JP2012039931A - Observation device, observation method, and method of producing culture material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an observation method for accurately identifying an area of a drop of culture medium in a culturing container.SOLUTION: The observation method includes: irradiating the culturing container internally including the drop of the culture medium which is used for culturing a culture material from a plurality of lighting directions (step S1); capturing images of the lighted culturing container at each of the plurality of lighting directions (step S2); generating a composite image of the culturing container by overlapping the captured images of the culturing container at the plurality of lighting directions (step S5); and identifying the area of the drop of culturing medium in the culturing container from the generated composite images (step S6).

Description

  The present invention relates to an observation apparatus and an observation method for detecting a region of a medium used for culturing a culture from a culture vessel, and further relates to a culture production method using such an observation apparatus and an observation method. .

  In recent years, with the development of assisted reproduction technology (ART), it has been practiced to observe the growth state of cultured fertilized eggs by in vitro fertilization. A culture microscope is mentioned as an example of the apparatus which observes the condition of cultures, such as a fertilized egg (for example, refer patent document 1). The culture microscope includes a culture apparatus (invecutor) that forms a suitable environment for culturing fertilized eggs, and a microscopic observation system that microscopically observes the state of the fertilized eggs in the culture container housed in the culture apparatus. The observation image of the fertilized egg is acquired at regular intervals, and the user can recognize the fertilized egg by visual observation and automatically perform observation, recording, management, etc. of the fertilized egg. The

  In such an apparatus, when observing the growth state of a fertilized egg in a culture container, it is necessary to first detect the fertilized egg to be observed. As a detection procedure, generally, the entire culture container is photographed. After detecting the medium (medium drop) immersed in mineral oil from the inside of the culture vessel based on the whole observation image, a plurality of microscopic observation images of the medium drop are acquired while changing the observation visual field position in the medium drop. Based on this microscopic observation image, a fertilized egg to be observed that is injected one by one (or a plurality) into the medium drop is discriminated from other foreign substances and detected. Thus, in order to recognize a fertilized egg, microscopic observation in a high magnification field is generally required. However, as the observation magnification increases, the observation field (observation range) becomes narrower. In contrast, a large number of microscopic observation images need to be taken. Therefore, if the medium drop area can be detected in advance by macro observation prior to detection of the fertilized egg by microscopic observation as described above, the area for obtaining the observation image in the microscopic observation is naturally limited, and the entire observation time is reduced. It becomes possible to shorten.

  In the automatic observation of fertilized eggs, as a method for limiting the exploration area of the medium drop, a method for mechanically specifying the exploration area and coloring the phenol added for checking the culture environment are used. There are ways to separate the colors. However, in the method of mechanically specifying the exploration area, since the user manually creates a medium drop in the dish, the exploration can only be performed by specifying the initial exploration area almost on the entire dish surface, and the effect of limiting the area is effective. I can't get it. In addition, although the color separation method can efficiently limit the search area, the color strength after coloring is unknown depending on the environment, and depending on the user, observation or the like may be performed without phenol. In addition, there are not a few users who write on the upper or lower surface of the dish with a pen. In the extraction by color separation, the detection accuracy of the medium drop region changes depending on the color density or the presence or absence of writing.

JP 2004-229619 A

  Therefore, a measure for extracting a medium drop region from an observation image in a wide observation field of view without depending on color information is desired.

  The present invention has been made in view of such problems, and an object of the present invention is to provide an observation apparatus, an observation method, and a culture production method capable of accurately identifying a region of a medium from a culture container. To do.

  In order to achieve such an object, according to an embodiment of the present invention, an illumination unit capable of illuminating a culture vessel having a culture medium used for culture of a culture from a plurality of illumination directions, and the illumination unit An imaging unit that images the illuminated culture vessel in each of the plurality of illumination directions, and a composite image of the culture vessel by superimposing images of the culture vessel in the plurality of illumination directions that are respectively imaged in the imaging unit A culture observation apparatus comprising: an image synthesis unit that generates a medium; and an image analysis unit that identifies the medium in the culture vessel from the synthesized image generated by the image synthesis unit. Is provided.

  Further, according to an embodiment illustrating the present invention, a culture container having a medium used for culture of the culture is illuminated from a plurality of illumination directions, and the illuminated culture container is illuminated in the plurality of illumination directions. Each of the images is taken, and the image of the culture vessel for the plurality of illumination directions taken is superimposed to generate a composite image of the culture vessel, and the culture medium in the culture vessel is generated from the generated composite image. A method for observing a culture characterized by distinguishing is provided.

  In addition, according to an embodiment illustrating the present invention, the culture is cultured under a predetermined environmental condition, and the observation apparatus according to the present invention is used from a culture container having a medium in which the culture is cultured. A method for producing a culture is provided that identifies the medium.

  Further, according to an embodiment illustrating the present invention, a culture is cultured under a predetermined environmental condition, and a culture container having a medium in which the culture is cultured is illuminated from a plurality of illumination directions, and the illumination is performed. The culture vessel is imaged for each of the plurality of illumination directions, and a composite image of the culture vessel is generated by superimposing the captured images of the culture vessel for the plurality of illumination directions, and the generated A method for producing a culture is provided, wherein the culture medium in the culture vessel is identified from a synthesized image.

  According to the present invention, it is possible to accurately identify a medium region from a culture container.

It is a flowchart which shows the outline | summary of the observation method. It is a general | schematic block diagram of the culture observation system shown as an example of application of this invention. It is a block diagram of the said culture observation system. (A) is a top view of a culture container, (B) is a perspective view which shows a dish. It is a schematic block diagram of a 1st illumination part. It is a schematic diagram which shows the process which produces | generates a synthesized image. 1 is a block diagram illustrating a schematic configuration of an image processing apparatus. It is a flowchart which shows the outline | summary of the manufacturing method of a fertilized egg.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. As an example of a system to which the observation apparatus according to the present embodiment is applied, a schematic configuration diagram and a block diagram of a culture observation system are shown in FIGS. 2 and 3, respectively.

  The culture observation system BS roughly observes the culture chamber 2 provided in the upper part of the housing 1, the shelf-like stocker 3 that accommodates and holds a plurality of culture containers 10, and the sample in the culture container 10. An observation unit 5, a transfer unit 4 for transferring the culture vessel 10 between the stocker 3 and the observation unit 5, a control unit 6 for comprehensively controlling the operation of the system, and an operation panel 7 provided with an image display device. Etc.

The culture chamber 2 is a chamber for forming a culture environment, and is kept sealed after the sample is charged in order to prevent environmental changes and contamination. Along with the culture chamber 2, a temperature adjustment device 21 that raises and lowers the temperature in the culture chamber 2, a humidifier 22 that adjusts humidity, and a gas supply device 23 that supplies a gas such as CO ₂ gas or N ₂ gas. A circulation fan 24 for making the entire environment of the culture chamber 2 uniform, an environmental sensor 25 for detecting the temperature, humidity, carbon dioxide concentration, etc. of the culture chamber 2 are provided. The operation of each device is controlled by the control unit 6, and the culture environment defined by the temperature, humidity, carbon dioxide concentration, etc. of the culture chamber 2 is maintained in a state that matches the culture conditions set on the operation panel 7.

  The stocker 3 is formed in a shelf shape that is partitioned into a plurality of parts in the front-rear direction and the vertical direction in FIG. A unique address is set for each shelf. For example, when the front-rear direction is A to C rows and the vertical direction is 1 to 7 rows, the A-row 5 rows shelf is set as A-5. The

  As the culture vessel 10, an appropriate one such as a flask, dish, or well plate is selected according to the type and purpose of the culture. In this embodiment, as shown in FIG. A configuration including five formed dishes 11 (consisting of a container 11a and a lid 11b) having a diameter of about 35 mm and a holder 12 that holds the dish 11 is illustrated. As shown in FIG. A fertilized egg J as a culture is injected into each dish 11 together with a medium drop D containing a pH indicator such as phenol red that changes color according to pH. On the bottom surface of the dish 11, one or more medium drops D of about 20 [μl] dropped by a pipette or the like are formed (three are shown in FIG. 4B). It is in a state immersed in the colorless and transparent mineral oil O inside. In each medium drop D, for example, one (or more) fertilized eggs J collected at the same time from the same mother for external fertilization are inserted. The culture vessel 10 is assigned a code number and is stored in association with the designated address of the stocker 3.

  The transfer unit 4 is provided inside the culture chamber 2 so as to be movable in the vertical direction and is moved up and down by the Z-axis drive mechanism. The transfer unit 4 is attached to the Z stage 41 so as to be movable in the front-rear direction and is moved by the Y-axis drive mechanism. The Y stage 42 that is moved back and forth, the X stage 43 that is attached to the Y stage 42 so as to be movable in the left-right direction and is moved left and right by the X-axis drive mechanism, and the like, is supported by lifting the culture vessel 10 to the tip side of the X stage 43 A support arm 45 is provided. The transport unit 4 has a moving range in which the support arm 45 can move between the entire shelf of the stocker 3 and the observation unit 5. The X-axis drive mechanism, the Y-axis drive mechanism, and the Z-axis drive mechanism are configured by, for example, a servo motor with a ball screw and an encoder, and the operation thereof is controlled by the control unit 6.

  The observation unit 5 includes a first illumination unit 51 that illuminates the sample from the lower side of the sample stage 15, a second illumination unit 52 that illuminates the sample from above the sample stage 15 along the optical axis of the microscopic observation system, and a sample from below. 3, a macro observation system 54 that performs macro observation of the sample, a micro observation system 55 that performs micro observation of the sample, an image processing apparatus 100 (see FIG. 2), and the like. The sample stage 15 is made of a light-transmitting material and has a transparent window 16 in the observation area. The sample stage 15 is composed of a fine drive stage that can be moved in the XY direction (horizontal plane direction) and the Z direction (vertical direction) by operation control from the control unit 6, and the culture vessel 10 placed on the upper surface thereof. Is moved in the XY directions, so that the culture vessel 10 can be inserted on the optical axis of the macro observation system 54 or on the optical axis of the microscopic observation system 55.

  As shown in FIG. 5, the first illumination unit 51 includes a ring illumination device 51 a and the like provided on the lower frame 1 b side, and illuminates the culture vessel 10 from below the sample stage 15. The ring illumination device 51a is configured to include a plurality of light emitting devices 51b such as LEDs arranged in a circle, and by causing some of the plurality of light emitting devices 51b to emit light, the illumination σ is small. In the system, the culture vessel 10 is illuminated from the illumination direction inclined with respect to the optical axis of the macro observation system 54. In addition, the ring illumination device 51a changes the light emission position of the light emitting device 51b and rotationally displaces the illumination direction with respect to the culture vessel 10 about the optical axis of the macro observation system 54, so that the culture vessel 10, the macro observation system 54, etc. The culture vessel 10 can be illuminated from a plurality of illumination directions without rotating.

  The second illumination unit 52 includes a light source such as an LED and an illumination optical system including a phase ring, a condenser lens, and the like. The second illumination unit 52 is provided in the culture chamber 2 and receives light from the microscopic observation system 55 from above the sample stage 15. The sample in the culture vessel 10 is illuminated along the axis. The third illumination unit 53 superimposes light emitted from each of the light sources, such as a plurality of LEDs and mercury that emit light having a wavelength suitable for epi-illumination observation and fluorescence observation, on the optical axis of the microscopic observation system 55. An illumination optical system composed of a beam splitter, a fluorescent filter, and the like to be disposed in the lower frame 1b located below the culture chamber 2, and the light of the microscopic observation system 55 from below the sample stage 15 The sample in the culture vessel 10 is illuminated along the axis.

  The macro observation system 54 includes an observation optical system 54 a and an imaging device 54 c such as a CCD camera that captures an image of the sample imaged by the observation optical system 54 a and is positioned above the first illumination unit 51. Provided in the culture chamber 2. The macro observation system 54 captures an image of transmitted light of the sample (culture vessel 10) backlit by the first illuminating unit 51, and acquires the entire observation image (macro image) as a color image.

  The microscopic observation system 55 includes an observation optical system 55a composed of an objective lens, an intermediate zoom lens, a fluorescent filter, and the like, and an imaging device 55c such as a cooled CCD camera that takes an image of a sample imaged by the observation optical system 55a. And disposed inside the lower frame 1b. The second illumination unit 52 and the microscopic observation system 55 constitute a phase difference observation microscope. A plurality of objective lenses and intermediate zoom lenses are provided, and are configured to be set to a plurality of magnifications using a displacement mechanism such as a revolver or a slider (not shown in detail). In this embodiment, at least two magnifications for low-magnification observation and high-magnification observation are switched so that magnification can be changed. The microscopic observation system 55 displays the sample in the culture vessel 10 such as a phase difference image by the transmitted light of the sample illuminated by the second illumination unit 52 and a fluorescence image by the fluorescence emitted from the sample illuminated by the third illumination unit 53. A microscopic observation image (micro image) is taken.

  The image processing apparatus 100 performs A / D conversion on signals input from the imaging device 54c of the macro observation system 54 and the imaging device 55c of the microscopic observation system 55, and performs various image processing to perform an entire observation image or a microscopic observation image. Image data is generated. Further, the image processing apparatus 100 performs image composition and image analysis on the image data of these observation images (entire observation image and microscopic observation image), and identifies the medium drop D and the fertilized egg J. Specifically, the image processing apparatus 100 is constructed by executing an image processing program stored in the ROM of the control unit 6 described below. The image processing apparatus 100 will be described in detail later.

  The control unit 6 includes a CPU 61 that executes processing, a ROM 62 that stores and stores control programs and control data of the culture observation system BS, a RAM 63 that temporarily stores observation conditions and image data, and the like. Control operation. Therefore, as shown in FIG. 3, the constituent devices of the culture chamber 2, the transport device 4, the observation unit 5, and the operation panel 7 are connected to the control unit 6. In the RAM 63, the environmental conditions of the culture chamber 2 according to the observation program, the observation schedule, the observation type and observation position in the observation unit 5, the observation magnification, and the like are set and stored. Further, the RAM 63 is provided with an image data storage area for recording image data photographed by the observation unit 5, and index data including the code number of the culture vessel 10 and photographing date / time are associated with the image data and recorded. Is done.

  The operation panel 7 is provided with an operation panel 71 provided with input / output devices such as a keyboard and a switch, and a display panel 72 for displaying an operation screen, image data, and the like. An operation command or the like is input. The communication unit 65 is configured in accordance with a wired or wireless communication standard, and data can be transmitted to and received from a computer or the like externally connected to the communication unit 65.

  In the culture observation system BS schematically configured as described above, the CPU 61 controls the operation of each part based on the control program stored in the ROM 62 according to the setting conditions of the observation program set on the operation panel 7, and the culture vessel 10 The sample inside is automatically captured. That is, when the observation program is started by a panel operation on the operation panel 71 (or a remote operation via the communication unit 65), the CPU 61 reads each condition value of the environmental conditions stored in the RAM 63, and from the environment sensor 25. The environmental state of the culture chamber 2 to be input is detected, and the temperature adjustment device 21, the humidifier 22, the gas supply device 23, the circulation fan 24, etc. are operated according to the difference between the condition value and the actual measurement value. Feedback control is performed on the culture environment such as temperature, humidity, and carbon dioxide concentration.

  Further, the CPU 61 reads the observation conditions stored in the RAM 63 and operates the X, Y, Z stages 41, 42, 43 of the transport unit 4 based on the observation schedule to observe the culture vessel 10 to be observed from the stocker 3. The sample is transported to the sample stage 15 of the unit 5 and observation by the observation unit 5 is started. For example, when the observation set in the observation program is macro observation, the culture vessel 10 transported from the stocker 3 by the transport unit 4 is positioned on the optical axis of the macro observation system 54 and placed on the sample stage 15. Then, the light emitting device 51b of the first illumination unit 51 (ring illumination device 51a) is turned on to irradiate illumination light (transmitted light) obliquely from below the culture vessel 10, and thus the optical axis of the macro observation system 54 A whole observation image is taken by the imaging device 54c from above the culture vessel 10 illuminated from the illumination direction inclined with respect to the direction. The signal input from the imaging device 54c to the control unit 6 is processed by the image processing device 100 to generate a whole observation image, and the image data is stored in the image data storage area of the RAM 63 together with index data such as the shooting date and time. Is done.

  At this time, the first illumination unit 51 (ring illumination device 51a) changes the light emission position of the light emitting device 51b and rotationally displaces the illumination direction with respect to the culture vessel 10 about the optical axis of the macro observation system 54, thereby culturing. The container 10 is illuminated from a plurality of preset illumination directions (for example, four types of illumination directions), and the imaging device 54c of the macro observation system 54 is cultivated in any illumination direction by the first illumination unit 51. The entire observation image of the container 10 is imaged for each of the set illumination directions. Therefore, a plurality of image data respectively corresponding to a plurality of illumination directions are stored in the image data storage area of the RAM 63, respectively.

  In addition, when the observation set in the observation program is micro observation of the sample at a specific position in the culture vessel 10, the specific position in the culture vessel 10 that has been transported by the transport unit 4 is indicated by the light of the microscopic observation system 55. Position on the axis and place on the sample stage 15, turn on the light source of the second illumination unit 52 or the third illumination unit 53, and cause the imaging device 55c to take a microscopic observation image by transmitted illumination, epi-illumination, and fluorescence. . A signal photographed by the imaging device 55c and inputted to the control unit 6 is processed by the image processing device 100 to generate a microscopic observation image (phase difference image, fluorescent image, etc.), and the image data is an index such as a photographing date / time. Stored in the image data storage area of the RAM 63 together with the data.

  The CPU 61 sequentially executes the above-described whole observation image photographing and microscopic observation image photographing based on the observation schedule set in the observation program. The image data stored in the RAM 63 is read from the RAM 63 in response to an image display command input from the operation panel 71. For example, an entire observation image, a microscopic observation image at a specified time, an analysis result of image analysis, and the like are displayed on the display panel 72. Is displayed.

  In the culture observation system BS configured as described above, when observing the growth state of the fertilized egg J in the culture container 10 (dish 11), the fertilized egg to be observed must be detected from the culture container 10. However, as a series of detection procedures, generally, the culture vessel 10 is photographed by the macro observation system 54 to obtain a whole observation image (macro image), and after detecting the medium drop D from this whole observation image, A flow in which the medium drop D is photographed by the microscopic observation system 55, and the fertilized eggs to be observed injected into the medium drop D one by one (or plural pieces) are distinguished from other foreign substances to recognize the fertilized eggs. It has become. In order to detect a fertilized egg, microscopic observation in a high-magnification visual field is generally required. However, as the observation magnification increases, the observation visual field (observation range) becomes narrower. On the other hand, it is necessary to capture and acquire a large number of images. Therefore, as described above, prior to detection of the fertilized egg by microscopic observation, if the area of the medium drop D containing the fertilized egg can be detected in advance by macro observation, the area for acquiring a high-magnification image in the microscopic observation is also limited thereafter. It becomes possible to shorten the entire observation time.

  However, the color of the medium drop D ranges from red to colorless and transparent, and it is not easy to detect the area of the medium drop D from the tint and brightness density using the entire observation image (macro image). Therefore, in the present embodiment, an entire observation image with a difference in brightness is taken and acquired at the periphery of the medium drop D using the lens effect of the medium drop D and the oblique illumination method. For example, as shown in FIG. In addition, the four overall observation images G1, G2, G3, and G4 obtained and captured in the four types of illumination directions are superimposed to generate a composite image GX of the overall observation image, and the culture vessel 10 is generated from the generated composite image GX. Identify (identify) the area of medium drop D inside. As described above, if the combined image GX of the overall observation images G1, G2, G3, and G4 respectively captured and acquired in a plurality of illumination directions is used, a color difference is created in the entire peripheral portion of the medium drop D. Regardless of the condition, the area of the medium drop D can be accurately identified. Note that the four overall observation images G1, G2, G3, and G4 shown in FIG. 6 are images acquired by rotating and illuminating the culture vessel 10 about the optical axis of the macro observation system 54 by 45 degrees. is there.

  Detection (identification) of the region of the medium drop D using the composite image GX is performed by a predetermined image processing method using the feature that the medium drop D is generally formed in a circular shape. For example, it is possible to detect (identify) the region of the medium drop D by performing a known differentiation process on the composite image GX to generate a differential image, and detecting a circle from the differential image by Hough transform. . Further, for example, the region of the medium drop D is detected by performing a circular extraction by performing integration in the θ direction of the polar coordinates for all the points of the differential image and extracting points showing peaks in a predetermined radius range ( Identification). Further, for example, the region of the medium drop D may be detected (identified) by extracting a circular shape from the differential image using a so-called level set method (dynamic contour method). At this time, the size of the image of the medium drop D (for example, about 7 μm in diameter of about 20 μl) to be imprinted on the image is almost determined by the image acquisition conditions (observation magnification, etc.). The area of a possible object (region surrounded by the extracted circular outline) is calculated by a known image processing method, and deviates from the setting range (upper limit and lower limit) determined as a discrimination criterion by area Is excluded.

  Image processing for detecting (identifying) the area of the medium drop D is executed in the image processing apparatus 100 of the culture observation system BS. As illustrated in FIG. 7, the image processing apparatus 100 acquires and stores an entire observation image (macro image) obtained by capturing the culture container 10 to be observed by the imaging device 54c, and the image storage unit 110. An image synthesizing unit 120 that superimposes the entire observation images captured and acquired in a plurality of illumination directions and generates a synthesized image, and a medium drop D based on the synthesized image generated by the image synthesizing unit 120 An image analysis unit 130 to detect, and an output unit 140 that outputs the composite image generated by the image synthesis unit 120 and the determination result analyzed by the image analysis unit 130 to the outside, and includes the image synthesis unit 120 and the image analysis unit 130. For example, the medium drop D image and the detection result determined by the above are output to the display panel 72 and displayed. The image processing apparatus 100 is configured such that an image processing program set and stored in advance in the ROM 62 is read by the CPU 61 and processing based on the image processing program is sequentially executed by the CPU 61.

  An observation method in the culture vessel 10 using the culture observation system BS configured as described above will be described with reference to the flowchart shown in FIG. As described above, in the culture observation system BS, observation in the culture vessel 10 designated every predetermined time is performed according to the observation conditions set in the observation program. Specifically, the CPU 61 operates each stage of the transport unit 4 to transport the culture vessel 10 to be observed from the stocker 3 to the observation unit 5 (in this embodiment, disposed on the optical axis of the macro observation system 54). A macro image is taken by the imaging device 54c.

  At this time, first, the CPU 61 operates the first illumination unit 51 to illuminate the culture vessel 10 from the illumination direction inclined with respect to the optical axis of the macro observation system 54 (step S1), and the imaging device 54c of the macro observation system 54 Is activated to capture the entire observation image of the culture vessel 10 (step S2). Here, the CPU 61 determines whether or not the entire observation image of the culture vessel 10 has been taken for all preset illumination directions (step S3). If the determination in step S3 is NO, the process proceeds to step S4, where the CPU 61 changes the light emission position of the light emitting device 51b and rotationally displaces the illumination direction with respect to the culture vessel 10 about the optical axis of the macro observation system 54. The processes from steps S1 to S2 are repeated. At this time, the image processing apparatus 100 acquires the entire observation image (macro image) captured by the imaging device 54c in step S2, and uses the acquired image as the code number, the observation position, the observation time, and the like of the culture vessel 10. Are stored in the image storage unit 110 together with the index data.

  On the other hand, when the determination in step S3 is YES, the process proceeds to step S5, and the image composition unit 120 superimposes the entire observation images captured and acquired in the plurality of illumination directions stored in the image storage unit 110, respectively ( A composite image of the entire observation image (for the culture vessel 10) is generated. At this time, the output unit 140 displays a composite image of the entire observation image (bird view image) generated by the image combining unit 120 on the display panel 72 of the operation panel 7 or the like.

  In step S6, the image analysis unit 130 detects (identifies) the region of the medium drop D in the dish 11 from the combined image generated by the image combining unit 120 using the circle detection method described above. At this time, a detection result indicating the region of the medium drop D detected by the image analysis unit 130 is output from the output unit 140.

  The detection result output from the output unit 140 is displayed on the display panel 72 of the operation panel 7, and an outline indicating the region of the medium drop D is displayed in the composite image or the entire observation image, or the medium drop D is displayed. For example, the symbol “D” indicating “” is displayed, or the outline of the medium drop D is displayed with a hue or brightness different from those of other regions.

  The detection data output from the output unit 140 is transmitted to a computer or the like externally connected via the communication unit 65 to display a similar image, or the color change (pH change) of the medium drop D or fertilization It can be configured to be used as basic data for observing the growth state of the egg J.

  Thus, the user can immediately recognize the medium drop D by referring to the image displayed on the display panel 72 or the image displayed on a monitor such as an externally connected computer. Only for, a microscopic observation image by the microscopic observation system 55 is acquired (avoid unnecessary shooting of only the background), and the target fertilized egg J can be efficiently detected from the medium drop D.

  Next, a method for producing a fertilized egg as a culture will be briefly described with reference to FIG. First, in step S110, a fertilized egg (culture) is injected into the culture vessel 10 (dish 11) together with the medium drop D, and the culture chamber 2 is maintained in an environmental condition suitable for culturing the fertilized egg. The fertilized egg is cultured under the environmental conditions. The environmental conditions are adjusted in the control unit 6 according to the culture environment of the fertilized egg such as the temperature, humidity, and carbon dioxide concentration in the culture chamber 2.

  In step S120, steps S1 to S6 (see FIG. 1) of the observation method described above are executed as a pre-stage of observation of the fertilized egg in the culture container 10, and the region of the medium drop D is identified from the dish 11. Next, in step S130, a microscopic observation image (for example, a phase difference image) of the medium drop D is taken by the microscopic observation system 55 using the second illumination unit 52, and fertilization is performed from among a plurality of objects that are imprinted on the observation image. Identify eggs. At this time, one (or more) fertilized eggs are identified for each medium drop D in the culture container 10 (dish 11).

  Subsequently, in step S140, a plurality of fertilized eggs identified for each medium drop D are sorted based on a predetermined sorting criterion. As a selection standard for a fertilized egg, the grade of the fertilized egg is determined based on the timing of cleavage, the shape of the blastomere, and the like, and a good one satisfying this selection standard is selected. For example, it is performed based on whether or not the timing at which cleavage occurs in all egg cells in the egg is in the same period as having passed through a good growth state. That is, regarding the cleavage of normal fertilized eggs, cells of the same generation divide at almost the same timing, and only cells of the same generation exist in the embryo. On the other hand, regarding the cleavage of an abnormal fertilized egg, even when cells are of the same generation, the division timing is shifted, and cells of different generations are mixed in the embryo.

  In step S150, the selected fertilized eggs (good fertilized eggs that have grown to a state called a blastocyst) are collected and stored frozen in, for example, liquid nitrogen at minus 196 ° C. The fertilized egg (blastocyst) is returned to the mother (embryo transfer) at a predetermined cycle. The fertilized eggs to be cultured may be fertilized eggs such as humans, cows, horses, pigs and mice. In addition, the fertilized egg may be stored in a blastocyst state or may be stored in a divided phase (4-cell stage embryo, 8-cell stage embryo).

  As described above, according to the present embodiment, since the entire peripheral portion of the culture medium drop D can be differentiated by using a composite image of the entire observation image captured and acquired in a plurality of illumination directions, color information is provided. Independently of this, the region of the medium drop D in the culture vessel 10 (dish 11) can be accurately identified.

  Further, by illuminating the culture container 10 from a plurality of illumination directions inclined with respect to the imaging direction with respect to the culture container 10 (dish 11) (that is, the optical axis of the macro observation system 54), the lens effect of the medium drop D can be obtained. By utilizing this, it is possible to capture and acquire the entire observation image of the culture vessel 10 with a difference in brightness at the periphery of the medium drop D, so that a composite image with a difference in brightness in the entire periphery of the medium drop D can be reliably obtained. Obtainable.

  Further, if the region of the medium drop D is identified by detecting a circle from the differential image obtained by performing the differentiation process on the composite image, the region of the medium drop D is accurately identified by a simple method. be able to.

  In the above-described embodiment, the four overall observation images G1, G2, G3, and G4 respectively captured and acquired in the four types of illumination directions are superimposed to generate the overall observation image composite image GX. For example, a total image may be generated by superimposing eight whole observation images captured and acquired in eight types of illumination directions, or a plurality of whole images acquired and acquired in a plurality of illumination directions. What is necessary is just to produce | generate a synthesized image by superimposing an observation image. Furthermore, you may use the whole observation image which changed the illumination direction continuously within integration time.

  In the above-described embodiment, the first illumination unit 51 changes the light emission position of the light emitting device 51b and rotationally displaces the illumination direction with respect to the culture vessel 10 about the optical axis of the macro observation system 54, thereby causing the culture vessel 10 to move. However, the present invention is not limited to this. For example, as shown by a two-dot chain line in FIG. 5, the ring illumination device 51a is provided with a shutter member 51c that allows only a part of the light emitting devices 51b to pass therethrough, and the shutter member 51c is macro-observed. A configuration may be adopted in which the illumination direction is changed by rotationally displacing the optical axis of the system 54. Further, in the configuration in which the light emitting position of the light emitting device 51b is changed and the illumination direction with respect to the culture vessel 10 is rotationally displaced about the optical axis center of the macro observation system 54, it is not necessary to rotate the culture vessel 10, the macro observation system 54, or the like. Although it is possible to superimpose the entire observation image without positioning, a configuration in which the macro observation system 54 is rotated around the optical axis may be used if image rotation correction and perspective correction are performed.

  Further, in the above-described embodiment, a differential image is generated after generating a composite image by superimposing a plurality of overall observation images. However, the present invention is not limited to this. For the overall observation image before image synthesis, Then, after performing differential processing, they may be superimposed to generate a composite image of the differential image. Furthermore, an image accumulated while rotating the illumination direction may be used.

BS culture observation system GX composite image D medium drop J fertilized egg 5 observation unit 6 control unit 7 control panel 10 culture vessel 11 dish 51 first illumination unit 51a ring illumination device 51b light emitting device 51c shutter member 54 macro observation system 54c imaging device 61 CPU 62 ROM
63 RAM
DESCRIPTION OF SYMBOLS 100 Image processing apparatus 120 Image composition part 130 Image analysis part 140 Output part

Claims (9)

An illumination unit capable of illuminating a culture vessel having a culture medium used for culture of the culture from a plurality of illumination directions;
An imaging unit that images each of the plurality of illumination directions of the culture vessel illuminated by the illumination unit;
An image compositing unit that generates a composite image of the culture vessel by superimposing images of the culture vessel for the plurality of illumination directions respectively captured by the imaging unit;
An apparatus for observing a culture, comprising: an image analysis unit for identifying the culture medium in the culture vessel from the synthesized image generated in the image synthesis unit.   The illumination unit illuminates the culture vessel from an illumination direction inclined with respect to the optical axis of the imaging unit, and rotationally displaces the illumination direction about the optical axis of the imaging unit, whereby the plurality of illumination directions The culture observation apparatus according to claim 1, wherein illumination is possible.   The said image analysis part identifies the said culture medium in the said culture container by detecting a circle from the differential image which performed the differential process with respect to the said synthetic | combination image, The said culture medium is characterized by the above-mentioned. Culture observation equipment. Illuminate the culture vessel with the medium used for culturing the culture inside from multiple illumination directions,
Imaging each of the illuminated culture vessels for each of the plurality of illumination directions;
Generating a composite image of the culture vessel by superimposing the images of the culture vessel for the plurality of captured illumination directions;
A culture observation method, wherein the culture medium in the culture vessel is identified from the generated composite image.   The culture observation method according to claim 4, wherein the culture container is illuminated from the plurality of illumination directions inclined with respect to the imaging direction with respect to the culture container.   The culture observation method according to claim 4 or 5, wherein the culture medium in the culture vessel is identified by detecting a circle from a differential image obtained by performing differential processing on the composite image. . Culturing the culture under the specified environmental conditions,
The culture medium, wherein the culture medium is identified by using the observation apparatus according to any one of claims 1 to 3 from a culture container having a culture medium in which the culture is cultured. Method. Culturing the culture under the specified environmental conditions,
Illuminating a culture vessel having a medium in which the culture is cultured from a plurality of illumination directions;
Imaging each of the illuminated culture vessels for each of the plurality of illumination directions;
Generating a composite image of the culture vessel by superimposing the images of the culture vessel for the plurality of captured illumination directions;
A method for producing a culture, wherein the medium in the culture vessel is identified from the generated composite image. Selecting a culture contained in the identified medium based on a predetermined selection criterion;
The method for producing a culture according to claim 7 or 8, wherein the selected culture is collected from the culture vessel and stored.