阅读说明：本技术 自动对焦装置和具备其的光学装置以及显微镜 (Autofocus device, optical device provided with same, and microscope ) 是由 安井真人 广岛通夫 上田昌宏 于 2019-01-24 设计创作，主要内容包括：自动对焦装置,包括：支撑载置试样(20)的玻璃部件(19)的载物台(ST)；观察试样(20)的放大光学系统(L2、HM、DM、OL)；经过放大光学系统对试样(20)发射光的光源装置(11、M、13)；配置在和放大光学系统的试样(20)相反的位置,限制从光源装置发射的发射光的光圈(IR)；经由放大光学系统接收反射光的AF用相机(22),该反射光为经由光圈(IR)以及放大光学系统到达玻璃部件(19)的发射光在反射面(S)所反射的反射光。光源装置相对于放大光学系统的轴以非零的角度(φ)发射光。控制装置(100)调整载物台(ST)的位置,以使被拍摄的遮蔽物的像的位置和目标位置一致。通过设置这种结构,能够实现高速的自动对焦。(An auto-focusing apparatus comprising: a Stage (ST) for supporting a glass member (19) on which a sample (20) is placed; a magnifying optical system (L2, HM, DM, OL) for observing the sample (20); light source devices (11, M, 13) for emitting light to the sample (20) via the magnifying optical system; an aperture (IR) disposed at a position opposite to the sample (20) of the magnifying optical system and limiting the emitted light emitted from the light source device; and an AF camera (22) which receives, via the magnifying optical system, reflected light which is reflected by the reflecting surface (S) of the emitted light reaching the glass member (19) via the aperture (IR) and the magnifying optical system. The light source device emits light at a non-zero angle (phi) with respect to an axis of the magnifying optical system. The control device (100) adjusts the position of the Stage (ST) so that the position of the image of the shield object to be captured matches the target position. With this configuration, high-speed autofocus can be realized.)

1. An autofocus device for an optical device having a stage for supporting a transparent member on which an observation target object is placed, and a magnifying optical system for observing the observation target object, the autofocus device comprising:

a light source device that emits light to the observation target object through the magnifying optical system;

a shielding object arranged at a position opposite to the observation object with respect to the magnifying optical system, and configured to limit the emitted light emitted from the light source device;

a light detection device that receives, via the magnifying optical system, reflected light that is reflected by a reflection surface of the transparent member from the light source device, the reflected light reaching the reflection surface via the shield and the magnifying optical system;

a control device that controls a position of the stage or the magnifying optical system;

the control device adjusts the position of the stage or the magnifying optical system based on reflected light of the shielding object obtained by causing the emitted light from the light source device limited by the shielding object to enter the observation object under a plurality of different conditions.

2. The autofocus device according to claim 1, wherein the light source device is configured to variably adjust an angular distribution of light emitted from the shield.

3. The autofocus device of claim 1, further comprising an optical element that reflects a portion of the emitted light from the light source device that is limited by the shield;

the light emitted from the light source device reflected by the optical element is incident on the observation target.

4. The autofocus device of claim 1, further comprising an optical element that masks, obscures, or reflects a portion of the emitted light from the light source device that is limited by the shield;

the emitted light from the light source device that passes without being shielded, dimmed, or reflected by the optical element is incident on the observation target.

5. The autofocus apparatus according to any one of claims 2 to 4, wherein the control device determines a control target value based on the positions of the reflected images of the shielding object obtained under the plurality of different conditions, and adjusts the position of the stage or the magnifying optical system.

6. The autofocus apparatus according to any one of claims 2 to 4, wherein the control device determines a control target value based on light intensity accumulated by dividing the image of the shielding object obtained under the plurality of different conditions into a plurality of images, and adjusts the position of the stage or the magnifying optical system.

7. An autofocus device for an optical device having a stage for supporting a transparent member on which an observation target object is placed, and a magnifying optical system for observing the observation target object, the autofocus device comprising:

a light source device that emits light to the observation target object through the magnifying optical system;

a shielding object arranged at a position opposite to the observation object with respect to the magnifying optical system, and configured to limit the emitted light emitted from the light source device;

an imaging device that receives reflected light, which is reflected by a reflection surface of the transparent member from the light source device and reaches the reflection surface via the shield and the magnifying optical system, via the magnifying optical system;

a control device that controls a position of the stage or the magnifying optical system;

the light source device emits light at a non-zero angle with respect to an axis of the magnifying optical system;

the control device adjusts the position of the stage or the magnifying optical system so that the position of the image of the shielding object captured by the capturing device coincides with the target position.

8. The autofocus apparatus according to claim 7, wherein the control device adjusts a position of the stage or the magnifying optical system so that a position of the opening of the shielding object in the image of the shielding object captured by the imaging device matches the target position.

9. The autofocus apparatus according to claim 7 or 8, wherein the control device separates the inside and the outside of the opening in the image of the shield by performing image processing on the image obtained by the imaging device.

10. The autofocus device according to any one of claims 7 to 9, wherein the light source device is configured to vary an angle of light emitted to the shield;

the control device adjusts the position of the stage or the magnifying optical system so that a difference between a first position and a second position is a target value, the first position being a position of the image of the shielding object when the light source device emits light at a first angle, and the second position being a position of the image of the shielding object when the light source device emits light at a second angle different from the first angle.

11. The autofocus device according to any one of claims 7 to 9, wherein the light source device is configured to vary an angle of light emitted to the shield;

the control device coarsely adjusts the position of the stage or the magnifying optical system according to a first position, and finely adjusts the position of the stage or the magnifying optical system according to a second position, wherein the first position is a position of the image of the shielding object when the light source device emits light at a first angle, and the second position is a position of the image of the shielding object when the light source device emits light at a second angle larger than the first angle.

12. The autofocus device of claim 10 or 11, the light source device comprising:

a light source that emits light having high straightness;

an electromotive optical element configured to receive the light emitted from the light source and change an angle at which the light emitted from the light source device is incident on the shield;

the control device changes the angle of the electro-optical element when the angle of the light emitted from the light source device is the first angle and when the angle of the light is the second angle.

13. The autofocus device of any one of claims 7 to 12, the magnifying optical system comprising:

an objective lens;

a half mirror;

a light source side imaging lens arranged on one of an optical path transmitted by the half mirror and an optical path reflected by the half mirror;

a camera-side imaging lens disposed on the other of the optical path transmitted by the half mirror and the optical path reflected by the half mirror.

14. The autofocus device of any of claims 7 to 12, the light source device emitting polarized light;

the magnifying optical system includes:

an objective lens;

a quarter-wave plate;

a polarizing beam splitter;

an imaging lens disposed between the quarter wave plate and the polarizing beamsplitter.

15. The autofocus device according to any one of claims 7 to 12, wherein the control device adjusts the position of the stage or the magnifying optical system based on coordinates of the center of gravity of the image of the opening in the image of the shield.

16. The autofocus device according to any one of claims 7 to 14, wherein the control device adjusts the position of the stage or the magnifying optical system based on coordinates of an edge of the image of the opening in the image of the shield.

17. An optical device, comprising:

the object stage;

the magnifying optical system;

the autofocus device of any one of claims 1 to 16.

18. A microscope, comprising:

the object stage;

the magnifying optical system;

the autofocus device of any one of claims 1 to 16.

Technical Field

The present disclosure relates to an autofocus device, and an optical device and a microscope provided with the autofocus device.

Background

Generally, it is known that a microscope determines resolution by Numerical Aperture (NA). Although the resolution of the obtained image becomes better if the numerical aperture is large, the focal depth becomes short, and as a result, focusing becomes difficult.

Automation of large numerical aperture microscopes such as super resolution imaging has not been widespread. Why automation has not been widespread, that is because there is no auto-focusing technique that combines high precision, wide range, and high speed.

Although there are many conventional methods for automatic focusing, the methods are mainly classified into the following two methods. In any of these autofocus techniques, there is a problem in one of accuracy, range, and speed.

Fig. 30 is a diagram for explaining a conventional first method. The first method is a method of observing the contrast of an image of a diaphragm captured by a camera. In this method, as shown in fig. 30, the diaphragm 502 is disposed at a position conjugate to the sample located on the bottom surface of the glass container 501, and an image of the diaphragm 502 is formed at the position of the sample (the bottom surface of the glass container). Then, the camera 503 captures an image of the aperture reflected from the glass surface. The object is moved up and down by scanning the stage to focus the image of the sample on the aperture. If the first method is used, even a microscope with a large numerical aperture can be automatically brought into focus with high accuracy.

The first method has a problem in that scanning of the stage is required at the time of focusing, which takes time. Although depending on the speed of the stage scan, it takes about several tens of seconds or so. Further, even if the speed is increased by forming a multi-focus system by providing two cameras to eliminate stage scanning, there is a problem that the range in which an image of a diaphragm is visible is narrow.

Fig. 31 is a diagram for explaining a conventional second method. The second method is a method of observing the reflection position of the glass surface to focus. Light from LED561 passes through objective lens 523 and is incident angularly toward sample 522. Then, the position of the reflected light is obtained by the CCD camera 564. The second method can always maintain the focus because the control can be performed such that the position of the light beam reaches the center.

The second method has a problem in that the accuracy of focusing is poor. The phenomenon of deterioration in accuracy is caused by skew of the optical system. Due to the shift of the optical system, if the position of the light beam is shifted by X, the position of the incident light is shifted by X/(multiple). Since such a deviation is generated by various external factors such as heat and vibration, adjustment for compensation is required every time. In addition, the range and accuracy of focusing depends on the angle at which the light beam is incident toward the glass surface S. Thus, a dilemma arises as to which of precision and range takes precedence.

Disclosure of Invention

Problems to be solved by the invention

At present, it is difficult to realize an automatic focusing technique having high precision, wide range and high speed at the same time. The reason why these three elements are required for automation of a microscope with a high numerical aperture is as follows. First, since the depth of focus of a microscope with a large numerical aperture is short, the image is blurred if it is not autofocus with high accuracy. In addition, if the accuracy is not maintained for a long time, focusing is required each time, which hinders automation. Second, a shift occurs between the observation sample and the objective lens when the stage moves. If the offset is not within the range of automatic focusing, automatic focusing cannot be performed. Third, in order to efficiently capture a sample, a technique for quickly focusing on a focus is required. As described above, for automation of microscopic observation, an autofocus technique having high precision, a wide range, and high speed is essential.

The object of the present invention is to provide an autofocus device capable of automatically focusing with high accuracy, a wide range, and high speed even in a microscope having a large numerical aperture, and an optical device and a microscope provided with the autofocus device.

Means for solving the problems

In one aspect, the present disclosure relates to an autofocus device for an optical device having a stage that supports a transparent member on which an observation target object is placed, and a magnifying optical system that observes the observation target object. The automatic focusing device includes: a light source device that emits light to an observation target object through a magnifying optical system; a shielding object which is arranged at a position opposite to the observation object relative to the magnifying optical system and limits the emitted light emitted from the light source device; a light detection device that receives, via the magnifying optical system, reflected light that reaches the reflecting surface of the transparent member via the shielding object and the magnifying optical system and is reflected by the reflecting surface from the light source device; and a control device for controlling the position of the stage or the magnifying optical system. And a control device for adjusting the position of the stage or the magnifying optical system based on the reflected light of the shielding object obtained by making the emitted light from the light source device limited by the shielding object enter the observation object under a plurality of different conditions.

Preferably, the light source device is configured to be capable of variably adjusting the angular distribution of light emitted to the shield.

Preferably, the autofocus device further includes an optical element that reflects a portion of the emitted light from the light source device that is confined by the shield. The light emitted from the light source device reflected by the optical element is incident on the observation target.

Preferably, the autofocus device further includes an optical element that shades, dims, or reflects a portion of the emitted light from the light source device that is limited by the shade. The light emitted from the light source device that passes without being blocked, attenuated, or reflected by the optical element enters the observation target.

More preferably, the control device determines the control target value based on the positions of the reflected images of the shielding object obtained under a plurality of different conditions, and adjusts the position of the stage or the magnifying optical system.

More preferably, the control device determines the control target value based on light intensities integrated by dividing the image of the shielding object obtained under a plurality of different conditions into a plurality of images, and adjusts the position of the stage or the magnifying optical system.

In other aspects, the present disclosure relates to an autofocus device for an optical device having a stage supporting a transparent member on which an observation target object is placed, and a magnifying optical system for observing the observation target object. The automatic focusing device includes: a light source device that emits light to an observation target object through a magnifying optical system; a shielding object which is arranged at a position opposite to the observation object relative to the magnifying optical system and limits the emitted light emitted from the light source device; an imaging device that receives, via an enlarging optical system, reflected light that reaches the reflecting surface of the transparent member via the shielding object and the enlarging optical system and is reflected by the reflecting surface from the light source device; and a control device for controlling the position of the stage or the magnifying optical system. The light source device emits light at a non-zero angle with respect to an axis of the magnifying optical system. The control device adjusts the position of the stage or the magnifying optical system so that the position of the image of the shielding object captured by the imaging device matches the target position.

Preferably, the control device adjusts the position of the stage or the magnifying optical system so that the position of the opening of the shielding object in the image of the shielding object captured by the imaging device coincides with the target position.

Preferably, the control device separates the inside and the outside of the opening in the image of the shield by performing image processing on the image obtained by the imaging device.

Preferably, the light source device is configured to vary an angle of light emitted to the shield. The control device adjusts the position of the stage or the magnifying optical system so that the difference between a first position where the light source device emits light at a first angle and a second position where the light source device emits light at a second angle different from the first angle is a target value.

Preferably, the light source device is configured to vary an angle of light emitted to the shield. The control device roughly adjusts the position of the stage or the magnifying optical system based on a first position where the light source device emits light at a first angle and finely adjusts the position of the stage or the magnifying optical system based on a second position where the light source device emits light at a second angle larger than the first angle.

More preferably, the light source device includes: a light source that emits light having high straightness; and an electromotive optical element configured to receive the light emitted from the light source and change an angle at which the light emitted from the light source device is incident on the shield. The control device changes the angle of the electro-optical element when the angle of the light emitted from the light source device is a first angle and when the angle of the light is a second angle.

Preferably, the magnifying optical system includes: the imaging lens is arranged on the light source side of one of the light path transmitted by the semitransparent mirror and the light path reflected by the semitransparent mirror, and the imaging lens is arranged on the camera side of the other of the light path transmitted by the semitransparent mirror and the light path reflected by the semitransparent mirror.

Preferably, the light source device emits polarized light; the magnifying optical system includes: an objective lens, a quarter-wave plate, a polarizing beam splitter, and an imaging lens disposed between the quarter-wave plate and the polarizing beam splitter.

Preferably, the control device adjusts the position of the stage or the magnifying optical system based on coordinates of the center of gravity of the image of the opening in the image of the shielding object.

Preferably, the control device adjusts the position of the stage or the magnifying optical system based on coordinates of an edge of the image of the opening in the image of the shielding object.

The disclosure, in other aspects, relates to an optical device comprising: an object stage; an amplifying optical system; the autofocus device of any of the above.

The disclosure further relates in other aspects to a microscope comprising: an object stage; an amplifying optical system; the autofocus device of any of the above.

Effects of the invention

According to the present invention, it is possible to realize automatic focusing that can perform automatic focusing with high accuracy, a wide range, and high speed. By using the invention, the microscope shooting with large numerical aperture can be more easily automated.

Drawings

Fig. 1 is a diagram showing a configuration of an optical system of a microscope according to the present embodiment.

Fig. 2 is a block diagram showing an object controlled by the control device.

Fig. 3 is a diagram showing the principle of autofocus performed in embodiment 1.

Fig. 4 is a diagram for explaining a modification in the case where a slit is used instead of the diaphragm in embodiment 1.

Fig. 5 is a diagram showing a relationship between the position of the center of gravity of the aperture image of the aperture and the position of the stage when the incident angle is Φ L.

FIG. 6 is a view showing the relationship between the position of the center of gravity of an aperture image of a diaphragm and the position of a stage when the incident angle is φ H (> φ L).

Fig. 7 is a schematic diagram showing an optical system of the automatic focusing apparatus according to the present embodiment.

Fig. 8 is a flowchart for explaining the control of autofocus in embodiment 1.

Fig. 9 is a diagram showing the principle of autofocus performed in embodiment 2.

Fig. 10 is a diagram for explaining a modification in the case of using a slit instead of a diaphragm in embodiment 2.

Fig. 11 is a flowchart for explaining the control of autofocus in embodiment 2.

Fig. 12 is an image of a slit captured by an AF camera.

Fig. 13 is a diagram showing a configuration of an optical system of a microscope according to embodiment 3.

Fig. 14 is an image when the incident angle is a positive angle.

Fig. 15 is an image when the incident angle is a negative angle.

Fig. 16 is a diagram for explaining the detection of an edge.

Fig. 17 is a flowchart for explaining the control of autofocus according to embodiment 3.

Fig. 18 is a diagram showing a configuration of an optical system of a microscope according to embodiment 4.

Fig. 19 is a diagram showing the positional relationship between the shape of the turning mirror RM1 and the light B in fig. 18.

Fig. 20 is a view showing a state in which the rotating mirror RM1 is rotated by 180 ° in the microscope 201 shown in fig. 18.

Fig. 21 is a diagram showing a positional relationship between the state of the turning mirror RM1 shown in fig. 20 and the light B.

Fig. 22 is a diagram showing a configuration of an optical system of a microscope according to embodiment 5.

Fig. 23 is a diagram showing the positional relationship between the shape of the rotating cover RM2 and the light B in fig. 22.

Fig. 24 is a view showing a state in which the rotating cover RM2 is rotated by 180 ° in the microscope 251 shown in fig. 22.

Fig. 25 is a diagram showing a state of the rotating cover RM2 shown in fig. 24 and a positional relationship of the light B.

Fig. 26 is a diagram showing a configuration of an optical system of a microscope according to embodiment 6.

Fig. 27 is a diagram showing the shape of the rotating cover RM3 of fig. 26 and the positional relationship of the light B.

Fig. 28 is a view showing a state in which the rotating cover RM3 is rotated by 180 ° in the microscope 271 shown in fig. 26.

Fig. 29 is a diagram showing the positional relationship between the state of the rotating cover RM3 shown in fig. 28 and the light B.

Fig. 30 is a diagram for explaining a conventional first method.

Fig. 31 is a diagram for explaining a conventional second method.

Detailed Description

Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the drawings, the same or corresponding portions are denoted by the same reference numerals, and description thereof will not be repeated.

[ summary ]

The present embodiment relates to an autofocus device having high precision, wide range, and high speed. Fig. 1 is a diagram showing a configuration of an optical system of a microscope according to the present embodiment. The light from the AF laser 11 is reflected by the motor mirror M and enters the diaphragm IR. Since the diaphragm IR and the galvano mirror M are in a conjugate relationship, the angle θ of the galvano mirror M can control the incident angle Φ of the light toward the diaphragm IR. The light from the diaphragm IR passes through the imaging lens L2 and the objective lens OL, reaches the surface (glass surface S) of the glass 19 on which the sample 20 is placed, and is reflected. The reflected light passes through the objective lens OL and the imaging lens L1, is imaged by the camera for auto focusing (camera for AF) 22, and an image of the diaphragm IR is projected by the camera for AF 22.

The present embodiment is characterized in that the shaped light is irradiated onto the glass surface S on which the sample 20 is placed on the stage ST at an angle, and the reflected image is observed.

In contrast, in the first method disclosed in patent document 1, light is not irradiated perpendicularly to the sample at an angle, and a reflected image is observed to be blurred. The present embodiment is different in that the reflected image is observed at a position where the light is incident at an angle phi. In the present embodiment, by measuring the position of the image of the diaphragm IR in the reflected image with incidence at an angle Φ, it is possible to know in which direction and to what extent the focal point is shifted from the glass surface S. This is associated with a high speed of focusing.

On the other hand, in the second method disclosed in patent document 2, the light is incident without being shaped, and the position of the center of gravity of the light is observed. The technique of the present embodiment is different in that incident light is shaped using the diaphragm IR. Since the shape of the image is determined by the diaphragm IR, the reflected image from the sample can be eliminated by image processing. This is associated with high precision of auto-focusing. Further, patent document 2 is also different in that the straightness of light irradiated to the glass surface is low. If the straightness of light is low, the reflected light is easily lost and the range is reduced because the image changes significantly due to the change in focus. Further, the present method is different in that the incident angle can be adjusted. By adjusting the angle of incidence, the balance between range and accuracy can be adjusted.

The outline of the procedure of using the autofocus apparatus according to the present embodiment is as follows. First, the user determines how far away from the glass surface S the user focuses. The position of the diaphragm IR in the direction D is moved according to the position determined by the user. Then, the angle θ of the mirror M is set so as to provide the incident angle Φ L, and the reflected image of the diaphragm IR is captured by the AF camera 22, and the position of the diaphragm IR on the image is determined. The Z stage, or the objective lens OL or the position of another lens (a lens before the camera or the diaphragm), or both, are moved so that the position of the diaphragm IR coincides with the target position. The above steps are repeated until the difference between the target positions is below a specified value. After the automatic focusing is completed at the incident angle phi L, the automatic focusing is performed at the incident angle phi H larger than the incident angle phi L in the same manner as the above-described sequence. This method enables high-precision, wide-range, and high-speed autofocus as compared with conventional methods.

[ embodiment 1]

The automatic focusing device of the embodiment is beneficial to more easily realizing the automation of the microscope. The microscope 1 shown in fig. 1 includes a stage ST, an optical system for auto-focusing, and an optical system for observation.

The microscope 1 includes a light source, a diaphragm IR, an imaging lens L2, a half mirror HM, a dichroic mirror DM, an objective lens OL, an imaging lens L1, and an AF camera 22 as an optical system for auto focusing, wherein the light source includes an AF laser 11, a galvano mirror M, and a kepler beam expander 13.

Further, the microscope 1 includes, as optical systems for observation, an excitation filter 23, a dichroic mirror 24 for observation, absorption filters 25 and 28, an imaging lens 26, and a camera 27 for observation.

In the optical system for auto-focusing, the laser beam reflected by the motor mirror M passes through the beam expander 13 and enters the diaphragm IR. The incidence angle phi of the laser light toward the diaphragm IR can be controlled by the angle theta of the motorized mirror M. Since the diaphragm IR and the glass surface S are at conjugate positions, the shadow of the diaphragm IR is imaged on the glass surface S. The image of the diaphragm IR is reflected and projected onto the AF camera 22.

In the optical system for observation, light having a wavelength that passes through the dichroic mirror DM for AF and the absorption filter 28 can be used. Specifically, the sample 20 can be irradiated with light through the dichroic observation mirror 24. Then, the fluorescence or reflected light can be observed by the observation camera 27. The absorption filter 28 absorbs only light from the AF light source, and prevents leakage light from the AF light source from entering the observation camera 27.

The microscope 1 further includes a control device 100, and the control device 100 controls the angle θ of the galvano-mirror M, the position of the diaphragm IR in the D direction, and the position of the stage ST. The control device 100 may control the position of the objective lens OL or other lens (lens before the camera or the diaphragm) or both of them without controlling the position of the stage ST. Hereinafter, for the sake of explanation, the control device 100 will be described as controlling the position of the stage ST.

Fig. 2 is a block diagram showing an object controlled by the control device. The control device 100 adjusts the position of the diaphragm IR in the D direction by the diaphragm position adjustment unit 101. The control device 100 adjusts the angle θ of the galvano mirror M by the mirror angle adjusting unit 102. The control device 100 drives the stage position adjustment unit 103 based on the position in the image of the diaphragm IR captured by the AF camera 22, and controls the Z-direction position of the stage ST.

Fig. 3 is a diagram showing the principle of autofocus performed in embodiment 1. The laser beam is incident on the sample 20 at an incident angle corresponding to the incident angle phi toward the aperture IR. At this time, in the AF camera 22, an image of the diaphragm IR is observed as shown in the camera image P1. The image of the diaphragm IR is also shifted in position in the image according to the Z position of the glass surface S from the focal position.

A target position XT of an image of the diaphragm IR corresponding to a pre-focusing position is set, and a difference dx between a position X1 of the image of the diaphragm IR obtained from a camera image P1 and the target position XT is calculated. When the difference dx is zero, it is the in-focus position. Since the direction and the amount of movement of the stage ST on which the glass surface S is mounted are known from the difference dx, continuous focusing can be performed at high speed.

Further, although a sensor such as a divided photodiode may be used instead of the AF camera 22, the accuracy is poor. This is because the positioning accuracy of the image of the diaphragm IR is deteriorated by the reflected image.

The aperture portion of the iris IR allows a sample image such as a cell to be observed. Therefore, since the screen of the opening portion of the diaphragm IR is an image of the sample depending on the cell or the like, the reflection of the diaphragm IR is not uniform. Therefore, if the position of the image of the diaphragm IR is determined by the weighted center of gravity of the divided photodiode, the focus shifts depending on the image of the sample. In order to solve this problem and improve the accuracy of the automatic focusing, it is preferable to perform image separation by image processing (for example, binarization processing or contour extraction processing) so that the opening is a white image and the shielded portion by the diaphragm IR is a black image. Thus, high-precision auto-focusing independent of the sample can be realized.

Fig. 4 is a diagram for explaining a modification in the case of using a slit instead of a circular diaphragm in embodiment 1. In the case of fig. 3, the difference dx is calculated from the position of the weighted center of gravity of the image of the diaphragm IR, but in this case of the slit, the difference dx may be calculated by calculating X1 by simple calculation or by directly using the X coordinate of the image. The shape of the diaphragm IR may be various shapes such as a star shape and a polygon shape, in addition to the circular shape in fig. 3 and the slit shape in fig. 4.

In the present embodiment, autofocus can be performed by fixing the incident angle Φ to a predetermined angle other than zero. The angle of incidence phi is fixed and the position of the image of the aperture IR is determined. Then, the stage ST is moved to adjust the focus so that the position of the image of the diaphragm IR moves to the target position. In this method, since the angle of the galvano mirror M is not changed, it is possible to perform autofocus at high speed and prevent wear of the driving portion of the galvano mirror M.

On the other hand, by changing the angle of incidence Φ toward the diaphragm IR at the time of auto-focusing, the accuracy and range can be adjusted. This relationship is compared between the case where the incident angle phi is large and the case where the incident angle phi is small, and the explanation is made. Fig. 5 is a diagram showing a relationship between the position of the center of gravity of the aperture image of the aperture and the position of the stage when the incident angle is Φ L. FIG. 6 is a view showing the relationship between the position of the center of gravity of an aperture image of a diaphragm and the position of a stage when the incident angle is φ H (> φ L). In fig. 5 and 6, the horizontal axis represents the pixel position (px: pixel) indicating the center of gravity of the aperture image of the diaphragm IR, and the vertical axis represents the position (μm) in the Z direction of the stage that moves the glass surface of the sample.

When the incident angle Φ is Φ H (when the incident angle Φ is large), the amount of beam shift increases, and the accuracy increases (approximately 50 nm/px). However, the larger the amount of movement, the narrower the range (the automatically-focusing possible Z-direction range). On the other hand, when the incident angle Φ is equal to Φ L (when the incident angle Φ is small), the amount of movement of the light beam is small, and the accuracy is large (about 760 nm/px). However, the range (the automatically-focusing possible Z-direction range) becomes wide.

(order of automatic focusing)

In embodiment 1, the following method can achieve both a wide range and high accuracy in autofocus. Initially, autofocus was started using a wide range of incident angles (Φ ═ Φ L), and some degree of coarse focus. Then, focusing is performed with high accuracy using a narrow range of incident angles (phi ═ phi H). Thus, the automatic focusing can be performed in a wide range and with high accuracy.

Fig. 7 is a schematic diagram showing an optical system of the automatic focusing apparatus according to the present embodiment. Fig. 8 is a flowchart for explaining the control of autofocus in embodiment 1.

As a premise of the automatic focusing control, the user sets how far from the glass surface S the focusing is performed. The position of the focal point and the position of the diaphragm IR in the optical axis direction are linearly related, and when the diaphragm IR is located at the focal point of the imaging lens L2, the focus is formed on the glass surface S. Therefore, the distance of the diaphragm IR is uniquely determined to be DT from the distance from the glass surface S. As other parameters set in advance, there are angles θ L and θ H of the galvano mirror corresponding to the stage control of the coarse movement and the fine movement, respectively, and convergence determination values L and H at the time of determining the focusing corresponding to the angles θ L and θ H, respectively. These are basically user-independent, and are certain values determined by the developer of the autofocus apparatus.

In step S1, the control device 100 sets the position D of the diaphragm IR to the position DT. Then, in step S2, the control device 100 determines to set the angle of incidence Φ toward the diaphragm IR to Φ L of coarse accuracy.

In step S3, the control device 100 sets the angle θ of the galvano mirror M to the angle formed by the incident angle Φ (═ Φ L). In addition, the determination convergence value is set to (═ L). Then, in step S4, the control device 100 acquires a reflected image by the AF camera 22, and calculates the position X1 of the center of gravity of the diaphragm IR.

Next, in step S5, the control device 100 calculates a difference dx (X1-XT) between the X coordinates of the center of gravity, and in step S6, moves the stage ST in the Z direction by a movement amount corresponding to the direction corresponding to the difference dx. In step S7, the control device 100 determines whether the difference dx is smaller than the determination convergence value. In step S7, if not the difference dx < (NO at S7), steps S3 to S6 are executed again.

In step S7, if it is the difference dx < (YES at S7), then in step S8, it is determined whether coarse focusing has been performed (whether the setting of the angle of incidence Φ is Φ L). If the rough focus is performed in step S8 (YES in S8), control device 100 changes the incident angle to Φ H and the determination convergence value to H in step S9, and then executes the processing of steps S3 to S7 to perform the high-precision auto focus. In step S8, if the focus is not the rough focus (Φ ≠ Φ L), the control device 100 ends the autofocus since the high-precision autofocus has been executed (NO in S8).

According to the present embodiment, since the moving direction and the moving amount of the stage can be immediately calculated from the position of the image of the diaphragm IR by setting the incident angle Φ to a non-zero angle, high-speed auto-focusing can be performed.

Although the autofocus is possible even with a fixed incident angle Φ, the accuracy and range of the autofocus can be adjusted by controlling the incident angle Φ of the light toward the sample 20 to Φ L and Φ H with the galvano-mirror M. When the angle of incidence φ to the stop IR is large, if the glass surface S is moved in the vertical direction, the center of gravity of the image of the stop IR on the glass surface S is largely moved. As a result, although the image of the diaphragm IR is likely to deviate from the AF camera 22 and the range is narrowed, the positional change of the image is large, and therefore the accuracy is high. Conversely, if the incident angle Φ toward the diaphragm IR is small, the range becomes wide although the accuracy becomes poor. The incident angle phi is first reduced and autofocus is performed in a wide range, and then the incident angle phi is increased and autofocus is performed with high accuracy. Thus, automatic focusing with both a wide range and high accuracy can be achieved.

[ embodiment 2]

Although the embodiment 1 can realize high-speed and high-range autofocus, the accuracy is not high because the capability of resisting the optical system shift is weak. Therefore, compensation adjustment is required before or during each observation. In embodiment 2, since autofocus is performed by making light incident from a plurality of directions, the ability to resist optical system misalignment is strong, and stable autofocus can be performed even for a long time.

The outline of the procedure of using the autofocus apparatus according to the present embodiment is as follows. First, the user determines how far from the glass surface S the user focuses. The position of the diaphragm IR in the direction D is moved according to the position determined by the user. Then, the angle θ of the galvano mirror M is set so as to provide the incident angle Φ L, the reflected image of the diaphragm IR is captured by the AF camera 22, and the position X1 of the center of gravity is calculated. Then, the angle θ of the galvano mirror M is set so as to provide an incident angle — Φ L, and the reflected image is again captured, and the position X2 of the center of gravity is calculated. A difference dx between the positions X1 and X2 of the center of gravity is obtained, and the Z stage is moved so that the difference dx becomes zero. The above steps are repeated until the difference dx is below a specified value. After the automatic focusing is completed at the incident angle phi L, the automatic focusing is performed at the incident angle phi H larger than the incident angle phi L in the same manner as the above-described sequence. This method can perform autofocus with high accuracy, a wide range, and high speed, and does not require a preset target position of the diaphragm, as compared with conventional methods.

Fig. 9 is a diagram showing the principle of autofocus performed in embodiment 2. The laser beam B1 is incident on the specimen 20 at an incident angle corresponding to the incident angle phi toward the aperture IR. At this time, in the AF camera 22, an image of the diaphragm IR is observed as shown in the camera image P1. The image of the diaphragm IR is also shifted in position in the image according to the change in the Z position from the focal position of the glass surface S.

The laser beam B2 is made to enter the sample by changing the angle of the galvano mirror M so that the angle of incidence on the diaphragm IR is set to-phi opposite to the angle of incidence phi. At this time, if the Z position of the glass surface S from the focal position changes, the image of the diaphragm IR also changes its position in the image, similarly to the angle Φ. However, unlike at the angle φ, the position of the image of the aperture IR is shifted in the opposite direction.

The in-focus position is when the difference dx between the incident angle phi and the position of the image of the aperture IR at the incident angle-phi is zero. Since the direction and amount of movement of the stage ST on which the glass surface S is mounted are known from the difference dx between the positions of the images of the diaphragm IR in the camera image P1 and the camera image P2, continuous focusing can be performed at high speed.

Further, although a sensor such as a divided photodiode may be used instead of the AF camera 22, the accuracy is poor. Since the positioning accuracy of the image of the diaphragm IR is deteriorated by the reflected image.

Further, the opening portion of the diaphragm IR allows a sample image such as a cell to be observed to be seen. Therefore, since the screen of the opening portion of the diaphragm IR is an image of the sample depending on the cell or the like, the reflection of the diaphragm IR is not uniform. Therefore, if the position of the image of the diaphragm IR is determined by the weighted center of gravity of the divided photodiode, the focus shifts depending on the image of the sample. In order to solve this problem and improve the accuracy of the automatic focusing, it is preferable to perform image separation by image processing (for example, binarization processing or contour extraction processing) so that the opening is a white image and the shielded portion by the diaphragm IR is a black image. Thus, high-precision auto-focusing independent of the sample can be realized.

In embodiment 2, as a method of aligning the image of the diaphragm IR with the target position XT, it is possible to use captured images at an incident angle Φ and an incident angle- Φ. By thus using the taken images at the angle of incidence phi and the angle of incidence-phi, the user does not need to set the target position XT of the image of the iris IR in advance.

In the case of embodiment 2, the accuracy and the range can be adjusted by changing the incident angle Φ toward the diaphragm IR at the time of autofocus. Since the adjustment of the accuracy and the range is explained in fig. 4 and 5, the explanation is not repeated here.

Fig. 10 is a diagram for explaining a modification in the case of using a slit instead of a diaphragm in embodiment 2. In the case of fig. 9, the difference dx is calculated from the position of the weighted center of gravity of the image of the diaphragm IR, but in this case of the slit, the difference dx may be calculated by calculating X1 and X2 by simple calculation using the X coordinate of the image as it is. The shape of the diaphragm IR may be any of various shapes such as a star shape and a polygon shape, in addition to a circular shape and a slit shape.

(order of automatic focusing)