JP6312410B2 - Alignment apparatus, microscope system, alignment method, and alignment program - Google Patents

Description

  The present invention relates to an alignment apparatus that acquires information about an inclination generated between a mounting surface of an object and an imaging apparatus, a microscope system to which the alignment apparatus is applied, an alignment method, and an alignment program.

  In general, the depth of focus of a microscope is several tens of μm, which is very shallow. Therefore, even if the stage on which the specimen to be observed is placed is slightly inclined, a blurred area is generated in the field of view of the microscope, and a correct image cannot be acquired. Such blurring in the field of view is more noticeable when a high-magnification observation with a shallow depth of focus is performed, and as a result, the image quality of the microscope observation image is deteriorated, the accuracy of sample measurement is reduced, and the like.

  Normally, the level of the microscope stage is maintained at the time of shipment from the factory. However, depending on the use situation thereafter, the stage fixing screw may be loosened or deteriorated, and the stage may be inclined. In such a case, the user adjusts the tilt of the stage by manually retightening the stage fixing screw or using an attitude adjustment mechanism of the electric stage provided in the microscope. However, it is very difficult to detect the amount of inclination and the direction of inclination at a level of several μm and perform adjustment with high accuracy.

  As a technique related to stage adjustment, Patent Document 1 discloses that a detection point (pattern mark) provided with a highly reflective material on a slide glass is irradiated with light from a measurement light source, and is detected via a cylindrical lens. A technique for detecting and adjusting the inclination of the slide glass based on the shape of the image formed by receiving the reflected light from is disclosed.

JP 2010-101959 A

  However, in the case of the above-mentioned Patent Document 1, it is necessary to provide a dedicated mechanism such as a cylindrical lens or a measurement light source in order to detect the inclination of the slide glass. Therefore, the apparatus configuration is complicated and cannot be applied to a general-purpose microscope as it is. Moreover, in patent document 1, only the presence or absence of inclination is detected, and detailed inclination information such as an inclination angle and an inclination start position cannot be obtained. Therefore, after detecting the presence or absence of inclination, fine adjustment must be repeated to correct the inclination.

  The present invention has been made in view of the above, and an alignment apparatus capable of acquiring detailed tilt information regarding the tilt occurring between the object mounting surface and the imaging device without providing a dedicated mechanism, It is an object to provide a microscope system, an alignment method, and an alignment program to which the alignment apparatus is applied.

  In order to solve the above-described problems and achieve the object, the alignment apparatus according to the present invention acquires an image obtained by capturing an image of the region of the subject that enters the field of view of an observation optical system provided facing the subject. Based on information on the focus area detected by the acquisition section, the focus area detection means for detecting the focus area from the image, and the focus area detection means, between the observation optical system and the subject And an inclination information calculating means for calculating the inclination information.

  A microscope system according to the present invention includes the alignment apparatus, the observation optical system, and a stage on which the subject is placed.

  The alignment method according to the present invention includes an image acquisition step of acquiring an image in which a region of the subject entering the field of view of an observation optical system provided opposite to the subject is acquired, and a focus detection of the focus region from the image. A focus area detecting step, and a tilt information calculating step of calculating tilt information between the observation optical system and the subject based on information on the focus area detected in the focus area detecting step. It is characterized by that.

  An alignment program according to the present invention includes an image acquisition step of acquiring an image in which a region of the subject entering the field of view of an observation optical system provided opposite to the subject is acquired, and a focus detection of the in-focus region from the image. A focus area detecting step; and a tilt information calculating step of calculating tilt information between the observation optical system and the subject based on information on the focus area detected in the focus area detecting step. It is made to perform.

  According to the present invention, since the tilt information between the observation optical system and the subject is calculated based on the information on the focus area detected from the image, the subject can be mounted without providing a dedicated mechanism for alignment. Detailed and accurate tilt information between the mounting surface and the imaging device can be acquired.

FIG. 1 is a block diagram showing a configuration example of a microscope system according to Embodiment 1 of the present invention. FIG. 2 is a schematic diagram illustrating a configuration example of the microscope apparatus illustrated in FIG. 1. FIG. 3 is a schematic diagram for explaining the function of the imaging control unit shown in FIG. FIG. 4 is a schematic diagram for explaining the operation of the control unit shown in FIG. FIG. 5 is a flowchart showing the operation of the alignment apparatus shown in FIG. FIG. 6 is a schematic diagram illustrating an example of a pattern formed on a subject. FIG. 7 is a flowchart showing a focus area detection process executed by the focus area detection unit shown in FIG. FIG. 8 is a schematic diagram showing an example in which an image is divided into 16 small regions. FIG. 9 is a schematic diagram of an edge image showing edges extracted from one small region shown in FIG. FIG. 10 is a schematic diagram for explaining a method of selecting a focus area. FIG. 11 is a schematic diagram illustrating a result of selecting a focus area. FIG. 12 is a flowchart for explaining the inclination information calculation processing executed by the inclination information calculation unit shown in FIG. FIG. 13 is a schematic diagram for explaining the inclination information calculation processing according to Embodiment 1 of the present invention. FIG. 14 is a schematic diagram for explaining the inclination information calculation processing according to Embodiment 1 of the present invention. FIG. 15 is a schematic diagram for explaining the inclination information calculation processing in the first embodiment of the present invention. FIG. 16 is a schematic diagram for explaining the inclination information calculation processing according to Embodiment 1 of the present invention. FIG. 17 is a schematic diagram for explaining the inclination information calculation processing according to Embodiment 1 of the present invention. FIG. 18 is a schematic diagram for explaining the inclination information calculation processing in Embodiment 1 of the present invention. FIG. 19 is a schematic diagram for explaining the inclination information calculation processing according to Embodiment 1 of the present invention. FIG. 20 is a schematic diagram showing an inclination characteristic map that is an example of display of inclination information. FIG. 21 is a flowchart showing a focus area detection process in the first modification of the first embodiment of the present invention. FIG. 22 is a schematic diagram showing an observation image and a reference image used in the focus area detection process in the first modification of the first embodiment of the present invention. FIG. 23 is a block diagram illustrating a configuration example of a microscope system according to Embodiment 2 of the present invention. FIG. 24 is a flowchart showing the operation of the alignment apparatus shown in FIG. FIG. 25 is a block diagram showing a configuration example of a microscope system according to Embodiment 3 of the present invention. FIG. 26 is a flowchart showing the operation of the alignment apparatus shown in FIG. FIG. 27 is a schematic diagram illustrating an example of an image to be processed in the third embodiment of the present invention. FIG. 28 is a flowchart showing the determination processing of whether or not the visual field movement is necessary, which is executed by the visual field movement determination unit shown in FIG. FIG. 29 is a schematic diagram for explaining a process for determining whether or not the visual field movement is necessary. FIG. 30 is a schematic diagram for explaining the determination process of whether or not the visual field movement is necessary. FIG. 31 is a schematic diagram for explaining the process of determining whether or not the visual field movement is necessary. FIG. 32 is a block diagram showing a configuration example of a microscope system according to Embodiment 4 of the present invention. FIG. 33 is a flowchart showing the operation of the alignment apparatus shown in FIG. FIG. 34 is a flowchart showing a focus area detection process executed by the focus area detection unit shown in FIG. FIG. 35 is a schematic diagram for explaining the focus area detection processing. FIG. 36 is a flowchart showing a non-focus area search determination process executed by the non-focus area search determination section shown in FIG. FIG. 37 is a schematic diagram for explaining the out-of-focus area search determination processing. FIG. 38 is a schematic diagram for explaining the out-of-focus area search determination process. FIG. 39 is a schematic diagram for explaining the out-of-focus area search determination process. FIG. 40 is a schematic diagram for explaining the out-of-focus area search determination process. FIG. 41 is a schematic diagram for explaining the out-of-focus area search determination processing. FIG. 42 is a schematic diagram for explaining the out-of-focus area search determination processing. FIG. 43 is a schematic diagram for explaining the out-of-focus area search determination processing. FIG. 44 is a block diagram showing a configuration example of a microscope system according to Embodiment 5 of the present invention. FIG. 45 is a schematic diagram showing an imaging method according to Embodiment 5 of the present invention. FIG. 46 is a schematic diagram showing an imaging method according to Embodiment 5 of the present invention. FIG. 47 is a flowchart showing the operation of the alignment apparatus shown in FIG. FIG. 48 is a schematic diagram showing an observation image and a reference image. FIG. 49 is a flowchart showing the inclination information calculation process executed by the inclination information calculation unit shown in FIG. FIG. 50 is a schematic diagram for explaining the inclination specifying calculation process. FIG. 51 is a schematic diagram for explaining the inclination specifying calculation process. FIG. 52 is a schematic diagram for explaining the inclination specifying calculation process.

  Hereinafter, embodiments of an alignment apparatus, a microscope system, an alignment method, and an alignment program according to the present invention will be described in detail with reference to the drawings. Note that the present invention is not limited to these embodiments. Moreover, in description of each drawing, the same code | symbol is attached | subjected and shown to the same part.

  Further, in the following embodiment, an example in which the present invention is applied to a microscope system that captures a subject placed on a stage and acquires a microscope observation image will be described. The present invention can be applied to any apparatus or system that can acquire an image of a subject.

(Embodiment 1)
FIG. 1 is a diagram showing a configuration of a microscope system according to Embodiment 1 of the present invention. As shown in FIG. 1, the microscope system 1 according to the first embodiment aligns the microscope apparatus 10 based on the microscope apparatus 10 that generates an enlarged image of the subject and the enlarged image generated by the microscope apparatus 10. Alignment device 20 is provided.

  FIG. 2 is a schematic diagram illustrating a configuration example of the microscope apparatus 10. As shown in FIG. 2, the microscope apparatus 10 includes a substantially C-shaped arm 100, a lens barrel 102 and an eyepiece unit 103 supported on the arm 100 via a trinocular tube unit 101, and the arm 100. The incident illumination unit 110 and the transmitted illumination unit 120, the electric stage unit 130 including the stage 131 on which the subject S is placed, and the stage 131 facing the stage 131 through the trinocular tube unit 101 on one end side of the lens barrel 102. An objective lens 140 that forms an image of observation light from the subject S, and a stage tilt adjustment unit 150.

  The trinocular tube unit 101 branches the observation light incident from the objective lens 140 in the direction of an eyepiece unit 103 for the user to directly observe the subject S and an imaging unit 211 described later.

  The epi-illumination unit 110 includes an epi-illumination light source 111 and an epi-illumination optical system 112, and irradiates the subject S with epi-illumination light. The epi-illumination optical system 112 condenses the illumination light emitted from the epi-illumination light source 111 and guides it in the direction of the optical axis L of the observation optical system 104 (filter unit, shutter, field stop, aperture stop). Etc.).

  The transmitted illumination unit 120 includes a transmitted illumination light source 121 and a transmitted illumination optical system 122 and irradiates the subject S with transmitted illumination light. The transmission illumination optical system 122 includes various optical members (filter unit, shutter, field stop, aperture stop, etc.) that collect the illumination light emitted from the transmission illumination light source 121 and guide it in the direction of the optical axis L.

  Either one of the epi-illumination unit 110 and the transmission illumination unit 120 is selected and used according to the spectroscopic method. The microscope apparatus 10 may be provided with only one of the epi-illumination unit 110 and the transmission illumination unit 120.

  The electric stage unit 130 includes a stage 131, a stage driving unit 132 that drives the stage 131, and a position detection unit 133. The stage drive unit 132 is configured by a motor, for example, and is a stage moving mechanism that moves the stage 131 in a plane orthogonal to the optical axis of the objective lens 140 (that is, in the XY plane) under the control of the imaging control unit 22 described later. is there. By moving the stage 131 in this way, the field of view of the objective lens 140 changes. Further, the stage driving unit 132 causes the objective lens 140 to focus on the subject S by moving the stage 131 along the Z axis.

  In the first embodiment, the stage 131 can be moved by electrical control. However, the stage 131 may be manually moved by an adjustment knob or the like.

  In the first embodiment, the position of the observation optical system 104 including the objective lens 140 and the lens barrel 102 is fixed and moved on the stage 131 side. However, the position of the stage 131 is fixed and the observation optical system 104 is fixed. You may move the side. Alternatively, both the stage 131 and the observation optical system 104 may be moved in opposite directions. That is, any configuration may be used as long as the observation optical system 104 and the subject S are relatively movable.

  The position detection unit 133 is configured by an encoder that detects the amount of rotation of the stage driving unit 132 made of, for example, a motor, and detects the position of the stage 131 and outputs a detection signal. Instead of the stage drive unit 132 and the position detection unit 133, a pulse generation unit and a stepping motor that generate pulses in accordance with the control of the imaging control unit 22 described later may be provided.

  The objective lens 140 is attached to a revolver 142 that can hold a plurality of objective lenses having different magnifications (for example, the objective lenses 140 and 141). The imaging magnification can be changed by rotating the revolver 142 and changing the objective lenses 140 and 141 facing the stage 131. FIG. 2 shows a state in which the objective lens 140 faces the stage 131. The revolver 142 is provided with an encoder, and the output value of the encoder is input from the microscope apparatus 10 to the alignment apparatus 20 at the timing when the image acquisition unit 21 described later performs imaging. The alignment apparatus 20 can acquire the numerical aperture and magnification of the objective lens 140 facing the subject S based on this output value.

  The stage tilt adjustment unit 150 includes an adjustment knob 151 that adjusts the tilt of the stage 131 with respect to the XY plane, and an adjustment knob drive unit 152 that operates the adjustment knob 151 by electrical control.

  Referring to FIG. 1 again, the alignment apparatus 20 includes an image acquisition unit 21 that acquires an image by imaging the subject S, an imaging control unit 22 that controls the imaging operation of the image acquisition unit 21, and the alignment apparatus 20. Information on the inclination of the subject S relative to the focal plane of the objective lens 140, in other words, the inclination of the stage 131 (hereinafter also referred to as inclination information), based on the image acquired by the image acquisition unit 21. As a result of processing by the control unit 23, a storage unit 24 that stores various information such as image data and a control program, an input unit 25 for inputting instructions and information to the alignment device 20, and a result of processing by the control unit 23 And an output unit 26 for outputting other predetermined information.

The image acquisition unit 21 includes an imaging unit 211 and a memory 212.
The imaging unit 211 includes an imaging element 211a made of, for example, a CCD or a CMOS, and pixel levels (pixel values) in each band of R (red), G (green), and B (blue) in each pixel included in the imaging element 211a. It is configured using a camera capable of capturing a color image having Or you may comprise the imaging part 211 using the camera which can image the monochrome image which outputs the luminance value Y as a pixel level (pixel value) in each pixel.

  Such an imaging unit 211 is provided at one end of the observation optical system 104 (lens barrel 102) so that the optical axis L passes through the center of the light receiving surface of the image sensor 211a. Image data corresponding to the field of view of the observation optical system 104 is generated by photoelectrically converting the incident observation light. Hereinafter, the center position of the light receiving surface of the image sensor 211a is also referred to as an imager center.

  The memory 212 includes a recording device such as a flash memory that can be updated and recorded, a semiconductor memory such as a RAM, and a ROM, and temporarily stores the image data generated by the imaging unit 211.

  FIG. 3 is a schematic diagram for explaining the function of the imaging control unit 22, and shows the stage 131 and the objective lens 140 shown in FIG. 2 in an enlarged manner. As illustrated in FIG. 3, the imaging control unit 22 outputs a control signal to the microscope apparatus 10 and moves the stage 131 to change the relative position of the observation optical system 104 with respect to the subject S, and the imaging unit. By causing 211 to perform imaging, control is performed to acquire an image of the area of the subject S that enters the field of view V of the observation optical system 104.

  The control unit 23 is configured by hardware such as a CPU, for example, and by reading a program stored in the storage unit 24, based on various parameters stored in the storage unit 24, information input from the input unit 25, and the like, The overall operation of the alignment apparatus 20 and the microscope system 1 is controlled. In addition, the control unit 23 generates an image by performing predetermined image processing on the image data input from the image acquisition unit 21, and acquires the tilt information of the stage 131 based on the image.

  FIG. 4 is a schematic diagram for explaining the operation of the control unit 23, and shows the stage 131 and the objective lens 140 shown in FIG. 2 in an enlarged manner. Normally, in the microscope apparatus 10, as shown in FIG. 3, the inclination of the stage 131 is adjusted so that the mounting surface 131 a of the stage 131 is orthogonal to the optical axis L of the objective lens 140. For example, when the optical axis L is parallel to the Z direction, the placement surface 131a is adjusted to be parallel to the XY plane. However, depending on how the microscope apparatus 10 is used, the stage 131 may be inclined with respect to the XY plane as shown in FIG. In such a case, the control unit 23 calculates the tilt direction and the tilt angle α of the stage 131 as tilt information of the stage 131 with respect to the XY plane.

  The control unit 23 calculates tilt information between the objective lens 140 and the stage 131 based on the focus region detection unit 231 that detects the focus region from the image in which the field of view V is captured, and information on the focus region. An inclination information calculation unit 232 that performs the above-described operation.

  The storage unit 24 includes a recording device such as a flash memory, RAM, and ROM that can be updated and recorded, a recording medium such as a hard disk, MO, CD-R, and DVD-R that is built-in or connected by a data communication terminal, and the recording medium. And a recording device including a reading device that reads information recorded on the recording medium. The storage unit 24 stores a parameter used for calculation in the control unit 23, a control program for causing the alignment apparatus 20 to perform a predetermined operation, and causes the control unit 23 to calculate inclination information. A program storage unit 242 for storing the alignment program and the like. The parameter storage unit 241 stores, for example, parameters such as the numerical aperture and magnification of each of the objective lenses 140 and 141 exchangeable in the microscope apparatus 10 and the image magnification based on the magnification.

  The input unit 25 includes input devices such as a keyboard, various buttons, and various switches, a pointing device such as a mouse and a touch panel, and the like, and inputs signals corresponding to operations on these devices to the control unit 23.

  The output unit 26 is an external interface that outputs an image based on the image data acquired by the image acquisition unit 21 and tilt information calculated by the control unit 23 to an external device such as the display device 27.

The display device 27 is configured by, for example, an LCD, an EL display, a CRT display, and the like, and displays an image, tilt information, and the like output from the output unit 26 on the screen.
Although the display device 27 is provided outside the alignment device 20 in the first embodiment, it may be provided inside the alignment device 20.

  Such an alignment apparatus 20 can be configured by combining a general-purpose digital camera via an external interface (not shown) with a general-purpose apparatus such as a personal computer or a workstation.

Next, the operation of the alignment apparatus 20 will be described. FIG. 5 is a flowchart showing the operation of the alignment apparatus 20.
In the following description, it is assumed that the optical axis L of the objective lens 140 is the Z direction, the plane orthogonal to the optical axis L is the XY plane, and the tilt information of the mounting surface 131a of the stage 131 with respect to the XY plane is calculated. . In the description of each step and the drawings, only the inclination occurring in the X direction is mentioned, but the inclination occurring in the Y direction can be calculated by the same processing.

  When calculating the tilt information of the stage 131, it is preferable to use, as the subject S, an object with guaranteed flatness, such as a transmission optical evaluation chart (test chart). This is because by using the subject S with guaranteed flatness, the tilt information calculated based on the image in which the subject S is captured can be used as it is as the tilt information of the placement surface 131a. When a test chart is used as the subject S, the pattern formed on the chart is not particularly limited, and a general-purpose chart can be used. In the following description, it is assumed that the subject S on which the pattern shown in FIG. 6 is formed is used as an example.

  First, in step S <b> 10, the alignment apparatus 20 captures an image of the subject S placed on the stage 131 by the image acquisition unit 21 and acquires an image of the subject S that enters the field of view V of the observation optical system 104. At this time, the control unit 23 receives the output value of the encoder provided in the revolver 142 from the microscope apparatus 10, and based on the output value of the encoder, the numerical aperture, the magnification, and the magnification of the objective lens 140 in use The parameter such as the image magnification based on is acquired from the parameter storage unit 241.

  In subsequent step S11, the focus area detection unit 231 detects a focus area from the image acquired in step S10. FIG. 7 is a flowchart showing a focus area detection process executed by the focus area detection unit 231.

In step S111 shown in FIG. 7, the in-focus area detection unit 231 calculates a cutoff frequency Fc given by the following equation (1) based on the numerical aperture NA and the image magnification B of the objective lens 140.
In the formula (1), the symbol λ indicates the wavelength of light irradiated on the subject S (for example, 550 nm).

  In subsequent step S112, the in-focus area detection unit 231 removes a frequency component higher than the cutoff frequency Fc calculated in step S111 from the image of the visual field V acquired in step S10. More specifically, the focusing area detection unit 231 uses a Hamming window or the like based on the cutoff frequency calculated in step S111 and the pixel pitch of the imaging element 211a provided in the imaging unit 211. Filter design is performed by a known method. And a frequency space image is produced | generated by performing a Fourier transform with respect to the image of the visual field V, and the high frequency component is cut by applying the filter designed to this frequency space image. Thereafter, an inverse Fourier transform is performed on the frequency space image to obtain an image from which noise is removed.

  In subsequent step S113, the in-focus area detection unit 231 divides the image M1 from which the high frequency component has been removed into a plurality of small areas. FIG. 8 is a schematic diagram illustrating an example in which the image M1 in which the field of view V is captured is divided into 16 small regions m1 to m16. Note that the number of divisions of the image M1 is not limited to 16. Increasing the number of divisions can improve the calculation accuracy of the slope information, but the amount of computation also increases. Therefore, if the number of divisions is appropriately determined in consideration of the computation speed of the hardware, required accuracy, etc. good.

In subsequent step S114, the focus area detection unit 231 calculates an evaluation value for evaluating the focus degree in each of the small areas m1 to m16. Hereinafter, this evaluation value is referred to as an image evaluation value. Various known evaluation values can be used as the image evaluation value. In Embodiment 1, for example, an evaluation value based on edge strength is used. Specifically, the focus area detection unit 231 performs Sobel filter processing on each of the small areas m1 to m16, thereby providing edges in the X direction and Y direction (hereinafter also referred to as horizontal edges and vertical edges). Extract. 9, as an example, with respect to the small area m3 shown in FIG. 8, obtained by applying the Sobel filter Sb _x for extracting the horizontal edge edge image m3 and (x), Sobel filter for extracting vertical edges is a schematic diagram showing an edge image m3 (y) obtained by applying a sb _y.

The focused area detection unit 231 normalizes the pixel value (that is, edge strength) in the edge image with the maximum value of the pixel value in the edge image for each of the small areas m1 to m16. For example, the pixel value (edge intensity) of the pixel _pn (n = 1 to N, N is the number of pixels of the edge image) in the edge image in the X direction of a certain small region is set to Ex (p _n ), When the maximum value of the pixel values and Ex _max, pixel values Ex of the pixel p _n of the normalized (p _n) 'is given by the following equation (2).
Ex (p _n ) ′ = Ex (p _n ) / Ex _max (2)

Then, the average value of the normalized pixel value Ex (p _n ) ′ is set as an image evaluation value EX given by the following equation (3).

The image evaluation value EY in the Y direction of the small area is similarly calculated using the pixel value of the edge image in the Y direction.
Note that the edge extraction method is not limited to the method using the Sobel filter described above, and a known edge extraction filter such as a LOG filter may be used. Further, the calculation method of the image evaluation value is not limited to the method shown in the formula (3).

  In subsequent step S115, the focus area detection unit 231 selects a focus area based on the image evaluation values EX and EY in the X direction and the Y direction of the small areas m1 to m16 calculated in step S114.

  FIG. 10 is a schematic diagram for explaining a method of selecting a focus area. The graph shown below the image M1 shown in FIG. 10 shows, as an example, the image evaluation value EX in the X direction of the small regions m5 to m8 (portions surrounded by a thick frame) in the second row from the top of the image M1. Note that the X coordinates of the points plotted on the graph are the center coordinates of the small regions m5 to m8. The graph shown on the right side of the image M1 shows, as an example, the image evaluation value EY in the Y direction of the small regions m3, m7, m11, and m15 (portions surrounded by a thick frame) in the third column from the left of the image M1. Show. The Y coordinates of the points plotted on the graph are the center coordinates of the small areas m3, m7, m11, and m15.

  The focused area detection unit 231 creates a graph of the image evaluation values EX and EY illustrated in FIG. 10 for each row and column of the small areas m1 to m16, and sets the image evaluation values EX and EY as threshold values Tx and Ty. Compare each. Here, the threshold value Tx in the X direction is set based on the average value or standard deviation of the edge intensity (pixel value of the edge image) in the X direction of all the pixels in the image M1. Similarly, the threshold value Ty in the Y direction is set based on the average value or standard deviation of the edge strength (pixel value of the edge image) in the Y direction of all the pixels in the image M1.

  The focus area detection unit 231 selects a small area having the image evaluation value EX larger than the threshold value Tx and the image evaluation value EY larger than the threshold value Ty as the focus area. For example, in the case of FIG. 10, since the image evaluation values EX and EY of the small area m7 are larger than the threshold values Tx and Ty, they are selected as in-focus areas.

As a result of such evaluation, in the following, as shown in FIG. 11, small regions m2, m3, m6, m7, m10, m11, m14 included in the second and third columns from the left in the image M1, A description will be given assuming that m15 is detected as the in-focus area.
After the focus area is detected, the operation of the alignment device 20 returns to the main routine.

  In step S12 following step S11, the control unit 23 determines whether or not all the small areas m1 to m16 obtained by dividing the image M1 are in-focus areas. When all the small areas m1 to m16 are in-focus areas (step S12: Yes), the alignment apparatus 20 causes the subject S, that is, the stage 131a of the stage 131 to be parallel to the XY plane (perpendicular to the optical axis L). ) And the operation is terminated.

  On the other hand, if any of the small areas m1 to m16 is not the in-focus area (step S12: No), the inclination information calculation unit 232 reads various parameters from the parameter storage unit 241, and the in-focus detected in step S11. The inclination information of the subject S (mounting surface 131a) is calculated using the position and width of the region (step S13). FIG. 12 is a flowchart showing the inclination information calculation process executed by the inclination information calculation unit 232. 13 to 19 are schematic diagrams for explaining the inclination information calculation processing.

  13A shows the positional relationship between the objective lens 140 and the subject S in the XZ plane, and FIG. 13B shows the image of the subject S that enters the field of view of the objective lens 140 in the case of the positional relationship. M1 is shown in the XY plane. The same applies to FIGS. 14 to 18. In FIGS. 13 to 18, the subject S and the field of view V are illustrated on the assumption that the subject S is a plane having no thickness in order to facilitate understanding.

Further, as shown in FIG. 13A, in the following, the field size of the observation optical system 104 (objective lens 140) is Fov (μm), the depth of field is DOF (μm), and the observation optical system 104 is combined. The angle between the focal plane and the subject S is the tilt angle α, the height of the tilt occurring in the region of the subject S included in the field of view V is z (μm), and the size of the image M1 corresponding to the field size Fov (image size) ) _Is W _image (pixel), and the width of the in-focus region R _focus1 in the image M1 in the X direction and Y direction is W _x (pixel) and W _Y (pixel), respectively. Among these, parameters (field size Fov, depth of field DOF, image size W _image ) depending on the specifications of the microscope apparatus 10 and the imaging unit 211 are stored in advance in the parameter storage unit 241 and read out as necessary. It is.

First, in step S131, the tilt information calculation unit 232 specifies the tilt direction of the subject S from the position of the focus area R _focus1 detected in step S11. Here, as shown in FIG. 13, the inclination direction of the subject S in the XZ plane can be considered to be two directions, that is, upward (FIG. 13 (a)) and downward (FIG. 14 (a)).

The depth of field DOF of the objective lens 140 is a predetermined range in the Z direction with the focal plane as the center. Of the field of view V, a region R ₀ included in the depth of field DOF is detected as a focused region of the image M1. Therefore, even if the subject S is inclined in any of the directions shown in FIGS. 13A and 14A, the same area (small area) is detected as the focus area R _{focus1 in} the image M1. The

Therefore, the tilt information calculation unit 232 causes the imaging control unit 22 to transmit a control signal to the microscope apparatus 10 and slightly changes the distance in the Z direction between the objective lens 140 and the subject S. For example, when the objective lens 140 and the subject S are brought closer by moving the objective lens 140 downward (minus Z direction) or by moving the stage 131 upward (plus Z direction), as shown in FIG. If the subject S is inclined upward, the focus area R _focus1 in the image M1 moves to the left (minus X direction) in the figure. On the other hand, as shown in FIG. 16, if the subject S is inclined downwardly to the right, the focus area R _focus1 in the image M1 moves to the right (plus X direction) in the figure.

Further, for example, when the objective lens 140 and the subject S are moved away by moving the objective lens 140 upward (plus Z direction) or by moving the stage 131 downward (minus Z direction), as shown in FIG. On the other hand, if the subject S is inclined upward, the focus area R _focus1 in the image M1 moves to the right (plus X direction) in the figure. On the other hand, as shown in FIG. 18, if the subject S is inclined downwardly to the right, the focus area R _focus1 in the image M1 moves to the left (minus X direction) in the figure.

The detection of the focus area R _focus1 when the distance between the objective lens 140 and the subject S is changed may be performed in the same manner as in step S11. The inclination information calculation unit 232 _acquires the moving direction of the focusing area R _focus1 by comparing the positions of the focusing area R _focus1 before and after changing the relative distance, and based on the moving direction, the _inclination of the subject S Specify the tilt direction. After specifying the tilt direction, the distance between the objective lens 140 and the subject S may be returned to the original.

Similarly, the inclination information calculation unit 232 specifies the inclination direction of the subject S in the YZ plane. As shown in FIG. 11, when the focus area R _focus1 detected from the image M1 is distributed over the entire Y direction, it may be determined that there is no inclination in the Y direction.

In the subsequent step S132, the inclination information calculation unit 232 calculates the inclination angle α of the subject S in the X direction and the Y direction from the widths W _X and W _{Y of the} focus area detected in step S11. More specifically, the inclination information calculation unit 232 generates the visual field V by substituting the width W _X of the in-focus region R _focus1 shown in FIG. _13B for the width W _Focal of the following equation (4). A tilt amount z in the X direction (difference in the Z coordinate between one end and the other end of the visual field V in the X direction) is calculated.

Subsequently, the inclination angle α of the subject S in the X direction is calculated by substituting the inclination amount z into the following equation (5).

Similarly, the tilt information calculation unit 232 calculates the tilt amount z in the Y direction generated in the field of view V by substituting the width W _Y of the focus area R _focus1 into the width W _Focal of Expression (4). Then, by substituting the inclination amount z into the equation (5), the inclination angle α of the subject S in the Y direction is calculated.

In subsequent step S133, the tilt information calculation unit 232 calculates the shift amount of the tilt fulcrum from the position and width of the focus area R _focus1 . Here, the tilt fulcrum is the center position of the focusing area R _focus1 and corresponds to the point where the subject S and the focusing plane intersect. More specifically, gradient information calculating unit 232, as shown in FIG. 19, and detects the center point P _M1 of the center point P _Focus1 and image M1 of focus area R _Focus1, these center points _P focus1, P _M1 The distance d between them is calculated in units of pixels. The tilt information calculator 232, when the center point P _Focus1 the focus area R _Focus1 exists in the positive X direction (or positive Y direction) than the center point P _M1 images M1, the sign of the distance d plus And the sign of the distance d is negative if it exists on the negative X direction (or negative Y direction) side. This signed distance d is the amount of deviation of the tilt fulcrum.

In subsequent step S134, the tilt information calculation unit 232 stores the tilt information acquired in steps S131 to S133, that is, the tilt direction, the tilt angle, and the shift amount of the tilt fulcrum in the parameter storage unit 241 as tilt information.
Thereafter, the operation of the alignment apparatus 20 returns to the main routine.

In step S14 following step S13, the control unit 23 outputs the tilt information acquired in step S13 and causes the display device 27 to display the tilt information. As a specific display example of the inclination information, for example, an inclination characteristic map shown in FIG. The inclination characteristic map shown in FIG. 20 is a graph in which the inclination (deviation from the focal plane) at each XY coordinate on the subject S entering the field of view V of the observation optical system 104 is mapped based on the inclination information stored in step S134. It is. The user adjusts the inclination of the stage 131 by operating an adjustment knob 151 provided in the microscope apparatus 10 while referring to such an inclination characteristic map.
Thereafter, the operation of the alignment apparatus 20 ends.

  As described above, according to the first embodiment, the tilt information between the objective lens 140 and the subject S can be acquired only from the image M1 in which the subject S is captured in the microscope apparatus 10. That is, detailed and accurate tilt information can be acquired with a simple apparatus configuration without providing a dedicated mechanism for alignment in the microscope apparatus 10. Therefore, by adjusting the tilt of the stage 131 based on the tilt information, a high-quality microscope image can be acquired.

  Further, according to the first embodiment, since detailed tilt information such as a tilt characteristic map is displayed on the display device 27, the user can adjust the tilt of the stage 131 without being confused about the adjustment direction and the adjustment angle. it can.

(Modification 1)
Next, a first modification of the first embodiment of the present invention will be described.
The focus area detection process in step S11 shown in FIG. 5 is not limited to the process described above. For example, when a state in which no tilt is generated between the objective lens 140 and the stage 131 is guaranteed as in a factory shipment, the subject S is imaged to obtain a reference image (hereinafter simply referred to as a reference image). And when the microscope system 1 is used, the in-focus area may be detected by comparing an image of the same subject S with a reference image.

  FIG. 21 is a flowchart showing a focus area detection process according to the first modification. It is assumed that the parameter storage unit 241 stores in advance image data of a reference image acquired by imaging the subject S at the time of factory shipment.

  First, in step S <b> 121, the focus area detection unit 231 acquires a reference image based on the image data stored in the parameter storage unit 241. At this time, the in-focus area detection unit 231 enlarges or reduces the reference image based on the imaging magnification in the microscope apparatus 10, thereby performing an observation on the image acquired in step S10 (hereinafter referred to as an observation image). Adjust the size of the reference image.

  In subsequent step S122, the in-focus area detection unit 231 performs alignment between the observation image and the reference image, and aligns the position of the visual field. As a positioning method, a known method such as a phase only correlation method can be used.

In step S123, the focus area detection unit 231 divides each of the observation image M1 and the reference image _Mbase into a plurality of small areas as shown in FIG. For example, in FIG. 22, the observation image M1 and the reference image M _base, respectively, 16 small regions M1 to M16, are divided into m1'~m16 '.

  In step S124, the focus area detection unit 231 calculates an image evaluation value for evaluating the focus degrees of the small areas m1 to m16 and m1 'to m16'. The calculation method of the image evaluation value is the same as that in the first embodiment (see step S114 in FIG. 7).

In step S125, the focus area detection unit 231 performs the observation image M1 and the reference image _Mbas.
The image evaluation values of the corresponding small areas (small areas m1 and m1 ′, small areas m2 and m2 ′,...) are compared with _e, and the focus area is selected based on the comparison result. Specifically, the focus area detection unit 231 calculates a difference in image evaluation values between corresponding small areas, and calculates an average value of the differences in all small areas. Then, a small area in which the difference between the image evaluation values is equal to or less than the average value of the differences is selected as the focusing area.

(Embodiment 2)
Next, a second embodiment of the present invention will be described.
FIG. 23 is a block diagram illustrating a configuration of a microscope system according to the second embodiment. As shown in FIG. 23, the microscope system 2 according to the second embodiment includes a microscope apparatus 10 and an alignment apparatus 30. Among these, the configuration and operation of the microscope apparatus 10 are the same as those in the first embodiment (see FIG. 2).

  The alignment apparatus 30 further includes an inclination adjustment control unit 31 with respect to the alignment apparatus 20 shown in FIG. The configuration and operation of each unit of the alignment apparatus 30 other than the inclination adjustment control unit 31 are the same as those in the first embodiment (see FIG. 1).

  The tilt adjustment control unit 31 outputs a control signal to the adjustment knob drive unit 152 provided in the microscope apparatus 10 based on the tilt information calculated by the tilt information calculation unit 232 to adjust the tilt of the stage 131.

  Next, the operation of the alignment apparatus 30 will be described. FIG. 24 is a flowchart showing the operation of the alignment apparatus 30. Note that steps S10 to S14 shown in FIG. 24 are the same as those in the first embodiment (see FIG. 5).

  In step S15 following step S14, the inclination adjustment control unit 31 reads out the inclination information (inclination direction, inclination angle, and inclination fulcrum deviation amount) calculated by the inclination information calculation unit 232 from the parameter storage unit 241, and the inclination information Is generated and output to the microscope apparatus 10 to adjust the tilt of the stage 131.

  More specifically, the tilt adjustment control unit 31 first adjusts the tilt direction and tilt angle of the subject S at the center position of the visual field V. For example, as shown in FIG. 13A, when the subject S moves up to the right with respect to the in-focus plane, a control signal is output to the adjustment knob driving unit 152 (see FIG. 2), and the stage 131 is moved in the XZ plane. The stage 131 is rotated clockwise with respect to the center of the stage 131 (position through which the optical axis L passes; hereinafter referred to as stage center). On the other hand, as shown in FIG. 14A, when the subject S is lowered to the right with respect to the in-focus plane, a control signal is output to the adjustment knob driving unit 152, and the stage 131 is set to the center of the stage in the XZ plane. Rotate counterclockwise.

Subsequently, the tilt adjustment control unit 31 moves the stage 131 in the direction opposite to the sign of the shift amount by the shift amount based on the shift amount of the tilt fulcrum. For example, when the sign of the amount of deviation in the X direction is positive, a control signal is output to the stage driving unit 132, and the stage 131 is moved in the negative X direction. Thereby, the tilt fulcrum coincides with the center of the visual field of the observation optical system 104 (that is, the position through which the optical axis L passes), and an in-focus image with an appropriate visual field can be acquired.
The adjustment method in the Y direction is the same.

  As described above, according to the second embodiment, the tilt of the stage 131 can be easily and accurately adjusted based on the tilt information calculated by the tilt information calculation unit 232. Therefore, the microscope system 2 can acquire a high-quality microscope observation image.

(Modification 2)
Next, a second modification of the second embodiment of the present invention will be described.
In the first embodiment and the second embodiment, the inclination generated between the focusing surface of the observation optical system 104 and the subject S by fixing the observation optical system 104 side and adjusting the stage 131 side. Was corrected. However, the tilt occurring between the two may be corrected by fixing the stage 131 side and adjusting the observation optical system 104 side. In this case, the microscope apparatus 10 may be provided with a tilt adjustment mechanism that integrally adjusts the tilt of the observation optical system 104.

  When performing the adjustment, if the subject S is raised to the right with respect to the focal plane (see FIG. 13A), the observation optical system 104 is rotated counterclockwise with respect to the imager center in the XZ plane. On the other hand, when the subject S is lowered to the right with respect to the focal plane (see FIG. 14A), the observation optical system 104 is rotated in the XZ plane so as to be clockwise with respect to the imager center. The adjustment of the tilt of the observation optical system 104 may be performed manually by the user as in the first embodiment, or may be performed automatically by electrical control over the tilt adjustment mechanism as in the second embodiment. . The adjustment method in the Y direction is the same.

(Embodiment 3)
Next, a third embodiment of the present invention will be described.
FIG. 25 is a block diagram showing a configuration of a microscope system according to Embodiment 3 of the present invention. As shown in FIG. 25, the microscope system 3 according to the third embodiment includes a microscope apparatus 10 and an alignment apparatus 40. Among these, the configuration and operation of the microscope apparatus 10 are the same as those in the first embodiment (see FIG. 2).

  The alignment apparatus 40 includes a control unit 41 instead of the control unit 23 in the alignment apparatus 30 according to the second embodiment. The configuration and operation of each part of the alignment apparatus 40 other than the control unit 41 are the same as those in the second embodiment (see FIG. 23).

  The control unit 41 further includes a visual field movement determination unit 411 with respect to the control unit 23 shown in FIG. The visual field movement determination unit 411 determines whether or not to perform imaging again by moving the visual field V based on the detection result of the focused region by the focused region detection unit 231. The operations of the focus area detection unit 231 and the inclination information calculation unit 232 are the same as those in the second embodiment.

  Next, the operation of the alignment apparatus 40 will be described. FIG. 26 is a flowchart showing the operation of the alignment apparatus 40. Note that steps S10 and S11 shown in FIG. 26 are the same as those in the first embodiment. In the following description, it is assumed that the image M2 shown in FIG. 27 has been acquired in step S10. FIG. 27 shows a state in which the image M2 in which the area of the subject S entering the visual field V (see FIG. 6) is divided into a plurality of small areas (see step S113 in FIG. 7).

  In step S31 following step S11, the visual field movement determination unit 411 classifies the inside of the image M2 into an in-focus area and an out-of-focus area from the detection result of the in-focus area in step S11, and based on the classification result, It is determined whether or not the visual field movement of the observation optical system 104 with respect to the subject S is necessary.

  FIG. 28 is a flowchart showing the determination processing of whether or not the visual field movement is necessary, which is executed by the visual field movement determination unit 411. FIGS. 29 to 31 are schematic diagrams for explaining the determination process of whether or not the visual field movement is necessary.

  First, in step S311, the visual field movement determination unit 411 determines the position of the out-of-focus area in the X direction of the image M2. More specifically, as shown in FIG. 27, the field-of-view movement determination unit 411 determines the image in the X direction for each row of the small region from the image evaluation value EX of each small region calculated by the focused region detection unit 231. Create an evaluation value distribution graph. And the intersection of this image evaluation value distribution graph and threshold value Tx is calculated | required. The graph of the image evaluation value EX shown in FIG. 27 shows the distribution of the image evaluation value EX in the small area in the second row from the top of the image M2.

  When the number of intersections is zero or an even number, the visual field movement determination unit 411 determines that the out-of-focus areas are located at the left and right ends of the image M2. On the other hand, as shown in the second row of FIG. 27, when the number of intersection points is an odd number (for example, one), it is determined that the out-of-focus area is located only on one side of the image M2.

  In subsequent step S312, the visual field movement determination unit 411 determines the position of the out-of-focus region in the Y direction of the image M2, similarly to step S311. That is, an image evaluation value distribution graph is created for each column of small regions, and the position of the out-of-focus region is determined based on the number of intersections between the graph and the threshold value Ty. Note that the graph of the image evaluation value EY shown in FIG. 27 shows the distribution of the image evaluation value EY in the small region in the third column from the left of the image M2.

As a result, in the case of the image M2, as shown in FIG. 29, the _non-focus area R _non-focus2 is located only at the end in the minus X direction, and the focus area R _focus2 is at the end in the plus X direction. You can see that it is biased. Note that the method for determining the position of the out-of-focus area is not limited to the above-described method, and whether or not the out-of-focus area is positioned so as to sandwich the in-focus area for each row or column of the small area. It only has to be detected.

In step S313, the visual field movement determination unit 411 determines whether or not the visual field movement is necessary from the position of the out-of-focus area determined in steps S311 and S312. Specifically, in each of the X direction and the Y direction of the image M2, when the out-of-focus area is located only on one side, it is determined that the visual field movement is necessary, and the out-of-focus area is located on both sides. If there is no out-of-focus region, it is determined that visual field movement is unnecessary. For example, in the case of the image M2 shown in FIG. 29, in the X direction, since the _non-focused region R _non-focus2 is located only on the left end side, it is determined that the visual field movement in the X direction is necessary. On the other hand, in the Y direction, since there is no out-of-focus area (contact point = zero), it is determined that visual field movement is unnecessary. The necessity of such visual field movement is determined for each row and column of the small area, and if there is at least one row or column of the small area where the out-of-focus area exists only on one side, the visual field movement is not performed. Judge as necessary. Also, the visual field movement determination unit 411 stores the image evaluation values EX and EY of each small region in the parameter storage unit 241 when it is determined that the visual field movement is necessary.

  If the result of determination in step S31 is that visual field movement is not required in either the X direction or the Y direction (step S32: No), the operation of the alignment apparatus 40 proceeds to step S13.

On the other hand, if the result of determination in step S31 is that visual field movement is necessary (step S32: Yes), the microscope system 3 moves the visual field V of the observation optical system 104 relative to the subject S (step S33). More specifically, the visual field movement determination unit 411 determines the relative movement direction of the visual field V based on the image evaluation values EX and EY of each small region stored in the parameter storage unit 241. The direction is determined to be the opposite direction. In response to this, the imaging control unit 22 outputs a control signal indicating the relative moving direction of the visual field V to the stage driving unit 132 (see FIG. 2). The stage drive unit 132 relatively changes the visual field V by moving the stage 131 in the XY plane according to this control signal. For example, in the case of the image M2 shown in FIG. 29, since the _non-focused region R _non-focus2 exists only at the end portion in the minus X direction, the visual field V is moved in the plus X direction. In this case, actually, in the microscope apparatus 10, the stage 131 on which the subject S is placed is moved in the minus X direction.
Thereafter, the operation of the alignment apparatus 40 proceeds to step S10.

  The alignment apparatus 40 performs such steps S10, S11, and S31 to S33 until it is determined that the visual field movement is unnecessary (No in step S32), that is, from the image acquired after the visual field V is moved. Repeat until is detected. When it is determined that the field of view should be moved in both the X direction and the Y direction, first, the field of view V is moved in the X direction and steps S10, S11, S31 to S33 are repeated, and the field of view is moved in the X direction. Is determined to be unnecessary, and then returns to the first visual field position, from which the visual field V is moved in the Y direction, and these steps S10, S11, S31 to S33 are repeated.

Here, in step S31 after the second round, the necessity of further visual field movement is determined including the image evaluation values EX and EY calculated for the images acquired so far. For example, in step S10 of the first round, an image M2 in which the visual field V shown in FIG. 6 is captured is obtained (see FIG. 29), and the visual field is moved in the plus X direction based on the detection result of the focus area. In this case, in step S10 of the second round, an image M3 (see FIG. 30) of the area adjacent to the visual field V in the subject S is acquired. After the focus area R _focus3 and the _non- focus area R _non-focus3 are detected by the focus area detection process for the image M3 (see step S11), in the subsequent step S31, as shown in FIG. A determination is made based on the image evaluation value EX calculated for the images M2 and M3.

  For example, the graph of the image evaluation value EX shown in FIG. 31 shows the distribution of the image evaluation value EX in the eight small regions included in the third row from the top of the images M2 and M3. Since the number of intersections between this graph and the threshold value Tx is an even number (two), it is determined that further visual field movement is unnecessary. Similarly, regarding the Y direction, whether or not further visual field movement is necessary is determined from the graphs of the image evaluation values EY of all the images acquired by visual field movement in the Y direction.

When it is determined in step S32 that visual field movement is not necessary (step S32: No), the subsequent processing in step S13 is the same as that in the first embodiment. However, when a plurality of images are acquired by moving the field of view, the tilt information is calculated using information about all the acquired images. For example, as shown in FIG. 30, when two images M2 and M3 are acquired by moving the field of view in the X direction, the entire area including the images M2 and M3 is set as the image size and detected from the images M2 and M3. The tilt angle and tilt fulcrum are calculated with the final focus region set as the focus region R _focus2 and R _focus3 combined. Specifically, two image sizes W _{(M2, M3)} are applied as the image size W _image in the expression (4), and the total width of the focus areas R _focus2 and R _focus3 is _set as the focus area width W _Focal. W _{f (M2, M3)} is applied.

  The subsequent steps S14 and S15 are the same as in the second embodiment. Instead of executing step S15, the user may manually adjust the tilt of the stage 131 with reference to the tilt information displayed in step S14.

  As described above, according to the third embodiment, since the out-of-focus area exists only on one side of one field of view as in one-sided blur, the width of the in-focus area cannot be specified. Even in such a case, by moving the field of view and capturing an image, it is possible to grasp the whole image of the in-focus area and calculate the tilt information between the objective lens 140 and the subject from only the image. Further, according to the third embodiment, the direction in which the out-of-focus area exists can be accurately estimated from the distribution of the image evaluation values of the small areas in the image. Thus, the tilt information can be calculated reliably and efficiently.

(Embodiment 4)
Next, a fourth embodiment of the present invention will be described.
FIG. 32 is a block diagram showing a configuration of a microscope system according to Embodiment 4 of the present invention. As shown in FIG. 32, the microscope system 4 according to the fourth embodiment includes a microscope apparatus 10 and an alignment apparatus 50. Among these, the configuration and operation of the microscope apparatus 10 are the same as those in the first embodiment (see FIG. 2).

  The alignment apparatus 50 includes a control unit 51 instead of the control unit 23 in the alignment apparatus 30 according to the second embodiment. The configuration and operation of each part of the alignment apparatus 50 other than the control unit 51 are the same as those in the second embodiment.

The control unit 51 includes an in-focus area detection unit 510 including an area classification unit 511 and an out-of-focus area search determination unit 512.
The area classification unit 511 divides the image in which the subject S entering the visual field V (see FIG. 6) is divided into a plurality of small areas, and determines whether each area is an in-focus area. Classification into in-focus areas and out-of-focus areas.

  The out-of-focus area search determination unit 512 determines whether or not it is necessary to perform re-imaging by moving the visual field V with respect to the subject S based on the classification result by the area classification unit 511 and moves the visual field V. Determine the direction of movement.

  Next, the operation of the alignment apparatus 50 will be described. FIG. 33 is a flowchart showing the operation of the alignment apparatus 50. Note that step S10 shown in FIG. 33 is the same as that in the first embodiment.

  In step S41 following step S10, the focus area detection unit 510 classifies the image acquired in step S10 into a focus area and a non-focus area. FIG. 34 is a flowchart showing a focus area detection process executed by the focus area detection unit 510. Note that steps S111 to S114 shown in FIG. 34 are the same as those in the first embodiment (see FIG. 7). FIG. 35 is a schematic diagram for explaining the focus area detection processing. In the following description, it is assumed that the image M4 shown in FIG. 35 has been acquired in step S10. FIG. 35 shows a state in which the image M4 in which the area of the subject S entering the visual field V (see FIG. 6) is divided into a plurality of small areas (see step S113).

In step S115 subsequent to step S114, the region classification unit 511 classifies each small region into a focused region and a non-focused region based on the image evaluation value calculated in step S114. For example, in the case of the image M4 shown in FIG. 35, the image evaluation value calculated for each small region m _i (i is a natural number of 2 or more) obtained by dividing the image M4 is compared with a predetermined threshold, and the image evaluation value is threshold value above focus the small region m _i which is the region _R focus4, image evaluation values to classify small subregion m _i than the threshold value as out-of-focus region _{R non-focus4.} Note that the threshold determination method is the same as in the first embodiment.

Then, the area classification part 511, the respective small regions m _i, to impart a label for region identification. Thereby, the common label is applied to all the pixels included in each small area m _i. Each subregion m _i, for each focus area, or out-of-focus area, are sequentially assigned a consecutive number. For example, in an image M4 shown in FIG. 35, because the small region m _i in the first row is classified as non-focus area, these small areas m _i, indicating that it is out-of-focus area A non-focused area label D _j (j = 1, 2,...) Is given. On the other hand, since the small areas m _{i in the} second to fourth columns are classified as in-focus areas, these small areas m _i are in-focus area labels F _k (k = k = 1, 2, ...) are given.

  In subsequent step S42, the out-of-focus area search determination unit 512 determines whether or not an additional search for the out-of-focus area is necessary. FIG. 36 is a flowchart showing a non-focus area search determination process executed by the non-focus area search determination unit 512. FIGS. 37 to 43 are schematic diagrams for explaining the out-of-focus area search determination processing.

  First, in step S421, the out-of-focus area search determination unit 512 acquires an imaging number label indicating the number of times the subject S has been imaged so far. In the first round of processing, the imaging count label is 1.

In subsequent step S422, the non-focus area search determination unit 512 determines the connectivity with the adjacent area for the area to which the non-focus area label D _j is assigned (hereinafter referred to as the D _j area), A connected non-focused region is generated by integrating a plurality of connected regions. At this time, as shown in FIG. 37, the out-of-focus area search determination unit 512 determines the connection condition for the small area in the image and the pixels in each small area in the raster order. That is, in the image M4, the determination is made from the upper left D _j region to the right, and when the determination of the first row is completed, the left end D _j region of the lower row is set as the determination target, and again from left to right. Make a judgment. In each D _j region, the determination is made from the upper left pixel toward the right, and when the determination in the first row is completed, the left end pixel in the lower row is set as the determination target, and again from the left toward the right. Make a decision. For example, the determination for the four corners of the pixel a1, b1, c1, d1 of the D ₁ in a region shown in FIG. 38 (a) is performed in the order of pixel a1 → pixel b1 → pixel c1 → pixel d1.

Coupling of D _j region is determined based on the connectivity of the four corners of the pixels in each D _j region. Out-of-focus region searching determination unit 512, first, as shown in FIG. 38 (a), for each of D ₁ 4 corners of pixels in the area a1, b1, c1, d1, neighboring pixels (peripheral pixels) is non It is determined whether or not the pixel is in the focus area. For example, since the position both neighboring pixels to D ₁ in the region of the pixel a1, connected with the other non-focus area is determined not. Further, since the neighboring pixel of the pixel b1 is located in the D ₁ region or in the adjacent in-focus region (F ₁ region), it is determined that there is no connectivity with other out-of-focus regions. On the other hand, in the case of the pixel c1, among the four neighboring pixels p11, p12, p13, and p14, the neighboring pixel p13 is included in the D ₂ region, so that it is determined that there is connectivity between the D ₁ region and the D ₂ region. Is done.

Thus, when it is determined that there is connectivity, the determination process for the D ₁ region ends. That is, the determination for the remaining pixel d1 is skipped. Then, the same connected non-focused label C _n (n = 1, 2,...) Is given to the D ₁ region and the D ₂ region determined to have connectivity. At this time, if the connected non-focused label C _n has already been assigned to the area (D ₂ area) of the connection partner for the area to be processed (D ₁ area), the connection non-focus label of the connection partner. C _n is assigned to the area to be processed. On the other hand, if the connected non-focused label C _n is not yet given to the connected partner area, a new connected non-focused label C _n is set. For example, in the case of FIG. 38 (a), because it is linked counterpart D still connected unfocused label C _n in the _second region is not granted, D ₁ region and D ₂ connected to the region-of-focus initial value C of the label ₁ is given (see FIG. 38B). Note that the initial setting value of the connected non-focused label C _n is C ₀ , and if the label number n is newly determined to be connected, the connected non-focused label C _n obtained so far The maximum label number is set to +1.

The determination is performed in the same manner for the subsequent D ₂ region to be processed. Here, as shown in FIG. 38 (c), the neighboring pixel p15 of the pixel a2 is located in the D ₁ area, but is already connected to itself (D ₂ area), so the processing is skipped. The same applies to the pixel b2. On the other hand, the pixel c2 includes four neighboring pixels p21, p22, p23, of p24, for neighboring pixels p23 is included in the D ₃ regions, it is determined that there is connectivity between the D ₂ region and D ₃ regions The In this case, the same unfocused label C ₁ as in the D ₂ area is given to the D ₃ area.

Here, D _j region of the connection partner, if different connecting unfocused label C _n and D _j region to be processed is already granted, fit the label C _n focusing connecting non focus towards label number of young It is done. For example, as shown in FIG. 38 (d), in the determination with respect to D ₁₁ regions, labeled C ₁ focus coupled non if the D ₁₄ area by the determination that there is connectivity with the D ₁₁ region is applied, then, the judgment for D ₁₃ area connected unfocused label C ₂ is applied, if it is determined that there is connectivity with the D ₁₃ regions D ₁₄ region, the same as D ₁₃ regions and the D ₁₃ area The connected non-focused label in the region D ₁₂ to which the connected non-focused label C ₂ is assigned is converted into a connected non-focused label C ₁ .
In addition, since the pixels at the four corners of the D _j region may be connected to different regions, the connection determination is performed at least once for each D _j region.

In subsequent step S423, the out-of-focus area searching determining unit 512, connected unfocused label C _n is assigned region (hereinafter, connected as a non-focus area) on the basis of the position of the other non-focus area Determine whether there is a need to search. If a search is necessary, the search direction is also determined.

More specifically, as shown in FIG. 39, the non-focus area search determination unit 512 counts the number of connected non-focus areas in the image M4. The number of regions, the maximum value of the connection-focus label C _n label number. For example, in the case of the image M4, the number of areas is 1.

When the number of connected non-focus areas is 1, the non-focus area search determination unit 512 determines that a search for non-focus areas is necessary. In this case, subsequently, out-of-focus area searching determining unit 512 obtains the position and the position of the center of gravity pixel F _g of focus areas R _Focus4 centroid pixel D _g of the coupling-of-focus area. Then, as shown in FIG. 40, connecting the direction of a vector towards the center of gravity pixel F _g-of-focus areas R _Focus4 focus from the center of gravity pixel D _g regions, determined as the search direction. A case where the number of connected out-of-focus areas is two or more will be described in detail later.

  When it is determined in step S42 that a search for an out-of-focus area is necessary (step S43: Yes), the microscope system 4 moves the field of view V of the observation optical system 104 with respect to the subject S in the search direction determined in step S423. (Step S44). More specifically, the out-of-focus area search determination unit 512 outputs vector information indicating the moving direction of the visual field V to the imaging control unit 22. In response to this, the imaging control unit 22 outputs a control signal indicating the relative moving direction of the visual field V to the stage driving unit 132 (see FIG. 2). The stage drive unit 132 relatively changes the visual field V by moving the stage 131 in the XY plane according to this control signal.

In addition, the out-of-focus area search determination unit 512 increments the imaging count flag by 1 with respect to the imaging count performed so far. For example, when the process proceeds to step S44 after steps S10 and S41 to S43 in the first round, the imaging count flag is 2. Further, the out-of-focus area search determining unit 512 sets the maximum value (1 in FIG. 40) of the label number C _n of the connected non-focused label as the maximum label number of the connected non-focused label in the parameter storage unit 241. Remember.

The alignment apparatus 50 repeats such steps S10 and S41 to S44 until it is determined that the search for the out-of-focus area is unnecessary (step S43: No).
Here, in the out-of-focus area linking process in step S42 after the second round (see step S422 in FIG. 36), the non-focus area search determining unit 512 stores the connected out-of-focus label stored in the parameter storage unit 241. The number obtained by adding 1 to this is used as the initial value of the unfocused label. For example, in the above-described example, since the maximum label number in the first round is 1, C ₂ is set as the initial value of the connected unfocused label in the second round.

  Further, in the second and subsequent rounds, the necessity and search direction of the additional search for the non-focused region and the search direction are determined using the result of the non-focused region connection process for the images acquired so far. Here, a case where the number of connected non-focused areas is two or more will be described.

For example, the image M4 shown in FIG. 35 is acquired in step S10 of the first round, and the visual field V is moved in the plus X direction based on the necessity of additional search for the out-of-focus region and the determination result of the search direction. In step S10 in the second round, an image M5 of the area adjacent to the visual field V shown in FIG. 6 in the subject S is acquired (see FIG. 41). By the classification process of the small area for the image M5, the small area in the image M5 is classified into the focused area R _focus5 and the _non-focused area R _non-focus5, and by the connection process for the _non-focused area R _non-focus5 , _Assume that a connected non-focused label C ₂ is given to each small area in the _non-focused area R _non-focus5 (see FIG. 42).

In this case, the out-of-focus area searching determination section 512 first acquires connecting the centroid pixel D _g1-of-focus label C ₁ is assigned region, and the centroid pixel of focus area. Among these, for the focus area, the barycentric pixel F _{g (M4, M5) of} the area R _{focus (M4, M5)} obtained by combining the focus areas R _focus4 and R _focus5 detected from the images M4 and M5, respectively _, is acquired. The

Subsequently, as shown in FIG. 43, the non-focused area search determining unit 512 connects the non-focused area search determining unit 512 with the smaller label number to the center pixel F _{g (M4, M5)} of the area R _{focus (M4, M5).} determining a centroid pixel D _g1 and symmetrical pixel a in region focusing label C ₁ has been applied. When the label given to the pixel A is a focus label, the non-focus area search determination unit 512 determines that an additional search for the non-focus area is necessary, and the centroid pixel F _g from the centroid pixel D _g1. The direction of the vector toward _{(M4, M5)} is defined as the search direction (the field of view movement direction).

On the other hand, when the label given to the pixel A is not the in-focus label, the out-of-focus area search determination unit 512 determines that the additional search for the out-of-focus area is unnecessary. For example, in the case of FIG. 43, since the connecting unfocused label C ₂ is applied to the pixel A, the search for additional out-of-focus region is determined to be unnecessary.

  When it is determined in step S43 that no further in-focus area search is necessary (step S43: No), subsequent steps S13 to S15 are the same as those in the third embodiment. Note that the user may manually adjust the tilt of the stage 131 with reference to the tilt information displayed in step S14 without executing step S15.

  As described above, according to the fourth embodiment, since the out-of-focus area exists only on one side of one field of view as in one-sided blur, the width of the in-focus area cannot be specified. Even in such a case, by moving the field of view and capturing an image, it is possible to grasp the whole image of the in-focus area and calculate the tilt information between the objective lens 140 and the subject from only the image. Further, according to the fourth embodiment, the direction in which the non-focused area exists can be accurately estimated from the positional relationship between the centroid of the connected non-focused area and the centroid of the focused area. It is possible to calculate the inclination information reliably and efficiently by specifying the direction to be performed. For example, even if there is a non-focused region in the oblique direction of the visual field shown in the acquired image, the moving direction of the visual field can be specified by one process.

(Embodiment 5)
Next, a fifth embodiment of the present invention will be described.
FIG. 44 is a block diagram showing a configuration of a microscope system according to Embodiment 5 of the present invention. As shown in FIG. 44, the microscope system 5 according to the fifth embodiment includes a microscope apparatus 10 and an alignment apparatus 60. Among these, the configuration and operation of the microscope apparatus 10 are the same as those in the first embodiment (see FIG. 2).

  The alignment apparatus 60 includes a control unit 61 instead of the control unit 23 in the alignment apparatus 30 according to the second embodiment. The configuration and operation of each part of the alignment apparatus 60 other than the controller 61 are the same as those in the second embodiment.

  The control unit 61 includes a focus area detection unit 231, a focus position comparison unit 611, and a tilt information calculation unit 612. Among these, the operation of the focus area detection unit 231 is the same as that of the first embodiment.

  45 and 46 are schematic diagrams illustrating a method of capturing the subject S in the fifth embodiment. In the fifth embodiment, as shown in FIGS. 45 and 46, the subject S is inclined in advance by a known inclination angle γ with respect to the focal plane of the observation optical system 104 including the objective lens 140. Take an image with. In order to incline both relatively, for example, as shown in FIG. 45, the subject S is placed on the placement surface 131a of the stage 131 installed horizontally, and the focusing surface is inclined with respect to the subject S. The observation optical system 104 may be installed so as to be inclined by the angle γ. In this case, an inclination adjustment driving unit 105 that changes the inclination of the observation optical system 104 by electrical control may be further provided. Alternatively, as shown in FIG. 46, the observation optical system 104 is installed so that the optical axis L is orthogonal to the mounting surface 131a, and an inclined pedestal 106 is installed on the mounting surface 131a. The subject S may be arranged. Note that the mechanism for relatively tilting the optical axis L and the subject S is not limited to these examples. In the following description, as shown in FIG. 45, imaging is performed with the observation optical system 104 side inclined.

  In the fifth embodiment, the focused surface of the observation optical system 104 is placed on the subject S in a state in which it is guaranteed that neither the stage 131 nor the observation optical system 104 is tilted as in the factory shipment. (Hereinafter, this state is referred to as a reference state) when the object S is focused on the observation optical system 104 (hereinafter referred to as a reference focus region). Hereinafter, the reference focus information) is stored in the parameter storage unit 241 in advance. Specifically, the reference focusing information is information on the position and width of the reference focusing area detected from an image acquired by performing imaging in the reference state (hereinafter referred to as a reference image). Alternatively, the reference image itself may be stored in the parameter storage unit 241. Alternatively, the position and width of the reference focus area may be calculated from various parameters stored in the parameter storage unit 241. This is because the position and width of the reference focus area are defined by parameters such as the size of the light receiving surface (imager size) of the image sensor 211a, the depth of field DOF of the objective lens 140, and the tilt angle γ.

The focus position comparison unit 611 calculates a difference by comparing the position and width of the focus area detected from the observation image of the subject S with the position and width of the reference focus area.
The tilt information calculation unit 612 calculates the tilt information of the subject S from the difference between the position and width of the focus area calculated by the focus position comparison unit 611.

Next, the operation of the alignment apparatus 60 will be described. FIG. 47 is a flowchart showing the operation of the alignment apparatus 60. Note that steps S10 and S11 shown in FIG. 47 are the same as those in the first embodiment. In the following description, it is assumed that the image M6 shown in FIG. 48A has been acquired in step S10. FIG. 48A shows a state in which the image M6 in which the area of the subject S entering the visual field V (see FIG. 6) is divided into a plurality of small areas (see step S113 in FIG. 7). In addition, it is assumed that the position and width of the reference focus area R _{focus (b)} detected from the reference image M _base shown in FIG. 48B is stored in the parameter storage unit 241 in advance.

In step S51 subsequent to step S11, the in-focus position comparison unit 611 reads information on the reference in-focus area R _{focus (b)} stored in the parameter storage unit 241 and is detected from the image M6 acquired in step S10. A difference is calculated by comparing the position and width of the in-focus area R _{focus6 with} the position and width of the reference in-focus area R _{focus (b)} . When the reference image M _base is stored in the storage unit 24, the in-focus position comparison unit 611 causes the in-focus area detection unit 231 to detect the reference in-focus area R _{focus (b)} from the reference image. The position and width of the reference focusing area R _{focus (b)} are acquired. Alternatively, the focus position comparison unit 611 reads parameters such as the imager size, the depth of field DOF of the objective lens 140 being used, and the tilt angle γ from the parameter storage unit 241, and the reference focus region R _{focus (b)} The position and width may be calculated.

More specifically, the in-focus position comparison unit 611 _calculates a _difference between the width W _focus6 of the in-focus area R _focus6 in the image M6 and the width W _{focus (b)} of the reference in-focus area R _{focus (b)} (hereinafter, difference) ΔW = W _{focus (b)} −W _focus6 is calculated. When the width of the focus area in the observation image is narrower than the width of the reference focus area, the sign of the difference width ΔW is positive, and the width of the focus area of the observation image is wider than the width of the reference focus area. The sign of the difference width ΔW is negative.

Moreover, the focus position comparator unit 611, the coordinates of the center point P ₆ in the focus area R _Focus6 in the image M6 (hereinafter, referred to as the fulcrum position) of the center point P _base the reference focus area R _{focus (b)} coordinate A difference (hereinafter referred to as a difference fulcrum) ΔC (pixel) from (same as above) is calculated. The difference fulcrum ΔC represents the displacement of the focus area R _focus6 from the center of the image. When the focus area R _focus6 is biased to the left side of the image M6, the sign is negative and the focus area R _focus6 is on the right side of the image M6. If it is biased, the sign is positive.

  The in-focus position comparison unit 611 stores the difference width ΔW and the difference fulcrum ΔC in the parameter storage unit 241.

  In subsequent step S52, the tilt information calculation unit 612 reads various parameters from the parameter storage unit 241, and calculates the tilt information of the observation optical system 104 using the difference width ΔW and the difference fulcrum ΔC calculated in step S51. FIG. 49 is a flowchart showing the inclination information calculation processing executed by the inclination information calculation unit 612. 50 to 52 are schematic diagrams for explaining the inclination specifying calculation processing.

  50A shows the positional relationship between the objective lens 140 and the subject S in the XZ plane, and FIG. 50B shows the image of the subject S that enters the field of view of the objective lens 140 in the case of the positional relationship. M6 is shown in the XY plane. The same applies to FIGS. 51 and 52. 50 to 52, for easy understanding, the subject S and the field of view V are illustrated on the assumption that the subject S is a plane having no thickness.

Further, as shown in FIG. 50A, in the following, the field size of the observation optical system 104 (objective lens 140) is Fov (μm), the subject depth is DOF (μm), and the in-focus plane of the observation optical system 104 And the subject S is the tilt angle β (rad), the height of the tilt occurring in the region of the subject S included in the field of view V is z (μm), and the size of the image M6 corresponding to the field size Fov (image Size) is W _image (pixel), and the width of the focus area R _focus6 in the image M6 is W _Focal (pixel). Among these, parameters (field size Fov, depth of field DOF, image size W _image ) depending on the specifications of the microscope apparatus 10 and the imaging unit 211 are stored in advance in the parameter storage unit 241 and read out as necessary. It is.

FIG. 50 shows a reference state in which there is no deviation in inclination between the focusing surface of the observation optical system 104 and the subject S. That is, the inclination angle β = γ, and the reference image M _base is acquired when imaging is performed in this state, and the region R ₀ included in the depth of field DOF in the field of view V is focused on the image M _base . It is detected as a region R _{focus (b)} . On the other hand, FIG. 51 and FIG. 52 show a state in which an inclination shift occurs between the focusing surface and the subject S.

First, in step S521, the tilt information calculation unit 612 calculates the tilt direction of the observation optical system 104 from the difference width ΔW. When the sign of the difference width ΔW is positive, that is, as shown in FIG. 51, the inclination deviation direction is when the width of the focusing area R _focus6 is narrower than the width of the reference focusing area R _{focus (b)} . It is determined that the observation optical system 104 is the same as the tilt direction. This indicates that the inclination angle β is larger than the reference state shown in FIG. On the other hand, when the sign of the difference width ΔW is negative, that is, as shown in FIG. 52, _when the width of the focusing area R _focus6 is wider _than the width of the reference focusing area R _{focus (b)} , the direction of the inclination shift Is determined to be opposite to the tilt direction of the observation optical system 104. This indicates that the inclination angle β is smaller than the reference state shown in FIG.

Further, the inclination information calculation unit 612 calculates an inclination angle deviation amount α ′ given by the following equation (6).
α ′ = γ−β (6)

In the equation (6), the inclination angle β is given by the following equation (7).
The sign of the inclination angle β is the same sign as the difference width ΔW.

In the equation (7), the inclination height z is given by the following equation (8).

  In subsequent step S522, the inclination information calculation unit 612 determines the difference fulcrum ΔC as the deviation amount of the inclination fulcrum. Here, when the difference fulcrum ΔC = 0, the center of the visual field V becomes the tilt fulcrum. In addition, when the sign of the difference fulcrum ΔC is plus, it indicates that the tilt fulcrum is shifted in the plus X direction from the center of the field of view V. When the sign of the difference fulcrum ΔC is minus, the tilt fulcrum is the center of the field of view V. Indicates that it is shifted in the minus X direction.

  In subsequent step S523, the inclination information calculation unit 612 stores the inclination information acquired in steps S521 and S522, that is, the inclination angle deviation direction, the inclination angle deviation amount α ′, and the inclination fulcrum deviation amount as parameter information. Stored in the unit 241.

Thereafter, the operation of the alignment apparatus 60 returns to the main routine.
Step S14 following step S52 is the same as that in the second embodiment.

  In step S53 following step S14, the tilt adjustment control unit 31 reads the tilt information calculated in step S52 from the parameter storage unit 241, generates a control signal based on the tilt information, and outputs the control signal to the microscope apparatus 10. The inclination of the stage 131 or the observation optical system 104 is adjusted.

The tilt adjustment control unit 31 first adjusts the tilt direction and tilt angle of the focal plane with respect to the subject S at the center position of the visual field V.
For example, as shown in FIG. 45, in the reference state, when the mounting surface 131a of the stage 131 is installed horizontally and the observation optical system 104 is tilted, the tilt adjustment control unit 31 sends a control signal to the tilt adjustment drive unit 105. Is output, and the tilt of the observation optical system 104 is returned in the direction opposite to the previously tilted direction by the tilt angle γ (see FIG. 50) in the reference state.

  Subsequently, the tilt adjustment control unit 31 adjusts the tilt of the stage 131. Specifically, as shown in FIG. 51, when the subject S moves up to the right with respect to the in-focus plane, a control signal is output to the adjustment knob driving unit 152 (see FIG. 2), and the stage 131 is moved in the XZ plane. Rotate clockwise around the stage center. On the other hand, as shown in FIG. 52, when the subject S is lowered to the right with respect to the in-focus plane, a control signal is output to the adjustment knob driving unit 152, and the stage 131 is counterclockwise with respect to the stage center in the XZ plane. Rotate around.

  On the other hand, as shown in FIG. 46, in the reference state, when the observation optical system 104 is installed so that the optical axis L is vertical and the subject S side is tilted, the subject S is removed by removing the base 106 or the like. Returning to the horizontal state, the inclination of the observation optical system 104 is adjusted. Specifically, when the subject S is raised to the right with respect to the in-focus plane, a control signal is output to the tilt adjustment driving unit 105, and the observation optical system 104 is counterclockwise with respect to the imager center in the XZ plane. Rotate like so. On the contrary, when the subject S is lowered to the right with respect to the in-focus plane, a control signal is output to the tilt adjustment driving unit 105 and the observation optical system 104 is rotated in the XZ plane so as to be clockwise with respect to the imager center. Let

Subsequently, the tilt adjustment control unit 31 moves the stage 131 in the direction opposite to the sign of the shift amount by the shift amount based on the shift region of the tilt fulcrum. For example, when the sign of the shift amount in the X direction is positive, a control signal is output to the stage driving unit 132, and the stage 131 is moved in the negative direction. On the other hand, when the sign of the shift amount is negative, a control signal is output to the stage drive unit 132, and the stage 131 is moved in the positive X direction. As a result, the tilt fulcrum coincides with the center of the visual field of the observation optical system 104 (that is, the position through which the optical axis L passes), and an in-focus image with an appropriate visual field can be acquired.
The adjustment method in the Y direction is the same.

  As described above, according to the fifth embodiment, imaging is performed by tilting the observation optical system 104 or the subject S in advance by a known angle, so that the observation optical system 104 or the stage 131 is not moved in the Z direction. The inclination direction can be specified. Further, even when one blur occurs, all the tilt information can be acquired without moving the visual field V. Therefore, the alignment of the microscope apparatus 10 can be performed at high speed and efficiently. Further, when imaging is performed with the observation optical system 104 or the subject S tilted, the contrast between the in-focus area and the out-of-focus area can be adjusted so that the detection accuracy of the in-focus area is improved. It is also possible.

  The first to fifth embodiments and the modifications described above are not limited as they are, and various inventions are formed by appropriately combining a plurality of components disclosed in the embodiments and the modifications. be able to. For example, some components may be excluded from all the components shown in the embodiment. Or you may form combining the component shown in different embodiment suitably.

1-5 Microscope system 10 Microscope device 20, 30, 40, 50, 60 Alignment device 21 Image acquisition unit 22 Imaging control unit 23, 41, 51, 61 Control unit 24 Storage unit 25 Input unit 26 Output unit 27 Display device 31 Tilt Adjustment control unit 100 Arm 101 Trinocular tube unit 102 Lens tube 103 Eyepiece unit 104 Observation optical system 105 Tilt adjustment drive unit 106 Base 110 Epi-illumination unit 111 Epi-illumination light source 112 Epi-illumination optical system 120 Trans-illumination unit 121 Trans-illumination unit Light source 122 Transmission illumination optical system 130 Electric stage unit 131 Stage 132 Stage drive unit 133 Position detection unit 140, 141 Objective lens 142 Revolver 150 Stage tilt adjustment unit 152 Adjustment knob drive unit 211 Imaging unit 212 Memory 23 , 510 Go focus area detection unit 232,612 gradient information calculating unit 241 parameter storage unit 242 program storage unit 411 view movement determination section 511 area classification unit 512 unfocused region searching determination section 611 focus position comparator unit

Claims (8)

An image acquisition unit for acquiring an image in which the region of the subject entering the field of view of an observation optical system provided opposite to the subject is captured;
A focusing area detecting means for detecting a focusing area from the image;
Inclination information calculation means for calculating inclination information between the observation optical system and the subject based on information on the in-focus area detected by the in-focus area detection means;
A determination unit for determining whether to perform imaging by moving a field of view of the observation optical system with respect to the subject based on a positional relationship between a focus area detected from the image and an area other than the focus area;
Equipped with a,
When it is determined that the determination unit moves the field of view and performs imaging, the image acquisition unit acquires a second image in which the field of view of the observation optical system with respect to the subject is different from the image,
The focus area detection means further detects a focus area from within the second image,
The tilt information calculating means calculates the tilt information based on information about the in-focus area detected from the image and the second image, respectively.
An alignment apparatus characterized by that.   The alignment apparatus according to claim 1, wherein the information related to the focus area is a position and a width of the focus area in the image.   3. The tilt information according to claim 1, wherein the tilt information is at least one of a tilt direction and a tilt angle generated between a focusing surface of the observation optical system and the subject. Alignment device.   The apparatus further comprises a tilt adjustment control unit that performs control to adjust a tilt generated between the subject and the observation optical system based on the tilt information calculated by the tilt information calculation unit. Item 4. The alignment apparatus according to any one of Items 1 to 3.   The alignment apparatus according to claim 1, further comprising display means for displaying the inclination information calculated by the inclination information calculation means. An alignment apparatus according to any one of claims 1 to 5 ,
The observation optical system ;
A stage on which the subject is placed;
A microscope system comprising: An image acquisition step of acquiring an image in which a region of the subject entering the field of view of an observation optical system provided facing the subject is captured;
A focus area detection step for detecting a focus area from the image;
A determination step of determining whether to perform imaging by moving a field of view of the observation optical system with respect to the subject based on a positional relationship between a focus area detected from the image and an area other than the focus area;
A second image acquisition step of acquiring a second image in which the field of view of the observation optical system for the subject is different from the image when it is determined that the field of view is moved in the determination step;
A second focus area detection step of detecting a focus area from within the second image;
A tilt information calculating step of calculating tilt information between the observation optical system and the subject based on information on the in-focus area detected from the image and the second image, respectively;
An alignment method comprising: An image acquisition step of acquiring an image in which a region of the subject entering the field of view of an observation optical system provided facing the subject is captured;
A focus area detection step for detecting a focus area from the image;
A determination step of determining whether to perform imaging by moving a field of view of the observation optical system with respect to the subject based on a positional relationship between a focus area detected from the image and an area other than the focus area;
A second image acquisition step of acquiring a second image in which the field of view of the observation optical system for the subject is different from the image when it is determined that the field of view is moved in the determination step;
A second focus area detection step of detecting a focus area from within the second image;
A tilt information calculating step of calculating tilt information between the observation optical system and the subject based on information on the in-focus area detected from the image and the second image, respectively;
An alignment program for causing a computer to execute.