JP5909902B2 - Autofocus device, microscope device - Google Patents

Description

  The present invention relates to an autofocus device for a microscope apparatus and a microscope apparatus having the same.

  In a biological microscope, illuminate the specimen with illumination light, and use the reflected light to position the specimen's observation position at the front focal point of the objective lens (hereinafter also referred to as “focus”) and detect the focus. An auto-focus device that moves the front focus of the objective lens in the direction of the optical axis with respect to the specimen by moving the focus position adjustment lens provided in the optical system along the optical axis of the focus detection optical system (so-called perfect focus) Apparatus) and a microscope apparatus having the same are known (for example, see Patent Document 1).

JP 2004-70276 A

  With the advancement of high-resolution observation technology of microscopes, it is necessary to accurately know the focal position (also referred to as Z position) on the observation object in the perfect focus device, and an autofocus device corresponding to this, Is required.

  The present invention has been made in view of the above problems, and has an autofocus device that can grasp the relationship between the movement of the focal position adjustment lens and the observation position and know the focal position on the observation object, and the same. An object is to provide a microscope apparatus.

In order to solve the above problems, the present invention provides:
An objective lens;
An observation optical system that forms an image of the object through the objective lens;
A focusing optical system for projecting and detecting a focusing optical image on the object via the objective lens;
An imaging position adjusting means arranged in the focusing optical system, for adjusting an offset amount between the imaging position of the observation optical system and the imaging position of the focusing optical system;
Z position detecting means for detecting a focal position of the objective lens with respect to the observation optical system;
X position detection means for detecting the position of the imaging position adjustment means;
A storage unit for storing a coordinate table in which the X position information from the X position detection unit and the Z position information from the Z position detection unit are associated;
A control unit,
The controller is
The relationship between the X position information and the Z position information acquired immediately before first observing the object, and the relationship between the X position information and the Z position information acquired before observing the object after a predetermined time has elapsed. Are stored in the coordinate table,
Comparing the initial relationship with the relationship after the lapse of the predetermined time , and based on the XZ relationship in which the deviation of the relationship between the X position information and the Z position information due to the time change is corrected , The autofocus device is characterized in that the Z position after the elapse of the predetermined time is positioned at the initial Z position .

  The present invention also provides a microscope apparatus comprising the autofocus device.

  ADVANTAGE OF THE INVENTION According to this invention, the autofocus apparatus which can grasp | ascertain the relationship between the movement of a focus position adjustment lens and an observation position, and can know the focus position on an observation target object, and a microscope apparatus which has this can be provided.

The figure which shows typically the structure of the optical system and control system of the microscope apparatus concerning embodiment. An example of a change in the focal position adjusting lens position (X position) and the front focal position (Z position) due to a temperature change is shown. An example of the coordinate table which matched the focal position adjustment lens position (X position) and the front side focal position (Z position).

  A microscope apparatus having an autofocus device according to an embodiment of the present application will be described below with reference to the drawings. The following embodiments are only for facilitating the understanding of the invention, and excluding additions and substitutions that can be performed by those skilled in the art without departing from the technical idea of the present invention. It is not intended.

  FIG. 1 is a schematic configuration diagram of a microscope apparatus equipped with an autofocus device according to an embodiment. This microscope apparatus forms an enlarged image of a specimen that is an observation object and uses it for observation or image acquisition. The specimen 48 that is an observation object is covered with a medium such as water. It is placed on the stage 11 between the glass 14 and the slide glass 15.

  In FIG. 1, the optical system of the microscope apparatus according to the present embodiment is an optical system of the observation optical system 3 disposed at the upper part of the specimen and the autofocus apparatus according to the present embodiment disposed on the side thereof. A focusing illumination optical system 5 and a focusing imaging optical system 7 are included.

  The focusing illumination optical system 5 includes, on its optical axis, in order from the LED light source 20 side, a first collector lens 21, a slit plate 22, a second collector lens 23, a first pupil limiting mask 24, a first half mirror 25, A focal position adjusting lens 8 and a visible light cut filter 10 are arranged. A rectangular elongated slit aperture 22a is formed at the center of the slit plate 22, and 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 is collected by the first collector lens 21 and enters the slit plate 22, and is the focal position of the infrared light. The first pupil passes through the sample surface (the boundary surface between the cover glass 14 and the medium in which the sample is immersed) and the slit aperture 22a of the slit plate 22 arranged at the conjugate position, and is converted into parallel light by the second collector lens 23. The restriction mask 24 is irradiated.

  The first pupil limiting mask 24 shields half of the pupil, and half of the pupil is shielded along the longitudinal center line of the slit-shaped infrared light around the optical axis of the focusing illumination optical system 5. It is arranged like this. The infrared light La that has passed through the first pupil restriction mask 24 passes through the first half mirror 25. The first half mirror 25 is disposed at a position where the optical axes of the focusing illumination optical system 5 and the focusing imaging optical system 7 intersect, and reflects a part of the infrared light, and the other A part of the light is transmitted through the focusing imaging optical system 7 as will be described later.

  A dichroic mirror 16 is disposed at a position 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 functions to reflect infrared light and transmit visible light and fluorescence.

  The infrared light La that has passed through the first half mirror 25 passes through the focus position adjusting lens 8, and then the visible light component contained in the infrared light La is removed by the visible light cut filter 10, and then the objective is taken by the dichroic mirror 16. The light is reflected in the direction of the lens 12 (infrared light Lb) and is collected on the sample surface by the objective lens 12. The objective lens 12 is shared by the observation optical system 3 as will be described later. The focus position adjusting lens 8 will be described later.

  The observation optical system 3 includes an objective lens 12, a dichroic mirror 16, an infrared light cut filter 18, a second half mirror 17, and a second objective lens 13 in order from the sample 48 side. Although not shown, an eyepiece lens is disposed at the tip of the two objective lens 13.

  Although not shown, an illuminating device that illuminates the specimen 48 placed on the stage 11 is provided. 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.

  In the case of transmitted illumination, the light emitted from the illumination device passes through the specimen 48 to become observation light, passes through the objective lens 12, passes through the dichroic mirror 16, and the infrared light is removed by the infrared light cut filter 18. , Enters the second half mirror 17.

  A portion of the observation light incident on the second half mirror 17 is reflected, and an observation image of the specimen 48 is formed by the second objective lens 13 and the eyepiece, and is used for observation. Further, a part of the observation light transmitted through the second half mirror 17 passes through the camera objective lens 36 and the camera relay lens 37 and forms an image on the imaging surface of the camera CCD sensor 38, and the camera signal processing unit. The sample image is projected on a monitor (not shown) by processing at 39.

  The focusing imaging optical system 7 receives slit-like infrared light that is irradiated and reflected on the sample surface on the stage 11 by the focusing illumination optical system 5. Here, since the specimen 48 on the stage 11 is covered with the cover glass 14, the focus detection infrared light imaged by the objective lens 12 is generated on the surface of the cover glass 14, the cover glass 14, and the specimen 48. Reflects at the boundary surface (sample surface). The infrared light reflected by the cover glass 14 or the sample surface is converted into parallel light by the objective lens 12 (infrared light Lc), reflected by the dichroic mirror 16 (infrared light Ld), and further, the visible light cut filter 10. Then, the light passes through the focal position adjusting lens 8, enters the first half mirror 25 that is disposed at an angle of approximately 45 degrees with respect to the optical axis of the focusing illumination optical system 5, and is partially reflected by the first half mirror 25. Then, the light enters the focusing imaging optical system 7.

  The focusing imaging optical system 7 includes a first half mirror 25, an autofocus objective lens 26, an autofocus relay lens 27, and a second pupil restriction mask 28 in order from the focusing illumination optical system 5 side along the optical axis. An autofocus relay lens 27, a cylindrical lens 29, and an autofocus CCD sensor 30 are provided.

  The infrared light Ld reflected by the first half mirror 25 is condensed by the autofocus objective lens 26 and converted into imaging light to form a slit image. The autofocus relay lenses 27, 27 relay the slit image (infrared light Le) formed by the autofocus objective lens 26, pass through the cylindrical lens 29, and slit on the imaging surface of the autofocus CCD sensor 30. 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. 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 functions to form an image on 30 imaging surfaces. The autofocus CCD sensor 30 can be composed of 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.

  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 aperture 22 a of the slit plate 22 and the image of the slit aperture 22 a is irradiated onto the sample surface. This is because when spot light is used, if there is a step on the sample surface, the reflected light is scattered and an ideal light quantity signal cannot be obtained. It is also possible to eliminate the plate 22 and perform autofocus control by irradiating the sample surface 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.

  The focus position adjusting lens 8 used in the autofocus device according to the present invention will be described. As shown in FIG. 1, the focal position adjusting lens 8 is positioned on the common optical path of the focusing illumination optical system 5 and the focusing imaging optical system 7 between the dichroic mirror 16 and the first half mirror 25, and is an afocal system. It is arranged.

  The focal position adjusting lens 8 is provided with a focal position adjusting lens driving unit 9a. Although not shown, the focal position adjusting lens DC motor is capable of moving the focal position adjusting lens 8 back and forth along the optical axis. And an electric turret for a focus position adjusting lens that can exchange a plurality of focus position adjusting lenses 8 having different magnifications. The movement of the focal position adjusting lens driving unit 9a is controlled via a focal position adjusting lens movement control unit of the CPU 41 described later.

  The focal position adjusting lens 8 is provided with an X position detecting unit 9b for detecting the position (X position) of the focal position adjusting lens 8, and the detected X position information is transmitted to the CPU 41.

  Further, limit sensors HL1 (−) and HL2 (+) (not shown) are provided for preventing collision between the objective lens 12 and the specimen 48 when the focal position adjusting lens 8 is moved in the optical axis direction. .

  In addition, a focus position adjusting lens operation dial 51 for moving the focus position adjusting lens 8 along the optical axis and a focus position adjusting lens switching switch (not shown) are disposed in the input unit of the microscope apparatus described later. When the focus position adjusting lens operation dial 51 is operated, the focus position adjusting lens 8 can be reciprocated along the optical axis based on a signal from an encoder (not shown) coupled thereto. Further, an arbitrary focal position adjusting lens 8 can be selected and switched from a plurality of focal position adjusting lenses 8 mounted on the focal position adjusting lens electric turret by the focal position adjusting lens switching switch.

  The operation of the focal position adjustment lens 8 will be described. In FIG. 1, the autofocus illumination light is indicated by a solid line, and the observation light in the observation optical system 3 is indicated by a dotted line.

  The focal position adjusting lens 8 shifts the imaging position of the slit image (autofocus illumination light) condensed and irradiated on the sample surface by the objective lens 12 along the optical axis of the focus illumination optical system 5 and simultaneously the sample surface. The image forming position of the slit image reflected and re-imaged on the imaging surface of the autofocus CCD sensor 30 is shifted along the optical axis of the focusing imaging optical system 7.

  Hereinafter, a method for adjusting the front focal point (hereinafter referred to as “focal point f”) of the observation optical system 3 of the objective lens 12 to the position where the specimen 48 is actually observed while performing autofocus control will be described. The focal position adjusting lens 8 includes a convex lens 8a and a concave lens 8b, and one lens is fixed on the optical axis, and the other lens is arranged to be movable along the optical axis. As a modification, the convex lens 8a and the concave lens 8b may be configured to be movable along the optical axis. In the following description, a case will be described in which the convex lens 8a is fixed on the object side, and the concave lens 8b is disposed behind (on the LED light source 20 side) so as to be movable along the optical axis.

  In the state in which the focus f of the objective lens 12 by the observation optical system 3 is aligned with the boundary surface between the cover glass 14 and the sample 48 (hereinafter referred to as the sample surface), the result of the autofocus slit image irradiated through the slit aperture 22a is obtained. The focal position is adjusted so that the image position (which is the focal position of the objective lens 12 by the illumination optical system 5 for focusing) is also aligned with the sample surface, and the focus of the reflected image is aligned with the imaging surface of the autofocus CCD sensor 30. The position (X position) of the lens 8 (concave lens 8b) is adjusted. In this state, the focal point f of the objective lens 12 by the observation optical system 3 coincides with the focal point (specimen surface) of the objective lens 12 by the focusing illumination optical system 5 and the focusing imaging optical system 7. This position is called “offset zero position” of the slit image by the focal position adjusting lens 8 (corresponding to X = 0 and Z = 0 in FIG. 2). In this state, the slit image is formed on the focal point f of the objective lens 12 by the observation optical system 3, that is, the sample surface, and the reflected light is formed on the imaging surface of the autofocus CCD sensor 30. In this state, the focus position adjusting lens 8 is simply a telephoto system, and the autofocus illumination light is a parallel light beam before and after the focus position adjusting lens 8. When autofocus control described later is applied in this state, the slit image is always the specimen surface, which is the focal point of the objective lens 12 by the focusing illumination optical system 5 and the focusing imaging optical system 7, and the focal point of the objective lens 12 by the observation optical system 3. The state is controlled so that f matches.

  Next, the focal position adjusting lens 8 (concave lens 8b) is moved back and forth along the optical axis, and the autofocus slit image is moved from the sample surface, and the imaging position of the autofocus slit image and the observation optical system 3 are used. The position of the focal point f of the objective lens 12 is shifted (offset). For example, when the concave lens 8b is moved backward (in a direction away from the convex lens 8a) by a distance X1, the imaging position of the autofocus slit image is a predetermined distance from the specimen surface toward the objective lens 12 (this distance is referred to as “offset amount Z1. ”). In this state, when autofocus control, which will be described later, is performed, the sample surface moves because the stage 11 moves, and the image formation position of the autofocus slit image coincides with the sample surface. At this time, the focal point f of the objective lens 12 by the observation optical system 3 moves into the sample 48 by the offset amount Z1 (see X = X1, Z = Z1 in FIG. 2). In this state, the autofocus slit image is on the sample surface, and the focal point f of the objective lens 12 by the observation optical system 3 is in the sample 48 by the offset amount. As a result, the position of the focal point f of the objective lens 12 by the observation optical system 3 is moved from Z = 0 to Z by moving the focal position adjusting lens 8 from the offset zero position (X = 0 position) by a distance (X = X1). = Z1 can be moved. Thereafter, the autofocus device operates to maintain this state.

  Thus, the amount of movement of the focal position adjusting lens 8 and the amount of movement of the focal point f of the objective lens 12 by the observation optical system 3 are related, and the change in the position (X value) accompanying the movement of the focal position adjusting lens 8 is as follows. By monitoring, the change (Z value) of the position of the focal point f of the objective lens 12 by the observation optical system 3 can be detected.

  At this time, the position X1 of the concave lens 8b constituting the focal position adjusting lens 8 and the offset amount Z1 by which the position of the focal point f of the objective lens 12 by the observation optical system 3 moves from the sample surface are the magnification (focal length) of the objective lens 12. ) The offset amount necessary for the microscope apparatus is required to be about 50 μm due to the configuration of the specimen. In the case of an immersion objective lens, the reflectance of the lower surface of the cover glass is almost 0 when the medium is oil, and the same reflectance as that of the upper surface (specimen surface) is obtained when water is used. However, since the focal depth on the specimen side is very shallow, the reflection on the lower surface of the cover glass does not hinder the autofocus control.

  In addition, the autofocus device according to the present invention can cope with a general so-called dry objective lens, but in the case of a dry system, the reflectance of the lower surface of the cover glass is 10 times or more that of the upper surface, and the depth of focus is relatively high. Since it is deep, it is difficult to set the upper surface of the original cover glass as the autofocus reference surface. Therefore, it is appropriate to use the lower surface of the cover glass 10 times or more as a signal as a reference surface. In this case, the offset amount is much larger than the offset amount (50 μm) of the high magnification / immersion objective lens (for example, the cover glass thickness 170 μm + 50 μm). A large offset amount can be set.

  In this way, by forming the focal position adjusting lens 8 with the convex lens 8a and the concave lens 8b, the focus f of the objective lens 12 by the observation optical system 3 can be moved by moving the imaging position of the autofocus illumination light with a simple configuration. Can be offset.

  Note that the distance that can be shifted (offset) by the focal position adjusting lens 8 is physically limited by the focal length of the focal position adjusting lens 8, so that it is desired to shift beyond the physical limit. Can be handled by exchanging the focal position adjusting lens 8. For example, a long offset amount can be realized by replacing the focal length with a long focal length. When the focal position adjusting lens 8 is replaced with a lens having a different focal length, it is necessary to adjust the position of the focal position adjusting lens 8 in order to determine the position of zero offset.

  In the above configuration, the focal position adjusting lens 8 is disposed between the dichroic mirror 16 and the first half mirror 25, that is, on the common optical path of the focusing illumination optical system 5 and the focusing imaging optical system 7, and is disposed on the sample surface. The focal point of both the slit image that is focused and irradiated and the slit image that is reflected by the specimen surface and re-imaged on the imaging surface of the autofocus CCD sensor 30 is shifted in the optical axis direction. The focus position adjusting lens is disposed between the first pupil limiting mask 24 and the first pupil limiting mask 24, that is, the focusing illumination optical system 5, and the focal position of the slit image focused and irradiated on the specimen surface is shifted in the optical axis direction. It is also possible to realize with. In addition, a focus position adjusting lens is disposed between the first half mirror 25 and the second autofocus objective lens 26, that is, in the focusing image forming optical system 7, so that the imaging surface of the autofocus CCD sensor 30 is re-applied. It can also be realized by shifting the position of the focus of the slit image to be formed in the optical axis direction.

  The control system of the microscope apparatus includes an autofocus signal processing unit 31 for detecting a focus position, a stage driving unit 34a for moving the stage 11 up and down, a Z position detecting unit 34b for detecting the vertical position (Z position) of the stage 11, It comprises an electric revolver drive unit 35 for driving an electric revolver for exchanging the objective lens 12, a CPU 41 for controlling them, a memory 42, and an input unit 43.

  The slit image signal detected by the autofocus CCD sensor 30 is output to the autofocus signal processing unit 31 and processed by the CPU 41 to detect the focus information of the sample surface with respect to the objective lens 12. A signal related to this focus information is sent to the stage drive unit 34 by the CPU 41, and the specimen is positioned at the focus f of the observation optical system 3 of the objective lens 12 by moving the position of the stage 11 up and down along the optical axis.

  It should be noted that the position where the slit image is formed on the imaging surface of the autofocus CCD sensor 30 is adjusted in accordance with the vertical movement of the stage 11 along the optical axis when the position of the sample surface or the cover glass 14 changes. Move in the short direction of the slit image. The moving direction of the stage 11 is controlled from the slit image detected by the autofocus CCD sensor 30.

  Although not shown in FIG. 1, the stage drive unit 34 includes a stage drive DC motor attached to the stage 11, a stage drive motor driver that rotates the stage drive DC motor, and a rotation angle of the stage drive DC motor. A rotary encoder to be detected (Z position detection 34b) and an up / down counter for counting the vertical movement of the stage 11 based on the detection result of the rotary encoder.

  The autofocus control is processed by the CPU 41, and the control signals are output to the stage drive motor driver as a vertical movement control signal and a speed control signal, and the stage drive DC motor is driven based on these signals. The count result of the up / down counter is output to the CPU 41 as a vertical movement position signal. When the stage driving DC motor rotates, the stage 11 moves up and down along the optical axis according to the rotation angle. Then, the specimen placed on the stage 11 also moves up and down together with the cover glass 14 and the slide glass 15, and the positional relationship between the specimen and the objective lens 12 is adjusted, and the Z position of the in-focus position is detected.

  The CPU 41 is connected to a temperature detector 44 arranged at a predetermined position of the microscope apparatus so that the ambient temperature around the microscope apparatus can be detected. The detected temperature information is used to know or reevaluate the relationship between the position of a focus position adjusting lens 8 described later and the focus position of the objective lens 12 in the sample.

  Although only one objective lens 12 is shown in FIG. 1, the microscope apparatus of the present embodiment can be configured by a plurality of objective lenses 12 having different magnifications. Although not shown, the plurality of objective lenses 12 are attached to an electric revolver, and the electric revolver is connected to an electric revolver driving unit 35 that rotationally drives the objective lens. Although not shown, the electric revolver driving unit 35 includes an electric revolver driving DC motor attached to the electric revolver, and an electric revolver driving motor driver that rotates the electric revolver driving DC motor based on a rotation control signal from the CPU 41. Is provided. The electric revolver rotates according to the rotation of the electric revolver driving DC motor. The plurality of objective lenses 12 mounted on the electric revolver also rotate together, and any one objective lens 12 is positioned on the observation optical path of the microscope apparatus. The electric revolver driving unit 35 has a sensor (not shown) for detecting the number (1-6) of the revolver holes positioned on the observation optical path of the microscope apparatus among the revolver holes (for example, six) of the electric revolver. Is provided.

  The input unit 43 includes a focus position adjustment lens operation dial 51, although not shown, a keyboard, an objective lens changeover switch, an autofocus control start switch, a focus position storage switch, an up / down fine adjustment switch, and the above-described focus position adjustment lens. The switch etc. which operate 8 are provided.

  The keyboard is used when inputting information of the plurality of objective lenses 12 set in the electric revolver. Each data of the plurality of objective lenses 12 input from the keyboard is stored in the memory 42. In-focus position information acquired by the in-focus position storage switch is also stored in the memory 42.

  The objective lens switching switch is used when switching the objective lens 12 positioned on the observation optical path of the microscope apparatus to another objective lens 12. The CPU 41 controls the electric revolver driving unit 35 based on the switching signal input from the objective lens switching switch, and positions the objective lens specified by the switching signal on the observation optical path of the microscope apparatus.

  The autofocus control start switch is used when instructing the start of autofocus control in the microscope apparatus. When the autofocus control start switch is operated, the CPU 41 starts executing the slit projection type autofocus control described above, and the sample 48 is positioned at the focal point of the objective lens 12.

  The up / down fine adjustment switch is used to finely adjust the vertical movement of the stage 11 by manual operation. The CPU 41 positions the stage 11 based on the fine adjustment signal input from the up / down fine adjustment switch. The up / down fine adjustment switch is operated while the operator observes the image of the specimen through the second objective lens 13 and the eyepiece of the microscope apparatus. Then, when an image with high contrast can be satisfactorily observed for the operator, the operation of the up / down fine adjustment switch is finished, and the stage 11 is positioned. At this time, in the microscope apparatus of this embodiment, an arbitrary surface in the specimen coincides with the focal point of the objective lens 12.

  According to such a configuration, the objective lens by the observation optical system 3 is moved by moving the focal position adjusting lens 8 back and forth along the optical axis while performing autofocus control from the position of zero offset (Z = 0). The position of the 12 focal points f can be freely moved to any position regardless of the amount of reflection of the autofocus slit image on the specimen surface. In addition, since the focus of the objective lens 12 by the observation optical system 3 can always be focused at a certain distance from the specimen surface in the optical axis direction, the specimen 48 is moved on the stage and another part of the specimen 48 is moved. Can be performed efficiently, or when exchanging with another specimen 48 for observation.

  In the autofocus device according to the present embodiment, for example, by changing the observation method by changing objective lenses, switching between dark field, bright field, fluorescence observation, etc., for example, by changing the observation environment condition such as temperature, the above-described focus It has been found that the relationship between the offset amount (Z position) and the movement amount (X position) of the distance lens 8 is deviated. On the other hand, in order to obtain a higher-resolution sample image due to recent advances in observation technology, it is necessary to more accurately know the focal position of the objective lens 12 by the observation optical system 3 with respect to the sample with respect to the position change of the focal position adjusting lens 8. It has become.

  Therefore, in the autofocus device according to the present embodiment, the position of the focus f of the objective lens 12 by the observation optical system 3 with respect to the change in the position (X position) of the focus position adjusting lens 8 even when the above-described situation change occurs. This makes it possible to know (Z position) more accurately.

  FIG. 2 shows the relationship between the movement (X position) of the focal position adjusting lens 8 and the position (Z position) of the focal point f of the objective lens 12 by the observation optical system 3 when the environmental temperature (temperature of the microscope apparatus) is 25 ° C. and 30 ° C. (Hereinafter referred to as “XZ relationship”).

  As can be seen from FIG. 2, the observation optical system of the specimen 48, the autofocus illumination optical system 5, the focus imaging optical system 7, and the like change due to the change in the environmental temperature, and the XZ relationship changes. When the temperature is 25 ° C., X = X1 and Z = Z1 change to Z = Z1 ′ at a temperature of 35 ° C. In this case, even if the position of the focal position adjusting lens 8 is the same, the position of the sample 48 being observed is different.

  Therefore, the autofocus device according to the present embodiment stores the (XZ) coordinate table corresponding to the observation condition as shown in FIG. 3 in the storage unit 42, and the CPU 41 corresponds to the observation condition XZ. It is possible to know the observation position (Z position) of the specimen 48 from the position (X position) of the focus position adjusting lens 8 by referring to the value of the coordinate table for the relationship. By storing the acquired specimen image together with the Z position of the specimen 48 corresponding to the X position of the focal position adjusting lens 8, it can be used for later analysis.

  In FIG. 3, two types of objective lenses A and B and two types of environmental temperatures of 25 ° C. and 35 ° C. are shown as data stored in advance. These data can be increased as necessary. Other observation conditions can also be stored as data (for example, filters).

  The remeasurement data is data stored by measuring the relationship between the X position of the focal position adjusting lens 8 and the focal position (Z position) of the objective lens 12 before the observer observes the sample. The remeasurement data is obtained by the CPU 41 after the observer operates the microscope apparatus to align the focus slit image with the focus f of the objective lens 12 by the observation optical system 3 on the sample surface at the offset zero position, and then the CPU 41 sets the focus position. The adjusting lens 8 is moved along the optical axis. At this time, the CPU 41 detects the X position of the focus position adjusting lens 8 and the position (Z position) of the focus f of the objective lens 12 by the observation optical system 3 via the X position detection unit 9b and the Z position detection unit 34b, respectively. Then, the XZ coordinate table shown in FIG. 3 is created and stored in the storage unit 42 (for example, Rb in FIG. 3). Thus, by re-measuring and storing the XZ relationship immediately before observation, the relationship between the X position of the focus position adjusting lens 8 and the focus position (Z position) of the objective lens 12 can be grasped more accurately. it can.

  As described above, the autofocus device according to the present embodiment measures and stores in advance the relationship between the position (X position) of the focus position adjusting lens 8 and the focus position (Z position) of the objective lens. Alternatively, by measuring and storing immediately before observation, it is possible to know the focal position of the objective lens 12 more accurately by moving the focal position adjusting lens 8. The Z position obtained from the XZ relationship (XZ coordinate table) is obtained by shifting the focal position of the objective lens 12 while shifting the position of the focal position adjusting lens 8 in the sample and acquiring an image with the imaging optical system. It is possible to memorize the sample image that has been earned. The image having the Z position acquired in this way can be effectively used for image analysis or the like at a later date.

  In addition, when performing time-lapse observation using this microscope apparatus, after acquiring and storing a sample image once, when acquiring a sample image again after a predetermined time has elapsed, before starting the acquisition, XZ related coordinates The table is re-measured and added to the coordinate table, and the X-Z relationship in the first coordinate table is compared with the X-Z relationship after the re-measurement, so that the deviation of the X-Z relationship due to the change in time is changed. A corrected sample image can be obtained.

  As described above, the autofocus device according to the present embodiment includes a processing routine in which the CPU 41 detects and stores the relationship between the position of the focus position adjusting lens and the focus position of the objective lens via the position detection unit disposed in each. Thus, even when the observation condition changes, the XZ relationship can be re-evaluated, and a more accurate focal position (Z position) can be acquired. The processing routine here is executed by operating related members according to a predetermined procedure by a program stored in the CPU.

  The re-evaluation may be configured to automatically execute a re-evaluation routine when the CPU detects an objective lens change, observation condition change, temperature change, or time-lapse condition change. .

DESCRIPTION OF SYMBOLS 3 Observation optical system 5 Focus illumination optical system 7 Focus imaging optical system 8 Focus position adjustment lens 9a Focus position adjustment lens drive part 9b X position detection part (focus position adjustment lens position detection part)
DESCRIPTION OF SYMBOLS 10 Visible light cut filter 11 Stage 12 Objective lens 13 Second objective lens 14 Cover glass 15 Slide glass 16 Dichroic mirror 17 Second half mirror 18 Infrared light cut filter 20 LED light source 21 LED light source 22 Slit plate 23 Second collector lens 24 First pupil restriction mask 25 First half mirror 26 Autofocus objective lens 27 Autofocus relay lens 28 Second pupil restriction mask 29 Cylindrical lens 30 Autofocus CCD sensor 31 Autofocus signal processing unit 34a Stage drive unit 34b Z Position detector (stage position detector)
35 Electric Revolver Drive Unit 36 Camera Objective Lens 37 Camera Relay Lens 38 Camera CCD Sensor 41 CPU
42 Memory 43 Input unit 44 Temperature detector 48 Specimen 51 Focal position adjustment lens operation dial 61 Encoder 62 Rotation signal ON / OFF

Claims (4)

An objective lens;
An observation optical system that forms an image of the object through the objective lens;
A focusing optical system for projecting and detecting a focusing optical image on the object via the objective lens;
An imaging position adjusting means arranged in the focusing optical system, for adjusting an offset amount between the imaging position of the observation optical system and the imaging position of the focusing optical system;
Z position detecting means for detecting a focal position of the objective lens with respect to the observation optical system;
X position detection means for detecting the position of the imaging position adjustment means;
A storage unit for storing a coordinate table in which the X position information from the X position detection unit and the Z position information from the Z position detection unit are associated;
A control unit,
The controller is
The relationship between the X position information and the Z position information acquired immediately before first observing the object, and the relationship between the X position information and the Z position information acquired before observing the object after a predetermined time has elapsed. Are stored in the coordinate table,
Comparing the initial relationship with the relationship after the lapse of the predetermined time , and based on the XZ relationship in which the deviation of the relationship between the X position information and the Z position information due to the time change is corrected , An autofocus device , wherein the Z position after the predetermined time elapses is positioned at the first Z position .   The auto-focus device according to claim 1, wherein the coordinate table is acquired before starting observation.   The coordinate table is acquired again when at least one of the objective lens, the object, an observation environment temperature, or at least one immediately before the start of time-lapse observation occurs. Autofocus device.   A microscope apparatus comprising the autofocus device according to any one of claims 1 to 3.

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