Control apparatus, measurement apparatus, control method, and program

Abstract

To provide a control device capable of highly accurately acquiring information on a shape of a transparent sample.SOLUTION: The control device is a control device for controlling a measurement device including an illumination unit that illuminates a sample, an imaging unit that captures an image of the sample, and a focus change unit that changes a focus state, and includes a first acquisition unit that acquires a first image including the sample obtained at a position defocused by a first amount from an in-focus position by imaging using the imaging unit, and first information related to a region corresponding to the sample in the first image, and a second acquisition unit that acquires shape information related to a shape of the sample on the basis of the first information and the first amount.SELECTED DRAWING: Figure 16

Description

[Technical Field]

[0001] The present invention relates to a control device for acquiring information about the shape of a transparent sample. [Background technology]

[0002] In cell culture, information about the shape of cells, such as their size and shape, is an important indicator of cell activity and growth, and must be acquired appropriately. Furthermore, since cells are generally colorless and transparent, it is necessary to observe transparent samples. Non-Patent Document 1 discloses a configuration for observing colorless and transparent samples at a defocused position with high contrast, utilizing the property that when light with a phase distribution propagates, an intensity distribution corresponding to the phase distribution appears. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] U. Agero, LG Mesquita, BR a. Neves, RT Gazzinelli, and ON Mesquita, “Defocusing microscopy.” Microscopy research and technique, Vol. 65, No. 3, pp159-165, 16 December 2004, USA Summary of the Invention [Problem to be solved by the invention]

[0004] The intensity distribution generated by the propagation of light with a phase distribution becomes higher in contrast as the propagation distance increases. Therefore, in the configuration of Non-Patent Document 1, it is necessary to increase the amount of defocus in order to obtain a high-contrast image. However, increasing the amount of defocus causes light diffraction, which distorts the observed image from the shape of the sample itself. It is therefore difficult to obtain information about the shape with high precision from such an image.

[0005] An object of the present invention is to provide a control device that can acquire information about the shape of a transparent sample with high accuracy. [Means for solving the problem]

[0006] A control device according to one aspect of the present invention is a control device for controlling a measurement device that includes an illumination unit that illuminates a sample, an imaging unit that images the sample, and a focus change unit that changes the focus state, and is characterized by having a first acquisition unit that acquires a first image including the sample obtained at a position defocused by a first amount from the in-focus position by imaging using the imaging unit, first information regarding a region corresponding to the sample in the first image, and a second acquisition unit that acquires shape information regarding the shape of the sample based on the first information and the first amount. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a control device that can acquire information about the shape of a transparent sample with high precision. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a schematic diagram of a measurement device according to an embodiment of the present invention. [Figure 2] FIG. 1 is a schematic diagram of the samples of Examples 1 and 2. [Figure 3] FIG. 10 is a diagram illustrating positive and negative defocus. [Figure 4] FIG. 2 is a diagram showing an image acquired in the first embodiment. [Figure 5] FIG. 10 is a diagram showing the relationship between the defocus amount and the diameter of a bright part in the first embodiment. [Figure 6] FIG. 1 is a diagram showing the relationship between the estimated diameter and the true diameter in Example 1. [Figure 7] 1 is a flowchart showing a method for measuring a sample in Example 1. [Figure 8] 10 is a flowchart showing a method for measuring a sample in Example 2. [Figure 9]FIG. 10 is a diagram showing the relationship between the estimated diameter and the true diameter in Example 2. [Figure 10] FIG. 1 is a schematic diagram of a sample of Example 3. [Figure 11] FIG. 10 is a diagram showing an image acquired in Example 3. [Figure 12] FIG. 10 is a diagram showing the relationship between the estimated major and minor axis lengths and the true major and minor axis lengths in Example 3. [Figure 13] FIG. 1 is a schematic diagram of a sample of Example 4. [Figure 14] FIG. 10 is a diagram showing an image acquired in Example 4. [Figure 15] FIG. 10 is a schematic diagram showing a method for estimating the shape of a sample in Example 4. [Figure 16] 10 is a flowchart showing a method for measuring a sample in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0009] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings. In the drawings, the same reference numerals are used to designate the same components, and redundant explanations will be omitted.

[0010] 1 is a schematic diagram of a measurement device 1000 according to an embodiment of the present invention. The measurement device 1000 includes an illumination unit 1010, an imaging unit 1020, a focus change unit 1030, a sample holder 1040, a control unit 1050, and a calculation unit 1060. In the measurement device 1000, a sample 1070 is illuminated with illumination light emitted from the illumination unit 1010, and the imaging unit 1020 acquires an image using light transmitted through the sample 1070. Note that although the measurement device 1000 is a transmission microscope in this embodiment, it may also be a reflection microscope that uses light reflected by the sample.

[0011] The illumination light emitted from the illumination unit 1010 only needs to be approximately spatially coherent. As an example of a configuration for this purpose, the illumination unit 1010 has a light source 1011 and an illumination optical system 1012. An LED, a laser light source, or the like can be used as the light source 1011. It is also possible to guide light from the LED or laser through an optical fiber and use the end of the optical fiber as the light source 1011. A general lens can be used as the illumination optical system 1012. Note that the present invention also functions in a configuration in which the illumination optical system 1012 is not provided and the light from the light source 1011 is directly applied to the sample 1070.

[0012] The imaging unit 1020 has an objective lens 1021, an imaging lens 1022, and an image sensor 1023. A magnified image of the sample 1070 formed by the objective lens 1021 and the imaging lens 1022 is converted into image data by the image sensor 1023. In order to obtain images at different magnifications, the objective lens 1021 may be attached to a revolver on which multiple objective lenses can be installed.

[0013] The focus changing unit 1030 is an electric stage or the like that can drive the sample holding unit 1040 in the optical axis direction, and is used to change the focus state. It is sufficient if the focus can be changed when photographing the sample 1070, and it may be an electric stage that can drive the entire imaging unit 1020 in the optical axis direction, or an electric stage that drives the image sensor 1023 in the optical axis direction. An optical system or optical element for changing the focus may be provided within the imaging unit 1020.

[0014] The sample holder 1040 is a sample stage used in a general microscope or the like, and is not particularly limited as long as it has a configuration capable of holding or placing the sample 1070 thereon.

[0015] The control unit 1050 is connected to the imaging unit 1020 and the focus change unit 1030, and controls the change of defocus and the acquisition of images.

[0016] The calculation unit 1060 includes a first acquisition unit 1061 and a second acquisition unit 1062. The first acquisition unit 1061 acquires an image (first image) including the sample 1070 obtained at a position (defocus position) defocused by a defocus amount (first amount) from the in-focus position from the control unit 1050. The first acquisition unit 1061 also acquires an evaluation amount (first information) related to a region (first region) corresponding to the sample 1070 in the acquired image. The evaluation amount is, for example, information indicating the contour of the first region, such as a plurality of pieces of coordinate information indicating the contour of the first region, or information indicating the diameter of the first region, such as the diameter of the first region itself or the distance between peaks of light intensity within the first region. The region corresponding to the sample 1070 includes the amount of blur based on the imaging unit 1020, and bright and dark areas are used as described below. The second acquisition unit 1062 acquires (estimates) information (shape information) related to the shape of the sample 1070 based on the first information and the defocus amount. In this embodiment, the information about the shape of the sample 1070 is information corresponding to an evaluation amount (second information) about a region (second region) corresponding to the sample in an image (second image) including the sample 1070 obtained at a position closer to the in-focus position than the defocus position. The information corresponding to the second information may be the second information itself, or information after the amount of blurring based on the imaging unit 1020 has been corrected, which is included in the second information. Specifically, the information about the shape of the sample 1070 is, for example, information indicating the contour of the second region, such as a plurality of pieces of coordinate information indicating the contour of the second region, or information indicating the diameter of the second region, such as the diameter of the second region itself or the distance between peaks of light intensity within the second region. The calculation unit 1060 is exemplified by a calculation device (control device) such as a computer or a workstation. The control unit 1050 and the calculation unit 1060 may be integrated. Alternatively, the calculation unit 1060 may be a calculation system provided on a cloud, or may be connected to the control unit 1050, the imaging unit 1020, etc. via a communication means such as the Internet. The calculation unit 1060 may be equipped with functions such as data storage and display in addition to calculation processing.

[0017] The sample 1070 is exemplified by a transparent biological sample such as a cell or tissue slice. The present invention functions particularly effectively for cells in a culture solution, but is not limited to biological samples and can also be applied to transparent objects having a size or structure of about 1 μm to 100 μm, such as microbeads made of polystyrene, silica, or the like. [Example]

[0018] The measurement method of this embodiment will be explained using a simulation. To simplify the explanation, consider a spherical sample with a refractive index of n and a diameter of D that is located in a medium with a refractive index of n0, as shown in Figure 2, as an example of sample 1070. Let λ be the wavelength of light emitted by illumination unit 1010, NA be the numerical aperture of objective lens 1021, and M be the magnification of image capture unit 1020, and simulate images of sample 1070 acquired by image capture unit 1020 for multiple defocus positions.

[0019] The sign of defocus will now be explained using FIG. 3. FIG. 3 is a diagram illustrating positive and negative defocus. In FIG. 3, the dotted line indicates the optical path in a focused state, and the solid line indicates the optical path when defocus is applied. FIG. 3(a) shows a case where defocus is negative, in which the sample 1070 approaches the objective lens 1021 and the imaging position is located behind the image sensor 1023. FIG. 3(b) shows a case where defocus is positive, in which the sample 1070 moves away from the objective lens 1021 and the imaging position is located in front of the image sensor 1023. A similar definition can be used when defocus is applied by moving the image sensor 1023 or the imaging unit 1020. For example, when the image sensor 1023 is moved, the image sensor 1023 approaches the imaging lens 1022, causing the imaging position to be located behind the image sensor 1023, as in FIG. 3(a), resulting in negative defocus. The amount by which the sample 1070 moves along the optical axis from the in-focus state is defined as z. In the present invention, z is called the defocus amount. Even when the image sensor 1023 is moved, the defocus amount can be defined in the same way from the imaging relationship.

[0020] FIG. 4 shows images acquired in this example. FIG. 4(a) shows images acquired by changing the defocus amount z when the refractive index n0 is 1.33, the refractive index n is 1.34, the diameter D is 10 μm, the wavelength λ is 0.53 μm, the numerical aperture NA is 0.12, and the magnification M is 1. To make it easier to see the changes in the light and dark shapes that appear in the images, the brightness range of each image in FIG. 4(a) is adjusted. FIG. 4(b) shows a cross section of each image in FIG. 4(a) when y is 0. A light and dark ring pattern can be seen at the in-focus position (z=0), but the contrast is very low, as shown in the cross section of FIG. 4(b).

[0021] Generally, images contain optical shot noise, dark current noise, and even artifacts caused by the container in which the sample is placed, making it difficult to perform image processing using images near the focus point, which have low contrast. At negative defocus (z<0), a bright, white area with high intensity appears as a ring, and the contrast is higher than in an image taken at the in-focus position. In this example, the diameter of the sample is estimated from the bright ring area.

[0022] The diameter of the bright area is related to the diameter D of the sample 1070. However, as shown in Figure 4, the bright area also expands as the defocus amount z increases. Therefore, the diameter of the sample 1070 cannot be estimated simply by detecting the expansion of the bright area. Furthermore, because the image is acquired via an optical system, the image also contains optical system blur. In this example, the diameter of the sample is estimated by removing the expansion of the bright area due to defocus and the expansion due to optical system blur.

[0023] First, we separate the spread caused by defocus. The diameter of the bright area, D w is obtained from the images acquired at each defocus position. In this example, the distance between peaks in the one-dimensional cross section indicated by the arrow in FIG. 4(b) is simply taken as the diameter D of the bright part. w Diameter D w The method for obtaining the diameter is not limited to this, and it is also possible to extract the outline of the bright part by performing binarization processing or differentiation processing on the image, and then perform calculations such as obtaining the diameter.

[0024] Figure 5 shows the relationship between the defocus amount z and the diameter D of the bright area. w This figure shows the relationship between the diameter D and the defocus amount. w Conversely, the obtained diameter D w (z) from the diameter D when the defocus amount z is 0 w By extrapolating (0), the influence of the spread due to defocus can be eliminated. In this example, the diameter D obtained at each defocus position w (z) is fitted to a linear function with respect to the defocus amount z, and the diameter D when the defocus amount z is 0 is calculated from the obtained approximation line (dashed line in Figure 5). w Get (0).

[0025] Diameter D w (0) also includes the spread due to the blur caused by the objective lens 1021 and the imaging lens 1022. The spread that an aberration-free optical system has at the focal position is known as the Airy disc, and its radius R a is expressed by the following equation (1). (Number 1) Ra=0.61λ / NA (1) In this embodiment, the width of the bright part corresponds to this. In this embodiment, the interval between the peak positions of the bright part is the diameter D w To obtain the diameter D w (0) to radius R a The value obtained by subtracting 1070 is estimated as the estimated diameter D' of the sample 1070.

[0026] FIG. 6 is a diagram showing the relationship between the estimated diameter D' and the true diameter D. In FIG. 6, the "circle" indicates the estimated diameter D', and the dashed line is a straight line indicating the true diameter D. The estimated diameter D' is obtained so as to follow the dashed line. In other words, the diameter of the sample 1070 can be appropriately obtained using the method of this example.

[0027] In this example, the diameter of the bright area obtained for negative defocus is acquired, but the present invention is not limited to this. As shown in Figure 4(a), a ring-shaped dark area with low intensity appears for positive defocus, and the diameter of the sample 1070 can be estimated from this diameter. However, with positive defocus, high intensity appears near the center, making it difficult to adjust the brightness during imaging, and in some cases, brightness saturation may prevent accurate image acquisition. Therefore, it is preferable to acquire an image for negative defocus and estimate the diameter from the bright area that appears in the image.

[0028] A method for measuring the sample 1070 based on the above principle will be described below with reference to Fig. 7. Fig. 7 is a flowchart showing the method for measuring the sample 1070 in this embodiment.

[0029] In step S11, the control unit 1050 adjusts the focus on the sample 1070 held in the sample holder 1040 via the focus change unit 1030 in response to an instruction from the user. Specifically, the processing of this step is executed by the user causing the control unit 1050 to drive the focus change unit 1030 while monitoring a focusing index such as contrast acquired from image data sent from the image sensor 1023. Note that the processing of this step may be configured to be automatically performed by the control unit 1050.

[0030] In step S12, first, the focus changing unit 1030 moves the sample 1070 in the optical axis direction, and the image sensor 1023 acquires image data at each defocus position. The calculation unit 1060 acquires images at multiple defocus positions. The defocus position where measurement is performed is away from the in-focus position, and it is sufficient that a defocus amount z is given that provides high enough contrast to enable the shape to be acquired by image processing.

[0031] It is preferable that the defocus amount z [μm] satisfies the following conditional expression (2).

[0032] 10≦|z|≦3000 (2) Below the lower limit, it is difficult to obtain high contrast for biological samples such as cells in nutrient solution, whereas above the upper limit, the bright or dark areas may become too broad and overlap with bright or dark areas from other samples.

[0033] It is preferable that the numerical range of conditional expression (2) be within the range of the following conditional expression (2a).

[0034] 25≦z≦900 (2a) It is more preferable that the numerical range of conditional expression (2) be set to the range of the following conditional expression (2b).

[0035] 100≦z≦300 (2b) The processing of this step may be configured to be performed automatically by the control unit 1050.

[0036] In step S13, the calculation unit 1060 acquires an evaluation amount for a bright or dark area that is a region corresponding to the sample 1070 in the image acquired in step S12. For sample 1070 that is assumed to be spherical, such as floating cells or microbeads, the contour of a circular bright area that appears in the image is extracted, and its diameter is acquired as the evaluation amount. Alternatively, an evaluation amount such as a diameter may be acquired by acquiring the distance between peaks of light intensity without extracting the contour.

[0037] In step S14, the calculation unit 1060 linearly approximates the evaluation amount with respect to the defocus amount. A linear function is the simplest and is desirable for linear approximation because it accurately reproduces changes in the evaluation amount due to defocus. However, it is not necessarily limited to a linear function, and approximation can also be performed using a polynomial.

[0038] In step S15, the calculation unit 1060 obtains an evaluation amount at the in-focus position from the obtained approximate straight line. In this step, it is sufficient to obtain the value of the approximate straight line when the defocus amount z is 0. The essence of this step is to eliminate the influence of spread due to defocus by predicting an evaluation amount at a position closer to the in-focus position than the position where measurement was performed from an evaluation amount obtained at a position other than the in-focus position. Therefore, a predicted value may be obtained without using an approximate function by machine learning such as a neural network that has been trained in advance. However, in practice, a method using a linear function that has a small calculation load and can be executed using general-purpose processing is most useful.

[0039] In step S16, the calculation unit 1060 corrects the amount of blur of the optical system from the evaluation amount acquired in step S15. As described above, when the peak-to-peak distance of the bright area at negative defocus is used as the evaluation value, the correction can be performed by dividing the Airy radius Ra. When the diameter of the circle depicting the outside of the bright area is used as the evaluation value, the correction amount can be twice the Airy radius Ra. When the diameter of the circle depicting the inside of the bright area is used as the evaluation value, the correction amount can be omitted. The correction amount is changed appropriately depending on the method for acquiring the evaluation value. Note that the processing of step S16 may be performed before step S14 or step S15. Furthermore, when the user determines that the accuracy of the evaluation amount acquired in the processing of step S15 is sufficient, the processing of step S16 does not need to be performed. [Example]

[0040] The measurement method of this embodiment will be described using simulations. In this embodiment, similarly to the first embodiment, a spherical sample shown in FIG.

[0041] As shown in Figure 5, the spread of bright areas that appear with negative defocus does not depend on the diameter of the spherical sample, and changes with roughly the same slope relative to the defocus amount z. In other words, by obtaining the change in the evaluation amount for bright areas with respect to the defocus amount z (slope in linear approximation) through a prior simulation or the like, it is possible to predict the evaluation amount at the in-focus position using the slope obtained from the evaluation amount obtained at one defocus position.

[0042] A method for measuring the sample 1070 based on the above principle will be described below with reference to Fig. 8. Fig. 8 is a flowchart showing the method for measuring the sample 1070 in this embodiment.

[0043] The process of step S21 is similar to the process of step S11, and therefore a description thereof will be omitted.

[0044] In step S22, the calculation unit 1060 acquires an image at a predetermined defocus position. The processing of this step is performed by moving the sample 1070 in the optical axis direction using the focus change unit 1030 and acquiring image data at the predetermined defocus position using the image sensor 1023. The defocus position at which measurement is performed may be one position within the range 40 μm≦|z|≦1 mm described in step S12. Note that the processing of this step may be configured to be performed automatically by the control unit 1050.

[0045] The process of step S23 is similar to the process of step S13, and therefore a description thereof will be omitted.

[0046] In step S24, the calculation unit 1060 reads the gradient m of an approximate line that approximates the evaluation amount and defocus amount that have been acquired in advance. If the evaluation amount and defocus amount are to be approximated by a non-linear function system, the calculation unit 1060 reads a coefficient that determines the approximate function.

[0047] In step S25, the calculation unit 1060 calculates the evaluation amount D at the in-focus position from the measured evaluation amount Dw(z) and the slope m of the read approximation line. w (0) is obtained. For linear approximation, use equation D. w (0)=D w(z)-mz can be used to obtain the evaluation amount at the in-focus position. If the approximating function system is not linear, calculations tailored to each function system can be performed to obtain the evaluation amount at the in-focus position. As described in the first embodiment, the processing in this step is one means for removing the influence of spread due to defocus contained in the evaluation amount. The evaluation amount at the in-focus position may be predicted using machine learning such as a neural network that has been trained in advance. Furthermore, the evaluation amount does not necessarily have to be the exact in-focus position; if the evaluation amount is obtained at a position closer to the focus than the defocus position at which the image was obtained, the influence of spread due to defocus can be reduced.

[0048] The process of step S26 is the same as the process of step S16, and therefore description thereof will be omitted. Note that the process of step S26 may be performed before step S24 or step S25. Furthermore, if the user determines that the accuracy of the evaluation amount obtained in the process of step S25 is sufficient, there is no need to perform the process of step S26.

[0049] FIG. 9 is a diagram showing the relationship between the estimated diameter D' estimated from the bright areas appearing in an image acquired with a defocus amount z of -100 μm and the true diameter D. In FIG. 9, the average value of the slope of the approximation curve acquired in Example 1 when the true diameter D of the sample 1070 is 5 to 20 μm is used as the predicted slope m. In FIG. 9, "◯" indicates the estimated diameter D', and the dashed line is a straight line indicating the true diameter D. The estimated diameter D' is obtained so as to follow the dashed line. In other words, the diameter of the sample 1070 can be appropriately acquired using the method of this example. [Example]

[0050] The measurement method of this embodiment will be described using simulations. In this embodiment, the sample 1070 is an ellipsoidal sample shown in FIG. 10. In this embodiment, the sample 1070 has a major axis in the x direction and a shape that is rotationally symmetric with respect to the x direction. In this embodiment, the major axis length and minor axis length of the ellipse are acquired as evaluation quantities.

[0051] FIG. 11 is a diagram showing an image acquired in this example, in which the internal refractive index n is 1.34, the refractive index n0 of the medium is 1.33, and the length of the major axis D x is 20 μm, and the length of the minor axis is D y The images shown in Figure 11 are obtained with a defocus amount z of -40 μm and -120 μm when the center of gravity is 10 μm. Similar to the results for the sphere in Figure 5, a ring-shaped bright area appears in Figure 11. However, reflecting the fact that the sample 1070 is an ellipsoid, the shape of the bright area becomes elliptical. Furthermore, as with the sphere, increasing the defocus amount widens the ellipse of the bright area. Therefore, by obtaining the lengths of the elliptical bright area in the x and y directions as evaluation quantities and performing processing according to the flowchart in Figure 7, the lengths of the sample 1070 in the x and y directions can be obtained.

[0052] Figure 12 shows the estimated major axis length D x ' and the length of the minor axis D y ' and the true major axis length D x and the length of the minor axis D y 12. The "◯" and "×" in FIG. 12 represent the length D of the major axis, respectively. x ' and the length of the minor axis D y The dashed line is a straight line showing the true value. The estimated length is obtained along the dashed line. In other words, the method of this example makes it possible to appropriately obtain the length of the major axis and the length of the minor axis of the sample 1070. It is also possible to obtain the ellipticity by taking the ratio of the length of the major axis to the length of the minor axis. [Example]

[0053] In this example, a method for estimating information representing the shape itself, rather than information about the shape of the sample, as an evaluation quantity, is described. In this example, as an example of sample 1070, the shape of partially bonded spheres with a diameter D shown in FIG. 13 is considered.

[0054] Figure 14 shows images acquired in this example, where the internal refractive index n is 1.34, the medium refractive index n0 is 1.33, and the diameter D of one sphere is 10 μm, and the defocus amount z is -40 μm and -90 μm. Similar to the results shown in Figures 4 and 11, bright areas reflecting the contours of the sample 1070 appear, and the bright areas expand as the defocus amount increases. In other words, the same phenomenon occurs with any transparent sample, not just the symmetrical shapes of Examples 1 and 3.

[0055] The principle of estimating the shape of the sample 1070 will be described with reference to Fig. 15. Fig. 15 is a schematic diagram showing a method of estimating the shape of the sample 1070 in this embodiment. The x and y coordinates on the contour of a bright or dark area that appears in an image with a predetermined defocus amount z are expressed as p x (z),p y (z). Coordinate p x (z),p y (z) represents all coordinates detected on the contour of the bright or dark area, and does not represent a single coordinate. The contour of the bright or dark area can be extracted using coordinates determined by determining the position where the light intensity is locally maximum, a contour determined by edge extraction, or a shape such as a circle or ellipse and fitting it. Coordinate p x (z),p y Although (z) varies depending on the defocus amount z, from the simulation results so far, it changes linearly with the absolute value |z|. Therefore, p extracted from images acquired with multiple defocus amounts z x (z),p y From (z), the defocus amount z can be approximated by a linear function as shown by the dotted line in FIG. 15. The coordinate p x (0),p y (0), the outline of the sample 1070 is obtained. However, the coordinate p x (z),p y (z) is a coordinate different from the outline of the sample 1070 due to blurring caused by the optical system. As described in Example 1, the position where the light intensity in the image is locally maximized is p x (z),p yIf the sample 1070 is extracted as (z), it will be overestimated by the size of the Airy radius Ra. Therefore, the shape of the sample 1070 should be smaller than the estimated shape by the Airy radius Ra.

[0056] A method for measuring the sample 1070 based on the above principle will be described below with reference to Fig. 16. Fig. 16 is a flowchart showing the method for measuring the sample 1070 in this embodiment.

[0057] The processes in steps S41 and S42 are similar to those in steps S11 and S22, respectively, and therefore will not be described further.

[0058] In step S43, the calculation unit 1060 calculates coordinates p x (z),p y The method for extracting the contours of the bright and dark areas is not particularly limited.

[0059] In step S44, the calculation unit 1060 calculates the coordinate p x (z),p y (z) is linearly approximated with respect to the defocus amount. It is not necessarily limited to a linear function, and approximation by a polynomial is also acceptable.

[0060] In step S45, the calculation unit 1060 calculates the coordinate p representing the contour at the in-focus position from the obtained approximate straight line. x (0),p y As in the first embodiment, in this step, the contour p obtained at the position other than the in-focus position is obtained. x (z),p y The essence of this is to eliminate the influence of spread due to defocus by predicting the evaluation amount at a position closer to the in-focus position than the measurement position from (z). This does not necessarily have to be via a linear function, and predictions can also be made using machine learning, etc.

[0061] In step S46, the calculation unit 1060 calculates the predicted contour p x (0),p yThe amount of blur of the optical system is corrected from (0). As described above, it is sufficient to obtain a shape that is reduced by the Airy radius Ra. The processing of this step may be performed before step S44 or step S45. Furthermore, if the user determines that the accuracy of the evaluation amount obtained in the processing of step S45 is sufficient, there is no need to perform the processing of step S46.

[0062] As described above, the configuration of this embodiment makes it possible to predict the shape of a sample itself, not just specific evaluation quantities that indicate shape, such as diameter. Once the shape of a sample is determined, evaluation quantities such as diameter and major axis length can also be obtained, making obtaining evaluation quantities an example of sample shape measurement. However, the more complex the sample shape, the more the outline shape of the bright or dark areas differs from the original sample due to the effects of diffraction and overlap between samples. Therefore, the present invention is well suited to measuring the shape of single-layer tissue fragments, cells, well-dispersed microbeads, and the like. [Other Examples] The present invention can also be realized by supplying a program that realizes one or more functions of the above-described embodiments to a system or device via a network or a storage medium, and having one or more processors in the computer of the system or device read and execute the program. It can also be realized by a circuit (e.g., ASIC) that realizes one or more functions.

[0063] The disclosure of this embodiment includes the following configurations and methods. (Configuration 1) A control device for controlling a measurement device including an illumination unit that illuminates a sample, an imaging unit that images the sample, and a focus change unit that changes a focus state, a first acquisition unit that acquires a first image including the sample obtained at a position defocused by a first amount from a focused position by imaging using the imaging unit, and first information regarding a region in the first image corresponding to the sample; a second acquisition unit that acquires shape information regarding the shape of the sample based on the first information and the first amount. (Configuration 2) the second acquisition unit estimates, based on the first information and the first amount, second information regarding a region corresponding to the sample in a second image including the sample obtained at a position defocused by a second amount smaller than the first amount; The control device according to configuration 1, wherein the shape information corresponds to the second information. (Configuration 3) 3. The control device according to configuration 2, wherein the second information is information indicating a diameter of a region in the second image corresponding to the sample. (Configuration 4) the first information is information indicating a diameter of a region corresponding to the sample in the first image; 4. The control device according to configuration 3, wherein the shape information is information indicating a diameter of the sample. (Configuration 5) 3. The control device according to configuration 2, wherein the second information is information indicating the outline of a region in the second image corresponding to the sample. (Configuration 6) the first information is information indicating a contour of a region corresponding to the sample in the first image; 6. The control device according to configuration 5, wherein the shape information is information indicating an outline of the sample. (Configuration 7) 7. The control device according to any one of configurations 2 to 6, wherein the second information is information about a region corresponding to the sample in the second image obtained at the in-focus position. (Configuration 8) The control device according to any one of configurations 2 to 7, wherein the second acquisition unit acquires the shape information by correcting the amount of blur based on the imaging unit included in the second information. (Configuration 9) The first acquisition unit acquiring a plurality of first images obtained at a plurality of defocus positions that are separated from the in-focus position by a plurality of different defocus amounts by imaging using the imaging unit; acquiring a plurality of pieces of first information corresponding to the plurality of first images, The control device according to any one of configurations 1 to 8, wherein the second acquisition unit acquires the shape information based on the plurality of first information and the plurality of defocus amounts. (Configuration 10) The control device according to configuration 9, wherein the second acquisition unit obtains a function that represents a relationship between the plurality of pieces of first information and the plurality of defocus amounts, and acquires the shape information based on the function. (Configuration 11) 11. The control device according to configuration 10, wherein the function expresses the first information as a polynomial of the plurality of defocus amounts. (Configuration 12) The control device according to any one of configurations 1 to 8, wherein the second acquisition unit calculates a coefficient that determines a function that represents the relationship between the first information and the first quantity, and acquires the shape information based on the function. (Configuration 13) 13. The control device according to claim 12, wherein the function is characterized in that the first information is expressed as a polynomial of the first quantity. (Configuration 14) The control device according to any one of configurations 1 to 8, wherein the second acquisition unit acquires the shape information by machine learning based on the first information and the first quantity. (Configuration 15) 15. The control device of any one of configurations 1 to 14, wherein the first image is acquired at a negative defocus position. (Configuration 16) When the first amount is z [μm], 10≦|z|≦3000 16. The control device according to any one of configurations 1 to 15, wherein the following conditional expression is satisfied: (Configuration 17) A control device according to any one of configurations 1 to 16; an illumination unit that illuminates the sample; an imaging unit that images the sample; a focus change unit that changes the focus state; (Method 1) 1. A control method for controlling a measurement device including an illumination unit that illuminates a sample, an imaging unit that images the sample, and a focus change unit that changes a focus state, comprising: acquiring a first image including the sample obtained at a position defocused by a first amount from a focused position by imaging using the imaging unit, and first information regarding a region in the first image corresponding to the sample; and acquiring shape information relating to a shape of the sample based on the first information and the first amount. (Configuration 18) A program that causes a computer to execute the control method described in Method 1.

[0064] Although the preferred embodiments of the present invention have been described above, the present invention is not limited to these embodiments, and various modifications and changes are possible within the scope of the gist of the present invention. [Explanation of symbols]

[0065] 1000 Shape measurement device (measuring device) 1010 Lighting Department 1020 Imaging unit 1030 Focus change unit 1060 Calculation unit (control device) 1061 First Acquisition Section 1062 Second Acquisition Section 1070 Samples

Claims

1. A control device for controlling a measurement device including an illumination unit that illuminates a sample, an imaging unit that images the sample, and a focus change unit that changes a focus state, a first acquisition unit that acquires a first image including the sample obtained at a position defocused by a first amount from a focused position by imaging using the imaging unit, and first information regarding a region in the first image corresponding to the sample; a second acquisition unit that acquires shape information relating to a shape of the sample based on the first information and the first amount.

2. the second acquisition unit estimates, based on the first information and the first amount, second information regarding a region corresponding to the sample in a second image including the sample obtained at a position defocused by a second amount smaller than the first amount; The control device according to claim 1 , wherein the shape information corresponds to the second information.

3. 3. The control device according to claim 2, wherein the second information is information indicating a diameter of a region in the second image corresponding to the sample.

4. the first information is information indicating a diameter of a region corresponding to the sample in the first image; 4. The control device according to claim 3, wherein the shape information is information indicating a diameter of the sample.

5. 3. The control device according to claim 2, wherein the second information is information indicating the outline of a region in the second image corresponding to the sample.

6. the first information is information indicating a contour of a region corresponding to the sample in the first image; 6. The control device according to claim 5, wherein the shape information is information indicating an outline of the sample.

7. 7. The control device according to claim 2, wherein the second information is information relating to a region corresponding to the sample in the second image obtained at the in-focus position.

8. The control device according to claim 2 , wherein the second acquisition unit acquires the shape information by correcting an amount of blur based on the imaging unit and included in the second information.

9. The first acquisition unit acquiring a plurality of first images obtained at a plurality of defocus positions that are separated from the in-focus position by a plurality of different defocus amounts by imaging using the imaging unit; acquiring a plurality of pieces of first information corresponding to the plurality of first images, 3. The control device according to claim 1, wherein the second acquisition unit acquires the shape information based on the plurality of pieces of first information and the plurality of defocus amounts.

10. The control device according to claim 9 , wherein the second acquisition unit obtains a function that represents a relationship between the plurality of pieces of first information and the plurality of defocus amounts, and acquires the shape information based on the function.

11. 11. The control device according to claim 10, wherein the function expresses the first information as a polynomial of the plurality of defocus amounts.

12. The control device according to claim 1 or 2, characterized in that the second acquisition unit calculates a coefficient that determines a function that represents the relationship between the first information and the first quantity, and acquires the shape information based on the function.

13. 13. The control device according to claim 12, wherein the function expresses the first information as a polynomial of the first quantity.

14. The control device according to claim 1 or 2, wherein the second acquisition unit acquires the shape information by machine learning based on the first information and the first amount.

15. 3. The control device according to claim 1, wherein the first image is acquired at a negative defocus position.

16. When the first amount is z [μm], 10≦|z|≦3000 3. The control device according to claim 1, wherein the following condition is satisfied:

17. The control device according to claim 1 or 2; an illumination unit that illuminates the sample; an imaging unit that images the sample; a focus change unit that changes the focus state;

18. 1. A control method for controlling a measurement device including an illumination unit that illuminates a sample, an imaging unit that images the sample, and a focus change unit that changes a focus state, comprising: acquiring a first image including the sample obtained at a position defocused by a first amount from a focused position by imaging using the imaging unit, and first information regarding a region in the first image corresponding to the sample; and acquiring shape information relating to a shape of the sample based on the first information and the first amount.

19. A program causing a computer to execute the control method according to claim 18.

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