Observation device

An observation device includes: an illumination optical system that irradiates illumination light onto a sample, an objective optical system that has a phase modulation region and a light blocking region and that acquires an image of the illumination light transmitted through the sample, and an autofocus mechanism. The objective optical system selectively outputs the illumination light from a first output region disposed at a position where the illumination light is to be projected onto the phase modulation region and a second output region disposed at a position where a portion of the illumination light is to be projected onto the light blocking region. The autofocus mechanism causes the objective optical system to acquire an image of the illumination light while causing the illumination light to be output from the second output region, and detects the focus position of the objective optical system based on the contrast of the acquired image.

TECHNICAL FIELD

The present invention relates to observation devices, and particularly, to a phase contrast image observation device having an autofocus function.

BACKGROUND ART

A known observation device in the related art has an autofocus function and uses a phase contrast observation method (e.g., see Patent Literatures 1 and 2). An autofocus process involves measuring the contrast of an image while changing the distance between a sample and an objective lens in the optical axis direction, and detecting the position of the objective lens where the contrast is at a maximum as a focus position where the the objective lens is in focus with the sample.

In the case of the phase contrast observation method, the contrast has a plurality of peaks, and the position where the contrast is at a maximum is not necessary the focus position. Therefore, it is difficult to determine which one of the peaks is the true focus position. In Patent Literature 1, a peak that satisfies a predetermined condition is determined to be the peak corresponding to the true focus position. In Patent Literature 2, two images are respectively acquired at focal points deviated forward and rearward from the focal plane of the objective lens, and the true focus position is determined based on a contrast difference between the two images.

CITATION LIST

Patent Literature

SUMMARY OF INVENTION

An aspect of the disclosure provides an observation device including: a stage on which a sample is placed; an illumination optical system that irradiates illumination light onto the sample on the stage; an objective optical system that acquires an image of the illumination light transmitted through the sample; and an autofocus mechanism that detects a focus position, which is where the objective optical system is in focus with the sample, on a basis of a contrast of the image of the sample acquired by the objective optical system, wherein the illumination optical system includes a mask that limits output of the illumination light to a first output region and a second output region, and an output-region switching element that causes the illumination light to be output selectively from the first output region and the second output region, wherein the objective optical system includes a phase modulation region that is provided in a part of a pupil of the objective optical system and that modulates a phase of the illumination light, and also includes a light blocking region that surrounds the pupil and that blocks the illumination light, wherein the first output region outputs illumination light to be irradiated onto the sample along an optical axis of the objective optical system, and is disposed at a position where the output illumination light is projected onto the phase modulation region, wherein the second output region outputs illumination light to be irradiated onto the sample obliquely relative to the optical axis of the objective optical system, and is disposed at a position where a portion of the output illumination light is projected onto the light blocking region, and wherein the autofocus mechanism changes a relative position between the stage and the objective optical system in a direction of the optical axis while causing the output-region switching element to output the illumination light from the second output region so as to cause the objective optical system to form an image of the illumination light at a plurality of relative positions.

DESCRIPTION OF EMBODIMENTS

First Embodiment

An observation device100according to a first embodiment of the present invention will now be described with reference toFIGS. 1 to 13.

As shown inFIG. 1, the observation device100according to this embodiment includes a horizontal stage2on which a container1containing a sample X is placed, an illumination optical system3that is disposed above the stage2and that irradiates illumination light beams L1and L2onto the sample X, an objective optical system4that is disposed below the stage2and that acquires images of the illumination light beams L1and L2transmitted through the sample X, and an autofocus (AF) mechanism5that automatically adjusts the focal point of the objective optical system4to the sample X.

The container1is, for example, a cell culturing flask having a top plate1aand is entirely formed of optically transparent resin. The sample X is, for example, a cell within a liquid Y.

The stage2is formed of an optically transparent material (such as glass).

The illumination optical system3has two types of modes, namely, a phase contrast illumination in which an annular illumination light beam L1is irradiated onto the sample X and an oblique illumination in which an illumination light beam L2is obliquely irradiated onto the sample X relative to an optical axis A of the objective optical system4. The illumination optical system3includes a first light source61for phase contrast illumination (output-region switching element), a second light source62for oblique illumination (output-region switching element), a scattering plate7that scatters the illumination light beams L1and L2respectively emitted from the first light source61and the second light source62, an illumination mask (mask)8that limits passing of the illumination light beams L1and L2scattered by the scattering plate7, and a collimating lens (collimating optical system)10that converts the illumination light beams L1and L2passing through the illumination mask8into collimated light beams.

The first light source61is an LED array formed of a plurality of LED light sources arranged annularly around a central axis, and is disposed such that the central axis is aligned with the optical axis A of the objective optical system4. The LED light sources emit light toward the stage2along the optical axis A so that an annular illumination light beam L1is formed as a whole. The plurality of LED light sources may be densely arranged, as shown inFIG. 2, or may be arranged with intervals in the circumferential direction, as shown inFIG. 3.

The second light source62is an LED light source that emits the second illumination light beam L2toward the stage2along the optical axis A of the objective optical system4. The second light source62is disposed at the radially outer side of the first light source61and is located away from the optical axis A in the radial direction relative to the first light source61.

The illumination mask8is formed of a plate-like member having light blocking properties and is disposed substantially horizontally between the first and second light sources61and62and the stage2. The illumination mask8may be integrally formed with the scattering plate7at one surface of the scattering plate7, or may be a separate component from the scattering plate7.

As shown inFIG. 4, the illumination mask8has a first output region91that allows the first illumination light beam L1to pass therethrough and a second output region92that allows the second illumination light beam L2to pass therethrough. The first output region91is disposed such that the central axis thereof is aligned with the optical axis A of the objective optical system4and is an annular opening that faces the first light source61with the scattering plate7interposed therebetween. The second output region92is disposed at the radially outer side of the first output region91and is a circular opening that faces the second light source62with the scattering plate7interposed therebetween.

The first illumination light beam L1emitted from the first light source61and scattered by the scattering plate7is limited from passing through the illumination mask8by the first output region91, and is output toward the stage2from the first output region91. The second illumination light beam L2emitted from the second light source62and scattered by the scattering plate7is limited from passing through the illumination mask8by the second output region92, and is output toward the stage2from the second output region92.

As will be described later, the first light source61and the second light source62are controlled by the AF mechanism5such that one is turned on while the other is turned off, whereby the illumination light beams L1and L2are selectively output from the output regions91and92.

The collimating lens10is disposed between the illumination mask8and the objective optical system4such that the first output region91and the second output region92are located on the focal plane of the collimating lens10and such that the collimating lens10is coaxial with the optical axis A. Accordingly, the first illumination light beam L1that is gradually scattered after passing through the first output region91is converted into a substantially collimated light beam by the collimating lens10and is irradiated onto the sample X along the same axis as the optical axis A. On the other hand, the second illumination light beam L2that is gradually scattered after passing through the second output region92is converted into a collimated light beam by the collimating lens10and is irradiated onto the sample X obliquely relative to the optical axis A.

The objective optical system4includes an objective lens11that collects the first illumination light beam L1and the second illumination light beam L2transmitted through the sample X, a pupil modulation element12provided at a pupil plane of the objective optical system4, an imaging lens13that forms an image of the illumination light beam L1whose phase is modulated by the pupil modulation element12, and an imaging element14that photographs the image formed by the imaging lens13.

As shown inFIG. 5, the pupil modulation element12has an annular phase modulation region15that modulates the phase of the illumination light beam L1and an annular light blocking region16that is provided around the phase modulation region15and that blocks the illumination light beams L1and L2. The light blocking region16constitutes an aperture stop (also referred to as “aperture stop16” hereinafter) serving as a pupil of the objective optical system4. Therefore, the phase modulation region15is provided in a part of the pupil.

The phase modulation region15is formed of an annular phase film centered on the optical axis A and is configured to delay (or advance) the phase of the illumination light beam L1by ¼ of the wavelength and to simultaneously attenuate the illumination light beam L1. The phase modulation region15is provided at an optically conjugate position with respect to the first output region91. The first output region91is disposed such that, when the first illumination light beam L1is output from the first output region91in a state where nothing is placed on the stage2, the projection region of the first illumination light beam L1to be projected onto the pupil modulation element12coincides with the phase modulation region15. Accordingly, in the first illumination light beam L1output from the first output region91, direct light not diffracted by the sample X enters the phase modulation region15, whereas diffracted light diffracted by the sample X passes through a region at the inner or outer side of the phase modulation region15.

The light blocking region16is provided at the radially outer side of the phase modulation region15and is spaced apart from the phase modulation region15in the radial direction. The second output region92is disposed such that, when the second illumination light beam L2is output from the second output region92in a state where nothing is placed on the stage2, a portion of luninous flux of the second illumination light beam L2to be projected onto the pupil modulation element12overlaps the inner edge of the light blocking region16so as to be vignetted by the inner edge of the light blocking region16.

FIG. 6illustrates the effect of the oblique illumination.

As shown inFIG. 6, the second illumination light beam L2is irradiated from the collimating lens10onto the sample X obliquely from above. Light beams a and e transmitted through a region where there is no sample X and a light beam c orthogonally entering the surface of the sample X pass through the vicinity of the inner edge of the aperture stop16without being refracted, so that a bright image is formed. On the other hand, a light beam b transmitted through the left edge of the sample X inFIG. 6is refracted so as to be vignetted by the inner edge of the aperture stop16. Moreover, a light beam d transmitted through the right edge of the sample X inFIG. 6is refracted so as to pass through a region closer to the center of the aperture stop16, whereby a bright image is formed by the imaging lens13.

As a result, a high-contrast image of the sample X that is shaded and thus appears to be three-dimensional is acquired, as shown inFIG. 7.

The AF mechanism5controls a moving mechanism (not shown), which relatively moves the stage2and the objective optical system4along the optical axis A, and the light sources61and62, so as to execute an AF process shown inFIG. 8.

In the AF process, the AF mechanism5turns off the first light source61and turns on the second light source62so that, of the first illumination light beam L1and the second illumination light beam L2, only the second illumination light beam L2is irradiated onto the sample X (step S1). Then, the AF mechanism5causes the imaging element14to execute a photographing process a plurality of times while causing the moving mechanism to relatively move the stage2and the objective optical system4so as to change the position of the focal point of the objective optical system4in the direction of the optical axis A relative to the sample X (step S2). Consequently, a plurality of images of the sample X illuminated by the oblique illumination are acquired by the imaging element14.

Subsequently, the AF mechanism5measures the contrast of each of the images, identifies the image with the maximum contrast, and sets the relative position between the stage2and the objective optical system4when the identified image was acquired as a focus position (step S3). Then, the AF mechanism5causes the stage2and the objective optical system4to relatively move to the set focus position. Subsequently, the AF mechanism5turns on the first light source61and turns off the second light source62, so as to irradiate the first illumination light beam L1onto the sample X (step S4). Accordingly, a phase contrast image whose focal point is adjusted to the sample X is acquired by the imaging element14(step S5).

The AF mechanism5is realized by, for example, a computer equipped with a central processing unit (CPU), a main storage device, and an auxiliary storage device that stores an AF program for causing the CPU to execute the above-described AF process.

FIG. 9illustrates the relationship between the contrast of an image and the amount of deviation of the relative position between the stage2and the objective optical system4from the focus position. In a case where the phase contrast illumination is used, the contrast has peaks at other positions in addition to the focus position, as indicated by a broken line inFIG. 9. In contrast, in a case where the oblique illumination is used, the contrast has a single peak only at the focus position by reaching a maximum at the focus position and decreasing as the amount of deviation from the focus position increases, as indicated by a solid line inFIG. 9. Thus, the relative position where the contrast is at a maximum can be detected as the focus position.

Accordingly, in this embodiment, the oblique illumination alone is used in the AF process, and the switching between the oblique illumination and the phase contrast illumination is performed by simply turning on and off the two light sources61and62. Consequently, the focus position can be detected accurately and quickly without requiring a complicated process, which is advantageous in that the AF process can be performed quickly.

Although the second output region92is circular in this embodiment, the shape of the second output region92is not limited to this. For example, the second output region92may be rectangular, as shown inFIG. 10, or may be semicircular, as shown inFIG. 11.

Although the two light sources61and62that respectively emit the first illumination light beam L1and the second illumination light beam L2are provided in this embodiment, a single lamp light source63, such as a halogen lamp, may be used as an alternative, as shown inFIG. 12. Reference sign17denotes a collector lens that collects an illumination light beam emitted from the lamp light source63. It is clear fromFIG. 12that an illumination optical system31according to this modification uses Kohler illumination.

The illumination optical system31includes a movable light blocking plate (output-region switching element, limiting member)18that has an opening (passing region)18abetween the collector lens17and the illumination mask8and through which illumination light passes, and that causes the illumination light to pass through either the first output region91or the second output region92. Other sections of the light blocking plate18are formed of a light blocking material, and the passing of the illumination light through the light blocking plate18is limited to the opening18aalone.

As shown inFIG. 13, the light blocking plate18is slidable in the horizontal direction between a photographing position (upper section), which is where the first output region91is positioned in the opening18a,and an AF position (lower section), which is where the second output region92is positioned in the opening18a.When viewed in the direction of the optical axis A, the photographing position is a position where the first output region91is positioned in the opening18aand the second output region92is covered by the light blocking plate18. When viewed in the direction of the optical axis A, the AF position is a position where the second output region92is positioned in the opening18aand the first output region91is covered by the light blocking plate18. Instead of turning on and off the light sources61and62, the AF mechanism5sets the light blocking plate18at the AF position when performing AF and sets the light blocking plate18at the photographing position when photographing a phase contrast image.

Accordingly, it is possible to switch between the oblique illumination and the phase contrast illumination by simply sliding the light blocking plate18.

Second Embodiment

Next, an observation device200according to a second embodiment of the present invention will be described with reference toFIGS. 14 and 15.

In this embodiment, differences from the first embodiment will be mainly described, whereas components identical to those in the first embodiment will be given the same reference signs, and descriptions thereof will be omitted.

As shown inFIG. 14, the observation device200according to this embodiment is different from the first embodiment in that an illumination optical system32is provided below the stage2.

The first light source61surrounds the objective optical system4and is disposed such that the central axis thereof is aligned with the optical axis A of the objective optical system4. The second light source62is disposed at the radially outer side of the first light source61. The first light source61and the second light source62emit illumination light beams L1and L2toward the stage2along the optical axis A.

As shown inFIG. 15, an illumination mask81further has, in the center thereof, an opening81ain which the objective optical system4is to be disposed. The illumination mask81is disposed between the first and second light sources61and62and the stage2and surrounds the objective optical system4. The second output region92may alternatively have the shape shown inFIG. 10 or 11.

A collimating lens101has, in the center thereof, an opening101ain which the objective optical system4is to be disposed. The collimating lens101is disposed between the light sources61and62and the stage2, surrounds the objective optical system4, and is coaxial with the optical axis A of the objective optical system4.

The illumination light beam L1and the illumination light beam L2respectively passing through the first output region91and the second output region92are converted into collimated light beams by the collimating lens101and are output obliquely from the collimating lens101toward the optical axis A of the objective optical system4. Subsequently, the first illumination light beam L1and the second illumination light beam L2are transmitted through the stage2and the bottom wall of the container1, are reflected at the top plate1aof the container1, and are irradiated onto the sample X obliquely from above. The first illumination light beam L1and the second illumination light beam L2transmitted through the sample X, the bottom wall of the container1, and the stage2are collected by the objective lens11, are imaged by the imaging lens13, and are photographed by the imaging element14.

The first output region91is disposed such that the projection region of the first illumination light beam L1to be projected onto the pupil modulation element12coincides with the phase modulation region15. The second output region92is disposed such that a portion of the second illumination light beam L2to be projected onto the pupil modulation element12overlaps the inner edge of the light blocking region16so as to be vignetted by the inner edge of the light blocking region16.

Since the remaining components according to this embodiment and the functions thereof are the same as those in the first embodiment, descriptions thereof will be omitted.

In addition to the advantages of the first embodiment, this embodiment is advantageous in that it can reduce the size of the observation device200since both the illumination optical system32and the objective optical system4are disposed below the stage2.

From the above-described embodiments, the following aspects of the present disclosure are derived.

An aspect of the disclosure provides an observation device including: a stage on which a sample is placed; an illumination optical system that irradiates illumination light onto the sample on the stage; an objective optical system that acquires an image of the illumination light transmitted through the sample; and an autofocus mechanism that detects a focus position, which is where the objective optical system is in focus with the sample, on a basis of a contrast of the image of the sample acquired by the objective optical system, wherein the illumination optical system includes a mask that limits output of the illumination light to a first output region and a second output region, and an output-region switching element that causes the illumination light to be output selectively from the first output region and the second output region, wherein the objective optical system includes a phase modulation region that is provided in a part of a pupil of the objective optical system and that modulates a phase of the illumination light, and also includes a light blocking region that surrounds the pupil and that blocks the illumination light, wherein the first output region outputs illumination light to be irradiated onto the sample along an optical axis of the objective optical system, and is disposed at a position where the output illumination light is projected onto the phase modulation region, wherein the second output region outputs illumination light to be irradiated onto the sample obliquely relative to the optical axis of the objective optical system, and is disposed at a position where a portion of the output illumination light is projected onto the light blocking region, and wherein the autofocus mechanism changes a relative position between the stage and the objective optical system in a direction of the optical axis while causing the output-region switching element to output the illumination light from the second output region so as to cause the objective optical system to form an image of the illumination light at a plurality of relative positions.

According to this aspect, the image of the illumination light irradiated onto the sample on the stage from the illumination optical system and transmitted through the sample is acquired by the objective optical system, so that the image of the sample is acquired. In this case, the region where the illumination light passes through the mask is switched between the first output region and the second output region by the output-region switching element, so that the illumination irradiated onto the sample can be switched between a phase contrast illumination and an oblique illumination.

Specifically, in the phase contrast illumination, the illumination light is irradiated onto the sample from the first output region along the optical axis of the objective optical system. At the pupil of the objective optical system, the illumination light refracted by the sample is not transmitted through the phase modulation region, whereas the illumination light not refracted by the sample is transmitted through the phase modulation region so that the phase thereof is modulated. Accordingly, a phase contrast image of the sample is acquired by the objective optical system. In contrast, in the oblique illumination, the illumination light is irradiated obliquely relative to the optical axis onto the sample from the second output region. At the pupil of the objective optical system, the illumination light is partially vignetted by the light blocking region. Accordingly, a shaded three-dimensional sample image is acquired by the objective optical system.

In this case, in the autofocus process for detecting the focus position, the autofocus mechanism causes the objective optical system to acquire images multiple times by changing the position of the focal point of the objective optical system relative to the sample in the optical axis direction while illuminating the sample by the oblique illumination, so that a plurality of images with different contrasts are acquired. In the oblique illumination, a peak of the contrast of an image appears only at the focus position where the objective optical system is in focus with the sample. Therefore, the focus position can be accurately detected by simply comparing the magnitude of the contrast, whereby the autofocus process can be performed quickly.

In the above aspect, the second output region may be disposed at a radially outer side of the first output region relative to the optical axis of the objective optical system.

Accordingly, by setting the second output region at a position located away from the objective optical system in the radial direction, the illumination light to be output obliquely from the second output region can be made to enter the objective optical system from an edge opposite from the second output region.

In the above aspect, the illumination optical system may include a collimating optical system that converts the illumination light output from the first output region and the second output region into collimated light, and wherein the first output region and the second output region may be disposed in a focal plane of the collimating optical system.

Accordingly, the irradiation angle of the illumination light to the sample can be aligned.

In the above aspect, the illumination optical system may include a first light source disposed facing the first output region in front of the mask and a second light source disposed facing the second output region in front of the mask, and wherein the output-region switching element may turn on one of the first light source and the second light source and turn off the other one of the first light source and the second light source.

Accordingly, it is possible to switch between the phase contrast illumination and the oblique illumination by simply turning on and off the two light sources.

In the above aspect, the output-region switching element may include a limiting member that is disposed in front of the mask and that limits passing of the illumination light to a predetermined passing region, and may move the limiting member between two positions where the first output region and the second output region are respectively independently located in the predetermined passing region.

Accordingly, it is possible to switch between the phase contrast illumination and the oblique illumination by simply moving the limiting member between two positions.

According to the aforementioned aspects, an advantagesous effect is afforded in that an autofocus process can be performed simply and quickly.

REFERENCE SIGNS LIST