JP2013174709A - Microscope device and virtual microscope device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable faster and reliable performance of focusing processing.SOLUTION: An imaging element 1101 outputs an imaging signal based on an image formed on a first plane. An optical path difference sensor 1113 outputs a first contrast signal corresponding to an image formed on a second plane and outputs a second contrast signal corresponding to an image formed on a third plane. A stage drive part 103 changes a relative position of a stage 102 and an optical system 200. An AF signal processing part 112 drives the stage drive part 103 on the basis of the first and second contrast signals when each value of the first and second contrast signals to be output by the optical path difference sensor 1113 is equal to or greater than a predetermined value, and drives the stage drive part 103 on the basis of the imaging signal to be output by the imaging element 1101 when each value of the first and second contrast signals to be output by the optical path difference sensor 1113 is less than the predetermined value.

Description

  The present invention relates to a microscope apparatus and a virtual microscope apparatus.

  In recent years, a virtual microscope apparatus that captures an entire image of a slide glass (specimen slide) on which a sample is arranged is known in the field of pathology such as cell and tissue diagnosis. The virtual microscope apparatus captures a partial image of a specimen slide and synthesizes the captured partial images to generate one entire image. In addition, the virtual microscope apparatus is strongly required to acquire images with high resolution and high speed in order to improve the efficiency and accuracy of pathological diagnosis.

  FIG. 5 is a schematic diagram showing a partial image area captured by a conventionally known virtual microscope apparatus and an entire image area generated by synthesizing the partial images. In the illustrated example, a slide glass 470 and a sample 471 are shown. Also, partial image areas 401 to 412 and an entire image area 450 are shown in the area of the slide glass 470. In this case, the virtual microscope apparatus can capture the partial images of all the partial image regions 401 to 412 of the sample 471 by performing the imaging 12 times. Further, when the partial image areas 401 to 412 are combined, an entire image area 450 is obtained.

  FIG. 6 is a schematic diagram illustrating a partial image captured by a conventionally known virtual microscope apparatus and an entire image generated from the partial image. In the illustrated example, partial images 501 to 512 of partial image areas 401 to 412 captured by the virtual microscope apparatus are shown. Moreover, the whole image 550 which the virtual microscope apparatus produced | generated combining the partial images 501-512 is shown.

  In the example of the partial images 501 to 512 illustrated in FIG. 6, the marginal part is not illustrated, but the partial images 501 to 512 captured by the virtual microscope apparatus include the marginal part. For example, when the virtual microscope apparatus captures a partial image 501 of the partial image area 401, the partial image 501 including an image of an area around the partial image area 401 illustrated is captured. Similarly, when imaging the partial images 502 to 512 of the other partial image regions 402 to 412, the virtual microscope apparatus captures the partial images 502 to 512 including the images of the regions around the partial image regions 402 to 412.

  By the way, a pathological specimen that is an observation target of the virtual microscope apparatus is a sample that transmits light with low reflectance. For this reason, the autofocus (hereinafter referred to as AF) method used by the virtual microscope apparatus is mainly a so-called passive autofocus method that detects the contrast of an observation image and performs a focusing operation.

  FIG. 7 is a schematic diagram showing a configuration of a conventionally known virtual microscope apparatus. In the illustrated example, the virtual microscope apparatus 600 includes a stage 602, a stage drive unit 603, a light source 604, a condenser lens 605, an objective lens 606, an imaging lens 607, a camera 608, and an AF signal processing unit 609. And. Note that the camera 608 includes an image sensor 6081.

  The stage 602 is a table for placing a specimen slide 601 on which a pathological specimen or a biological tissue is placed on a slide glass. The stage 602 is disposed between the condenser lens 605 and the objective lens 606. The stage driving unit 603 drives the stage 602 in the vertical direction based on the AF signal (autofocus signal) output from the AF signal processing unit 609. The light source 604 generates light that illuminates the specimen slide 601.

  The condenser lens 605 collects the light emitted from the light source 604 and irradiates the sample slide 601. The objective lens 606 includes a plurality of lenses, and is disposed so as to face the sample slide 601. The objective lens 606 collects the light flux from the specimen slide 601 and irradiates the focused light to the imaging lens 607.

  The imaging lens 607 is disposed along the optical axis of the objective lens 606. The imaging lens 607 forms an image of the light condensed by the objective lens 606 on the imaging surface of the imaging element 6081 provided in the camera 608. As a result, light from the specimen slide 601 is guided to the image sensor 6081. An image sensor 6081 provided in the camera 608 receives light from the specimen slide 601 and photoelectrically converts the received light into an electrical signal corresponding to the intensity of the received light. The camera 608 generates image data of the specimen slide 601 based on the electrical signal photoelectrically converted by the image sensor 6081. The AF signal processing unit 609 calculates the contrast value of the image data generated by the camera 608 and outputs an AF signal so that the stage 602 moves to a position where the contrast value reaches a peak.

  Next, hill-climbing contrast AF performed by the virtual microscope apparatus 600 will be described. The camera 608 captures an image of the specimen slide 601. The AF signal processing unit 609 calculates a contrast value of an image captured by the image sensor 6081 and outputs an AF signal so that the stage 602 moves to a position where the contrast value reaches a peak. The stage driving unit 603 moves the stage 602 based on the AF signal output from the AF signal processing unit 609. As a result, the focus of the image sensor 6081 provided in the camera 608 is aligned with the specimen slide 601 (see, for example, Patent Document 1).

  A virtual microscope apparatus using AF different from the virtual microscope apparatus 600 shown in FIG. 7 is known. FIG. 8 is a schematic diagram showing a configuration of a conventionally known virtual microscope apparatus. In the illustrated example, the virtual microscope apparatus 700 includes a stage 602, a stage driving unit 603, a light source 604, a condenser lens 605, an objective lens 606, a camera 608, an AF signal processing unit 609, and a first half. A mirror 701, a first imaging lens 702, a second imaging lens 703, and an AF unit 704 are provided. Note that the camera 608 includes an image sensor 6081. The AF unit 704 includes a second half mirror 7041, a mirror 7042, and a line sensor 7043.

  The stage 602, the stage driving unit 603, the light source 604, the condenser lens 605, the objective lens 606, and the camera 608 are the same as the respective units shown in FIG. The first half mirror 701 is disposed between the objective lens 606 and the first imaging lens 702, and irradiates a part of light from the objective lens 606 to the second imaging lens 703. The remaining light is transmitted and applied to the first imaging lens 702.

  The first imaging lens 702 is disposed along the optical axis of the first half mirror 701. The first imaging lens 702 forms an image on the imaging surface of the imaging element 6081 included in the camera 608 of the light condensed by the objective lens 606 and transmitted through the first half mirror 701. Thereby, the light from the specimen slide 601 is guided to the camera 608. The second imaging lens 703 irradiates the AF unit 704 with light reflected by the first half mirror 701 out of the light collected by the objective lens 606. Thereby, the light from the specimen slide 601 is guided to the AF unit 704.

  The second half mirror 7041 is disposed between the second imaging lens 703 and the line sensor 7043. The second half mirror 7041 transmits a part of the light from the second imaging lens 703 and reflects a part thereof in the direction of the mirror 7042. Thereby, the second half mirror 7041 divides the light from the second imaging lens 703 into two in the direction of the line sensor 7043 and the direction of the mirror 7042. The mirror 7042 reflects the light reflected by the second half mirror 7041 toward the line sensor 7043.

  Note that an optical path of light transmitted through the second half mirror 7041 and incident on the line sensor 7043 is an optical path a. The optical path of the light reflected by the second half mirror 7041 and reflected by the mirror 7042 and entering the line sensor 7043 is defined as an optical path b. The mirror 7042 is disposed so that the optical path of the reflected light is substantially parallel to the optical path a. The optical path a is an optical path that is directly guided from the second half mirror 7041 to the line sensor 7043, and the optical path b is an optical path that is guided from the second half mirror 7041 to the line sensor 7043 via the mirror 7042. The optical path length of b is longer than the optical path length of the optical path a.

  The line sensor 7043 is disposed on the optical path a and the optical path b, receives light from the specimen slide 601 guided to the optical path a and light from the specimen slide 601 guided to the optical path b, and receives the received light. Is photoelectrically converted into an electrical signal corresponding to the intensity of light and output. A plane that is a light detection surface of the line sensor 7043 is a light receiving surface.

  The AF signal processing unit 609 is based on the electrical signal output from the line sensor 7043, and the contrast of light from the specimen slide 601 that is guided to the optical path a and incident on the line sensor 7043, and the linear sensor that is guided to the optical path b. A difference from the contrast of light from the specimen slide 601 incident on 7043 is detected. The AF signal processing unit 609 outputs an AF signal so that the stage 602 moves to a position where the detected contrast difference becomes zero. Here, the electrical signal obtained by photoelectrically converting the light guided to the optical path a by the line sensor 7043 is defined as a rear pin signal, and the electrical signal obtained by photoelectrically converting the light guided to the optical path b by the line sensor 7043 is a front pin. Defined as a signal.

  FIG. 9 is a graph showing changes in the front pin contrast signal and the rear pin contrast signal when the position of the specimen slide 601 is changed in the optical axis direction of the objective lens 606 in the conventionally known virtual microscope apparatus 700. is there. The horizontal axis of the illustrated graph indicates the distance (defocus amount) between the specimen slide 601 and the objective lens 606, and the vertical axis indicates the contrast signal value. A curve 801 indicates the relationship between the defocus amount and the front pin contrast signal value. A curve 802 indicates the relationship between the defocus amount and the rear pin contrast signal value. When the specimen slide 601 placed on the stage 602 is moved from a position (−Z) sufficiently far from the objective lens 606 to a position (+ Z) sufficiently close to the objective lens 606, first, the front pin contrast signal value is changed to the optical path b. It becomes maximum at the projected image focus position (front pin position) due to and then decreases. Subsequently, the rear pin contrast signal value becomes maximum at the projected image focusing position (rear pin position) along the optical path a and then decreases.

  FIG. 10 is a graph showing changes in the differential contrast signal value when the position of the specimen slide 601 is changed in the optical axis direction in the conventionally known virtual microscope apparatus 700. The horizontal axis of the illustrated graph indicates the distance (defocus amount) between the specimen slide 601 and the objective lens 606, and the vertical axis indicates the difference contrast signal value. A curve 850 indicates the relationship between the defocus amount and the difference contrast signal value. When the specimen slide 601 placed on the stage 602 is moved from a position (−Z) sufficiently far from the objective lens 606 to a position (+ Z) sufficiently close to the objective lens 606, the difference contrast signal value is obtained as a projection image by the optical path b. An S-shaped curve is drawn which becomes maximum at the in-focus position, then decreases sharply to zero, and becomes minimum at the in-focus position of the projected image by the optical path a.

  Note that the imaging surface of the imaging device 6081 provided in the camera 608 is disposed at a position where the image of the specimen slide 601 is formed when the differential contrast signal value is zero. Therefore, the stage driving unit 603 moves the sample slide 601 (stage 602) to a position where the differential contrast signal value becomes zero based on the AF signal input from the AF signal processing unit 609, so that the camera 608 is provided. Control can be performed so that the focus of the image sensor 6081 is aligned with the specimen slide 601. Note that the process of controlling the image sensor 6081 included in the camera 608 so that the focus of the image sensor 6081 is aligned with the sample slide 601 is referred to as a focusing process.

  Next, optical path difference AF by the virtual microscope apparatus 700 will be described. First, the line sensor 7043 receives the light from the specimen slide 601 guided to the optical path a and the light from the specimen slide 601 guided to the optical path b, and the received light according to the intensity of the light. Photoelectrically converted into electrical signals and output. The AF signal processing unit 609 is based on the electrical signal output from the line sensor 7043, and the contrast of light from the specimen slide 601 that is guided to the optical path a and incident on the line sensor 7043, and the linear sensor that is guided to the optical path b. A difference from the contrast of light from the specimen slide 101 incident on 7043 is detected. The AF signal processing unit 609 outputs an AF signal so that the stage 602 moves to a position where the detected contrast difference becomes zero. The stage driving unit 603 moves the stage 602 based on the AF signal output from the AF signal processing unit 609. Accordingly, the focus of the image sensor 6081 provided in the camera 608 is aligned with the specimen slide 601 (see, for example, Patent Document 2).

JP-A-5-236315 Japanese Patent Laid-Open No. 53-50851

  As described above, a virtual microscope apparatus is required to acquire an image of a specimen slide with high resolution and high speed. In the conventionally known hill-climbing contrast AF process, an image picked up by the image pickup device is used, so that the AF field of view is the same as the image pickup field of view. Therefore, since the subject is included in the AF field of view, the AF operation can be performed reliably. However, in the hill-climbing contrast AF process, in order to find the peak of the contrast value of the image captured by the image sensor, the decrease or increase of the contrast value is detected by moving the stage or the objective lens in the optical axis direction. The direction of movement of the stage or objective lens must be determined. Therefore, the hill-climbing contrast AF process has a problem that the focusing speed is slow, and the image of the specimen slide cannot be acquired at high speed.

  Further, in the conventionally known optical path difference AF processing, since the in-focus driving direction can always be determined by the sign of the difference contrast value, the in-focus speed is fast. Therefore, the image of the specimen slide can be acquired at high speed. However, the AF field of the optical path difference AF has a very narrow range compared to the imaging field.

  FIG. 11 is a schematic diagram showing the relationship between the AF field of view and the imaging field of view of the conventionally known optical path difference AF. In the illustrated example, an object 901, an AF field 902 for optical path difference AF, and an imaging field 903 are shown. Thus, the AF field 902 of the optical path difference AF is a part of the imaging field 903. Therefore, as shown in the drawing, the subject 901 is included in the imaging field 903, but the subject 901 may not be included in the AF field 902 of the optical path difference AF.

  As described above, when the subject 901 does not exist in the AF field 902 of the optical path difference AF, the focusing process cannot be performed in the optical path difference AF process. When the subject 901 does not exist in the AF field 902 for the optical path difference AF, the stage 602 may be moved so that the subject 901 is included in the AF field 902 for the optical path difference AF. However, in the virtual microscope apparatus 700, in order to optimize the imaging time and facilitate the image composition process, when capturing partial images, it is necessary to sequentially capture adjacent partial images at equal intervals. Therefore, depending on the arrangement and shape of the subject 901, the subject 901 may not exist in the AF field 902 of the optical path difference AF, and there is a possibility that the specimen slide 601 cannot be focused.

  In addition, it is conceivable to provide a plurality of AF units so that the focusing operation can be performed without fail, so that the AF field of the optical path difference AF always exists on the subject, but the apparatus becomes large and the cost increases. Not realistic.

  The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a microscope apparatus and a virtual microscope apparatus that can perform focusing processing at higher speed and with reliability.

  The present invention includes a stage on which a sample is placed, a light source that illuminates the sample, and an image of a first region of the sample region formed on a first plane, and is included in the first region. An optical system that forms an image of the second region on a second plane and a third plane; an image sensor that outputs an image signal based on the image formed on the first plane; The first contrast signal corresponding to the image formed on the second plane and the image formed on the third plane is disposed between the second plane and the third plane. An optical path difference sensor that outputs a second contrast signal, a drive unit that changes a relative position between the stage and the optical system, the first contrast signal output from the optical path difference sensor, and the second contrast. If the signal is greater than or equal to a predetermined value, the first contrast signal and the The drive unit is driven based on the contrast signal of 2 and the imaging device outputs when the first contrast signal and the second contrast signal output by the optical path difference sensor are less than a predetermined value. And a focusing processing unit that drives the driving unit based on the imaging signal.

  In the microscope apparatus of the present invention, the focusing control unit may be configured such that the first contrast signal and the second contrast signal output from the optical path difference sensor are equal to or greater than a predetermined value. Based on the contrast signal and the second contrast signal, the driving unit is driven using optical path difference autofocus, and the first contrast signal and the second contrast signal output from the optical path difference sensor are predetermined. When the value is less than the value, the driving unit is driven using hill-climbing contrast autofocus based on the imaging signal output by the imaging device.

  In the present invention, a stage on which a sample is placed, a light source that illuminates the sample, and an image of a first region of the sample region are formed on a first plane, and the first region is formed on the first region. An optical system that forms an image of the second region included in the second plane and the third plane, an imaging element that outputs an imaging signal based on the image formed on the first plane, A first contrast signal corresponding to an image formed between the second plane and the third plane and formed on the second plane; and an image formed on the third plane. An optical path difference sensor that outputs a second contrast signal corresponding to the above, a drive unit that changes the relative position of the stage and the optical system, the first contrast signal output by the optical path difference sensor, and the second The first contrast signal when the contrast signal is equal to or greater than a predetermined value. When the driving unit is driven based on the second contrast signal and the first contrast signal and the second contrast signal output from the optical path difference sensor are less than a predetermined value, the imaging device A focusing processing unit that drives the driving unit based on the imaging signal output from the virtual microscope apparatus.

  According to the present invention, the stage mounts a sample. The light source illuminates the sample. In addition, the optical system forms an image of the first region of the sample region on the first plane, and images of the second region included in the first region are the second plane and the third plane. And form an image. The imaging element outputs an imaging signal based on the image formed on the first plane. The optical path difference sensor is disposed between the second plane and the third plane, and forms a first contrast signal corresponding to an image formed on the second plane and an image on the third plane. And a second contrast signal corresponding to the image formed. The drive unit changes the relative position between the stage and the optical system. In addition, the focusing processing unit determines that the first contrast signal and the second contrast signal are output when the first contrast signal and the second contrast signal output from the optical path difference sensor are equal to or greater than a predetermined value. And driving the drive unit based on the imaging signal output from the image sensor when the first contrast signal and the second contrast signal output from the optical path difference sensor are less than a predetermined value. .

  As a result, the focusing processing unit moves the driving unit based on the signal output from the optical path difference sensor when the first contrast signal and the second contrast signal output from the optical path difference sensor are equal to or greater than a predetermined value. It can be driven. The focusing processing unit drives the driving unit based on the imaging signal output from the imaging element when the first contrast signal and the second contrast signal output from the optical path difference sensor are less than a predetermined value. be able to. Therefore, the focusing process can be performed reliably at a higher speed.

It is the schematic which showed the structure of the microscope apparatus in one Embodiment of this invention. It is the schematic which showed the relationship between the optical path difference AF visual field of the optical path difference sensor in this embodiment, and the imaging visual field of an image pick-up element. The front pin signal and rear pin signal when the subject is present in the field of view of the optical path difference AF unit in the present embodiment, and the front pin signal and rear pin signal when the subject is not present in the field of view of the optical path difference AF unit. It is the graph which showed the relationship. It is the flowchart which showed the operation | movement procedure of the microscope apparatus in this embodiment. It is the schematic which showed the partial image area which the conventionally known virtual microscope apparatus images, and the whole image area | region which synthesize | combines and produces | generates a partial image. It is the schematic which showed the partial image imaged with the conventionally known virtual microscope apparatus, and the whole image produced | generated from the partial image. It is the schematic which showed the structure of the conventionally known virtual microscope apparatus. It is the schematic which showed the structure of the conventionally known virtual microscope apparatus. It is the graph which showed the change of the front pin contrast signal and the back pin contrast signal when the position of a sample slide is changed in the optical axis direction of an objective lens in the conventionally known virtual microscope apparatus. It is the graph which showed the change of the difference contrast signal value when the position of a sample slide is changed in an optical axis direction in the conventionally known virtual microscope apparatus. It is the schematic which showed the relationship between AF visual field of the optical path difference AF known conventionally, and an imaging visual field.

  Hereinafter, an embodiment of the present invention will be described with reference to the drawings. FIG. 1 is a schematic diagram illustrating a configuration of a microscope apparatus 1 according to the present embodiment. For example, the microscope apparatus 1 is a virtual microscope apparatus that captures partial images of a specimen slide and synthesizes the captured partial images to generate one entire image. Note that the microscope apparatus 1 may be a microscope apparatus that captures one image.

  In the illustrated example, the microscope apparatus 1 includes a stage 102, a stage driving unit 103 (driving unit), a light source 104, a condenser lens 105, an objective lens 106, a first half mirror 107, and a first connection. An image lens 108, a second imaging lens 109, a camera 110, an optical path difference AF unit 111, an AF signal processing unit 112 (focus processing unit), and an image processing unit 113 are provided. Note that the camera 110 includes an image sensor 1101. The optical path difference AF unit 111 includes a second half mirror 1111, a mirror 1112, and an optical path difference sensor 1113. The objective lens 106, the first half mirror 107, the first imaging lens 108, the second imaging lens 109, the second half mirror 1111, and the mirror 1112 are used as the optical system 200. .

  The stage 102 is a table for placing a specimen slide 101 on which a pathological specimen or a biological tissue is placed on a slide glass. The stage 102 is disposed between the condenser lens 105 and the objective lens 106. The stage driving unit 103 drives the stage 102 in the horizontal and vertical directions based on the AF signal (autofocus signal) output from the AF signal processing unit 112. The light source 104 generates light that illuminates the specimen slide 101.

  The condenser lens 105 collects the light emitted from the light source 104 and irradiates the sample slide 101. The objective lens 106 is composed of a plurality of lenses and is disposed so as to face the specimen slide 101. The objective lens 106 condenses the light flux from the specimen slide 101 and irradiates the first half mirror 107 with the condensed light. The first half mirror 107 is disposed between the objective lens 106 and the first imaging lens 108, and irradiates a part of the light from the objective lens 106 to the second imaging lens 109. The remaining light is transmitted and applied to the first imaging lens 108.

  The first imaging lens 108 is disposed along the optical axis of the first half mirror 107. In addition, the first imaging lens 108 is on the imaging surface (first plane) of the imaging device 1101 provided in the camera 110 with the light transmitted through the first half mirror 107 out of the light collected by the objective lens 106. To form an image. Thereby, the light from the specimen slide 101 is guided to the camera 110. An image sensor 1101 provided in the camera 110 receives light from the specimen slide 101 and photoelectrically converts the received light into an electric signal corresponding to the intensity of the received light. The camera 110 outputs an electrical signal (imaging signal) photoelectrically converted by the imaging element 1101.

  The second imaging lens 109 irradiates the optical path difference AF unit 111 with the light reflected by the first half mirror 107 out of the light collected by the objective lens 106. Thereby, the light from the specimen slide 101 is guided to the optical path difference AF unit 111. The second half mirror 1111 is disposed between the second imaging lens 109 and the optical path difference sensor 1113. The second half mirror 1111 transmits part of the light from the second imaging lens 109 and reflects part of the light in the direction of the mirror 1112. As a result, the second half mirror 1111 divides the light from the second imaging lens 109 into two in the direction of the optical path difference sensor 1113 and the direction of the mirror 1112. The mirror 1112 reflects the light reflected by the second half mirror 1111 in the direction of the optical path difference sensor 1113.

  Note that an optical path of light transmitted through the second half mirror 1111 and incident on the optical path difference sensor 1113 is an optical path A. The optical path of the light that is reflected by the second half mirror 1111, reflected by the mirror 1112, and incident on the optical path difference sensor 1113 is an optical path B. The mirror 1112 is disposed so that the optical path of the reflected light is substantially parallel to the optical path A. Further, the optical path A is an optical path that is directly guided from the second half mirror 1111 to the optical path difference sensor 1113, and the optical path B is an optical path that is guided from the second half mirror 1111 to the optical path difference sensor 1113 via the mirror 1112. The optical path length of the optical path B is longer than the optical path length of the optical path A. Therefore, the imaging point of the light guided to the optical path A is the rear pin position 11 on the plane m (second plane) behind the optical path difference sensor 1113. Further, the image formation point of the light guided to the optical path B is a front pin position 12 on the plane n (third plane) in front of the optical path difference sensor 1113.

  The optical path difference sensor 1113 is a line sensor, for example. The optical path difference sensor 1113 is arranged on the optical path A and the optical path B, and receives and receives the light from the specimen slide 101 guided to the optical path A and the light from the specimen slide 101 guided to the optical path B. Light is photoelectrically converted into an electrical signal corresponding to the intensity of the light and output. That is, the optical path difference sensor 1113 forms an image at the front pin position 12 on the plane n and the electrical signal (first contrast signal) corresponding to the light (image) imaged at the rear pin position 11 on the plane m. An electrical signal (second contrast signal) corresponding to the emitted light (image) is output. A plane that is a light detection surface of the optical path difference sensor 1113 is a light receiving surface.

  The AF signal processing unit 112 is guided to the optical path A based on the electrical signals (first contrast signal and second contrast signal) output from the optical path difference sensor 1113 and is incident on the optical path difference sensor 1113. And the contrast of the light from the specimen slide 101 that is guided to the optical path B and enters the optical path difference sensor 1113 is calculated. The AF signal processing unit 112 also contrasts the light from the specimen slide 101 that is guided to the optical path A and is incident on the optical path difference sensor 1113, and the specimen slide 101 that is guided to the optical path B and is incident on the optical path difference sensor 1113. If the value of the contrast with the light from the optical path difference sensor 1113 is equal to or greater than a predetermined value, the stage drive unit 103 is driven using the optical path difference AF based on the electrical signal output from the optical path difference sensor 1113. That is, the AF signal processing unit 112 performs a focusing operation using the optical path difference AF. A specific example of the focusing operation using the optical path difference AF will be described later.

  The AF signal processing unit 112 also contrasts the light from the specimen slide 101 that is guided to the optical path A and is incident on the optical path difference sensor 1113, and the specimen slide 101 that is guided to the optical path B and is incident on the optical path difference sensor 1113. If the value of the contrast with the light from is less than a predetermined value, the stage driving unit 103 is driven using the hill-climbing contrast AF based on the imaging signal output from the imaging element 1101. That is, the AF signal processing unit 112 performs a focusing operation using hill-climbing contrast AF. A specific example of the focusing operation using the hill-climbing contrast AF will be described later. The predetermined value is, for example, the minimum contrast value at which the AF signal processing unit 112 can use the optical path difference AF. Further, the predetermined value may be a value larger than the maximum value of the contrast value, for example, by measuring the contrast signal by performing the optical path difference AF process on the slide glass on which the subject is not mounted.

  The image processing unit 113 generates an image based on the electrical signal photoelectrically converted by the image sensor 1101. When the microscope apparatus 1 is a virtual microscope apparatus, a partial image is generated based on the electrical signal photoelectrically converted by the imaging element 1101, and a plurality of partial images are combined to generate one whole image.

  Next, a specific example of the focusing operation using the optical path difference AF will be described. The AF signal processing unit 112 is guided to the optical path A based on the electrical signal output from the optical path difference sensor 1113 and the contrast of the light from the specimen slide 101 incident on the optical path difference sensor 1113 and the optical path B. A difference (difference contrast signal value) from the contrast of light from the specimen slide 101 incident on the optical path difference sensor 1113 is detected. Here, the electrical signal obtained by photoelectrically converting the light guided to the optical path A by the optical path difference sensor 1113 is defined as a rear pin signal, and the electrical signal obtained by photoelectrically converting the light guided by the optical path difference sensor 1113 to the optical path B. Defined as front pin signal.

  The imaging surface of the imaging device 1101 provided in the camera 110 is disposed at a position where the image of the specimen slide 101 is formed when the differential contrast signal value is zero. Therefore, the stage drive unit 103 moves the sample slide 101 (stage 102) to a position where the difference contrast signal value becomes zero based on the AF signal input from the AF signal processing unit 112, so that the camera 110 includes. Control can be performed so that the focus of the image sensor 1101 matches the specimen slide 101.

  Next, a specific example of a focusing operation using hill-climbing contrast AF will be described. The AF signal processing unit 112 calculates the contrast value of the imaging signal output from the imaging element 1101 and outputs an AF signal so that the stage 102 moves to a position where the contrast value reaches a peak. The stage driving unit 103 moves the stage 102 based on the AF signal output from the AF signal processing unit 112. Thereby, it can control so that the focus of the image pick-up element 1101 with which the camera 110 is provided suits the sample slide 101.

  Next, the relationship between the optical path difference AF field of the optical path difference sensor 1113 (optical path difference AF unit 111) and the imaging field of view of the image sensor 1101 (camera 110) will be described. FIG. 2 is a schematic diagram showing the relationship between the optical path difference AF field of the optical path difference sensor 1113 and the imaging field of the image sensor 1101 in the present embodiment. In the illustrated example, a subject 201, an optical path difference AF field 202 of the optical path difference sensor 1113, and an imaging field 203 of the image sensor 1101 are shown. Thus, the optical path difference AF visual field 202 of the optical path difference sensor 1113 is a part of the imaging visual field 203 of the image sensor 1101. Therefore, as shown in the drawing, the subject 201 is included in the imaging field 203 of the imaging device 1101, but the subject 201 may not be included in the optical path difference AF field 202 of the optical path difference sensor 1113.

  Next, a front pin signal and a rear pin signal when the subject exists in the field of view of the optical path difference AF unit 111, and a front pin signal and a rear pin signal when the subject does not exist in the field of view of the optical path difference AF unit 111, The relationship will be described. FIG. 3 shows a front pin signal and a rear pin signal when a subject is present in the field of view of the optical path difference AF unit 111 and a front pin signal when the subject is not present in the field of view of the optical path difference AF unit 111 in this embodiment. It is the graph which showed the relationship with a back pin signal.

  The horizontal axis of the illustrated graph indicates the distance (defocus amount) between the specimen slide 101 and the objective lens 106, and the vertical axis indicates the contrast signal value. A curve 301 indicates the relationship between the defocus amount and the front pin contrast signal value when a subject is present in the optical path difference AF field 202 of the optical path difference sensor 1113. A curve 302 indicates the relationship between the defocus amount and the rear pin contrast signal value when a subject is present in the optical path difference AF visual field 202 of the optical path difference sensor 1113. A curve 303 shows the relationship between the defocus amount and the front pin contrast signal value when no subject is present in the optical path difference AF field 202 of the optical path difference sensor 1113. A curve 304 shows the relationship between the defocus amount and the rear pin contrast signal value when no subject is present in the optical path difference AF field 202 of the optical path difference sensor 1113.

  In the illustrated example, when a subject is present in the optical path difference AF visual field 202 of the optical path difference sensor 1113, the contrast in the optical path difference AF visual field 202 is high, so that the contrast signal output from the optical path difference sensor 1113 is large and has a peak. Get higher. On the other hand, when no subject is present in the optical path difference AF field 202 of the optical path difference sensor 1113, the contrast in the optical path difference AF field 202 is low, so the contrast signal output from the optical path difference sensor 1113 is extremely small and no peak is generated. . Therefore, by comparing the magnitude of the front pin signal or the rear pin signal, or the magnitude of the sum signal of the front pin signal and the rear pin signal with a predetermined value (threshold value), the optical path difference AF visual field 202 of the optical path difference sensor 1113 is obtained. It can be determined whether or not there is a subject.

Next, an operation procedure of the microscope apparatus 1 in the present embodiment will be described. FIG. 4 is a flowchart showing an operation procedure of the microscope apparatus 1 in the present embodiment.
(Step S101) The stage drive unit 103 enables the stage 102 (specimen slide 101) so that the image sensor 1101 of the camera 110 can capture the first partial image (for example, the partial image in the partial image region 401 shown in FIG. 5). Is moved horizontally. Thereafter, the process proceeds to step S102.

  (Step S102) The stage drive unit 103 moves the stage 102 in the vertical direction so that the interval between the objective lens 106 and the stage 102 (specimen slide 101) is the initial interval. The initial interval is, for example, an interval at which the objective lens 106 and the stage 102 (specimen slide 101) are closest to each other, and an interval at which the in-focus state is sufficiently defocused. Thereafter, the process proceeds to step S103.

(Step S103) The stage drive unit 103 moves the stage 102 by a certain distance in the direction away from the objective lens 106, that is, in the in-focus direction. Thereafter, the process proceeds to step S104.
(Step S104) The optical path difference sensor 1113 receives the light from the specimen slide 101 guided to the optical path A and the light from the specimen slide 101 guided to the optical path B, and uses the received light as the light intensity. Photoelectrically converted into a contrast signal according to the output. Subsequently, the AF signal processing unit 112 outputs the contrast signal of light that is output from the optical path difference sensor 1113 to the optical path A and is incident on the optical path difference sensor 1113, and is guided to the optical path B and is incident on the optical path difference sensor 1113. A contrast sum signal is calculated by adding the value of the contrast signal of the light to be obtained. Thereafter, the process proceeds to step S104.

(Step S105) The AF signal processing unit 112 determines whether or not the contrast sum signal calculated in the process of Step S104 is a predetermined value or more. When the AF signal processing unit 112 determines that the contrast sum signal is equal to or greater than a predetermined value, that is, when an object is present in the optical path difference AF visual field 202, the process proceeds to step S107. The process proceeds to step S106.
(Step S106) The AF signal processing unit 112 determines whether or not the distance between the objective lens 106 and the stage 102 is equal to or less than the maximum interval value. The maximum distance value is a maximum distance value at which the objective lens 106 and the stage 102 can be separated. If the AF signal processing unit 112 determines that the distance between the objective lens 106 and the stage 102 is equal to or less than the maximum distance value, the process returns to step S103, and otherwise, the process proceeds to step S108.

  (Step S <b> 107) The AF signal processing unit 112 outputs the contrast signal of light that is output from the optical path difference sensor 1113 to the optical path A and is incident on the optical path difference sensor 1113, and the optical path difference sensor 1113 that is guided to the optical path B. Based on the contrast signal of the incident light, a focusing operation is performed using the optical path difference AF. Thereafter, the process proceeds to step S109.

  (Step S108) The image sensor 1101 provided in the camera 110 receives light from the specimen slide 101 and outputs an image signal. The AF signal processing unit 112 performs a focusing operation using the hill-climbing contrast AF based on the imaging signal output from the imaging element 1101. Thereafter, the process proceeds to step S109.

(Step S <b> 109) The image sensor 1101 included in the camera 110 captures a partial image region and outputs an image signal to the image processing unit 113. The image processing unit 113 generates a partial image based on the imaging signal output from the imaging element 1101. Thereafter, the process proceeds to step S110.
(Step S110) The image processing unit 113 determines whether all partial images have been generated. If the image processing unit 113 determines that all the partial images have been generated, the process proceeds to step S112. Otherwise, the process proceeds to step S111.

(Step S111) The stage drive unit 103, when the imaging device 1101 of the camera 110 captures the next partial image (for example, the partial image of the partial image region 401 shown in FIG. The stage 102 (specimen slide 101) is moved in the horizontal direction so that a partial image of the region 402 can be captured. Thereafter, the process returns to step S102.
(Step S112) The image processing unit 113 synthesizes the partial images generated in the process of step S109 to generate one whole image. Thereafter, the process ends.

  As described above, in the present embodiment, that is, when the first contrast signal and the second contrast signal output from the optical path difference sensor 1113 are greater than or equal to a predetermined value, the AF signal processing unit 112 performs the optical path difference sensor. The stage drive unit 103 is driven based on the signal output from 1113. The AF signal processing unit 112 drives the stage based on the imaging signal output from the imaging element 1101 when the first contrast signal and the second contrast signal output from the optical path difference sensor 1113 are less than a predetermined value. The unit 103 is driven.

  That is, when an object is present in the optical path difference AF field 202 of the optical path difference sensor 1113, the focusing operation is performed using the optical path difference AF based on the contrast signal output from the optical path difference sensor 1113. When there is no subject in the optical path difference AF visual field 202 of the optical path difference sensor 1113, the focusing operation is performed using the hill-climbing contrast AF based on the imaging signal output from the imaging element 1101 provided in the camera 110. Accordingly, since the focusing operation is performed based on the signal including the subject, it is possible to capture a partial image that is always in focus. Therefore, the focusing process can be performed reliably at a higher speed.

  Although one embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment, and includes design and the like within a scope not departing from the gist of the present invention.

  In the above-described embodiment, the AF signal processing unit 112 outputs the contrast signal of the light that is guided to the optical path A and is incident on the optical path difference sensor 1113 that is output from the optical path difference sensor 1113 and the optical path difference sensor that is guided to the optical path B. A contrast sum signal obtained by adding the value of the contrast signal of the light incident on 1113 is calculated. Then, the AF signal processing unit 112 determines that the subject exists in the optical path difference AF field 202 when the calculated contrast sum signal is equal to or greater than a predetermined value, and otherwise, the optical path difference AF field 202. However, the present invention is not limited to this. For example, a contrast signal of light that is guided to the optical path A and incident on the optical path difference sensor 1113 without calculating a contrast sum signal, and a contrast signal of light that is guided to the optical path B and incident on the optical path difference sensor 1113. It may be determined whether or not a subject is present in the optical path difference AF visual field 202 by comparing each of the values with a predetermined threshold value.

  In the above-described embodiment, when the relative position between the stage 102 (specimen slide 101) and the objective lens 106 is changed, the stage driving unit 103 moves the stage 102. However, the present invention is not limited to this. For example, a drive unit that moves the objective lens may be provided, and the drive unit may move the objective lens 106 when the relative position between the stage 102 (specimen slide 101) and the objective lens 106 is changed.

  DESCRIPTION OF SYMBOLS 1 ... Microscope apparatus, 11 ... Back pin position, 12 ... Front pin position, 101, 601 ... Sample slide, 102, 602 ... Stage, 103, 603 ... Stage drive part, 104,604 ... light source, 105,605 ... condenser lens, 106,606 ... objective lens, 107,701, ... first half mirror, 108,702 ... first imaging Lens, 109, 703 ... Second imaging lens, 110, 608 ... Camera, 111, 704 ... AF unit, 112, 609 ... AF signal processing unit, 113 ... Image processing unit , 200 ... Optical system, 201, 901 ... Subject, 202, 902 ... Optical path difference AF field, 203, 903 ... Imaging field, 401-412 ... Partial image region, 450 ... Whole picture Area, 470 ... slide glass, 471 ... sample, 501 to 512 ... partial image, 550 ... whole image, 600, 700 ... virtual microscope apparatus, 607 ... imaging lens, 1101 , 6081 ... Image sensor, 1111, 7041 ... Second half mirror, 1112, 7042 ... Mirror, 1113 ... Optical path difference sensor, 7043 ... Line sensor

Claims (3)

A stage on which a sample is placed;
A light source for illuminating the sample;
An image of a first area of the sample area is formed on a first plane, and an image of a second area included in the first area is formed on a second plane and a third plane. An optical system
An imaging element that outputs an imaging signal based on an image formed on the first plane;
A first contrast signal corresponding to an image formed between the second plane and the third plane and formed on the second plane; and an image formed on the third plane. An optical path difference sensor that outputs a second contrast signal corresponding to
A drive unit for changing a relative position between the stage and the optical system;
When the first contrast signal and the second contrast signal output from the optical path difference sensor are equal to or greater than a predetermined value, the driving unit is based on the first contrast signal and the second contrast signal. And when the first contrast signal and the second contrast signal output by the optical path difference sensor are less than a predetermined value, the drive unit is controlled based on the imaging signal output by the imaging element. A focusing processing unit to be driven;
A microscope apparatus comprising: When the first contrast signal and the second contrast signal output from the optical path difference sensor are equal to or greater than a predetermined value, the focusing control unit is configured to output the first contrast signal and the second contrast signal. If the first contrast signal and the second contrast signal output from the optical path difference sensor are less than a predetermined value by driving the driving unit using optical path difference autofocus based on The microscope apparatus according to claim 1, wherein the driving unit is driven using hill-climbing contrast autofocus based on the imaging signal output from the element. A stage on which a sample is placed;
A light source for illuminating the sample;
An image of a first area of the sample area is formed on a first plane, and an image of a second area included in the first area is formed on a second plane and a third plane. An optical system
An imaging element that outputs an imaging signal based on an image formed on the first plane;
A first contrast signal corresponding to an image formed between the second plane and the third plane and formed on the second plane; and an image formed on the third plane. An optical path difference sensor that outputs a second contrast signal corresponding to
A drive unit for changing a relative position between the stage and the optical system;
When the first contrast signal and the second contrast signal output from the optical path difference sensor are equal to or greater than a predetermined value, the driving unit is based on the first contrast signal and the second contrast signal. And when the first contrast signal and the second contrast signal output by the optical path difference sensor are less than a predetermined value, the drive unit is controlled based on the imaging signal output by the imaging element. A focusing processing unit to be driven;
A virtual microscope apparatus comprising:

Images