JP5655617B2 - microscope - Google Patents

Description

  The present invention relates to a microscope.

  When fluorescent observation of a biological specimen in a liquid is performed with an optical microscope (biological microscope), the focus may be shifted after a while after focusing on the position to be observed and the focus may be shifted. This is because the positional relationship between the stage on which the sample is placed and the objective lens changes due to vibration or temperature. In time-lapse observation in which observation is performed for a long time and changes with time are observed, it is a big problem that the focal position moves during observation. Even for short-time observations, if the focus position is moved again when attempting to observe again at the position where fluorescence observation has been performed once, focus adjustment will be required again, but fluorescence will fade. However, since the image disappears while focusing on the fluorescent image, an autofocus device that automatically tracks the focus from the observation position once adjusted has been desired.

  Until now, in the field of industrial microscopes, the focus position is determined by irradiating infrared light for autofocus with the observation position as the reflection surface, tracking the light returned from the reflection surface as a signal, and continuing to adjust the focus. There was an autofocus device that prevented the camera from moving. However, an apparatus having such a configuration cannot be used for a specimen having no reflecting surface as observed with a biological microscope. In view of this, an autofocus device in which an adjustment lens that adjusts the focal position of infrared light for autofocus is introduced (see, for example, Patent Document 1). Here, the focus position of the observation light and the focus position of the infrared light for autofocus are shifted by an adjustment lens (offset lens), and the focus position of the autofocus infrared light is aligned with the interface between the sample and the cover glass. The focus is adjusted based on the reflected light from there.

JP 2007-323094 A

  Recently, however, observation of a deeper position of the specimen has been demanded, and the high numerical aperture objective lens used for two-photon excitation observation is greatly affected by aberrations. When the focal position of the light and the focal position of the infrared light for autofocus are greatly shifted, the signal intensity of the infrared light becomes weak and the autofocus mechanism does not work well.

  The present invention has been made in view of such a problem, and the objective lens is focused on an arbitrary position of the specimen in order to observe the deeper part of the specimen using a high numerical aperture objective lens. Even if it is a case, it aims at providing the microscope which has an autofocus apparatus which continues following it.

In order to solve the above problems, a microscope according to the present invention includes an observation optical system for observing light irradiated on a specimen through an objective lens, and a drive unit that changes a relative position between the specimen and the objective lens. by controlling the operation, a microscope having an auto-focus device to maintain the focus of the objective lens to a desired position relative to the specimen, the autofocus device, via the objective lens to the specimen, the focus of the objective lens A focusing illumination optical system that forms a predetermined projected image at a position different from the position, and the reflected light from the sample of the projected image is received via the objective lens, and the reflected image of the projected image is connected to the imaging surface of the photoelectric converter. and focusing the imaging optical system to the image, based on the signal of the reflected image obtained by the photoelectric converter to chromatic signal output unit for outputting a signal for controlling the operation of the drive unit.

In such a microscope, the focusing illumination optical system includes an imaging position adjusting member that moves the imaging position of the projected image in the optical axis direction, and includes the focusing illumination optical system and the focusing imaging optical system. A variable aperture is provided on at least one of the apertures, and the aperture diameter of the variable aperture is controlled in accordance with the amount of movement of the imaging position by the imaging position adjusting member. It is characterized in that at least one of the numerical apertures of the optical system can be changed.

Such a microscope includes a movement amount detection unit that detects the movement amount of the imaging position by the imaging position adjustment member, and a control that changes the aperture diameter of the variable diaphragm according to the movement amount detected by the movement amount detection unit. Part.

In addition, such a microscope preferably includes a storage unit that stores the operation amount of the imaging position adjusting member, and a control unit that changes the aperture diameter of the variable diaphragm according to the operation amount.

In such a microscope, it is preferable that the control unit changes the light amount of the focus illumination light source for forming a projection image with the focus illumination optical system according to the aperture diameter of the variable stop.

  According to the present invention, even when observing the deep part of a specimen using a high numerical aperture objective lens, it automatically keeps track of the observation position so that time-lapse observation and the same position can be observed many times. Is possible.

It is explanatory drawing which shows the structure of the optical system and control system of a microscope which have an autofocus apparatus. It is explanatory drawing explaining operation | movement of a variable aperture at the time of immersion type | system | group objective lens use. It is explanatory drawing explaining operation | movement of a variable aperture at the time of using a dry system objective lens. It is explanatory drawing which shows the optical system of the microscope which concerns on 1st Example. It is explanatory drawing which shows the optical system of the microscope which concerns on 2nd Example.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. First, the configuration of the microscope 100 having the autofocus device 1 according to the present embodiment will be described with reference to FIG. The optical system of the microscope 100 according to the present embodiment includes an observation optical system 3 disposed on an upper part of a specimen 18 placed on a stage 11 via a slide glass 15, and an autofocus disposed on the side thereof. The optical system of the apparatus 1 includes a focusing illumination optical system 5 and a focusing imaging optical system 7.

  The focusing illumination optical system 5 includes an LED light source 20, a first collector lens 21, a slit plate 22, a second collector lens 23, a first pupil limiting mask 24, a variable aperture 2, and a first half mirror in order on the optical axis. 25, a focus position adjusting lens 8 as an imaging position adjusting member and a visible light cut filter 10 are provided. A rectangular elongated slit aperture 22a is formed at the center of the slit plate 22. The slit plate 22 is centered on the optical axis so that the longitudinal direction of the slit aperture 22a extends in a direction perpendicular to the paper surface in FIG. It is arranged.

  Infrared light (near infrared light) emitted from the LED light source 20 of the focusing illumination optical system 5 is collected by the first collector lens 21 and enters the slit plate 22, and the specimen surface (the cover glass 14 and the specimen). The first pupil limiting mask 24 is irradiated with light by being converted into substantially parallel light by the second collector lens 23 through the slit aperture 22a of the slit plate 22 arranged at a conjugate position with the medium 18). Is done. The first pupil restriction mask 24 shields half of the pupil, and is arranged so that half of the pupil is shielded along the longitudinal center line of the slit-shaped infrared light with the optical axis as the center. . The infrared light (infrared light La in FIG. 1) that has passed through the first pupil restriction mask 24 passes through the variable aperture 2 and then enters the first half mirror 25. The first half mirror 25 is disposed at a point where the optical axes of the focusing illumination optical system 5 and the focusing imaging optical system 7 intersect, and a part of infrared light (La and Ld in FIG. 1). Is reflected and transmitted through the other part, and is also shared by the focusing imaging optical system 7, as will be described later.

  A dichroic mirror 16 is disposed at a point where the optical axes of the focusing illumination optical system 5 and the observation optical system 3 intersect, and is shared by the observation optical system 3 as described later. The dichroic mirror 16 is disposed in an afocal system on the observation optical path of the observation optical system 3, and reflects infrared light (La and Lc) and transmits visible light (observation light indicated by a broken line in FIG. 1). do. The infrared light La that has passed through the first half mirror 25 passes through the focal position adjusting lens 8, and then enters the dichroic mirror 16 through the visible light cut filter 10 and is reflected downward (infrared light Lb). The sample 18 is condensed and irradiated by one objective lens 12. That is, an image of the slit aperture 22a of the slit plate 22 is irradiated to the specimen 18 through the first objective lens 12 by the focus illumination light emitted from the focus illumination light source including the LED light source 20 and the slit plate 22. The The first objective lens 12 is shared by the observation optical system 3 as will be described later.

  The observation optical system 3 includes a first objective lens 12, a dichroic mirror 16, an infrared light cut filter 19, a second half mirror 17, and a second objective lens for eyepiece 13, which are arranged in order from the side closer to the specimen 18. Further, although not shown, an eyepiece lens is disposed at the tip of the second objective lens 13 for eyepiece. Although not shown, the microscope 100 is provided with an illumination device for illuminating the specimen 18 placed on the stage 11. This illuminating device is of a transmission type or an epi-illumination type, and is disposed below the stage 11 in the case of the transmissive illumination device, and is disposed above the stage 11 in the case of the epi-illumination type illumination device. Visible light emitted from the illumination device passes through the specimen 18 to become observation light, passes through the first objective lens 12, passes through the dichroic mirror 16, and the infrared light is removed by the infrared light cut filter 19. 2 enters the half mirror 17. The second half mirror 17 reflects a part of the observation light and transmits the other part. The observation light incident on the second half mirror 17 is partially reflected and the second objective for eyepiece. An observation image of the specimen 18 is formed by the lens 13 and the eyepiece, and used for observation.

  Note that a part of the observation light transmitted through the second half mirror 17 passes through the second objective lens 36 for camera and the relay lens 37 for camera, and is imaged on the imaging surface of the CCD sensor 38 for camera. It is also possible to use the image of the specimen 18 by projecting it on the monitor (not shown) after being processed by the unit 39. The operation of the camera signal processing unit 39 is controlled by the CPU 41.

  Next, the focusing imaging optical system 7 will be described. As described above, the focusing imaging optical system 7 receives slit-shaped infrared light (projected reflection image of the slit aperture 22a) that is irradiated and reflected on the specimen 18 on the stage 11 by the focusing illumination optical system 5. It receives light. Here, since the specimen 18 on the stage 11 is covered with the cover glass 14, the infrared light Lb imaged by the first objective lens 12 is generated by the surface 14 a of the cover glass 14, the cover glass 14, the specimen 18, and the like. Is reflected at the boundary surface (specimen surface 18a). Infrared light reflected by the surface 14a of the cover glass 14 and the sample surface 18a is converted into substantially parallel light by the first objective lens 12 (infrared light Lc), enters the dichroic mirror 16 and is reflected laterally. (Infrared light Ld) and visible light (observation light from the sample surface 18a, which becomes noise) are further removed by the visible light cut filter 10, and then enter the first half mirror 25 through the focal position adjustment lens 8. .

  Part of the infrared light Ld incident on the first half mirror 25 is reflected upward and enters the focusing imaging optical system 7. The focusing imaging optical system 7 includes a first half mirror 25, an autofocus second objective lens 26, an autofocus relay lens 27, a second pupil restriction mask 28, and an autofocus relay lens 27 along the optical axis. A cylindrical lens 29 and an autofocus CCD sensor 30 which is a photoelectric converter are arranged. The infrared light Ld reflected by the first half mirror 25 is condensed by the second autofocus objective lens 26 and converted into imaging light to form a projected reflected image of the slit aperture 22a. The autofocus relay lens 27 relays the reflected image (infrared light Le) formed by the second autofocus objective lens 26, and reflects to the imaging surface of the autofocus CCD sensor 30 via the cylindrical lens 29. Reimage the image.

  The second pupil restriction mask 28 is disposed so as to shield half of the pupil, and the light-shielded area corresponds to the area shielded by the first pupil restriction mask 24 described above. . The cylindrical lens 29 is a lens having a refractive action only in a predetermined direction, and compresses the infrared light Le in a direction perpendicular to the paper surface (longitudinal direction of the slit image) in FIG. It acts to form an image on the imaging surface. Further, the autofocus CCD sensor 30 can be configured by a line sensor in which a plurality of light receiving units are arranged one-dimensionally or an area sensor arranged in a two-dimensional manner.

  The autofocus device 1 processes the projection reflection image of the slit aperture 22a acquired by the autofocus CCD sensor 30 by the autofocus signal processing unit 31 that is a signal output unit, thereby causing the sample 18 of the first objective lens 12 to be processed. Is calculated, and a signal for making the focal point of the first objective lens 12 coincide with a predetermined position with respect to the sample 18 is output. The CPU 41 moves the stage 11 or the first objective lens 12 in the optical axis direction by the focus actuator of the stage drive unit 34 or the objective lens drive unit 35 based on the signal output from the autofocus signal processing unit 31. Thus, the focal plane of the first objective lens 12 is controlled to be maintained at a desired position of the sample 18.

  In the above description, in the focusing illumination optical system 5, the light emitted from the LED light source 20 is formed into a slit shape through the slit opening 22 a of the slit plate 22, thereby irradiating the sample 18 with the image of the slit opening 22 a. . This is because, in the case of spot light, if there is a step portion on the sample surface 18a or the like, the reflected light is scattered and an ideal light amount signal cannot be obtained, but depending on the state of the sample surface 18a or the like. It is also possible to eliminate the slit plate 22 and perform autofocus control by irradiating the sample with the image of the LED light source 20 by the method described above. Further, this can be realized without the first collector lens 21.

  Next, the variable diaphragm 2 used in the autofocus device 1 according to the present embodiment will be described. In FIG. 1, the variable stop 2 is located between the first pupil restriction mask 24 and the first half mirror 25, that is, on the optical path of the focusing illumination optical system 5, and is disposed in an afocal system. As will be described later, the variable diaphragm 2 has a diameter of an opening (in accordance with the focal position of the autofocus infrared light moved by the focal position adjusting lens 8 (image position of the slit aperture 22a of the slit plate 22)). The numerical aperture of the autofocus infrared light is controlled by changing the size of the aperture diameter (changing the amount of aperture). The operation of the variable diaphragm 2 is controlled by a CPU 41 as a control unit in accordance with the focal position of the autofocus infrared light detected by the movement amount detection unit 32 (image position of the slit aperture 22a of the slit plate 22). Is controlled by the drive unit 9 based on this control signal, and the light quantity of the LED light source 20 similarly controlled by the CPU 41 is adjusted in conjunction therewith. For example, when the variable stop 2 is stopped, the amount of infrared light for focusing toward the sample 18 is reduced, so that the amount of light from the LED light source 20 is increased to secure the intensity of the signal reaching the autofocus CCD sensor 30. Information for control by the CPU 41 is stored in the memory 42, and instructions from the observer are input to the CPU 41 via the input unit 43. Further, the operation of the focal position adjusting lens 8 is determined by the operation amount calculated from the distance from the interface between the cover glass 14 and the sample 18 (sample surface 18a) to the observation surface, the refractive index of the sample, etc. When the CPU 41 uses the operation amount stored in the control unit), the amount of light of the variable aperture 2 and the LED light source 20 is adjusted from the operation amount of the focus position adjustment lens 8 by the CPU 41 without providing the movement amount detection unit 32. You may comprise so that it may do.

  Now, the operation of the variable diaphragm 2 will be described with reference to FIGS. 2 and 3, the infrared light for autofocus is indicated by a solid line or a dotted line, and the observation light is indicated by a double line.

  FIG. 2 schematically shows the vicinity of the observation position when an immersion objective lens is used as the first objective lens 12. When the immersion objective lens is used, the infrared light for autofocus is reflected at the interface between the cover glass 14 and the sample 18 (sample surface 18a). When observing a depth not far from the cover glass 14 as shown in FIG. 2A, the focus position of the infrared light for autofocusing by the focus position adjusting lens 8 is the interface between the cover glass 14 and the sample 18 ( It is placed on the specimen surface 18a). When observing a deep position away from the cover glass 14 as shown in FIG. 2B, even if the focus position of the infrared light for autofocus is adjusted by the focus position adjustment lens 8, the first objective lens 12 The autofocus mechanism does not work because the light is not collected well due to the influence of aberration and the reflected light signal cannot be received. This becomes more conspicuous than in the case of the first objective lens 12 having a high numerical aperture. Accordingly, the aperture of the variable aperture 2 is stopped, and the numerical aperture of the autofocus infrared light is reduced as shown in FIG. 2C, thereby suppressing the influence of the aberration of the first objective lens 12 and restoring the reflected light signal. it can.

  The same can be said when a dry objective lens is used as the first objective lens 12. FIG. 3 schematically shows the vicinity of an observation position when a dry objective lens is used. When a dry objective lens is used, the infrared light for autofocus is reflected at the interface (the surface 14a of the cover glass 14) between the cover glass 14 and the air layer on the first objective lens 12 side. The reason for reflecting at this position is that a larger reflected light can be obtained from a larger refractive index difference than the reflection at the interface between the cover glass 14 and the sample 18 (sample surface 18a). When observing a depth that is not so far from the cover glass 14 as shown in FIG. 3A, the focus position of the infrared light for autofocusing by the focus position adjusting lens 8 is the interface between the cover glass 14 and the air layer ( Placed on the surface 14a). When observing a deep position away from the cover glass 14 as shown in FIG. 3B, even if the focal position of the infrared light for autofocus is adjusted by the focal position adjusting lens 8, the first objective lens 12 The autofocus mechanism does not work because the light is not collected well due to the influence of aberration and the reflected light signal cannot be received. This becomes more noticeable when the first objective lens 12 has a high numerical aperture. Therefore, the aperture of the variable aperture 2 is stopped, and the numerical aperture of the autofocus infrared light is reduced as shown in FIG. 3C, thereby suppressing the influence of the aberration of the first objective lens 12 and restoring the reflected light signal. it can.

  In the autofocus device 1 according to the present embodiment, the variable aperture 2 shown in FIG. 1 shows a case where it is arranged on the optical path of infrared light that is substantially parallel to the second collector lens 23 of the focusing illumination optical system 5. However, it can also be arranged in a substantially parallel light beam between the first half mirror 25 of the focusing imaging optical system 7 and the second objective lens 26 for autofocusing. Hereinafter, examples in each case will be described.

[First embodiment]
FIG. 4 shows a first example of the optical system of the microscope 100 having the autofocus device 1 according to this embodiment. In FIG. 4, the autofocus infrared light is shown by a solid line, and the observation light is shown by a dotted line. In the first embodiment, the variable stop 2 is located between the first pupil limiting mask 24 and the first half mirror 25, that is, on the optical path of the focusing illumination optical system 5, and is disposed in an afocal system. Yes. FIG. 4 shows a state in which the numerical aperture of the autofocus infrared light in the vicinity of the sample position decreases when the variable stop 2 is stopped. In FIG. 4, constituent elements not related to the description are omitted.

  Here, when the immersion objective lens is used as the first objective lens 12, observation is performed at a position shifted by 25 μm and 50 μm from the interface between the cover glass 14 and the sample 18 (sample surface 18 a) to the inside of the sample by the focal position adjusting lens 8. Table 1 shows the respective diaphragm amounts of the variable diaphragm 2 when the surface is set. In Table 1, fo represents the focal length of the objective lens, and NAo represents the numerical aperture of the objective lens.

  When the variable stop 2 is opened (the numerical aperture of the autofocus infrared light is equivalent to the numerical aperture of the objective lens used), the autofocus infrared light signal peaks due to the deterioration of the aberration of the first objective lens 12. It cannot be detected. Therefore, the variable aperture 2 is stopped, and the numerical aperture of the autofocus infrared light irradiated to the interface (sample surface 18a) between the cover glass 14 and the sample 18 is 0.95 when the depth is 25 μm, and the depth is When it is 50 μm, it is adjusted to 0.80.

  In this case, the decrease in the light amount of the autofocus infrared light due to the reduction of the variable aperture 2 is adjusted by changing the light amount of the LED light source 20. Since the amount of light from the LED light source 20 is proportional to the area of the aperture of the variable aperture 2, that is, the square of the numerical aperture NA, the numerical aperture NA is 0.95, 0.00 when the objective lens with NAo = 1.40 is used. When the aperture is reduced to 80, the amount of light is 2.17 and 3.06, respectively, where 1 is the amount of light before the variable aperture 2 is reduced. Further, when the numerical aperture NA is reduced to 0.95 and 0.80 when the objective lens with NAo = 1.38 is used, the light amount is 2.11 when the light amount before the variable aperture 2 is reduced is 1. 2.98. Further, when the numerical aperture NA is reduced to 0.95 and 0.80 when the objective lens with NAo = 1.30 is used, the light amount is 2.09 when the light amount before the variable aperture 2 is reduced is 1. 2.64. As a result, the infrared light signal for autofocus is recovered to a level sufficient for the autofocus mechanism to work, and observation while following the observation position becomes possible. At this time, if the amount of aperture is too large, the depth of focus becomes deep and the accuracy of focus alignment is lowered, and the focus adjustment speed becomes slow due to the gentle signal peak. On the other hand, if the amount of aperture is too small, the signal will not be recovered and the autofocus mechanism will not work well. Here, a large aperture amount corresponds to a decrease in the numerical aperture, and a small aperture amount corresponds to a small decrease in the numerical aperture.

  Next, when the dry objective lens is used as the first objective lens 12, the observation surface is shifted to the inside of the sample from the interface (surface 14 a) between the cover glass 14 and the air layer by the focal position adjustment lens 8 by 170 μm and 220 μm. Table 2 shows the respective diaphragm amounts of the variable diaphragm 2 when set. However, 170 μm corresponds to the thickness of the cover glass 14, and the former at the shifted position is the interface between the cover glass 14 and the sample 18 (sample surface 18a), and the latter is the interface between the cover glass 14 and the sample 18 (sample surface). From 18a), the position is 50 μm inside the specimen. In Table 2, fo represents the focal length of the objective lens, and NAo represents the numerical aperture of the objective lens.

  When the variable stop 2 is opened (the numerical aperture of the autofocus infrared light is equivalent to the numerical aperture of the objective lens used), the autofocus infrared light signal peaks due to the deterioration of the aberration of the first objective lens 12. It cannot be detected. Therefore, the aperture stop 2 is stopped and the numerical aperture of the autofocus infrared light irradiated to the interface (surface 14a) between the cover glass 14 and the air layer is 0.60 when the depth is 170 μm, and the depth is 220 μm. In the case of, adjustment is made so that each becomes 0.50.

  In this case, the decrease in the light amount of the autofocus infrared light due to the variable aperture 2 being stopped is adjusted by changing the light amount of the LED light source 20. Since the amount of light from the LED light source 20 is proportional to the area of the aperture of the variable aperture 2, that is, the square of the numerical aperture NA, the numerical aperture NA when using an objective lens with NAo = 0.85 is set to 0.60, 0. When the aperture is reduced to .50, the amount of light is 2.01 and 2.89, respectively, where 1 is the amount of light before the variable aperture 2 is reduced. Further, when the numerical aperture NA is reduced to 0.60 and 0.50 when the objective lens with NAo = 0.75 is used, the light amount is 1.56 when the light amount before the variable stop 2 is stopped is 1. 2.25. As a result, the infrared light signal for autofocus is recovered to a level sufficient for the autofocus mechanism to work, and observation while following the observation position becomes possible. At this time, if the amount of aperture is too large, the depth of focus becomes deep and the accuracy of focus alignment is lowered, and the focus adjustment speed becomes slow due to the gentle signal peak. On the other hand, if the amount of aperture is too small, the signal will not be recovered and the autofocus mechanism will not work well. Here, a large aperture amount corresponds to a decrease in the numerical aperture, and a small aperture amount corresponds to a small decrease in the numerical aperture.

  The position of the variable stop 2 is not limited to the position between the first pupil restriction mask 24 and the first half mirror 25 described above, and is disposed anywhere on the optical path from the slit plate 22 to the dichroic mirror 16. Also good.

[Second Embodiment]
FIG. 5 shows a second example of the optical system of the microscope 100 having the autofocus device 1 according to this embodiment. In FIG. 5, the variable stop 2 'is positioned between the first half mirror 25 and the second auto-focus objective lens 26, that is, on the optical path of the focusing imaging optical system 7, and is disposed in the afocal system. Has been. When the variable stop 2 ′ is stopped, only a component having a small numerical aperture of the autofocus infrared light reflected by the cover glass 14 is shown reaching the focusing imaging optical system 7. In the second embodiment, the relationship between the first objective lens 12 to be used and the aperture amount of the variable aperture 2 ′ is the relationship between the first objective lens 12 and the aperture amount of the variable aperture 2 in the first embodiment. It becomes the same as Table 1 and Table 2, respectively.

  As shown in the first and second embodiments described above, the numerical aperture and reflected light of the focus illumination light corresponding to the position (the focus illumination optical system 5 or the focus imaging optical system 7) where the variable stop is disposed. By configuring such that at least one of the numerical apertures of the first and second numerical apertures can be changed, the observation position is automatically followed even when the deep portion of the specimen is observed using the first objective lens 12 having a high numerical aperture, and time-lapse observation or the same position It is possible to realize an autofocus device 1 capable of performing autofocus control so that the image can be observed many times.

DESCRIPTION OF SYMBOLS 1 Autofocus apparatus 2,2 'Variable stop 5 Focusing illumination optical system 7 Focusing imaging optical system 8 Focus position adjusting lens (imaging position adjusting member)
12 First objective lens (objective lens) 18 Sample 20 LED light source (focusing illumination light source)
22 Slit plate (focusing illumination light source)
30 CCD sensor for autofocus (photoelectric converter)
31 Signal processing unit for autofocus (signal output unit)
32 Movement amount detection unit 34 Stage drive unit 35 Objective lens drive unit 41 CPU (control unit) 42 Memory (storage unit) 100 Microscope

Claims (4)

An observation optical system for observing the light irradiated on the specimen through the objective lens;
An autofocus device that maintains the focal point of the objective lens at a desired position with respect to the specimen by controlling the operation of a drive unit that changes the relative position between the specimen and the objective lens,
The autofocus device is
A focusing illumination optical system for forming a predetermined projection image at a position different from the focal position of the objective lens via the objective lens on the specimen;
An imaging optical system for focus that receives reflected light from the specimen of the projected image through the objective lens and forms the reflected image of the projected image on an imaging surface of a photoelectric converter;
A signal output unit that outputs a signal for controlling the operation of the drive unit based on the signal of the reflected image obtained by the photoelectric converter;
The focusing illumination optical system includes an imaging position adjusting member that moves an imaging position of the projected image in an optical axis direction,
A variable stop is provided in at least one of the focus illumination optical system and the focus imaging optical system, and the aperture diameter of the variable stop is controlled according to the amount of movement of the imaging position by the imaging position adjusting member. Accordingly, at least one of the numerical aperture of the focusing illumination optical system and the numerical aperture of the focusing imaging optical system can be changed. A movement amount detection unit for detecting a movement amount of the imaging position by the imaging position adjusting member;
The microscope according to claim 1, further comprising: a control unit that changes an opening diameter of the variable diaphragm according to the movement amount detected by the movement amount detection unit. A storage unit for storing an operation amount of the imaging position adjusting member;
The microscope according to claim 1, further comprising: a control unit that changes an opening diameter of the variable diaphragm according to the operation amount. 4. The control unit according to claim 2, wherein the control unit changes a light amount of a focus illumination light source for forming the projection image by the focus illumination optical system according to an aperture diameter of the variable stop. 5. The microscope described.

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