JP2012147739A - Observation apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To perform excellent observation in an observation apparatus which observes a sample existing in liquid.SOLUTION: The observation apparatus includes: an illumination optical system 32 for irradiating the sample with illumination light; and an observation optical system 33 for collecting the light from the sample. The illumination optical system 32 and the observation optical system 33 are adjusted according to the kind of a culture container in which the sample is stored. An angle of the light axis of luminous fluxes of the illumination light collected on the sample from the vicinity with respect to the light axis of the luminous fluxes being the center of the illumination light can be changed by adjusting a position of a light axis direction of an aperture 42A. When the culture container is a well plate, the angle is adjusted to be narrow. When the culture container is a dish, the angle is adjusted to wide. The apparatus can be applied to a culture observation apparatus having an incubator.

Description

  The present invention relates to an observation apparatus, and more particularly, to an observation apparatus that enables good observation.

  Conventionally, fertilized eggs are cultured and observed in in vitro fertilization (IVF), microinsemination (ICSI: Intracytoplasmic Sperm Injection), and the like. The observation of a fertilized egg includes a medium drop observation method and an observation method using a well plate.

  In the medium drop observation method, the collected fertilized eggs are put one by one in a medium drop of about 20 μl made on a culture container (for example, 35 mm dish). The surface of the medium drop is coated with mineral oil, and in this state, the entire culture container is accommodated in a culture apparatus having a constant temperature and humidity environment called an incubator, and culture is performed.

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

  With reference to FIG. 5, the shadow that appears at the edge of the medium drop will be described. FIG. 5 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, an optically strong concave lens (at the boundary surface between the mineral oil O and the medium drop D) ( The same effect as f = -14) occurs. Therefore, the illumination light incident on the vicinity of the edge of the medium drop D is refracted outside 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.

  On the other hand, the surface tension of the mineral oil O on the inner wall surface of the culture vessel 1 also produces the same effect as the concave lens, which is a meniscus effect, at the boundary surface between the mineral oil O and the liquid surface. 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.

  Moreover, in the observation method using a well plate, for example, a culture medium is put in a hole in a 4-96 well plate, and a fertilized egg is put in the culture medium. At this time, the surface tension of the medium on the inner wall surface of the well plate hole causes a meniscus effect at the edge of the well plate hole. When observing the fertilized egg, the fertilized egg is hidden in the shadow. Observation becomes difficult.

  Therefore, the applicant of the present application has proposed a phase contrast microscope that can move the phase difference ring so as to cancel the influence of the meniscus effect due to the liquid surface or the boundary surface between the mineral oil O and the medium drop (see Patent Document 1). ).

JP 2009-122356 A

  By the way, the influence of the meniscus effect differs depending on the type of culture vessel, and even if the optical system is set so that good observation can be performed in one type of culture vessel, good observation is performed in other types of culture vessels There was something that could not be done. Therefore, there has been a demand for an observation apparatus that can perform good observation regardless of the type of culture vessel.

  The present invention has been made in view of such a situation, and enables a good observation.

  The observation apparatus according to the present invention is an observation apparatus for observing a sample present in a liquid, an optical system that irradiates the sample with illumination light over a certain angle range and collects light from the sample, and the sample It is characterized by comprising acquisition means for acquiring the type of the culture container in which is stored, and adjustment means for adjusting the optical system according to the type of the culture container.

  In the observation apparatus of the present invention, the type of the culture container in which the sample present in the liquid is stored together with the liquid is acquired, and the optical system is adjusted according to the type of the culture container.

  According to the observation apparatus of the present invention, good observation can be performed.

It is a figure which shows the structural example of one Embodiment of the observation unit with which the culture observation apparatus to which this invention is applied is provided. It is a figure explaining the illumination optical system and observation optical system for every observation condition. It is a flowchart explaining the process of a control part. It is a flowchart explaining an observation process. It is a figure explaining the shadow which appears in the edge of a culture medium drop.

  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 an observation unit provided in a culture observation apparatus to which the present invention is applied.

  In FIG. 1, the observation unit 11 is configured such that an arm part 13 and a stage 14 are arranged on the upper side of the unit body 12, and the arm part 13 and the stage 14 are held in an environment suitable for culturing fertilized eggs. It is arranged inside the incubator of the culture observation apparatus. Then, fertilized eggs are cultured in a plurality of culture containers (dish or well plate) inside the incubator. For example, the culture container 15 to be observed according to the observation schedule is placed on the stage 14 by a robot arm or the like, Observation of fertilized eggs cultured in the culture vessel 15 is performed.

  The arm portion 13 has a shape that extends upward from the side of the stage 14 and projects to the upper side of the stage 14, and houses an LED (Light Emitting Diode) light source 31 and an illumination optical system 32. The illumination light output from the LED light source 31 is applied to the culture vessel 15 via the illumination optical system 32.

  The unit main body 12 houses an observation optical system 33, an imaging unit 34, and an image processing unit 35. The observation optical system 33 is arranged on the optical axis of the illumination light irradiated from the illumination optical system 32 to the culture vessel 15, and the observation light is incident on the imaging unit 34 via the observation optical system 33. The imaging unit 34 is cultured. The fertilized egg in the container 15 is imaged.

  The image picked up by the image pickup unit 34 is subjected to image processing in the image processing unit 35. For example, in the observation unit 11, a process of observing the entire region of the observation range (entire medium drop in the dish or the whole hole of the well plate) at a low magnification is performed, and the image captured by the imaging unit 34 is displayed. A fertilized egg is searched by image processing. Then, the process which observes each fertilized egg at high magnification is performed, and the state of a fertilized egg is observed in detail.

  Further, in the observation unit 11, the illumination optical system 32 and the observation optical system 33 can be adjusted according to the type of the culture container 15, for example, depending on whether the culture container 15 is a dish or a well plate. it can. That is, the observation unit 11 includes a detection unit 21 that detects the type of the culture vessel 15, an illumination optical system adjustment unit 22 that adjusts the illumination optical system 32, an observation optical system adjustment unit 23 that adjusts the observation optical system 33, and a detection A control unit 24 that controls the illumination optical system adjustment unit 22 and the observation optical system adjustment unit 23 according to the detection result of the unit 21 is provided.

  The illumination optical system 32 includes a collector lens 41 that collects light beams from each point of the LED light source 31, and a stop 42A that can move in the optical axis direction of the illumination optical system 32 and that has a variable opening diameter (FIG. 2). When the diaphragm 42 is retracted from the optical path of the illumination optical system 32, an aperture diaphragm 43 (FIG. 2) serving as an aperture diaphragm and a condenser lens 44 that condenses the light flux that has passed through the diaphragm 42 or the opening of the aperture diaphragm 43. And a high magnification observation condenser lens 45 disposed on the optical path of the illumination optical system 32 during high magnification observation.

  The collector lens 41 makes the luminous flux from each point of the LED light source 31 parallel. The parallel light flux that has passed through the diaphragm 42 or the aperture diaphragm 42 is condensed by the condenser lens 44 or the condenser lens 45 for high magnification observation. The illumination optical system 32 exemplifies critical illumination for the sake of simplicity. However, the present invention is not limited to the illumination optical system in which the light source and the sample have a conjugate relationship, and the light source and the sample need not have a conjugate relationship.

  The detection unit 21 is a sensor that reads, for example, a label attached to the culture container 15. The label of the culture container 15 describes, for example, the type of the culture container 15 (dish or well plate), information about the fertilized egg being cultured, and the detection unit 21 reads the label and reads the culture container 15 The type is acquired and supplied to the control unit 24.

  As will be described later with reference to FIG. 2, the illumination optical system adjusting unit 22 is a condenser lens driving unit so that one of the lens elements constituting the condenser lens can be inserted and removed from the optical axis of the illumination optical system 32. In addition, the illumination optical system 32 is adjusted by switching the stop arranged at the focal position of the collector lens on the optical axis or moving the stop in the optical axis direction. Further, as will be described later with reference to FIG. 2, the observation optical system adjusting unit 23 switches an objective lens disposed on the optical axis, and inserts / removes a phase ring or a Hoffman modulation diaphragm on the optical axis. Then, the observation optical system 33 is adjusted.

  The control unit 24 performs the illumination optical system adjustment unit 22 and the observation optical system adjustment unit 23 based on whether the observation is performed at a low magnification or a high magnification and whether the culture vessel 15 is a dish or a well plate. The illumination optical system 32 and the observation optical system 33 are adjusted so that good observation can be performed.

  With reference to FIG. 2, the illumination optical system 32 and the observation optical system 33 that are adjusted so that good observation can be performed will be described.

  The illumination optical system 32 includes a collector lens 41, a stop 42A, a ring stop 42B, an MC stop 42C that is a Hoffman modulation stop, an aperture stop 43, a condenser lens 44, and a high magnification observation condenser lens 45. In the illumination optical system 32, the illumination optical system adjustment unit 22 can switch one of the stop 42A, the ring stop 42B, or the MC stop 42C that is inserted on the optical axis, and each can be switched in the optical axis direction. It is possible to move along.

  The observation optical system 33 includes objective lenses 51A to 51D that can be inserted into and removed from the optical axis by a revolver mechanism (not shown). Each of the objective lens 51C and the objective lens 51D includes a phase plate 52 in which a phase film is formed in a ring shape around the optical axis, or a modulator 53 for Hoffman modulation contrast observation method in the same lens barrel. Yes. Further, the revolver mechanism has a rotational position detection mechanism, and it is possible to detect which objective lens is disposed in the optical path of the observation optical system 33 according to the rotational position of the revolver. The rotational position detection mechanism is connected to the control unit 24, and the output of the rotational position detection mechanism is supplied to the control unit 24.

  Further, the observation optical system 33 includes the second objective lens 54 illustrated in FIG. 1, and the light beam condensed by any of the objective lenses 51A, 51B, 51C, and 51D is collected by the second objective lens 54. By doing so, an image of the sample is formed.

  The objective lens 51A has a low magnification of about 2 or 5 times and a high NA (Numerical Aperture), and the objective lens 51B has a low magnification of about 2 or 5 times and a normal NA (for example, NA). The objective lens 51C is for high-magnification and phase difference observation, and the objective lens 51D is for high-magnification and Hoffman modulation contrast observation. In addition, an objective lens that can perform bright field observation or dark field observation with high magnification may be separately prepared. In the observation optical system 33, among the objective lens 51A, the objective lens 51B, and the objective lens 51C, the one inserted on the optical axis can be switched by the observation optical system adjustment unit 23, and the phase plate 52 and The modulator 53 can be inserted into and removed from the optical axis.

  FIG. 2A shows the illumination optical system 32 and the observation optical system 33 under the observation conditions in which the observation magnification is low and the culture vessel 15 is a well plate (96 well). The illumination optical system 32 includes an LED light source 31, a collector lens 41 that is a focal position at the position of the LED light source 31, a stop 42A that blocks a part of the light beam collected by the collector lens 41, and a light beam that has passed through the stop 42A. A condenser lens 44 for condensing the light. At this time, for example, the diameter of the observation range is about 6.4 mm. Moreover, the depth of the hole of a well plate is about 10 mm, and the depth to the liquid level of the culture solution in a well plate is 2.5 mm. Accordingly, the distance from the inlet of the well plate hole to the liquid level is approximately 7.5 mm.

  By the way, when it is assumed that the side wall of the well plate is parallel to the optical axis of the illumination optical system, the angle of the principal ray of the illumination light beam at each position of the illumination field is determined from the opening of the well plate to the culture solution in the well plate. It is necessary to set the angle so as not to be kicked by the portion serving as the side wall of the well plate.

  Under such observation conditions, the position of the stop 42A is arranged in a region closer to the collector lens 41 than the focal position of the condenser lens 44 on the light source side. As a result, the principal ray of each light beam incident on the entrance of the well plate propagates in a direction converging toward the optical axis of the illumination optical system 32. The principal ray is refracted in the direction of divergence by the meniscus effect generated at the liquid level of the culture solution in the well plate. Therefore, it is possible to illuminate the entire area at the bottom of the well plate. As a result, the chief ray of the illumination beam is not blocked by the side wall of the well plate. On the other hand, as shown in FIG. 2A, the chief ray of each light beam illuminating the bottom of the well plate reaches the objective lens while propagating in the direction of divergence. In the objective lens 51A, an objective lens 51A having a low magnification and a high NA (for example, about NA = 0.25) is arranged on the optical axis so that these light beams can be sufficiently collected. Thus, in order to ensure uniformity in the illumination field, the position of the stop 42A is adjusted in the optical axis direction.

  On the other hand, it is necessary to prevent the marginal rays of the light beams reaching the liquid level in the well plate at a position away from the optical axis from being blocked by the side wall of the well plate. The angular range of the marginal ray can be adjusted by the size of the opening of the stop 42A. In addition, the illumination NA of the illumination optical system is made smaller than the NA of the objective lens 51A so that all of the illumination light beam that illuminates the outer peripheral area with the objective lens 51A can be collected.

  By adjusting the position on the optical axis of the stop 42A and the size of the opening of the stop 42A by the illumination optical system 32 having such a configuration, the inner diameter of a container for placing a sample such as a cell such as a well plate is adjusted. Even in the case of an observation sample whose liquid level is deeper than the above, uniform illumination can be achieved. Furthermore, by replacing the objective lens having a large NA with the observation optical system 33, light that is refracted toward the outside on the surface of the culture medium in each concave portion of the well plate that is the culture vessel 15 can be introduced into the observation optical system 33. it can.

  That is, due to the meniscus effect caused by the concave surface of the liquid generated by the side wall of the well plate, even if the illumination light reaching the outer periphery of the well plate recess is further refracted radially from the center of the well plate, the light beam transmitted through the well plate By adopting the objective lens 51A of the observation optical system having a sufficient numerical aperture to receive the optical system, it becomes possible to simultaneously observe the entire culture region, which is the bottom surface of the well plate, and near the wall surface of the well plate hole. It is avoided that shadows occur. Thereby, it is possible to perform better observation without hiding the fertilized egg to be observed. In such observation, it is preferable to perform coherent low NA illumination in order to illuminate the entire region of the culture vessel 15 without unevenness.

  FIG. 2B shows the illumination optical system 32 and the observation optical system 33 under the observation condition where the observation magnification is low, the culture vessel 15 is a dish, and the observation sample is a medium drop. At this time, for example, the diameter of the observation range is about 6 mm.

  Under such observation conditions, even if the illumination light beam is incident from the side, it is not kicked by the side wall unlike the well plate. Therefore, the illumination light beam that illuminates the outer periphery of the medium drop is largely deflected from the outside toward the optical axis by the condenser lens 44 so as to enter the medium drop. Therefore, the objective lens 51B having a low magnification and a normal NA can be used.

  In order to achieve this with the same illumination optical system, the position of the stop 42A is more than that in the observation condition of FIG. 2A (observation condition in which the observation magnification is low and the culture vessel 15 is a well plate). Is also moved toward the collector lens 41 along the optical axis direction.

  With the illumination optical system 32 and the observation optical system 33 configured as described above, the illumination light beam is introduced from the outside onto the surface of the culture medium drop in the dish that is the culture vessel 15, thereby introducing the illumination light beam into the observation optical system 33. Can do. That is, the optical system cancels the convex meniscus due to the interface between the medium drop and the oil, and the occurrence of a shadow near the edge of the medium drop is avoided. As described above, since there is no difference in the amount of light between the central part and the terminal part of the medium drop, the fertilized egg to be observed is not hidden in the shadow, and better observation can be performed.

  Here, when FIG. 2A is compared with FIG. 2B, the optical axis of the luminous flux of the illumination light condensed on the sample from the periphery (the optical axis indicated by the broken line) is obtained by moving the stop 42A along the optical axis direction. ) With respect to the optical axis of the luminous flux at the center of the illumination light (the optical axis indicated by the alternate long and short dash line). That is, when the culture vessel 15 is a well plate (FIG. 2A), this angle is narrowed, and when the culture vessel 15 is a dish (FIG. 2B), this angle is adjusted to be wide.

  FIG. 2C illustrates an illumination state when the observation magnification is higher than that in FIGS. 2A and 2B and the culture vessel 15 is a well plate (96 wells). In addition, an illumination optical system 32 and an observation optical system 33 under observation conditions for performing phase difference observation are shown.

  Since phase difference observation is performed under such observation conditions, a phase difference observation ring stop 42B is used instead of the normal stop 42A. In addition, since the observation is performed at a high magnification (for example, 10 times or more), the visual field to be observed is limited. What is necessary is to secure an illumination field that only illuminates the field of view. Therefore, it is preferable to illuminate with a large illumination NA in the limited illumination field. Therefore, in FIG. 2C, in order to achieve a larger illumination NA, a high-magnification observation condenser lens 45 having a positive refractive power is further added. On the other hand, in order to prevent the illumination light flux from being blocked by the side wall of the well plate, the ring diaphragm 42B is positioned closer to the collector lens 41 side than the focal position of the collector lens 41 on the sample side, as described with reference to FIG. It is set.

  By doing in this way, the sample in the liquid in the well plate 15 can be observed with the illumination NA corresponding to the NA of the objective lens 51C at a high magnification.

  FIG. 2D illustrates an illumination state when the observation magnification is high and the culture vessel 15 is a dish as compared to the cases of FIGS. 2A and 2B. Further, the illumination optical system 32 and the observation optical system 33 under the observation conditions for performing the Hoffman modulation observation are shown.

  Even under such observation conditions, the MC diaphragm 42C for Hoffman modulation is arranged at a position closer to the collector lens 41 than the focal position on the sample side of the collector lens 41 as in the phase difference observation. However, since the Hoffman modulation contrast observation method is an observation method in which contrast is provided according to the angle of the surface of the phase object, the interface of the medium drop also has contrast. Therefore, the position of the MC stop 42 on the optical axis is set so that the illumination light beam has an incident angle to the medium drop that does not give contrast in a shape other than the sample due to the medium drop.

  Specifically, the angle is set such that the light beam that illuminates the field of view of the objective lens 51D is perpendicularly incident at the interface of the medium drop. By doing in this way, it can prevent that contrast is given by a culture medium drop. Further, the modulator 53 is also provided with a light shielding portion (transmittance: 0%) so as to shield the light beam that has not been refracted by the sample and has traveled straight. A portion other than the light shielding portion is formed with a modulator 53 so as to have a different transmittance for each place. For example, the modulator 53 is formed with a transmittance of 8% where the light beam that has passed through the aperture of the MC stop 42C is condensed, and a portion other than the portion with the transmittance of 8% and the light shielding portion is formed with a transmittance of 100%. Has been.

  As described above, depending on the type of container in which the sample is placed or the environment in which the sample is placed, the illumination optical system or the observation optical system is set up, and this setup is made appropriate depending on the observation method. be able to.

  For this purpose, in the embodiment of the present invention, the control unit 24 uses the label attached to the culture vessel 15 by the detection unit 21 to determine the type of the culture vessel and whether or not there is a medium drop when the culture vessel is a dish. On the other hand, information on the objective lens selected by the user is obtained from the rotational position detection mechanism. Then, the control unit 24 controls the illumination optical system adjustment unit 22 so as to move the variable diaphragm so as to obtain an optimal illumination state from the obtained information and insert the high magnification observation condenser lens 45 into the optical path. ing.

  Thereby, even if the kind of culture container changes, the illumination conditions which can observe almost all the culture | cultivation area | regions can be set.

  Next, FIG. 3 is a flowchart for explaining the processing of the control unit 24.

  If a culture container is arrange | positioned at the stage 14, the detection part 21 will read the label attached | subjected to the culture container in step S1. The type of culture vessel is specified from the information on the label. In addition, when the culture container is a dish, whether the medium drop is observed or whether the dish is simply filled with the culture solution is also recorded on the label, and the information is also read by the detection unit 21.

  Next, in step S2, the position of the rotational position detection mechanism is detected in order to detect the objective lens arranged in the optical path of the observation optical system 33. The rotational position information of the revolver mechanism and the magnification and type of the objective lens arranged at that position are registered in the control unit 24 in advance. The control unit 24 recognizes the selected objective lens based on the output from the rotational position detection mechanism.

  Next, in step S3, the position of the stop 42A is changed so as to obtain an optimal illumination state or the ring stop is changed from the stop 42A according to the culture container, the culture state, and the objective lens arranged in the observation optical system 33. Replace with 42B, or replace with the MC aperture 42C instead of the aperture 42A. The determination criteria are as shown in FIG. According to FIG. 2, whether or not to replace the diaphragm 42A with the ring diaphragm 42B or the MC diaphragm 42C depending on whether the phase difference observation or the Hoffman modulation contrast observation is selected or the normal observation method. Determine whether.

  Next, in the case of a normal observation method, the position of the stop 42A is determined according to whether the selected objective lens is high or low.

  In the above description, the example in which the user selects the objective lens is illustrated. However, the control unit 24 may automatically select the objective lens depending on the culture container. An example of the processing is shown in FIG. FIG. 4 is a flowchart for explaining an observation process in which the observation unit 11 in FIG. 1 observes a fertilized egg in the culture vessel 15.

  For example, the process starts when the culture vessel 15 is placed on the stage 14 according to a preset observation schedule. In step S11, the detection unit 21 acquires the type of the culture vessel 15 and supplies it to the control unit 24. The control unit 24 determines whether the culture container 15 is a dish or a well plate.

  In step S11, when the control unit 24 determines that the culture vessel 15 is a dish, the process proceeds to step S12.

  In step S <b> 12, the control unit 24 controls the illumination optical system adjustment unit 22 and the observation optical system adjustment unit 23 so that the observation magnification is a low magnification and the culture container 15 has a configuration corresponding to the observation condition. Take control. Thereby, for example, as shown in FIG. 2B, the illumination optical system adjustment unit 22 adjusts the illumination optical system 32, and the observation optical system adjustment unit 23 adjusts the observation optical system 33. That is, the optical system is adjusted so as to cancel the convex meniscus due to the medium drop.

  After the process of step S12, the process proceeds to step S13. In the observation unit 11, observation at a low magnification is performed, and a fertilized egg in the medium drop is searched. At this time, generation of a shadow due to the meniscus effect is avoided, so that a fertilized egg can be easily searched. And if the position of the fertilized egg in a culture medium drop is grasped, processing will progress to Step S14.

  In step S <b> 14, the control unit 24 controls the illumination optical system adjustment unit 22 and the observation optical system adjustment unit 23 so that the observation magnification is a high magnification and the culture container 15 has a configuration according to the observation condition. Take control. Thereby, for example, as shown in FIG. 2D, the illumination optical system adjustment unit 22 adjusts the illumination optical system 32, and the observation optical system adjustment unit 23 adjusts the observation optical system 33. That is, the optical system is adjusted such that the MC diaphragm 42C, the high magnification observation condenser lens 45, and the modulator 53 are added.

  After the process of step S14, the process proceeds to step S15, and the observation unit 11 performs observation at a high magnification and performs detailed observation of the fertilized egg. After the process of step S15, the process is terminated.

  On the other hand, if the control unit 24 determines in step S11 that the culture vessel 15 is a well plate, the process proceeds to step S16.

  In step S <b> 16, the control unit 24 has the illumination optical system adjustment unit 22 and the observation optical system adjustment unit 23 so that the observation magnification is low and the configuration is in accordance with the observation conditions in which the culture vessel 15 is a well plate. Control over. Thereby, for example, as shown in FIG. 2A, the illumination optical system adjustment unit 22 adjusts the illumination optical system 32, and the observation optical system adjustment unit 23 adjusts the observation optical system 33. That is, the optical system is adjusted so as to cancel the concave meniscus due to the well plate hole.

  After the process of step S16, the process proceeds to step S17, and the observation unit 11 performs observation at a low magnification and searches for a fertilized egg in the medium drop. At this time, generation of a shadow due to the meniscus effect is avoided, so that a fertilized egg can be easily searched. Further, since the influence of image quality deterioration varies depending on the R-plane shape of the meniscus for each well plate, adjustment is made so as to reduce the influence of image quality deterioration by adjusting the position of the stop 42A. And if the position of the fertilized egg in a culture medium drop is grasped, processing will progress to Step S18.

  In step S18, the control unit 24 sets the illumination optical system adjustment unit 22 and the observation optical system adjustment unit 23 so that the observation magnification is high and the configuration is in accordance with the observation conditions in which the culture vessel 15 is a well plate. Control over. Thereby, for example, as shown in FIG. 2C, the illumination optical system adjustment unit 22 adjusts the illumination optical system 32, and the observation optical system adjustment unit 23 adjusts the observation optical system 33. That is, the optical system is adjusted such that the ring diaphragm 42B, the high magnification observation condenser lens 45, and the phase plate 52 are added.

  After the process of step S18, the process proceeds to step S19, and the observation unit 11 performs observation at a high magnification and performs detailed observation of the fertilized egg. After the process of step S19, the process is terminated.

  As described above, in the observation unit 11, the illumination optical system 32 and the observation optical system 33 are adjusted so as to be an optical system suitable for each culture container 15 according to whether the culture container 15 is a dish or a well plate. Therefore, good observation can be performed regardless of the type of the culture vessel 15. That is, it is possible to recognize a fertilized egg in the entire observation area, and to ensure a high contrast image quality.

  Further, for example, by enabling the stop 42A to move along the optical axis direction, even if the curvature of the liquid level varies depending on various conditions, illumination corresponding to the variation can be performed.

  Further, for example, when performing phase difference observation, when the observation position is a medium drop terminal, the ring diaphragm 42B and the phase plate due to the meniscus effect are moved by moving the ring diaphragm 42B in a direction orthogonal to the optical axis. The positional relationship with 52 can be corrected. Such correction is described in detail in Japanese Patent Application Laid-Open No. 2009-122356 filed by the applicant of the present application.

  Further, when the control unit 24 stores the center coordinates of the medium drop and moves the ring diaphragm 42B in the direction orthogonal to the optical axis, the difference in position is observed when observing the end part of the medium drop. The ring diaphragm 42B can be configured to automatically move by the amount of movement corresponding to. Further, a subtle difference in meniscus shape occurs depending on the type of the culture vessel 15, but such a difference can be dealt with by adjusting the position of the ring diaphragm 42B in the optical axis direction.

  In the present embodiment, the optical system for observing a fertilized egg has been described. However, the present invention can also be applied to normal cell observation. Further, for example, a differential interference observation method, a dark field observation method, a polarization observation method, or the like may be applied.

  In addition to the configuration for reading the label of the culture vessel 15, the detection unit 21 may be configured to acquire the type of the culture vessel 15 input by the user, for example.

  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 Observation unit, 12 unit main body, 13 arm part, 14 stage, 15 culture container, 21 detection part, 22 illumination optical system adjustment part, 23 observation optical system adjustment part, 24 control part, 31 LED light source, 32 illumination optical system, 33 observation optical system, 34 imaging unit, 35 image processing unit, 41 collector lens, 42A aperture, 42B ring aperture, 42C MC aperture, 43 aperture aperture, 44 condenser lens, 45 condenser lens for high magnification observation, 51A to 51D objective lens, 52 phase plate, 53 modulator, 54 second objective lens

Claims (5)

An optical system for irradiating illumination light to a sample housed in a culture container together with a liquid,
Obtaining means for obtaining the type of culture vessel in which the sample is stored;
An observation device comprising: an adjusting unit that adjusts an illumination state of the sample by the optical system according to a type of the culture vessel. The optical system includes an illumination optical system for illuminating the sample with illumination light,
The illumination optical system has changing means for changing an angle of a principal ray of a luminous flux of illumination light reaching the sample from a position away from the optical axis of the illumination optical system with respect to the optical axis of the illumination optical system. And
The observation apparatus according to claim 1, wherein the adjusting unit controls the changing unit according to the culture vessel. The observation apparatus according to claim 2, wherein the changing unit includes a diaphragm member driving unit configured to move a diaphragm member arranged in the illumination optical system in an optical axis direction of the illumination optical system. The optical system further includes an observation optical system that forms an image of the sample illuminated by the illumination optical system,
The observation optical system includes any one objective lens held by an objective lens exchange mechanism that can hold a plurality of objective lenses,
The adjusting means selects one of a plurality of objective lenses held by the objective lens exchanging mechanism according to the culture container, and the selected objective lens is on the optical axis of the observation optical system. The observation apparatus according to claim 3, wherein the objective lens exchange mechanism is controlled so as to be arranged. The observation apparatus according to claim 4, wherein the adjusting unit further controls the changing unit according to the objective lens.

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