JP2015152649A - phase-contrast microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable position alignment of an aperture image and a phase film in a phase-contrast microscope using a simple configuration.SOLUTION: Tilting a stage 27 relative to illumination light allows for making a bottom of a culture container 12 act as a prism. An aperture image, or an image at an aperture of a phase diaphragm, is thus shifted on a phase plate to correct for an aperture image shift caused by the meniscus of a culture medium inside the culture container 12, thereby aligning positions of the aperture image and a phase film 37A. The present invention is applicable, for example, to phase-contrast microscopes.

Description

  The present invention relates to a phase contrast microscope, and more particularly to a phase contrast microscope that suppresses image quality degradation due to a meniscus.

  When observing a sample such as a cell using a phase contrast microscope, a liquid called a medium for culturing the sample and the sample is generally placed in a culture container such as a dish (dish) or well plate (microplate), In this, the sample is cultured and observed.

  In this case, a meniscus in which the liquid level of the medium in the culture vessel becomes a concave curved surface due to surface tension is generated, and the image quality of the observed image of the sample is deteriorated due to the influence of the meniscus. Specifically, the illumination light that has passed through the phase stop having the ring-shaped opening is refracted by the meniscus of the culture medium, and the shape of the image of the phase stop (hereinafter referred to as the stop image) is distorted. As a result, the amount of direct light transmitted through the sample is incident on the ring-shaped phase film disposed on the exit pupil plane of the objective lens is reduced, and the contrast of the observation image is reduced.

  Therefore, conventionally, at the time of alignment before observation, the displacement of the aperture image due to the meniscus is corrected by moving the phase stop, and the correction position of the phase stop and the position of the stage are recorded as observation information, and the observation information is used. It has been proposed to observe a sample after setting the phase stop to an appropriate position (see, for example, Patent Document 1).

JP 2009-122356 A

  However, in the invention described in Patent Document 1, it is necessary to provide a dedicated stage for moving the phase stop, which makes the configuration complicated and increases the cost.

  The present invention has been made in view of such a situation, and makes it possible to align a diaphragm image and a phase film of a phase contrast microscope with a simple configuration.

  A phase contrast microscope according to one aspect of the present invention is a phase contrast microscope for observing a sample in a container disposed on a stage by irradiating illumination light. The phase contrast microscope is disposed on a condenser lens and an incident side of the condenser lens. An aperture, an objective lens, a phase film, a phase plate disposed on an exit pupil plane of the objective lens or a pupil conjugate plane conjugate with the exit pupil plane, and an inclination of the container with respect to the illumination light Adjusting mechanism.

  In one aspect of the present invention, the relative position between the image of the stop on the phase plate and the phase film is adjusted by the inclination of the container with respect to the illumination light.

  According to one aspect of the present invention, it is possible to align the diaphragm image of the phase contrast microscope and the phase film with a simple configuration.

It is a figure which shows the structural example of the external appearance of one Embodiment of the phase-contrast microscope to which this invention is applied. It is a figure which shows the structural example of 1st Embodiment of the optical system of the phase-contrast microscope to which this invention is applied. It is a figure which shows the example of the moving direction of the aperture image by a deviation prism unit. It is a block diagram which shows the structural example of the function of the part which controls and drives a phase-contrast microscope. It is a block diagram which shows the structural example of the function of 1st Embodiment of the position alignment control part. It is a flowchart for demonstrating the observation process performed with a phase-contrast microscope. 5 is a flowchart for explaining details of a first embodiment of aperture image position adjustment processing; It is a flowchart for demonstrating the detail of 2nd Embodiment of an aperture image position adjustment process. It is a block diagram which shows the structural example of the function of 2nd Embodiment of the position alignment control part. It is a flowchart for demonstrating the detail of 3rd Embodiment of an aperture image position adjustment process. It is a figure which shows the example of a phase plate image typically. It is a block diagram which shows the structural example of the function of 3rd Embodiment of the position alignment control part. It is a flowchart for demonstrating the detail of 4th Embodiment of an aperture image position adjustment process. It is a block diagram which shows the structural example of the function of 4th Embodiment of the position alignment control part. It is a flowchart for demonstrating the detail of 5th Embodiment of an aperture image position adjustment process. It is a flowchart for demonstrating 1st Embodiment of the time lapse observation process performed with a phase-contrast microscope. It is a figure which shows the structural example of 2nd Embodiment of the optical system of the phase-contrast microscope to which this invention is applied. It is a figure which shows the structural example of 3rd Embodiment of the optical system of the phase-contrast microscope to which this invention is applied. It is a figure for demonstrating the method of inclining a stage and aligning an aperture image. It is a figure for demonstrating an example of the method of inclining a stage. It is a figure for demonstrating the problem at the time of tilting a stage and aligning an aperture image. It is a figure for demonstrating the method of aligning an aperture image combining the tilt of a stage and the shift of a phase stop. It is a figure for demonstrating the method of aligning an aperture image combining the deviation prism unit or a parallel plate glass, and the shift of a phase stop. It is a figure for demonstrating the case where an observation optical system and a measurement optical system are provided. It is a block diagram which shows the structural example of the function of 6th Embodiment of the position alignment control part. It is a flowchart for demonstrating 2nd Embodiment of the time-lapse observation process performed with a phase-contrast microscope. It is a block diagram which shows the structural example of the function of 7th Embodiment of the position alignment control part. It is a block diagram which shows the structural example of a computer.

Hereinafter, modes for carrying out the present invention (hereinafter referred to as embodiments) will be described. The description will be given in the following order.
1. Embodiment 2. FIG. Modified example

<1. Embodiment>
[Configuration Example of Phase-Contrast Microscope 11]
1 and 2 show an embodiment of a phase contrast microscope to which the present invention is applied. FIG. 1 shows an external configuration example of the phase contrast microscope 11, and FIG. 2 shows a configuration example of an optical system of the phase contrast microscope 11.

  The phase contrast microscope 11 is provided with a stage 27, and a culture vessel 12 (FIG. 2) such as a well plate or a petri dish is disposed on the stage 27. A sample (not shown) such as a cell to be observed and a culture medium are placed in the culture container 12.

  Further, a light source 21 that emits illumination light for irradiating the sample is provided above the phase-contrast microscope 11 in FIG. The type of the light source 21 is not particularly limited. For example, the illumination uniformity and long life, and the phenol red in the medium changes color due to the deterioration of the medium and absorbs light in a wide visible range. Considering that the brightness changes depending on the culture state, a red LED is used.

  Illumination light emitted from the light source 21 enters the condenser lens 25 via an optical system shown in FIG. The condenser lens 25 collects the illumination light and irradiates the sample in the culture vessel 12 on the stage 27.

  The stage 27 is moved in a direction perpendicular to the optical axis of the condenser lens 25 (hereinafter referred to as X direction and Y direction) and a direction parallel to the optical axis of the condenser lens 25 (hereinafter referred to as Z direction). Can do. Further, the stage 27 can be tilted with respect to the optical axis of the condenser lens 25 as will be described later.

  The illumination light applied to the sample passes through the sample or the medium, or diffracts in the sample and enters the objective lens 28. That is, the observation light incident on the objective lens 28 from the sample irradiated with the illumination light and the surroundings of the sample are diffracted in the direct light transmitted through the sample or the medium, and the inside of the sample and the boundary between the sample and the medium. It consists of diffraction light including shape information. The observation light is collected by the objective lens 28 and enters the camera 39 and the eyepiece lens 43 through an optical system described later.

  The camera 39 captures an image of the observation light incident from the objective lens 28 and supplies the obtained image to, for example, another device (not shown) to display it or store it in a storage device. A user who is an observer can also observe the sample by looking at an image displayed on another device.

  The eyepiece 43 condenses the observation light incident from the objective lens 28 and forms an image by the observation light. Thereby, the user can observe the sample through the eyepiece lens 43.

  Here, the details of the optical system of the phase-contrast microscope 11 will be described with reference to FIG. In FIG. 2, the optical system from the phase plate 37 to the eyepiece 43 is not shown.

  The illumination light emitted from the light source 21 passes through the lens 22, is then collected by the collector lens 23, and forms a front focal plane of the condenser lens 25 (an entrance pupil plane of the objective lens 28 or a plane conjugate to the entrance pupil plane). The diameter of the light beam is reduced by the arranged phase stop 24. The illumination light that has passed through the substantially circular opening of the phase stop 24 passes through the condenser lens 25, is changed in optical path by the mirror 26, and is irradiated onto the sample in the culture vessel 12.

  The illumination light applied to the sample becomes observation light composed of direct light and diffraction light as described above, and enters the objective lens 28. The observation light is condensed by the objective lens 28, passes through the second objective lens 29, the optical path is changed by the mirror 30, and an image is formed on the primary image plane P1.

  In the vicinity of the primary image plane P1, a deviation prism unit 31 including a pair of deviation prism 31a and deviation prism 31b is disposed. The deviation prism 31a and the deviation prism 31b face each other across the primary image plane P1, and are arranged so that the optical axis of the objective lens 28 and the like coincide. The deviation prism 31a and the deviation prism 31b can be rotated independently about the optical axis, respectively, and at least one of the deviation prism 31a and the deviation prism 31b is rotated to deflect the observation light emitted from the deviation prism unit 31. Thereby, as will be described later, the incident position of the observation light on the phase plate 37 is adjusted, and the image formation position of the image of the opening of the phase stop 24 (hereinafter referred to as a diaphragm image) by the observation light can be adjusted. it can.

  Here, the deviation prism is a prism that is also called a deviation prism. The shape of the deviation prism is various. In the present embodiment, for example, a wedge-shaped deviation prism can be used. The wedge shape is a shape obtained by cutting the cylinder obliquely from a direction perpendicular to the axis connecting the center of the upper surface of the cylinder and the center of the lower surface.

  For example, in the case of the deviation prism unit 31 of FIG. 2, the two deviation prisms 31a and 31b each having a wedge shape as described above are used in parallel with each other along the optical axis. Further, the deviation prism 31a and the deviation prism 31b are arranged in a state where the surfaces corresponding to the upper surface and the lower surface of the cylindrical member are opposed to each other.

  The deviation prism that can be used in the present embodiment is not limited to the above.

  The observation light emitted from the deviation prism unit 31 passes through the lens 32 and the lens 33, the optical path is changed by the mirror 34, passes through the lens 35 and the lens 36, and enters the phase plate 37.

  The phase plate 37 is disposed on the pupil conjugate plane P2 conjugate with the exit pupil plane of the objective lens 28 (the rear focal plane of the objective lens 28), and the image of the opening of the phase stop 24 on the phase plate 37 ( That is, a diaphragm image is formed.

  The phase plate 37 is provided with a circular phase film 37A centered on the optical axis. It is empirically known that the area of the phase film 37A is preferably set to about 10% of the visual field of the pupil conjugate plane P2. By setting this area, even if the shape of the aperture image is deformed by the meniscus of the culture medium in the culture vessel 12, the aperture image can be stored in the phase film 37A.

  Of the illumination light incident on the phase plate 37, the observation light incident on the phase film 37A is weakened in light amount and shifted in phase by a predetermined amount (for example, a quarter wavelength). Note that the phase of the observation light may be advanced or delayed by the phase film 37A. On the other hand, the observation light incident on the portion of the phase plate 37 other than the phase film 37A is transmitted through the phase plate 37 as it is.

  Accordingly, if the aperture image is adjusted so as to be within the phase film 37A, almost all of the observation light incident on the phase plate 37 is incident on the phase film 37A and is attenuated, and the phase shifts. To do. On the other hand, most of the diffracted light is incident on a portion of the phase plate 37 other than the phase film 37 </ b> A and passes through the phase plate 37 as it is. As a result, the interference effect between direct light and diffracted light can be enhanced, and a high-contrast and high-quality sample observation image can be obtained.

  The alignment of the aperture image and the phase film 37 </ b> A is performed using the deviation prism unit 31. Specifically, as described above, the deviation prism unit 31 deflects the observation light, and the position of the aperture image on the pupil conjugate plane P2 (phase plate 37) is moved in the direction perpendicular to the optical axis. Matching is done.

  For example, when the deviation prism 31a and the deviation prism 31b are rotated around the optical axis at different angular velocities, the aperture image I moves spirally on the pupil conjugate plane P2, as shown in FIG. Although not shown, by rotating the deviation prism 31a and the deviation prism 31b in the same direction around the optical axis at the same angular velocity, the aperture image is on the circumference around the optical axis on the pupil conjugate plane P2. Move in the circumferential direction. Further, although not shown in the drawings, by rotating the deviation prism 31a and the deviation prism 31b in the opposite directions around the optical axis at the same angular velocity, the aperture image is radially formed on the pupil conjugate plane P2 with the optical axis as the center. Move on a straight line.

  Then, by appropriately adjusting the positions of the deviation prism 31a and the deviation prism 31b in the rotation direction, it is possible to perform alignment so that the aperture image is within the phase film 37A.

  The adjustment of the position of the deviation prism 31a and the deviation prism 31b in the rotation direction can be performed manually or electrically. However, in the case where the aperture image and the phase film 37A are automatically aligned, it is necessary to adjust at least electrically.

  Hereinafter, the fact that the aperture image fits in the phase film 37A is expressed as the position of the aperture image and the phase film 37A being matched, and in particular, the aperture image and the center of the phase film 37A are in agreement. And the position of the phase film 37 </ b> A match.

  The observation light transmitted through the phase plate 37 is imaged by an imaging lens 38 on an image sensor 39A (imaging surface thereof) in the camera 39 disposed on an image plane conjugate with the primary image plane P1. Then, in the image sensor 39A, direct observation light and diffracted light interfere to form an observation image of the sample (hereinafter referred to as a sample image).

  The camera 39 captures a sample image or the like formed on the image sensor 39A. In addition, the camera 39 supplies an image obtained as a result of shooting to another device (not shown), displays it, supplies it to the controller 72 (FIG. 4), or stores it in a storage device (not shown). To do.

  The Bertrand lens 40 is inserted at a predetermined position on the optical path between the phase plate 37 and the imaging lens 38 as necessary. When the Bertrand lens 40 is inserted, an image of the pupil conjugate plane P2, that is, an image of the phase plate 37 is formed on the image sensor 39A (imaging plane thereof). Since an aperture image is formed on the phase plate 37, as a result, an image of the phase plate 37 including an aperture image and an image of a phase film (hereinafter referred to as a phase film image) can be taken by the camera 39. it can.

  The mirror 41 is inserted at a predetermined position on the optical path between the phase plate 37 and the imaging lens 38 as necessary, and in the inserted state, the observation light after passing through the phase plate 37 is detected by a photodetector. 42 is incident.

  The photodetector 42 is configured by a sensor capable of detecting the amount of incident light, such as a PMT (photomultiplier tube). The photodetector 42 detects the amount of observation light that is reflected by the mirror 41 and enters, and supplies a signal indicating the result (hereinafter referred to as a light detection signal) to the controller 72 (FIG. 4).

  Although not shown in FIG. 2, the eyepiece 43 condenses the observation light transmitted through the phase plate 37 and forms an image of the observation light. Thereby, the user can observe the sample through the eyepiece lens 43.

  FIG. 4 shows a configuration example of functions of a part that controls and drives the phase-contrast microscope 11.

  The input unit 71 includes, for example, operation members such as buttons, switches, keys, input devices, and the like. The user can perform various operations of the phase-contrast microscope 11 or input data via the input unit 71. For example, the user can adjust the position and inclination of the stage 27 and adjust the position of the deviation prism unit 31 in the rotation direction via the input unit 71.

  The control unit 72 controls each unit of the phase-contrast microscope 11 based on an image supplied from the camera 39, a light detection signal supplied from the photodetector 42, a command or data supplied from the input unit 71, and the like. . For example, the control unit 72 controls the drive unit 73 to adjust the position and inclination of the stage 27 and adjust the position of the deviation prism unit 31 in the rotation direction. For example, the control unit 72 controls the photographing process of the camera 39.

  The drive unit 73 includes, for example, a mechanism including an actuator, a motor, and the like for driving movable parts of the phase-contrast microscope 11 such as the stage 27 and the deviation prism unit 31.

  FIG. 5 shows a configuration example of the function of the alignment control unit 101a which is a part of the function of the control unit 72.

  The alignment control unit 101a controls alignment between the aperture image and the phase film 37A of the phase plate 37. The alignment control unit 101a is configured to include a luminance detection unit 111 and an adjustment unit 112.

  The luminance detection unit 111 detects the luminance of the image supplied from the camera 39 and supplies the detection result to the adjustment unit 112.

  The adjustment unit 112 controls the driving unit 73 based on the detection result of the luminance of the image, and adjusts the position of the deviation prism unit 31 in the rotation direction so that the aperture image and the phase film 37A are aligned.

[Processing of phase-contrast microscope 11]
Next, processing of the phase contrast microscope 11 will be described.

(Observation process)
First, the observation process executed by the phase-contrast microscope 11 will be described with reference to the flowchart of FIG.

  In step S1, the phase contrast microscope 11 moves the culture vessel 12 to the observation position. Specifically, the control unit 72 of the phase-contrast microscope 11 controls the drive unit 73 so that the sample to be observed in the culture vessel 12 is arranged on the optical axis of the objective lens 28. Is moved in the horizontal direction (XY direction).

  The movement of the stage 27 may be performed manually by a user operation, or may be automatically performed by the phase-contrast microscope 11 based on a preset setting value.

  In step S2, the phase-contrast microscope 11 performs focus adjustment. The focus adjustment method is not particularly limited. For example, the control unit 72 of the phase-contrast microscope 11 uses the autofocus function to focus on the sample to be observed. Adjust the position in the vertical direction (Z direction).

  In step S3, the phase-contrast microscope 11 executes aperture image position adjustment processing. Here, the details of the aperture image position adjustment process will be described with reference to the flowcharts of FIGS.

  In this aperture image position adjustment process, either an image obtained by photographing a sample image (hereinafter referred to as a sample image) or an image obtained by photographing an image of the phase plate 37 (hereinafter referred to as a phase plate image). To be executed.

(First Embodiment of Aperture Image Position Adjustment Process)
First, the case where the aperture image position adjustment process is executed using a sample image will be described with reference to the flowchart of FIG.

  In step S21, the phase-contrast microscope 11 acquires a sample image. Specifically, this processing is performed in a state where neither the belt run lens 40 nor the mirror 41 is inserted on the optical path between the phase plate 37 and the imaging lens 38, and the phase sensor 37 A has a phase on the image sensor 39 A of the camera 39. A sample image is formed by the observation light after passing through the plate 37. The camera 39 captures a sample image under the control of the control unit 72 and supplies the obtained sample image to the luminance detection unit 111.

  In step S22, the luminance detector 111 detects the luminance of the entire image (sample image).

  In step S23, the luminance detection unit 111 determines whether the luminance is equal to or less than a threshold value. Specifically, as the area where the aperture image and the phase film 37A overlap on the phase plate 37 increases, the direct light included in the observation light is not attenuated, and thus the luminance of the sample image decreases. On the other hand, the smaller the area where the aperture image and the phase film 37A overlap, the lower the direct light contained in the observation light, so the luminance of the sample image increases. Therefore, it is possible to grasp the degree of overlap between the aperture image and the phase film 37A based on the luminance of the sample image. Further, by appropriately setting the threshold value, it is possible to determine whether the aperture image is within the phase film 37A or is not within the phase film 37A based on the luminance of the sample image. become.

  When the luminance detection unit 111 determines that the luminance of the entire sample image exceeds the threshold value, that is, when it is assumed that the aperture image is not within the phase film 37A, the process proceeds to step S24.

  In step S24, the alignment control unit 101a adjusts the position of the aperture image. Specifically, the luminance detection unit 111 supplies the detection result of the luminance of the sample image to the adjustment unit 112. The adjustment unit 112 adjusts the position of the deviation prism 31a and the deviation prism 31b in the rotation direction via the drive unit 73 in a direction to reduce the luminance of the entire sample image. As a result, the aperture image moves on the phase plate 37 in a direction that fits in the phase film 37A.

  Thereafter, the process returns to step S21, and the processes of steps S21 to S24 are repeatedly executed until it is determined in step S23 that the luminance of the sample image is equal to or less than the threshold value. As a result, the rotational position of the deviation prism 31a and the deviation prism 31b is adjusted so that the luminance of the sample image is lowered.

  It is assumed that the direction in which the luminance of the entire sample image decreases is unknown at the start of the aperture image position adjustment process. In this case, for example, first, the direction in which the luminance of the sample image decreases may be specified while changing the rotation direction and the rotation amount of the deviation prism 31a and the deviation prism 31b.

  Further, for example, the sample image is divided into a predetermined number of regions, the luminance of each region is detected, and the direction in which the aperture image is shifted from the phase film 37A is specified based on the position of the brightest region. It may be. As a result, it is possible to estimate the rotation direction and the amount of rotation of the deviation prism 31a and the deviation prism 31b necessary for accommodating the aperture image in the phase film 37A, and it is possible to align the aperture image more quickly.

  On the other hand, if it is determined in step S23 that the luminance of the sample image is equal to or less than the threshold value, the aperture image position adjustment process ends.

  In this way, the displacement of the diaphragm image due to the meniscus of the medium in the culture vessel 12 is automatically corrected.

(Second Embodiment of Aperture Image Position Adjustment Processing)
Next, with reference to the flowchart of FIG. 8, the case where a diaphragm image position adjustment process is performed using a phase plate image is demonstrated.

  In step S <b> 41, the phase contrast microscope 11 acquires an image of the phase plate 37. Specifically, first, the drive unit 73 drives a turret (not shown), for example, and inserts the belt run lens 40 at a predetermined position on the optical path between the phase plate 37 and the imaging lens 38. As a result, an image of the phase plate 37 including the aperture image and the phase film image is formed on the image sensor 39A of the camera 39. In this state, the camera 39 takes an image of the phase plate 37 under the control of the control unit 72, and supplies the obtained phase plate image to the luminance detection unit 111.

  The belt run lens 40 may be inserted manually by a user operation, or may be automatically performed by the phase-contrast microscope 11.

  In the processing of Steps S42 to S44, the same processing as Steps S22 to S24 of FIG. 6 is performed. In Step S43, the processing of Steps S41 to S44 is performed until it is determined that the luminance of the phase plate image is equal to or less than the threshold value. Repeatedly executed.

  On the other hand, when it is determined in step S43 that the luminance of the phase plate image is equal to or lower than the threshold value, the aperture image position adjustment process ends.

  The belt run lens 40 is removed from the optical path between the phase plate 37 and the imaging lens 38 after the stop image position adjustment process is completed. The removal of the belt run lens 40 may be performed manually by a user operation, or may be automatically performed by the phase-contrast microscope 11.

  In this manner, similarly to the case of using the sample image, the displacement of the aperture image due to the meniscus of the medium in the culture vessel 12 is automatically corrected. In addition, since the phase plate image has a larger amount of change in luminance accompanying the change in the position of the aperture image than the sample image, the position of the aperture image can be more accurately aligned with the phase film 37A.

  Note that the position of the aperture image may be adjusted using the luminance of a part of the image, such as the luminance of a predetermined range in the center of the image, instead of the luminance of the entire sample image or phase plate image.

  Further, for example, the position of the deviation prism 31a and the deviation prism 31b in the rotation direction may be adjusted so that the luminance of the sample image or the phase plate image is minimized without using the threshold value.

  Returning to FIG. 6, in step S <b> 4, the phase-contrast microscope 11 captures a sample. For example, the camera 39 captures a sample image under the control of the control unit 72, and supplies the obtained sample image to another device (for example, a personal computer, a display, etc.) (not shown) for display. A user observes a sample by an image displayed on another device. Alternatively, the user observes the sample image by the observation light transmitted through the phase plate 37 through the eyepiece lens 43.

  Thereafter, the observation process ends.

  As described above, alignment of the aperture image and the phase film 37A can be performed simply, quickly and appropriately. Further, a high-contrast and high-quality sample image can be obtained without being affected by the meniscus of the medium in the culture vessel 12.

  Furthermore, the aperture image and the phase film 37A can be aligned by a simple and inexpensive configuration and method that only rotates the deviation prism unit 31 without providing a stage for shifting the phase stop 24.

  In addition, since the area irradiated with illumination light is limited to a narrow area at the center of the optical axis and the effective numerical aperture (NA) of the objective lens 28 is reduced, it can be used in a wide range from low magnification to high magnification. can do. Moreover, when observing the sample of the edge part of the culture container 12, interference with illumination light and the wall surface of the culture container 12 becomes difficult to generate | occur | produce.

  Further, in the culture vessel 12 having a large ratio of the wall height to the hole diameter, such as a 96-well plate, for example, when the aperture image is aligned by shifting the phase aperture 24, the phase aperture 24 has a slight amount. The illumination light interferes with the wall surface of the culture vessel 12 due to the movement, and the observable range becomes narrow. On the other hand, in this method, since the position of the aperture image is adjusted after the objective lens 28, such a phenomenon does not occur.

  In the above description, the example in which the position of the aperture image is aligned using the luminance of the image in step S3 of FIG. 6 is shown, but it is possible to use other than the luminance of the image.

(Third embodiment of aperture image position adjustment processing)
First, a third embodiment of the aperture image position adjustment process in step S3 of FIG. 6 will be described with reference to FIGS.

  FIG. 9 shows an example of the functional configuration of the alignment control unit 101b which is the second embodiment of the alignment control unit.

  The alignment control unit 101b is configured to include a position detection unit 131 and an adjustment unit 132.

  The position detection unit 131 detects the positions of the aperture image and the phase film image in the phase plate image supplied from the camera 39 and supplies the detection result to the adjustment unit 132.

  The adjusting unit 132 controls the driving unit 73 based on the detection result of the position of the aperture image and the phase film image, and the position of the deviation prism unit 31 in the rotation direction so that the position of the aperture image and the phase film 37A matches. Adjust.

  Next, a third embodiment of the aperture image position adjustment process in step S3 of FIG. 6 will be described with reference to the flowchart of FIG.

  In step S <b> 61, a phase plate image is acquired as in the process of step S <b> 41 in FIG. 8, and the acquired phase plate image is supplied from the camera 39 to the position detection unit 131.

  In step S62, the position detector 131 detects the positions of the aperture image and the phase film image from the phase plate image.

  FIG. 11 is a diagram schematically illustrating an example of a phase plate image. The aperture image Ia and the phase film image Ib are both substantially circular and their sizes are known. Further, the aperture image Ia is smaller than the phase film image Ib and has higher luminance. Therefore, even if the diaphragm image Ia is distorted due to the meniscus of the culture medium, it is easy to detect two images individually from the phase plate image and detect the position of each image by a method such as shape recognition. is there.

  Therefore, the position detector 131 individually detects the aperture image and the phase film image from the phase plate image by a predetermined method, and detects the position (for example, the center position) of each image. Based on this detection result, it is possible to grasp the degree of overlap between the aperture image and the phase film 37A.

  In step S63, the position detection unit 131 determines whether the aperture image is within the phase film image based on the result of step S62. If it is determined that the aperture image does not fit in the phase film image, the process proceeds to step S64.

  In step S64, the alignment control unit 101b adjusts the position of the aperture image. Specifically, the position detection unit 131 supplies the detection results of the positions of the aperture image and the phase film image to the adjustment unit 132. The adjustment unit 132 adjusts the rotational position of the deviation prism 31a and the deviation prism 31b via the drive unit 73 so that the diaphragm image moves in a direction in which the center of the diaphragm image coincides with the center of the phase film image.

  Thereafter, the process returns to step S61, and the processes of steps S61 to S64 are repeatedly executed until it is determined in step S63 that the aperture image is within the phase film image.

  On the other hand, if it is determined in step S63 that the aperture image is within the phase film image, the aperture image position adjustment process ends.

  Similar to the aperture image position adjustment process of FIG. 8, the belt run lens 40 is removed from the optical path between the phase plate 37 and the imaging lens 38 after the aperture image position adjustment process is completed.

  In this way, the displacement of the diaphragm image due to the meniscus of the medium in the culture vessel 12 is automatically corrected. In addition, since the relative position of the aperture image and the phase film 37A is actually detected and the aperture image is aligned, the alignment is performed more accurately than in the case of adjusting based on the luminance of the image described above. be able to.

(Fourth Embodiment of Aperture Image Position Adjustment Process)
Next, a fourth embodiment of the aperture image position adjustment process in step S3 of FIG. 6 will be described with reference to FIGS.

  FIG. 12 shows an example of the functional configuration of the alignment control unit 101c, which is the third embodiment of the alignment control unit.

  The alignment control unit 101 c is configured to include a sample detection unit 151, a contrast detection unit 152, and an adjustment unit 153.

  The sample detection unit 151 detects a sample and its details in the sample image. The sample detection unit 151 supplies the detection result to the contrast detection unit 152 together with the sample image.

  The contrast detection unit 152 detects the contrast intensity of the sample in the sample image, and supplies the detection result to the adjustment unit 153.

  The adjustment unit 153 controls the drive unit 73 based on the detection result of the contrast intensity of the sample in the sample image, so that the position of the aperture image and the phase film 37 </ b> A matches the rotational direction of the deviation prism unit 31. Adjust the position.

  Next, a fourth embodiment of the aperture image position adjustment process in step S3 in FIG. 6 will be described with reference to the flowchart in FIG.

  In step S <b> 81, a sample image is acquired in the same manner as in step S <b> 21 of FIG. 7, and the acquired sample image is supplied from the camera 39 to the sample detection unit 151.

  In step S82, the sample detection unit 151 detects a sample in the image. For example, when the sample to be observed is a cell, the sample detection unit 151 detects the nucleus of the cell, the cell membrane, and the like in the sample image using known cell morphology information. The sample detection unit 151 supplies the detection result to the contrast detection unit 152 together with the sample image.

  In step S <b> 83, the contrast detection unit 152 detects the contrast intensity of the sample in the sample image and supplies the detection result to the adjustment unit 153.

  In step S84, the contrast detection unit 152 determines whether or not the contrast intensity is greater than or equal to a threshold value. Specifically, as the area where the aperture image and the phase film 37A overlap on the phase plate 37 increases, the interference between the direct light and the indirect light in the sample image increases, and the contrast of the sample image in the sample image increases. Conversely, as the area where the aperture image and the phase film 37A overlap on the phase plate 37 becomes smaller, the interference between the direct light and the indirect light in the sample image becomes smaller and the contrast of the sample image in the sample image becomes weaker. Therefore, the degree of overlap between the aperture image and the phase film 37A can be grasped based on the contrast of the sample image. Further, by appropriately setting the threshold value, it is determined whether the aperture image is within the phase film 37A or is not within the phase film 37A based on the contrast intensity of the sample image. Is possible.

  When the contrast detection unit 152 determines that the contrast intensity is less than the threshold value, that is, when it is assumed that the aperture image is not within the phase film 37A on the phase plate 37, the process proceeds to step S85. move on.

  In step S85, the alignment control unit 101d adjusts the position of the aperture image. Specifically, the contrast detection unit 152 supplies the detection result of the contrast of the sample image to the adjustment unit 153. The adjustment unit 153 adjusts the positions of the deviation prism 31a and the deviation prism 31b in the rotation direction via the drive unit 73 in a direction in which the contrast of the sample image is increased.

  Thereafter, the process returns to step S81, and the processes of steps S81 to S85 are repeatedly executed until it is determined in step S84 that the contrast intensity of the sample image is equal to or greater than the threshold value. As a result, the positions of the deviation prism 31a and the deviation prism 31b in the rotational direction are adjusted so that the contrast of the sample image is increased.

  It is assumed that the direction in which the contrast of the sample image increases is unknown at the start of the aperture image position adjustment process. In this case, for example, the direction in which the contrast of the sample image is increased may be specified while changing the rotation direction and the rotation amount of the deviation prism 31a and the deviation prism 31b.

  On the other hand, if it is determined in step S84 that the contrast of the sample image is equal to or greater than the threshold value, the aperture image position adjustment process ends.

  In this way, the displacement of the diaphragm image due to the meniscus of the medium in the culture vessel 12 is automatically corrected.

  Note that, for example, the position of the deviation prism 31a and the deviation prism 31b in the rotation direction may be adjusted so that the contrast of the sample image is maximized without using the threshold value.

(Fifth embodiment of aperture image position adjustment processing)
Next, a fifth embodiment of the aperture image position adjustment process in step S3 of FIG. 6 will be described with reference to FIGS.

  FIG. 14 illustrates a configuration example of functions of an alignment control unit 101d which is the fourth embodiment of the alignment control unit.

  The alignment control unit 101d is configured to include an adjustment unit 171.

  The adjustment unit 171 controls the drive unit 73 based on the light detection signal supplied from the light detector 42, and the position of the deviation prism unit 31 in the rotation direction so that the position of the aperture image and the phase film 37A is matched. Adjust.

  Next, a fifth embodiment of the aperture image position adjustment process in step S3 in FIG. 6 will be described with reference to the flowchart in FIG.

  In step S101, the phase-contrast microscope 11 detects the amount of observation light. Specifically, first, the drive unit 73 drives a turret (not shown), for example, and inserts the mirror 41 at a predetermined position on the optical path between the phase plate 37 and the imaging lens 38. Thereby, the observation light after passing through the phase plate 37 enters the photodetector 42. The photodetector 42 detects the amount of incident observation light and supplies a light detection signal indicating the detection result to the adjustment unit 171.

  In step S102, the adjustment unit 171 determines whether or not the amount of observation light is equal to or less than a threshold value. Specifically, as the area where the aperture image and the phase film 37A overlap on the phase plate 37 increases, the amount of direct light included in the observation light is reduced, so the light amount of the observation light decreases. Conversely, as the area where the aperture image and the phase film 37A overlap is smaller, the amount of direct light included in the observation light is reduced, so that the amount of observation light increases. Therefore, it is possible to grasp the degree of overlap between the aperture image and the phase film 37A based on the amount of observation light. In addition, by appropriately setting the threshold value, it is possible to determine whether the aperture image is within the phase film 37A or is not within the phase film 37A based on the amount of observation light. become.

  If the adjustment unit 171 determines that the amount of observation light exceeds the threshold, that is, if it is assumed that the aperture image is not within the phase film 37A, the process proceeds to step S103.

  In step S103, the adjustment unit 171 adjusts the position of the aperture image. Specifically, the adjustment unit 171 adjusts the positions of the deviation prism 31a and the deviation prism 31b in the rotation direction via the drive unit 73 in the direction in which the amount of observation light decreases. As a result, the aperture image moves on the phase plate 37 in a direction that fits in the phase film 37A.

  Thereafter, the process returns to step S101, and the processes of steps S101 to S103 are repeatedly executed until it is determined in step S102 that the amount of observation light is equal to or less than the threshold value. Thereby, the positions of the deviation prism 31a and the deviation prism 31b in the rotation direction are adjusted so that the amount of observation light is reduced.

  It is assumed that the direction in which the amount of observation light decreases is unknown at the start of the aperture image position adjustment process. In this case, for example, the direction in which the amount of observation light decreases may be specified while changing the rotation direction and the rotation amount of the deviation prism 31a and the deviation prism 31b.

  Further, for example, the photodetector 42 is configured by a plurality of sensors, and the light amount of the observation light is divided and detected for each predetermined region, and the aperture image is detected from the phase film 37A based on the position of the region where the light amount is the largest. You may make it pinpoint the direction which has shifted | deviated. As a result, it is possible to estimate the rotation direction and the amount of rotation of the deviation prism 31a and the deviation prism 31b necessary for accommodating the aperture image in the phase film 37A, and it is possible to align the aperture image more quickly.

  On the other hand, if it is determined in step S102 that the amount of observation light is equal to or less than the threshold value, the aperture image position adjustment process ends.

  In this way, the displacement of the diaphragm image due to the meniscus of the medium in the culture vessel 12 is automatically corrected.

  For example, the rotational position of the deviation prism 31a and the deviation prism 31b may be adjusted so that the amount of observation light is minimized without using the threshold value.

(Time-lapse observation process)
Next, the time lapse observation process executed by the phase contrast microscope 11 will be described with reference to the flowchart of FIG. Here, the time-lapse observation refers to observing a change in the time series of a sample by, for example, photographing the sample at a predetermined time based on a preset photographing schedule. Note that, in the shooting schedule, for example, the position of the stage 27 corresponding to each observation position, the shooting time, and the like are set.

  In the following description, a case where shooting is performed at a plurality of observation positions at one shooting timing will be described as an example. This is, for example, a case where a sample in each well of the well plate is photographed, a case where the sample is divided into a plurality of regions, and tiling photographing is performed.

  In step S201, the phase-contrast microscope 11 moves the culture vessel 12 to the next observation position. Specifically, the control unit 72 of the phase contrast microscope 11 moves the culture vessel 12 to the next observation position by moving the stage 27 in the horizontal direction to the next position set in the imaging schedule.

  In step S202, focus adjustment is performed in the same manner as in step S2 of FIG.

  In step S203, the aperture image position adjustment process is executed as in the process of step S3 of FIG. Note that any of the aperture image position adjustment processes described above with reference to FIGS. 7, 8, 10, 13, or 15 can be employed. However, in consideration of the processing time, it is desirable to adopt the processing of FIG. 7 or FIG. 13 that does not require insertion / removal of the belt run lens 40 and the mirror 41.

  Hereinafter, an example in which the alignment control unit 101a in FIG. 5 and the aperture image position adjustment process in FIG. 7 are employed will be described.

  In step S204, the adjustment unit 112 stores the deviation correction amount. For example, the adjustment unit 112 calculates the difference between the position in the rotation direction of the deviation prism 31a and the deviation prism 31b and the predetermined reference position after aligning the aperture image in the process of step S203, at the current observation position. The correction amount is stored in a storage device (not shown). That is, in this case, the deviation correction amount represents the rotation direction and the rotation amount from the reference position of the deviation prism 31a and the deviation prism 31b necessary for matching the aperture image with the phase film 37A. For example, when the deviation prism 31a and the deviation prism 31b are driven by a stepping motor, the deviation correction amount is expressed by a step position from the origin of the stepping motor.

  In step S205, the phase-contrast microscope 11 performs imaging. Specifically, the camera 39 captures a sample image under the control of the control unit 72, and transmits the obtained sample image to another device (not shown) or stores it in a storage device.

  In step S206, the control unit 72 determines whether or not photographing of all observation positions has been completed. If it is determined that imaging at all observation positions has not been completed yet, the process returns to step S201.

  Thereafter, the processes in steps S201 to S206 are repeatedly executed until it is determined in step S201 that photographing at all observation positions has been completed. As a result, after the aperture images are aligned at all observation positions, the sample images are taken. In each observation position, a deviation correction amount necessary for setting the aperture image to an appropriate position is detected and stored.

  On the other hand, if it is determined in step S206 that shooting of all observation positions has been completed, the process proceeds to step S207.

  In step S207, the control unit 72 determines whether or not the observation period has ended based on the shooting schedule. If it is determined that the observation period has not yet ended, the process proceeds to step S208.

  In step S208, the control unit 72 determines whether or not the shooting time has come based on the shooting schedule. The determination process of step S208 is repeatedly executed at predetermined intervals until it is determined that the shooting time has come, and when it is determined that the shooting time has come, the process proceeds to step S209.

  In step S209, the control unit 72 determines whether or not the state of the medium level has changed. If it is determined that the state of the liquid level of the medium has not changed, the process proceeds to step S210.

  In step S210, the culture container 12 moves to the next observation position, as in the process of step S201.

  In step S211, focus adjustment is performed in the same manner as in step S2 of FIG.

  In step S212, the adjustment unit 112 aligns the aperture image based on the stored deviation correction amount. Specifically, the adjustment unit 112 reads the deviation correction amount at the current observation position from a storage device (not shown). Then, the adjustment unit 112 controls the driving unit 73 to rotate the deviation prism 31a and the deviation prism 31b from the predetermined reference position by a direction and an angle represented by the read deviation correction amount.

  Thereby, it is possible to appropriately and quickly align the aperture image without performing the aperture image position adjustment process as in the process of step S203.

  In step S213, imaging is performed in the same manner as in step S205.

  In step S214, it is determined whether imaging at all observation positions has been completed, as in the process of step S206. If it is determined that shooting of all observation positions has not been completed yet, the process returns to step S210.

  Thereafter, the processes in steps S210 to S214 are repeatedly executed until it is determined in step S214 that photographing at all observation positions has been completed. As a result, the aperture images are aligned using the stored deviation correction amounts at all the observation positions, and then sample images are taken. Therefore, a high-contrast and high-quality sample image can be quickly taken at each observation position.

  On the other hand, if it is determined in step S214 that imaging at all observation positions has been completed, the process returns to step S207, and the processes after step S207 are executed.

  Moreover, when it determines with the state of the liquid level of a culture medium having changed in step S209, a process returns to step S201. This is the case, for example, when the medium is exchanged between the end of the previous shooting and the current shooting.

  Thereafter, the processes in steps S201 to S206 are repeatedly executed until it is determined in step S206 that photographing at all observation positions has been completed. That is, when the state of the liquid level of the medium changes, photographing is performed again while adjusting the position of the aperture image at each observation position, and the deviation correction amount at each observation position is stored.

  On the other hand, if it is determined in step S207 that the observation period has ended, the time lapse observation process ends.

  Similarly, when performing time-lapse observation as described above, a high-contrast and high-quality sample image can be obtained without being affected by the meniscus of the medium in the culture vessel 12.

<2. Modification>
Hereinafter, modifications of the above-described embodiment of the present technology will be described.

[Modified example of aperture image alignment method]
In the above description, the example in which the position of the aperture image is aligned using the deviation prism unit 31 has been described, but other methods may be employed.

(Method using parallel flat glass (harving))
For example, as shown in FIG. 17, a parallel plate glass 201 can be used instead of the deviation prism unit 31.

  The parallel flat glass 201 is disposed, for example, in the vicinity of the primary image plane P1 (including on the primary image plane P1) so that the optical axis coincides with the objective lens 28 and the like. The parallel flat glass 201 is installed so that the inclination with respect to observation light can be adjusted with the drive part 73, for example.

  Alternatively, the parallel flat glass 201 can be arranged, for example, in the vicinity of the incident side of the phase plate 37 so that the optical axis coincides with the phase plate 37. More precisely, the parallel plate glass 201 can be arranged in the vicinity of the incident side of the lens 36 arranged on the incident side of the phase plate 37 so that the optical axis of the lens 36 and the phase plate 37 coincides. It is.

  Even when the parallel flat glass 201 is installed at any position, the parallel flat glass 201 is tilted with respect to the observation light so that the parallel flat glass 201 is separated from the parallel flat glass 201 with respect to the central axis of the observation light incident on the parallel flat glass 201. The central axis of the emitted observation light can be shifted in parallel. Therefore, by adjusting the inclination of the parallel flat glass 201, the incident position of the observation light on the phase plate 37 can be adjusted, and as a result, the position of the aperture image on the phase plate 37 can be adjusted.

  Therefore, the aperture image and the phase film 37A can be aligned by a simple and inexpensive configuration and method in which the parallel plate glass 201 is simply tilted.

  It is also possible to arrange the parallel flat glass 201 both near the primary image plane P1 and near the incident side of the phase plate 37.

  Further, the adjustment of the inclination of the parallel flat glass 201 can be performed either manually or electrically. However, when the aperture image and the phase film 37 </ b> A are automatically aligned as described above, it is necessary to adjust at least electrically.

(Method of tilting the culture vessel 12)
Further, for example, the diaphragm image can be aligned by tilting the culture vessel 12 directly or indirectly with respect to the illumination light and causing the bottom surface of the culture vessel 12 to function as a prism. Here, tilting the culture vessel 12 directly means, for example, tilting the culture vessel 12 by directly moving it. On the other hand, tilting the culture container 12 indirectly means tilting the culture container 12 by tilting the stage 27 on which the culture container 12 is placed, for example.

  FIG. 18 shows a configuration example of the optical system of the phase-contrast microscope 11 when the method of tilting the culture vessel 12 is adopted. The optical system of FIG. 18 has a configuration in which the deviation prism unit 31 is deleted from the optical system of FIG.

  Here, with reference to FIG. 19, a method for aligning the aperture image in the phase-contrast microscope 11 having the optical system of FIG. 18 will be described.

  FIG. 19A schematically shows a state before the stage 27 is tilted with respect to the observation light, and FIG. 19B schematically shows a state after the stage 27 is tilted with respect to the observation light.

  As shown in FIG. 19A, by tilting the stage 27 with respect to the illumination light via the drive unit 73, the bottom surface of the culture vessel 12 is inclined with respect to the illumination light, and the illumination light is refracted at the bottom surface of the culture vessel 12. . Therefore, for example, as shown in FIG. 19B, by tilting the bottom surface of the culture vessel 12 diagonally to the left (right diagonally downward), the traveling direction of the observation light is shifted to the right as compared to before tilting. On the other hand, for example, in contrast to FIG. 19B, by tilting the bottom surface of the culture vessel 12 to the upper right (lower left), the traveling direction of the observation light is shifted to the left as compared to before tilting.

  Therefore, by changing the direction and angle of tilting the stage 27, the incident position of the observation light on the phase plate 37 can be adjusted, and as a result, the position of the aperture image on the phase plate 37 can be adjusted. Then, by appropriately adjusting the direction and angle of tilting the stage 27, the position of the aperture image can be matched with the phase film 37A.

  Therefore, the aperture image and the phase film 37A can be aligned by a simple and inexpensive configuration and method in which the stage 27 is tilted.

  It should be noted that the stage 27 can be tilted while the focus position of the objective lens 28 is adjusted to the observation position by tilting the stage 27 around the position of the sample to be observed in the culture vessel 12 (that is, the observation position). it can. This eliminates the need for focus adjustment after the stage 27 is tilted.

  Here, an example of a method of tilting the stage 27 around the vicinity of the observation position will be described with reference to FIG.

  FIG. 20A shows a state before the stage 27 is tilted. The stage 27 is configured by an inclined stage supported at three points P1 to P3, and can be tilted with any one of the three points as a fulcrum. A point Q indicates an observation position. It is assumed that the positional relationship between the points P1 to P3 and the point Q is known.

  FIG. 20B shows a state where the stage 27 is tilted in a desired direction by a desired angle with the point P1 as a fulcrum. By tilting the stage 27, the point Q moves from the initial position shown in FIG. 20A.

  Here, since the positional relationship between the points P1 to P3 and the point Q is known, the shift amounts of the point Q in the X direction, the Y direction, and the Z direction can be calculated based on the tilt amount of the stage 27, respectively. Therefore, the point Q can be returned to the original position (position in FIG. 20A) by translating the entire stage 27 in the X direction, Y direction, and Z direction in the direction opposite to the shift direction of the point Q.

  As a result, as shown in FIG. 20C, as a result, the stage 27 can be tilted to a desired direction and angle around the point Q. As a result, the state in which the focus position of the objective lens 28 matches the point Q is maintained before and after the stage 27 is tilted, and the focus adjustment after adjusting the tilt of the stage 27 becomes unnecessary.

  In the above description, the example in which the culture container 12 is tilted indirectly by tilting the stage 27 has been described, but the culture container 12 may be tilted directly as described above. For example, it is possible to adjust the inclination of the culture vessel 12 by supporting the culture vessel 12 with a plurality of pins provided on the stage 27 and individually moving each pin in the vertical direction.

  The inclination of the stage 27 or the culture vessel 12 can be adjusted either manually or electrically. However, when the aperture image and the phase film 37 </ b> A are automatically aligned as described above, it is necessary to adjust at least electrically.

  Further, when the diaphragm image is aligned by inclining the culture vessel 12, the phase plate 37 can be disposed on the exit pupil plane of the objective lens.

(Method of combining the tilt of the culture vessel 12 and the shift of the phase stop 24)
For example, when the sample at the end of the culture vessel 12 is to be observed, if the inclination of the culture vessel 12 (stage 27) increases as shown schematically in FIG. Territory is limited. In order to prevent this, for example, in addition to tilting the stage 27 or the culture vessel 12, it is conceivable to shift the phase stop 24 in a direction perpendicular to the optical axis.

  FIG. 22 schematically shows optical paths of illumination light and observation light from the phase stop 24 to the phase film 37A. In FIG. 22, illustration of optical components other than the condenser lens 25 and the objective lens 28 between the phase stop 24 and the phase film 37 </ b> A and bending of the optical path are omitted.

  FIG. 22A shows the optical paths of illumination light and observation light before alignment. In this figure, the imaging position of the aperture image by the observation light protrudes outside the phase film 37A due to the meniscus of the culture medium.

  Therefore, first, regardless of the occurrence of interference between the illumination light and the wall surface of the culture vessel 12, the phase stop 24 is shifted to align the position of the stop image with the phase film 37A. If interference between the illumination light and the wall surface of the culture vessel 12 does not occur at this point (if a part of the illumination light does not hit the wall surface of the culture vessel 12), the alignment ends here.

  On the other hand, as shown in FIG. 22B, after the phase stop 24 is shifted, the illumination light and the wall surface of the culture vessel 12 interfere with each other, and vignetting of the illumination light occurs, as shown in FIG. 22C. In addition, by tilting the stage 27, interference between the illumination light and the wall surface of the culture vessel 12 is eliminated. At this time, the stage 27 is tilted in the opposite direction to the case where only the stage 27 described above with reference to FIG. 19 is tilted to align the aperture image.

  Then, when the culture vessel 12 is tilted together with the stage 27, the position of the aperture image is shifted. As a result, when the aperture image protrudes from the phase film 37A, the phase aperture 24 is shifted again, and the position of the aperture image may be adjusted so that it is within the phase film 37A.

  This makes it possible to observe the sample at the end of the culture vessel 12 without causing interference between the illumination light and the wall surface of the culture vessel 12.

  The position of the phase stop 24 can be adjusted either manually or electrically. However, when the aperture image and the phase film 37 </ b> A are automatically aligned as described above, it is necessary to adjust at least electrically.

(Method of combining deviation prism unit 31 or parallel flat glass 201 and shift of phase stop 24)
Further, in the optical system of FIG. 2 or FIG. 17, the phase stop 24 may be shifted. That is, it is possible to align the aperture image by combining the deviation prism unit 31 or the parallel flat glass 201 and the shift of the phase stop 24.

  For example, as shown by the dotted line in FIG. 23, first, regardless of whether or not the interference between the illumination light and the wall surface of the culture vessel 12 has occurred, the deviation prism unit 31 or the parallel plate glass 201 is used to stop the aperture. The position of the image is adjusted to the phase film 37A. If there is no interference between the illumination light and the wall surface of the culture vessel 12, the alignment ends here.

  On the other hand, when interference between the illumination light and the wall surface of the culture vessel 12 occurs, the phase stop 24 is shifted in a direction in which the optical axis of the phase stop 24 approaches the center of the culture vessel 12. As a result, as shown by the solid line in FIG. 23, the interference between the illumination light and the wall surface of the culture vessel 12 is eliminated, while the influence of the meniscus is increased, and the phase diaphragm 24 is shifted on the phase plate 37. The aperture image is shifted in the opposite direction. As a result, when the aperture image protrudes from the phase film 37A, the position of the aperture image may be adjusted again using the deviation prism unit 31 or the parallel plate glass 201.

  This makes it possible to observe the sample at the end of the culture vessel 12 without causing interference between the illumination light and the wall surface of the culture vessel 12.

  A fly-eye lens for reducing luminance unevenness of illumination light is provided between the phase stop 24 and the condenser lens 25, and instead of the phase stop 24, the fly-eye lens is shifted in a direction perpendicular to the optical axis. The position of the aperture image may be adjusted.

  It is also possible to align the aperture image and the phase film 37A using only the shift of the phase stop 24.

[Example of providing multiple optical systems]
In the above description, an example in which only one optical system is provided in the phase-contrast microscope 11 has been described, but two or more optical systems may be provided.

(When multiple optical systems are installed)
For example, a plurality of optical systems shown in FIG. 2, FIG. 17, or FIG. 18 can be provided. In this case, each optical system can be made completely independent, or a part of them can be shared. When sharing a part, for example, it is conceivable to share only the stage. Further, for example, it is conceivable that an illumination system from the light source 21 to the condenser lens 25 and the objective lens 28 are individually provided for each optical system, and parts after the objective lens 28 are shared. When the optical system of FIG. 18 is adopted, it is desirable to provide a stage for each optical system in order to adjust the position of the aperture image according to the tilt of the stage.

  For example, when two identical optical systems are provided, one optical system (hereinafter referred to as optical system A) observes a sample in one well of the well plate, while the other optical system (Hereinafter referred to as optical system B), the aperture image can be aligned with the well to be observed next. Then, as soon as the observation by the optical system A is finished, the observation by the optical system B is started, and in parallel, the aperture image can be aligned with the well to be observed next by the optical system A. By repeating this, it is possible to improve the image quality of the sample while rapidly observing the sample in each well of the well plate.

  In addition, when the optical system A and the optical system B are completely independent, it is possible to observe the sample completely in parallel, and to share the role in each optical system. Is also possible. In the latter case, for example, the detection of the displacement correction amount is detected by the optical system A, the aperture image and the phase film 37A are aligned using the displacement correction amount detected by the optical system B, and the sample is observed. Is possible.

(When an optical system for detecting the deviation of the luminous flux due to the meniscus is provided independently)
In addition, for example, as schematically shown in FIG. 24, a measurement optical system for measuring the deviation of the light flux due to the meniscus of the medium in the culture vessel 12 separately from the observation optical system 301 for observing the sample. It is also possible to provide 302 independently.

  Hereinafter, an example in which the well plate 303 is used as the culture container 12 will be described.

  As the observation optical system 301, any of the optical systems described above with reference to FIG. 2, FIG. 17, or FIG. 18 can be used. However, the observation optical system 301 does not detect data indicating the degree of overlap between the aperture image and the phase film 37A as described above.

  Hereinafter, an example in which the optical system of FIG. 2 is adopted as the observation optical system 301 will be described.

  The measurement optical system 302 is an optical system for measuring the deviation of the light flux caused by passing through the medium in each well of the well plate 303 using the measurement light, that is, the deviation of the light flux caused by the meniscus of the culture medium.

  As the measurement light, it is desirable to use light having excellent directivity and a sufficiently small diameter. Further, the same light source 21 as that of the observation optical system 301 may be used as the light source of the measurement light, or another light source may be used. When the same light source 21 as the observation optical system 301 is used, for example, measurement light is generated from illumination light emitted from the light source 21 using a pinhole or a lens, and the measurement light is collected using an optical fiber or the like. It is conceivable that the light is incident on the lens 23.

  Then, the measurement light is emitted from the condenser lens 25 in a direction perpendicular to the stage 27 in a horizontal state without being inclined, and at the observation position of each well of the well plate 303 on the stage 27 with respect to the bottom surface of the well. Incident vertically. Then, when the observation position is substantially in the center of the well, it goes straight as it is like the measurement light L1 in FIG. 24, and when the observation position is close to the wall surface of the well, it is refracted by the meniscus of the medium like the measurement light L2. The

  The measurement light transmitted through the well plate 303 and the stage 27 is directly incident on the image sensor 39A without passing through optical components such as the objective lens 28 of the observation optical system 301. Note that the mirror for changing the optical path of the measurement light and guiding it to the image sensor 39A may be shared with that of the observation optical system 301 (for example, the mirror 30 and the mirror 34 in FIG. 2), or the observation optical system. It may be provided separately from the system 301.

  Further, an optical component, a stage, and an image sensor may be provided individually, and the observation optical system 301 and the measurement optical system 302 may be made independent. Even when the observation optical system 301 and the measurement optical system 302 are made independent, the light source can be shared.

  FIG. 25 shows a configuration example of the function of the alignment control unit 351 which is a part of the function realized by the control unit 72 (FIG. 4) when the optical system of FIG. Yes.

  The alignment control unit 351 is configured to include a deviation detection unit 361 and an adjustment unit 362.

  The deviation detection unit 361 detects an amount of deviation (center axis) of the measurement light based on an image obtained by capturing an image of the measurement light supplied from the camera 39. Specifically, as shown in FIG. 24, when the optical path (center axis) of the measurement light is not refracted by the meniscus of the culture medium in the well like the measurement light L1, the predetermined reference position of the image sensor 39A It is adjusted to be incident on (pixel). Therefore, the deviation detection unit 361 detects a difference between the incident position of the measurement light to the image sensor 39A and the reference position as a deviation amount of the measurement light. The deviation detection unit 361 supplies the detection result to the adjustment unit 362.

  The adjustment unit 362 controls the drive unit 73 based on the amount of deviation of the measurement light, and adjusts the position of the deviation prism unit 31 in the rotation direction so that the aperture image and the phase film 37A are aligned.

  Therefore, in this optical system, in parallel with the observation of the sample in one well of the well plate 303, it is possible to detect the amount of measurement light deviation in the well to be observed. Thereby, while observing a some sample rapidly, the image quality of each sample can be improved.

  Similarly, for example, when a sample in a well of a petri dish or a well plate is divided into a plurality of regions and tiling photography is performed, the measurement light at the observation position to be photographed from now is photographed while photographing one observation position. Quantity detection can be performed. Accordingly, it is possible to quickly perform tiling shooting and improve the image quality of an image obtained by tiling shooting.

  When the observation optical system 301 and the measurement optical system 302 are made independent, for example, while observing one well plate using the observation optical system 301, the measurement optical system 302 is used to measure the measurement light in another well plate. The amount of deviation can be detected. Further, for example, while performing tiling photography using the observation optical system 301, the measurement optical system 302 can be used to detect the amount of measurement light misalignment at each observation position in tiling photography of another culture vessel. .

  Here, the time lapse observation process executed by the phase contrast microscope 11 when the optical system of FIG. 24 is adopted in the phase contrast microscope 11 will be described with reference to the flowchart of FIG.

  In step S301, the culture vessel 12 is moved to the next observation position in the same manner as in step S201 of FIG.

  In step S302, the phase-contrast microscope 11 detects the amount of deviation of the measurement light. Specifically, under the control of the control unit 72, the camera 39 performs imaging in a state where only measurement light is irradiated without irradiating illumination light, and supplies the obtained image to the shift detection unit 361.

  The deviation detection unit 361 detects the position of the image of the measurement light in the image, and detects the difference between the position of the image and a predetermined reference position as the amount of deviation of the measurement light. The reference position is set to a position on the image of the measurement light image when the measurement light is incident on the image sensor 39A without being refracted.

  In step S303, the alignment control unit 351 stores the amount of deviation. Specifically, the deviation detection unit 361 supplies the calculation result of the deviation amount to the adjustment unit 362. The adjustment unit 362 associates the current observation position with the amount of deviation and stores them in a storage device (not shown).

  In step S304, the deviation detection unit 361 determines whether or not a deviation amount has been detected at all observation positions. If it is determined that the amount of deviation has not been detected at all the observation positions, the process returns to step S301.

  Thereafter, the processes in steps S301 to S304 are repeatedly executed until it is determined in step S304 that the shift amount has been detected at all the observation positions. Thereby, before performing time-lapse observation, the deviation | shift amount of measurement light is detected in all the observation positions.

  On the other hand, if it is determined in step S304 that the amount of deviation has been detected at all observation positions, the process proceeds to step S305.

  In step S305, it is determined whether or not the state of the liquid surface of the medium has changed as in the process of step S209 in FIG. If it is determined that the state of the liquid level of the medium has not changed, the process proceeds to step S306.

  In step S306, as in the process of step S208 in FIG. 16, it is determined whether or not the shooting time has come. If it is determined that the shooting time has not come, the process returns to step S305.

  Thereafter, the processes in steps S305 and S306 are repeatedly executed until it is determined in step S305 that the state of the medium level has changed or in step S306 it is determined that the photographing time has come.

  On the other hand, if it is determined in step S306 that the shooting time has come, the process proceeds to step S307.

  In step S307, the culture vessel 12 is moved to the next observation position in the same manner as in step S201 of FIG.

  In step S308, focus adjustment is performed in the same manner as in step S2 of FIG.

  In step S309, the adjustment unit 362 aligns the aperture image based on the stored shift amount. Specifically, the adjustment unit 362 calculates the amount of deviation between the aperture image on the phase plate 37 and the phase film 37A based on the amount of deviation at the current observation position. The amount of deviation between the aperture image and the phase film 37A is represented, for example, by the difference between the center of the aperture image and the center of the phase film 37A.

  Since the characteristics and positions of the parts constituting the observation optical system 301 and the measurement optical system 302 are known, using these data, the difference between the center of the aperture image and the phase film 37A is based on the amount of measurement light deviation. The amount can be easily calculated.

  Further, the adjusting unit 362 calculates the rotation direction and the rotation amount of the deviation prism 31a and the deviation prism 31b necessary for matching the aperture image with the phase film 37A based on the shift amount of the aperture image. Then, the adjusting unit 362 controls the driving unit 73 to rotate the deviation prism 31a and the deviation prism 31b by the rotation direction and the rotation amount calculated from the predetermined reference position, respectively. Thereby, the aperture image and the position of the phase film 37A coincide.

  Note that the amount of deviation between the aperture image and the phase film 37A at each observation position may be calculated in advance and stored in place of the amount of deviation of the optical axis, and used for alignment of the aperture image.

  In step S310, imaging is performed in the same manner as in step S205 in FIG.

  In step S311, it is determined whether or not photographing of all observation positions has been completed, similar to the processing in step S206 of FIG. If it is determined that shooting at all observation positions has not been completed, the process returns to step S307.

  Thereafter, the processes in steps S307 to S311 are repeatedly executed until it is determined in step S311 that photographing at all observation positions has been completed.

  On the other hand, if it is determined in step S311 that photographing at all observation positions has been completed, the process proceeds to step S312.

  In step S312, it is determined whether or not the observation period has ended, similar to the process in step S208 of FIG. If it is determined that the observation period has not yet ended, the process returns to step S305, and the processes after step S305 are executed.

  On the other hand, when it is determined in step S305 that the state of the liquid level of the medium has changed, the process returns to step S301, and the processes after step S301 are executed. That is, when the state of the liquid level of the culture medium is changed, the amount of measurement light deviation at each observation position is re-detected.

  If it is determined in step S312 that the observation period has ended, the time lapse observation process ends.

  As described above, when performing time-lapse observation, a high-contrast and high-quality sample image can be obtained without being affected by the meniscus of the medium.

  In addition, when the state of the liquid level of the medium changes due to medium exchange or the like, since the amount of measurement light deviation is re-detected before the imaging process is performed, the throughput of the imaging process is improved and imaging per time is performed. Time can be shortened.

  Note that the position information and light amount information of the sample may be acquired during the detection processing of the amount of measurement light deviation. Thereby, it is possible to shorten the processing time for positioning and light amount adjustment at the time of shooting, and it is possible to further improve the throughput of the shooting process.

  For example, when performing tiling photography, the difference in the amount of measurement light is considered to be small because the refractive index of the light beam due to the meniscus does not change significantly at observation positions close to each other. Therefore, when performing tiling photography, for example, the amount of deviation is detected by appropriately thinning the observation position, and the amount of deviation at the undetected observation position is obtained using various methods such as interpolation. May be.

  Furthermore, in the measurement optical system 302, when measurement light is incident from below the culture vessel 12, the measurement light is similarly refracted by the meniscus of the culture medium. Therefore, it is also possible to detect the amount of measurement light by inputting measurement light perpendicularly to the bottom surface of the culture vessel 12 and receiving the measurement light by the image sensor above the culture vessel 12. It is.

  In the above description, an example in which an image sensor is used as a light receiving unit for detecting the incident position of measurement light has been described. However, for example, a plurality of optical sensors may be used.

[Example of positioning the aperture image based on the shape of the liquid surface of the medium]
Further, for example, the shape of the liquid level of the culture medium (R shape) is calculated based on the amount of deviation of the measurement light detected using the measurement optical system 302, and the position of the aperture image is calculated based on the shape of the liquid level of the culture medium. You may make it match.

  FIG. 27 shows a configuration example of the function of the alignment control unit 371 that is a part of the function realized by the control unit 72 (FIG. 4) when the aperture image is aligned according to the shape of the liquid level of the culture medium. ing.

  The alignment control unit 371 is configured to include a deviation detection unit 381, a liquid surface shape calculation unit 382, and an adjustment unit 383.

  The deviation detection unit 381 has the same function as the deviation detection unit 361 in FIG. 25, detects the amount of measurement light deviation at a plurality of positions on the liquid level of the culture medium, and detects the detection result as the liquid level shape calculation unit 382. To supply.

  The liquid surface shape calculation unit 382 calculates the shape of the liquid surface of the culture medium based on the amount of deviation of the measurement light at each position on the liquid surface of the culture medium, and supplies the result to the adjustment unit 383. For example, the refraction direction and refraction angle of the measurement light at each position on the liquid surface of the medium can be obtained from the amount of deviation of the measurement light, and the shape of the liquid surface of the medium can be easily detected from the result.

  The adjusting unit 383 calculates the amount of deviation between the diaphragm image on the phase plate 37 and the phase film 37A based on the shape of the liquid level of the culture medium, and controls the driving unit 73 to correct the amount of deviation. The deviation prism 31a and the deviation prism 31b are rotated to align the aperture image.

  As a result, the amount of calculation of the displacement amount of the aperture image can be reduced and the processing time for alignment of the aperture image can be shortened compared to the case where the alignment of the aperture image is performed based on the displacement amount of the measurement light. it can.

  For example, when using the same culture container and the same culture medium, the shape of the liquid level of the culture medium is basically almost the same. Therefore, once the shape of the liquid level of the medium is obtained, the data can be diverted when the same culture vessel and the same medium are used thereafter, and re-detection of the shape of the liquid level of the medium becomes unnecessary. .

[Modification of the method for acquiring the liquid level information of the medium]
Information on the liquid level of the culture medium (hereinafter referred to as liquid level information) may be acquired by another method, and the position of the aperture image may be adjusted based on the acquired liquid level information. As such liquid level information, for example, it is conceivable to use the height and curvature of the liquid level.

  As a method of measuring the height of the liquid level, for example, the following two methods are conceivable.

1. 1. Method of measuring the time until the reflected light from the bottom surface of the culture vessel 12 reaches the detector by irradiating the pulse laser in the vertical direction from the liquid surface. Method of directly measuring the liquid depth by inserting a conductive probe into the medium

  As a method for measuring the curvature of the liquid surface, for example, the following six methods are conceivable.

1. A method in which a conductive probe is brought into direct contact with the liquid surface and information on the height direction of the contact position is mapped. In this method, it is possible to simultaneously measure the height and curvature of the liquid surface.
2. 2. A method for obtaining from a luminance distribution of a macro image of an entire container photographed by shining light from an angle. 3. A method for calculating an approximate R plane from a two-dimensional array of luminance information of a micro image 4. Method of reading the R shape and height from the side of the culture vessel 12 5. A method for calculating and performing two-dimensional mapping of specularly reflected light incident on the liquid surface at an incident angle of 0 <θ <90 °. A method of calculating from the propagation pattern of ripples based on the relationship that the distance between ripples becomes smaller as the curvature increases by blowing wind from above the liquid surface

  It should be noted that the liquid surface height and curvature of the medium may be appropriately thinned and measured, and the liquid surface height and curvature at the thinned position may be obtained using various methods such as an interpolation method.

  Then, for example, the measured liquid level information is stored in association with the position on the stage 27, and based on the position on the stage 27 of the sample to be observed, the height of the liquid level of the medium at the sample position is stored. The refraction direction of the illumination light at the position can be calculated from the curvature and the curvature. Furthermore, the amount of displacement of the aperture image on the phase plate 37 can be calculated based on the result, and the alignment of the aperture image can be performed based on the calculated amount of displacement.

  For example, standard liquid level information may be calculated and used based on the shape and size of the culture vessel 12 holding the sample without actually measuring the liquid level information. In this case, information on the shape and size of the culture vessel 12 and the position of the center of the culture vessel 12 on the stage 27 (hereinafter also simply referred to as the center position) is required.

  For example, the following three methods are conceivable as methods for acquiring each piece of information.

1. Information including the shape and size of all the culture vessels 12 that are assumed to be used in advance (hereinafter referred to as vessel information) is acquired and stored together with the installation position (installation coordinates) on the stage 27; Method for determining the culture container 12 to be used and obtaining the corresponding container information

  In addition, as a method of discriminating the culture container 12 to be used, for example, the following two methods are conceivable.

1a. Method for allowing user to input product information such as manufacturer and product name of culture vessel 12

1b. A method of capturing a micro image while moving from the end of the stage 27, detecting the contour of the entire culture vessel 12, and collating the shape of the detected contour with the stored vessel information. As a method for obtaining the outline of a container from a micro image, for example, a two-dimensional map of brightness values or contrast differences between adjacent minute spaces can be created and obtained based on the distribution.

  In addition, when the culture container 12 is comprised by a well plate, it is possible to detect the outline (outer shape and well part) of a container and the center position of each well by taking a micro image continuously. In this case, the outline of the entire container may be actually measured, or the whole value may be obtained by measuring a part of the outline and multiplying the same.

2. A method of photographing the entire culture container 12 by macro photography and detecting the shape and size of the container and the center position based on the obtained image. In this method, it is possible to apply a pattern matching technique using a template image.

3. Method of detecting measurement light such as laser light on the culture vessel 12 and detecting the shape and center position of the vessel from the bottom of the vessel

  When the culture container 12 is made of plastic, a convex structure is formed on the bottom surface at the end of the container due to the molding characteristics of the plastic container. Therefore, when laser light passing through the center of the objective lens 28 is applied to the bottom surface of the container, the surface of the bottom surface is not flat at the container end, and thus the return light is reduced. On the other hand, since the bottom surface is flat at the center of the container, the container end and the center position can be detected by two-dimensionally scanning the entire container and mapping the intensity of the return light. The shape and size can be determined.

  The measurement light does not necessarily have to pass through the objective lens 28. For example, the measurement light may be incident from the vicinity of the objective lens 28 and the reflected light may be detected by a separately provided detector.

  Further, even when the bottom surface is made of glass, the flatness of the bottom surface itself is uniform, but since there is a side surface (wall surface) portion, this third method can be applied.

  Then, for example, the shape and size of the culture vessel 12 and the center position are obtained by any one of the three methods described above. Further, the height and curvature of the liquid level of the culture medium are estimated based on the shape and size of the culture vessel 12. Further, based on the position of the observation position of the sample on the stage 27 and the center position of the culture container 12, the position of the observation position in the culture container 12 is obtained, and from the height and curvature of the liquid level of the culture medium at that position. The refraction direction of the illumination light at the position can be calculated. Based on the result, the displacement amount of the aperture image on the phase plate 37 can be calculated, and the aperture image can be aligned based on the calculated displacement amount.

[Image correction using measurement light misalignment]
It is also possible to provide an image processing unit that corrects the sample image based on the amount of deviation of the measurement light.

  For example, the illumination light is refracted by the meniscus of the culture medium, thereby causing unevenness of the brightness of the aperture image on the exit pupil plane and the pupil conjugate plane of the objective lens 28, thereby shading on the sample image taken by the image sensor 39A. Will occur.

  Therefore, for example, the shading direction can be specified based on the amount of measurement light shift and the direction at each observation position. Therefore, by performing shading correction in the specified direction, the luminance unevenness of the sample image can be determined. Can be corrected.

[Modification of shape of phase stop and phase film]
In the above description, an example in which the shape of the opening of the phase stop 24 and the shape of the phase film 37A is circular is shown, but other shapes are also possible. For example, it is possible to form a ring like a conventional phase contrast microscope.

  In addition, when comparing the case where it is circular and the case where it is ring-shaped, if the area of the opening of the phase stop 24 is the same so that the amount of illumination light applied to the sample is the same, the diameter of the opening The ring shape is larger than the circular shape. Therefore, in the circular shape, the distortion of the shape due to the meniscus of the medium is smaller than in the ring shape, the alignment between the aperture image and the phase film 37A is facilitated, and the contrast of the sample image becomes clearer. On the other hand, since the substantial numerical aperture (NA) is larger in the ring shape than in the circular shape, the resolution of the sample image is improved.

[Modified Example of Positioning Method of Aperture Image and Phase Film 37A]
In the above description, the aperture image is moved to align the aperture image and the phase film 37A. However, the phase plate 37 (phase film 37A) or the aperture image and phase plate 37 (phase film 37A) are shown. ) May be moved to adjust the relative position of the aperture image and the phase film 37A.

  When the phase plate 37 is moved, the phase plate 37 can be arranged not on the pupil conjugate plane but on the exit pupil plane of the objective lens 28.

  In addition to the method of moving the phase film 37 </ b> A by physically moving the phase plate 37, for example, the phase plate 37 is configured by an acousto-optic element such as AOM or AOTF, and the electronic phase within the phase plate 37. The film 37A may be generated and moved.

  A series of processes of the control unit 72, the alignment control units 101a to 101d, the alignment control unit 351, and the alignment control unit 371 described above can be executed by hardware or can be executed by software. it can. When a series of processing is executed by software, a program constituting the software is installed in the computer. Here, the computer includes, for example, a general-purpose personal computer capable of executing various functions by installing various programs by installing a computer incorporated in dedicated hardware.

  FIG. 28 is a block diagram illustrating an example of a hardware configuration of a computer that executes the above-described series of processes using a program.

  In a computer, a CPU (Central Processing Unit) 501, a ROM (Read Only Memory) 502, and a RAM (Random Access Memory) 503 are connected to each other by a bus 504.

  An input / output interface 505 is further connected to the bus 504. An input unit 506, an output unit 507, a storage unit 508, a communication unit 509, and a drive 510 are connected to the input / output interface 505.

  The input unit 506 includes a keyboard, a mouse, a microphone, and the like. The output unit 507 includes a display, a speaker, and the like. The storage unit 508 includes a hard disk, a nonvolatile memory, and the like. The communication unit 509 includes a network interface or the like. The drive 510 drives a removable medium 511 such as a magnetic disk, an optical disk, a magneto-optical disk, or a semiconductor memory.

  In the computer configured as described above, the CPU 501 loads the program stored in the storage unit 508 to the RAM 503 via the input / output interface 505 and the bus 504 and executes the program, for example. Is performed.

  The program executed by the computer (CPU 501) can be provided by being recorded on a removable medium 511 as a package medium or the like, for example. The program can be provided via a wired or wireless transmission medium such as a local area network, the Internet, or digital satellite broadcasting.

  In the computer, the program can be installed in the storage unit 508 via the input / output interface 505 by attaching the removable medium 511 to the drive 510. Further, the program can be received by the communication unit 509 via a wired or wireless transmission medium and installed in the storage unit 508. In addition, the program can be installed in the ROM 502 or the storage unit 508 in advance.

  The program executed by the computer may be a program that is processed in time series in the order described in this specification, or in parallel or at a necessary timing such as when a call is made. It may be a program for processing.

  Furthermore, the embodiments of the present technology are not limited to the above-described embodiments, and various modifications can be made without departing from the gist of the present technology.

  In addition, each step described in the above flowchart can be executed by being shared by a plurality of apparatuses in addition to being executed by one apparatus.

  Further, when a plurality of processes are included in one step, the plurality of processes included in the one step can be executed by being shared by a plurality of apparatuses in addition to being executed by one apparatus.

  11 phase contrast microscope, 12 culture vessel, 21 light source, 23 collector lens, 24 phase stop, 25 condenser lens, 27 stage, 28 objective lens, 29 second objective lens, 31 deviation prism unit, 31a, 31b deviation prism, 37 phase Plate, 37A phase film, 38 imaging lens, 39 camera, 39A image sensor, 40 Bertrand lens, 41 mirror, 42 photodetector, 43 eyepiece lens, 72 control unit, 73 drive unit, 101a to 101d alignment control unit, 111 brightness detection unit, 112 adjustment unit, 131 position detection unit, 132 adjustment unit, 151 sample detection unit, 152 contrast detection unit, 153 adjustment unit, 171 adjustment unit, 201 parallel plate glass, 301 observation optics System, 302 measurement optical system, 303 well plate, 351 alignment control unit, 361 deviation detection unit, 362 adjustment unit, 371 alignment adjustment unit, 381 deviation detection unit, 382 liquid level gauge calculation unit, 383 adjustment unit

Claims (6)

In a phase contrast microscope that observes a sample in a container placed on the stage by illuminating it with illumination light,
A condenser lens,
A diaphragm disposed on the incident side of the condenser lens;
An objective lens;
A phase plate provided with a phase film and disposed on an exit pupil plane of the objective lens or a pupil conjugate plane conjugate with the exit pupil plane;
A phase contrast microscope comprising: a mechanism for adjusting an inclination of the container with respect to the illumination light. The phase contrast microscope according to claim 1, wherein the mechanism adjusts the tilt of the container with respect to the illumination light by tilting the stage. The phase contrast microscope according to claim 1, wherein the aperture and the phase film have a circular shape centered on the optical axis. The adjustment part which adjusts the inclination of the said container with respect to the said illumination light via the said mechanism, and adjusts the relative position of the said image of the said aperture | diaphragm on the said phase plate, and the said phase film is further provided. The phase contrast microscope according to any one of the above. The phase contrast microscope according to claim 1, wherein the diaphragm is movable in a direction perpendicular to the optical axis. After adjusting the position of the diaphragm and adjusting the relative position between the image of the diaphragm on the phase plate and the phase film, the illumination light and the wall surface of the container interfere with each other via the mechanism. The phase contrast microscope according to claim 5, further comprising an adjustment unit that adjusts an inclination of the container with respect to illumination light to eliminate interference between the illumination light and a wall surface of the container.

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