JP2014238466A - Image acquisition device and image acquisition method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an image acquisition device and an image acquisition method, capable of acquiring the excellent image of an object to be inspected reduced in blur, even when an objective optical system having high resolution is used.SOLUTION: An image acquisition device 1000 including a stage 20 which is movable in such a state that an object to be inspected 30 including a cover glass 301 and a sample 302 being in contact with the cover glass 301 are held, an objective optical system 400 forming the image of the object to be inspected 30, and an imaging element 50 imaging the object to be inspected 30 through the objective optical system 400 includes an arithmetic part 4 controlling the stage 20, so that the surface of the sample 302 is within the depth of field of the objective optical system 400, based on the measurement value of the thickness of the cover glass 301.

Description

  The present invention relates to an image acquisition device, and is suitable, for example, for a microscope that acquires image data of a pathological specimen.

  In the field of pathology and the like, a digital image (virtual slide image) is acquired by imaging a test object (preparation) including a sample using an image acquisition device (microscope), and the digital image is displayed at a high resolution. An image acquisition system to be displayed on the screen attracts attention.

  Patent Document 1 proposes a microscope using an objective optical system having a high resolving power in order to acquire a high-resolution image of a preparation. Patent Document 2 discloses a microscope that can efficiently detect a position where the contrast value is maximized by setting a search range based on the thickness of a sample when performing automatic focus adjustment. Has been.

JP 2009-003016 A JP 2009-223164 A

  However, it is known that the thickness of the cover glass varies by about 30 μm to 50 μm for each slide. Therefore, when an objective optical system having a high resolving power is used, the depth of focus becomes shallow, and thus a focus shift due to variations in the thickness of the cover glass for each preparation cannot be ignored.

  In the microscopes according to Patent Documents 1 and 2, since the influence of the cover glass thickness on the focus is not considered, it is difficult to obtain a good digital image when an objective optical system having high resolution is used. Become.

  Therefore, an object of the present invention is to provide an image acquisition apparatus and an image acquisition method that can acquire an image of a good test object with less blur even when an objective optical system having high resolution is used. .

  In order to achieve the above object, an image acquisition apparatus according to an aspect of the present invention includes a stage that can hold and move a test object including a cover glass and a sample in contact with the cover glass; An objective optical system that forms an image; and an imaging device that captures the test object via the objective optical system, and the surface of the sample is based on a measured value of the thickness of the cover glass. It has a calculating part which controls the stage so that it may be in the depth of field of the objective optical system.

  Further objects and other features of the present invention will become apparent from the preferred embodiments described below with reference to the accompanying drawings.

  According to the present invention, it is possible to provide an image acquisition device and an image acquisition method that can acquire a good image of a test object with less blur even when an objective optical system having high resolution is used.

1 is a schematic diagram of an image acquisition device according to Embodiment 1. FIG. 1 is a schematic diagram of a preparation (test object) according to Example 1. FIG. FIG. 2 is a diagram illustrating an arrangement of a reflection mirror, a re-imaging optical system, and an image sensor according to Example 1. 3 is a flowchart illustrating an image acquisition method according to the first embodiment. It is a figure for demonstrating the measuring method of the focus position of an objective optical system. FIG. 6 is a diagram for explaining a focus adjustment method according to the first embodiment. 10 is a flowchart illustrating an image acquisition method according to the second embodiment. It is a figure for demonstrating the measuring method of the thickness of the cover glass which concerns on Example 2. FIG. FIG. 10 is a diagram for explaining a focus adjustment method according to the second embodiment. FIG. 10 is a schematic diagram of a main part of an image acquisition apparatus according to a third embodiment. 10 is a flowchart illustrating an image acquisition method according to the third embodiment. FIG. 10 is a diagram for explaining a focus adjustment method according to a third embodiment.

  Hereinafter, preferred embodiments of the present invention will be described with reference to the drawings. In addition, in each figure, the same reference number is attached | subjected about the same member and the overlapping description is abbreviate | omitted.

[Example 1]
FIG. 1 is a schematic diagram of a main part of an image acquisition apparatus 1000 according to the present embodiment. The image acquisition apparatus 1000 includes an imaging unit 1, a measurement unit 2, a detection unit 3, a calculation unit 4, and a stage 20. The imaging unit 1 includes an illumination optical system 10, an imaging optical system 40, a reflection mirror 70, an objective optical system 400 including a re-imaging optical system 60, an imaging device 50, a first measuring instrument 100, and a second measuring device. It has a measuring instrument 601. The measuring unit 2 includes a surface shape measuring instrument 90 and a third measuring instrument 901, and the detecting unit 3 includes a camera 80 and an imaging stand 82. Based on a program stored in the memory 500, the calculation unit 4 acquires each data described later, calculates a drive command value, and controls the stage 20.

  The stage 20 is configured to be movable while holding a preparation 30 as an object to be observed, and moves between the imaging unit 1 and the measurement unit 2 in accordance with a drive command from the calculation unit 4. Can do. The stage 20 includes an XY stage 23 that can change the position of the preparation 30 in the XY direction, and a Z tilt stage 24 that can change the positions of the preparation 30 in the Z direction, θx direction, and θy direction. Here, the Z direction corresponds to the optical axis direction of the imaging optical system 40, the XY direction corresponds to the direction perpendicular to the optical axis, the θx direction corresponds to the rotational direction around the X axis, and the θy direction corresponds to the rotational direction around the Y axis. To do. The stage 20 can adjust the position and orientation of the preparation 30 by the XY stage 23 and the Z tilt stage 24. The stage 20 includes a drive mechanism (not shown). As the drive mechanism, a swing mechanism such as a commercially available gonio stage or a rotation mechanism such as a rotary stage may be used.

  Examples of means for holding the preparation 30 in the stage 20 include a leaf spring, vacuum suction, and electrostatic suction. For example, when using a leaf spring, a method of pressing the non-imaging region of the slide 30 from the Z direction or pressing the side surface of the slide 30 from the XY direction can be considered. In the case of using vacuum suction or electrostatic suction, a method of sucking the non-imaging region of the preparation 30 from the back surface of the preparation 30 can be considered. With these holding means, the stage 20 can keep the holding state of the preparation 30 constant even when moving between the imaging unit 1 and the measuring unit 2. Note that the XY stage 23 and the Z tilt stage 24 are provided with openings for illuminating the preparation 30 by allowing light from the illumination optical system 10 in the imaging unit 1 to pass therethrough.

  In the present embodiment, a preparation 30 as shown in FIG. 2 is used as a specimen to be observed. The preparation 30 has a configuration in which a sample 302 (such as a biological sample such as a tissue section) disposed on a slide glass 303 is tightly fixed by a cover glass 301 and sealed with an adhesive 304. A label 333 on which information necessary for managing the preparation 30 (sample 302), such as an identification number of the slide glass 303 and a thickness of the cover glass 301, is recorded on the slide glass 303. Good.

  Hereinafter, a procedure for acquiring the image of the preparation 30 in the image acquisition apparatus 1000 will be described with reference to FIG.

  First, the preparation 30 stored in the stocker 200 is transported to the imaging stand 82 in the detection unit 3 by transporting means (not shown). In the detection unit 3, the preparation 30 is imaged by the camera 80, and a region (sample region) in which the sample is present in the preparation 30 is detected. Note that the camera 80 can capture at least the entire area of the cover glass in the preparation 30. According to the detection unit 3, the sample region information 81 can be acquired prior to measurement by the measurement unit 2 described later and image acquisition by the imaging unit 1. Therefore, since only the region where the sample exists can be set as a measurement and imaging target, the entire processing in the image acquisition apparatus 1000 can be speeded up.

  Next, the preparation 30 is placed on the stage 20 located in the measurement unit 2 by the moving unit 250. In the measurement unit 2, the surface shape of the preparation 30 is measured by the surface shape measuring instrument 90. Specifically, the surface shape measuring instrument 90 measures the position of the cover glass or the surface of the sample in the Z direction (Z position) while moving the XY stage 23 in the XY direction (scan driving). By calculating this measurement value by the calculation unit, surface shape information of the entire area of the cover glass or the sample can be acquired.

  In this embodiment, a distance sensor is used as the surface shape measuring instrument 90. However, for example, a Shack-Hartmann sensor or the like that can collectively measure the surface shape of a cover glass or a sample may be used. At this time, the calculation unit 4 efficiently measures the surface shape of the sample region of the preparation 30 by sending a measurement command to the surface shape measuring instrument 90 based on the sample region information 81 acquired by the detection unit 3. be able to.

  As shown in FIG. 1, the measuring unit 2 includes a third measuring instrument 901 for measuring the position and orientation of the Z tilt stage 24 with respect to the surface shape measuring instrument 90. In the present embodiment, three distance sensors 901a to 901c are provided as the third measuring device 901 (only two of them are shown in FIG. 1). Each of the distance sensors 901a to 901c measures the distance to the stage reference plane A, which is the upper surface of the Z tilt stage 24, and acquires information on the position and orientation of the stage reference plane A from each positional relationship and measurement value. Can do. That is, by removing the influence of the posture of the Z tilt stage 24 from the measurement result obtained by the surface shape measuring instrument 90, the accurate surface shape (surface shape information 91) of the cover glass or the sample can be acquired.

  Next, the slide 30 on which the surface shape measurement has been performed is moved to the measurement position (under the first measuring instrument 100) in the imaging unit 1 by the stage 20, and the thickness of the cover glass is measured. The first measuring instrument 100 according to the present embodiment is supported by the same structure as the imaging optical system 40 and performs measurement using a spectral interference method. Specifically, the first measuring instrument 100 has a reference reflection surface inside the sensor head, causes the reflected light from the test object and the reflected light from the reference reflection surface to interfere, and the interference light has a wavelength. Each has a structure in which the light is dispersed and detected by a sensor. With this configuration, the distance to the test object can be measured based on the intensity of the interference light of each wavelength. At this time, the thickness of the cover glass can be measured by measuring both the distance to the surface of the cover glass and the distance to the surface of the sample by the first measuring instrument 100.

  Thereafter, the stage 30 moves the preparation 30 to the imaging position in the imaging unit 1 (below the imaging optical system 40). In the imaging unit 1, the preparation 30 is illuminated by the illumination optical system 10, and the light beam from the preparation 30 is condensed near the reflection mirror 70 by the imaging optical system 40. Further, the light beam reflected by the reflecting mirror 70 is collected by the re-imaging optical system 60, and an enlarged image of the sample is formed on the imaging surface of the imaging device 50. At this time, the calculation unit 4 sends a measurement command to the imaging unit 1 based on the sample region information 81 and the surface shape information 91 acquired by the detection unit 3 and the measurement unit 2, respectively. The imaging according to can be performed.

  In the present embodiment, as shown in FIG. 3, four reflection mirrors 70, a re-imaging optical system 60, and an image sensor 50 are arranged four by four so as not to interfere with each other. Here, in order to fill a gap between areas (imaging areas) that can be imaged by each imaging element 50 by one imaging, the XY stage 23 is moved and imaging is performed a plurality of times (step imaging). An entire image of the sample 302 can be acquired. Note that the number and arrangement of the reflecting mirror 70, the re-imaging optical system 60, and the image sensor 50 are appropriately determined according to the field of view of the imaging optical system 40, the size of the imaging area of each image sensor 50, and the like. be able to.

  And the digital information of the preparation 30 is acquirable by processing the imaging information acquired in the imaging part 1 in the calculating part 4. FIG. When the image acquisition operation in the imaging unit 1 is completed, the stage 20 holding the preparation 30 moves below the surface shape measuring instrument 90 in the measurement unit 2, and the preparation 30 is moved to the imaging stage 82 by the moving unit 250. The preparation 30 is stored in the stocker 200 by a transport unit 201 (not shown).

  Here, as shown in FIG. 1, a second measuring instrument 601 for measuring the position and orientation of the Z tilt stage 24 is attached to the imaging optical system 40. Each of the first measuring instrument 100 and the second measuring instrument 601 is calibrated so that a distance from a reference plane (imaging reference plane B) perpendicular to the optical axis of the imaging optical system 40 can be measured. In this embodiment, the incident surface of the objective optical system 400 is the imaging reference plane B. However, the present invention is not limited to this. For example, a surface used as a reference when assembling the objective optical system 400 is set as the imaging reference plane B. Also good. The Z tilt stage 24 can be positioned according to each measurement result (details will be described later). The second measuring instrument 601 includes three distance sensors 601a to 601c, and the position and inclination of the stage reference plane A with respect to the imaging reference plane B are determined from the measurement information of these distance sensors 601a to 601c and the positional relationship of each sensor. It can be measured.

  Hereinafter, the image acquisition method including the positioning method (focus adjustment method) of the Z tilt stage 24 according to the present embodiment will be described in detail with reference to the flowchart shown in FIG. Note that the processing of the flowchart shown in FIG. 4 is performed by the calculation unit 4 unless otherwise specified. The image acquisition apparatus 1000 according to the present embodiment is configured to store a program according to the flowchart of FIG. 4 in the memory 500 and execute the program by the calculation unit 4. Note that the arithmetic unit 4 indicates a CPU in a PC, a microcomputer board, an industrial controller, or the like, for example.

  First, the calculation unit 4 performs a process (preparation process) of acquiring the focus position of the objective optical system 400 based on the reference preparation 31. Note that the surface of the reference preparation 31 is flat (has flatness enough to ignore the influence on the focus), and a pattern for detecting the in-focus state is drawn on the surface. Specifically, first, as shown in FIG. 5A, the Z tilt stage 24 on which the reference preparation 31 is installed is driven in the Z direction so that the imaging section 1 images the reference preparation 31 a plurality of times. The computing unit 4 detects a focus image (an image having a contrast value that is an allowable value) from the plurality of acquired images (step S401).

  Further, as shown in FIG. 5B, the Z tilt stage 24 is positioned at the position when the focus image is acquired. Further, as shown in FIG. 5C, the XY stage 23 is driven and positioned so that the point P0 on the surface of the reference preparation 31 becomes the measurement position of the first measuring instrument 100 (step S402). At this time, only the XY stage 23 is driven so as not to change the posture of the reference preparation 31 and the position in the Z direction. Then, the computing unit 4 causes the first measuring instrument 100 to measure the distance from the imaging reference plane B to the point P0, and stores the measured value at this time as the focus position Z′0 (step S403).

  Note that the above-described focus image refers to an image acquired when the surface of the reference preparation 31 enters the depth of field of the objective optical system 400. In this embodiment, the image having the maximum contrast value. Is a focus image (best focus image). Therefore, the focus position Z′0 according to the present embodiment is a position (best focus position) at which the surface of the reference preparation 31 becomes the best focus.

  Next, the calculating part 4 performs the process which measures the position and thickness of the cover glass 301 (1st process). First, as shown in FIG. 6A, the XY stage 23 on which the preparation 30 to be observed is placed so that the surface of the cover glass 301 is placed at the measurement position of the first measuring instrument 100 ( Step S404). The calculation unit 4 causes the first measuring instrument 100 to measure the position of the cover glass 301 (the distance from the imaging reference plane B to the point P ′ on the surface of the cover glass 301) Z ′ and the thickness d of the cover glass 301. (Step S403).

Further, the calculation unit 4 performs processing (second step) for positioning the Z tilt stage 24 at the focus position of the objective optical system 400. First, as shown in FIG. 6B, the XY stage 23 is driven to move the preparation 30 to the imaging position. At this time, the calculation unit 4 calculates a target position Z′ref expressed by the following equation (1) based on the focus position Z′0 and the thickness d of the cover glass 301 (step S406).
Z'ref = Z'0-d / N (1)

  In equation (1), the refractive index N of the cover glass 301 stored in advance is considered with respect to the thickness d of the cover glass 301. Further, the calculation unit 4 calculates a difference errZ 'between the position Z' of the cover glass 301 and the target position Z'ref (step S407).

  Then, the calculation unit 4 calculates the command value Zcom so that the position Z ′ of the cover glass 301 matches the target position Z′ref, that is, the movement distance of the Z tilt stage 24 matches the difference errZ ′. . At this time, the measurement value Z (distance from the imaging reference plane B to the stage reference plane A) of the second measuring instrument 601 is referred to. By moving the Z tilt stage 24 by errZ ′ in the Z direction according to the command value Zcom, the distance from the imaging reference plane B to the point P on the surface of the sample 302 can be matched with the focus position Z′0. it can. In this way, the position of the Z tilt stage 24 is adjusted and the position is fixed. Thereby, as shown in FIG.6 (c), the surface of the sample 302 in the preparation 30 can be positioned in the focus position of the objective optical system 400 (step S408).

  As described above, in the image acquisition apparatus according to the present embodiment, the surface of the sample falls within the depth of field of the objective optical system based on the measurement value of the thickness of the cover glass measured by the first measuring instrument by the calculation unit. So that the stage is controlled. Thereby, since the position of the stage can be adjusted in consideration of the thickness of the cover glass in the preparation, the surface of the sample can be positioned at the focus position of the objective optical system. Therefore, even when an objective optical system having a high resolving power is used, a good sample image with little blur can be acquired.

[Example 2]
Hereinafter, a second embodiment of the present invention will be described. The same or equivalent components as those of the first embodiment are denoted by the same reference numerals, and the description thereof will be simplified or omitted.

  In the first embodiment described above, it is assumed that the thickness of the cover glass 301 is uniform, and only the thickness at one place (one point) of the cover glass 301 is measured to position the Z tilt stage 24. However, it is known that the thickness of a general cover glass has unevenness (variation) of about 4 μm to 10 μm for each position in the XY direction. Therefore, in the image acquisition apparatus 1000 including the objective optical system 400 having a high resolving power, it is more desirable to position the Z tilt stage 24 in consideration of thickness unevenness of the cover glass 301.

  Therefore, in this embodiment, the thickness is measured at a plurality of positions (a plurality of points) of the cover glass 301, and the Z tilt stage 24 is positioned in consideration of the thickness unevenness of the cover glass 301. Hereinafter, the image acquisition method including the positioning method (focus adjustment method) of the Z tilt stage 24 according to the present embodiment will be described in detail with reference to the flowchart shown in FIG.

  First, as in the first embodiment, the calculation unit 4 performs a process of acquiring the focus position Z′0 of the objective optical system 400 based on the reference preparation 31 (preparation steps: steps S701 to S703). And the calculating part 4 performs the process which measures the position and thickness in several places in the cover glass 301 (1st process). In this embodiment, the preparation 30 including the cover glass 301 in which the thickness unevenness as shown in FIG.

  First, as shown in FIG. 9A, the XY stage 23 on which the preparation 30 to be observed is placed is positioned so that the surface of the cover glass 301 in the preparation 30 is placed at the measurement position of the first measuring instrument 100. (Step S704). Further, the calculation unit 4 causes the first measuring instrument 100 to detect the position of the cover glass 301 (the distance from the imaging reference plane B to the point P1 ′ on the surface of the cover glass 301) Z1 ′ and the point P1 ′ of the cover glass 301. The thickness d1 is measured. Further, the computing unit 4 moves the XY stage 23 in the XY direction, and causes the first measuring instrument 100 to measure the thickness d2 of the cover glass 301 at a point P2 'different from the point P1' (step S705).

Next, the calculation unit 4 performs processing (second step) for positioning the Z tilt stage 24 at the focus position of the objective optical system 400. First, the calculation unit 4 calculates the target position Z1′ref represented by the following expressions (2) and (3) based on the focus position Z′0 and the thicknesses d1 and d2 of the cover glass 301 described above. Z2′ref is calculated (step S706).
Z1′ref = Z′0−d1 / N (2)
Z2′ref = Z′0−d2 / N (3)

  In the equations (2) and (3), the refractive index N of the cover glass 301 stored in advance is considered for each of the thicknesses d1 and d2 of the cover glass 301. In addition, the calculation unit 4 calculates a difference errZ1 'between the position Z1' and the target position Z1'ref in at least one place on the cover glass 301 (step S707).

Further, the calculation unit 4 calculates the target posture θy′ref based on the target positions Z1′ref and Z2′ref and the distance dLx between at least two locations (points P1 ′ and P2 ′) on the cover glass 301. Calculation is performed (step S708). The target posture θy′ref is a target posture around the Y axis of the Z tilt stage 24 expressed by the following equation (4).
θy′ref = (Z2′ref−Z1′ref) / dLx
= (D1-d2) / NdLx (4)

  The distance dLx is calculated by the calculation unit 4 based on the moving distance of the XY stage 23 when measuring the thickness d2 after measuring the thickness d1. As shown in FIG. 8, the target posture θy′ref is composed of a line connecting the points P1 ′ and P2 ′ on the surface of the cover glass 301 and a line connecting the points P1 and P2 on the surface of the sample 302. Corresponds to the angle.

  Further, as shown in FIG. 9B, the calculation unit 4 drives the XY stage 23 to move the preparation 30 to the imaging position. Then, the Z tilt stage 24 is driven so that the position Z1 ′ at at least one position on the cover glass 301 matches the target position Z1′ref and the attitude of the Z tilt stage 24 matches the target attitude θy′ref. . Specifically, first, the measurement value Z (distance from the imaging reference plane B to the stage reference plane A) of the second measuring instrument 601 and the measurement value θy (inclination around the Y axis of the stage reference plane A with respect to the imaging reference plane B). ) To calculate the command value Zcom.

  In response to the command value Zcom, the Z tilt stage 24 is driven by errZ1 'in the Z direction and is driven by θy'ref around the Y axis. In this way, the position and posture of the Z tilt stage 24 are adjusted, and the position and posture are fixed. Accordingly, as shown in FIG. 9C, the surface of the sample 302 in the preparation 30 can be positioned at the focus position of the objective optical system 400 (step S709). Note that the tilt around the X axis of the Z tilt stage 24 can be positioned similarly to the tilt around the Y axis.

  As described above, in the image acquisition apparatus according to the present embodiment, the surface of the sample is the object field of the objective optical system based on the measured thickness values at a plurality of locations in the cover glass measured by the first measuring instrument. The stage is controlled to enter the depth. Thereby, since the position and posture of the stage can be adjusted in consideration of the thickness variation of the cover glass in the preparation, the surface of the sample can be positioned at the focus position of the objective optical system. Therefore, even when an objective optical system having a high resolving power is used, a good sample image with little blur can be acquired.

[Example 3]
Hereinafter, a third embodiment of the present invention will be described. The same or equivalent components as those of the first and second embodiments are denoted by the same reference numerals, and the description thereof will be simplified or omitted.

  In the above-described first and second embodiments, the surface shape and thickness of the cover glass 301 are separately measured by the surface shape measuring instrument 90 and the first measuring instrument 100, respectively. Cost. Further, the second embodiment includes a step of measuring the thickness unevenness, and the XY stage 23 is step-driven in each of the surface shape measurement and the thickness unevenness measurement. Will reduce the overall throughput.

  Furthermore, in the first and second embodiments, the first measuring instrument 100 measures the focus position of the objective optical system 400 and the position of the cover glass 301. Therefore, an operation of moving the XY stage 23 from the measurement position of the first measuring instrument 100 to the imaging position of the imaging unit 1 and calculation of the difference between the position of the cover glass 301 and the target position are necessary, each of which requires time. End up.

  Therefore, in this embodiment, as shown in FIG. 10, instead of the surface shape measuring instrument 90 in the measuring unit 2, a fourth measuring instrument 100 ′ having the same configuration as the first measuring instrument 100 is provided, thereby covering the surface. The surface shape and thickness of the glass 301 can be measured simultaneously. Further, in the present embodiment, the fourth measuring instrument 100 ′ and the third measuring instrument 901 are calibrated so that the distance from the common measurement reference plane C can be measured (so as to obtain the same distance measurement value). ing.

  Thereby, the relative position of the cover glass 301 with respect to the position of the stage reference plane A can be acquired simultaneously with the acquisition of the surface shape information. As a result, it is possible to suppress a decrease in throughput as described above, and it is not necessary to provide the first measuring instrument 100 in the imaging unit 1, thereby realizing downsizing and cost reduction of the apparatus.

  Hereinafter, the image acquisition method including the positioning method (focus adjustment method) of the Z tilt stage 24 according to the present embodiment will be described in detail with reference to the flowchart shown in FIG.

  First, as in the first embodiment, the calculation unit 4 performs a process of acquiring the focus position Z0 of the objective optical system 400 based on the reference preparation 31 (preparation step). First, as shown in FIG. 12A, the calculation unit 4 drives the Z tilt stage 24 on which the reference preparation 31 is installed in the Z direction so that the imaging unit 1 captures images a plurality of times, and focuses from the acquired plurality of images. An image is detected (step S1101). Then, as shown in FIG. 12B, the Z tilt stage 24 is positioned at the position when the focus image is acquired. At this time, the calculation unit 4 causes the second measuring device 601 to measure the distance from the imaging reference plane B to the stage reference plane A, and stores the measured value as the focus position Z0 (step S1102).

  Next, the calculating part 4 performs the process which measures the thickness and surface shape of the cover glass 301 (1st process). First, as shown in FIG. 10, the XY stage 23 on which the observation target preparation 30 is installed is positioned so that the surface of the cover glass 301 is arranged at the measurement position of the fourth measuring instrument 100 ′ in the measurement unit 2. (Step S1103). Then, the calculation unit 4 causes the fourth measuring instrument 100 'to measure the surface shape and the thickness d of the cover glass 301 (step S1104).

Further, the calculation unit 4 performs processing (second step) for positioning the Z tilt stage 24 at the focus position of the objective optical system 400. First, as shown in FIG. 12C, the XY stage 23 is positioned so that the surface of the cover glass 301 in the preparation 30 is disposed at the imaging position. At this time, the calculation unit 4 calculates the target position Zref expressed by the following equation (5) based on the focus position Z0 and the thickness d of the cover glass 301 as shown in FIG. 10 (step S1105). ).
Zref = Z0−d / N (5)

  Then, the calculation unit 4 calculates the command value Zcom so that the measurement value Z (distance from the imaging reference plane B to the stage reference plane A) of the second measuring instrument 601 matches the target position Zref. That is, by moving the Z tilt stage 24 in the Z direction according to the command value Zcom, the position Z of the Z tilt stage 24 can be matched with the target position Zref. Thereby, as shown in FIG.12 (d), the surface of the sample 302 in the preparation 30 can be positioned in the focus position of the objective optical system 400 (step S1106).

  Also in this embodiment, focus adjustment may be performed in consideration of thickness unevenness of the cover glass 301 as in the second embodiment. Further, the measurement by the fourth measuring instrument 100 ′ may be performed before or after the above-described measurement of the focus position, or may be performed simultaneously with the measurement of the focus position.

  As described above, in the image acquisition apparatus according to the present embodiment, the surface of the sample falls within the depth of field of the objective optical system based on the measurement value of the thickness of the cover glass measured by the fourth measuring instrument by the calculation unit. So that the stage is controlled. Thereby, since the position of the stage can be adjusted in consideration of the thickness of the cover glass in the preparation, the surface of the sample can be positioned at the focus position of the objective optical system. Further, by adopting a configuration capable of simultaneously measuring the surface shape and thickness of the cover glass in the fourth measurement unit, it is possible to improve the throughput at the time of focus adjustment.

[Modification]
As mentioned above, although the preferable Example of this invention was described, it cannot be overemphasized that this invention is not limited to these Examples, A various deformation | transformation and change are possible within the range of the summary.

  For example, in each of the embodiments described above, focus adjustment is performed only by moving the stage, but in addition, focus adjustment is performed by driving the reflecting mirror and the image sensor according to the surface shape information of the cover glass. You may do it. Furthermore, the posture of the Z tilt stage may be adjusted based on the surface shape information of the cover glass.

  In each of the above-described embodiments, when the surface of the cover glass or the sample is wavy, the above-described focus adjustment may be applied after obtaining an approximate plane of the surface by the calculation unit. The focus adjustment described above may be performed at a plurality of points on the surface of the sample in the preparation, or may be performed only on the center point on the surface (approximate plane).

  Further, the above-described memory may be mounted inside the image acquisition apparatus, or information may be transmitted to the calculation unit by communication as an external memory. Here, the focus position of the objective optical system need not be measured by the image acquisition device if it is stored in advance in the calculation unit or memory. For example, information on a focus position acquired by an external device may be received by a calculation unit.

  Further, although the configuration in which each of the second measuring instrument and the third measuring instrument has three distance sensors has been described, each measuring instrument is not limited to this configuration. That is, since it is only necessary to acquire the above-described posture information from the measurement value of the measuring instrument, it is not necessary to limit the number of distance sensors to three, and sensors other than the distance sensor may be used as the measuring instrument.

  In the first and second embodiments, the first measuring instrument and the second measuring instrument may be used together. For example, one of the distance sensors in the second measuring instrument may be used as the first measuring instrument. Good. Or you may make it measure each of the position and thickness of the cover glass which a 1st measuring device measures with a separate measuring device.

  In Example 2, although the thickness in two places in a cover glass is measured, the number of the places which measure thickness is not restricted to this, What is necessary is just two or more places. In addition, when measuring the thickness at three or more locations, if the target position in at least one location is calculated and the target posture is calculated from the distance between at least two locations, the thickness variation of the cover glass is taken into account. Focus adjustment can be performed.

  In each embodiment, step imaging is performed when imaging the entire sample, but the present invention is also applicable to a configuration that scans the entire sample. Further, the image acquisition apparatus according to the present invention is not limited to a microscope that magnifies and observes a sample using the entire objective optical system as an enlargement system. For example, appearance inspection of a substrate or the like (inspection of foreign matter, scratches, etc.) It is also suitable as an inspection apparatus for performing the above.

4 arithmetic unit 20 stage 30 test object 50 imaging device 301 cover glass 302 sample 400 objective optical system 1000 image acquisition device

Claims (25)

A stage capable of holding and moving a test object including a cover glass and a sample in contact with the cover glass, an objective optical system for forming an image of the test object, and the test object via the objective optical system An image acquisition device comprising:
An image acquisition apparatus comprising: a calculation unit that controls the stage so that the surface of the sample falls within a depth of field of the objective optical system based on a measurement value of the thickness of the cover glass. .   The calculation unit calculates a target position of the cover glass based on a measured value of the thickness of the cover glass and a focus position of the objective optical system, and the position of the cover glass matches the target position. The image acquisition apparatus according to claim 1, wherein the position of the stage is controlled as described above.   2. The image acquisition according to claim 1, wherein the calculation unit controls the stage based on measured thickness values at a plurality of locations in the cover glass and a focus position of the objective optical system. apparatus.   The calculation unit calculates a target position of the cover glass based on a measured thickness value at at least one of the plurality of positions in the cover glass and a focus position of the objective optical system, The image acquisition apparatus according to claim 3, wherein the position of the stage is controlled so that a position at at least one of a plurality of positions on the glass matches the target position.   The calculation unit controls the posture of the stage based on a measured value of thickness at a plurality of locations in the cover glass and a measured value of a distance between at least two of the plurality of locations. The image acquisition apparatus according to claim 3, wherein the image acquisition apparatus is an image acquisition apparatus.   The calculation unit calculates a target posture of the stage based on a measurement value of a thickness at a plurality of locations in the cover glass and a measurement value of a distance between at least two of the plurality of locations, The image acquisition apparatus according to claim 5, wherein the posture of the stage is controlled so that the posture of the stage matches the target posture.   The said calculating part controls the attitude | position of the said stage based on the measured value of the thickness of the said cover glass, the focus position of the said objective optical system, and the surface shape of the said cover glass. The image acquisition device according to any one of 1 to 6.   The image acquisition apparatus according to claim 1, further comprising a first measuring instrument that measures a position of the cover glass.   The image acquisition apparatus according to claim 8, wherein the stage is moved, and the position of the cover glass at a plurality of locations in the cover glass is measured by the first measuring instrument.   The image acquisition apparatus according to claim 8, wherein the first measuring instrument measures a thickness at a location where the position of the cover glass is measured.   11. The image acquisition apparatus according to claim 8, wherein the position of the cover glass is a distance from an imaging reference plane of the objective optical system to a surface of the cover glass.   The image acquisition apparatus according to claim 8, further comprising a measuring instrument that measures a distance from the same reference as the first measuring instrument.   The image acquisition apparatus according to claim 1, further comprising a second measuring instrument that measures the position of the stage.   The image acquisition apparatus according to claim 13, wherein the position of the stage is a distance from an imaging reference plane of the objective optical system to a stage reference plane of the stage.   The second measuring device measures positions at a plurality of locations on the stage, and the calculation unit calculates an inclination of the stage reference surface with respect to the imaging reference surface based on a measurement value of the second measuring device. The image acquisition apparatus according to claim 14, wherein the image acquisition apparatus calculates the posture.   The image acquisition apparatus according to claim 1, wherein the image acquisition apparatus is a microscope, and the objective optical system is an enlargement system. A stage capable of holding and moving a test object including a cover glass and a sample in contact with the cover glass, an objective optical system for forming an image of the test object, and the test object via the objective optical system An image acquisition method in an image acquisition device comprising:
A first step of measuring the thickness of the cover glass;
A second step of controlling the stage so that the surface of the sample falls within the depth of field of the objective optical system based on the measured value of the thickness of the cover glass;
An image acquisition method comprising:   In the second step, the target position of the cover glass is calculated based on the measured value of the thickness of the cover glass and the focus position of the objective optical system, and the position of the cover glass becomes the target position. The image acquisition method according to claim 17, wherein the position of the stage is controlled so as to match.   The image acquisition method according to claim 17, wherein in the first step, thicknesses at a plurality of locations in the cover glass are measured.   In the second step, the target position of the cover glass is calculated based on the thickness measurement value at at least one of the plurality of positions in the cover glass and the focus position of the objective optical system, The image acquisition method according to claim 19, wherein the position of the stage is controlled so that a position of at least one of the plurality of locations on the cover glass matches the target position.   In the second step, the posture of the stage is controlled based on a measured value of thickness at a plurality of locations in the cover glass and a measured value of a distance between at least two of the plurality of locations. 21. The image acquisition method according to claim 19 or 20, wherein:   In the second step, the target posture of the stage is calculated based on the thickness measurement values at a plurality of locations in the cover glass and the distance measurement value between at least two of the plurality of locations. The image acquisition method according to claim 21, wherein the posture of the stage is controlled so that the posture of the stage matches the target posture.   In the second step, the posture of the stage is controlled based on a measured value of the thickness of the cover glass, a focus position of the objective optical system, and a surface shape of the cover glass. The image acquisition method according to any one of claims 17 to 22.   The image acquisition method according to any one of claims 17 to 23, further comprising a step of acquiring a focus position of the objective optical system.   The step of acquiring the focus position of the objective optical system includes setting a reference preparation on the stage, driving the stage in the optical axis direction of the objective optical system, and imaging the reference preparation a plurality of times. Detecting the focus image from the image, positioning the stage at the position when the focus image was acquired, measuring the distance from the imaging reference plane of the objective optical system to the surface of the reference preparation, The method according to claim 24, further comprising: acquiring a value as the focus position.

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