Holography device and cell evaluation method

Abstract

A holography device 10 has an optical system 200 that forms an interference image resulting from interference between objective light L1 and reference light L2. The optical system 200 includes: a phase element 30 disposed on an optical path for the objective light or the reference light; and a rotational mechanism 31 that causes the phase element 30 to rotate such that the optical axis of the objective light L1 or reference light L2 serves as the axis of rotation. The holography device 10 includes an image-capture unit 40 that captures the interference image; and a control unit 50 that derives an evaluation value for the interference image captured by the image-capture unit 40, and controls the rotation position of the rotational mechanism 31 on the basis of the evaluation value.

Description

[Technical Field]

[0001] The disclosed technology relates to a holography device and a method for evaluating cells using the holography device. [Background technology]

[0002] The following techniques are known as techniques relating to cell evaluation methods using digital holography technology. For example, International Publication No. 2019 / 176427 describes a method for generating a phase-contrast image of a cell from a hologram capturing an image of a cell that is an aggregate of multiple cells, and determining the state of the cell based on the phase-contrast image and a shape index value corresponding to the shape of the cell.

[0003] Furthermore, the following technique is known as a technique for avoiding a decrease in the coherence between the object beam and the reference beam. For example, Japanese Patent Application Laid-Open No. 2003-15509 describes an image exposure recording device comprising: an exposure / recording means for irradiating a hologram recording medium with laser beams as object beams and reference beams to expose and record a hologram image; a polarization state detection means for detecting the polarization state of the laser beam that has passed through the hologram recording medium; and a polarization state varying means for varying the polarization state of the laser beam incident on the hologram recording medium based on the detection result by the polarization state detection means so as to maximize the coherence between the object beam and the reference beam on the recording layer of the hologram recording medium. The polarization state detection means uses a polarizing plate, which is an optical element, and a photodetector, which is an intensity detection means. Summary of the Invention [Problem to be solved by the invention]

[0004] In cell evaluation using digital holography technology, for example, object light is irradiated onto cells cultured on a light-transmitting substrate such as a petri dish. An interference image (hologram) is generated by the interference between the object light that has passed through the cells and the substrate and a reference light that is coherent with the object light, and the image is captured by an imaging device. Numerical calculations based on light propagation are performed on the image data of the interference image to reconstruct the wavefront of the light wave that has passed through the cells. Digital holography technology makes it possible to obtain three-dimensional information about cells.

[0005] The inventors have discovered that the contrast of an interference image acquired using holography technology decreases depending on the position on the substrate, i.e., the contrast of the interference image varies greatly. Low contrast in an interference image means that the difference in brightness between the interference fringes is small. If the decrease in contrast is due to the optical system, the interference image cannot be said to accurately represent cell information, and it is considered inappropriate to evaluate cells based on such an interference image. In particular, the increase in measurement error is a major problem when evaluating cells, such as embryonic cells, which require individual evaluation.

[0006] After extensive research, the inventors discovered that the variation in contrast of the interference image is due to birefringence of a substrate, such as a petri dish, on which cells are cultured. As shown in FIG. 1 , the interference image is formed by combining an object beam L1 that has passed through a cell 60 and a substrate 61 with a reference beam L2 that is coherent with the object beam. To cause interference between the object beam L1 and the reference beam L2, the object beam L1 and the reference beam L2 must each be linearly polarized and have the same polarization direction (polarization axis). However, when the object beam L1 passes through the substrate 61, birefringence occurs, causing the object beam to become elliptically polarized. This causes the polarization axis of the object beam L1 to rotate, causing the polarization direction of the object beam L1 and the polarization direction of the reference beam L2 to become misaligned. As a result, the coherence between the object beam L1 and the reference beam L2 decreases, resulting in a decrease in the contrast of the interference image. Because the magnitude of birefringence varies depending on the position on the substrate 61, the contrast of the interference image changes depending on the irradiation position of the object beam L1 on the substrate 61.

[0007] The disclosed technology has been made in consideration of the above points, and aims to suppress variations in contrast of interference images acquired using holography technology. [Means for solving the problem]

[0008] The holography device according to the disclosed technology is an optical system that forms an interference image due to interference between object light and reference light, and includes an optical system including a phase shifter arranged on the optical path of the object light or the reference light, and a rotation mechanism that rotates the phase shifter around the optical axis of the object light or the reference light as the rotation axis; an imaging unit that images the interference image; and a control unit that derives an evaluation value for the interference image imaged by the imaging unit and controls the rotation position of the rotation mechanism based on the evaluation value.

[0009] The evaluation value may be a standard deviation or variance of pixel values ​​of the interference image, and the control unit may control the rotation position of the rotation mechanism so as to maximize the evaluation value. The retarder may be a half-wave plate.

[0010] The optical system may further include a polarizer arranged on the optical path of the object light and an attenuator arranged on the optical path of the reference light for attenuating the amount of light of the reference light. The control unit may control the amount of attenuation in the attenuator so that the difference between the luminance of an image based on the object light captured by the imaging unit and the luminance of an image based on the reference light falls within a predetermined range.

[0011] The cell evaluation method according to the disclosed technology is a cell evaluation method using the above-described holography device, and includes arranging cells cultured on a substrate that is transparent to object light in the optical path of the object light, obtaining an interference image due to interference between the object light that has passed through the substrate and the cells and a reference light, generating a phase image from the interference image, and evaluating the cells using the phase image.

[0012] The phase image is an interference image caused by the interference between the object light and the reference light that have passed through the substrate and the cells, and is preferably generated from an interference image captured while controlling the rotational position of the rotation mechanism so that the standard deviation or variance of the pixel values ​​of the interference image is maximized. [Effects of the Invention]

[0013] According to the disclosed technology, it is possible to suppress variations in contrast of interference images acquired using holography technology. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a diagram showing an example of a process for generating an interference image obtained using holography technology. [Figure 2] FIG. 1 is a diagram illustrating an example of the configuration of a holography device according to an embodiment of the disclosed technology. [Figure 3] 10 is a flowchart illustrating an example of a flow of a process executed by a control unit according to an embodiment of the disclosed technology. [Figure 4] 10 is a graph showing an example of the relationship between the rotational position of a phase shifter according to an embodiment of the disclosed technique and the standard deviation of pixel values ​​of image data of an interference image. [Figure 5] FIG. 1 is a diagram illustrating an example of the configuration of a holography device according to an embodiment of the disclosed technology. [Figure 6] FIG. 1 is a diagram illustrating an example of the configuration of a holography device according to an embodiment of the disclosed technology. [Figure 7] FIG. 1 is a diagram illustrating an example of the configuration of a holography device according to an embodiment of the disclosed technology. [Figure 8] 10 is a flowchart illustrating an example of a flow of a process executed by a control unit according to an embodiment of the disclosed technology. [Figure 9] 10 is a graph showing the results of comparing the standard deviation for each well number corresponding to the imaging position of the interference image between the devices. [Figure 10] 10 is a graph showing the results of comparing the average value, maximum value, and minimum value of the standard deviation of pixel values ​​obtained at 36 locations on a substrate between devices. [Figure 11A] FIG. 10 is a diagram showing an example of an interference image of an aggregate. [Figure 11B] FIG. 10 is a diagram showing an example of a Fourier transform image of an aggregate. [Figure 11C]FIG. 10 is a diagram showing an example of a phase contrast image of an aggregate before unwrapping. [Figure 11D] FIG. 10 shows an example of a phase contrast image of an aggregate after unwrapping. [Figure 12] 1A and 1B are diagrams illustrating the concept of a phase contrast image according to an embodiment of the disclosed technique. [Figure 13] 10 is a graph showing the results of acquiring correlation characteristics between the volume of aggregates and the total phase amount. DETAILED DESCRIPTION OF THE INVENTION

[0015] An example of an embodiment of the present invention will be described below with reference to the drawings. In the drawings, the same or equivalent components and parts are designated by the same reference numerals, and redundant description will be omitted where appropriate.

[0016] [First embodiment] FIG. 2 is a diagram illustrating an example of the configuration of a holography device 10 according to a first embodiment of the disclosed technology. The holography device 10 includes a laser light source 20, an optical system 200, an imaging unit 40, and a control unit 50. The optical system 200 is configured to form an interference image due to the interference between object light L1 and reference light L2, and includes a splitter 21, reflecting mirrors 22 and 24, an objective lens 23, an imaging lens 25, a combiner 26, a retarder 30, and a rotation mechanism 31. The holography device 10 according to this embodiment can be used to evaluate cells. A cell 60 to be evaluated is cultured on a substrate 61 and positioned between the reflecting mirror 22 and the objective lens 23 while either adhering to the substrate 61 or floating above the substrate 61. The type of cell 60 is not particularly limited, and may be, for example, an embryonic cell, for which individual evaluation of each cell is required.

[0017] The laser light source 20 may be, for example, a HeNe laser with a wavelength of 632.8 nm. 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 may be used as the splitter 21. The object light L1 passes through a phase shifter 30 held by a rotation mechanism 31 and 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 a cell 60 on a substrate 61.

[0018] The substrate 61 may be a container commonly used in cell culture, such as a petri dish or a well plate. The substrate 61 is made of a material that is transparent to the object light L1, such as polystyrene. The substrate 61 may be made of plastic other than polystyrene or glass. An image formed by the object light L1 that has passed through the cell 60 and the substrate 61 is enlarged by the objective lens 23. The object light L1 that has passed through the objective lens 23 has its traveling direction bent by the reflecting mirror 24 and enters the beam combiner 26 via the imaging lens 25. Meanwhile, the reference light L2 also enters the beam combiner 26. The object light L1 and the reference light L2 are combined by the beam combiner 26 and an image is formed on the imaging plane of the imaging unit 40. A beam splitter can be used as the beam combiner 26.

[0019] An interference image (hologram) generated by the interference between the object light L1 and the reference light L2 is captured by the imaging unit 40. The imaging unit 40 includes an imaging element such as a CMOS (Complementary Metal-Oxide-Semiconductor) image sensor, and generates image data of the interference image. The image data of the interference image is supplied to the control unit 50.

[0020] The retarder 30 is disposed between the demultiplexer 21 and the reflecting mirror 22 on the optical path of the object light L1. The retarder 30 has the function of changing the polarization direction of the object light L1. That is, the retarder 30 changes the polarization direction of the incident light according to the rotation position of the retarder 30 and outputs the light. The retarder 30 may be, for example, a half-wave plate (λ / 2 plate). The rotation mechanism 31 rotates the retarder 30 around the optical axis of the object light L1 as the rotation axis based on a control signal supplied from the control unit 50. By rotating the retarder 30, it is possible to arbitrarily set the polarization direction of the object light L1 that has passed through the retarder 30.

[0021] The interference image of the object light L1 and the reference light L2 is formed by combining the object light L1 and the reference light L2 that have passed through the cell 60 and the substrate 61. For the object light L1 and the reference light L2 to interfere with each other, the object light L1 and the reference light L2 must each be linearly polarized and have the same polarization direction (polarization axis). However, if the substrate 61 is made of a material that generates birefringence, birefringence occurs when the object light L1 passes through the substrate 61, and the object light L1 becomes elliptically polarized. This causes the polarization axis of the object light L1 to rotate, causing the polarization direction of the object light L1 and the polarization direction of the reference light L2 to become misaligned. As a result, the coherence between the object light L1 and the reference light L2 is reduced, resulting in a decrease in the contrast of the interference image. However, by rotating the retarder 30 to change the polarization direction of the object light L1, the polarization direction of the object light L1 can be aligned with the polarization direction of the reference light L2, thereby suppressing a decrease in the contrast of the interference image.

[0022] The control unit 50 derives an evaluation value for the interference image captured by the imaging unit 40 and controls the rotation position of the rotation mechanism 31 (and the retarder 30) based on the evaluation value. The evaluation value is an index value indicating the contrast of the interference image and may be, for example, the standard deviation or variance of pixel values ​​in the image data of the interference image. The pixel value means a numerical value indicating the brightness of the pixel. The higher the coherence between the object light L1 and the reference light L2, the higher the contrast of the interference image and the larger the standard deviation or variance as the evaluation value. The evaluation value can also be said to be an index value indicating the coherence between the object light L1 and the reference light L2. The control unit 50 controls the rotation position of the rotation mechanism 31 (and the retarder 30) so as to maximize the evaluation value. Specifically, the control unit 50 is configured to include a computer equipped with a processor (not shown) and performs the following processes.

[0023] 3 is a flowchart showing an example of the flow of processing executed by the control unit 50. In step S1, the control unit 50 sets the rotation position of the rotation mechanism 31 (and the phase shifter 30) to an initial position. In step S2, the control unit 50 acquires image data of the interference image from the imaging unit 40. In step S3, the control unit 50 derives an evaluation value for the image data of the interference image acquired in step S2. The control unit 50 derives, for example, the standard deviation of pixel values ​​as the evaluation value and stores it in a memory (not shown).

[0024] In step S4, the control unit 50 determines whether or not the derivation of evaluation values ​​for all predetermined rotation positions has been completed. If the control unit 50 determines that the derivation of evaluation values ​​for all rotation positions has not been completed, the control unit 50 proceeds to step S5. If the control unit 50 determines that the derivation of evaluation values ​​for all rotation positions has been completed, the control unit 50 proceeds to step S6.

[0025] In step S5, the control unit 50 rotates the rotational position of the rotation mechanism 31 (and the retarder 30) by one step. As a result, the retarder 30 rotates by one step (e.g., 5°) around the optical axis of the object light L1 as the rotation axis. Thereafter, the control unit 50 returns the process to step S2. That is, the control unit 50 acquires image data of the interference image in a state in which the rotational position of the retarder 30 has rotated by one step, and derives an evaluation value for the image data. The control unit 50 repeats the processes from step S2 to step S5 until the amount of change in the polarization direction caused by the retarder 30 from the initial position reaches, for example, 180°. If the retarder 30 is a half-wave plate (λ / 2 plate), changing the retarder by 90° from the initial position changes the polarization direction of the object light L1 by 180°.

[0026] In step S6, the control unit 50 supplies a control signal to the rotation mechanism 31, thereby positioning the rotation mechanism 31 (and the phase shifter 30) at a rotation position where the evaluation value stored in the memory is maximized.

[0027] As described above, the holography device 10 according to the embodiment of the disclosed technique includes an optical system 200 that forms an interference image due to the interference between the object light L1 and the reference light L2. The optical system 200 includes a retarder 30 disposed on the optical path of the object light L1 and a rotation mechanism 31 that rotates the retarder 30 around the optical axis of the object light L1. The holography device 10 also includes an imaging unit 40 that captures the interference image and a control unit 50 that derives an evaluation value for the interference image captured by the imaging unit 40 and controls the rotation position of the rotation mechanism 31 (and the retarder 30) based on the evaluation value. The evaluation value used is an index value that indicates the contrast state of the interference image, such as the standard deviation or variance of pixel values ​​in image data of the interference image. The control unit 50 controls the rotation position of the rotation mechanism 31 (and the retarder 30) so as to maximize the evaluation value.

[0028] According to the holography device 10 of the embodiment of the disclosed technique, even in a situation where the contrast of the interference image is reduced due to birefringence in the substrate 61, the rotational position of the rotation mechanism 31 (and the phase shifter 30) is controlled so as to maximize the evaluation value, thereby aligning the polarization direction of the object light L1 with the polarization direction of the reference light L2 and suppressing the reduction in contrast of the interference image. In other words, according to the holography device 10, the influence of birefringence in the substrate 61 can be suppressed. When an interference image is acquired for a cell present at each position on the substrate 61, the rotational position of the rotation mechanism 31 (and the phase shifter 30) is controlled each time, thereby suppressing variations in the contrast of the interference image.

[0029] FIG. 4 is a graph showing an example of the relationship between the rotational position of the retarder 30 and the standard deviation of pixel values ​​of image data of an interference image. FIG. 4 shows the relationship between the case where a substrate 61 is placed (with substrate) and the case where a substrate 61 is not placed (without substrate). As shown in FIG. 4, by placing a substrate 61 that generates birefringence on the optical path of the object beam L1, the standard deviation at a rotational position of the retarder 30 of 0° is reduced compared to the case where the substrate 61 is not placed. However, it was confirmed that the standard deviation increases by changing the rotational position of the retarder 30 and reaches a maximum value at a specific rotational position (around 35°). Furthermore, the maximum value of the standard deviation is the same when the substrate 61 is placed and when the substrate 61 is not placed. This indicates that by controlling the rotational position of the retarder 30, the contrast of the interference image can be improved to an extent that the influence of the birefringence of the substrate 61 can be offset.

[0030] 5 and 6 are diagrams showing an example of the configuration of a holography device 10 according to a modified example. As shown in FIG. 5, it is also possible to arrange the phase shifter 30 and the rotation mechanism 31 on the rear side of the cell 60 and the substrate 61 in the traveling direction of the object light L1. In the example shown in FIG. 5, the phase shifter 30 and the rotation mechanism 31 are arranged between the reflecting mirror 24 and the imaging lens 25. In this case, too, it is possible to suppress variations in the contrast of the interference image. Furthermore, as shown in FIG. 6, the phase shifter 30 and the rotation mechanism 31 may be arranged on the optical path of the reference light L2. In this case, too, it is possible to suppress variations in the contrast of the interference image.

[0031] [Second embodiment] FIG. 7 is a diagram showing an example of the configuration of a holography device 10A according to a second embodiment of the disclosed technology. Holography device 10A according to this embodiment differs from holography device 10 according to the first embodiment (see FIG. 2) in that it further includes a polarizer 27, an attenuator 28, a first shutter 29A, and a second shutter 29B. Polarizer 27 is disposed on the optical path of object light L1. In the example shown in FIG. 7, polarizer 27 is disposed between reflecting mirror 24 and imaging lens 25. Polarizer 27 is a polarizing filter that has the function of removing polarization components unnecessary for forming an interference image, which are contained in elliptically polarized light generated when object light L1 passes through substrate 61.

[0032] The attenuator 28 is disposed on the optical path of the reference light L2. The attenuator 28 has a function of attenuating the reference light L2 by an amount of attenuation corresponding to a control signal supplied from the control unit 50. When the object light L1 passes through the polarizer 27, the amount of light of the object light L1 decreases. If the amount of light of the reference light L2 is not attenuated, the amount of light of the object light L1 and the reference light L2 will be uneven. Therefore, the control unit 50 controls the amount of attenuation in the attenuator 28 so that the average pixel value (average luminance value) of the image data of the image formed only by the object light L1 captured by the imaging unit 40 and the average pixel value (average luminance value) of the image data formed only by the reference light L2 become the same.

[0033] 7, first shutter 29A is disposed between imaging lens 25 and multiplexer 26, but may be disposed anywhere on the optical path of object light L1. First shutter 29A switches between passing and blocking object light L1 in response to a control signal supplied from control unit 50.

[0034] The second shutter 29B is disposed on the optical path of the reference light L2. In the example shown in Fig. 7, the second shutter 29B is disposed between the attenuator 28 and the multiplexer 26, but may be disposed anywhere on the optical path of the reference light L2. The second shutter 29B switches between passing and blocking the reference light L2 in response to a control signal supplied from the control unit 50.

[0035] 8 is a flowchart showing an example of the flow of processing executed by the control unit 50 according to this embodiment. Each processing shown in FIG. 8 is executed after the processing shown in FIG.

[0036] In step S11, the control unit 50 controls the first shutter 29A to be in an open state and the second shutter 29B to be in a closed state. This causes the imaging unit 40 to capture an image formed only by the object light L1. In step S12, the control unit 50 acquires image data of the image formed only by the object light L1 from the imaging unit 40. In step S13, the control unit 50 derives an average pixel value (average luminance value) of the object light image represented by the image data acquired in step S12 and stores this in memory (not shown).

[0037] In step S14, the control unit 50 controls the first shutter 29A to be closed and the second shutter 29B to be open. This causes the imaging unit 40 to capture an image formed only by the reference light L2. In step S15, the control unit 50 acquires image data of the image formed only by the reference light L2 from the imaging unit 40. In step S16, the control unit 50 derives the average pixel value (average luminance value) of the reference light image represented by the image data acquired in step S15 and stores this in memory (not shown).

[0038] In step S17, the control unit 50 determines whether the average pixel value (average luminance value) of the object light image stored in memory matches the average pixel value (average luminance value) of the reference light image. If the control unit 50 determines that the average pixel values ​​(average luminance values) do not match, it proceeds to step S18, and if it determines that the average pixel values ​​(average luminance values) match, it proceeds to step S19.

[0039] In step S18, the control unit 50 increases the attenuation of the attenuator 28 by one step, and returns the process to step S15. That is, the control unit 50 acquires image data for the reference light image in a state in which the attenuation of the attenuator 28 has been increased by one step, stores the image data in memory, and derives the average pixel value (average luminance value). The control unit 50 repeats the processes from step S15 to step S18 until the average pixel value (average luminance value) of the object light image stored in memory matches the average pixel value (average luminance value) of the reference light image. In step S19, the control unit 50 controls the first shutter 29A to an open state, and also controls the second shutter 29B to an open state.

[0040] In holography device 10A according to this embodiment, polarization components unnecessary for forming an interference image, which are contained in the elliptically polarized light generated when object light L1 passes through substrate 61, are removed by polarizer 27. In addition, the attenuation amount of attenuator 28 is controlled so that the light intensity of object light L1 and the light intensity of reference light L2 are equal. This enhances the effects of suppressing a decrease in contrast of the interference image and suppressing variations in contrast of the interference image.

[0041] In this embodiment, the example has been given of controlling the amount of attenuation in the attenuator 28 so that the brightness of the image formed by the object light L1 captured by the imaging unit 40 matches the brightness of the image formed by the reference light L2, but the amount of attenuation in the attenuator 28 may also be controlled so that the difference between the brightness of the image formed by the object light L1 and the brightness of the image formed by the reference light L2 falls within a predetermined range.

[0042] [Example] Interference images were formed using the holography device 10 according to the first embodiment (see FIG. 2), the holography device 10A according to the second embodiment (see FIG. 7), and a holography device according to a comparative example (not shown), and the contrast and variation of the interference images were compared.

[0043] The components of each holography device are shown in Table 1 below. Holography device 10 according to the first embodiment is configured without the polarizer and attenuator of the components shown in Table 1. The holography device according to the comparative example is configured without the phase shifter, rotation mechanism, polarizer, and attenuator of the components shown in Table 1. In other words, the holography device according to the comparative example is configured without a means for correcting fluctuations in the polarization direction of object light caused by birefringence of substrate 61. [Table 1]

[0044] Embryonic cells were cultured in 12 wells on the substrate. Each holography device acquired image data of the embryonic cell interference images for 12 locations on each substrate, for a total of three substrates. In other words, each holography device acquired 36 interference images. The standard deviation of pixel values ​​was calculated for each of the acquired interference image data.

[0045] Fig. 9 is a graph showing the results of comparing the standard deviation for each well number corresponding to the imaging position of the interference image between the devices. Fig. 10 is a graph showing the results of comparing the average, maximum, and minimum values ​​of the standard deviation for 36 points between the devices. In Figs. 9 and 10, Example 1 corresponds to holography device 10 according to the first embodiment, Example 2 corresponds to holography device 10A according to the second embodiment, and Comparative Example corresponds to the holography device according to the comparative example.

[0046] 9 and 10, it was confirmed that the standard deviation of the pixel values ​​of the interference image increases and the variation in the standard deviation decreases when the holography device is provided with a retarder as a means for correcting the variation in the polarization direction of the object light caused by the birefringence of the substrate 61. This indicates that correction of the polarization direction by the retarder suppresses the decrease in contrast of the interference image caused by the birefringence of the substrate 61 and suppresses the variation in contrast.

[0047] [Cell evaluation method] A cell evaluation method according to an embodiment of the disclosed technique will be described below. The cell evaluation method according to this embodiment uses the holography device 10 according to the first embodiment or the holography device 10A according to the second embodiment.

[0048] The cell evaluation method according to this embodiment includes placing cells 60 cultured on a substrate 61 that is transparent to object light L1 in the optical path of the object light L1, obtaining an interference image between the object light L1 that has passed through the substrate 61 and the cells 60 and a reference light L2, generating a phase image from the interference image, and evaluating the cells using the phase image.

[0049] An example of a method for acquiring a phase image from an interference image will be described below with reference to Figures 11A to 11D. Figures 11A to 11D were acquired for an aggregate (sphere) of iPS cells (induced pluripotent stem cells).

[0050] First, the interference image (hologram) of the cell shown in FIG. 11A obtained by the imaging unit 40 is trimmed to a size of, for example, 2048 × 2048, and then subjected to a two-dimensional Fourier transform. FIG. 11B shows an example of a Fourier transform image of the cell obtained by this process. FIG. 11B shows images based on direct light, object light, and conjugate light.

[0051] 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 aperture of a radius of 250 pixels.

[0052] Next, for example, an angular spectrum method is applied to restore an image showing the phase of a cell at an arbitrary spatial position. Specifically, the angular spectrum U(f x ,f y ;0) is calculated. 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)).

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[0053] Next, as shown in the following equation (2), the wavefront U(f x ,f y By performing an inverse Fourier transform on x, y; z, the solution u(x, y; z) at position z is derived.

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[0054] Next, a phase image is generated by deriving the phase φ for u(x, y; z) as shown in the following equation (3): Figure 11C shows an example of a phase image before unwrapping obtained by the above processes.

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[0055] The phase in the phase difference image before unwrapping shown in Figure 11C is convoluted to values ​​between 0 and 2π. Therefore, by applying a phase unwrapping (unwrapping) method such as Unweighted Least Squares or Flynn's Algorithm to join the parts greater than 2π, a final phase image such as the one shown in Figure 11D can be obtained. Note that many unwrapping methods have been proposed, and it is sufficient to select an appropriate one that does not cause phase mismatch.

[0056] The phase image will be explained below. Figure 12 shows the phase image I P The lower part of Fig. 12 shows the concept of phase image I P The upper part of Fig. 12 shows the phase image I P The phase amount at each pixel k is shown on a plane in gray scale.

[0057] where the phase image I P The phase of the background (area without cells) present in the same focal plane is P B The phase of the region where the cells exist is P SThen, the phase image I P The phase amount P in is expressed by the following equation (4): Furthermore, 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.

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[0058] Phase Image I P The phase amount P at each pixel k k can be expressed by the following equation (5): k is the phase image I P is the refractive index of the cell at the site corresponding to each pixel k, and d k is the phase image I P is the thickness of the cell at the site corresponding to each pixel k, and λ is the wavelength of the object light in the hologram optical system.

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[0059] A phase image of a cell is an image that shows the optical path length distribution of the object light that has passed through the cell. Since the optical path length within the cell is equivalent to the product of the refractive index and thickness of the cell, the phase image of a cell contains information on the refractive index and thickness (shape) of the cell, as shown in equation (5).

[0060] An example of a method for evaluating cells using phase images is the total phase amount P A The total phase amount P A is expressed by the following equation (6): where s is the area of ​​each pixel k in the phase image, and v k is the volume of the cell at the location corresponding to each pixel k in the phase image. As shown in equation (6), the total phase amount P A is the phase amount P for each pixel of the phase image of the cell k The pixel value of the phase image corresponds to the sum of the phase amount P k It corresponds to.

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[0061] Figure 13 shows the relationship between the volume of iPS cell aggregates (spheres) and the total phase volume P A 13 is a graph showing the correlation characteristics of the total phase amount P A It was confirmed that the volume of the aggregate is proportional to the total phase amount P A A trend line L showing the measure of correlation between S The regression line derived from each plot shown in Figure 13 is shown as the trend line L S was applied as.

[0062] Figure 13 shows the trend line L S The phase images of the aggregates corresponding to the plots a1 and a2 above, and the trend line L S The phase images of the aggregates corresponding to plots a3, a4, and a5, which are located at positions deviated from the trend line L, are shown. S For the aggregates corresponding to plots a1 and a2 on the top, phase images with uniform brightness were obtained throughout the aggregate. This indicates that the multiple cells that make up the aggregate are homogeneous, and that the density of cells within the aggregate is uniform. On the other hand, the trend line L S For the aggregates corresponding to plots a3 and a4, which are located at positions away from the trend line L, a phase image was obtained in which the brightness of the center was lower than that of other areas. This indicates that the multiple cells that make up the aggregate are heterogeneous, and that the density of cells within the aggregate is non-uniform. S For the aggregate corresponding to plot a5, which is located at a position away from the center, a phase contrast image was obtained in which the contour line of the aggregate was noticeably uneven. This indicates that abnormalities have occurred in the cells that make up the aggregate.

[0063] From the above results, the total phase amount P AIt can be said that it is possible to judge the state of the aggregates by using the correlation between the total phase amount P A The trend line L showing the correlation between the volume of aggregates and S and the total phase amount P for the aggregate to be determined A The correlation between the volume of the aggregate and the concentration of the aggregate is compared, and the trend line L S It can be said that it is possible to judge the state of the aggregate depending on the degree of deviation from the value of the total phase amount P. Specifically, the volume of the aggregate is plotted on one axis, and the total phase amount P is plotted on the other axis. A The trend line L of the plot for the aggregate to be judged plotted on the graph S Therefore, for example, the total phase amount P A Trend line L S For aggregates whose negative width from is greater than or equal to a threshold, it can be determined that there is an abnormality in at least one of the density, homogeneity, and external shape of the spheres of the multiple cells contained in the aggregate.

[0064] In evaluating cells using phase images, it is preferable to use phase images generated from high-contrast interference images, i.e., phase images generated from interference images captured while controlling the rotational position of the rotation mechanism 31 (and the phase shifter 30) so that the standard deviation or variance of pixel values ​​in the interference image is maximized.

[0065] The disclosure of Japanese Patent Application No. 2021-066769, filed on April 9, 2021, 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 was specifically and individually indicated to be incorporated by reference.

Claims

1. an optical system that forms an interference image due to interference between an object beam and a reference beam, the optical system including: a phase shifter disposed on an optical path of the object beam or the reference beam; and a rotation mechanism that rotates the phase shifter around an optical axis of the object beam or the reference beam as a rotation axis; an imaging unit that captures the interference image; a control unit that derives an evaluation value for the interference image captured by the imaging unit and controls a rotation position of the rotation mechanism based on the evaluation value; 1. A holographic device comprising:

2. the evaluation value is a standard deviation or a variance of pixel values ​​of the interference image, The control unit controls the rotation position of the rotation mechanism so as to maximize the evaluation value.

10. The holographic device of claim 1.

3. The retarder is a half-wave plate 10. The holographic device of claim 1.

4. The optical system comprises: a polarizer disposed on the optical path of the object beam; an attenuator disposed on an optical path of the reference light and configured to attenuate the amount of the reference light; Further includes 4. The holographic device according to claim 1.

5. The control unit controls the amount of attenuation in the attenuator so that the difference in luminance between the image formed by the object light and the image formed by the reference light captured by the imaging unit falls within a predetermined range.

5. The holographic device of claim 4.

6. A method for evaluating a cell using the holography device according to any one of claims 1 to 5, comprising: Cells cultured on a substrate that is transparent to the object light are placed on an optical path of the object light, and an interference image is obtained by interference between the object light that has passed through the substrate and the cells and a reference light; generating a phase image from the interference image; Evaluating the cells using the phase images Evaluation method.

7. The phase image is an interference image caused by interference between the object light and the reference light that have passed through the substrate and the cells, and is generated from an interference image captured while controlling the rotation position of the rotation mechanism so that the standard deviation or variance of pixel values ​​of the interference image is maximized. The evaluation method according to claim 6.

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