Analysis method and analysis device

Abstract

To provide an analysis method and analysis apparatus capable of obtaining a more accurate analysis result of a sample than that in a case where either two-dimensional photographing or three-dimensional photographing is performed solely.SOLUTION: An analysis apparatus comprises a two-dimensional photographing section 30, a three-dimensional photographing section 40, and a computer 50 as an analysis section. The two-dimensional photographing section 30 two-dimensionally photographs a sample to acquire a two-dimensional image D2. The three-dimensional photographing section 40 three-dimensionally photographs the sample to acquire a three-dimensional image D3. The computer 50 analyzes the two-dimensional image D2 and outputs a two-dimensional analysis result R2. Subsequently, the computer 50 analyzes the three-dimensional image D3 and outputs a three-dimensional analysis result R3. At that time, the computer 50 analyzes the three-dimensional image D3 using an indicator included in the two-dimensional analysis result R2. Consequently, the accuracy of the three-dimensional analysis result R3 can be improved.SELECTED DRAWING: Figure 4

Description

[Technical Field]

[0001] The present invention relates to an analytical method and an analytical device for analyzing a sample in which a plurality of objects to be observed are distributed three-dimensionally. [Background technology]

[0002] Conventionally, in pathology and cell culture, biological samples in which a plurality of cells are distributed three-dimensionally are photographed, and the biological samples are analyzed based on the obtained images. Known methods for photographing biological samples include a method of performing two-dimensional photography using an optical microscope and a method of performing three-dimensional photography using optical coherence tomography (OCT). For example, Patent Document 1 describes a technique for obtaining three-dimensional images of an embryo using optical coherence tomography. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Japanese Patent Application Publication No. 2019-133429 Summary of the Invention [Problem to be solved by the invention]

[0004] Two-dimensional imaging using an optical microscope can obtain images with higher resolution than three-dimensional imaging. However, with two-dimensional imaging, it is not possible to grasp the three-dimensional structure of a biological sample from a single image. For this reason, multiple images are taken while changing the focal position of the optical microscope, and the biological sample is analyzed based on the multiple two-dimensional images obtained.

[0005] On the other hand, three-dimensional imaging such as optical coherence tomography (OCT) has the advantage that it is easier to grasp the three-dimensional structure of biological samples than two-dimensional imaging. However, the resolution of the images obtained with three-dimensional imaging is often lower than that of two-dimensional imaging, making it difficult to obtain accurate analysis results for detailed structures.

[0006] The present invention has been made in consideration of the above circumstances, and aims to provide an analysis method and an analysis device that can obtain more accurate analysis results of a sample than when two-dimensional or three-dimensional imaging is performed alone. [Means for solving the problem]

[0007] In order to solve the above problems, a first invention of the present application is an analytical method for analyzing a sample in which a plurality of objects to be observed are distributed three-dimensionally, the method comprising: a) a step of acquiring a two-dimensional image by two-dimensionally photographing the sample; b) a step of acquiring a three-dimensional image by three-dimensionally photographing the sample; c) a step of outputting a two-dimensional analysis result by analyzing the two-dimensional image; and d) a step of outputting a three-dimensional analysis result by analyzing the three-dimensional image, wherein in the step d), the three-dimensional image is analyzed using an index included in the two-dimensional analysis result. The sample is an embryo, the object under observation is a cell contained in the embryo, the two-dimensional analysis result is the number of the cells contained in the embryo, and the three-dimensional analysis result is a shape identification result of a plurality of the cells contained in the embryo, and in step d), the number of regions for calculating the shape identification result of the cell is determined based on the number of the cells obtained in step c). .

[0010] The first part of this application 2 The invention is First Invention The analysis method of (1), further comprising the step of: e) after the step d), outputting a Veeck classification result representing the state of the embryo based on the three-dimensional analysis result.

[0011] The first part of this application 3 The present invention provides an analytical method for analyzing a sample in which a plurality of objects to be observed are distributed three-dimensionally, the method comprising the steps of: a) acquiring a two-dimensional image by photographing the sample two-dimensionally; b) acquiring a three-dimensional image by photographing the sample three-dimensionally; c) outputting a two-dimensional analysis result by analyzing the two-dimensional image; d) outputting a three-dimensional analysis result by analyzing the three-dimensional image; and e) outputting an integrated analysis result based on the two-dimensional analysis result and the three-dimensional analysis result. The sample is an embryo, and the two-dimensional analysis result, the three-dimensional analysis result, and the integrated analysis result are Veeck classification results that represent the state of the embryo. .

[0013] The first part of this application 4 The inventions are the first invention to the second invention. 3An analysis method according to any one of the preceding inventions, wherein in step a), a plurality of two-dimensional images are obtained by performing bright-field photography while changing the focal position, and in step c), the two-dimensional analysis results are obtained by analyzing the plurality of two-dimensional images.

[0014] The first part of this application 5 The inventions are the first invention to the second invention. 4 In the analysis method of any one of the above aspects, in the step b), the three-dimensional image is obtained by optical coherence tomography.

[0015] The first part of this application 6 The invention is an analytical device for analyzing a sample in which a plurality of objects to be observed are distributed three-dimensionally, the device comprising: a two-dimensional imaging unit that acquires two-dimensional images by photographing the sample two-dimensionally; a three-dimensional imaging unit that acquires three-dimensional images by photographing the sample three-dimensionally; and an analysis unit that analyzes the sample based on the two-dimensional images and the three-dimensional images, the analysis unit executes two-dimensional analysis processing that analyzes the two-dimensional images to output two-dimensional analysis results; and three-dimensional analysis processing that analyzes the three-dimensional images to output three-dimensional analysis results, and the three-dimensional analysis processing analyzes the three-dimensional images using indexes included in the two-dimensional analysis results. The sample is an embryo, the object under observation is a cell contained in the embryo, the two-dimensional analysis result is the number of cells contained in the embryo, the three-dimensional analysis result is a shape identification result of a plurality of the cells contained in the embryo, and the three-dimensional analysis process determines the number of regions for calculating the shape identification result of the cell based on the number of cells acquired by the two-dimensional analysis process. .

[0016] The first part of this application 7 The present invention provides an analytical device for analyzing a sample in which a plurality of objects to be observed are distributed three-dimensionally, the analytical device comprising: a two-dimensional imaging unit that acquires two-dimensional images by photographing the sample two-dimensionally; a three-dimensional imaging unit that acquires three-dimensional images by photographing the sample three-dimensionally; and an analysis unit that analyzes the sample based on the two-dimensional images and the three-dimensional images, wherein the analysis unit performs two-dimensional analysis processing that analyzes the two-dimensional images and outputs two-dimensional analysis results; three-dimensional analysis processing that analyzes the three-dimensional images and outputs three-dimensional analysis results; and integrated analysis processing that outputs integrated analysis results based on the two-dimensional analysis results and the three-dimensional analysis results. The sample is an embryo, and the two-dimensional analysis result, the three-dimensional analysis result, and the integrated analysis result are Veeck classification results that represent the state of the embryo. . [Effects of the Invention]

[0017] Original wish First Invention According to the method, three-dimensional images can be analyzed using indices that can be obtained with high accuracy through two-dimensional analysis, thereby obtaining highly accurate three-dimensional analysis results.

[0018] Also , the present application First Invention According to the paper, information on the number of cells, which can be obtained more accurately by two-dimensional analysis than by three-dimensional analysis, is used in three-dimensional analysis, thereby enabling the acquisition of highly accurate three-dimensional analysis results.

[0019] Also , the present application First Invention According to the method, the results of three-dimensional analysis of cells can be calculated with high accuracy based on the number of cells obtained by two-dimensional analysis.

[0020] In addition, Third Invention According to the method, two analytical results obtained from two approaches, two-dimensional analysis and three-dimensional analysis, are used to output an integrated analytical result, which allows for accurate integrated analytical results to be obtained.

[0021] Also , the present application 3 According to the present invention, two Veeck classification results obtained by two approaches, two-dimensional analysis and three-dimensional analysis, are integrated to output a final Veeck classification result, thereby obtaining a highly accurate Veeck classification result.

[0022] In particular, 4 According to the present invention, by analyzing a plurality of two-dimensional images, it is possible to obtain highly accurate two-dimensional analysis results.

[0023] In particular, 5 According to the invention, three-dimensional images can be obtained with high accuracy by optical coherence tomography.

[0024] In addition, 6According to the present invention, three-dimensional images can be analyzed using indices that can be obtained with high accuracy through two-dimensional analysis, thereby providing highly accurate three-dimensional analysis results.

[0025] In addition, 7 According to the present invention, two analytical results obtained by two approaches, two-dimensional analysis and three-dimensional analysis, are used to output an integrated analytical result, thereby obtaining an integrated analytical result with high accuracy. [Brief explanation of the drawings]

[0026] [Figure 1] FIG. 1 is a diagram showing the configuration of an analysis device. [Figure 2] FIG. 1 shows an example of an embryo. [Figure 3] FIG. 2 is a control block diagram of the analysis device. [Figure 4] FIG. 2 is a block diagram conceptually showing the functions of a computer for realizing imaging and analysis processing according to the first embodiment. [Figure 5] 1 is a flowchart showing the flow of imaging and analysis processing according to the first embodiment. [Figure 6] FIG. 10 is a block diagram conceptually showing the functions of a computer for realizing imaging and analysis processing according to the second embodiment. [Figure 7] 10 is a flowchart showing the flow of imaging and analysis processing according to the second embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0027] Hereinafter, an embodiment of the present invention will be described with reference to the drawings.

[0028] <1. Configuration of the analysis device> 1 is a diagram showing the configuration of an analytical device 1 according to one embodiment of the present invention. This analytical device 1 is a device that photographs a sample held in a sample container 90 and analyzes the state of the sample based on the obtained image.

[0029] In this embodiment, a case will be described in which the sample to be analyzed is a human embryo 9. FIG. 2 is a diagram showing an example of an embryo 9. The embryo 9 is formed by the cleavage of a fertilized egg. As shown in FIG. 2, the embryo 9 has a spherical zona pellucida 91 and a plurality of cells 92, which are the object to be observed, distributed three-dimensionally. Each cell 92 is transparent or translucent.

[0030] As shown in FIG. 1, the analysis device 1 includes a stage 10, a light irradiation unit 40, a two-dimensional imaging unit 20, a three-dimensional imaging unit 30, and a computer 50.

[0031] The stage 10 is a support base that supports the sample container 90. For example, a well plate is used as the sample container 90. The well plate has multiple wells (recesses). The sample, that is, an embryo 9, is held in each well together with a culture medium. However, the sample container 90 may also be a dish having only one recess. The material of the sample container 90 is a transparent resin that transmits light. The stage 10 has an opening 11 that penetrates in the vertical direction. The sample container 90 is supported horizontally while fitted into the opening 11 of the stage 10. Therefore, the upper and lower surfaces of the sample container 90 are exposed and not covered by the stage 10.

[0032] The light irradiation unit 40 is disposed above the sample container 90 supported by the stage 10. The light irradiation unit 40 has a light-emitting element such as an LED. When imaging is performed by the two-dimensional imaging unit 20, the light-emitting element of the light irradiation unit 40 emits light. As a result, light is irradiated from the light irradiation unit 40 toward the sample container 90 below.

[0033] The two-dimensional imaging unit 20 is a unit that takes two-dimensional images of the embryo 9 in the sample container 90 using an optical microscope. The two-dimensional imaging unit 20 is disposed below the sample container 90 supported on the stage 10. As shown in FIG. 1 , the two-dimensional imaging unit 20 has an imaging optical system 21, a camera 22, and a focal point movement mechanism 23. The imaging optical system 21 has multiple optical components including an objective lens 211. The objective lens 211 is a lens for adjusting the focus of the camera 22 on the embryo 9 in the sample container 90. The camera 22 has an imaging element such as a CCD or CMOS.

[0034] When performing two-dimensional imaging, the camera 22 captures an image of the embryo 9 in the sample container 90 in a bright field while irradiating the sample container 90 with light from the light irradiation unit 40. This allows a two-dimensional image of the embryo 9 to be acquired as digital data. The acquired two-dimensional image is input from the camera 22 to the computer 50. The two-dimensional image is composed of a plurality of pixels arranged on a two-dimensional coordinate system, and the data has a defined brightness value for each pixel.

[0035] The focal point shifting mechanism 23 is a mechanism that changes the focal point position of the camera 22. The focal point shifting mechanism 23 uses an actuator such as a motor or a piezoelectric element to slightly shift the position of the objective lens 211 along the optical axis. This causes the focal point of the camera 22 to slightly move in the vertical direction. The two-dimensional imaging unit 20 photographs the embryo 9 multiple times (multi-focus photography) while changing the focal point position using the focal point shifting mechanism 23. This allows multiple two-dimensional images with different focal points to be acquired.

[0036] Furthermore, the two-dimensional imaging unit 20 can be moved horizontally together with the light irradiation unit 40 by a movement mechanism (not shown). This allows the field of view of the two-dimensional imaging unit 20 to be switched among multiple wells.

[0037] The three-dimensional imaging unit 30 is a unit that takes three-dimensional images of the embryo 9 in the sample container 90. The three-dimensional imaging unit 30 is disposed below the sample container 90 that is supported on the stage 10. The three-dimensional imaging unit 30 uses an optical coherence tomography (OCT) device that can take tomographic images of the embryo 9.

[0038] 1, the three-dimensional imaging unit 30 has a light source 31, an object optical system 32, a reference optical system 33, a detection unit 34, and an optical fiber coupler 35. The optical fiber coupler 35 has a first optical fiber 351 to a fourth optical fiber 354 connected at a connection unit 355. The light source 31, the object optical system 32, the reference optical system 33, and the detection unit 34 are connected to one another via an optical path formed by the optical fiber coupler 35.

[0039] The light source 31 has a light-emitting element such as an LED. The light source 31 emits low-coherence light containing a wide band of wavelength components. In order for the light to reach the inside of the embryo 9, it is desirable that the light emitted from the light source 31 be near-infrared. The light source 31 is connected to a first optical fiber 351. The light emitted from the light source 31 enters the first optical fiber 351 and is branched at a connection portion 355 into light that enters a second optical fiber 352 and light that enters a third optical fiber 353.

[0040] The second optical fiber 352 is connected to the object optical system 32. Light traveling from the connection part 355 to the second optical fiber 352 enters the object optical system 32. The object optical system 32 has a plurality of optical components including a collimator lens 321 and an objective lens 322. The light emitted from the second optical fiber 352 passes through the collimator lens 321 and the objective lens 322 and is irradiated onto the embryo 9 in the specimen container 90. At this time, the objective lens 322 converges the light toward the embryo 9. Then, the light reflected by the embryo 9 (hereinafter referred to as "observation light") passes through the objective lens 322 and the collimator lens 321 and enters the second optical fiber 352 again.

[0041] 1, the object optical system 32 is connected to a scanning mechanism 323. The scanning mechanism 323 moves the object optical system 32 minutely in the vertical and horizontal directions in accordance with commands from a computer 50. This allows the incident position of light on the embryo 9 to be moved minutely in the vertical and horizontal directions.

[0042] The three-dimensional imaging unit 30 can be moved horizontally by a movement mechanism (not shown), which allows the field of view of the three-dimensional imaging unit 30 to be switched among a plurality of wells.

[0043] The third optical fiber 353 is connected to the reference optical system 33. Light traveling from the connection part 355 to the third optical fiber 353 is incident on the reference optical system 33. The reference optical system 33 has a collimator lens 331 and a mirror 332. The light emitted from the third optical fiber 353 passes through the collimator lens 331 and is incident on the mirror 332. Then, the light reflected by the mirror 332 (hereinafter referred to as "reference light") passes through the collimator lens 331 and is incident on the third optical fiber 353 again.

[0044] 1, the mirror 332 is connected to a reciprocating mechanism 333. The reciprocating mechanism 333 moves the mirror 332 slightly in the optical axis direction in accordance with a command from the computer 50. This makes it possible to change the optical path length of the reference light.

[0045] The fourth optical fiber 354 is connected to the detection unit 34. The observation light incident on the second optical fiber 352 from the object optical system 32 and the reference light incident on the third optical fiber 353 from the reference optical system 33 are joined at the connection unit 355 and then incident on the fourth optical fiber 354. The light emitted from the fourth optical fiber 354 then enters the detection unit 34. At this time, interference occurs between the observation light and the reference light due to a phase difference. The spectrum of this interference light differs depending on the height of the reflection position of the observation light.

[0046] The detection unit 34 includes a spectrometer 341 and a photodetector 342. The interference light emitted from the fourth optical fiber 354 is separated into its wavelength components by the spectrometer 341 and enters the photodetector 342. The photodetector 342 detects the separated interference light and outputs the detection signal to the computer 50. The computer 50 obtains the vertical light intensity distribution of the observation light by performing a Fourier transform on the detection signal obtained from the photodetector 342. Furthermore, the computer 50 can obtain the light intensity distribution of the observation light at each coordinate in three-dimensional space by repeatedly calculating the above-described light intensity distribution while moving the object optical system 32 horizontally using the scanning mechanism 323. As a result, the computer 50 can obtain a three-dimensional image of the embryo 9. The three-dimensional image is data composed of multiple pixels arranged on three-dimensional coordinates, with a specified brightness value for each pixel.

[0047] The computer 50 functions as a control unit that controls the operation of each unit in the analysis device 1. The computer 50 also functions as an analysis unit that analyzes the state of the embryo 9 based on the two-dimensional image input from the two-dimensional imaging unit 20 and the three-dimensional image input from the three-dimensional imaging unit 30.

[0048] 3 is a control block diagram of the analysis device 1. As conceptually shown in FIG. 3, a computer 50 has a processor 51 such as a CPU, a memory 52 such as a RAM, and a storage unit 53 such as a hard disk drive. Stored in the storage unit 53 are a control program P1 for controlling the operation of each unit in the analysis device 1, and an analysis program P2 for analyzing the state of the embryo 9 based on the two-dimensional image D2 input from the two-dimensional imaging unit 20 and the three-dimensional image D3 input from the three-dimensional imaging unit 30.

[0049] 3, the computer 50 is communicatively connected to the above-described light irradiation unit 40, camera 22, focal point movement mechanism 23, light source 31, scanning mechanism 323, advance / retract mechanism 333, photodetector 342, and display unit 70 (described later). The computer 50 controls the operation of each of the above-described units in accordance with a control program P1. This allows the image capture process of the embryo 9 held in the specimen container 90 to proceed. The computer 50 also analyzes the state of the embryo 9 by processing the two-dimensional image input from the two-dimensional imaging unit 20 and the three-dimensional image input from the three-dimensional imaging unit 30 in accordance with an analysis program P2.

[0050] <2. Analysis processing> <2-1. First embodiment> Next, a first embodiment of the photographing and analyzing process of the embryo 9 in the above-mentioned analyzing device 1 will be described.

[0051] Fig. 4 is a block diagram conceptually showing the functions of the computer 50 for realizing the imaging and analysis processing according to the first embodiment. As shown in Fig. 4, the computer 50 has a two-dimensional analysis unit 62 and a three-dimensional analysis unit 63. The functions of the two-dimensional analysis unit 62 and the three-dimensional analysis unit 63 are realized by the processor 51 of the computer 50 operating in accordance with the above-mentioned analysis program P2.

[0052] 5 is a flowchart showing the flow of the imaging and analysis process according to the first embodiment. When imaging and analyzing an embryo 9 in the analysis device 1, first, a sample container 90 is set on the stage 10 (step S11). The sample container 90 holds the embryo 9 together with a culture medium.

[0053] Next, the analysis device 1 performs two-dimensional imaging of the embryo 9 using the two-dimensional imaging unit 20 (step S12). As a result, a two-dimensional image D2 of the embryo 9 is acquired. Specifically, while irradiating light from the light irradiation unit 40 toward the sample container 90, the camera 22 images the embryo 9 in the sample container 90 in a bright field. Furthermore, the focus shifting mechanism 23 performs the above imaging multiple times while changing the position of the focus. As a result, multiple two-dimensional images D2 with different focus positions are acquired. The acquired two-dimensional images D2 are input from the camera 22 to the computer 50.

[0054] Next, the analysis device 1 performs three-dimensional imaging of the embryo 9 using the three-dimensional imaging unit 30 (step S13). In this embodiment, a three-dimensional image D3 of the embryo 9 is acquired by optical coherence tomography. Specifically, light is emitted from the light source 31, and the object optical system 32 is slightly moved by the scanning mechanism 323, while the interference light of the observation light and the reference light is detected by the photodetector 342 for each wavelength component. The computer 50 calculates the light intensity distribution at each coordinate position of the embryo 9 based on the detection signal output from the photodetector 342. As a result, a three-dimensional image D3 of the embryo 9 is obtained.

[0055] In this way, a plurality of two-dimensional images D2 and three-dimensional images D3 are acquired for the same embryo 9 in steps S12 and S13.

[0056] The order of the two-dimensional imaging in step S12 and the three-dimensional imaging in step S13 may be reversed. That is, the three-dimensional imaging of the embryo 9 may be performed by the three-dimensional imaging unit 30, and then the two-dimensional imaging of the embryo 9 may be performed by the two-dimensional imaging unit 20. The two-dimensional imaging in step S12 and the three-dimensional imaging in step S13 may be performed simultaneously. Because the state of the embryo 9 changes over time, it is desirable to perform steps S12 and S13 simultaneously or almost simultaneously (sequentially).

[0057] Next, the two-dimensional analysis unit 62 of the computer 50 performs two-dimensional analysis of the embryo 9 based on the two-dimensional image D2 acquired by the two-dimensional imaging unit 20 (step S14). In this embodiment, the two-dimensional analysis unit 62 counts the number of cells 92 contained in the embryo 9 based on the two-dimensional image D2. Compared to three-dimensional imaging, two-dimensional imaging can acquire images with higher resolution. Therefore, it is easier to capture the outlines of the cells 92 in the two-dimensional image D2 than in the three-dimensional image D3. Therefore, the number of cells 92 can be counted more accurately in the two-dimensional image D2 than in the three-dimensional image D3.

[0058] A possible method for counting the number of cells 92 is, for example, a method using deep learning. Specifically, a learning model is prepared in advance by machine learning, with the two-dimensional image D2 of the embryo 9 as an input variable and the number of cells 92 contained in the embryo 9 as an output variable. Then, by inputting the two-dimensional image D2 captured by the two-dimensional imaging unit 20 into the learning model, an estimated value for the number of cells 92 can be output from the learning model. For example, a classification network can be used as the deep learning algorithm.

[0059] Alternatively, a two-dimensional Gaussian distribution having a peak at the center of the cell 92 may be learned as a correct image, and a two-dimensional image D2 captured by the two-dimensional imaging unit 20 may be input to the resulting learning model. In this case, an image having a two-dimensional Gaussian distribution corresponding to each cell 92 is output from the learning model. Therefore, the number of cells 92 can be estimated by counting the number of peaks in the output image.

[0060] The two-dimensional analysis unit 62 stores the number of cells 92 obtained by analyzing the two-dimensional image D2 as a two-dimensional analysis result R2 in the storage unit 53. The two-dimensional analysis unit 62 also displays the two-dimensional analysis result R2 on the display unit 70, such as a liquid crystal display.

[0061] Next, the three-dimensional analysis unit 63 of the computer 50 performs a three-dimensional analysis of the embryo 9 based on the three-dimensional image D3 acquired by the three-dimensional imaging unit 30 (step S15). In this embodiment, the three-dimensional analysis unit 63 calculates the volume of the plurality of cells 92 contained in the embryo 9 based on the three-dimensional image D3. Specifically, the three-dimensional analysis unit 63 classifies the three-dimensional image D3 into cellular regions corresponding to the plurality of cells 92 and regions other than the cells 92 (for example, regions corresponding to fragments). As a method for classifying such regions, for example, a known local thickness method may be used.

[0062] The three-dimensional analysis unit 63 then identifies the boundaries of each cell 92 in the above-mentioned cell region included in the three-dimensional image D3. This divides the cell region into regions for each cell 92. A known watershed algorithm, for example, may be used as a method for identifying the boundaries of the cells 92. The three-dimensional analysis unit 63 then calculates the volume of each of the multiple regions divided for each cell 92.

[0063] At this time, by using information on the number of cells 92, which is the two-dimensional analysis result R2, the boundaries of the cells 92 can be identified with greater accuracy. For example, in the Watershed algorithm, markers are specified in the three-dimensional image D3 as indicators of the position of each cell 92. The markers can be specified so that the number of markers matches the two-dimensional analysis result R2. This allows the number of markers to match the number of cells 92 contained in the embryo 9 with high accuracy. In other words, the number of regions for which the volume is calculated can match the number of cells 92 contained in the embryo 9 with high accuracy. Therefore, the boundaries of the cells 92 can be identified with high accuracy, and the volumes of the multiple cells 92 contained in the embryo 9 can be output with high accuracy.

[0064] The three-dimensional analysis unit 63 stores the volume of the cell 92 obtained by analyzing the three-dimensional image D3 as a three-dimensional analysis result R3 in the storage unit 53. The three-dimensional analysis unit 63 also displays the three-dimensional analysis result R3 on the display unit 70, such as a liquid crystal display.

[0065] As described above, this analysis device 1 analyzes the three-dimensional image D3 using the indices included in the two-dimensional analysis result R2. Therefore, the three-dimensional image D3 can be analyzed using indices that can be obtained with higher accuracy in two-dimensional analysis than in three-dimensional analysis. This allows for the acquisition of a highly accurate three-dimensional analysis result R3.

[0066] In particular, in this embodiment, multiple two-dimensional images D2 are acquired for one embryo 9 by multi-focus photography. Then, two-dimensional analysis is performed based on the multiple two-dimensional images D2. Therefore, it is possible to obtain a two-dimensional analysis result R2 with higher accuracy than when two-dimensional analysis is performed based on a single two-dimensional image D2.

[0067] Furthermore, in this embodiment, information on the number of cells 92, which can be obtained more accurately by two-dimensional analysis than by three-dimensional analysis, is used in the three-dimensional analysis. This allows the three-dimensional analysis to be performed with high accuracy. In particular, when calculating the volume of cells 92 in three-dimensional analysis, information on the number of cells 92 is important reference information. Therefore, by using information on the number of cells 92 that can be obtained with high accuracy by two-dimensional analysis, the volume of cells 92 can be calculated with high accuracy.

[0068] <2-2. Second embodiment> Next, a second embodiment of the photographing and analyzing process of the embryo 9 in the above-mentioned analyzing device 1 will be described.

[0069] Fig. 6 is a block diagram conceptually showing the functions of the computer 50 for realizing the imaging and analysis processing according to the second embodiment. As shown in Fig. 4, the computer 50 has a two-dimensional analysis unit 62, a three-dimensional analysis unit 63, and an integrated analysis unit 64. The functions of the two-dimensional analysis unit 62, the three-dimensional analysis unit 63, and the integrated analysis unit 64 are realized by the processor 51 of the computer 50 operating in accordance with the above-mentioned analysis program P2.

[0070] 7 is a flowchart showing the flow of the imaging and analysis process according to the second embodiment. In the second embodiment, the analysis device 1 first sets the sample container 90 on the stage 10 (step S21), then performs two-dimensional imaging of the embryo 9 using the two-dimensional imaging unit 20 (step S22), and then performs three-dimensional imaging of the embryo 9 using the three-dimensional imaging unit 30 (step S23). The processes in steps S21 to S23 are the same as steps S11 to S13 in the first embodiment described above, and therefore will not be described again.

[0071] Next, the two-dimensional analysis unit 62 of the computer 50 performs a two-dimensional analysis of the embryo 9 based on the two-dimensional image D2 acquired by the two-dimensional imaging unit 20 (step S24). In this embodiment, the two-dimensional analysis unit 62 performs a Veeck classification of the embryo 9 based on the two-dimensional image D2. The Veeck classification expresses the state of the embryo 9 at the cleavage stage in five grades, from grade 1 to grade 5. In the Veeck classification, the state of the embryo 9 is evaluated based on the uniformity of the sizes of the cells 92 contained in the embryo 9 and the amount of fragments contained in the embryo 9.

[0072] In step S24, the two-dimensional analysis unit 62 calculates the size uniformity of the cells 92 and the amount of fragments based on the multiple two-dimensional images D2 obtained by multi-focus photography. Then, based on these calculated index values, the Veeck classification result of the embryo 9 is determined and output. Note that in step S24, deep learning may be used to categorize the size uniformity of the cells 92 and the amount of fragments (high / low). Furthermore, the two-dimensional analysis unit 62 may determine the Veeck classification result directly from the multiple two-dimensional images D2 using deep learning such as a classification network, without calculating the size uniformity of the cells 92, the amount of fragments, etc.

[0073] The two-dimensional analysis unit 62 stores the Veeck classification result obtained by analyzing the two-dimensional image D2 as a two-dimensional analysis result R2 in the storage unit 53. The two-dimensional analysis unit 62 also displays the two-dimensional analysis result R2 on a display unit 70 such as a liquid crystal display.

[0074] Next, the three-dimensional analysis unit 63 of the computer 50 performs a three-dimensional analysis of the embryo 9 based on the three-dimensional image D3 acquired by the three-dimensional imaging unit 30 (step S25). In this embodiment, the three-dimensional analysis unit 63 performs Veeck classification of the embryo 9 based on the three-dimensional image D3. Specifically, the three-dimensional analysis unit 63 calculates the size uniformity of the cells 92 and the amount of fragments based on the three-dimensional image D3. Then, based on these calculated index values, the three-dimensional analysis unit 63 determines and outputs the Veeck classification result of the embryo 9. However, the three-dimensional analysis unit 63 may also determine the Veeck classification result directly from the three-dimensional image D3 using deep learning such as a classification network, without calculating the size uniformity of the cells 92, the amount of fragments, etc.

[0075] The three-dimensional analysis unit 63 stores the Veeck classification result obtained by analyzing the three-dimensional image D3 as a three-dimensional analysis result R3 in the storage unit 53. The three-dimensional analysis unit 63 also displays the three-dimensional analysis result R3 on the display unit 70, such as a liquid crystal display.

[0076] In this manner, in this embodiment, in steps S24 and S25, the Veeck classification result is obtained for the same embryo 9 using two different approaches.

[0077] The order of the two-dimensional analysis in step S24 and the three-dimensional analysis in step S25 may be reversed. That is, Veeck classification of the embryo 9 may be performed by the three-dimensional analysis, and then Veeck classification of the embryo 9 may be performed by the two-dimensional analysis. Furthermore, the two-dimensional analysis in step S24 and the three-dimensional analysis in step S25 may be performed simultaneously.

[0078] Thereafter, the integrated analysis unit 64 of the computer 50 outputs the Veeck classification result as the integrated analysis result R4 based on the Veeck classification result obtained by the two-dimensional analysis and the Veeck classification result obtained by the three-dimensional analysis (step S26). For example, the integrated analysis unit 64 outputs the Veeck classification result as the integrated analysis result R4 only when the Veeck classification result as the two-dimensional analysis result R2 and the Veeck classification result as the three-dimensional analysis result R3 match.

[0079] Furthermore, if the Veeck classification result as the two-dimensional analysis result R2 and the Veeck classification result as the three-dimensional analysis result R3 do not match, both results may be output, or the user may be allowed to select which of the two-dimensional analysis result R2 and the three-dimensional analysis result R3 will be used as the integrated analysis result R4. Furthermore, if the Veeck classification result as the two-dimensional analysis result R2 and the Veeck classification result as the three-dimensional analysis result R3 do not match, the integrated analysis unit 64 may automatically select either the two-dimensional analysis result R2 or the three-dimensional analysis result R3, and add information to the integrated analysis result R4 indicating that the reliability of the result is low.

[0080] The integrated analysis unit 64 stores the Veeck classification result obtained by the integrated analysis in step S26 as an integrated analysis result R4 in the storage unit 53. The integrated analysis unit 63 also displays the integrated analysis result R4 on the display unit 70, such as a liquid crystal display.

[0081] As described above, in this embodiment, two Veeck classification results obtained by two approaches, two-dimensional analysis and three-dimensional analysis, are used to output a Veeck classification result as an integrated analysis result R4, thereby making it possible to obtain a highly accurate Veeck classification result.

[0082] The size (volume) of the cells 92, which is an index of the Veeck classification, can be calculated more accurately from the three-dimensional image D3 than from the two-dimensional image D2. Meanwhile, the amount of fragments, another index of the Veeck classification, can be calculated more accurately from the two-dimensional image D2 than from the three-dimensional image D3. Therefore, the amount of fragments calculated based on the two-dimensional image D2 may be set as the two-dimensional analysis result R2, and the size uniformity of the cells 92 calculated based on the three-dimensional image D3 may be set as the three-dimensional analysis result R3. Based on these results, the integrated analysis unit 64 may output a Veeck classification result as an integrated analysis result R4. In this way, the advantages of both two-dimensional and three-dimensional analyses can be utilized to output a highly accurate Veeck classification result.

[0083] <3. Modifications> Although one embodiment of the present invention has been described above, the present invention is not limited to the above embodiment.

[0084] In the first embodiment described above, the number of cells 92 contained in the embryo 9 is output in the two-dimensional analysis in step S14. However, other indicators may be output in the two-dimensional analysis. For example, in the two-dimensional analysis in step S14, the amount of fragments contained in the embryo 9, the position of the cells 92 in the embryo 9, the spread of the cells 92 in the embryo 9, the Veeck classification result, etc. may be output as the two-dimensional analysis result R2.

[0085] In the first embodiment, the volume of the cells 92 contained in the embryo 9 is output in the three-dimensional analysis in step S15. However, in the three-dimensional analysis, the shapes of the multiple cells 92 may be identified based on the number of cells 92 obtained by the two-dimensional analysis, and the shape identification results of the cells 92 may be output as the three-dimensional analysis result R3. In the three-dimensional analysis, other indicators may be output while using the two-dimensional analysis result R2. For example, in the three-dimensional analysis in step S15, the amount of fragments contained in the embryo 9, the Veeck classification result, etc. may be output. In the first embodiment, after the three-dimensional analysis in step S15, the computer 50 may output the Veeck classification result based on the three-dimensional analysis result.

[0086] In the second embodiment described above, the Veeck classification result is output as the integrated analysis result R4 in step S26. However, an index other than the Veeck classification result may be output as the integrated analysis result R4. For example, the volume of the cell 92, the amount of fragments, etc. may be output as the integrated analysis result R4.

[0087] In the above embodiment, the two-dimensional imaging unit 20 performs multi-focus imaging in a bright field. However, the two-dimensional imaging unit 20 may acquire the two-dimensional image D2 by other imaging methods.

[0088] In the above embodiment, the three-dimensional imaging unit 30 performs optical coherence tomography (OCT). However, the three-dimensional imaging unit 30 may acquire the three-dimensional image D3 by another imaging method.

[0089] In the above embodiment, the analysis device 1 outputs the two-dimensional analysis result R2, the three-dimensional analysis result R3, and the integrated analysis result R4 to the display unit 70. However, the analysis device 1 may output the two-dimensional analysis result R2, the three-dimensional analysis result R3, and the integrated analysis result R4 as data to another computer.

[0090] In the above embodiment, the sample to be analyzed is a human embryo 9, and the objects under observation are cells 92 contained in the embryo 9. However, the "sample" in the present invention is not limited to an embryo 9, and may be any object in which a plurality of objects under observation are distributed three-dimensionally.

[0091] Furthermore, the elements appearing in the above-described embodiments and modifications may be combined as appropriate within the scope of not causing any contradiction. [Explanation of symbols]

[0092] 1 Analysis device 9 Embryos 10 stages 20 Two-dimensional imaging unit 21 Imaging optical system 22 Camera 23 Focus movement mechanism 30 Three-dimensional imaging unit 31 Light source 32 Object optical system 33 Reference optical system 34 Detector 35 Optical Fiber Coupler 40 Light irradiation unit 50 Computers 62 Two-dimensional analysis section 63 Three-dimensional analysis department 64 Integrated Analysis Department 70 Display section 90 Sample container 91 Zona pellucida 92 cells D2 2D image D3 3D image R2 2D analysis results R3 3D analysis results R4 integrated analysis results

Claims

1. An analysis method for analyzing a sample in which a plurality of objects under observation are distributed three-dimensionally, comprising: a) acquiring a two-dimensional image by two-dimensionally photographing the sample; b) acquiring a three-dimensional image by three-dimensionally photographing the sample; c) analyzing the two-dimensional image to output a two-dimensional analysis result; d) analyzing the three-dimensional image to output a three-dimensional analysis result; and In the step d), the three-dimensional image is analyzed using an index included in the two-dimensional analysis result; the sample is an embryo; the object to be observed is a cell contained in the embryo, the result of the two-dimensional analysis is the number of cells contained in the embryo; the three-dimensional analysis result is a shape identification result of the plurality of cells contained in the embryo; In the step d), the number of regions for calculating the shape identification results of the cells is determined based on the number of the cells obtained in the step c).

2. The analysis method according to claim 1, e) after step d), outputting a Veeck classification result representing the state of the embryo based on the three-dimensional analysis result. The analysis method further comprises:

3. An analysis method for analyzing a sample in which a plurality of objects under observation are distributed three-dimensionally, comprising: a) acquiring a two-dimensional image by two-dimensionally photographing the sample; b) acquiring a three-dimensional image by three-dimensionally photographing the sample; c) analyzing the two-dimensional image to output a two-dimensional analysis result; d) analyzing the three-dimensional image to output a three-dimensional analysis result; e) outputting an integrated analysis result based on the two-dimensional analysis result and the three-dimensional analysis result; and the sample is an embryo, An analysis method, wherein the two-dimensional analysis result, the three-dimensional analysis result, and the integrated analysis result are Veeck classification results that represent the state of the embryo.

4. The analysis method according to any one of claims 1 to 3, In the step a), a plurality of the two-dimensional images are acquired by performing bright-field photography while changing a focal position; In the step c), the two-dimensional analysis result is obtained by analyzing a plurality of the two-dimensional images.

5. The analysis method according to any one of claims 1 to 4, In the step b), the three-dimensional image is obtained by optical coherence tomography.

6. An analysis apparatus for analyzing a sample in which a plurality of objects to be observed are distributed three-dimensionally, a two-dimensional imaging unit that acquires a two-dimensional image by two-dimensionally photographing the sample; a three-dimensional imaging unit that acquires a three-dimensional image by three-dimensionally photographing the sample; an analysis unit that analyzes a sample based on the two-dimensional image and the three-dimensional image; Equipped with The analysis part is a two-dimensional analysis process for analyzing the two-dimensional image and outputting a two-dimensional analysis result; a three-dimensional analysis process for analyzing the three-dimensional image and outputting a three-dimensional analysis result; Run In the three-dimensional analysis process, the three-dimensional image is analyzed using an index included in the two-dimensional analysis result; the sample is an embryo, the object to be observed is a cell contained in the embryo, the result of the two-dimensional analysis is the number of cells contained in the embryo; the three-dimensional analysis result is a shape identification result of the plurality of cells contained in the embryo; In the three-dimensional analysis process, the analysis device determines the number of regions for calculating the shape identification results of the cells based on the number of the cells obtained by the two-dimensional analysis process.

7. An analysis apparatus for analyzing a sample in which a plurality of objects to be observed are distributed three-dimensionally, a two-dimensional imaging unit that acquires a two-dimensional image by two-dimensionally photographing the sample; a three-dimensional imaging unit that acquires a three-dimensional image by three-dimensionally photographing the sample; an analysis unit that analyzes a sample based on the two-dimensional image and the three-dimensional image; Equipped with The analysis part is a two-dimensional analysis process for analyzing the two-dimensional image and outputting a two-dimensional analysis result; a three-dimensional analysis process for analyzing the three-dimensional image and outputting a three-dimensional analysis result; an integrated analysis process for outputting an integrated analysis result based on the two-dimensional analysis result and the three-dimensional analysis result; Run the sample is an embryo, An analysis device, wherein the two-dimensional analysis result, the three-dimensional analysis result, and the integrated analysis result are Veeck classification results that represent the state of the embryo.

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