X-ray illumination microscope and method for capturing image of luminescent substance

Abstract

In order to improve the resolution of a light-emitting substance image excited by X-rays, provided is a microscope (100) provided with an X-ray irradiation unit and an observation optical system. The X-ray irradiation unit (10) generates a patterned X-ray sheet beam (1) for illuminating an object to be observed (7). The patterned X-ray sheet beam has an intensity pattern (2) in which the intensity varies repeatedly with respect to a position in a certain direction included in the spread in a partial region (32) in the sheet region (3) having the spread. The observation optical system (50) receives radiation having an observation wavelength from an observation region (4) disposed so as to include at least a part of the partial region. The embodiment of the invention also provides a shooting method of the light-emitting substance image.

Description

Technical Field

[0001] The present invention relates to an X-ray illumination microscope and a method for forming an image of a luminescent substance. More specifically, the present invention relates to an X-ray illumination microscope and a method for forming an image of a luminescent substance that can achieve image super-resolution by improving X-ray illumination. Background Art

[0002] There is a great demand for the advancement of microscopy. For example, if an image with a resolution that can distinguish two tiny points close to each other by about 20 nm can be obtained, it will be possible to observe phenomena at the synaptic cleft of nerve cells. To achieve such a high goal, research and development that effectively utilizes various technologies has accumulated. In microscopy in the optical wave region, image super-resolution methods have been developed. For example, structured illumination microscopy (SIM) has attracted attention because it can reduce the overall PSF (overall pointspread function), a performance indicator of the image resolution limit size, by improving the illumination optical system (Non-Patent Document 1). Microscopy that utilizes the high focusing properties of X-rays with a wavelength shorter than that in the optical wave region has also been developed. Some of the inventors of the present invention have focused on promoting the development of microscopy by developing an X-ray light sheet microscope called MAXWELL technology (Microscopy by Achromatic X-rays With Emission of Laminar Light Technique) (Non-Patent Document 2).

[0003] Prior art literature

[0004] Non-patent literature

[0005] Non-patent document 1: Bi-Chang Chen et al., “Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution,” Science, 346, 6208, 1257998-1-1257998-10 (2014), DOI: 10.1126 / science.125799

[0006] Non-patent document 2: Y. Kohmura et al., “The new X-ray / visible microscopy MAXWELL technique for fast three-dimensional nanoimaging with isotropic resolution”, Scientific Reports, 12: 9668 (2022), DOI: 10.1038 / s41598-022-13377-w

[0007] Non-patent document 3: JH Bruning et al., "Digital wavefront measuring interferometer for testing optical surfaces and lenses," Appl. Opt., 13, 2693-2703 (1974), DOI: 10.1364 / AO.13.002693

[0008] Non-patent document 4: A. Maiden et al., “Further improvements to the ptychographical iterative engine”, Optica, Vol. 4, No. 7, pp. 736-745 (2017), DOI: 10.1364 / OPTICA.4.000736

[0009] Non-patent document 5: S. Dong et al., “High-resolution fluorescence imaging via pattern-illuminated Fourier ptychography”, OPTICS EXPRESS Vol. 22, No. 17, pp. 20856-20870 (2014), DOI: 10.1364 / OE.22.020856 Summary of the Invention

[0010] Problems to be solved by the invention

[0011] In an X-ray sheet microscope that uses X-rays for illumination, the luminescent image of a luminescent substance excited by the sheet of X-rays is observed and visualized using an observation optical system. As a result, a high image resolution (resolution) that cannot be obtained by illumination using a light wavelength range is achieved in the X-ray sheet microscope. One of the reasons for the high image resolution of the X-ray sheet microscope is the general properties of X-rays. For X-rays with short wavelengths, it can be expected that they will achieve ideal focusing properties at the diffraction limit. In X-rays that show high transmittance and are not easily refracted, their focusing properties are also maintained within the observed object. Therefore, X-rays themselves are advantageous as illumination for microscopy. In fact, in terms of the image resolution achieved in previous X-ray sheet microscopes (non-patent document 2), the concentrated area where the thickness of the X-ray sheet beam (X-ray sheet beam) becomes the smallest is about 65nm thick (full width at half maximum). Conventional X-ray light sheet microscopes have successfully excited and imaged luminescent materials only within a range extremely thin compared to the depth of field of the observation optical system by aligning the optical axis of the observation optical system with the thickness direction of the X-ray sheet beam.

[0012] While conventional X-ray light-sheet microscopes can achieve a resolving power far exceeding the depth of field in the direction of the observation system's optical axis, their resolving power in the direction transverse to the observation system's optical axis—that is, in the direction that extends the field of view and captures the image—is limited to the level of the observation system itself. The resolving power of a typical observation system is diffraction-limited (e.g., 400 nm at wavelengths near visible light). Even in X-ray light-sheet microscopes using X-rays as illumination, further improvements in image resolution in the direction transverse to the observation system's optical axis are being pursued.

[0013] The present invention aims to solve at least a part of the related problems and contribute to the improvement of microscopy by providing a new X-ray illumination microscopy method and a method for forming an image of a luminescent substance.

[0014] Means for solving problems

[0015] The inventors of the present application conceived of applying a structured illumination microscopy method using patterned illumination to an X-ray light sheet microscopy method, thereby completing the invention according to the present invention.

[0016] That is, in one embodiment of the present invention, a microscope is provided, comprising: an X-ray irradiation unit that generates a patterned X-ray sheet beam for illuminating an observation object, the patterned X-ray sheet beam having an intensity pattern in which the intensity in a partial region of a sheet region having an extension repeatedly varies with respect to a position in a certain direction included in the extension; and an observation optical system that receives radiation having an observation wavelength from an observation region, the observation region being configured so as to include at least a portion of the partial region.

[0017] In addition, in one embodiment of the present invention, a method for photographing an image of a luminescent substance is also provided, which is a method for photographing an image of a luminescent substance using the above-mentioned microscope, and the aforementioned photographing method includes: a stage of arranging a marked portion of the observation object marked by the X-ray excited luminescent substance in the aforementioned partial area; an irradiation stage of generating the aforementioned patterned X-ray sheet beam using the aforementioned X-ray irradiation part; and a photographing stage of receiving radiation of the observation wavelength from the aforementioned X-ray excited luminescent substance using the aforementioned observation optical system.

[0018] In the present invention, an X-ray sheet beam refers to an X-ray beam that has high intensity in a sheet-like region of limited thickness within space and is a traveling wave or standing wave that travels in any direction of the sheet's expansion. The thickness of the X-ray sheet beam may not be constant or uniform. A patterned X-ray sheet beam refers to an X-ray beam that is similar to an X-ray sheet beam as a whole but has an intensity pattern with repeated intensities in at least a portion of its sheet-like region. It should be noted that the shape of an X-ray sheet beam or a patterned X-ray sheet beam is determined by the local intensity of the X-rays and can be determined by selecting a portion with high intensity, determining its contour, and calculating or predicting it using the action of an optical element used to generate it. In the present invention, an X-ray irradiation unit refers to a functional element that generates an X-ray beam for illumination of a target. The X-ray irradiation unit may or may not include a light source that generates X-rays. For example, an optical system that converts an external X-ray beam into an X-ray sheet beam using an X-ray optical system such as a Wolter mirror and an optical system for generating a patterned X-ray sheet beam having an intensity pattern from the X-ray sheet beam can be combined to form an X-ray irradiation unit.

[0019] The observation optical system in the present invention can include any element for acquiring any form of data (image data) for forming an image. A sheet-like region is not a mathematically two-dimensional surface, but rather a region having thickness and extension. A partial region is any portion of the sheet-like region. In the present invention, when the term "repeat" is used in relation to space, the repetition does not need to be completely periodic. In addition, a stripe pattern is a pattern formed with stripes and does not need to be completely periodic or infinitely extended. A typical observation optical system in the present invention includes an imaging optical system and light-receiving elements such as an electronic imager and film. The observation optical system can include, as needed, optical elements appropriately employed in conventional microscopes, such as reflectors, apertures, relay optical systems, and filters. The observation area in an observation optical system generally refers to the area from which information about radiation from the observation optical system is acquired through the observation optical system. In addition, the optical axis in the observation optical system is the axis that determines the main direction in which radiation from the observation area is received. The field of view is intentionally set based on the size of the imaging optical system and imager of the observation optical system, or for other purposes, and has an extension in a direction transverse to the optical axis. Depth of field (DOF) refers to the range along the optical axis over which an observation optical system can achieve image resolution consistent with its intended purpose. Therefore, typically, the field of view determines the angle of view, while the depth of field determines the focus range. The observation area is the spatial region defined by the extension of the field of view and the depth of field. The observation wavelength is typically determined by selecting a single wavelength or a range of wavelengths within the optical wavelength range and determining it based on the characteristics of the optical elements.

[0020] In the present invention, radiation refers to electromagnetic radiation of any energy, that is, any wavelength. The term "light wave region" mainly refers to the wavelength range including infrared rays (including terahertz waves), visible light, and ultraviolet rays. Therefore, typically, the observation wavelength is a single wavelength or a range of wavelengths determined in the wavelength region including infrared rays (including terahertz waves), visible light, and ultraviolet rays. As mentioned above, in the present invention, terms from the field of optics are generally adopted, especially terms from the field of optical instruments including microscopes. These terms are sometimes used for electromagnetic radiation of wavelengths other than visible light (including soft X-rays and hard X-rays). For example, the term "illumination" can also include radiation of wavelengths different from the observation wavelength. Therefore, irradiation with radiation that is used to excite or photosensitize substances that can be accompanied by electron migration, and irradiation with radiation that does not expect direct brightness changes is sometimes called illumination. In addition, as in Non-Patent Document 2, the term "light" is sometimes used for X-rays, such as the expression "light sheet" in structured illumination microscopy in the light wave region. Unless otherwise specified, the wavelength refers to the wavelength in a vacuum. Expressions such as "structured", "having a structure", and "exhibiting a structure" of a value corresponding to space (for example, the distribution of X-ray intensity corresponding to a position) describe that the value is not constant with respect to space but exhibits variation.

[0021] Effects of the Invention

[0022] In any aspect of the present invention, a high-resolution microscopy method using X-rays and a method for capturing an image of a luminescent substance can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] [ Figure 1 ] Figure 1 This is a perspective view showing a schematic configuration of an X-ray illumination microscope in an embodiment of the present invention.

[0024] [ Figure 2A-B ] Figure 2A-B 2A is a schematic diagram illustrating the relationship between a partial region and an observation region in the sheet-like region according to the embodiment of the present invention, and is a plan view ( FIG. 2A ) and a front view ( FIG. 2B ).

[0025] [ Figure 3A-B ] Figure 3A-B 3A and 3B are a plan view and a front view showing a schematic configuration of a sample (observation object) and X-ray illumination in a conventional X-ray sheet microscope.

[0026] [ Figure 4A-C ] Figure 4A-C4A, a beam having a stripe pattern repeated in the x-direction, a beam having a stripe pattern repeated in the y-direction ( FIG. 4B ), and a structure capable of improving image resolution in both the x-direction and the y-direction ( FIG. 4C ).

[0027] [ Figure 5 ] Figure 5 This is an explanatory diagram showing another example of a two-dimensional grid pattern that can form a patterned X-ray sheet beam in the X-ray illumination microscope according to the embodiment of the present invention.

[0028] [ Figure 6 ] Figure 6 This is a perspective view illustrating a patterned X-ray sheet beam having a multilayer intensity pattern in an X-ray illumination microscope according to an embodiment of the present invention.

[0029] [ Figure 7A-B ] Figure 7A-B 7A and 7B illustrate how sheet-like X-ray beams are arranged to intersect and interfere to generate a fringe pattern in an embodiment of the present invention, showing a case where the intersection angle is large ( FIG. 7A ) and a case where the intersection angle is small ( FIG. 7B ).

[0030] [ Figure 8A-B ] Figure 8A-B 8A is a diagram showing an example configuration of an X-ray irradiation unit that generates an intensity pattern of a patterned X-ray sheet beam in an embodiment of the present invention, including a configuration using an X-ray beam focusing element (X-ray beam focusing unit) and a diffraction grating ( FIG. 8A ), and a configuration using an X-ray branching element (X-ray branching unit) and an X-ray interferometer ( FIG. 8B ).

[0031] [ Figure 9A-B ] Figure 9A-B FIG9A is a plan view and a cross-sectional view ( FIG9B ) showing a state of a patterned X-ray sheet beam when a fringe pattern is realized using the Talbot effect of a transmission diffraction grating as a specific configuration of an embodiment of the present invention.

[0032] [ Figure 10A-B ] Figure 10A-B 10A and 10B are diagrams showing a configuration in which two X-ray sheet beams are interfered with by wavefront splitting interference using a prism in an embodiment of the present invention. The diagrams include a configuration in which two X-ray sheet beams are formed using two prisms ( FIG. 10A ) and a configuration in which two X-ray sheet beams are formed using one prism ( FIG. 10B ).

[0033] [ Figure 11 ] Figure 11This is a diagram showing a configuration in which three X-ray sheet beams are interfered with each other by wavefront division interference using a prism in an embodiment of the present invention.

[0034] [ Figure 12A-B ] Figure 12A-B This is a schematic diagram showing an example of calculation of interference patterns based on three X-ray sheet beams in the embodiment of the present invention.

[0035] [ Figure 13A-C ] Figure 13A-C These are explanatory diagrams illustrating a situation envisioned in an image super-resolution algorithm in which the position of a luminous point excited by illumination with a patterned X-ray sheet beam having a stripe pattern is precisely determined in an embodiment of the present invention. The diagrams include a diagram showing the shift of the X-ray intensity pattern ( FIG. 13A ), a diagram showing the amount of light obtained by shifting the phase of the pattern ( FIG. 13B ), and a diagram showing the position of an image obtained by image super-resolution within a period of the X-ray intensity pattern ( FIG. 13C ).

[0036] [ Figure 14 ] Figure 14 This is an explanatory diagram showing the concept of phase correction based on unwrap calculation in the embodiment of the present invention.

[0037] [ Figure 15 ] Figure 15 This is a flowchart illustrating a method for capturing an image of a luminescent substance according to an embodiment of the present invention.

[0038] [ Figure 16A-B ] Figures 16A and 16B are flowcharts illustrating the details of the shooting stage (Figure 16A) and the image reconstruction stage (Figure 16B) in the method for shooting an image of a luminescent substance according to an embodiment of the present invention.

[0039] [ Figure 17 ] Figure 17 This is a diagram showing an exemplary configuration of an X-ray irradiation unit that generates an intensity pattern of a patterned X-ray sheet beam in an embodiment of the present invention, using a pinhole array and a two-dimensional Wolter mirror.

[0040] [ Figure 18 ] Figure 18 This is a perspective view showing the configuration of an X-ray illumination microscope used in an observation experiment according to an embodiment of the present invention.

[0041] [ Figure 19A-C ] Figure 19A-C is a microscope image showing a scintillation image captured in an embodiment of the present invention.

[0042] [ Figure 20 ] Figure 2019A to 19C are graphs showing the average brightness within the square frame indicated by the white lines in FIG. 19A to FIG. 19C relative to the shift amount of the diffraction grating in the embodiment of the present invention.

[0043] [ Figure 21A-B 21A and 21B are diagrams illustrating a process of determining image super-resolution performance based on brightness changes, and show the range of the translation position of the diffraction grating. Figure 20 21A ) and a graph showing the difference in brightness ( FIG. 21B ). DETAILED DESCRIPTION

[0044] The following describes embodiments of the X-ray illumination microscope and the method for photographing luminescent material images of the present invention. Common parts or elements are designated by common reference numerals unless otherwise specified in the description. Furthermore, the elements of the various embodiments in the figures are not necessarily shown to scale with one another.

[0045] 1. Implementation Method

[0046] 1-1. Structure of an X-ray Illumination Microscope

[0047] Figure 1 : is a stereoscopic diagram showing the schematic structure of the X-ray illumination microscope 100 in the present embodiment. The X-ray illumination microscope 100 in the present embodiment includes an X-ray irradiation unit 10 and an observation optical system 50. For the observation optical system 50, an existing optical element for an optical microscope is used, and the image resolution of the observation optical system 50 is enhanced by improving the X-ray irradiation of the X-ray irradiation unit 10. The observation object 7 is illuminated by a patterned X-ray sheet beam 1. The patterned X-ray sheet beam 1 is generated by the X-ray irradiation unit 10 in a manner having an intensity pattern 2. The intensity pattern 2 of the patterned X-ray sheet beam 1 is repeated in at least a partial area 32 of the sheet area 3 having an extension including the first direction and the second direction. In Figure 1 In the example of , the expansion of the patterned X-ray sheet beam 1 is made consistent with the xy plane, and the first and second directions can be set as, for example, the x and y directions of Cartesian coordinates determined relative to the observation object 7 and the observation optical system 50. In this case, the sheet area 3 refers to the range where the patterned X-ray sheet beam 1 expands in the xy plane, and the partial area 32 refers to a portion of such sheet area 3. The partial area 32 can also be defined as the range where the intensity pattern 2 is visible in the patterned X-ray sheet beam 1. Figure 1 In FIG. 1 , the intensity pattern 2 of the patterned X-ray sheet beam 1 is shown in a range wider than the partial area 32 as a pattern having positions that are stronger than the representation of the black elongated stripes and positions that are weaker than the representation of the unblackened blanks therebetween. That is, Figure 1 The illustrated intensity pattern 2 is a stripe pattern that repeats in the x-direction and extends in the y-direction. It should be noted that in this embodiment, the intensity of the intensity pattern including the stripe pattern is not necessarily limited to a binary intensity or an intensity that is completely zero at a weak position. Therefore, the stripe pattern of the patterned X-ray sheet beam 1 may also include, for example, Figure 1 The pattern shown as a sinusoidal intensity I corresponding to the position in the x-direction is a pattern whose value varies in relation to the position in the x-direction and is repeated to vary the intensity. In this embodiment, even though a stripe pattern is described, it is not limited to a pattern with no variation in the y-direction. It should be noted that, typically, the intensity distribution of the patterned X-ray sheet beam 1 in the z-direction, which is orthogonal to both the x- and y-directions, exhibits an intensity concentrated at a specific location. Therefore, while the patterned X-ray sheet beam 1 is generally sheet-shaped, it may have a finite thickness in the direction normal to the surface on which the intensity pattern 2 is formed.

[0048] The observation optical system 50 is provided for receiving radiation of the observation wavelength from the observation area 4 and forming an image in the light wavelength range. The observation wavelength is not limited to a single wavelength, but may be a wavelength range. The specific value of the observation wavelength is appropriately determined according to the purpose of observation and can be set using a filter or the like as needed. For example, the observation object 7 contains an X-ray excited luminescent substance 8 ( Figure 1 In the case of an X-ray excitation light source (not shown in the figure), a range of wavelengths that overlap with the luminescent wavelength of the luminescent substance can be selected as the observation wavelength. For the present embodiment, the observation wavelength can be, for example, electromagnetic radiation (hereinafter referred to as "observation light") of a wavelength suitable for observation, such as ultraviolet light, visible light, infrared light (including terahertz waves). If there are multiple types of X-ray-excited luminescent substances, the observation wavelength is set to a plurality of wavelength ranges, or to any wavelength contained therein. Typically, the observation optical system 50 includes an imaging optical system 52 such as an objective lens and an imaging element 54. The observation optical system 50 can include optical elements not shown in the figure, such as optical filters, reflectors, and relay lenses, which are configured as needed.

[0049] The observation optical system 50 is configured, for example, with its optical axis oriented in a third direction (z-direction) perpendicular to both the first direction (x-direction) and the second direction (y-direction). The observation area 4 is the spatial range within which the observation optical system 50 is expected to form an image using its inherent image resolution. Therefore, the actual range of the observation area 4 depends on the specific design of the observation optical system 50. The observation area 4 is configured to include at least a portion of the aforementioned partial area 32 where the intensity pattern repeats, that is, to overlap with the aforementioned partial area 32.

[0050] 1-2. Observation area

[0051] Figures 2A and 2B are schematic diagrams illustrating the relationship between a partial area 32 of the sheet-shaped area 3 and the observation area 4 in this embodiment, showing a top view (Figure 2A) and a front view (Figure 2B). The observation area 4 is defined, for example, by a depth of field 44 in the direction along the optical axis (z-direction) and a field of view 42 extending transversely to the optical axis. Typically, it is a flat plate-shaped area having this thickness and extension. Here, the field of view 42 is the range within which an image is formed by the optical system and light-receiving elements of the observation optical system 50, or the range within which such image formation is expected. The observation area 4 is a region having this thickness near the object-side image plane of the observation optical system 50. For purposes of illustration, it is assumed that the observation light has a wavelength range from ultraviolet light to visible light to infrared light (including terahertz waves), and that the observation optical system 50 includes an imaging optical system 52 and an imaging element 54. This imaging element 54 includes a group of light-receiving elements with an array of pixels formed at an imaging position on the image side relative to the imaging optical system 52. In this scenario, observation light emitted from a certain range on the object side is incident on one of the light-receiving elements to form an image, and the output data of the multiple light-receiving elements becomes the image data. In this case, the depth of field 44 can be determined as the range within which observation light from a position within it can be imaged with the target image resolution using the light-receiving element group that forms the pixel arrangement, and observation light from a position deviating from this position can be judged as blurred. Therefore, either the size of the light-receiving element or the diffraction limit of the optical system of the observation optical system 50 will determine the depth of field 44. It should be noted that the depth of field 44 is an indicator of the observation optical system 50 at the wavelength of the observation light. In other words, the depth of field 44 provides a range in the optical axis direction within which the observation optical system 50 can obtain an image generated by the observation light with the image resolution at its ability level without any problems. The depth of field 44 can also be defined as the range in the z direction where the PSF (point spread function) has a value.

[0052] Typically, the observation object 7, which is the object of observation, includes an X-ray excited luminescent substance. The X-ray excited luminescent substance is any substance that is excited by X-rays and emits observation light by any luminescence mechanism including fluorescence and phosphorescence. For example, it includes tiny particles such as NaGdF4:Eu, NaGdF4:Tb, CsPbBr3, etc., which are called SciNPs (scintillating nanoparticles). When the observation object 7 is an object such as biological tissue, the X-ray excited luminescent substance may specifically accumulate in tiny areas inside it, such as molecular sizes, which are optically indistinguishable and cannot be observed. If such an observation object 7 is irradiated with X-rays that extend to a thickness exceeding the depth of field 44 for the above-mentioned observation optical system 50 and only show slow intensity changes within the depth of field 44, the image resolution of the image obtained by the observation optical system 50 will be determined by the performance of the observation optical system 50. However, in the X-ray illumination microscope 100 of this embodiment and conventional X-ray sheet microscopes (Non-Patent Document 2), illumination is performed using X-rays, which have a better focusing property than observation light. Therefore, the resolving power in the optical axis direction (z direction) is determined by the thickness of the patterned X-ray sheet beam (X-ray illumination microscope 100) and the X-ray sheet (Non-Patent Document 2). To determine these thicknesses, an indicator such as the full width at half maximum of the intensity can be used.

[0053] 1-3. Patterned X-ray Sheet Beam

[0054] 3A and 3B are a top view (FIG. 3A) and a front view (FIG. 3B) showing the schematic configuration of a sample (observation object) and X-ray illumination in a conventional X-ray sheet microscope. They are a view showing the relationship between X-ray illumination and the observation object in a conventional X-ray sheet microscope 900 as viewed from the optical axis (FIG. 3A), and a view cut along a plane including the optical axis and the irradiation direction of the X-rays (FIG. 3B). The Cartesian coordinate representation is maintained as Figure 1 In the conventional X-ray sheet microscope 900, in order to Figure 1For the purpose of X-ray illumination, the patterned X-ray sheet beam 1 shown irradiates a single sheet of X-ray sheet beam 91. X-ray sheet beam 91 corresponds to an X-ray sheet (Non-Patent Document 2). X-ray sheet beam 91 exhibits intensity only within a narrow range in the thickness direction, along the optical axis (z direction in Figures 3A and 3B ) relative to the observation optical system 50. In the direction perpendicular to the optical axis (xy direction in the same figure), intensity varies little within the scale of the observation range. For example, the observation object 7 contains an X-ray-excited luminescent substance 8. As shown in Figure 3B , in an X-ray sheet microscope 900, a thin X-ray sheet beam 91 is irradiated at a position passing through the observation object 7 supported by glass 72. Thus, even if X-ray-excited luminescent substance 8 is scattered within the observation object 7, the illumination range for luminescence is limited to an extremely narrow range in the optical axis direction relative to the objective lens 52 of the observation optical system 50. If the thickness of the X-ray sheet beam 91 is thinner than the depth of field 44 shown in FIG. 2 , then in the z-direction of the observation optical system 50, it is possible to resolve two or more luminous points that are closer in size than the optical image resolution limit, thereby achieving image super-resolution. It should be noted that, if the detailed structure is observed, as reported in non-patent document 2, the X-ray sheet beam 91 has a concentrated portion with a waist-shaped thickness relative to its position in the direction of travel (y-direction). This concentrated portion has a range in the y-direction that is determined by the optical system that generates the X-ray sheet beam, and it extends roughly in the x-direction. Therefore, the intensity of the X-ray sheet beam 91 is the same regardless of the position in the x-direction. When the observation range of the observation optical system 50 is positioned near the center of this concentrated portion, a good luminescent image of the X-ray-excited luminescent material 8 can be obtained. The thickness of the X-ray sheet beam 91 in this concentrated portion is about 65 nm thick (full width at half maximum) in the example reported in non-patent document 2, and is less than 350 nm in a wide range of about 600 μm that can cover the observation field of the observation optical system 50 in the y direction. That is, in the X-ray light sheet microscope 900, the thickness of the X-ray sheet beam 91 is concentrated only in a range smaller than the optical depth of field in the direction of the optical axis of the observation optical system 50 (non-patent document 2, Fig. 1a). In this way, image super-resolution can be achieved in the direction of the optical axis by utilizing the fact that the thickness of the X-ray sheet beam 91 is smaller than the depth of field of the objective lens 52 of the observation optical system 50. Specifically, the depth of field D is calculated according to D=n(0.61λ / (NA) 2) is provided. Here, n corresponds to the refractive index of the lens medium (e.g., 1.52), λ corresponds to the observation wavelength (e.g., 550 nm), and NA corresponds to the numerical aperture. With a 20x objective lens (NA = 0.7), the depth of field D is sub-μm, while with a 5x objective lens (NA = 0.15), the depth of field D exceeds 10 μm. However, in conventional X-ray light sheet microscopes 900, the image resolution in the xy plane is still at the level of the performance of the observation optical system 50 itself.

[0055] 1-3-1. Stripe pattern

[0056] In contrast, in the X-ray illumination microscope 100 provided by the present invention, typically, Figure 1 As shown, the patterned X-ray sheet beam 1 is directed to at least one direction ( Figure 1 The intensity changes in the x-direction. This is described in more detail. FIG4A shows a schematic configuration of the patterned X-ray sheet beam 1 in the X-ray illumination microscope 100 of this embodiment. The coordinate representation maintains Figure 1 The intensity pattern of the patterned X-ray sheet beam 1 is shown in FIG. Figure 1 The same is true, but here the stripes are shown without blackening. That is, in a typical example, the patterned X-ray sheet beam 1 exhibits a repeating intensity pattern in the x-direction and exhibits the same degree of variation in the y-direction as the aforementioned X-ray light sheet microscope 900. As indicated by the hollow arrows, the patterned X-ray sheet beam 1 travels generally in the y-direction. In the X-ray illumination microscope 100, by utilizing the intensity pattern of the patterned X-ray sheet beam 1, it is possible to improve image resolution in one direction within the xy plane, namely, the x-direction in FIG. 4A , which is not achieved in the X-ray light sheet microscope 900, thereby achieving image super-resolution. Other configurations that can be implemented by the X-ray illumination microscope 100 of this embodiment are further summarized.

[0057] In this embodiment, the direction in which the patterned X-ray sheet beam is irradiated on the observation object 7 and the observation optical system 50 may be any direction. FIG4B shows an embodiment of the X-ray illumination microscope of the present invention configured in a manner having a repeated stripe pattern in the y direction. As shown in the figure, the patterned X-ray sheet beam 1a is a beam that changes the relative direction relationship with the observation object 7 by 90° in the xy plane from the relationship shown in FIG4A and is configured in a manner having an intensity pattern in the y direction. In this case, as indicated by the hollow arrow, the direction in which the patterned X-ray sheet beam 1a travels is approximately the x-direction in which the stripe pattern extends. Figure 1In the patterned X-ray sheet beam 1 of FIG4A , the X-ray intensity has an intensity pattern that repeats in the x-direction and extends in the y-direction. In contrast, in the patterned X-ray sheet beam 1a of FIG4B , the X-ray intensity pattern extends in the x-direction and repeats in the y-direction, thereby improving image resolution in the y-direction. FIG4C illustrates a configuration that can improve image resolution in both the x- and y-directions. FIG4C , based on the configuration of FIG4A , incorporates X-ray reflectors 18a and 18b for reflecting X-rays. These X-ray reflectors 18a and 18b have the function of aligning the propagation direction of the patterned X-ray sheet beam 1 in the x-direction at the position of the observation object 7 and are inserted into the X-ray optical path as needed. As shown in FIG4C , the patterned X-ray sheet beam 1 can be emitted as shown in FIG4A at one time, and at another time, the X-ray reflectors 18a and 18b can be inserted into the optical path to emit the patterned X-ray sheet beam 1a as shown in FIG4B . As such, by changing the relative orientation between the fringe pattern and the observation object 7 in the xy plane and then irradiating the observation object 7, image super-resolution can be achieved for two-dimensional coordinates in the xy plane while maintaining a fixed relative position between the observation object 7 and the observation optical system 50 (not shown in Figures 4A-4C). In other words, even if the intensity pattern of the patterned X-ray sheet beam has a one-dimensional fringe structure, image super-resolution can be achieved for two-dimensional coordinates in the xy plane. Therefore, using the patterned X-ray sheet beam of this embodiment not only improves image resolution in the z direction but also enables image super-resolution in all directions.

[0058] 1-3-2. Two-dimensional grid pattern

[0059] The patterned X-ray sheet beam in the X-ray illumination microscope of this embodiment is not limited to the beam having a one-dimensional structure in the xy plane as shown in FIG. 4A and FIG. 4B , or the beam shown in FIG. 4C which is a combination of these beams at different time points. Figure 5 This is an explanatory diagram showing another example of a two-dimensional grid pattern that can become a patterned X-ray sheet beam in the X-ray illumination microscope of the present embodiment. The two-dimensional grid pattern of the patterned X-ray sheet beam 1b has repeated intensity changes in one direction (x direction) in the xy plane and in another direction (y direction) in the xy plane that is not parallel thereto. In this way, by adopting a two-dimensional grid pattern, image super-resolution can be achieved for the two-dimensional coordinates in the xy plane while the relative configuration of the observation object 7 and the observation optical system 50 is fixed. Since image super-resolution can be achieved for the two-dimensional coordinates in the xy plane, it is possible to improve the image resolution in the z direction and achieve image super-resolution in all directions. It should be noted that, Figure 5The two-dimensional grid pattern of the patterned X-ray sheet beam 1 b shown is a square grid shape for illustration. The two-dimensional grid pattern for the patterned X-ray sheet beam 1 b in this embodiment includes any other grid shape.

[0060] 1-3-3. Multi-layer pattern

[0061] Furthermore, the patterned X-ray sheet beam in the X-ray illumination microscope of the present embodiment may be a sheet beam having multiple layers arranged in the thickness direction. Figure 6 It is a stereoscopic diagram illustrating a patterned X-ray sheet beam 1c in which the intensity pattern in the X-ray illumination microscope is a multilayer pattern. The illustrated multilayer pattern includes a plurality of single-sheet regions 341 and 342 that form a stripe pattern with repeated intensity changes in one direction (x direction) within the xy plane. Each of the single-sheet regions 341 and 342 has an extension parallel to the xy plane, has an orientation with a direction (z direction, thickness direction) that crosses both the x direction and the y direction as a normal, and is separated from each other. Each single-sheet region 341 and 342 corresponds to each layer of the multilayer pattern, which here means alternating odd numbers and even numbers. In a partial area of ​​each single-sheet region 341 and 342, an intensity pattern of a stripe pattern that repeats in the x direction and extends in the y direction as shown in FIG. 4A is realized. As shown in FIG. Figure 6 As shown, regarding the stripe patterns of the individual monolithic regions 341 and 342, when one of the monolithic regions 341 and one of the monolithic regions 342 are selected and projected in the thickness direction, they can be arranged relative to each other so as to be offset in the stripe repeating direction (x direction). Figure 6 In FIG. 3 , the intensity pattern of a single-piece region 341 and the intensity pattern of another single-piece region 342 adjacent to it in the thickness direction are reversed in the x direction, as shown by the intensity I versus x graph. Figure 6 In this example, the intensity range of each stripe representing the stripe pattern is defined as a stripe with width and no thickness. These stripes, arranged parallel to each other in the xy plane, represent the intensity pattern of each monolithic region. Furthermore, to clarify the three-dimensional arrangement, each stripe forming the stripes is labeled with a sand pattern. In real multilayer patterns, intensity is determined as a function of position both in the xy plane and in the z direction, and can take continuous values. A pattern with an intensity that is sinusoidal with respect to the z axis is also an example of a multilayer pattern in this embodiment.

[0062] It can be considered Figure 6The increase and decrease pattern of the single-layer regions 341 and 342 shown is a pattern in which, when projected in the thickness direction of the stacked single-layer patterns, the odd-numbered single-layer patterns 341 and the even-numbered single-layer patterns 342 are arranged relative to each other in a manner such that the intensity pattern repeats in a staggered manner. Figure 6 The reversal of the pattern shown can be expressed as a shift of the periodic intensity variation in the monolithic regions 341, 342 by 1 / 2 of the repetition period in the x-direction. Figure 6 The multilayer pattern shown can also be expressed as follows: for the periodic intensity variation in the single-piece regions 341 and 342, the repetition period of the intensity in the single-piece regions adjacent to each other in the z direction is reversed. As another expression, Figure 6 The multilayer pattern shown can also be expressed as follows: with respect to the periodic intensity variation in the single-piece regions 341 and 342, the repetition of the intensity in the single-piece regions adjacent to each other in the z direction becomes opposite in phase. Figure 6 In the figure, a combination of intensity patterns of a single-piece area is shown as two shift positions with a shift amount of 1 / 2 in the repetition period in the x direction, but in this embodiment, a combination of intensity patterns with different shift amounts or a combination of intensity patterns of a single-piece area with a shift amount of 0 can also be used.

[0063] 1-4. Generation of Patterned X-ray Sheet Beams

[0064] Next, methods for generating the various patterned X-ray sheet beams described above are described. Figures 7A and 7B illustrate the generation of a fringe pattern by interfering X-ray sheet beams (the first and second X-ray sheet beams) in a cross configuration, showing a case with a large intersection angle (Figure 7A) and a case with a small intersection angle (Figure 7B). X-rays are preferably monochromatic, linearly polarized light generated by a radiation device, for example, and narrowed to any one coherent (interferable) direction. It should be noted that, as shown in Non-Patent Document 2, methods for generating sheet beams are known, such as passing the beam through a slit, etc., and then concentrating the beam in the thickness direction using a Wolter mirror, etc. From at least one X-ray sheet beam generated in this manner, two sheet beams (the first and second X-ray sheet beams) are generated while maintaining mutual coherence. These two sheet beams have the same wavelength, and share the z direction as the thickness direction. Unless otherwise specified, this narrowed direction will continue to be referred to as the z direction and also as the thickness direction. Figures 7A and 7B schematically illustrate the interference fringes and intensity variations in the x-direction for the first and second X-ray sheet beams when they form a large angle and a small angle, respectively. The wavenumber vectors of the first and second X-ray sheet beams are k1 and k2, respectively. It should be noted that although these are vectors, arrows and boldface are omitted. The grating vector K that defines the interference fringes satisfies K = k2 - k1. Here, Λ, which satisfies |K| = 2π / Λ, provides the pitch (period of the intensity pattern) of the interference fringes as the grating. When the first and second X-ray sheet beams have no curvature and the sum of vectors k1 and k2 is oriented toward the y-axis, the intensity of the X-rays after interference becomes constant along a straight line parallel to the y-axis. When this is observed in the x-direction, the X-ray oscillating electric field becomes strong and weak at locations of constructive and destructive interference, respectively, as indicated by the solid and dashed lines, forming interference fringes. As shown in Figures 7A and 7B, the period Λ of the intensity pattern can be easily controlled by the directions of vectors k1 and k2, that is, the angles of the first and second X-ray sheet beams. Furthermore, the spacing of the interference fringes can be varied by changing the wavelength of the X-rays. While the principle of forming a fringe pattern using two beams has been described here, a two-dimensional grid pattern can be achieved by utilizing the interference of three beams.

[0065] Figures 8A and 8B are diagrams illustrating an example configuration of an X-ray irradiation unit that generates an intensity pattern of a patterned X-ray sheet beam in this embodiment, illustrating a more specific configuration of the X-ray irradiation unit for generating the intensity pattern shown in Figures 7A and 7B through interference. Figure 8A is a perspective view illustrating an example configuration of an X-ray irradiation unit 10 that generates intensity pattern 2 using an X-ray focusing element (X-ray focusing unit) 12 and a diffraction grating 14. The X-ray focusing unit 12 narrows the thickness of X-rays emitted from the X-ray source and passing through the slit of the X-ray limiting element 11. This narrowed thickness is an expansion in a direction corresponding to the slit width of the X-ray limiting element 11, corresponding to the thickness of the sheet-shaped X-ray sheet beam 13 in the Z direction. The X-ray focusing unit 12 can be, for example, a one-dimensional Wolter mirror reflective optical system comprising a combination of a hyperbolic reflector with a surface that is hyperbolic in the yz plane and has no curvature in the x-direction, and an ellipsoidal reflector with a surface that is parabolic in the yz plane and has no curvature in the x-direction. As such, the X-ray focusing unit 12 in this embodiment is not limited to a single element and can also be a combination of multiple X-ray optical elements. The X-ray sheet beam 13 thus generated is a traveling wave that has an in-plane spread, a thickness in the direction normal to the surface, and travels in a single direction within the surface. It should be noted that the X-ray sheet beam 13 at this stage has a uniform intensity with almost no intensity variation in the xy plane within the X-ray sheet beam, maintaining the coherence and polarization of the X-ray source. A typical example of an X-ray source that maintains coherence and polarization is synchrotron radiation. The X-ray sheet beam 13 is a sufficiently thin sheet beam having a beam waist thickness of approximately 65 nm when directly propagating, for example, and corresponds to the X-ray sheet beam 91 shown in FIG. 3A and FIG. 3B .

[0066] Next, all or part of the X-ray sheet beam 13 is incident on the diffraction grating 14 provided in the optical path. It should be noted that the diffraction grating 14 may be a crystal having a grating constant with a period that causes the X-rays to diffract. By passing through the diffraction grating 14, the direction of at least a part of the X-ray sheet beam can be changed and interference can occur. That is, by directing at least two X-ray sheet beams that maintain coherence and change their directions toward the same space, diffraction occurs, and therefore any position illuminated by both of the at least two X-ray sheet beams becomes the range of the patterned X-ray sheet beam. In this way, by utilizing the effect of the diffraction grating 14, a light having the following characteristics can be generated: Figure 1 4A shows a patterned X-ray sheet beam having an intensity pattern. FIG8A shows how two X-ray sheet beams 13A and 13B interfere with each other immediately after passing through a diffraction grating 14 to form a fringe pattern. The diffraction grating 14 may be, for example, a π phase grating.

[0067] Figures 9A and 9B are a top view (Figure 9A) and a cross-sectional view (Figure 9B) illustrating a patterned X-ray sheet beam when a fringe pattern is achieved using the Talbot effect of a phase diffraction grating, as a specific configuration of this embodiment. Figure 9A shows the configuration of a diffraction grating 14V, which is constructed by artificially providing concave and convex fringes on a flat plate made of a material having a refractive index other than unity with respect to X-rays, and the resulting pattern. The diffraction grating 14V can be a transmission-type phase diffraction grating in which portions that produce a phase advance of π / 2 or π with respect to X-rays are arranged in a fringe pattern. The diffraction grating 14V shown here is oriented so that the fringe pattern that provides this phase advance is oriented in the thickness direction of the X-ray sheet beam (the z direction in Figures 9A and 9B), allowing the X-ray sheet beam to pass through the flat-plate transmission-type diffraction grating in the thickness direction (the y direction in the same figure). When such a diffraction grating 14V is used, as shown in FIG9A , a pattern of periodic intensity variations in the x-direction due to the Talbot effect can be achieved in a portion of the sheet area of ​​the patterned X-ray sheet beam 1 (not shown in FIG9A ), and the observation area 4 can be configured to include this pattern. It should be noted that in the patterned X-ray sheet beam 1 in FIG9A and FIG9B , the X-ray intensity is represented by shades, with darker areas indicating higher intensities. Furthermore, as shown in FIG9B , the thickness of the patterned X-ray sheet beam in the z-direction at the sample position can be set to approximately 60 nm, for example. It should be noted that the Talbot effect is a phenomenon in which the self-image formed by the interference of the beams diffracted by the diffraction grating 14V exhibits an intensity pattern that increases and decreases within the diffraction plane (xy plane). In the case of a π-phase grating, the intensity pattern in the x-direction exhibits an intensity fluctuation period of d / 2, which is half the pitch d of the diffraction grating stripes. The principle is that the self-imaging of the diffraction grating can form interference fringes with a sharpness close to 1 and a very large contrast between light and dark. The measurement method of scanning the interference fringes with the edge sharpness of the bright part set to less than tens of nanometers can also achieve image super-resolution performance. In addition, in the intensity pattern of the patterned X-ray sheet beam after diffraction, the sharp and unclear positions appear alternately depending on the position in the y direction. For example, in the case of an amplitude grating, the sharpness of the bright part is set to less than d 2 / λ (where λ is the wavelength of the X-rays) appear periodically and alternately. Therefore, it is useful to select the configuration of the observation area 4 to be precisely set as shown in the figure. The phase diffraction grating 14V shown in Figures 9A and 9B is an example of the diffraction grating 14 shown in Figure 8A.

[0068] The patterned X-ray sheet beam 1 of the present invention can also adopt the configuration shown in FIG8B . FIG8B illustrates an exemplary configuration of an X-ray irradiation unit 1 that utilizes an X-ray branching element (X-ray branching unit) 15 and an X-ray interferometer. FIG8B illustrates a scenario in which at least two X-ray sheet beams 13A and 13B, generated by branching through the X-ray branching element 15, which can function as a diffraction grating or partially reflective mirror, further interfere with each other through the function of X-ray interferometers 16a and 16b, which can function as mirrors. In this case, in addition to the X-ray focusing unit 12 (not shown in FIG8B ), an X-ray branching element (X-ray branching unit) 15 is employed, positioned in the optical path of the X-ray sheet beam 13. The X-ray branching element 15 generates a first X-ray sheet beam 13A and a second X-ray sheet beam 13B, both narrowed in one direction. FIG8B illustrates how these beams are separated using a diffraction grating. In addition to the components shown here, the X-ray branching element may also employ a reflector that deflects only a portion of the X-ray sheet beam 13 in the width direction. The first X-ray sheet beam 13A and the second X-ray sheet beam 13B are further oriented by the X-ray interferometers 16a and 16b to become the first X-ray sheet beam 13C and the second X-ray sheet beam 13D, respectively. These interfere again to form a patterned X-ray sheet beam having a fringe pattern. FIG8B illustrates the reflection process using the reflectors serving as the X-ray interferometers 16a and 16b. The X-ray branching element in this embodiment is not limited to a single element such as the X-ray branching element 15; it may also be a combination of multiple X-ray optical elements. It should be noted that, as shown in FIG8B , due to the difference in the direction of the beam before and after incident, the reflector functions to generate a pattern based on interference fringes near its own reflection surface. This interference of X-rays before and after reflection on the reflection surface can also be used to form the pattern in this embodiment.

[0069] In addition, in order to obtain Figure 5 The two-dimensional grid pattern shown may be a configuration in which three or more X-ray sheet beams are generated by an X-ray branching unit and interfered with each other, in addition to the configuration utilizing the Talbot effect shown in FIG. 9A .

[0070] The interference of this embodiment can also be implemented by a method of wavefront splitting interference using a prism. Figures 10A and 10B are diagrams showing the structure of the structure of interfering two X-ray sheet beams by wavefront splitting interference using a prism in this embodiment, which are a structure of forming two X-ray sheet beams using two prisms (Figure 10A) and a structure of forming two X-ray sheet beams using one prism (Figure 10B). The patterned X-ray sheet beam 1 of the present invention can also adopt the structure shown in Figure 10A. In addition to the X-ray focusing unit 12 (not shown in Figure 10A), an X-ray prism 17 is also used, which is arranged in the optical path of the X-ray sheet beam. The X-ray prism 17 generates at least one of the first X-ray sheet beam 13A and the second X-ray sheet beam 13B that propagate non-parallel to each other. Figure 10A shows a structure in which the thickness passed through varies depending on the position in the x-direction. The patterned X-ray sheet beam 1 of the present invention can also adopt the structure shown in Figure 10B. That is, a configuration may be employed in which only a portion of the wide X-ray sheet beam 13 is deflected by the X-ray prism 172 to form a first X-ray sheet beam 13A, which interferes with the remaining undeflected portion of the X-ray sheet beam 13. As shown in Figures 10A and 10B, in a material having a refractive index less than 1 with respect to X-rays, the beam is refracted toward the side opposite to the prism.

[0071] In addition, in order to obtain Figure 5 In addition to using the prisms shown in Figures 10A and 10B, prisms or diffraction gratings can also be used to generate three or more X-ray sheet beams and cause them to interfere. In this case, an X-ray focusing element (X-ray focusing unit) is positioned in the optical path of X-rays emitted from an X-ray source and passing through a slit, generating at least one X-ray sheet beam narrowed in a certain direction. Furthermore, an X-ray branching unit is positioned in the optical path of the X-ray sheet beams, generating at least one of a first X-ray sheet beam, a second X-ray sheet beam, and a third X-ray sheet beam narrowed in a certain direction. When the X-ray interferometer adjusts the directions of the first, second, and third X-ray sheet beams so that they propagate nonparallel to each other, interference generates a patterned X-ray sheet beam having a two-dimensional grid pattern.

[0072] Figure 11This is a diagram showing the configuration of the present embodiment in which three X-ray sheet beams are interfered with by wavefront splitting interference using a prism. The X-ray irradiation unit includes an X-ray focusing unit (not shown) and X-ray prisms 174a and 174b. The X-ray focusing unit is, for example, the same as that in FIG8A . The X-ray prisms 174a and 174b are disposed in the optical path of the generated X-ray sheet beam 13 to generate at least one of the first X-ray sheet beam 13A, the second X-ray sheet beam 13B, and the third X-ray sheet beam 13, each narrowed in a certain direction. These first to third X-ray sheet beams 13A, 13B, and 13 propagate non-parallel to one another. Thus, a patterned X-ray sheet beam having a two-dimensional grid pattern is generated by interference.

[0073] Figures 12A and 12B are schematic diagrams showing an example of calculation based on the interference pattern of three X-ray sheet beams. Figure 12A (crossing angle 30°) and Figure 12B (crossing angle 60°) are diagrams showing the calculation under the condition that three light waves of equal intensity interfere with each other, with the wavelength fixed and the crossing angle of the light waves changed. Figure 11 The arrangements of the first to third X-ray sheet beams 13A, 13B, and 13 in the case where the wave number vectors are k1, k2, and k3, respectively, are collectively shown in the respective figures. Figure 11 The pattern shown as a schematic in the figure can be transformed into an intensity pattern such as those in Figures 12A and 12B. It has been confirmed that the larger the intersection angle, the smaller the spatial period of the pattern and the finer the pattern. In this way, by interfering three X-ray sheet beams, a two-dimensional grid pattern can be generated.

[0074] It should be noted that two-dimensional grid patterns such as those shown in Figures 12A and 12B can also be generated using a diffraction grating. In this case, it is effective to use a diffraction grating with a non-rectangular depth profile, for example, and adjusted for multi-wave interference conditions, in the position of the diffraction grating 14 in Figure 8A. Furthermore, the Talbot effect pattern based on the phase diffraction grating shown in Figure 9A also exhibits repeated intensity variations not only in the x-direction but also in the y-direction. Therefore, by adjusting the structure of the phase diffraction grating, it can be applied to produce the two-dimensional grid patterns shown in Figures 12A and 12B.

[0075] 1-5. Principles of Image Super-Resolution

[0076] 1-5-1. Principle of Image Super-Resolution in the Thickness Direction

[0077] The reason why X-ray illumination microscope 100 and X-ray light sheet microscope 900 achieve image super-resolution in the optical axis direction (z direction) is because the thickness (z-direction width) of the patterned X-ray sheet beam 1 and X-ray sheet beam 91 is smaller than the depth of field 44 ( FIG. 2 ). In other words, the X-ray illumination microscope 100 also reflects the property of the X-ray sheet beam capable of focusing light in the thickness direction. By slightly moving the sheet region 3 shown in FIG. 2B in the z direction within the depth of field 44 using, for example, a piezoelectric element, the z coordinate of the patterned X-ray sheet beam 1 irradiating the observation object 7 can be changed. In this case, even if the relationship between the observation object 7 and the observation optical system 50 is fixed, since X-ray excitation is achieved only within the range encompassed by the sheet region 3, rather than throughout the depth of field 44, variations in luminescence intensity caused by the X-ray excitation of minute locations of the luminescent material can be observed. Therefore, the X-ray illumination microscope 100 preferably further includes a relative arrangement moving unit (not shown) that changes the relative arrangement of the sheet-like region 3 and the observation object 7 in a manner that includes displacement of the sheet-like region 3 in the normal direction. The observation optical system 50 captures an image at each slightly moved position, and a volume image is reconstructed using the plurality of captured images, thereby achieving image super-resolution exceeding the image resolution limit of the observation optical system 50 in the optical axis direction.

[0078] 1-5-2. Example of In-Plane Image Super-Resolution

[0079] In the X-ray illumination microscope 100, by utilizing the intensity pattern 2 of the patterned X-ray sheet beam 1, image super-resolution can be achieved in the xy plane. As shown in Figures 4A to 4C, in the xy plane, if image super-resolution is achieved in one direction within the plane having the intensity pattern, in principle, image super-resolution can also be achieved in other directions within the xy plane. In addition, the principle of image super-resolution that is established in the stripe pattern used for the patterned X-ray sheet beam is also established for patterned X-ray sheet beams with patterns other than stripes. Here, for a stripe pattern ( Figure 1 4A), the description is based on PMI (Phase Measurement Interferometry, non-patent document 3), which is a fringe scanning calculation algorithm for achieving image super-resolution.

[0080] Figures 13A to 13C are explanatory diagrams illustrating the conditions envisioned in an image super-resolution algorithm that precisely determines the position of a light-emitting point emitted by illumination excitation of a patterned X-ray sheet beam 1 having a fringe pattern. They are a diagram showing the shift of the X-ray intensity pattern (Figure 13A), a diagram showing the amount of light obtained by shifting the phase of the pattern (Figure 13B), and a diagram showing the position of an image obtained by image super-resolution within a period of the X-ray intensity pattern (Figure 13C). In Figure 13A, the x-axis shows the coordinate in the x-direction, the vertical axis shows the intensity of the sine wave of the patterned X-ray sheet beam 1, and the shifting of the patterned X-ray sheet beam 1 relative to the x-coordinate is shown. It should be noted that the x-coordinate is fixed relative to the observation object 7. In the patterned X-ray sheet beam 1 having an ideal fringe pattern, the relative configuration of the intensity change and the observation object 7 (i.e., the x-coordinate) can be determined using a phase having a domain of 0 to 2π corresponding to the range of one repeated period of its own intensity change. It should be noted that the phase The phase is not the phase of the electromagnetic wave itself, which is a wave of electric field and magnetic field, but the phase when the intensity (electromagnetic wave) pattern is expressed by the wave. The phase is shown in FIG13A. The intensity when the phase is 0, 2π / 5 (= 72°), 4 / 5π, ..., 2π. and phase In this case, it cannot be distinguished. The change in is equivalent to shifting the fringe pattern in the x-direction within the range of the X-ray intensity period d1. It should be noted that the X-ray intensity period d1 is the same as the intensity pattern period Λ (Figures 7A and 7B) in the fringe pattern. For the luminous point located at an unknown position within the range of x0 and x0+d1, if the patterned X-ray sheet beam 1 is shifted relative to the x-coordinate, the luminous intensity relative phase is shown in the bar graph of Figure 13B. The curve in Figure 13B is a graph showing the phase The sine wave of this change at a finite number of sample points. This sine wave can be regarded as making the phase As the number of samples increases, the curve approaches, so it is considered that the sine wave is accurately determined by a finite number of luminous intensity samples (the value of the histogram), providing the phase of the peak position of the sine wave. The position correspondence between the luminous point x0 and x0+d1 is reasonable. The range of x0 and x0+d1 is not the same as the original, but it corresponds to the range of x0 and x0+d1, and is marked accordingly in the figure. That is, as shown in Figure 13C, the luminous point should exist somewhere between x0 and x0+d1. If the X-ray intensity at this position is reflected in the light intensity of the bar graph in Figure 13B, the position of the luminous point can be determined in more detail than within the range of x0 and x0+d1.

[0081] It should be noted that changing the phase Such shifting of the intensity pattern can be achieved by fixing the observation object 7 and the imaging optical system 50 and slightly moving the position of the patterned X-ray sheet beam 1, or by fixing the positions of the observation object 7, the imaging optical system 50, and the patterned X-ray sheet beam 1 as a whole and slightly moving the intensity pattern 2 within the patterned X-ray sheet beam 1. These slight shifts can be readily achieved using existing means such as piezoelectric elements, which have good controllability and can achieve minute displacements. Furthermore, slight shifting of the intensity pattern 2 within the patterned X-ray sheet beam 1 can be achieved using various methods, preferably inserting an object with a refractive index not strictly equal to 1, rotating the object to change the optical path length, or rotating the diffraction grating 14 relative to the X-ray sheet beam 1. Therefore, a functional component that can be referred to as a pattern shifter (pattern shifter) includes any mechanical element or optical element capable of achieving displacement or optical path length change. In particular, when the pattern shifter does not change the relative arrangement between the observation area 4 of the observation optical system and the observation object 7 , the position of the observation object 7 does not move in the plurality of element images acquired by the imaging optical system 50 , facilitating subsequent processing.

[0082] The algorithm is explained using a mathematical formula. As described above, the intensity of the observation light is measured by shifting the stripe pattern that repeats in the x-direction in the direction of repetition. Considering that an X-ray sheet beam focused in a sufficiently narrow range is also used in the z-direction, it is estimated that the algorithm for the sine wave in FIG. 13B corresponds to the case where I i (x, z) is the intensity observed at each sample position (x, z) at each shift position specified by subscript i (i is 0 or more and N1-1 or less, and N1 is 3 or more),

[0083] [Mathematical formula 1]

[0084]

[0085] φ(x, z) = tan -1 (S1(x,z) / C1(x,z)) (1.3)

[0086] Perform the calculations in [Formula 1] to determine (x, z), that is, tan -1(S1 / C1) (Formula (1.3)). Calculate from this value That is, dl×tan -1 When (S1 / C1) / 2π, as shown in FIG13C, the position can be determined more accurately within the range of x0 and x0+d1. That is, if the description is made based on FIG13A to FIG13C, the position can be determined more accurately within the range of x0 and x0+d1. The obtained sample intensity (histogram) of the observation light in FIG13B determines the sine wave in FIG13B and determines the position of the peak. This is equivalent to setting N1 to 5 in equations (1.1) and (1.2) and calculating the phase according to equation (1.3). The peak position is determined by measuring the relative position of the patterned X-ray sheet beam 1 and the observation object 7 in the z direction to resolve the image.

[0087] Here, tan is calculated according to formula (1.3) -1 When the value of To solve this problem, a phase correction (expansion calculation) of an integer multiple of π is required. Figure 14 This is an explanatory diagram showing the concept of phase correction based on the expansion calculation. The straight line group L1 to L3 is shown as tan -1 The phase (vertical axis) obtained by the value of is relative to the phase (horizontal axis) corresponding to the distance in the actual space (Figure 13C). As shown by the range of the vertical axis of the straight line group L1 to L3, the phase (vertical axis) obtained by the value of is relative to the phase (horizontal axis) corresponding to the distance in the actual space (Figure 13C). -1 Only values ​​of -π / 2 to π / 2 can be obtained. Therefore, in order to determine the phase corresponding to the range of one fringe, for example, 0 to 2π, the obtained tan -1 The value of is determined by which line in the set of lines L1 to L3 is used to obtain the value corresponding to line L, which represents the phase of 0 to 2π corresponding to the distance in real space. It should be noted that when determining the phase in this way, a reference particle indicating the origin position can be selected. The integer used in this phase correction corresponds to, for example, twice the number obtained by adding one to the number of fringes between the reference particle and the particle whose phase is to be determined, or a number obtained by subtracting one from the number obtained by adding one to the number of fringes between the reference particle and the particle whose phase is to be determined.

[0088] If the above method can realize image super-resolution for the x-coordinate, as shown in FIG. 4A to FIG. 4C , image super-resolution can be performed in the x-direction and the y-direction respectively.

[0089] Next, the adoption Figure 5The case of the two-dimensional grid pattern shown in FIG is described. When the two-dimensional grid pattern is a pattern formed by combining sinusoidal stripe patterns, by expanding equations (1.1) to (1.3) to combine them for two coordinate axes, two-dimensional image super-resolution can be achieved. Specifically, when I i When (x, y, z) is the intensity observed at the sample position (x, y, z) as a shift position determined by subscript i (i is 0 or greater and N1-1 or 0 or greater and N2-1, with N1 and N2 both being 3 or greater), periods d1 and d2 are respectively the repetition periods of the intensity pattern in the patterned X-ray sheet beam in the x-direction and y-direction.

[0090] [Mathematical formula 2]

[0091]

[0092] φ y (x, y, z) = tan -1 (S 2y (x, y, z) / C 2y (x, y, z)) (2.6)

[0093] Implement the calculation of [Mathematical formula 2]. For x and y coordinates, When determined, the coordinates of the X-ray-excited luminescent material can be determined with higher accuracy than the pitch of the two-dimensional grid pattern formed in the patterned X-ray sheet beam 1, enabling image super-resolution. Phase correction (expansion calculation) using integer multiples of π can be performed for both the x- and y-coordinates in the same manner as for a one-dimensional fringe pattern.

[0094] It should be noted that the periods d1 and d2 described above are patterns formed in the X-ray sheet beam and can therefore be determined independently of or in association with the image resolution criteria of the observation optical system 50. Here, the image resolution criteria of the observation optical system 50 refer to, for example, the xy-plane dimensions of the range on the object side of the incident light incident on a single light-receiving element when the observation optical system 50 employs an imaging element 54, the size of the Airy disk of the imaging optical system 52, and the PSF (point spread function) of the imaging optical system. These are arbitrary dimensions that serve as indicators of the image resolution of the observation optical system 50 itself.

[0095] If the periods d1 and d2 are set smaller than the image resolution standard of the observation optical system 50, the observation optical system 50 will not be able to distinguish fine structures with periods d1 and d2. Nevertheless, the above-described image super-resolution method can achieve higher image resolution within the xy plane of the observation optical system 50 in the X-ray illumination microscope 100 as a whole.

[0096] Even if the periods d1 and d2 are set to the same level as the standard of the image resolution of the observation optical system 50, the observation optical system 50 does not have the ability to distinguish fine structures with periods d1 and d2. Nevertheless, the above-described image super-resolution method can achieve image resolution in the xy plane in the X-ray illumination microscope 100 as a whole, higher than the standard of the image resolution of the observation optical system 50. Furthermore, in this case, when intensity sample values ​​such as those in Figure 13B are obtained in the fringe pattern, the ability of the observation optical system 50 can be used to determine in which of the multiple intensity bands the X-rays excite the luminescent material to emit light.

[0097] When the periods d1 and d2 are set to be larger than the standard of the image resolution of the observation optical system 50, the observation optical system 50 can perform image resolution on fine structures having the periods d1 and d2. However, if the position shown in FIG13C can be determined with a higher accuracy than the image resolution of the observation optical system 50, then in the entire X-ray illumination microscope 100, the above-mentioned image super-resolution method can obtain a higher image resolution within the xy plane of the observation optical system 50. It should be noted that a finite number of sample points as shown in FIG13B can be determined according to each period of the X-ray intensity. For example, when the image resolution of the observation optical system 50 is the same as that of the pixels of the imaging element 54 and the pixel size matches the period of the X-ray intensity, the intensity curve can be determined for each pixel as shown in FIG13B and FIG13C, thereby determining the position.

[0098] It should be noted that the changes in the periods d1 and d2 can be implemented, for example, by adjusting the conditions for forming the intensity pattern 2, such as by changing the interference conditions shown in Figures 7A and B. Furthermore, the standard size of the image resolution of the observation optical system 50 can be changed by changing the imaging optical system 52, such as adjusting the magnification of the objective lens, changing the imaging element 54, or changing the range of integrating the outputs of multiple light-receiving elements in the imaging element 54 (adjusting the image resolution of the electronic image).

[0099] 1-6. Method for Capturing Luminescent Material Images

[0100] Next, a method of capturing an image of a luminescent substance using the aforementioned X-ray illumination microscope 100 will be described. Figure 15 16A and 16B are flowcharts illustrating details of the imaging phase and the image reconstruction phase in the imaging method.

[0101] Figure 15 The overall flow of the imaging method is shown in FIG. In this imaging method, the above-mentioned X-ray illumination microscope 100 is used. The observation object 7 is marked with an X-ray excited luminescent substance 8 selected according to the observation purpose. As the beginning of imaging, the marked portion (marked portion) is arranged in the partial area 32 (S02). Next, the X-ray irradiation unit 10 generates a patterned X-ray sheet beam 1 and starts irradiation (S04). Then, the observation optical system 50 receives and captures the radiation of the observation wavelength from the X-ray excited luminescent substance 8 (S10). When the imaging is completed, the irradiation of the patterned X-ray sheet beam 1 ends (S20), and image reconstruction is performed as needed (S30). In the example shown here, the irradiation stage is performed in a manner that overlaps with the imaging stage (S10) in time from the start of irradiation (S04) to the end of irradiation (S20). The irradiation of the observation object 77 with X-rays can be continuous between S04 and S20, or it can be intermittent. In addition, the image reconstruction ( S30 ) can be executed at any time point as long as the imaging ( S10 ) is completed.

[0102] The photographing stage (S10) is implemented as shown in FIG16A, for example. Specifically, in the photographing stage (S10), the observation optical system 50 performs image capture (S12) multiple times. In each instance, the image capture (S12) may be performed as shown in FIG13A. In this way, the intensity pattern 2 of the patterned X-ray sheet beam 1 is shifted (S18). The captured image is not the final observed image, so it is called an element image in this embodiment. The element image is recorded (S14) and it is determined whether it includes the phase that needs to be shifted (S16). It should be noted that when the intensity pattern 2 is a stripe pattern, the shift here is a shift in the repetitive direction. A typical shift is to divide one period of the stripe into multiple periods and perform it in a manner that includes all of them. The recorded element image is obtained in a style that can be used for reconstruction by any method such as associating it with the shift amount of the pattern and is saved in the image recording unit 56.

[0103] Image reconstruction (S30) is performed, for example, as shown in FIG16B. Specifically, first, the element image associated with the shift amount is retrieved from the image recording unit 56 (S32). Next, based on the retrieved element image, a process for generating an observation image is performed (S34). When the intensity pattern 2 is a stripe pattern, the process is performed in accordance with equations (1.1) to (1.3). When the intensity pattern 2 is a two-dimensional grid pattern, the process is performed in accordance with equations (2.1) to (2.6). The generated observation image is recorded as needed (S36).

[0104] 2. Modification

[0105] This embodiment can be modified in various ways in addition to the above-described configuration. Here, the configuration of the intensity pattern (2-1), the method of generating the intensity pattern (2-2), the reconstruction method (2-3), and other modifications (2-4) are described.

[0106] 2-1. Modification of the Configuration of the Intensity Pattern

[0107] The composition of intensity patterns such as stripe patterns and two-dimensional grid patterns has been described above, and the method for generating intensity patterns has mainly been described as a method based on interference. The intensity pattern of this embodiment is not limited to such a composition and generation method. For example, in addition to a pattern in which the intensity changes in a sinusoidal wave shape, a pattern in which the intensity changes in a rectangular wave shape in a manner that essentially takes two values, or a pattern in which the intensity changes in a sawtooth wave shape can also be used. When using these patterns, a set of orthogonal functions is appropriately selected when expressed as a series of products with orthogonal functions, such as equations (1.1) to (1.3) and equations (2.1) to (2.6).

[0108] In addition, methods such as ghost imaging and compressed sensing using random structured illumination can be used.

[0109] 2-2. Method for Generating Intensity Pattern

[0110] Regarding the method for generating an intensity pattern, a method for forming a sheet-shaped beam using the X-ray confinement element 11 and the X-ray focusing unit 12, and then generating an X-ray intensity pattern using the diffraction grating 14 and the X-ray prisms 17 and 172, is described in conjunction with Figures 8A, 10A, and 10B. In this description, the X-ray focusing unit 12 is described as a reflective optical system such as a Wolter mirror, a combination of a hyperbolic mirror and an ellipsoidal mirror, with imaging capabilities in the xz plane. In contrast, imaging is possible if a Wolter mirror capable of two-dimensional imaging (a two-dimensional Wolter mirror) is employed for the X-ray confinement unit 12, or a mirror having a reflective surface in the shape of an ellipsoid (an ellipsoidal mirror) is employed for the X-ray confinement unit. In this case, the emission surface of the X-ray confinement element 11 (Figure 8A) can be optically conjugated with a surface parallel to the xz plane near the observation area 4 (Figure 9B). Figure 17 This is a structural diagram showing an example of the structure of an X-ray irradiation unit when a pinhole array and a two-dimensional Wolter mirror are used. A plurality of pinholes for passing an X-ray beam from an X-ray source are provided in a predetermined arrangement in the X-ray limiting element 11p. Figure 17 In the figure, pinhole arrays 111 and 112 are schematically depicted as an example. The arrangement of the pinholes can be any arrangement that can be formed in the X-ray limiting element 11p. Typically, the X-ray focusing unit 12p is a two-dimensional Wolter mirror or a rotating ellipsoidal reflector, which is an X-ray reflecting element capable of two-dimensional imaging formed into an appropriate shape. A pattern corresponding to the arrangement of the pinholes in the X-ray limiting element 11p is formed in the X-ray sheet beam 13p. However, due to the long-field distribution of the X-ray source and the diffraction of each pinhole, the X-ray beam corresponding to each pinhole in the X-ray sheet beam 13p will expand with the distance from the X-ray limiting element 11p. In addition to having the deflection function of reflecting the direction of the X-ray sheet beam 13p as a whole toward the y-axis direction, the X-ray focusing unit 12p also has the function of forming an image in the xz plane after reflection. Figure 17 , which are conjugate images of the pinhole array 191 and 192 formed on the focal plane 19 parallel to the xz plane. Figure 17: In the figure, straight lines are shown connecting the pinholes of the corresponding pinhole arrays 111 and 112 and the images of the pinhole array conjugate images 191 and 192 passing through the X-ray focusing unit 12p. The X-rays that actually pass through each pinhole have the above-mentioned expansion at the position before being reflected by the X-ray focusing unit 12p. After the reflection, they are focused by the action of the X-ray focusing unit 12p. These straight lines are straight lines showing two beams of light representing this expansion for one pinhole. In addition, the conjugate images at the positions of the X-ray limiting element 11p and the focal plane 19 are shown by hollow arrows. In this way, the X-rays that pass through the pinholes of the pinhole arrays 111 and 112 form the pinhole array conjugate images 191 and 192, respectively. In addition, the distance from the X-ray limiting element 11p to the focal plane 19 can be adjusted according to the specific design of the X-ray focusing unit 12p. For the purpose of explanation, the imaging of X-rays onto the pinhole array conjugate images 191 and 192 is described. However, X-ray patterns corresponding to the pinhole array conjugate images 191 and 192 are also formed at positions shifted in the y-axis direction from the focal plane 19 focused in this imaging. Figure 17 A sufficiently practical intensity variation can be achieved within a range not shown in the figure). Therefore, by adopting such an X-ray limiting element 11p and an X-ray focusing unit 12p, an intensity pattern 2 can be formed in the sheet region 3, which has both intensity variation in the z direction and intensity variation in the x direction corresponding to the pinhole array conjugate images 191 and 192. The pinhole array conjugate image 191 and the pinhole array conjugate image 192 respectively constitute a single sheet region, thereby forming two single sheet regions that overlap in the z-axis direction, that is, in the thickness direction. With respect to the X-ray focusing unit, in Figure 17 A Wolter mirror utilizing reflection is shown in FIG. 1 , but a rotating ellipsoidal reflector may be used instead of the X-ray focusing unit 12 p .

[0111] In this way, when the above-mentioned two-dimensional Wolter mirror and rotating ellipsoid are used for the X-ray focusing unit 12, due to its two-dimensional imaging effect, it is possible to form an intensity pattern 2 with intensity variations in the x-direction and z-direction in the sheet-like area 3 according to the pattern of the X-ray transmitting portion in the X-ray limiting element. In this case, in addition to the structure having a pinhole array such as the X-ray limiting element 11p, by using a slit array (not shown), it is possible to form a pattern based on imaging reflecting the arrangement of these slits at the position of the observation area 4. The slit array can be arranged, for example, Figure 17 The positions of the pinhole arrays 111 and 112 shown in the example are each provided with one slit.

[0112] As described above, the configuration employing a two-dimensional Wolter mirror or ellipsoid for the X-ray focusing unit 12p advantageously enables structured X-ray illumination without the insertion of a diffraction grating, and allows for the formation of a flexible intensity pattern by the X-ray transmitting portion formed in the X-ray confinement element. Furthermore, in this configuration, by mechanically moving the X-ray confinement element to relatively shift the positions of the slit, slit array, pinhole, pinhole array, and observation object 7, the X-ray intensity pattern can be shifted.

[0113] 2-3. Modification of the reconstruction method

[0114] In Section 1-5-2, the calculation algorithm of the fringe scanning method is described as an example of a reconstruction method for realizing image super-resolution. The reconstruction method for image super-resolution in this embodiment is not limited to such a method. In this embodiment, as an example, a reconstruction calculation method of a light field (intensity information and phase information) can be applied. In this reconstruction calculation method, an image that has lost the phase information obtained by structured illumination microscopy is used as the object, and the Fourier ptychographical iterative engine (see non-patent documents 4 and 5) is effectively utilized to apply an iterative algorithm to perform image super-resolution and phase recovery. In addition, a method can be used to significantly reduce the amount of calculation by using the difference in resolution between X-ray illumination and light observation and using the constraints of convergence operations.

[0115] 2-4. Others

[0116] While the example shows a configuration in which the observation optical system 50 has its optical axis oriented in a third direction (z-direction) perpendicular to both the first direction (x-direction) and the second direction (y-direction), the present invention is not limited to this configuration of observation optical systems. In the X-ray illumination microscope of the present invention, for example, multiple observation optical systems can be configured for observation. In a configuration employing multiple observation optical systems, for example, by employing two observation optical systems tilted from the third direction (z-direction), stereoscopic observation is possible, enabling simultaneous observation at multiple wavelengths.

[0117] 3. Examples

[0118] As an example of this embodiment, Figure 1 9A and 9B , and details of an observation experiment conducted to verify the X-ray illumination microscope 100 will be described. Figure 18 : is a perspective view showing the structure of the X-ray illumination microscope 100 used in the observation experiment of this embodiment. Figure 18 In the figure, it is schematically shown Figure 1The following description involves an actual configuration example of the X-ray illumination microscope 100. Figure 18 The coordinate system is consistent with that described previously. The observation experiments in this example were conducted in Experimental Chamber 3 of the undulator beamline BL29XU at the synchrotron radiation facility SPring-8 (Sayō-cho, Hyōgo Prefecture, Japan). The X-ray energy used was monochromatized from 8 keV X-rays, using a two-crystal spectrometer.

[0119] To build Figure 1 9A and 9B, i.e., an X-ray sheet showing intensity variations in the x-direction, first, an X-ray sheet 13 showing no intensity variations in the x-direction is generated ( FIG. 9A ). A one-dimensional Wolter mirror (focal length of approximately 200 mm) is used as an X-ray lens for one-dimensional focusing in the vertical direction (z direction). This X-ray lens is an optical element equivalent to the X-ray focusing unit 12. Figure 18 The same symbols are used in the text, so the same symbols are marked. In addition, as a slit equivalent to the X-ray limiting element 11 (for example, Figure 8A), a slit with an opening width of 10 to 30 μm is used. This opening becomes a virtual light source of a reduced image formed on an imaging surface parallel to the zx plane in the observation area (observation area 4 in Figure 9A) due to the action of the X-ray lens 12. The vertical width (z-direction thickness) of the one-dimensional focused X-ray beam formed is about 65 to 100 nm, similar to the example reported in non-patent document 2. This one-dimensional focused X-ray beam is equivalent to the X-ray sheet beam 13 in Figure 9A.

[0120] Furthermore, in order to generate the one-dimensional focused X-ray beam as the X-ray sheet beam 13 as shown in FIG. Figure 1 9A , a patterned X-ray sheet beam 1 showing intensity variation also in the horizontal direction (X direction) is formed. A diffraction grating similar to the diffraction grating 14V in FIG. 9B is adjusted on the downstream side of the X-ray beam of the X-ray lens 12 so as to have a grating vector in the x direction (described later). Figure 18) and are arranged in the direction of the X-ray beam (not shown). As shown in FIG9A , the patterned X-ray sheet beam 1 at the downstream of the X-ray beam passing through the diffraction grating is an interference structure of self-imaging formed based on the Talbot effect, and shows an intensity change in the x-direction. As for the diffraction grating, in order to form a π / 2 phase diffraction grating relative to 8keV monochromatic light, a tantalum film of about 1 μm thick is vapor-deposited on a SiC / SiN thin film on a silicon substrate. The period length (pitch = P) of the diffraction grating and the self-imaging in the horizontal direction is 5 μm. As for the composition of the diffraction grating, the L / S (line / space) width is set to P / 2. As for the positional relationship between the diffraction grating and the sample position, the observation area 4 ( Figure 18 (not shown) is set to a position 8 cm downstream of the diffraction grating 14V. This position corresponds to half the Talbot distance at 8 keV.

[0121] The object to be observed, or the observation target, is a substance containing X-ray scintillating nanoparticles and CsPbBr3 quantum dots smaller than 25 nm in size in a toluene solution. The CsPbBr3 quantum dots can efficiently generate visible scintillating light when irradiated with X-rays.

[0122] The observation utilized a two-dimensional test pattern created to align the visual microscope observation system with the X-ray optical axis. A two-step pattern with height differences was created on the surface of an optical microscope cover glass primarily composed of borosilicate glass using a resist called SU8. A toluene solution containing the aforementioned quantum dots was dripped onto the surface and dried, allowing the CsPbBr3 scintillating nanoparticles to adhere.

[0123] In the observation example using the X-ray illumination microscope 100 employing the patterned X-ray sheet beam 1 shown in FIG9A , in order to obtain the data shown in the bar graph of FIG13B , the diffraction grating 14V itself is translated relative to the other components in the x-axis direction by a feed amount of 250 nm. The feed amount of 250 nm is a value obtained by dividing the pitch P (5 μm) of the diffraction grating 14V into 20 parts. Due to the translation of the diffraction grating 14V, the self-image formed by the Talbot effect also translates in the x-axis direction. Figure 18 , the position of the diffraction grating and the position of its self-image seen in the patterned X-ray sheet beam 1 are schematically shown before irradiation with the observation object 7 and inside the observation object 7 .

[0124] 19A to 19C are microscope images showing scintillation images captured in the observation object of this embodiment. Figure 20Graphs showing the average brightness within the square frame indicated by white lines in Figures 19A-C relative to the translation amount of the diffraction grating 14V in this embodiment. Figures 19A-C show images of the same position of the first observation object continuously captured while the step of translating the diffraction grating 14V itself in the x-axis direction at a feed amount of 250 nm is continuously performed. Positions with high scintillation intensity appear brighter. At each position in Figures 19A-C, the brightness of each portion varies depending on the translation amount of the diffraction grating 14V. For example, when observing the area within the square frame indicated by white lines, a clear bright spot is observed in Figures 19A and 19C, but almost no bright spot is observed in Figure 19B. This reflects the repeated intensity fluctuations of X-rays relative to the scintillating nanoparticles present at that location. As described above, it was confirmed that according to this embodiment, the X-ray illumination microscope 100 of this embodiment is capable of actually operating, and in particular, the luminescence intensity generated by the scintillating nanoparticles responds sensitively to the intensity change of the X-rays caused by the translation of the diffraction grating 14V, that is, the patterned X-ray sheet beam 1.

[0125] In addition, Figure 20 19A to 19C , the translation positions of the diffraction grating 14V are shown by arrows A to C. Figure 20 In the curve, a clear brightness change is observed, especially for the translation of the diffraction grating 14V of about 0.5 μm shown by the two dotted lines. Figures 21A and 21B are diagrams illustrating the process of determining the image super-resolution performance based on brightness changes, and are diagrams showing the range of the translation position of the diffraction grating. Figure 20 21A and a graph showing the difference in brightness (FIG. 21B). The horizontal axis of each graph is the translation position of the diffraction grating 14V. When the difference (differential intensity) is calculated from the brightness of FIG. 21A, the solid line of FIG. 21B is obtained. At the peak position of the differential intensity, it can be inferred that the X coordinate of the boundary portion where the intensity change is large in the self-imaging of the Talbot effect caused by the diffraction grating is consistent with the X coordinate of the scintillating nanoparticle. As shown in the dotted line of FIG. 21B, by fitting a curve assuming a Gaussian distribution to the differential intensity distribution, it is possible to perform an analysis of the error evaluation of the position of the scintillating nanoparticle. In the example of FIG. 21B, it is shown that excellent image super-resolution performance exceeding 100 nanometers is obtained. In this way, the example confirms the possibility of achieving image super-resolution imaging exceeding about 400 nm (which is the image resolution limit of a visible light microscope) in the X-ray illumination microscope 100 of this embodiment.

[0126] 4. Conclusion

[0127] The above specifically describes the embodiments of the present invention. The above embodiments, modifications, and examples are described for the purpose of illustrating the invention to which the present invention relates, and the scope of the invention of the present invention should be determined based on the description of the claims. Modifications that include other combinations of the embodiments and exist within the scope of the present invention are also included in the claims of the present invention. That is, those skilled in the art can make various changes, combinations, sub-combinations, and substitutions to the constituent elements of the above embodiments within the technical scope of the present invention or a scope equivalent thereto.

[0128] Industrial applicability

[0129] The present invention can be used in a microscope that performs observation using X-rays for illumination or excitation of substances.

[0130] Description of Reference Numerals

[0131] 100X-ray illumination microscope

[0132] 1. 1a. 1b. Patterned X-ray sheet beam

[0133] 1c Patterned X-ray sheet beam (multi-layer pattern)

[0134] 10X-ray irradiation department

[0135] 11. 11p X-ray confinement element

[0136] 111, 112 pinhole array

[0137] 12, 12p X-ray focusing unit, X-ray lens

[0138] 13, 13A, 13B, 13C, 13D, 13p X-ray sheet beam

[0139] 14. 14V diffraction grating

[0140] 15X-ray branching element

[0141] 16a, 16b X-ray interferometer

[0142] 17, 172, 174a, 174b X-ray prisms

[0143] 18a, 18b X-ray reflectors

[0144] 19 focal planes

[0145] Conjugate image of pinhole arrays 191 and 192

[0146] 2 Intensity Pattern

[0147] 3 sheet areas

[0148] 32 Partial area

[0149] 341, 342 single-piece area

[0150] 4 Observation area

[0151] 42 Field of View

[0152] 44 Depth of Field

[0153] 50 Observation Optical System

[0154] 52 Imaging Optical System

[0155] 54 Imaging Elements

[0156] 56 Image Recording Unit

[0157] 7 Observation Objects

[0158] 72 Glass

[0159] 8X-rays excite luminescent materials

[0160] 900 X-ray light sheet microscope

[0161] 91 X-ray sheet beam

Claims

1. A microscope having: an X-ray irradiation section for generating a patterned X-ray sheet beam for illuminating an observation object, the patterned X-ray sheet beam having an intensity pattern in which the intensity in a partial region of a sheet region having an expansion repeatedly varies with respect to a position in a certain direction included in the expansion; and The observation optical system receives radiation having an observation wavelength from an observation region configured to include at least a portion of the partial region.

2. The microscope according to claim 1, wherein The observation optical system is an optical system whose optical axis is oriented in the normal direction of the expansion of the sheet-shaped region.

3. The microscope according to claim 1, wherein The intensity pattern is generated by interference of X-rays.

4. The microscope according to claim 1, wherein The intensity pattern is a stripe pattern having an intensity that repeats in one direction and extends in another direction that is not parallel to the one direction, and is included in the extension of the sheet-like region.

5. The microscope according to claim 4, wherein: The X-ray irradiation unit includes: An X-ray focusing unit is provided in an optical path of X-rays emitted from an X-ray source and passing through the slit, and generates at least one X-ray sheet beam narrowed in a certain direction; and A diffraction grating is disposed in the optical path of the X-ray sheet beam and generates the patterned X-ray sheet beam having the fringe pattern.

6. The microscope according to claim 4, wherein: The X-ray irradiation unit includes: An X-ray focusing unit is provided in the optical path of X-rays emitted from the X-ray source and passing through the slit, and generates at least one X-ray sheet beam narrowed in a certain direction; An X-ray branching section is provided in an optical path of the X-ray sheet beam and generates a first X-ray sheet beam and a second X-ray sheet beam that are both narrowed in a certain direction; and The X-ray interferometer adjusts the directions of the first X-ray sheet beam and the second X-ray sheet beam so that they propagate non-parallel to each other, thereby generating the patterned X-ray sheet beam having the fringe pattern through interference.

7. The microscope according to claim 4, wherein: The X-ray irradiation unit includes: An X-ray focusing unit is provided in the optical path of X-rays emitted from the X-ray source and passing through the slit, and generates at least one X-ray sheet beam narrowed in a certain direction; and An X-ray prism is disposed in the optical path of the X-ray sheet beam and generates at least one of a first X-ray sheet beam and a second X-ray sheet beam that are both narrowed in a certain direction. The first X-ray sheet beam and the second X-ray sheet beam are configured to propagate non-parallel to each other and generate the patterned X-ray sheet beam having the fringe pattern through interference.

8. The microscope according to claim 1, wherein The X-ray irradiation unit includes: an X-ray limiting element disposed in an optical path of X-rays emitted from an X-ray source and having a predetermined transmission pattern; and The X-ray focusing unit is provided downstream of the X-ray limiting element in the optical path of the X-rays and exhibits focusing properties on the X-rays in two directions intersecting the optical path of the X-rays.

9. The microscope according to claim 1, wherein: The intensity pattern is a two-dimensional grid pattern in the extension of the patchy area.

10. The microscope according to claim 1, wherein: The sheet-like region includes a plurality of single-sheet regions each having an extension, and the single-sheet regions are arranged to be stacked on top of each other. The intensity pattern of the patterned X-ray sheet beam is a multilayer pattern formed by laminating separate single-layer patterns having expansion in each single-sheet region. The single layer pattern has an intensity pattern with repeated intensities in a partial region of each single-piece region.

11. The microscope according to claim 1, wherein: The sheet-like region has a thickness in the normal direction of the extension, The intensity pattern of the patterned X-ray sheet beam has a repetitive intensity with respect to the normal direction in a partial region within a range determined by the spread and the thickness.

12. The microscope according to claim 1, further comprising a pattern shifter that shifts the relative arrangement of the intensity of the intensity pattern with respect to the observation object in at least one of a repetitive direction of intensity or a normal direction of the expansion of the sheet-shaped region.

13. A method for photographing an image of a luminescent substance using the microscope according to claim 1, the method comprising: a step of arranging a labeled portion of the observation object labeled with an X-ray-excited luminescent substance in the partial region; an irradiation stage of generating the patterned X-ray sheet beam by using the X-ray irradiation unit; and The imaging stage is a stage in which the observation optical system receives radiation of the observation wavelength from the X-ray excited luminescent substance.

14. The method for capturing an image of a luminescent substance according to claim 13, further comprising a pattern shifting stage, wherein the relative arrangement of the intensity of the intensity pattern relative to the observation object in the irradiation stage is shifted in a direction of repetition of the intensity of the intensity pattern. In the photographing method, the photographing phase is repeatedly performed multiple times with the pattern shifting phase interposed therebetween.

15. The method for capturing an image of a luminescent substance according to claim 14, wherein: The observation optical system receives radiation of the observation wavelength a plurality of times corresponding to the plurality of imaging stages, and records a plurality of element images in the image recording unit. The imaging method further includes an image reconstruction step of reconstructing an observation image based on the plurality of element images recorded in the image recording unit.

16. The method for photographing an image of a luminescent substance according to claim 13, further comprising a pattern rotation stage, wherein a relative direction of a repetition direction of the intensity of the intensity pattern with respect to the observation object is changed in the pattern rotation stage.

Images