Phase image acquisition method and quantitative data acquisition method

Abstract

The present invention acquires a hologram image of an object in a state in which the object is accommodated in a recess in a container, a bottom surface of the recess being curved. A phase image is generated from the hologram image. Processing for subtracting, from the phase image, a phase component that is derived from a polynomial indicating the shape of the bottom surface of the recess is performed to acquire a processed phase image.

Description

Phase image acquisition method and quantitative data acquisition method

[0001] The disclosed technology relates to a phase image acquisition method and a quantitative data acquisition method.

[0002] The following techniques are known as techniques for monitoring cells using holography. For example, JP 2018-516591 A describes a cell culture incubator that includes an incubator cabinet that includes an internal chamber for incubating cells in a cell culture vessel, and a holographic imager configured to capture images of the cells in the internal chamber.

[0003] A phase image generated based on a hologram image (interference image) formed by interference between object light transmitted through a cell and reference light coherent with respect to the object light is an image showing the phase distribution of the object light transmitted through the cell, and reflects the state of the cell. Therefore, it is possible to evaluate the quality of a cell based on the phase image. For example, the total phase amount obtained by integrating the phase amount for each pixel of the phase image can be used as quantitative data for evaluating the cell.

[0004] A phase image of a cell can be acquired by placing the cell in a well of a dish (container) having a well with an inclined bottom. A phase component resulting from the inclination of the well bottom is superimposed on the phase image of the cell acquired in this state. The phase component resulting from the well bottom is, so to speak, noise, and can reduce the accuracy of quantitative data obtained from the phase image.

[0005] The disclosed technology has been made in consideration of the above points, and aims to appropriately remove unnecessary phase components from a phase image of an object.

[0006] The phase image acquisition method according to the disclosed technology includes acquiring a hologram image of an object placed in a recess of a container having a curved bottom surface, generating a phase image from the hologram image, and subtracting a phase component derived from a polynomial that represents the shape of the bottom surface of the recess from the phase image to obtain a processed phase image.

[0007] The degree of the polynomial is preferably not less than 2 and not more than 7. The amount of change in height of the bottom surface of the recess in the phase image is preferably not less than 10 μm.

[0008] The object may be, for example, a cell or a cell cluster, and the cell or cell cluster may be a fertilized animal egg.

[0009] In the phase image acquisition method according to the embodiment of the disclosed technique, a phase image of a cell or cell aggregate cultured in a state of being contained in a well may be acquired.

[0010] The method for acquiring quantitative data according to the disclosed technology is a method for acquiring quantitative data using the phase image acquisition method described above, and includes deriving quantitative data indicating the state of a cell or cell aggregate based on a processed phase image.

[0011] According to the disclosed technology, it is possible to appropriately remove unnecessary phase components from a phase image of an object.

[0012] FIG. 1 is a diagram showing an example of a culture state of a fertilized egg according to an embodiment of the disclosed technology. FIG. 2 is a diagram showing an example of the configuration of a holography device according to an embodiment of the disclosed technology. FIG. 3 is a diagram showing an example of a phase image of a fertilized egg before unwrapping according to an embodiment of the disclosed technology. FIG. 4 is a diagram showing the concept of a phase image according to an embodiment of the disclosed technology. FIG. 5 is a diagram showing an example of function fitting performed on the shape of the bottom surface of a recess according to an embodiment of the disclosed technology. FIG. 6 is a diagram showing an example of a processed phase image according to an embodiment of the disclosed technology. FIG. 7 is a diagram showing an example of a processed phase image according to an embodiment of the disclosed technology. FIG. 8 is a diagram showing an example of the configuration of a container according to a comparative example. FIG. 9 is a diagram showing the amount of change in height of the bottom surface of a recess according to an embodiment of the disclosed technology.

[0013] Hereinafter, an example of an embodiment of the disclosed technology will be described with reference to the drawings. In each drawing, the same or equivalent components and parts are given the same reference numerals, and redundant description will be omitted.

[0014] A phase image acquisition method according to an embodiment of the disclosed technology includes a first step of acquiring a hologram image of an object while the object is contained in a recess of a container having a recess with a curved bottom surface, a second step of generating a phase image from the hologram image, and a third step of performing a process (background removal process) of subtracting a phase component derived from a polynomial corresponding to the shape of the bottom surface of the recess from the phase image to acquire a processed phase image.

[0015] Each of the first to third steps will be described in detail below. In this embodiment, the object from which a phase image is acquired will be described as a fertilized egg of an animal. The fertilized egg may be one that has developed to an embryonic state. The phase image acquisition method according to the disclosed technology can be used for purposes such as quality control, quality evaluation, quality inspection, or quality prediction of the fertilized egg.

[0016] <First Step: Acquiring a Hologram Image> FIG. 1 is a diagram showing an example of the culture state of a fertilized egg 200. The fertilized egg 200 is cultured in a state where it is contained in a recess 110 of a container 100 having recesses 110. The container 100 may include a plurality of recesses 110, and a fertilized egg 200 may be contained in each of the plurality of recesses 110. The container 100 may be, for example, a commercially available dish or petri dish. The recess 110 may be a well provided in the dish or petri dish. For example, a 38Special GPS (registered trademark) manufactured by CooperSurgical can be suitably used as the container 100.

[0017] The recess 110 contains a culture medium 210 together with the fertilized egg 200, and the fertilized egg 200 is cultured in the recess 110 while immersed in the culture medium 210. The bottom surface 120 of the recess 110 is curved, with the height at the center 121 being the lowest and sloping gently upward from the center 121 outward. The shape of the bottom surface 120 of the recess 110 may be spherical.

[0018] When acquiring a hologram image of the fertilized egg 200, it is necessary to capture the fertilized egg 200 with a microscope. In order to keep the fertilized egg 200 within the field of view of the microscope, it is preferable to suppress the amount of movement of the fertilized egg 200. If the fertilized egg 200 were placed on a flat surface, the fertilized egg 200 would sway due to convection caused by heating of the culture medium, making it difficult to keep the fertilized egg 200 within the field of view of the microscope. By sloping the bottom surface 120 of the recess 110 so that the height of the center portion 121 is the lowest, the fertilized egg 200 can be placed near the center portion 121, and further, the amount of movement of the fertilized egg 200 can be suppressed.

[0019] In the first step, a hologram image of the fertilized egg 200 is acquired. The hologram image of the fertilized egg 200 is acquired in a state where the fertilized egg 200 is placed in the recess 110. Fig. 2 is a diagram showing an example of the configuration of the holography device 10 used to acquire the hologram image.

[0020] The holography device 10 includes a splitter 21, reflecting mirrors 22 and 24, an objective lens 23, an imaging lens 25, a combiner 26, and an imaging device 30. The fertilized egg 200 to be imaged is placed between the reflecting mirror 22 and the objective lens 23 while being accommodated in the recess 110 of the container 100 together with the culture medium.

[0021] The laser light source 20 can be, for example, a HeNe laser with a wavelength of 632.8 nm. The linearly polarized laser light L0 emitted from the laser light source 20 is split into two laser lights by a splitter 21. One of the two laser lights is designated as object light L1, and the other is designated as reference light L2. A beam splitter can be used as the splitter 21. The object light L1 is incident on a reflecting mirror 22. The object light L1, whose traveling direction has been bent by the reflecting mirror 22, is irradiated onto the fertilized egg 200. The object light L1 is irradiated in a direction penetrating the bottom surface 120 of the recess 110.

[0022] An image formed by the object light L1 that has passed through the fertilized egg 200 is enlarged by the objective lens 23. The direction of travel of the object light L1 that has passed through the objective lens 23 is bent by the reflecting mirror 24, and the light is incident on the combiner 26 via the imaging lens 25. Meanwhile, the reference light L2 also enters the combiner 26. The object light L1 and the reference light L2 are combined by the combiner 26, and an image is formed on the imaging surface of the imaging device 30. A beam splitter can be used as the combiner 26.

[0023] A hologram image, which is an interference image generated by the interference between the object light L1 and the reference light L2, is captured by the imaging device 30. The imaging device 30 includes an imaging element such as a CMOS (Complementary Metal-Oxide-Semiconductor) image sensor, and generates image data of the hologram image.

[0024] <Second Step: Acquiring a Phase Image> An example of a method for acquiring a phase image from a hologram image is described below. First, the hologram image of the fertilized egg 200 acquired by the imaging device 30 is trimmed to a size of, for example, 2048 x 2048, and then subjected to a two-dimensional Fourier transform. The Fourier transform image obtained by this process may include images based on direct light, object light, and conjugate light.

[0025] Next, the position of the object light is identified by determining the amount of deviation of the object light from the direct light in the Fourier transform image, and the complex amplitude component of only the object light is extracted by frequency filtering using, for example, a mask with a circular opening with a radius of 250 pixels.

[0026] Next, for example, an angular spectrum method is applied to restore an image showing the phase of the fertilized egg 200 at an arbitrary spatial position. Specifically, the angular spectrum U(f ｘ , f ｙ Next, the angular spectrum U(f ｘ , f ｙ ;0) to the transfer function H(f ｘ , f ｙ;z), the wavefront at any position z in the optical axis direction (z direction) is reconstructed. Here, the transfer function H(f ｘ , f ｙ ;z) is the frequency response function (Fourier transform of the impulse response function (Green's function)).

[0027] Next, as shown in the following equation (2), the wavefront U(f ｘ , f ｙ ;z) to derive the solution u(x,y;z) at position z.

[0028] Next, as shown in the following equation (3), a phase image is generated by deriving the phase φ for u(x, y; z).

[0029] The phases in the phase image before unwrapping obtained by the above process are convoluted to values ​​between 0 and 2π. Therefore, a phase unwrapping (unwrapping) method such as Unweighted Least Squares or Flynn's Algorithm is applied to unwrap the parts of 2π or more. Note that many unwrapping methods have been proposed, and an appropriate one that does not cause phase mismatch can be selected.

[0030] 3 is a diagram showing an example of a phase image of the embryo 200 before unwrapping. A phase component resulting from the inclination of the bottom surface 120 of the well 110 is superimposed on the phase image. The phase component resulting from the bottom surface 120 of the well 110 is, so to speak, noise, and causes a decrease in the accuracy of the quantitative data obtained from the phase image. The phase component resulting from the bottom surface 120 of the well 110 is removed by a background removal process described below.

[0031] <Third step: background removal> FIG. 4 shows the phase image I Ｐ The lower part of FIG. 4 shows the concept of the phase image I Ｐ The upper part of Fig. 4 shows the phase image I ＰThe phase amount at each pixel j is shown on a plane in gray scale.

[0032] Here, the phase image I Ｐ The phase amount at each pixel j is P Ｓ and the phase of the background (area where the fertilized egg 200 does not exist) existing in the same focal plane is P Ｂ In this case, the background removal process can be expressed by the following equation (4): Note that the term "phase" in this specification refers to the phase of the electric field amplitude when light is regarded as an electromagnetic wave, and is used in a more general sense.

[0033] Background phase P Ｂ is a phase component originating from the bottom surface 120 of the recess 110. Therefore, by specifying the shape of the bottom surface 120 of the recess 110, the phase P Ｂ It is possible to calculate the function of the bottom surface 120 of the recess 110. Because the bottom surface 120 of the recess 110 is a curved surface, it is possible to perform function fitting using a polynomial of degree 2 or greater on the shape of the bottom surface 120 of the recess 110. The function fitting can be performed, for example, by obtaining a profile of the shape of the bottom surface 120 of the recess 110 by actual measurement and searching for a polynomial that approximates the obtained profile.

[0034] FIG. 5 is a diagram showing an example of function fitting performed on the shape of the bottom surface 120 of the recess 110. In FIG. 5, the horizontal axis indicates the position in the planar direction of the recess 110, and the vertical axis indicates the position in the height direction of the recess 110. In FIG. 5, the dotted line is a curve defined by a second-order polynomial that represents the shape of the bottom surface 120 of the recess 110, and the solid line is a profile of the shape of the bottom surface 120 of the recess 110 obtained by actual measurement. It can be seen that the solid line and the dotted line are approximately coincident. In this way, the shape of the bottom surface 120 of the recess 110 can be described by a polynomial of degree two or more.

[0035] Background phase P Ｂ , that is, the phase component originating from the bottom surface 120 of the recess 110 can be derived from a polynomial that represents the shape of the bottom surface 120 of the recess 110.Ｂ is expressed by the following equation (5). In equation (5), x is the distance from the center 121 of the recess 110, and m is the degree of the polynomial. ｋ are coefficients of each term, and can be obtained from a polynomial that represents the shape of the bottom surface 120 of the recess 110.

[0036] When the polynomial representing the shape of the bottom surface 120 is C(x), the phase of the background P Ｂ In equation (6), n ｓ is the refractive index of the container 100, and n ｍ is the refractive index of the medium, and λ is the wavelength of the object light in the holographic optical system.

[0037] In the background removal process, as shown in equation (4), a phase component derived from a polynomial that represents the shape of the bottom surface 120 of the recess 110 is subtracted from the phase amount of each pixel in the phase image obtained in the second step. This allows a phase image to be obtained from which the phase component derived from the bottom surface 120 of the recess 110 has been removed. Ｂ In other words, a phase image from which the phase component originating from the bottom surface 120 of the recess 110 has been removed will be referred to as a "processed phase image" hereinafter.

[0038] 6A, 6B, and 6C are diagrams showing examples of processed phase images. FIG. 6A is a processed phase image obtained by performing function fitting using a polynomial representing the shape of the bottom surface 120 of the recess 110 with a degree of 3. FIG. 6B is a processed phase image obtained by performing function fitting using a polynomial representing the shape of the bottom surface 120 of the recess 110 with a degree of 5. FIG. 6C is a processed phase image obtained by performing function fitting using a polynomial representing the shape of the bottom surface 120 of the recess 110 with a degree of 7. As shown in FIGS. 6A to 6C, by performing background removal processing, phase components derived from the bottom surface 120 of the recess 110 are removed, and processed phase images in which only the fertilized egg 200 is extracted can be obtained.

[0039] 7 is a diagram showing an example of a container 100X according to a comparative example. The container 100X differs from the container 100 according to the embodiment of the disclosed technique described above (see FIG. 1 ) in that the bottom surface 120 of the recess 110X is a non-curved surface. That is, the slope of the bottom surface 120X of the recess 110X according to the comparative example is linear.

[0040] In the container 100X according to the comparative example, the height of the center 121X of the bottom surface 120X of the recess 110X changes abruptly, so that when function fitting is performed on the shape of the bottom surface 120X, an error is large. The error in function fitting is due to the background phase P Ｂ Therefore, when the container 100X according to the comparative example is used, it becomes difficult to appropriately remove the phase component originating from the bottom surface 120X of the recess 110X.

[0041] On the other hand, the bottom surface 120 of the recess 110 of the container 100 according to the embodiment of the disclosed technology is a curved surface that has the lowest height at the center 121 and is gently inclined outward from the center 121, so that it is possible to perform highly accurate function fitting on the shape of the bottom surface 120 of the recess 110 (see FIG. 5 ). That is, the phase image acquisition method according to the embodiment of the disclosed technology makes it possible to appropriately remove unnecessary phase components from a phase image of an object.

[0042] It is preferable that the degree of the polynomial representing the shape of the bottom surface 120 of the recess 110 is 2 or more and 7 or less. By setting the degree of the polynomial to 2 or more, it becomes possible to describe a curved surface using a polynomial. By setting the degree of the polynomial to 7 or less, it becomes possible to perform highly accurate function fitting for the shape of the bottom surface 120 of the recess 110, and it becomes possible to appropriately remove phase components originating from the bottom surface 120 of the recess 110. When the degree of the polynomial is 8 or more, errors tend to occur more easily in function fitting. It is also possible to perform fitting with degree 8 or more, but the contribution of the coefficient (a ｋ ×x ｋ ) is the sum of the contributions of the second to seventh order coefficients (Σk = 2 to 7 a ｋ ×x ｋ), it is considered that the fitting is substantially seventh order. Furthermore, it is preferable that the variation Δh (see FIG. 8 ) of the height of the bottom surface 120 of the recess 110 in the phase image (within the imaging range of the phase image) is 10 μm or more. When the variation Δh is 10 μm or more, it becomes possible to perform function fitting of the shape of the bottom surface 120 of the recess 110 with high accuracy, and it becomes possible to appropriately remove the phase component originating from the bottom surface 120 of the recess 110.

[0043] <Acquisition of Quantitative Data> A method for deriving quantitative data according to an embodiment of the disclosed technology uses the phase image acquisition method described above. That is, the quantitative data derivation method derives quantitative data indicating the state of the fertilized egg 200 based on a processed phase image acquired by carrying out the phase image acquisition method described above. The quantitative data derivation method according to this embodiment will be described in detail below.

[0044] The phase amount P at each pixel j of the processed phase image ｊ can be expressed by the following formula (7): ｊ is the refractive index of the fertilized egg 200 at the portion corresponding to each pixel j of the processed phase image, and d ｊ is the thickness of the fertilized egg 200 at the location corresponding to each pixel j of the processed phase image, and λ is the wavelength of the object light in the hologram optical system.

[0045] The processed phase image of the fertilized egg 200 is an image showing the phase distribution of the object light L1 that has passed through the fertilized egg 200, and is also an image showing the optical path length distribution of the object light that has passed through the fertilized egg 200. Since the optical path length within the fertilized egg 200 corresponds to the product of the refractive index of the fertilized egg 200 and the thickness of the fertilized egg 200, the processed phase image of the fertilized egg 200 contains information on the refractive index and thickness (shape) of the fertilized egg 200, as shown in equation (7). The processed phase image of the fertilized egg 200 reflects the internal state of the fertilized egg 200, so it is possible to evaluate the quality of the fertilized egg 200 based on the processed phase image. Specifically, the total phase amount P Ａcan be used as quantitative data indicating the state of the fertilized egg 200. In equation (8), s is the area of ​​each pixel j of the processed phase image, and v ｊ is the volume of the fertilized egg 200 at the location corresponding to each pixel j of the processed phase image. As shown in equation (8), the total phase amount P Ａ is the phase amount P for each pixel of the processed phase image ｊ The pixel value of the processed phase image corresponds to the sum of the phase amount P ｊ It corresponds to.

[0046] For example, the total phase amount P derived based on the processed phase image Ａ By performing a threshold determination on this, it is possible to determine whether the embryo 200 is good or bad. By deriving quantitative data indicating the state of the embryo 200 based on the processed phase image acquired by carrying out the phase image acquisition method according to the embodiment of the disclosed technology, the accuracy of the quantitative data can be improved, and the embryo 200 can be appropriately evaluated.

[0047] The quantitative data derived based on the phase image is the total phase amount P Ａ For example, the pixel value of the processed phase image (phase amount P ｊ ) can be used as quantitative data. Ａ The phase density, which is the value obtained by dividing the density by the volume of the fertilized egg 200, can also be used as quantitative data.

[0048] In the above description, the object for which a phase image is acquired is a fertilized egg, but this is not limiting. The object may be a cell or cell aggregate other than a fertilized egg. For example, the disclosed technology can be applied to acquiring a phase image of an iPS cell (induced pluripotent stem cell) or a spheroid, which is an aggregate of iPS cells. Furthermore, the object for which a phase image is acquired may be an artificial object.

[0049] [Examples] Mouse embryos were cultured for 5 days using the dishes shown in Table 1 below. The shape of the bottom surface of the recess (well) provided in each dish is shown in Table 1. The bottom surfaces of the recesses in the dishes of Examples 1 and 2 are curved as shown in FIG. 1. The dish of Comparative Example 1 is a dish with a flat bottom surface without a recess. The bottom surface of the recess in the dish of Comparative Example 2 is a non-curved surface with a linear slope as shown in FIG. 7. With a mouse embryo contained in each dish, a hologram image of the mouse embryo was acquired, and a phase image was obtained from the hologram image.

[0050] <Measurement of change in bottom height Δh> A cross-sectional image was taken of each dish, and the change in bottom height Δh of the recess (well) in the phase image was measured (see FIG. 8). The measured values ​​of the change Δh are shown in Table 1.

[0051] <Background Removal Processing> Background removal processing was performed on the phase images of mouse embryos acquired for each Example and Comparative Example. For Examples 1 and 2, in which the bottom surface of the well was curved, function fitting using a fifth-order polynomial was performed on the shape of the bottom surface of the well. For Comparative Example 2, in which the bottom surface of the well was uncurved, function fitting using a first-order polynomial was performed on the shape of the bottom surface of the well. The phase components derived from these polynomials were removed from the phase image to obtain a processed phase image. Note that for Comparative Example 1, in which the bottom surface of the dish was flat, background removal processing was not performed.

[0052] <Measurement of Movement Amount> The movement amount of the mouse embryo during the culture period was measured for each Example and Comparative Example. The position of the mouse embryo at the start of culture was defined as the initial position, and the distance from the initial position to the position of the mouse embryo when it moved to the farthest position from the initial position during the culture period was defined as the movement amount. The measured values ​​of the movement amount are shown in Table 1. The dishes according to Example 1, Example 2, and Comparative Example 2 have a recess with an inclined bottom, and therefore the movement amount of the mouse embryo is suppressed. On the other hand, when the dish according to Comparative Example 1, which has a flat bottom, was used, the movement amount of the mouse embryo was the largest.

[0053] <Measurement of Phase Measurement Error> The phase measurement error was obtained for the processed phase images obtained for each Example and Comparative Example. For the same mouse embryo, the total phase amount P Ａ The measurement was repeated 100 times. Ａ The width of the phase amount where the variation of the total phase amount P Ａ The phase measurement error was calculated by dividing the error by the average value of the phase measurement error. σ is the standard deviation. The measured values ​​of the phase measurement error are shown in Table 1.

[0054] In the dishes according to Examples 1 and 2, in which the bottom surfaces of the recesses (wells) are curved, it is possible to perform highly accurate function fitting for the shape of the bottom surfaces of the recesses, and therefore background removal processing can be performed appropriately. As a result, the phase measurement error was reduced to 5% or less. On the other hand, in the dish according to Comparative Example 2, in which the bottom surfaces of the recesses are non-curved, it is difficult to perform highly accurate function fitting for the shape of the bottom surfaces of the recesses, and it is difficult to perform appropriate background removal processing. As a result, the phase measurement error was significantly larger than in Examples 1 and 2.

[0055] As described above, by performing background removal processing using the dishes according to Examples 1 and 2, the amount of movement of mouse embryos was suppressed, and quantitative data with small errors (total phase amount P Ａ ) was obtained.

[0056] The following supplementary notes are further disclosed with respect to the above-described embodiments: (Supplementary Note 1) A phase image acquisition method comprising: acquiring a hologram image of an object placed in a recess of a container having a curved bottom surface; generating a phase image from the hologram image; and performing a process of subtracting a phase component derived from a polynomial that represents the shape of the bottom surface of the recess from the phase image, thereby acquiring a processed phase image.

[0057] (Supplementary Note 2) The phase image acquisition method according to Supplementary Note 1, wherein the degree of the polynomial is 2 or more and 7 or less.

[0058] (Supplementary Note 3) The phase image acquisition method according to Supplementary Note 1 or Supplementary Note 2, wherein a change in height of a bottom surface of the recess in the phase image is 10 μm or more.

[0059] (Supplementary Note 4) The phase image acquisition method according to any one of Supplementary Note 1 to Supplementary Note 3, wherein the object is a cell or a cell aggregate.

[0060] (Supplementary Note 5) The phase image acquisition method according to Supplementary Note 4, wherein the cell or the cell aggregate is a fertilized egg of an animal.

[0061] (Supplementary Note 6) The phase image acquiring method according to Supplementary Note 4 or Supplementary Note 5, further comprising acquiring a phase image of the cell or cell aggregate cultured in the well.

[0062] (Supplementary Note 7) A method for acquiring quantitative data using the phase image acquisition method described in Supplementary Note 4 or Supplementary Note 5, comprising deriving quantitative data indicating a state of the cell or cell aggregate based on the processed phase image.

[0063] The disclosure of Japanese Patent Application No. 2022-091056, filed on June 3, 2022, is incorporated herein by reference in its entirety. In addition, all documents, patent applications, and technical standards described herein are incorporated herein by reference to the same extent as if each individual document, patent application, and technical standard were specifically and individually indicated to be incorporated by reference.

Claims

1. A phase image acquisition method comprising the steps of: acquiring a hologram image of an object placed in a recess of a container having a curved bottom surface; generating a phase image from the hologram image; and subtracting a phase component derived from a polynomial that represents the shape of the bottom surface of the recess from the phase image, thereby obtaining a processed phase image.

2. The phase image acquisition method according to claim 1, wherein the degree of the polynomial is between 2 and 7.

3. The phase image acquisition method according to claim 2, wherein the change in height of the bottom surface of the recess in the phase image is 10 μm or more.

4. The phase image acquisition method according to any one of claims 1 to 3, wherein the object is a cell or a cell aggregate.

5. The phase image acquisition method according to claim 4, wherein the cell or cell aggregate is a fertilized egg of an animal.

6. The phase image acquisition method according to claim 4, wherein a phase image of the cell or cell aggregate cultured in the well is acquired.

7. A method for acquiring quantitative data using the phase image acquisition method according to claim 4, wherein quantitative data indicating the state of the cell or cell aggregate is derived based on the processed phase image.