JP2011017964A - Culture observation device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To surely detect an observation object.SOLUTION: An observation unit 32 includes: a microobservation part 44 for imaging a culture vessel 14 in an incubator part 12 through a twofold power objective lens 61; and a high-NA illumination unit 49 which is disposed in a position facing the microobservation part 44 and irradiates the observation object in the culture vessel 14 with illumination light. The numerical aperture of the illumination light emitted from the high-NA illumination unit 49 is higher than that of the objective lens 61of the microobservation part 44.

Description

  The present invention relates to a culture observation apparatus, and more particularly, to a culture observation apparatus capable of reliably detecting an observation target.

  Conventionally, in assisted reproductive technology (ART), for example, in vitro fertilization (IVF) and microinsemination (ICSI: Intracytoplasmic Sperm Injection), 5 to 20 samples collected from one maternal body at a time. The in vitro fertilized eggs are put in a medium drop of about 20 μl made on the culture container. The surface of the medium drop is coated with mineral oil, and the whole culture container is accommodated in the culture apparatus in that state. In this environment, the fertilized egg begins to divide.

  The person in charge of culture observes the growth state of the fertilized egg at regular intervals, and performs medium exchange and scoring of the fertilized egg as necessary. A few days later, a good blastocyst that has grown to a state called a blastocyst is returned to the mother's body. Other blastocysts are stored frozen.

  For example, by applying the culture apparatus described in Patent Document 1 to IVF or ICSI that follows the above procedure, the fertilized egg is automatically observed, recorded, and managed while suppressing damage to the fertilized egg due to environmental exposure. Can do. Patent Document 2 discloses a technique for tracking cells floating in an observation field.

  By the way, when trying to observe a fertilized egg existing in the culture drop of the culture container, if the fertilized egg has moved to the edge of the medium drop, it is hidden behind the shadow appearing on the edge of the medium drop, and the fertilized egg Eggs may not be detected.

  With reference to FIG. 10, the shadow that appears on the edge of the medium drop will be described. FIG. 10 shows a plan view and a cross-sectional view inside the culture vessel.

  The culture container 1 consists of what is called a dish, and the internal diameter is about 35 mm. On the bottom surface of the culture vessel 1, a plurality of medium drops D of about 20 μl (eight in the example of FIG. 10) are formed so as to be evenly arranged along the circumference of the culture vessel 1.

  The surface and interval of each medium drop D are filled with colorless and transparent mineral oil O to prevent drying. In each medium drop D, fertilized eggs collected from a common mother at a common time are included. Contains one a. The diameter of each medium drop D is about 7 mm, and the diameter of each fertilized egg a is about 100 μm. In addition, the medium drop D is attached to the bottom surface of the culture container 1, but the fertilized egg a may float inside the medium drop D.

  Thus, when observing the fertilized egg a in the medium drop D, illumination light is irradiated from the upper side, and an image is picked up through the objective lens arranged on the lower side.

  At this time, the meniscus effect is generated due to the difference in refractive index between the medium drop D and the mineral oil O. For example, when the refractive index of the medium drop D is about 1.3 and the refractive index of the mineral oil O is about 1.4 to 1.6, the medium drop D functions as a concave lens (around f = -14) that is optically strong. The illumination light incident on the vicinity of the edge of the medium drop D is refracted to the outside of the medium drop D. This makes it difficult for the illumination light incident on the vicinity of the edge of the medium drop D to enter the objective lens, so that a shadow appears on the entire periphery of the edge of the medium drop D.

  Furthermore, the meniscus effect is also generated at the edge of the culture vessel 1 due to the surface tension of the mineral oil O on the inner wall surface of the culture vessel 1. This makes it difficult for the illumination light incident on the vicinity of the edge of the culture vessel 1 to enter the objective lens, so that a shadow having a crescent-like shape on the inner wall surface of the culture drop 1 (the culture drop D on the culture vessel 1). Asymmetric shadows appear on the inside and outside.

JP 2007-6841 A JP 2001-264318 A

  As described above, in the conventional observation apparatus, when a shadow appears on the edge of the medium drop in the culture container and the fertilized egg to be observed moves to the edge of the medium drop, the fertilized egg may not be detected. It was.

  The present invention has been made in view of such a situation, and makes it possible to reliably detect an observation target.

  The culture observation apparatus of the present invention is a culture observation apparatus for observing an observation target in a culture container, the observation means for imaging the culture container through an objective lens having a predetermined magnification, and the observation means And an illuminating means for irradiating the observation object in the culture vessel with illumination light, and the numerical aperture of the illumination light from the illumination means is larger than the numerical aperture of the objective lens of the observation means It is characterized by.

  In the culture observation apparatus of the present invention, illumination light having a numerical aperture larger than the numerical aperture of the objective lens of the observation means is irradiated to the observation target in the culture vessel by the illumination means. As a result, the appearance of a shadow is suppressed, and the observation target can be reliably detected.

  According to the culture observation apparatus of the present invention, an observation target can be reliably detected.

It is a figure which shows the structural example of one Embodiment of the culture observation apparatus to which this invention to which this invention is applied is applied. It is a figure which shows the structural example of an observation unit. It is a flowchart explaining the observation process in which a culture observation apparatus observes a fertilized egg in IVF mode. It is a figure explaining whole image I _whole . It is a figure explaining the process of step S103. It is a diagram illustrating a partial drop image I _ia ~I _id. It is a figure explaining the process of step S107. It is a figure explaining the relationship between fertilized egg a _i and bubble b _i . It is a diagram illustrating an egg image I ₁ '~I _7'. It is a figure explaining the shadow which appears on the edge of the culture medium drop in a culture container.

  Hereinafter, specific embodiments to which the present invention is applied will be described in detail with reference to the drawings.

  FIG. 1 is a diagram showing a configuration example of an embodiment of a culture observation apparatus to which the present invention is applied. FIG. 1A shows a perspective view of the culture observation apparatus, and FIG. 1B shows a front view of the culture observation apparatus with the door removed.

  As shown in FIG. 1, the culture observation apparatus 11 includes an incubator unit 12 that is maintained in an environment for culturing cells, and a gantry unit 13 in which various devices are stored, and the incubator unit 12 is provided above the gantry unit 13. Is arranged and configured. In the culture observation apparatus 11, cells are cultured and observed in the culture container 14 accommodated in the incubator unit 12.

  The incubator unit 12 includes an incubator housing 21 and an incubator door 22, and an internal space (constant temperature chamber) sealed by the incubator housing 21 and the incubator door 22 maintains an environment suitable for culturing cells. To be controlled.

  For example, the incubator housing 21 includes a temperature control device using a Peltier device, a spray device that ejects mist, a gas introduction unit connected to an external carbon dioxide cylinder, and an environmental sensor that detects the environment of the internal space (all (Not shown). Moreover, the incubator housing 21 and the incubator door 22 each have a heat insulating material inside. The internal space of the incubator housing 21 is an environment suitable for culturing cells, such as a temperature of 37 ° C., a humidity of 90%, and a carbon dioxide concentration of 5% (hereinafter referred to as a cell culture environment as appropriate). Maintained.

  The incubator door 22 is attached to the incubator housing 21 so as to be openable and closable, and an operation panel 23 used for operating the culture observation apparatus 11 is provided on the surface thereof.

  The operation panel 23 is, for example, a touch panel that displays an image and is operated by a user. An image in the culture vessel 14 to be observed and a schedule for capturing cells at a predetermined interval are displayed on the display panel. Display the schedule setting screen to set the. Further, the operation panel 23 supplies an operation signal corresponding to a user operation to the control unit 31 housed in the gantry 13.

  The gantry 13 includes a housing 24 and doors 25A and 25B. The doors 25A and 25B are attached to the housing 24 so as to be openable and closable. Further, inside the gantry 13 are housed a control unit 31 that controls each part of the culture observation apparatus 11 and a lower part of an observation unit 32 (see FIG. 2) for observing cells in the cell culture environment. . The control unit 31 includes a storage unit 31a configured with a hard disk, a nonvolatile memory, or the like, and stores various types of data to be described later.

  Further, inside the incubator unit 12, an upper part of the observation unit 32, a stocker unit 33 that accommodates the plurality of culture containers 14, and a transport unit 34 that transports the culture container 14 between the observation unit 32 and the stocker unit 33. Is stored. The stocker unit 33 includes a plurality of shelves, and a loading / unloading table 33A on which the culture vessel 14 loaded / unloaded into / from the incubator unit 12 is placed is provided at the lowest level, and corresponds to the position of the loading / unloading table 33A. The incubator door 22 is provided with a loading / unloading port (not shown). The transport part 34 has a support part 34 </ b> A that supports the culture container 14 when transporting the culture container 14.

  In the culture observation apparatus 11 configured as described above, the control unit 31 controls each unit according to the observation schedule stored in the storage unit 31a, and the culture container 14 to be observed is stored in the stocker unit by the transport unit 34. It is conveyed from 33 to the stage of the observation unit 32, and the culture vessel 14 is observed.

  Next, FIG. 2 is a diagram illustrating a configuration example of the observation unit 32. FIG. 2A shows the configuration of the entire observation unit 32, and FIG. 2B shows a part of the observation unit 32 in an enlarged manner.

  As shown in FIG. 2, the observation unit 32 is configured by arranging an arm part 42 and a stage 43 on the upper side of the unit main body 41, and the unit main body 41 is arranged inside the gantry part 13 of FIG. 1. The arm part 42 and the stage 43 are arranged inside the incubator part 12.

  The unit main body 41 includes a microscopic observation unit 44 for observing the culture container 14 from the lower side, an illumination unit 45 for irradiating illumination light when observing the entire culture container 14 from the upper side, and the observation unit 32. An image processing unit 46 that performs image processing on the acquired image is housed.

  The arm part 42 has a shape that extends upward from the side of the stage 43 and protrudes to the upper side of the stage 43, and a container observation part for observing the entire culture container 14 from the upper side in the protruding part. 47 and an illuminating unit 48 for irradiating illumination light when observing the culture vessel 14 by magnifying it from the lower side are housed. A high NA illumination unit 49 is mounted on the lower surface of the projecting portion of the arm portion 42.

  And the container observation part 47 of the arm part 42 and the illumination part 45 of the unit main body 41 are arrange | positioned in the mutually opposing position, and comprise the observation system for whole observation (for wide field observation).

  Further, the microscopic observation unit 44 of the unit main body 41, the illumination unit 48 of the arm unit 42, and the high NA illumination unit 49 are arranged at positions facing each other, and are used for phase difference observation or high NA illumination observation (microscopic observation). A microscope for visual observation). Here, the high NA illumination unit 49 is configured to be able to be inserted into and removed from the optical path for microscopic observation including the microscopic observation unit 44, and the high NA illumination unit 49 is inserted into the optical path. Sometimes, a microscope for high NA illumination observation is configured by the microscopic observation unit 44 and the high NA illumination unit 49. When the high NA illumination unit 49 is extracted from the optical path, phase difference observation is performed by the microscopic observation unit 44 and the illumination unit 48. A microscope is constructed.

  The stage 43 is made of a translucent material, and moves the culture vessel 14 placed thereon in the horizontal direction (XY direction) and the vertical direction (Z direction) with high accuracy. For example, in the stage 43, the culture container 14 has an optical path of a microscope for observing a phase difference (microscopic observation unit 44, illumination unit 48), and an optical path of a microscope for high NA illumination observation (microscopic observation unit 44, high NA illumination unit 49). The culture vessel 14 is moved in the horizontal direction so as to be inserted into the optical path of the entire observation system (container observation unit 47 and illumination unit 45).

  Here, in the observation unit 32, the whole image of the culture container 14 can be captured in color in a state where the culture container 14 is inserted into the optical path of the observation system for the whole observation. Hereinafter, an image acquired by this imaging is referred to as an “entire image”. Further, in the observation unit 32, a part of the enlarged phase difference image of the culture vessel 14 can be taken with the culture vessel 14 inserted into the optical path of the microscope for phase difference observation. Hereinafter, an image acquired by this imaging is referred to as a “phase difference image”. In the observation unit 32, a bright field image illuminated with light having a large illumination NA can be taken by taking the phase filter 62 out of the optical path with the high NA illumination unit 49 inserted in the optical path. Hereinafter, an image acquired by this imaging is referred to as a “high NA illumination image”.

  In addition, the stage 43 changes the positional relationship in the Z direction between the culture vessel 14 and a microscope for phase difference observation and a microscope for high NA illumination observation, so that the microscope for phase difference observation and high NA illumination observation are used. Microscope focus adjustment can be performed. In addition, the stage 43 changes the positional relationship in the XY direction between the culture vessel 14 and the microscope for phase difference observation and the microscope for high NA illumination observation, so that the microscope for phase difference observation and for high NA illumination observation are used. The observation point of the microscope can be changed.

  The image processing unit 46 performs various kinds of image processing on the image acquired by the observation unit 32 (any one of the whole image, the low magnification phase difference image, the high NA illumination observation, and the high magnification phase difference image). Image processing that can be executed by the image processing unit 46 includes color detection processing (described later) for detecting a colored region in an input image, and ring search processing (described later) for searching a circular pattern from the input image. ) And blastocyst discrimination processing (described later) for discriminating whether or not the input image is an image of a blastocyst.

Note that the combination of the objective lens 61 and the phase filter 62 of the microscopic observation unit 44 can be switched between at least two types of combinations having different observation magnifications. Here, it is assumed that switching is performed between a combination for 2 × observation (objective lens 61 _low and phase filter 62 _low ) and a combination for 10 × observation (objective lens 61 _high and phase filter 62 _high ).

Therefore, the observation unit 32 may obtain the magnification twice the phase difference image (low magnification phase contrast image) in a state of setting the objective lens 61 _low and the phase filter 62 _low in the optical path, an objective lens 61 _high and the phase A phase difference image (high magnification phase difference image) with a magnification of 10 can be acquired with the filter 62 _high set in the optical path. The magnification of the low-magnification phase difference image by the observation unit 32 is higher than the magnification of the whole image described above.

In the observation unit 32, the high NA illumination unit 49 is used when acquiring a high NA illumination image by a combination for observing twice high NA illumination (objective lens 61 _low, no phase filter). That is, as described with reference to FIG. 10 described above, when a shadow appears in the vicinity of the edge of the medium drop D (a shadow over the entire periphery of the edge and a crescent-shaped shadow), the fertilized egg a is hidden in the shadow. Thus, the fertilized egg a may not be detected.

  Therefore, in the observation unit 32, when acquiring an image so that such a shadow does not occur, the high NA illumination unit 49 is inserted in the middle of the optical path of the illumination light from the illumination unit 48, and the microscopic observation unit 44 and Observation is performed by a high NA illumination observation microscope including the high NA illumination unit 49.

As shown in FIG. 2B, the high NA illumination unit 49 includes a light source 71, a mirror 72, and a Fresnel lens 73. The illumination light emitted from the light source 71 is reflected by the mirror 72 and then collected by the Fresnel lens 73. The illumination light having a NA larger than the NA (Numerical Aperture) of the objective lens 61 _low is the culture vessel 14. The medium drop D is irradiated.

Thus, since the NA of the illumination light irradiated by the high NA illumination unit 49 is large, even if the illumination light is refracted by the meniscus effect in the vicinity of the edge of the medium drop D, the vicinity of the edge of the medium drop D is irradiated. The illumination light is incident on the objective lens 61 _low . Similarly, even if the illumination light is refracted due to the meniscus effect in the vicinity of the inner wall surface of the culture vessel 14, the illumination light irradiated on the inner wall surface side of the culture vessel 14 of the medium drop D is incident on the objective lens 61 _low . To do.

  Therefore, by using the high NA illumination unit 49, it is possible to suppress the occurrence of a shadow over the entire circumference of the medium drop D and a crescent-shaped shadow on the inner wall surface side of the culture container 14 of the medium drop D. Thereby, even if the fertilized egg a moves to the edge of the medium drop D, it is not hidden by the shadow, so the fertilized egg a can be reliably detected.

Further, as a result of calculating the change of the illumination light flux due to the medium drop D with respect to the NA of the illumination light from the high NA illumination unit 49, when the NA of the objective lens 61 _low is 0.2, 0.3 or more, more preferably 0.35 or more It turned out to be necessary. In other words, if the NA of the illumination light is 0.3 or more, good observation can be performed in combination with the objective lens 61 _{low at} any part of the medium drop D. Therefore, the high NA illumination unit 49 is designed so that the NA of the illumination light is 0.3 or more, more preferably 0.35 or more. Specifically, the upper limit of NA of illumination light is sufficient to be about 0.5.

  Here, the high NA illumination unit 49 can be additionally attached to the culture observation apparatus 11 conventionally used in order to achieve compatibility with other general phase difference observations, for example. It is preferable to be designed. That is, in the culture observation apparatus 11, the interval between the arm part 42 and the stage 43 is already determined, for example, at about 100 mm. For this reason, the high NA lighting unit 49 can be mounted in the limited space, and it is considered to secure a space for transporting the culture vessel 14 by the support portion 34A of the transport portion 34. For example, the height is designed to be within 50 mm.

  Accordingly, the high NA illumination unit 49 employs an LED having a large light emitting area as the light source 71 in order to secure a working distance necessary for realizing a high NA and a wide illumination field within the height. A mirror 72 is used to increase the optical path length and a Fresnel lens 73 with a planar shape and a large area is employed.

  Next, an observation process in which the culture observation apparatus 11 observes a fertilized egg in the IVF mode will be described with reference to the flowchart of FIG.

  In step S <b> 101, the control unit 31 carries the culture container 14 into the incubator unit 12 in accordance with a user operation, and instructs the transport unit 34 to transport the culture container 14. The transport unit 34 transports the culture container 14 and places it on a predetermined position of the stage 43.

In step S102, the control unit 31 instructs the observation unit 32 to acquire the entire image of the culture vessel 14. The observation unit 32 arranges the culture vessel 14 in the optical path of the observation system for whole observation (the vessel observation unit 47 and the illumination unit 45), and acquires the whole image I _whole as shown in FIG. The whole image I _whole is displayed on the operation panel 23. In the whole image I _whole , seven media drop D images (drop images) I _D are shown.

In step S103, the control unit 31 instructs the image processing unit 46 to perform color detection processing on the entire image I _whole acquired in step S102. The image processing unit 46 detects a plurality of reddish areas (areas colored with phenol red) in the whole image I _whole . The control unit 31 assigns drop numbers 1 to 7 to the plurality of areas detected by the image processing unit 46 in the arrangement order. As a result, the seven drop images I _D are labeled. Hereinafter, the drop image I _D that drop number i is assigned is referred to as "I _Di" (see FIG. 5).

The control unit 31 then determines the position coordinates of the stage 43 in the XY direction and the whole image I _whole.
As shown in FIG. 5, the coordinates (x ₁ , y ₁ ) to (x ₇ , y ₇ ) of the centers of the individual medium drops in the culture vessel 14 are based on the center coordinates of the individual drop images I _{D1 to} I _D7 . And the coordinates (x ₁ , y ₁ ) to (x ₇ , y ₇ ) are respectively associated with the drop numbers 1 to 7 and stored in the storage unit 31a as management data of the culture vessel 14.

  On the other hand, the control unit 31 causes the user to input an observation schedule (such as a time lapse observation time interval) of the culture vessel 14 and writes the observation schedule into the management data of the culture vessel 14.

  Thereafter, the control unit 31 instructs the transport unit 34 to store the culture vessel 14 in the stocker unit 33. The transport unit 34 stores the culture container 14 in the stocker unit 33. Thereby, the registration of the culture vessel 14 is completed.

  In step S104, the control unit 31 compares the observation schedule written in the management data of the culture vessel 14 with the current date and time to determine whether or not the observation start time of the culture vessel 14 has come. When the observation start time has come, the process proceeds to step S105, and when the observation start time has not come, the process waits.

  In step S <b> 105, the control unit 31 instructs the transport unit 34 to transport the culture vessel 14. The transport unit 34 unloads the culture vessel 14 from the stocker unit 33 and places it on a predetermined position of the stage 43. At this time, the drop number i is set to the initial value “1”.

In step S106, the control unit 31 obtains the coordinates (x _i , y _i ) corresponding to the current drop number i in order to obtain the medium drop image (drop image I _i ) corresponding to the current drop number i. The data is read from the management data of the culture vessel 14 and specified to the observation unit 32, and the observation unit 32 is instructed to acquire a high NA illumination image of the object located at the coordinates (x _i , y _i ).

  Here, the control unit 31 discharges the phase filter 62 out of the optical path while inserting the high NA illumination unit 49. That is, a combination of the microscope observation unit 44 and the high NA illumination unit 49 is used as a microscope for high NA illumination observation that acquires a low-magnification bright-field image.

The observation unit 32 arranges the culture vessel 14 in the optical path of the microscope for high NA illumination observation (microscopic observation unit 44 and high NA illumination unit 49), and doubles the combination of the objective lens 61 and the phase filter 62. Set for high NA illumination observation (objective lens 61 _low , no phase filter).

Here, the visual field size (4 mm × 4 mm) of the objective lens 61 _low is smaller than the size of one medium drop (diameter 7 mm). Therefore, the observation unit 32 moves the optical axis position of the objective lens 61 _low relative to the culture vessel 14 stepwise around the coordinates (x _i , y _i ), and performs low-magnification high NA illumination as shown in FIG. Images I _{ia to} I _id are acquired in order. The whole of these low-magnification high NA illumination images I _{ia to} I _id is the drop image I _i . Therefore, hereinafter, the low-magnification high NA illumination images I _{ia to} I _id are referred to as “partial drop images I _{ia to} I _id ”. Note that image acquisition may be performed at once by setting the visual field size of the objective lens 61 _{low to} the visual field size of one medium drop.

Note that the observation unit 32 performs autofocus control by a contrast method when acquiring individual images. This autofocus control adjusts the height (position in the Z direction) of the stage 43 while monitoring the contrast of the image, and stops the stage 43 at such a height that the contrast reaches a peak. Information on the focus position (the height of the focus surface) at the time of acquisition of each of the partial drop images I _{ia to} I _id is stored by the control unit 31.

Incidentally, the observation unit 32 can limit the focus adjustment range of the autofocus control according to an instruction from the control unit 31. However, in the autofocus control when the partial drop images I _{ia to} I _id of a certain drop number i are first acquired, the focus adjustment range is set sufficiently wide.

In step S107, the control unit 31 instructs the image processing unit 46 to perform a circular pattern search process on each of the partial drop images I _{ia to} I _id acquired in step S106. For example, as shown in FIG. 7, the image processing unit 46 detects the annular pattern Ic from each of the partial drop images I _{ia to} I _id by the annular pattern search process.

Based on the position coordinates of the stage 43 in the X and Y directions and the center coordinates of the annular pattern Ic in the drop image I _i , the control unit 31 performs all of the annular patterns (fertilized eggs and bubbles) shown in the drop image I _i. Center coordinates (x _i1 ′, y _i1 ′) to (x _im ′, y _im ′) are calculated. Here, for the sake of explanation, the number of annular patterns Ic detected from the drop image I _i is assumed to be “m”.

Usually, since the contour portion of the fertilized egg image appears as a bright ring on the image, the detected annular pattern Ic is likely to be an image of the fertilized egg. However, as shown in FIG. 8, there is a possibility that bubbles b _i may adhere to the surface of the culture vessel 14 or the interface between the medium drop D and the mineral oil O, and the outline of the image of the bubbles b _i is also an image. Appears as a bright ring on top. Therefore, the annular pattern Ic detected in this step is an image of the fertilized egg a _i or an image of the bubble b _i .

In step S108, the control unit 31 distinguishes between the image of the fertilized egg a _{i and} the image of the bubble b _i by the luminance dispersion value (or luminance differential value) inside each annular pattern Ic detected in step S107. Is calculated, and each is compared with a threshold value. Then, the control unit 31 regards only the annular pattern Ic that is equal to or greater than the threshold as an image of the fertilized egg a _i . Hereinafter, the coordinates of the center of the fertilized egg a _i in the culture container 14 are set to (x _i ′, y _i ′).

Since the number of fertilized eggs placed in one medium drop is 1, the number of images of fertilized eggs a _i detected in this step is 1 or less, but the partial drop images I _{ia to} I _id At the time of acquisition, as shown in FIG. 8, both the bubble b _i and the fertilized egg a _i exist in the field of view of the objective lens 61 _low , and the position of the bubble b _{i in} the optical axis direction (Z direction) is If the fertilized egg a _i is away from the position in the optical axis direction, and the bubble b _i is in focus, the number of the fertilized eggs a _i is zero.

In step S109, the control unit 31 determines whether or not an image of the fertilized egg a _i is detected in step S108. If an image of the fertilized egg a _i is detected, the process proceeds to step S111, where the fertilized egg a _i is detected. If no image is detected, the process _proceeds to step S110 in order to reacquire the drop image I _i (partial drop images I _{ia to} I _id ) of the drop number i.

In step S110, the control unit 31 instructs the observation unit 32 to exclude the range in the vicinity of the previous focus plane from the focus adjustment range when each of the partial drop images I _{ia to} I _id is acquired. After instructing the observation unit 32 to shift the imaging center of the image I _i (the center of the partial drop images I _{ia to} I _id ) by a predetermined distance (Δx, Δy) from the current coordinates (x _i , y _i ), the step Return to S106. Each of Δx and Δy is a distance corresponding to one fertilized egg. By changing the acquisition condition of the drop image I _i (partial drop image I _ia ~I _id) In this manner, an image of the embryo a _i can make it more likely to be detected.

In step S111, the control unit 31 specifies the coordinates (x _i ′, y _i ′) to the observation unit 32 and acquires a high-magnification phase difference image of the object located at the coordinates (x _i ′, y _i ′). To the observation unit 32. At this time, the control unit 31 instructs the observation unit 32 to extract the high NA illumination unit 49 inserted in step S106 from the optical path. That is, a combination of the microscopic observation unit 44 and the illumination unit 48 is used as a phase difference observation microscope for acquiring a high-magnification phase difference image.

And the observation unit 32 arrange | positions the culture container 14 to the optical path of the microscope (microscopic observation part 44 and the illumination part 48) for phase difference observation, and also combines the combination of the objective lens 61 and the phase filter 62 for 10 times observation. The combination (objective lens 61 _high and phase filter 62 _high ) is set.

Here, the size of the field of view of the objective lens 61 _high (0.8 mm × 0.8 mm) is larger than the size of one fertilized egg (diameter 100 μm). Therefore, the observation unit 32 matches the optical axis position of the objective lens 61 _high with respect to the culture vessel 14 to the coordinates (x _i ′, y _i ′), and in this state, for example, a high-magnification phase difference image I as shown in FIG. _i 'get. This high-magnification phase difference image I _i ′ is a detailed image of the fertilized egg a _i having the drop number i. Therefore, hereinafter, the high-magnification phase difference image I _i ′ is referred to as “egg image I _i ′”.

In obtaining the egg image I _i ′, the control unit 31 restricts the focus adjustment range of the autofocus control only to the vicinity of the focus surface of the partial drop image from which the image of the fertilized egg a _i is detected. As a result, the possibility of focusing on an object other than the fertilized egg a _i (such as bubbles b _i and scratches) is suppressed.

In step S112, the control unit 31 associates the egg image I _i ′ acquired in step S111 with the drop number i, and stores it in the storage unit 31a as observation data of the culture vessel 14. As a result, the egg image I _i ′ is managed by the drop number i.

In step S113, the control unit 31 determines whether or not the current drop number is the final value (in this case, “7”). If it is the final value, the process proceeds to step S114. After the drop number is incremented by 1, the process returns to step S106. Therefore, the processing of steps S106 to S112 is executed for all drop numbers 1 to 7. As a result, seven egg images I ₁ ′ to I ₇ ′ are accumulated in the observation data of the culture container 14.

  In step S114, the control unit 31 instructs the transport unit 34 to transport the culture vessel 14. The transport unit 34 transports the culture container 14 from the stage 43 to a predetermined storage position of the stocker unit 33.

In step S115, the control unit 31 reads the eggs image I ₁ '~I _7' stored in the step S112, the image so that for each of them eggs image I ₁ '~I _7' subjected to blastocyst胞判specific processing The processing unit 46 is instructed. The image processing unit 46 performs blastocyst discrimination processing by template matching or the like. Specifically, the image processing unit 46 compares the feature image extracted in advance from the high-magnification phase difference image of the blastocyst with the feature image extracted from the egg image I _i ′, and there is a certain correlation between them. The egg image I _i ′ is regarded as a blastocyst image. Since the blastocyst has directionality, this comparison is repeated while rotating the feature image.

In step S116, the control unit 31 determines whether or not there is an image of a blastocyst in the egg images I ₁ ′ to I ₇ ′. If there is, the control unit 31 returns to step S104 after executing step S117, If not, the process immediately returns to step S104. Therefore, steps S105 to S117 are executed every time the observation start time comes. As a result, the egg images I ₁ ′ to I ₇ ′ are accumulated in the observation data of the culture container 14 for each observation period.

  In step S117, the control unit 31 displays on the operation panel 23 that one or more blastocysts have been generated in the culture vessel 14. Therefore, the user can take appropriate measures for fertilized eggs in the culture container 14 at an early stage. In order to notify a user away from the culture observation apparatus 11 in real time, the control unit 31 may send an email to the user's personal computer (or mobile) at this timing. The mail is transmitted through a communication circuit (not shown) provided in the control unit 31 (step S117).

  As described above, since the culture observation apparatus 11 automatically detects the medium drop based on the entire image (step S103), it is possible to save the user from specifying the position of the medium drop. Further, since the medium drop detection (step S103) is performed based on the color distribution of the entire image, the accuracy is high. Moreover, since the culture observation apparatus 11 of this embodiment detects a fertilized egg automatically based on a drop image (step S107), the effort which a user designates the position of a fertilized egg can be saved.

  Further, the culture observation apparatus 11 of the present embodiment is based on the possibility that bubbles are detected together with the fertilized egg in the circular pattern search process (step S107), and the circular pattern detected in the circular pattern search process is an image of the fertilized egg. It is determined again whether or not (step S108). Therefore, the detection accuracy of a fertilized egg is high.

  Furthermore, the culture observation apparatus 11 of this embodiment considers the possibility that the fertilized egg cannot be focused, and if the image of the fertilized egg is not detected from the drop image (step S109 NO), the culture condition of the drop image is set. After the change (step S110), the drop image is reacquired. Therefore, there is little possibility that the detection of a fertilized egg will fail.

  Therefore, the user of the culture observation apparatus 11 stores the culture container 14 in the culture observation apparatus 11 and simply inputs an observation schedule (time-lapse observation time interval, etc.) to the culture observation apparatus 11 so that fertilization floats in the medium drop. A detailed image of the egg can be automatically acquired by the culture observation apparatus 11.

  Further, in the culture observation apparatus 11, the high NA illumination unit 49 is inserted on the optical axis of the microscopic observation unit 44 in step S 106, and a high NA with a low magnification is obtained by a microscope composed of a combination of the microscopic observation unit 44 and the high NA illumination unit 49. Since the illumination image is acquired, it is possible to suppress the appearance of a shadow near the edge of the medium drop D. Thereby, the fertilized egg a can be reliably detected in the process of step S108.

  In the present embodiment, the dish is used as the culture container 14, but a well plate having a plurality of wells may be used as the culture container 14. In particular, in a so-called 96-well plate having 96 wells, since the diameter of each well is small, the influence of the shadow due to the surface tension of mineral oil on the inner wall surface of the well becomes relatively large. Then, even if it is a 96 well plate, since it can suppress that the shadow appears, it can observe more favorably.

  Further, in the high NA illumination unit 49, in addition to adopting an LED as the light source 71, for example, an organic EL or an inorganic EL can be adopted to widen the light emitting surface. In addition to the Fresnel lens 73, the illumination light can be collected with a high NA, such as a DOE (Diffractive Optical Element) or an aspherical lens, in order to collect the illumination light from the light source 71. An optical element having a planar shape and a large area can be used.

  The culture observation apparatus 11 may operate the control unit 31 as follows. That is, the control unit 31 determines that it is impossible to detect a fertilized egg related to the current drop number when the number of executions of step S110 exceeds a predetermined number of times, and displays a display to that effect (warning display). )I do. In order to give a warning in real time to a user away from the culture observation apparatus 11, the control unit 31 may send an email to the user's personal computer (or mobile) at this timing.

  In addition, the culture observation apparatus 11 of any of the above-described embodiments is arranged on a partial drop image in order to reduce the calculation amount of the circular pattern search process (step S107) during the second and subsequent observations (after the second round). The search range may be limited only to the vicinity of the detection position in the previous round. Alternatively, instead of limiting the search range on the partial drop image, an area near the detection position in the previous round may be searched preferentially.

  Moreover, although the culture observation apparatus 11 automatically detected the medium drop from the culture container 14, instead of automatically detecting the medium drop, the user may specify the position of the medium drop.

  In addition, since the approximate thickness of the medium drop is known, the culture observation apparatus 11 sets the focus adjustment range when acquiring the drop image, egg image, and annular pattern image in the IVF mode to a range corresponding to the thickness of the medium drop. You may restrict.

  Further, although the phase difference observation method is applied to the microscopic observation unit 44 as a method of observing the cultured cells that are transparent objects, other observation methods that can observe the transparent objects, such as differential interference observation methods, A dark field observation method, a polarization observation method, or the like may be applied. However, when applying an observation method (differential interference observation method, polarization observation method, etc.) in which the fertilized egg image is circular instead of circular, a circular pattern search is used instead of the circular pattern search processing described above. Processing is applied. Incidentally, details of this circular pattern search process and the above-described annular pattern search process are also disclosed in Japanese Patent Laid-Open No. 2008-15714.

  The culture observation apparatus 11 is equipped with an IVF mode in which cells (here, fertilized eggs) included in each of a plurality of medium drops are observed, but other modes with different observation targets are available. It may be mounted. For example, a mode in which a plurality of cells included in one medium are observation targets may be installed.

  In addition, the image processing unit 46 may execute part of the processing by the control unit 31, or the control unit 31 may execute part or all of the processing by the image processing unit 46. In the culture observation apparatus 11, the control program of the culture observation apparatus 11 is executed by the control unit 31 built in the culture observation apparatus 11. A part or all of the control program is connected to the control unit 31. The device may execute.

  Note that the processes described with reference to the flowcharts described above do not necessarily have to be processed in time series in the order described in the flowcharts, but are performed in parallel or individually (for example, parallel processes or objects). Processing).

  The embodiment of the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention.

DESCRIPTION OF SYMBOLS 11 Culture observation apparatus, 12 Incubator part, 13 Base part, 14 Culture container, 21 Incubator case, 22 Incubator door, 23 Operation panel, 24 Case, 25A and 25B door, 31 Control unit, 31a Storage part, 32 Observation unit , 33 Stocker section, 34 Transport section, 34A Support section, 41 Unit body, 42 Arm section, 43 Stage, 44 Microscopic observation section, 45 Illumination section, 46 Image processing section, 47 Container observation section, 48 Illumination section, 49 High NA Illumination unit, 61, 61 _high and 61 _low objective lens, 62, 62 _high and 62 _low phase filter, 71 light source, 72 mirror, 73 Fresnel lens

Claims (5)

In the culture observation apparatus for observing the observation object in the culture container,
Observation means for imaging the culture vessel through an objective lens having a predetermined magnification,
An illuminating means arranged at a position facing the observing means, and irradiating illumination light to an observation target in the culture vessel,
The culture observation apparatus, wherein the numerical aperture of the illumination light from the illumination means is larger than the numerical aperture of the objective lens of the observation means. The culture observation apparatus according to claim 1, wherein the illuminating unit can be inserted into and extracted from an optical path for microscopic observation including the observation unit. The observation means includes a first objective lens that is used when imaging at a first magnification and a second objective lens that is used when imaging at a second magnification that is higher than the first magnification. By switching, you can get images of different magnification,
The culture observation apparatus according to claim 2, wherein the illuminating unit is inserted into the optical path during imaging using the first objective lens. The said illumination means has a planar shape and an optical element with a wide area for condensing illumination light to the observation object in the said culture container. The Claim 1 thru | or 3 characterized by the above-mentioned. Culture observation device. 5. The apparatus according to claim 1, further comprising: a search unit that searches for a target object having a predetermined characteristic in the observation target based on an image captured using the first objective lens. The culture observation apparatus described in 1.

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