, in order to prevent unevenness in the captured image surface, it is preferable that the amount of irradiation light 005 irradiated to the imaging area is uniform. From the above, it is preferable to select a light source that is mainly irradiated to the culture surface 001 and can irradiate the culture surface with a uniform amount of irradiation light 005. For example, it is preferable to use a highly directional line-type LED light source or a line light guide in which the output end of a bundle fiber is arranged in a line. By selecting such a light source, it is possible to efficiently irradiate only the vicinity of the culture surface 001. Since the irradiation light 005 from the light irradiation unit 004 is irradiated from the side of the culture vessel 002, if the divergence angle of the irradiation light 005 is large, it will diffuse, and a difference will occur in the intensity of the irradiation light 005 irradiated on the side of the culture surface 001 close to the light irradiation unit 004 and the side far from the light irradiation unit 004. In order to reduce this difference, it is preferable that the irradiated light 005 is nearly parallel light with a small spread angle. For this reason, it is effective to use a collimating means for the light irradiating portion 004. An example of the collimating means is a cylindrical lens.

[0020] Furthermore, in order to limit the irradiation range of the light irradiation unit 004 to the culture surface 001, it is effective to place a mask between the light irradiation unit 004 and the culture surface 001, for example, on the side surface of the culture vessel 002. When a mask is placed, the light from the light irradiation unit 004 may be wide-ranging, eliminating the need for the light irradiation unit 004 to be in a line shape, and expanding the options for the light irradiation unit 004.

[0021] (Spectral characteristics of irradiated light) A light source such as a halogen or LED can be used as the light irradiation unit 004. The irradiation light 005 irradiated by the light irradiation unit 004 needs to have spectral characteristics that cause little damage to cells. Furthermore, in order to control the variation in the amount of the irradiation light 005 caused by the light absorption of the medium in the culture vessel 002, it is preferable that the irradiation light 005 has spectral characteristics that are less absorbed by the medium in the culture vessel 002. By using light with a wavelength that is less absorbed by the medium as the irradiation light 005, it is possible to reduce the difference in the amount of the irradiation light 005 irradiated on the side closer to the light irradiation unit 004 and the side farther from the light irradiation unit 004 in the culture vessel 002, and to suppress the variation in the amount of the irradiation light 005 caused by the light absorption of the medium. In addition, when the absorption characteristics of the medium change depending on conditions such as pH, it is preferable to irradiate light with a wavelength that is less susceptible to the change, and it is possible to suppress the change in the amount of irradiation light caused by the change in conditions.

[0022] Cell culture media, which are common media, contain pH indicators such as phenol red, and their absorption characteristics change with changes in pH. Therefore, the wavelength λ1 [nm] is the wavelength at which the intensity is maximum in the spectral spectrum of the irradiated light, and the wavelength λ 2A [nm] is the wavelength at which absorption is maximum in the absorption spectrum of the medium when the medium is acidic, and the wavelength λ 2B If the medium is alkaline, and the wavelength at which absorption is maximum in the absorption spectrum of the medium is [nm], the irradiated light has a wavelength λ 2A [nm] and wavelength λ 2B [nm], and the intensity at wavelength λ 2A [nm] and wavelength λ 2B It is preferable that the intensity at wavelength λ1 [nm] is 50% or less of the intensity at wavelength λ1 [nm].

[0023] Figure 2 shows the absorption characteristics of phenol red as a function of pH. When the medium is fresh, it is alkaline and has a reddish purple color, but as it gradually becomes acidic through cultivation, it takes on a yellowish hue. In FIG. 2, 008 indicates the absorption spectrum of phenol red in an acidic state, 009 indicates the absorption spectrum of phenol red in a neutral state, and 010 indicates the absorption spectrum of phenol red in an alkaline state. As shown in FIG. 2, phenol red has a small absorption rate above 600 nm regardless of pH, so it is preferable to use light with a wavelength of 600 nm as the irradiated light 005, as shown in FIG. 4. In addition, even when the pH fluctuates, phenol red has a relatively low absorption rate around 480 nm and has a constant isosbestic point regardless of pH. As the irradiated light 005, it is also effective to use light with a wavelength near the isosbestic point of phenol red, as shown in FIG. 3, since the intensity of the irradiated light 005 is less susceptible to fluctuations due to changes in pH. In addition, 007 in FIG. 3 indicates the spectral spectrum of the irradiated light.

[0024] 3 and 4, the spectrum 007 of the irradiated light 005 is unimodal, but it may be bimodal or multimodal, and the irradiated light 005 may have maximum intensities near 480 nm and at or above 600 nm. As a recommended spectral characteristic, the peak wavelength at which the intensity is maximum is preferably in the range of 450 nm to 500 nm or 600 nm or above. It is even more preferable that the intensities at 430 nm and 560 nm are 50% or less of the maximum values.

[0025] When the medium contains a pH indicator other than phenol red, it is preferable to employ irradiated light 005 having spectral characteristics within the above range in accordance with the characteristics of each pH indicator. The pH indicator contained in the medium may be any known pH indicator without limitation, and examples thereof include phenol red, methyl violet, brilliant green, metanil yellow, 4-phenylazodiphenylamine, metacresol purple, thymol blue, 2,8-dinitrophenol, 2,4-dinitrophenol, methyl yellow, ethyl orange, bromophenol blue, Congo red, methyl orange, naphthyl red, alizarin red, bromocresol green, 2,5-dinitrophenol, methyl red, lacmoid, ethyl red, p-nitrophenol, bromocresol purple, bromophenol red, bromothymol blue, neutral red, rosolic acid, α-naphtholphthalein, cresol red, phenolphthalein, p-xylenol blue, α-cresolphthalein, p-naphtholbenzene, thymolphthalein, alizarin yellow G, alizarin yellow R, tetryl, tropaeoline O, indigo carmine, etc. The absorption characteristics of each indicator can be referred to the catalog of the sales company.

[0026] The effect of suppressing the variation in the light quantity caused by the light absorption of the medium, which is brought about by using the above-mentioned irradiating light 005, becomes more prominent as the imaging range becomes wider, that is, as the optical path difference in the imaging area becomes larger. A larger effect is produced when using a culture vessel 002 having a culture surface area of ​​a commonly used dish having a width of 30 mm or more. That is, when the imaging unit images an area of ​​30 mm x 30 mm or more, the cell observation system of this embodiment produces a more prominent effect.

[0027] (Imaging unit) The imaging unit 006 is only required to be able to capture images of the cells attached to the culture surface 001, and a so-called digital camera using a CMOS, a CCD, or the like can be used.

[0028] Second Embodiment A second embodiment of the present disclosure is a mounting portion on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; and An imaging unit that images the culture surface, A) controlling fluctuations in the amount of light irradiated onto the culture surface due to light absorption by the medium in the incubator; or a) correcting the influence caused by fluctuations in the amount of light irradiated onto the culture surface due to light absorption by the medium in the incubator; The present invention has at least one of the following characteristics: In the cell observation system, A cell observation system having the above feature (a) and further having an information processing unit, The information processing unit includes: an image information acquiring step of acquiring image information of an image captured by the imaging unit; an irradiation light fluctuation information acquisition step of acquiring irradiation light fluctuation information; an image processing step of generating a processed image based on the image information and the irradiation light fluctuation information, A cell observation system.

[0029] (Configuration of cell observation system) The cell observation system 200 of this embodiment will be described with reference to FIG. 5 to FIG. 7. As shown in FIG. 5, the cell observation system of this embodiment further includes an information processing unit 201 in addition to the first embodiment. As shown in FIG. 6, the information processing unit 201 acquires image information of an image captured by the imaging unit 006 (image information acquisition step 2001). On the other hand, it acquires an irradiation light fluctuation factor that causes a fluctuation in the intensity of the irradiation light 005 (irradiation light fluctuation factor acquisition step 2002), and calculates an estimated irradiation light fluctuation based on the acquired irradiation light fluctuation factor (estimated irradiation light fluctuation calculation step 2003). Then, it generates a processed image based on the image information and the estimated irradiation light fluctuation (image processing step 2004). The information processing unit 201 is preferably connected to the imaging unit 006 for sending and receiving information. Furthermore, it may be connected to the placement unit 003, the light irradiation unit 004, and the like. Note that the connection here means that information can be exchanged, and may be wired or wireless.

[0030] (Information Processing Department) The information processing unit 201 may have an image information acquisition unit for executing an image information acquisition step 2001, an irradiation light fluctuation factor acquisition unit for executing an irradiation light fluctuation factor acquisition step 2002, an estimated irradiation light fluctuation calculation unit for executing an estimated irradiation light fluctuation calculation step 2003, an image processing unit for executing an image processing step 2004, and a memory unit for storing irradiation light fluctuation factors, etc.

[0031] The information processing unit 201 has a function of a computer. An outline of the hardware configuration of the information processing unit 201 is illustrated in FIG. 7. The information processing unit 201 may be integrated with a desktop PC (Personal Computer), a laptop PC, a tablet PC, a smartphone, or the like. The information processing unit 201 includes a CPU (Central Processing Unit) 2006, a RAM (Random Access Memory) 2007, a ROM (Read Only Memory) 2008, and a HDD (Hard Disk Drive) 2009 to realize the function of a computer that performs calculations and storage. The information processing unit 201 also includes a communication I / F (Interface) 2010, a display device 2011, and an input device 2012. The CPU 2006, the RAM 2007, the ROM 2008, the HDD 2009, the communication I / F 2010, the display device 2011, and the input device 2012 are connected to each other via a bus 2013. Some of these functions may be configured by external devices. For example, the display device 2011 and the input device 2012 may be external devices separate from the parts constituting the functions of the computer including the CPU 2006 and the like. The CPU 2006 performs a predetermined operation according to a program stored in the RAM 2007, the HDD 2009, and the like, and also has a function of controlling each part of the information processing unit 201. The RAM 2007 is made of a volatile storage medium and provides a temporary memory area necessary for the operation of the CPU 2006. The ROM 2008 is made of a non-volatile storage medium and stores necessary information such as a program used in the operation of the information processing unit 201. The HDD 2009 is a storage device made of a non-volatile storage medium. The communication I / F 2010 is a communication interface based on standards such as Wi-Fi (registered trademark) and 4G, and is a module for communicating with other devices. The display device 2011 is a liquid crystal display, an OLED (Organic Light Emitting Diode) display, or the like, and is used to display moving images, still images, characters, and the like. The input device 2012 is a button, a touch panel, a keyboard, a pointing device, or the like, and is used by the user to operate the information processing unit 201 .The display device 2011 and the input device 2012 may be integrally formed as a touch panel.

[0032] The hardware configuration shown in FIG. 7 is an example, and other devices may be added, or some devices may not be provided. Some devices may be replaced with other devices having similar functions. Some functions may be provided by other devices via a network, and the functions constituting the present embodiment may be distributed and realized among a plurality of devices. For example, the HDD 2009 may be replaced with an SSD (Solid State Drive) using semiconductor elements such as flash memory, or may be replaced with cloud storage. The CPU 2006 loads a program stored in the ROM 2008 or the like into the RAM 2007 and executes it, thereby realizing an image information acquisition step 2001, an irradiation light fluctuation factor acquisition step 2002, an estimated irradiation light fluctuation calculation step 2003, and an image processing step 2004.

[0033] (Image information acquisition process) The image information is any information based on a captured image, and can use pixel data at a certain time point or multiple time points across the entire image of the culture surface. The image information can include, for example, color distribution, changes in color and density, the number and density of cultured cells and colonies, the size and shape of cells, average in-plane power distribution information, and information on reflected light from the culture vessel 002.

[0034] One or more pieces of image information can be obtained from one or more images. The multiple pieces of image information may be image information on multiple different regions in the culture surface, or may be image information on different regions or the same region at different time points. Information calculated from the difference or sum based on the multiple pieces of image information may be used as image information. In particular, the image information on each of different positions on the culture surface 001, or the image information on the same position at different time points, can be used as a clue to infer the color change or pH change of the medium by comparing them, so it is useful to use multiple pieces of image information. When using multiple pieces of image information, if the image information on the region closest to the light irradiation unit 004 and the region farthest from the culture surface 001 is used, the irradiation light fluctuation factor caused by the difference in distance from the light irradiation unit 004 can be efficiently obtained. Similarly, if multiple pieces of image information separated over time are used, the irradiation light fluctuation factor caused by the change over time can be efficiently obtained.

[0035] (Process for acquiring factors causing fluctuations in irradiation light) A factor that causes a change in the amount of light of the irradiation light 005 irradiated onto the culture surface 001 within the surface or over time is called an irradiation light change factor. The irradiated light fluctuation factors may include, for example, information on the irradiated light, information on the culture vessel, and information on the medium in the culture vessel. The irradiated light information may include the type of light source of the light irradiating unit 004, the intensity of the irradiated light 005, the spectral spectrum, the irradiation distribution, and the like. The culture vessel information may include the type, material, size, capacity, shape, manufacturer, product number, and the like of the culture vessel 002. The medium in the culture vessel information may include the type of dye contained in the medium, the medium or the absorption characteristics of the dye contained in the medium, the liquid volume of the medium, the pH of the medium, the time elapsed since the start of the culture, the type of cultured cells, the number of cultured cells, the growth state of the cultured cells, the culture conditions, and the like. The irradiated light fluctuation factors are recorded in the memory unit of the information processing unit 201, and this recording may be performed by inputting by the user or selecting from options. Alternatively, this recording may be performed by the cell observation system by automatic input. Examples of automatic input include determining the type of the culture vessel 002 from image information acquired by the imaging unit 006, attaching a weight sensor to the mounting unit 003, calculating the liquid volume of the medium, and inputting the results to the storage unit. Information regarding the correspondence between the captured image information and the irradiated light fluctuation factors may be stored in advance in the information processing unit 201, and the irradiated light fluctuation factors may be calculated from the information. Alternatively, an inference device may be provided, and the irradiated light fluctuation factors may be inferred from the captured image information using the inference device, and the inference result may be used as the irradiated light fluctuation factors. The inference device may use, for example, a learning model obtained using learning data in which image information is input data and the irradiated light fluctuation factors are output data.

[0036] (Estimated irradiation light fluctuation calculation process, image processing process) Based on the acquired irradiation light fluctuation factors, an estimated irradiation light fluctuation is calculated, which is the degree of fluctuation in the irradiation light 005 due to the irradiation light fluctuation factors on the culture surface 001 to be imaged. For example, when a change in pH of the medium over time is acquired as an irradiation light fluctuation factor, based on the accompanying color change in the medium over time, information on the spectral spectrum of the irradiation light 005, the width of the culture vessel 002 (the width from the side closer to the light irradiation unit 004 to the side farther from the light irradiation unit 004), and the transmittance of the irradiation light 005 estimated from the material of the culture vessel 002, it is possible to calculate how much the amount of light increase or decrease has occurred in each region of the culture surface 001 at each time point compared to the reference amount of the irradiation light 005, and use this as the estimated irradiation light fluctuation.

[0037] By adding the estimated irradiation light fluctuation information to the captured image, unevenness in the image and fluctuations in the image over time caused by fluctuations in the estimated irradiation light 005 can be removed, and an image that would be obtained if there was no fluctuation in the irradiation light 005 can be generated. The effect of suppressing the variation in the light quantity caused by the light absorption of the medium, which is brought about by using the above-mentioned irradiating light 005, becomes more prominent as the imaging range becomes wider, that is, as the optical path difference in the imaging area becomes larger. A larger effect is produced when using a culture vessel 002 having a culture surface area of ​​a commonly used dish having a width of 30 mm or more. That is, when the imaging unit images an area of ​​30 mm x 30 mm or more, the cell observation system of this embodiment produces a more prominent effect.

[0038] Third embodiment A third embodiment of the present disclosure comprises: a mounting portion on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; An imaging unit that images the culture surface, and An information processing unit is provided, The information processing unit includes: an image information acquiring step of acquiring image information of a captured image from the imaging unit; A characteristic calculation step of calculating characteristics of the medium in the incubator from the image information; and A characteristic output step of outputting characteristics of the medium in the incubator is carried out. A cell observation system.

[0039] (Configuration of cell observation system) The configuration of the cell observation system of this embodiment can refer to the cell observation system of the second embodiment shown in Fig. 5. As shown in Fig. 8, the information processing unit 201 acquires image information of an image captured by the imaging unit 006 (image information acquisition step 3001), and calculates the characteristics of the medium in the incubator from the image information (characteristic calculation step 3002). Furthermore, the information processing unit may acquire irradiance light fluctuation factors (irradiance light fluctuation factor acquisition step 3003), and execute an estimated irradiance light fluctuation calculation step (3004) for calculating an estimated irradiance light fluctuation based on the irradiance light fluctuation factors, and calculate the characteristics of the medium from the image information and the estimated irradiance light fluctuation.

[0040] (Spectral characteristics of irradiated light) In this embodiment, in order to calculate the characteristics of the medium, it is preferable to obtain a large variation in the light intensity of the irradiated light 005 caused by the light absorption of the medium in the culture vessel 002, and it is particularly preferable that the variation caused by the pH of the medium is large. Therefore, it is preferable that the irradiated light 005 has spectral characteristics that provide a high absorption rate by the medium in the culture vessel 002.

[0041] The absorption maximum wavelength of the medium when the medium is acidic is λ 2A [nm], and the absorption maximum wavelength of the medium when the medium is alkaline is λ 2B [nm], the wavelength of the irradiated light 005 is λ 2A -25[nm] or more λ 2A +25[nm] or less or λ 2B -25[nm] or more λ 2BIt is preferable that the intensity is maximum in the range of +25 [nm] or less. When the pH indicator of the medium is phenol red, it is preferable that the intensity of the irradiated light 005 is maximum in the wavelength range of 400 nm to 450 nm or in the wavelength range of 525 nm to 575 nm in the spectroscopic spectrum. When the medium contains a pH indicator other than phenol red, it is preferable to adopt irradiated light 005 having spectral characteristics in the above ranges according to the characteristics of each pH indicator.

[0042] (Characteristics calculation process / characteristics output process) The image information acquisition step 3001 is the same as that of the second embodiment. The characteristic calculation step 3002 will be described below. The characteristic calculation step is a step of calculating the characteristics of the medium in the culture vessel. The characteristics of the medium refer to the characteristics of the medium in the culture vessel 002, and may include the liquid volume of the medium, the pH of the medium, the time elapsed since the start of culture, the number of cultured cells, the growth state of the cultured cells, etc., and more preferably, the pH of the medium, the number of cultured cells, and the growth state of the cultured cells. The information processing unit 201 calculates these characteristics based on the image information of the captured image.

[0043] For example, when the medium is acidic, the absorption maximum wavelength is λ 2A [nm], and the absorption maximum wavelength when the medium is alkaline is λ 2B [nm], and λ as the irradiated light 005 2A When light having a wavelength of λ [nm] is irradiated, if the difference in brightness of the image between the area close to the light irradiated portion 004 and the area far from the light irradiated portion 004 is higher than a certain value, it is understood that the irradiated light 005 is absorbed by the medium as it travels across the width of the culture vessel 002. 2A [nm], i.e., the medium is acidic.

[0044] In this embodiment, the information processing unit may execute an irradiation light fluctuation factor acquisition step and an estimated irradiation light fluctuation calculation step in addition to the characteristic calculation step and the characteristic output step. However, these steps may not be performed. The irradiation light fluctuation factors are as described in the second embodiment. However, since this embodiment is for calculating the characteristics of the medium, in this embodiment, it is preferable that the irradiation light fluctuation factors do not include those related to the characteristics of the medium to be calculated. As the characteristics of the medium, preferred examples of the objects to be calculated include the pH of the medium, the number of cultured cells, and the state of proliferation of the cultured cells. In contrast, the irradiation light fluctuation factors may include the type of light source of the light irradiation unit 004 as information related to the irradiation light, the intensity, spectroscopic spectrum, irradiation distribution, etc. of the irradiation light, and the type, material, size, capacity, shape, manufacturer, product number, etc. of the culture vessel as information related to the culture vessel. When information related to the medium in the culture vessel is used as the irradiation light fluctuation factor, the type of pigment contained in the medium, the elapsed time from the start of culture, the type of cultured cells, the culture conditions, etc., which can be easily input by the user, are applicable.

[0045] Information regarding the correspondence between captured image information and medium characteristics is stored in advance in the information processing unit 201, and the medium characteristics can be calculated from the captured image information. In this case, it is also possible to add an irradiation light fluctuation factor and store information regarding the correspondence between the captured image information, the irradiation light fluctuation factor, and medium characteristics in advance, and calculate the medium characteristics from the captured image information and the irradiation light fluctuation factor based on this.

[0046] Alternatively, an inference device may be provided, and the characteristics of the medium may be inferred from the captured image information using the inference device. The inference device may use, for example, a learning model obtained using learning data in which the image information is input data and the characteristics of the medium are output data. In this case, a learning model obtained using learning data in which, in addition to the captured image information, the irradiation light fluctuation factors are input data and the characteristics of the medium are output data may be used.

[0047] (Characteristics output process) The calculated characteristics are output to a medium such as paper via a monitor screen, tablet terminal, smartphone screen, writable optical drive, or printer so that the user can recognize them. By checking the medium characteristics displayed in this characteristic output unit, the user of the cell observation system can confirm that unevenness in the image and fluctuations over time in the image are due to the progress of the culture and are not a malfunction of the cell observation system itself. In addition, the user can grasp changes in the state of the medium and obtain information to determine the timing of the next culture process, such as changing the culture medium.

[0048] Furthermore, the feature of this system, in which light is irradiated from the side, is a great advantage in calculating the medium properties, as it allows a long optical path even with a small amount of medium, thereby improving the accuracy of the calculation of the medium properties.

[0049] If the characteristics of the medium (e.g., pH of the culture medium) can be calculated, the distribution of the amount of irradiated light within the culture surface can be calculated. The image processing unit calculates such a distribution of the amount of irradiated light, and by processing the image information acquired by the imaging unit 006, it is possible to correct unevenness in the amount of light. In addition, it is also possible to add information on the characteristics of the medium to the image information, which is effective for managing images. For example, it is possible to write the pH of the medium in the image, or write the pH information in the header section or file name.

[0050] The wider the imaging range, that is, the larger the optical path difference in the imaging area, the more noticeable the fluctuation in the amount of light due to the characteristics of the medium. That is, when using a culture vessel 002 having a culture surface area of ​​a commonly used dish with a width of 30 mm or more, a larger effect is obtained. That is, when the imaging unit images an area of ​​30 mm x 30 mm or more, the cell observation system of the present disclosure has a more noticeable effect.

[0051] (Programs and media) The present disclosure includes a program for causing an information processing unit to execute each step in each embodiment of the cell observation system described above, and a recording medium on which the program is stored in a computer-readable format.

[0052] The present disclosure can also be realized by a process in which a program for implementing one or more functions of the above-described embodiments is supplied to a system or device via a network or a storage medium, and one or more processors in a computer of the system or device read and execute the program. It can also be realized by a circuit (e.g., ASIC) that implements one or more functions. Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above-mentioned embodiments. Inventions modified within the scope of the present invention and inventions equivalent to the present invention are also included in the present invention. In addition, the above-mentioned embodiments can be appropriately combined within the scope of the present invention.

[0053] The embodiments of the present disclosure include the following configurations. (Configuration 1) a mounting portion on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; and An imaging unit that images the culture surface, A) controlling the variation in the amount of light irradiated onto the culture surface caused by the light absorption of the medium in the incubator; or a) correcting the effect caused by the variation in the amount of light irradiated onto the culture surface due to the light absorption of the medium in the incubator; The present invention has at least one of the following characteristics: Cell observation system. (Configuration 2) The above-mentioned feature (a) is present, The irradiation light has a wavelength λ 2A [nm] and wavelength λ 2B The cell observation system according to configuration 1, wherein the intensity at wavelength λ1 [nm] is 50% or less of the intensity at wavelength λ1 [nm]; however, In the spectrum of the irradiation light, the intensity is maximum at a wavelength λ1 [nm], When the medium is acidic, the wavelength λ2A The absorption maximum is at [nm], When the medium is alkaline, the wavelength λ in the absorption spectrum of the medium 2B [nm] has maximum absorption. (Configuration 3) The above-mentioned feature (a) is present, 3. The cell observation system according to claim 1 or 2, wherein the intensity of the irradiating light is maximum in the wavelength range of 450 nm or more and 500 nm or less, or 600 nm or more, in the spectrum of the irradiating light. (Configuration 4) The above-mentioned feature (a) is present, 4. The cell observation system according to any one of configurations 1 to 3, wherein the intensity of the irradiating light at wavelengths of 430 nm and 560 nm in its spectrum is 50% or less of the maximum intensity. (Configuration 5) A cell observation system having the above feature (a) and further having an information processing unit, The information processing unit includes: an image information acquiring step of acquiring image information of an image captured by the imaging unit; an irradiation light fluctuation factor acquisition step of acquiring an irradiation light fluctuation factor; an estimated irradiation light fluctuation calculation step of calculating an estimated irradiation light fluctuation based on the irradiation light fluctuation factor; and an image processing step of generating a processed image based on the image information and the estimated irradiation light variation, A cell observation system according to configuration 1. (Configuration 6) 6. The cell observation system according to configuration 5, wherein the irradiated light fluctuation factor is acquired based on at least one selected from the group consisting of information about the irradiated light, information about the culture vessel, and information about the medium in the culture vessel. (Configuration 7) 7. The cell observation system according to claim 6, wherein the information about the medium includes information about the pH of the medium. (Configuration 8) 8. The cell observation system according to any one of configurations 5 to 7, characterized in that in the irradiation light fluctuation factor acquisition step, information about the medium is acquired using the image information about a plurality of different regions within the culture surface. (Configuration 9) a mounting portion on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; An imaging unit that images the culture surface, and An information processing unit is provided, The information processing unit includes: an image information acquiring step of acquiring image information of a captured image from the imaging unit; A characteristic calculation step of calculating characteristics of the medium in the incubator from the image information; and A characteristic output step of outputting characteristics of the medium in the incubator is carried out. Cell observation system. (Configuration 10) Furthermore, the information processing unit An irradiation light fluctuation factor acquisition step of acquiring an irradiation light fluctuation factor; an estimated irradiation light fluctuation calculation step of calculating an estimated irradiation light fluctuation based on the irradiation light fluctuation factor; 10. A cell observation system according to claim 9, (Configuration 11) The absorption maximum wavelength of the medium when the medium is acidic is defined as λ 2A [nm], The absorption maximum wavelength of the medium when the medium is alkaline is defined as λ 2B [nm], The irradiation light has a wavelength of λ 2A -25[nm] or more λ 2A +25[nm] or less or λ 2B -25[nm] or more λ 2B 11. The cell observation system according to configuration 9 or 10, wherein the intensity is maximum in the range of +25 [nm] or less. (Configuration 12) A cell observation system described in any one of configurations 9 to 11, characterized in that the intensity of the irradiated light is greatest in the wavelength range of 400 nm or more and 450 nm or less, or in the wavelength range of 525 nm or more and 575 nm or less, in its spectral spectrum. (Configuration 13) 13. The cell observation system according to any one of configurations 9 to 12, wherein in the characteristic calculation step, the characteristics of the medium are calculated using the image information relating to a plurality of different regions within the culture surface. (Configuration 14) 14. The cell observation system according to any one of configurations 9 to 13, wherein the property of the medium is the pH of the medium. (Configuration 15) 15. The cell observation system according to any one of configurations 1 to 14, wherein the imaging unit images an area of ​​30 mm x 30 mm or more. (Configuration 16) A program for causing the information processing unit to execute the image information acquisition process, the irradiance light fluctuation factor acquisition process, the estimated irradiance light fluctuation calculation process, and the image processing process in the cell observation system described in any one of configurations 5 to 8. (Configuration 17) 15. A program for causing the information processing unit to execute the image information acquisition step and the characteristic output step in the cell observation system according to any one of configurations 9 to 14. (Configuration 18) A program for causing the information processing unit to execute the image information acquisition step, the irradiance light fluctuation factor acquisition step, the estimated irradiance light fluctuation calculation step, and the characteristic calculation step in the cell observation system according to any one of configurations 10 to 14. (Configuration 19) A recording medium storing the program according to any one of configurations 16 to 18 in a computer-readable format. [Explanation of symbols]

[0054] 001 Culture surface 002 Culture container 003 Placement part 004 Light irradiation unit 005 Irradiation light 006 Imaging unit 007 Spectral spectrum of irradiated light 008 Absorption spectrum of phenol red (acidic) 009 Absorption spectrum of phenol red (neutral) 010 Absorption spectrum of phenol red (alkaline) 100 Cell Culture Systems 200 Cell Observation System 201 Information Processing Section 2001 Image information acquisition process 2002 Irradiation light fluctuation factor acquisition process 2003 Estimated light fluctuation calculation process 2004 Image processing 2006 CPU 2007 RAM 2008 ROM 2009 HDD 2010 Communication I / F 2011 Display device 2012 Input Device 2013 Bus 3001 Image information acquisition process 3002 Characteristic calculation process 3003 Irradiation light fluctuation factor acquisition process 3004 Estimated irradiation light fluctuation calculation process

Claims

1. a mounting portion on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; and An imaging unit that images the culture surface, A) controlling the variation in the amount of light irradiated onto the culture surface caused by the light absorption of the medium in the incubator; or a) correcting the effect caused by the variation in the amount of light irradiated onto the culture surface due to the light absorption of the medium in the incubator; The present invention has at least one of the following characteristics: Cell observation system.

2. The above-mentioned feature (a) is present, The irradiation light has a wavelength λ ２Ａ [nm] and wavelength λ ２Ｂ The intensity at wavelength λ [nm] is １ The cell observation system according to claim 1, wherein the intensity is 50% or less of the intensity at [nm]. however, In the spectrum of the irradiation light, the wavelength λ １ The intensity is maximum at [nm], When the medium is acidic, the wavelength λ ２Ａ The absorption is maximum at [nm], When the medium is alkaline, the wavelength λ in the absorption spectrum of the medium ２Ｂ The absorption is maximum at [nm].

3. The above-mentioned feature (a) is present, 2. The cell observation system according to claim 1, wherein the intensity of the irradiating light is maximum in the wavelength range of 450 nm to 500 nm or 600 nm or more in the spectrum of the irradiating light.

4. The above-mentioned feature (a) is present, 2. The cell observation system according to claim 1, wherein the intensity of the irradiating light at wavelengths of 430 nm and 560 nm in its spectrum is 50% or less of the maximum intensity.

5. A cell observation system having the above feature (a) and further having an information processing unit, The information processing unit includes: an image information acquiring step of acquiring image information of an image captured by the imaging unit; an irradiation light fluctuation factor acquisition step of acquiring an irradiation light fluctuation factor; an estimated irradiation light fluctuation calculation step of calculating an estimated irradiation light fluctuation based on the irradiation light fluctuation factor; and an image processing step of generating a processed image based on the image information and the estimated irradiation light variation, The cell observation system according to claim 1 .

6. The cell observation system according to claim 5 , wherein the irradiated light fluctuation factor is acquired based on at least one selected from the group consisting of information about the irradiated light, information about the culture vessel, and information about a medium in the culture vessel.

7. The cell observation system according to claim 6 , wherein the information about the medium includes information about a pH of the medium.

8. 7. The cell observation system according to claim 6, wherein in the irradiation light fluctuation factor acquisition step, information about the medium is acquired using the image information about a plurality of different regions within the culture surface.

9. a mounting portion on which a culture vessel having a culture surface on which cells can be cultured and an optically transparent side surface constituting a peripheral wall of the culture surface is mounted; a light irradiation unit that irradiates the culture surface with irradiation light from the side surface; An imaging unit that images the culture surface, and An information processing unit is provided, The information processing unit includes: an image information acquiring step of acquiring image information of a captured image from the imaging unit; A characteristic calculation step of calculating characteristics of the medium in the incubator from the image information; and A characteristic output step of outputting characteristics of the medium in the incubator is carried out. Cell observation system.

10. Furthermore, the information processing unit An irradiation light fluctuation factor acquisition step of acquiring an irradiation light fluctuation factor; an estimated irradiation light fluctuation calculation step of calculating an estimated irradiation light fluctuation based on the irradiation light fluctuation factor; The cell observation system according to claim 9, which executes the above.

11. The absorption maximum wavelength of the medium when the medium is acidic is defined as λ ２Ａ [nm], The absorption maximum wavelength of the medium when the medium is alkaline is defined as λ ２Ｂ As [nm], The irradiation light has a wavelength of λ ２Ａ -25 [nm] or more λ ２Ａ +25 [nm] or less or λ ２Ｂ -25 [nm] or more λ ２Ｂ The cell observation system according to claim 9, wherein the intensity is maximum in the range of +25 nm or less.

12. 10. The cell observation system according to claim 9, wherein the intensity of the irradiating light is greatest in the wavelength range of 400 nm to 450 nm or in the wavelength range of 525 nm to 575 nm in its spectrum.

13. The cell observation system according to claim 9, wherein in the characteristic calculation step, the characteristic of the medium is calculated using the image information relating to a plurality of different regions within the culture surface.

14. The cell observation system according to claim 9 , wherein the property of the medium is a pH of the medium.

15. The cell observation system according to claim 1 , wherein the imaging unit images an area of ​​30 mm×30 mm or more.

16. 6. A program for causing the information processing unit to execute the image information acquiring step, the irradiating light fluctuation factor acquiring step, the estimated irradiating light fluctuation calculating step, and the image processing step in the cell observation system according to claim 5.

17. 10. A program for causing the information processing unit to execute the image information acquiring step and the characteristic outputting step in the cell observation system according to claim 9.

18. 11. A program for causing the information processing unit to execute the image information acquiring step, the irradiating light fluctuation factor acquiring step, the estimated irradiating light fluctuation calculating step, and the characteristic calculating step in the cell observation system according to claim 10.

19. A recording medium storing the program according to any one of claims 16 to 18 in a computer-readable format.

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