Imaging device and imaging method

Abstract

This imaging device (10) comprises a substrate (1), a first optical system (2), and an imaging unit (3). A subject (110) is placed on the substrate (1). The first optical system (2) irradiates the subject (110) using a first emitted light (L1). The imaging unit (3) images an interferogram of a first reflected light (L2) and a second reflected light (L3). The first reflected light (L2) is obtained by the first emission light (L1) being reflected by a first interface (F1) conforming to the outer surface of the subject (110). The second reflected light (L3) is obtained by the first emission light being reflected by a second interface (F2) between the subject (110) and the substrate (1).

Description

Imaging device and imaging method

[0001] The present invention relates to an imaging device and an imaging method.

[0002] In research into cell biology, imaging devices are used to observe cell shapes using interference images obtained by light reflected from the cell surface (see, for example, Non-Patent Document 1). The imaging device is required to accurately grasp the three-dimensional shape of the object.

[0003] T. Yamauchi et.al., “Low coherent quantitative phase microscope for nanometer scale measurement of living cell morphology.”, Optics Express 2008, Vol.16, No.16, 12227-12238.

[0004] One aspect of the present invention is an imaging device comprising: a substrate on which an object is placed; a first optical system that irradiates first emitted light onto the object; and an imaging unit that captures an interference image between first reflected light of the first emitted light reflected at a first interface corresponding to an outer surface of the object, and second reflected light of the first emitted light reflected at a second interface between the object and the substrate.

[0005] One aspect of the present invention is an imaging method including placing an object on a substrate, irradiating the object with first emitted light, and capturing an interference image between first reflected light, which is the first emitted light reflected at a first interface corresponding to an outer surface of the object, and second reflected light, which is the first emitted light reflected at a second interface between the object and the substrate.

[0006] 12 is a schematic diagram showing an imaging device of an embodiment. FIG. 13 is a schematic diagram showing the structure of a gas supply unit. FIG. 14 is a schematic diagram showing a gas supply unit and a substrate. FIG. 15 is a schematic diagram showing an enlarged view of a substrate and an object. FIG. 16 is a schematic diagram showing a further enlarged view of a substrate and an object. FIG. 17 is an example of an interference image. FIG. 18 is a diagram showing the brightness of an interference image along line A-A' in FIG. 6. FIG. 19 is an example of an interference image including a transmission image. FIG. 19 is an enlarged view of FIG. 20. FIG. 21 is an explanatory diagram of an imaging method of an embodiment. FIG. 22 is an example of an interference image. FIG. 23 is a diagram showing the change over time in brightness along line A-A' in FIG. 22. FIG. 24 is a diagram showing the change over time in brightness at the center of the interference fringes along line B-B' in FIG. 23. FIG. 25 is a brightness profile along line C-C' in FIG. 23.

[0007] Hereinafter, embodiments of the present invention will be described in detail, with reference to the drawings where necessary. In the drawings, identical or corresponding parts are designated by identical or corresponding reference numerals, and redundant explanations will be omitted. The dimensional ratios in the drawings may be exaggerated for the purpose of explanation, and do not necessarily correspond to the actual dimensional ratios.

[0008] [Imaging Device] Fig. 1 is a schematic diagram showing an imaging device 10 of an embodiment. Fig. 2 is a schematic diagram showing the structure of a gas supply unit 4. Fig. 2 shows a cross-sectional view along the central axis of a hollow member 41 and a cross-sectional view taken along line II perpendicular to the central axis. Fig. 3 is a schematic diagram showing the hollow member 41 and the substrate 1. Fig. 4 is a schematic diagram showing an enlarged view of the bottom plate 11 of the substrate 1 and the object 110. Fig. 5 is a schematic diagram showing an even further enlarged view of the bottom plate 11 and the object 110. "Planar view" refers to a view from a direction perpendicular to the top surface 11a of the bottom plate 11.

[0009] As shown in FIG. 1 , the imaging device 10 of the embodiment includes a substrate 1, a first optical system 2, an imaging unit 3, a gas supply unit (substance supply unit) 4, a second optical system 5, and a stage 6.

[0010] As shown in Fig. 3, the substrate 1 includes a bottom plate 11 and side plates 12. The side plates 12 rise from the periphery of the bottom plate 11. The substrate 1 can store a liquid 100 in an internal space 1a defined by the bottom plate 11 and the side plates 12. The top surface 11a of the bottom plate 11 is formed flat. The substrate 1 can hold the liquid 100 and an object 110.

[0011] 3 includes a bottom plate 11 and side plates 12, the structure of the substrate is not limited to this. For example, a flat substrate consisting of only the bottom plate 11 may be used as long as it can hold a droplet and an object.

[0012] The bottom plate 11 is capable of transmitting the first output light L1 from the first optical system 2 (see FIG. 4). The difference in refractive index between the bottom plate 11 and the object 110 may be 0.1 or more. When the refractive index of the bottom plate 11 is within this range, the reflectance at the interface (second interface F2) between the bottom plate 11 and the object 110 can be increased. Either the refractive index of the bottom plate 11 or the refractive index of the object 110 may be higher. The refractive index of the bottom plate 11 may be, for example, 1.45 to 2.1. JIS K7142:2014 is an example of a method for measuring the refractive index.

[0013] Examples of materials for the substrate 1 include polystyrene, polycarbonate, polymethyl methacrylate (PMMA), polyethylene, polypropylene, poly-4-methylpentene-1, and glass. The refractive index of polystyrene is approximately 1.5. The refractive index of polycarbonate is approximately 1.6. The refractive index of PMMA is approximately 1.5. The refractive index of polyethylene and polypropylene is approximately 1.5. The refractive index of poly-4-methylpentene-1 is approximately 1.46. The refractive index of glass is, for example, 1.4 to 2.1.

[0014] The upper surface 11 a of the substrate 1 may be subjected to a surface treatment to enhance cell adhesion. Examples of the surface treatment include physical hydrophilization treatment such as plasma treatment, and application of a cell adhesive substance such as gelatin, fibronectin, collagen, or extracellular matrix.

[0015] There is no particular limitation on the shape of the substrate 1. The substrate 1 may be, for example, a petri dish; a well plate such as a 6-well, 12-well, 24-well, 48-well, or 96-well plate; a fluidic device; or a slide glass.

[0016] As shown in FIG. 1, the first optical system 2 includes a first light source 21, a bandpass filter 22, an aperture stop 23, a half mirror 24, an objective lens 25, and an aperture control unit 26.

[0017] The first light source 21 may be, for example, a mercury lamp, a halogen lamp, or a laser light source. The wavelength of the first emitted light L1 from the first light source 21 may include the near-infrared region. This makes it possible to suppress light absorption and scattering in the object 110 even when a substance that absorbs or scatters light in the visible light region is present inside the object 110. This reduces noise that affects the interference image. The wavelength of the near-infrared region is, for example, 650 nm to 1.4 μm.

[0018] The wavelength of the first output light L1 may include wavelengths equal to or greater than 650 nm. For example, if the object 110 is a cell, the first reflected light L2 can be obtained without being affected by intracellular substances (such as organelles) that absorb or scatter light in the visible light range. This allows for an interference image with less noise to be obtained. The wavelength of the first output light L1 may be, for example, equal to or less than 1.7 μm.

[0019] The wavelength width of the first outgoing light L1 may be 50 nm or less. By setting the wavelength width within this range, the coherence length can be increased. Because interference between the first reflected light and the second reflected light occurs within the range of the coherence length, the coherence length essentially becomes the upper limit of the measurable thickness of the object 110. By setting the wavelength width within the above range, the coherence length can be increased, so that a good interference image can be obtained even when the object 110 is thick. The wavelength width of the first outgoing light L1 may be, for example, 0.1 nm or more.

[0020] The wavelength width of the first output light L1 may be 1 / 10 or less of the wavelength of the first output light L1. Since the coherence length is determined by the wavelength and the wavelength width, it is preferable to appropriately determine the wavelength width relative to the wavelength. By making the wavelength width of the first output light L1 1 / 10 or less of the wavelength, a good interference image can be obtained even if the object 110 is thick. The wavelength width of the first output light L1 may be 1 / 1000 or more of the wavelength.

[0021] The first outgoing light L1 may include multiple wavelengths (i.e., multiple wavelength components) with a wavelength difference of 20 nm or less. This increases the effective central wavelength. If the thickness of the object 110 is contained within one interference fringe, information about the height of the outer surface of the object 110 can be obtained from the brightness of the interference image. Since information about the height of the outer surface of the object 110 can be obtained without analyzing complex interference fringes, it is possible to easily and accurately grasp the shape of the object 110. The first outgoing light L1 may include multiple wavelengths with a wavelength difference of 1 nm or more.

[0022] The bandpass filter 22 selectively transmits light having predetermined characteristics (for example, wavelength, wavelength width, etc.).

[0023] The half mirror 24 reflects the first emitted light L1 from the first light source 21 toward the substrate 1. The half mirror 24 transmits the first reflected light L2 and the second reflected light L3 toward the imaging unit 3.

[0024] The aperture stop 23 adjusts the diameter of the light beam of the first output light L1, thereby adjusting the numerical aperture of the illumination by the first light source 21. The aperture control unit 26 controls the diameter of the aperture stop 23. The aperture control unit 26 can reduce the numerical aperture of the illumination by, for example, reducing the diameter of the aperture stop 23. This allows an interference image with clear interference fringes to be obtained. On the other hand, since the illumination light illuminates the object 110 through the objective lens 25, if the numerical aperture of the objective lens 25 is smaller than the numerical aperture of the illumination, the numerical aperture of the objective lens 25 becomes the final numerical aperture of the illumination. Therefore, the numerical aperture of the illumination controlled by the aperture stop 23 may be equal to or less than the numerical aperture of the objective lens 25. The numerical aperture of the objective lens 25 may be 0.7 or less. Furthermore, the imaging numerical aperture is determined only by the numerical aperture of the objective lens 25. The imaging numerical aperture may be 0.7 or less.

[0025] The first optical system 2 can irradiate the first emitted light L1 from the first light source 21 onto the object 110 from below the substrate 1 through the bottom plate 11 (see FIG. 4).

[0026] The imaging unit 3 includes a detection unit 31 and an information processing unit 32. The detection unit 31 includes an imaging element such as a CCD or CMOS. The detection unit 31 captures an image of the target object 110. The imaging element may be an area sensor, a linear sensor, or a point sensor. A detection signal corresponding to the detected light is appropriately A / D converted by an A / D converter or the like and output to the information processing unit 32.

[0027] The information processing unit 32 processes information based on the detection signal from the detection unit 31 .

[0028] As shown in FIG. 2 , the gas supply unit 4 includes a hollow member 41 and a control unit 42. The hollow member 41 is formed, for example, in a circular pipe (or cylindrical) shape. The hollow member 41 has a flow path 43 through which the gas 40 flows. The hollow member 41 can release the gas 40 from the tip 41 a by feeding the gas 40 into the flow path 43. When the tip 41 a is in the liquid 100, bubbles 44 are formed at the tip 41 a (see FIG. 3 ). The gas 40 is an example of a “substance.” The gas 40 is, for example, air. The refractive index of the gas 40 may be 0.1 or more lower than that of the target object 110. The gas supply unit 4 can maintain the bubbles 44 by closing the flow path within the gas supply unit 4 or the flow path 43 to prevent the gas 40 from flowing back into the hollow member 41. The bubble 44 may be maintained by adjusting the amount of gas 40 drawn in or supplied from the gas supply unit 4, thereby adjusting at least one of the pressure and volume of the bubble 44.

[0029] The hollow member 41 may be configured to be movable in a direction perpendicular to the central axis of the hollow member 41 (horizontal direction) by a driving unit (not shown) such as a motor.

[0030] The control unit 42 may include, for example, a pump that supplies the gas 40. The control unit 42 can control the supply amount of the gas 40. Therefore, the control unit 42 can adjust the volume of the bubbles 44 (see FIG. 3).

[0031] The control unit 42 may include, for example, a pressure sensor that detects the pressure of the gas 40 (the pressure inside the flow path 43). The control unit 42 can control the pressure of the gas 40 by controlling the supply amount of the gas 40 using a pump that supplies the gas 40. Therefore, the control unit 42 can adjust the pressure of the bubbles 44 (see FIG. 3).

[0032] 1, the second optical system 5 includes a second light source 51 and a condenser lens 52. The second optical system 5 can irradiate the object 110 with the second outgoing light L4 from above.

[0033] The substrate 1 can be placed on the upper surface of the stage 6. The stage 6 can be moved in a direction perpendicular to the central axis of the hollow member 41 (horizontal direction) by a driving unit (not shown) such as a motor.

[0034] [Imaging Method] (First Embodiment) As shown in Fig. 3, an object 110 and a liquid 100 are accommodated in the internal space 1a of a substrate 1. The liquid 100 is, for example, water. The refractive index of water is approximately 1.33. The liquid 100 may be a liquid culture medium for culturing the object 110. The refractive index of the liquid culture medium is also approximately 1.33. The object 110 is placed on the substrate 1 (specifically, on the bottom plate 11).

[0035] The object 110 is placed in the liquid 100. That is, the object 110 is immersed in the liquid 100. The object 110 is, for example, a cell. The cell may be an animal cell, a plant cell, an insect cell, a bacterium, a yeast, or the like. The cell may be an adherent cell or a floating cell. Even if the cell is a floating cell, it can be imaged by leaving the substrate 1 stationary and allowing the cell to settle until it contacts the bottom plate 11. The refractive index of the cell is, for example, approximately 1.35 to 1.60.

[0036] The object 110 is not particularly limited, but a large difference in refractive index at the interface when light passes through the object 110 is preferable because it increases the reflectivity and produces a clear interference image. An object that has a small absorption coefficient but a refractive index distribution is called a phase object. In a phase object, a large difference in refractive index at the interface increases the reflectivity and produces clear interference fringes.

[0037] The hollow member 41 of the gas supply unit 4 is disposed so that the tip 41a is located in the liquid 100. The portion of the hollow member 41 including the tip 41a is immersed in the liquid 100. A control unit 42 (see FIG. 2 ) supplies gas 40 to a flow path 43 of the hollow member 41, forming bubbles 44 in the tip 41a. The refractive index of the gas 40 is different from that of the liquid 100.

[0038] As shown in Figures 4 and 5, the object 110 is in planar contact with the upper surface 11a of the bottom plate 11. More specifically, as shown in Figure 5, the entire area of ​​the lower surface 110b of the object 110 is in contact with the upper surface 11a of the bottom plate 11. In Figure 5, the object 110 has a bow shape formed by a straight line forming the lower surface 110b and an arc forming the upper surface 110a. The upper surface 110a of the object 110 is the outer surface of the object 110. The interface between the bottom plate 11 and the object 110 is a second interface F2.

[0039] When the hollow member 41 is brought closer to the object 110, the bubbles 44 cover the object 110. In a plan view, the bubbles 44 surround the object 110. At this time, part of the liquid 100 that was in contact with the object 110 is pushed outward by the bubbles 44.

[0040] A portion of the liquid 100 remains in a portion including the peripheral edge of the object 110. This remaining liquid 100 is referred to as a residual liquid 101. The residual liquid 101 forms a layer that gradually becomes thinner toward the outside in the radial direction of the object 110.

[0041] The remaining liquid 101 may also be present in the form of a thin layer in the central portion of the upper surface 110a of the object 110 in a planar view. In this case, the remaining liquid 101 is present spreading over the entire circular region centered on the object 110 in a planar view. The remaining liquid 101 may also remain only in an annular portion including the peripheral portion of the object 110. In this case, the remaining liquid 101 forms an annular shape surrounding the central portion of the object 110 in a planar view.

[0042] When the remaining liquid 101 exists spreading over the entire circular region centered on the object 110, the interface between the remaining liquid 101 and the gas 40 is the first interface F1. When the remaining liquid 101 exists in a ring shape, the first interface F1 is formed by the ring-shaped interface between the remaining liquid 101 and the gas 40 and the circular interface between the center of the object 110 and the gas 40. At least a portion of the first interface F1 is formed between the remaining liquid 101 and the gas 40 (a gas having a refractive index different from that of the remaining liquid 101).

[0043] Because the remaining liquid 101 is flowable and has surface tension, the upper surface 101a of the remaining liquid 101 has a smooth shape that conforms to the upper surface (outer surface) 110a of the object 110. Therefore, the first interface F1 has a smooth shape that conforms to the upper surface (outer surface) 110a of the object 110.

[0044] With the first interface F1 formed by the gas bubble 44, the control unit 42 can adjust the supply amount of gas 40 as needed, thereby adjusting the pressure of the gas 40 in the gas bubble 44. If the pressure of the gas 40 is too high, the object 110 (cell) may be deformed, but the control unit 42 can accurately grasp the three-dimensional shape of the object 110 by controlling the supply amount of gas 40 so that the deformation of the object 110 does not become too great.

[0045] With the first interface F1 formed by the bubble 44, the stage 6 (see FIG. 1 ) may be moved, as necessary, in a direction perpendicular to the central axis of the hollow member 41 (horizontally). This makes it possible to adjust the position of the tip 41 a relative to the object 110, thereby adjusting the shape of the first interface F1.

[0046] With the first interface F1 formed by the bubble 44, the hollow member 41 may be moved, as necessary, in a direction perpendicular to the central axis of the hollow member 41 (horizontally). This makes it possible to adjust the position of the tip portion 41a relative to the object 110, thereby adjusting the shape of the first interface F1.

[0047] As shown in Fig. 1, a first light source 21 emits a first output light L1. The first output light L1 is incident on a bandpass filter 22. The bandpass filter 22 selectively transmits light with predetermined characteristics. The diameter of the light beam of the first output light L1 that has passed through the bandpass filter 22 is adjusted by an aperture stop 23, and then the first output light L1 reaches a half mirror 24. The first output light L1 is reflected by the half mirror 24 and travels upward, passing through an objective lens 25 and incident on the bottom plate 11 of the substrate 1.

[0048] As shown in FIG. 4 , the first outgoing light L1 enters the bottom plate 11 from the lower surface 11b, passes upward through the bottom plate 11, and reaches the second interface F2. As shown in FIG. 5 , the refractive index of the bottom plate 11 (e.g., approximately 1.5) is different from that of the object 110 (e.g., approximately 1.35). Therefore, a portion of the first outgoing light L1 is reflected at the second interface F2. This reflected light is referred to as "second reflected light L3." As shown in FIG. 1 , the second reflected light L3 travels downward, passes through the objective lens 25 and the half mirror 24, and reaches the detection unit 31 of the imaging unit 3.

[0049] As shown in Figure 5, the first outgoing light L1 (see Figure 4) that is not reflected at the second interface F2 travels upward and reaches the first interface F1. The refractive index of the remaining liquid 101 (refractive index: e.g., approximately 1.33) or the object 110 (refractive index: e.g., approximately 1.35) is different from that of the gas 40 (refractive index: e.g., 1). Therefore, a portion of the first outgoing light L1 is reflected at the first interface F1. This reflected light is referred to as "first reflected light L2."

[0050] 1, the first reflected light L2 travels downward and again passes through the object 110 or the remaining liquid 101. The first reflected light L2 further travels downward through the bottom plate 11, passes through the objective lens 25 and the half mirror 24, and reaches the detection unit 31 of the imaging unit 3.

[0051] The first reflected light L2 and the second reflected light L3 are incident on the detection unit 31. Therefore, the imaging unit 3 captures an interference image based on the phase difference between the first reflected light L2 and the second reflected light L3. The detection unit 31 sends data on the obtained interference image to the information processing unit 32. The information processing unit 32 can obtain a three-dimensional image of the object 110 based on the interference image.

[0052] Fig. 6 is an example of an interference image. Fig. 7 is a diagram showing the brightness of the interference image along line A-A' in Fig. 6. The shape (height) of the first interface F1 can be calculated based on the brightness shown in Fig. 7. In this way, the three-dimensional shape of the object 110 can be grasped based on the interference image.

[0053] As shown in FIG. 5 , there is residual liquid 101 (liquid between the first interface F1 and the second interface F2) near the periphery of the object 110, and therefore interference fringes are observed in an area wider than the object 110 in the interference image (see FIG. 6 ). In order to grasp the shape of the object 110 in a planar view, heat may be generated by irradiation with the first outgoing light L1, causing at least a portion of the residual liquid 101 to evaporate. As a result, the first interface F1 assumes a shape that conforms to the outer surface of the object 110, and an interference image that conforms to the object 110 is obtained. This makes it possible to accurately grasp the shape of the object 110.

[0054] The imaging device 10 of the first embodiment includes an imaging unit 3 that captures an interference image between a first reflected light L2 at a first interface F1 and a second reflected light L3 at a second interface F2. The first interface F1 is the interface between the remaining liquid 101 (or the object 110) and the gas 40. The first interface F1 is the interface between two materials with a large difference in refractive index, and therefore has a high reflectance. As a result, a good interference image with clear interference fringes can be obtained. This allows the three-dimensional shape of the object 110 to be accurately grasped.

[0055] When the object 110 is a cell, intracellular organelles and the like are present within the cell. The intracellular organelles and the like have a higher refractive index than the cell nucleus, cytoplasm, and the like (e.g., mitochondria: approximately 1.4, lysosomes: 1.6). Therefore, the difference in refractive index between the intracellular organelles and the cell nucleus and the like causes irregular reflection within the cell, which can become a noise component in the interference fringes. Meanwhile, the first interface F1 has a very large refractive index difference between the cell and the substance (gas). Therefore, the reflectivity of the first interface F1 is high, and the reflected light from the first interface F1 becomes dominant, making it possible to ignore the influence of the reflected light from the intracellular organelles and the like. Therefore, good interference fringes are obtained. Therefore, the three-dimensional shape of the object 110 can be accurately grasped.

[0056] In contrast, when the object 110 is placed in water, the reflectance of the first interface (the interface between the water and the object 110) tends to be low because the difference in refractive index between the water and the object 110 is small. As a result, the reflected light at the first interface becomes weak, and the interference image may become unclear.

[0057] In the imaging method of the first embodiment, the first interface F1 is formed with high reflectivity and is less likely to cause disturbance of the plane wave, thereby obtaining high-quality first reflected light L2. As a result, a clear interference image of the first reflected light L2 and the second reflected light L3 is obtained. Therefore, the three-dimensional shape of the object 110 can be grasped with high accuracy.

[0058] 1 , in the imaging method of the second embodiment, similarly to the imaging method of the first embodiment, the substrate 1 and the object 110 are irradiated with first emitted light L1 from the first light source 21. The first reflected light L2 and the second reflected light L3 are incident on the imaging unit 3.

[0059] The imaging method of the second embodiment uses a second optical system 5. A second light source 51 of the second optical system 5 emits a second outgoing light L4. The second outgoing light L4 passes downward through the object 110 and the bottom plate 11. The second outgoing light L4 passes through the objective lens 25 and the half mirror 24 and enters the detection unit 31 of the imaging unit 3. In addition to the interference image, the imaging unit 3 obtains a transmission image of the object 110 by the second outgoing light L4. The transmission image is superimposed on the interference image.

[0060] The imaging method of the second embodiment obtains an interference image that includes information from the transmission image. Therefore, an interference image that includes information about the outer shape of the object 110 can be obtained. FIG. 8 is an example of an interference image that includes a transmission image. FIG. 9 is an enlarged view of FIG. 8. As shown in FIGS. 8 and 9, a transmission image can be seen inside the interference image that has interference fringes.

[0061] 4 and 5 , in the imaging method of the third embodiment, a hollow member 41 is positioned so that a gas bubble 44 covers the object 110. A control unit 42 increases or decreases the amount of gas 40 supplied to the hollow member 41. This changes the pressure of the gas 40. At the same time, the shape of the object 110 ascertained from the interference image acquired by the imaging unit 3 also changes. The information processing unit 32 can acquire viscoelastic information and surface tension of the object 110 from the pressure change of the gas 40, the shape change of the object 110, and the time required for the pressure change and the shape change.

[0062] Under certain conditions, the pressure (surface stress) applied to the object 110 can be considered the pressure of the gas 40, so there is no need to consider the tension of the first interface F1. However, there are cases where it is necessary to consider the tension inherent to the first interface F1. The surface stress applied to the object 110 by the first interface F1 can be calculated from the eigenvalue of the tension of the first interface F1 and the interface shape (the amount of deformation of the interface), and can therefore be calculated based on the shape of the first interface F1 ascertained from the interference image. For example, when the first interface F1 is an interface between air and liquid, it is known that stress (Laplace pressure) resulting from the surface tension and the radius of curvature of the interface occurs. This Laplace pressure can be obtained from the surface tension (eigenvalue) and the shape of the first interface F1 ascertained from the interference image (the radius of curvature of the interface). The information processing unit 32 of the imaging unit 3 can obtain the surface stress applied to the object 110 from this shape information and the physical properties of the interface.

[0063] For example, in a state where the first interface F1 is formed by the bubble 44, a first interference image when the pressure of the gas 40 is set to a first pressure and a second interference image when the pressure of the gas 40 is set to a second pressure (a pressure different from the first pressure) may be acquired. The information processing unit 32 of the imaging unit 3 can obtain viscoelastic information of the object 110 based on the first interference image and the second interference image. In this case, the time it takes for the pressure to change from the first pressure to the second pressure and the change in shape of the object may be used to analyze the viscoelastic information.

[0064] [Imaging Method] (Fourth Embodiment) FIG. 10 is an explanatory diagram of an imaging method according to a fourth embodiment. A substance in contact with an object may have a higher refractive index than the object. For example, as shown in FIG. 10 , a liquid 200 containing the object 110 has a higher refractive index than the object 110. The refractive index of the liquid 200 may be, for example, 0.1 or more higher than that of the object 110. The refractive index of the liquid 200 is, for example, 1.45 or more. Examples of the liquid 200 include an aqueous glycerin solution and an aqueous sugar solution. The first interface F1 is the interface between the liquid 200 and the object 110.

[0065] In this embodiment as well, the three-dimensional shape of the object 110 can be accurately grasped from the interference image of the first reflected light L2 at the first interface F1 and the second reflected light L3 at the second interface F2.

[0066] [Imaging Method] (Fifth Embodiment) FIG. 11 is an explanatory diagram of an imaging method according to the fifth embodiment. The substance in contact with the object 110 may be a thin film member 300. The thin film member 300 has a refractive index different from that of the object 110, or has the property of reflecting light. The thin film member 300 may have a refractive index higher than that of the object 110 by 0.1 or more, for example. The refractive index of the thin film member 300 is, for example, 1.45 or more. The thin film member 300 is formed of, for example, a resin. The thin film member 300 is overlaid on the upper surface 110a of the object 110. The first interface F1 is the interface between the thin film member 300 and the object 110.

[0067] The thin film member 300 may be formed in a bag or balloon shape that expands when gas is introduced through the hollow member 41. The thin film member 300 can be held at the tip 41a (see FIG. 3) of the hollow member 41. The position and shape of the thin film member 300 can be adjusted by adjusting the position of the tip 41a or the pressure inside the hollow member 41. Therefore, the pressure applied to the object 110 can be controlled, similar to the gas 40 (see FIGS. 4 and 5). Note that the position and shape of the thin film member 300 may be adjusted by controlling at least one of the pressure and volume of the gas supplied or drawn through the hollow member 41.

[0068] In this embodiment as well, the three-dimensional shape of the object 110 can be accurately grasped from the interference image of the first reflected light L2 at the first interface F1 and the second reflected light L3 at the second interface F2.

[0069] [Imaging Method] (Sixth Embodiment) To grasp a three-dimensional image of the object 110, it is desirable to set the position where the first interface F1 reaches the substrate 1 (specifically, the upper surface 11a of the bottom plate 11) as the reference position for the height of the object 110. However, if an attempt is made to expose the upper surface 11a of the bottom plate 11 by increasing the pressure of the gas 40 in the bubble 44 (see FIG. 5 ), the object 110 may be deformed by the pressure of the gas 40. Therefore, it is effective to grasp the shape of the object 110 in a state where the pressure of the gas 40 is not applied (original shape: shape before deformation) by the following method. Note that the position where the first interface F1 reaches the substrate 1 may be a part of the surface in a close proximity state where a small amount of water exists between the first interface F1 and the substrate 1 when the first interface F1 and the substrate 1 are brought sufficiently close to each other.

[0070] Fig. 12 is an example of an interference image. Fig. 13 is a diagram showing the change in brightness over time along line A-A' in Fig. 12. In Fig. 13, the horizontal axis represents time, and the vertical axis represents brightness along line A-A'. Fig. 14 is a diagram showing the change in brightness over time at the center of the interference fringes. Fig. 15 is a brightness profile along line C-C' in Fig. 13.

[0071] As shown in FIG. 13 , when the pressure of the gas 40 is applied to the object 110 by pressing the bubble 44 against the object 110 (see FIG. 5 ), interference fringes appear (point B). The interference fringes of the interference image change over time until the final point (point B'). As shown in FIG. 14 , the brightness of the center of the interference fringes increases and decreases. The change in the interference fringes indicates the deformation process of the object 110, and the brightness profile at the final point (point B') shown in FIG. 15 indicates the shape of the object 110 at that point. Therefore, based on information about the deformation process and the final shape, it is possible to predict and understand the original shape of the object 110 before the pressure of the gas 40 is applied.

[0072] On the other hand, the following method can be used to control the first interface F1 to reach the substrate 1 while maintaining a low pressure of the gas 40. By adjusting the affinity between the first interface F1 and the substrate 1, it is possible to expose the upper surface 11a of the bottom plate 11 without increasing the pressure of the gas 40. For example, when the liquid 100 is a liquid culture medium, replacing the liquid culture medium with a phosphate buffer solution with high surface tension increases the affinity of the gas 40 with the substrate 1, and the first interface F1 easily approaches or reaches the upper surface 11a of the bottom plate 11. Similarly, making the upper surface 11a of the bottom plate 11 water-repellent also increases the affinity of the gas 40 with the substrate 1, and the first interface F1 easily approaches or reaches the upper surface 11a of the bottom plate 11.

[0073] One embodiment of the present invention has been described in detail above with reference to the drawings, but the specific configuration is not limited to that described above, and various design changes and the like are possible within the scope that does not deviate from the gist of the present invention.

[0074] In the imaging device 10, the first optical system 2 for obtaining an interference image irradiates the object 110 with light from below. The second optical system 5 for obtaining a transmission image irradiates the object 110 with light from above. The configuration of the imaging device is not limited to this example. The first optical system may irradiate the object with light from below. The second optical system may irradiate the object with light from above.

[0075] REFERENCE SIGNS LIST 1 substrate 2 first optical system 3 imaging section 4 gas supply section (substance supply section) 5 second optical system 10 imaging device 21 first light source 23 aperture stop 26 aperture control section 31 detection section 32 information processing section 41 hollow member 43 flow path 44 air bubble 51 second light source F1 first interface F2 second interface

Claims

1. A substrate on which an object is placed; and a first optical system that irradiates the object with a first emitted light; and an imaging unit configured to capture an interference image between a first reflected light obtained by the first emitted light being reflected at a first interface corresponding to an outer surface of the object, and a second reflected light obtained by the first emitted light being reflected at a second interface between the object and the base material. Imaging device.

2. The substrate has a refractive index difference of 0.1 or more with respect to the object. The imaging device according to claim 1 .

3. The substrate is capable of holding a liquid and the object to be placed in the liquid. The imaging device according to claim 1 .

4. At least a portion of the first interface is formed between the liquid and a substance having a refractive index different from that of the liquid; a material supply unit that supplies the material so that the first interface has a shape corresponding to an outer surface of the object; The imaging device according to claim 3 .

5. The substance has a refractive index 0.1 or more lower than that of the object. The imaging device according to claim 4.

6. the substance is a gas; The substance supply unit is a gas supply unit; The imaging device according to claim 4.

7. the gas supply unit includes a hollow member having a tip end disposed in the liquid, and the first interface is formed by bubbles formed and maintained at the tip end in the liquid by the gas supplied through a flow path in the hollow member; The imaging device according to claim 6.

8. The wavelength of the first emitted light includes a near infrared region. The imaging device according to claim 1 .

9. The wavelength of the first emitted light includes 650 nm or more. The imaging device according to claim 8.

10. The wavelength width of the first emitted light is 50 nm or less. The imaging device according to claim 1 .

11. The wavelength width of the first emitted light is 1 / 10 or less of the wavelength of the first emitted light. The imaging device according to claim 1 .

12. the first optical system includes an aperture stop, an aperture control unit that controls a diameter of the aperture stop, and an objective lens; the aperture control unit adjusts a diameter of the aperture stop so that an illumination numerical aperture is equal to or smaller than an imaging numerical aperture; The imaging numerical aperture is 0.7 or less. The imaging device according to claim 1 .

13. The first outgoing light irradiated by the first optical system includes a plurality of wavelength components having a wavelength difference of 20 nm or less. The imaging device according to claim 1 .

14. A second optical system that irradiates the object with a second light, The imaging unit also captures a transmission image by the second light transmitted through the object. The imaging device according to claim 1 .

15. The imaging unit includes a detection unit that captures the interference image, and an information processing unit that acquires a stereoscopic image of the object based on the interference image. The imaging device according to claim 1 .

16. The imaging unit includes a detection unit that captures the interference image, and an information processing unit that acquires viscoelastic information of the object from a change in pressure of the substance, a change in shape of the object calculated from the interference image, and a time required for the change in pressure and the change in shape of the object. The imaging device according to claim 4.

17. The substance has a higher refractive index than the object. The imaging device according to claim 4.

18. The substance is a liquid that contains the object. The imaging device according to claim 17.

19. The material is a thin film material.

18. The imaging device according to claim 17.

20. The object is a phase object. The imaging device according to any one of claims 1 to 19.

21. Placing an object on the substrate; Irradiating the object with a first emitted light; capturing an interference image of a first reflected light obtained by the first outgoing light being reflected at a first interface corresponding to an outer surface of the object, and a second reflected light obtained by the first outgoing light being reflected at a second interface between the object and the base material; Imaging method.

22. The object is placed in a liquid; At least a portion of the first interface is an interface between the object and a gas in contact with the object. The imaging method according to claim 21.

23. supplying the gas so that the first interface forms a shape conforming to an outer surface of the object; The imaging method according to claim 22.

24. forming the first interface so as to conform to the object by evaporating at least a portion of the liquid between the first interface and the second interface; The imaging method according to claim 22.

25. The affinity between the gas or the liquid and the base material is adjusted to form the first interface so as to conform to the object. The imaging method according to claim 22.

26. a point at which the first interface reaches the base material is set as a reference position for the height of the object, and a three-dimensional image of the object is obtained from the interference image. The imaging method according to claim 21.

27. A tip of a hollow member having a flow path is placed in the liquid, supplying the gas into the flow path to form and maintain the first interface at the tip end of the liquid, and adjusting at least one of the amount of gas supplied and the position of the tip end in a state where the first interface is formed by the gas bubbles formed and maintained at the tip end of the liquid. The imaging method according to claim 22.

28. acquiring a first interference image when the pressure of the gas is set to a first pressure, and a second interference image when the pressure of the gas is set to a second pressure different from the first pressure; The imaging method according to any one of claims 22 to 25 and 27.

29. acquiring viscoelasticity information of the object based on the first interference image, the second interference image, a change in pressure of the gas, a change in shape of the object calculated from the first interference image and the second interference image, and the change in pressure and a time required for the change in shape of the object; 29. The imaging method of claim 28.