Observation method and observation device

Abstract

An observation method according to one aspect comprises: a step for disposing an object to be observed at a non-focal position different from a focal position of a condensing optical system on an optical path; a step for irradiating, via the condensing optical system, the object to be observed disposed at the non-focal position with light having a wavelength longer than the size of the object to be observed; and a step for receiving, by a light receiving surface, the light, which is irradiated to the object to be observed and attenuated by Rayleigh scattering, to generate an image of the object to be observed.

Description

Observation method and observation device

[0001] The present disclosure relates to an observation method and an observation device.

[0002] Optical microscopes using visible light are widely used to observe samples. The spatial resolution of optical microscopes is limited to approximately 200 nm due to the diffraction limit of visible light. When higher spatial resolution is required, samples are observed using X-rays or electron beams, which have shorter wavelengths than visible light. For example, Non-Patent Documents 1 to 3 describe techniques for observing samples at micrometer- or nanometer-scale resolution using X-ray microscopes or scanning electron microscopes. Generally, when observing a sample using an X-ray microscope or scanning electron microscope, the sample is encapsulated in a capsule that maintains atmospheric pressure, and the interior of the microscope body is evacuated to observe the sample in liquid. In addition, to reduce damage to the sample caused by the electron beam, pretreatment is often performed, such as coating the surface of the sample with gold or platinum or staining the sample with a heavy metal.

[0003] In recent years, super-resolution fluorescence microscopes have been developed that observe samples by labeling them with specific fluorescent labels, detecting the fluorescence, and performing various image processing. Super-resolution fluorescence microscopes enable the observation of samples with high resolution, exceeding the diffraction limit of optical microscopes.

[0004] S.C. Mayo,T.J. Davis, T.E. Gureyev, P.R. Miller, D. Paganin, A. Pogany, A.W. Stevenson,and S.W. Wilkins, "X-ray phase-contrast microscopy andmicrotomography", Optics Express, Vol. 11, Issue 19, pp. 2289-2302, 2003TakashiKimura, Yasumasa Joti, Akemi Shibuya, Changyong Song, Sangsoo Kim, KensukeTono, Makina Yabashi, Masatada Tamakoshi, Toshiyuki Moriya, Tairo Oshima,Tetsuya Ishikawa, Yoshitaka Bessho & Yoshinori Nishino, "Imaging livecell in micro-liquid enclosure by X-ray laser diffraction", NatureCommunications, volume 5, Article number 3052, 2014EugeniuBalaur, Guido A. Cadenazzi, Nicholas Anthony, Alex Spurling, Eric Hanssen,Jacqueline Orian, Keith A. Nugent, Belinda S. Parker & Brian Abbey,"Plasmon-induced enhancement of ptychographic phase microscopy viasub-surface nanoaperture arrays", Nature Photonics, volume 15, pages222-229,2021

[0005] As described above, general optical microscopes use the diffraction phenomenon of light to observe objects, and therefore have a resolution limit due to the diffraction of light. On the other hand, scanning electron microscopes and super-resolution fluorescence microscopes can observe objects with high spatial resolution on the micrometer or nanometer scale, but require pretreatment such as staining or fluorescent labeling of the objects, making it difficult to directly observe the objects.

[0006] Therefore, an object of the present disclosure is to provide an observation method and an observation device that are capable of optically observing an observation object with high resolution.

[0007] An observation method according to one embodiment includes the steps of placing an object to be observed at a non-focal position on an optical path that is different from the focal position of a focusing optical system, irradiating the object to be observed placed at the non-focal position with light having a wavelength longer than the size of the object to be observed via the focusing optical system, and receiving the light irradiated onto the object to be observed and attenuated by Rayleigh scattering at a light-receiving surface to generate an image of the object to be observed.

[0008] Conventional optical microscopes perform observation by focusing diffracted light generated when light is irradiated onto an object to be observed. According to Abbe's theory of diffraction limit, the resolution of an optical microscope is limited to the wavelength of light, making it difficult for conventional optical microscopes to observe an object using light with a wavelength longer than the size of the object. In contrast, the observation method according to the present embodiment irradiates an object to be observed, positioned at a non-focal position, with light with a wavelength longer than the size of the object. When light is irradiated onto a tiny object to be observed, positioned at a non-focal position, Rayleigh scattering occurs, and the light is attenuated by the energy of the scattered light and received by the light-receiving surface. Meanwhile, light that is not irradiated onto the object to be observed is received by the light-receiving surface without attenuation. Therefore, this observation method allows an image of the object to be obtained based on the contrast in light intensity between the light irradiated onto the object to be observed and the light that is not irradiated onto the object to be observed. In other words, in the above observation method, the object to be observed is observed using the attenuation of light caused by Rayleigh scattered light rather than diffracted light, so that the object to be observed can be optically observed with high resolution exceeding the diffraction limit without any special pre-processing.

[0009] The light irradiated onto the observation object may be visible light. By using visible light, the observation object can be observed under atmospheric pressure.

[0010] The light irradiated onto the object of observation may be laser light having a wavelength of 340 nm to 550 nm. The probability of Rayleigh scattering occurring is inversely proportional to the fourth power of the wavelength. Therefore, by irradiating the object of observation with light having a short wavelength among visible light, the attenuation effect due to Rayleigh scattering can be increased. As a result, a high-contrast image of the object of observation can be generated.

[0011] The non-focal position may be located closer to the light-receiving surface than the focal position. By locating the object to be observed closer to the light-receiving surface than the focal position, the object to be observed is irradiated with diffused light that widens as it moves from the focal position toward the rear of the optical path. As a result, a magnified image of the object to be observed can be observed on the light-receiving surface.

[0012] The optical path length between the focal position and the non-focal position may be 0.1 μm or more, in which case an enlarged image of the observation object can be observed on the light receiving surface.

[0013] The ratio of the optical path length between the focal position and the light-receiving surface to the optical path length between the focal position and the non-focal position may be 100 or more and 10,000 or less. The magnification of the image of the observation object observed on the light-receiving surface is proportional to the ratio of the optical path length between the focal position and the light-receiving surface to the optical path length between the focal position and the non-focal position. By making this ratio 100 or more, it is possible to observe an image of the observation object at a high magnification. By making this ratio 10,000 or less, it is possible to suppress a decrease in the brightness of the image observed on the light-receiving surface.

[0014] The observation method may further include the steps of moving the object to be observed in a direction intersecting the optical path, and periodically capturing images of the object to be observed received on the light-receiving surface while moving the object to be observed, thereby generating a plurality of images with different angles of incidence of light on the object to be observed. Since the angle of incidence of light changes depending on the position of the object to be observed, a plurality of images with different angles of incidence of light can be generated by periodically outputting images of the object to be observed while moving the object to be observed.

[0015] The observation method may further include a step of generating a three-dimensional model of the observed object by reconstructing the plurality of images taken at different angles of incidence. By generating the three-dimensional model by reconstructing the plurality of images taken at different angles of incidence, the three-dimensional structure of the observed object can be analyzed in detail.

[0016] An observation device according to one embodiment includes a light source that outputs light having a wavelength longer than the size of an object to be observed, a focusing optical system that is arranged on the optical path of the light and focuses the light at a focal position, a support having a support surface that supports the object to be observed, the support surface being arranged at a non-focal position that is different from the focal position on the optical path, and an image sensor that receives, at a light-receiving surface, transmitted light that has been attenuated by Rayleigh scattered light generated by irradiating the object to be observed with light, and generates an image of the object to be observed.

[0017] In the above observation device, the object to be observed is observed using the attenuation of light caused by Rayleigh scattered light rather than diffracted light, so that the object to be observed can be optically observed with a high resolution that exceeds the diffraction limit without any special pre-processing.

[0018] The light output from the light source may be visible light. By using visible light, an object to be observed can be observed under atmospheric pressure.

[0019] The light output from the light source may be laser light having a wavelength of 340 nm to 550 nm. By irradiating the object to be observed with light of a short wavelength, the attenuation effect due to Rayleigh scattering is increased, making it possible to generate an image with high contrast.

[0020] The non-focal position may be located closer to the light-receiving surface than the focal position. By locating the object to be observed closer to the light-receiving surface than the focal position, the object to be observed is irradiated with diffused light that widens as it moves from the focal position toward the rear of the optical path. As a result, a magnified image of the object to be observed can be observed on the light-receiving surface.

[0021] The optical path length between the focal position and the non-focal position may be 0.1 μm or more, in which case an enlarged image of the observation object can be observed on the light receiving surface.

[0022] The ratio of the optical path length between the focal position and the light-receiving surface to the optical path length between the focal position and the non-focal position may be 100 or more and 10,000 or less. By making the optical path length ratio 100 or more, it is possible to observe an image of an observation object at a high magnification. By making the optical path length ratio 10,000 or less, it is possible to suppress a decrease in the brightness of the image observed on the light-receiving surface.

[0023] The center line average roughness of the support may be 100 nm or less, which prevents the unevenness of the support from being seen when observing an object to be observed.

[0024] The support may have a flow path for a solution containing the object to be observed. By continuously or periodically observing and analyzing the object to be observed while flowing the solution containing the object to be observed through the flow path, it is possible to obtain information on the distribution of the object to be observed in the solution.

[0025] The observation device may further include a drive device that moves the support in a direction intersecting the optical axis, and the imaging element may periodically capture images of the observation object received on the light-receiving surface while moving the support, thereby generating multiple images with different angles of incidence of light on the observation object. Since the angle of incidence of light changes depending on the position of the observation object, multiple images with different angles of incidence of light can be generated by periodically capturing images of the observation object while moving the support.

[0026] The observation device may further include an image processing device that reconstructs a plurality of images to generate a three-dimensional model of the observation object, making it possible to analyze the three-dimensional structure of the observation object in detail.

[0027] According to the present disclosure, an object to be observed can be optically observed with high resolution.

[0028] 1 is a diagram schematically illustrating an observation device according to one embodiment; FIG. 2 is a diagram schematically illustrating laser light emitted from an objective lens; (a) is a diagram schematically illustrating an image generated on the light-receiving surface when a micrometer-scale observation object is observed, and (b) is a diagram schematically illustrating an image generated on the light-receiving surface when a nanometer-scale observation object is observed; and (b) is a flowchart illustrating an observation method according to one embodiment; (a) is a diagram illustrating the optical path of white light when a white light source is turned on; and (b) is a diagram illustrating the optical path of laser light when a laser light source is turned on. A diagram schematically illustrating a modified example of a support; and a diagram schematically illustrating another modified example of a support. A diagram schematically illustrating an observation device according to another embodiment; (a), (b), and (c) are diagrams illustrating the relationship between the position of an observation object and the angle of incidence of laser light. A flowchart illustrating an observation method according to another embodiment; (a), (b), (c), and (d) show images of polystyrene beads produced in a comparative experimental example; and (a) and (b) show images of a sunscreen agent containing fine particles of an ultraviolet reflecting agent produced in an experimental example.

[0029] Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. In the following description, the same or equivalent elements will be denoted by the same reference numerals, and redundant description will not be repeated. The dimensional ratios of the drawings do not necessarily correspond to those in the description.

[0030] Fig. 1 is a diagram schematically illustrating an observation device 1 according to one embodiment. The observation device 1 shown in Fig. 1 is a microscope that generates a magnified image of an observation object 2. The observation device 1 is capable of observing the observation object 2 with a spatial resolution on the micrometer or nanometer scale without subjecting the observation object 2 to pretreatment such as staining or fixation.

[0031] The observation object 2 is a minute nanoparticle or nanostructure on the nanometer scale. For example, the size of the observation object 2 is 1000 nm or less, 500 nm or less, 300 nm or less, 200 nm or less, or 100 nm or less. The size of the observation object 2 typically refers to the diameter of the observation object 2. If the planar shape of the observation object 2 is not circular, the size of the observation object 2 refers to the maximum width of the observation object 2 in the direction perpendicular to the optical path.

[0032] For example, the observation object 2 may be an organic sample such as bacteria, viruses, proteins, or protein complexes, or may be ceramic or metal particles. The observation object 2 may also be a liquid food such as milk or mayonnaise, a cosmetic product such as sunscreen or hand cream, or an industrial lubricant such as machine oil or gear oil.

[0033] In the following description, "optical path" refers to the path of light output from a light source, and "optical path length" refers to the distance between two objects along the optical path. In addition, the direction along the optical path closer to the light source is sometimes referred to as the "front side" of the optical path, and the direction along the optical path away from the light source is sometimes referred to as the "rear side" of the optical path.

[0034] As shown in Fig. 1, the observation device 1 includes a laser light source 11, a white light source 12, a focusing optical system 13, a support 14, a first image sensor 15, a second image sensor 16, and a control device 17. The observation device 1 shown in Fig. 1 is an upright microscope that observes an observation object 2 from above, but it may also be an inverted microscope that observes an observation object 2 from below. In this case, the components of the observation device 1 are arranged upside down.

[0035] The laser light source 11 outputs laser light L1 having a wavelength longer than the size of the observation object 2. For example, the wavelength of the laser light L1 may be at least twice the size of the observation object 2. The laser light L1 is used to observe the observation object 2. The laser light L1 output from the laser light source 11 is visible light with a single wavelength. For example, the laser light L1 has a wavelength of 340 nm to 550 nm.

[0036] The white light source 12 is a light source that outputs broadband white light L2. For example, a white LED or a halogen lamp light source is used as the white light source 12. The white light L2 is used to identify the focal position Pf of the objective lens 23, which will be described later.

[0037] The focusing optical system 13 is disposed on the optical paths of the laser light L1 and the white light L2, and includes a first beam splitter 21, a second beam splitter 22, and an objective lens 23. The first beam splitter 21 is disposed, for example, on the optical axis Z of the objective lens 23, and reflects the laser light L1 output from the laser light source 11 and outputs it toward the objective lens 23. The first beam splitter 21 also outputs the light that has been reflected by the observation object 2 and transmitted through the second beam splitter 22 toward the second image sensor 16.

[0038] The second beam splitter 22 is disposed, for example, on the optical axis Z of the objective lens 23, and reflects the white light L2 output from the white light source 12 and outputs it toward the objective lens 23. The second beam splitter 22 also transmits the light reflected by the observation object 2 and outputs it toward the first beam splitter 21.

[0039] The objective lens 23 is provided on the optical path of the laser light L1 reflected by the first beam splitter 21 and the white light L2 reflected by the second beam splitter 22, and is disposed so as to face the observation object 2. The objective lens 23 receives the laser light L1 or the white light L2 and focuses it at a focal position Pf. For example, the objective lens 23 is a focusing lens having a magnification of 40 times or more and 100 times or less and a numerical aperture of 0.8 or more and 1.5 or less. The objective lens 23 also transmits light reflected by the observation object 2 due to irradiation with the white light L2 and outputs it to the first beam splitter 21.

[0040] The first imaging element 15 is an image sensor having a two-dimensional pixel structure, such as a CCD image sensor or a CMOS image sensor. The first imaging element 15 has a light-receiving surface 15a that is arranged on the optical axis Z of the objective lens 23 so as to face the objective lens 23. The first imaging element 15 is irradiated with laser light L1, receives light that has passed through the observation object 2 on the light-receiving surface 15a, and generates an image of the observation object 2 that has been received. The first imaging element 15 captures the image of the observation object 2 that has been received, and outputs the image of the observation object 2.

[0041] The second imaging element 16 is an image sensor having a two-dimensional pixel structure, such as a CCD image sensor or a CMOS image sensor. The second imaging element 16 has a light receiving surface 16a that is positioned on the optical axis Z of the objective lens 23 so as to face the objective lens 23. A first beam splitter 21 and a second beam splitter 22 are disposed between the second imaging element 16 and the objective lens 23. The second imaging element 16 receives light that is reflected by the observation object 2 when irradiated with white light L2, and outputs an image according to the intensity of the received light.

[0042] The control device 17 is a computer equipped with a processor, a storage device, an input device, a display device, a communication device, etc., and controls the overall operation of the observation device 1. The control device 17 is communicatively connected to the first image sensor 15 and the second image sensor 16, and acquires images of the observation object 2 output from the first image sensor 15 and the second image sensor 16. The control device 17 may correct the acquired images by performing various image processing related to the observation of the observation object 2. The control device 17 is also communicatively connected to the laser light source 11, the white light source 12, and the support body 14, and can turn on or off the laser light source 11 and the white light source 12, and adjust the position of the support body 14 in a direction along the optical axis Z (hereinafter referred to as the "optical axis direction").

[0043] The support 14 has a support surface 14a that supports the observation object 2, and is disposed between the first image sensor 15 and the objective lens 23. Typically, the support surface 14a is disposed perpendicular to the optical axis Z so as to face the objective lens 23. The support 14 is made of a material that is highly transmissive to the laser light L1, such as thin-film glass or silicon nitride. The center line average roughness (Ra) of the support 14 may be 100 nm or less so that the irregularities of the support 14 are not visible when observing the observation object 2.

[0044] The support 14 may be movable in the optical axis direction of the objective lens 23. The position of the support 14 in the optical axis direction is controlled by, for example, the control device 17. When observing the observation object 2, the support surface 14a of the support 14 is positioned at a non-focal position Pn, which is different from the focal position Pf, on the optical path of the laser light L1 (see FIG. 2). For example, the non-focal position Pn where the support surface 14a is positioned is a position closer to the light receiving surface 15a of the first image sensor 15 than the focal position Pf. In other words, in the optical axis direction of the objective lens 23, the non-focal position Pn is positioned behind the focal position Pf.

[0045] 2 is a diagram schematically illustrating the laser light L1 emitted from the objective lens 23. As shown in Fig. 2, the laser light L1 emitted from the objective lens 23 is focused at a focal position Pf. Therefore, between the objective lens 23 and the focal position Pf, the laser light L1 becomes converging light whose beam diameter decreases as it approaches the focal position Pf, and between the focal position Pf and the light-receiving surface 15a, the laser light L1 becomes diverging light whose beam diameter increases as it moves away from the focal position Pf.

[0046] By arranging the support surface 14a on the rear side of the focal position Pf, the object of observation 2 supported on the support surface 14a is irradiated with diffused light of the laser light L1. This diffused light passes through the object of observation 2 and forms an enlarged image of the object of observation 2 on the light-receiving surface 15a arranged on the rear side in the optical axis direction.

[0047] 2, when the optical path length between the focal position Pf and the non-focal position Pn is D1 and the optical path length between the focal position Pf and the light-receiving surface 15a of the first image sensor 15 is D2, an image of the observation object 2 magnified at a magnification determined by the ratio (D2 / D1) of the optical path length D2 to the optical path length D1 is received on the light-receiving surface 15a of the first image sensor 15. For example, when the optical path length D1 is 10 μm and the optical path length D2 is 10 mm, an image of the observation object 2 placed at the non-focal position Pn magnified 1000 times is observed on the light-receiving surface 15a.

[0048] In one embodiment, the optical path length D1 between the focal position Pf and the non-focal position Pn may be 0.1 μm or more. By setting the optical path length D1 to 0.1 μm or more, diffused light of the laser light L1 can be irradiated onto the observation object 2, and a magnified image of the observation object 2 can be observed on the light-receiving surface 15a. The optical path length D1 may be 100 μm or less. Furthermore, D2 / D1 may be 100 or more and 10,000 or less. By setting D2 / D1 to 100 or more, an image of the observation object 2 can be observed at a high magnification. By setting D2 / D1 to 10,000 or less, a decrease in the brightness of the image observed on the light-receiving surface 15a can be suppressed.

[0049] FIG. 3( a) schematically illustrates an image observed on the light-receiving surface 15a when a micrometer-scale observation object 2 is observed, and FIG. 3( b) schematically illustrates an image observed on the light-receiving surface 15a when a nanometer-scale observation object 2 is observed. The micrometer-scale observation object 2 has a size larger than the wavelength of the laser light L1. Therefore, as shown in FIG. 3( a), when the micrometer-scale observation object 2 is irradiated with visible laser light L1, photons Ph of the laser light L1 are diffracted by the observation object 2, and interference fringes associated with the diffraction of light are generated around the image 2i of the observation object 2 observed on the light-receiving surface 15a. Therefore, the image output from the first image sensor 15 has low spatial resolution.

[0050] In contrast, as shown in FIG. 3B, when the laser light L1 is irradiated onto an observation object 2 on a nanometer scale, which is smaller than the wavelength of the laser light L1, the photons incident on the observation object 2 undergo Rayleigh scattering without diffraction, generating scattered light Ls. As a result, the light is attenuated by the energy of the scattered light Ls, and the intensity (number of photons) of the light received by the light-receiving surface 15a decreases. Note that Rayleigh scattering occurs when the size of the observation object 2 is sufficiently smaller than the wavelength of the incident light, and does not occur when observing an observation object 2 on a micrometer scale. On the other hand, photons that do not enter the observation object 2 travel straight without undergoing Rayleigh scattering. As a result, the light of the laser light L1 that does not enter the observation object 2 is received by the light-receiving surface 15a without being attenuated. Therefore, an image 2i of the observation object 2 is generated on the light-receiving surface 15a, which has a contrast in light intensity between the light that entered the observation object 2 and the light that did not enter the observation object 2. Therefore, it becomes possible to observe the observation object 2 on a nanometer scale with high spatial resolution.

[0051] The probability of Rayleigh scattering occurring is inversely proportional to the fourth power of the wavelength. Therefore, by irradiating the observation object 2 with visible light having a short wavelength, Rayleigh scattering is more likely to occur, and the attenuation effect due to Rayleigh scattering can be promoted. Therefore, the laser light source 11 may output laser light L1 having a wavelength of 340 nm to 550 nm. By using laser light L1 with such a short wavelength, the attenuation effect due to Rayleigh scattering is increased, and the contrast of the image 2i of the observation object 2 received by the light receiving surface 15a can be increased.

[0052] Conventional optical microscopes perform observation by collecting diffracted light that is generated when light is irradiated onto an observation object 2. According to Abbe's theory of the diffraction limit, the resolution of an optical microscope is limited to approximately the wavelength of light, so conventional optical microscopes have difficulty observing the observation object 2 using light with a wavelength longer than the size of the observation object 2. In contrast, the observation device 1 observes the observation object 2 using the light attenuation effect caused by Rayleigh scattered light, and therefore can optically observe the observation object 2 with a high resolution that exceeds the diffraction limit.

[0053] Next, a method for observing the observation object 2 using the observation device 1 will be described with reference to Fig. 4. Fig. 4 is a flowchart showing the observation method according to one embodiment.

[0054] 4, in an observation method according to one embodiment, the observation object 2 is first placed on the support surface 14a of the support 14 of the observation device 1 (step ST1). The observation object 2 placed on the support surface 14a is a minute nanoparticle or nanostructure on the nanometer scale. At this time, the observation object 2 is placed on the support surface 14a under atmospheric pressure without undergoing pretreatment such as staining. The observation object 2 may be placed on the support surface 14a in a state of being suspended in an aqueous solution.

[0055] Next, the user of the observation device 1 turns on the white light source 12 (step ST2). As a result, white light L2 is irradiated from the white light source 12 onto the second beam splitter 22, as shown in FIG. 5 . The white light L2 output from the white light source 12 is reflected by the second beam splitter 22 and irradiated onto the observation object 2 on the support surface 14a via the objective lens 23. The white light L2 irradiated onto the observation object 2 is reflected by the support surface 14a, passes through the objective lens 23, the second beam splitter 22, and the first beam splitter 21 in this order, and is received by the light-receiving surface 16a of the second image sensor 16. The second image sensor 16 generates an image according to the reflected light received by the light-receiving surface 16a.

[0056] Because the size of the observation object 2, which is on the nanometer scale, is below the resolution of a typical optical microscope, an image of the observation object 2 cannot be observed in the image output from the second image sensor 16. On the other hand, the support 14 can be observed in the image output from the second image sensor 16. The user of the observation device 1 moves the support 14 in the optical axis direction of the objective lens 23 to focus on the support surface 14a of the support 14, thereby identifying the focal position Pf of the objective lens 23 (step ST3).

[0057] Next, the user moves the support 14 in the optical axis direction to position the support surface 14 a at a non-focal position Pn different from the focal position Pf (step ST4). For example, the user moves the support surface 14 a 0.1 μm or more behind the focal position Pf in the optical path of the laser beam L1.

[0058] Next, the user turns off the white light source 12 and turns on the laser light source 11 (step ST5). As a result, as shown in FIG. 6 , the laser light source 11 irradiates the first beam splitter 21 with laser light L1. The laser light L1 output from the laser light source 11 is reflected by the first beam splitter 21 and irradiates the observation object 2 on the support surface 14a located at the non-focal position Pn via the objective lens 23. At this time, the observation object 2 is irradiated with diffused light of the laser light L1. This diffused light passes through the observation object 2 and is received by the light receiving surface 15a of the first image sensor 15 located on the rear side in the optical axis direction.

[0059] When the laser light L1 is irradiated onto the observation object 2, which is micrometer-sized and smaller than the wavelength of the laser light L1, photons incident on the observation object 2 undergo Rayleigh scattering without diffracting, generating scattered light. As a result, the light is attenuated by the energy of the scattered light, and the intensity (number of photons) of the light received by the light-receiving surface 15a decreases. On the other hand, photons that do not enter the observation object 2 travel straight without undergoing Rayleigh scattering. As a result, the light of the laser light L1 that does not enter the observation object 2 is received by the light-receiving surface 15a without being attenuated. Therefore, an image 2i of the observation object 2 is generated on the light-receiving surface 15a, which has a contrast in light intensity between the light that entered the observation object 2 and the light that did not enter the observation object 2.

[0060] The first image sensor 15 outputs an image corresponding to the intensity of the light received by the light receiving surface 15a (step ST6). The control device 17 acquires the image output from the first image sensor 15 and displays it on a display device (not shown). The image includes an image 2i of the observation object 2 magnified by a magnification determined by D2 / D1. The user can observe the observation object 2 using the image.

[0061] As described above, the observation method shown in Figure 4 observes the object 2 using the attenuation of light due to Rayleigh scattering rather than diffracted light, so the object 2 can be optically observed with a high resolution exceeding the diffraction limit without staining or fixation. Furthermore, high resolution can be achieved with a simple device configuration that adds a laser light source 11 and a first image sensor 15 to a conventional optical microscope. This makes it possible to directly observe various objects 2 with nano-level resolution, which can be used in a wide range of industrial fields, such as the development of nanomaterials, food analysis, and drug analysis.

[0062] Next, a modified example of the support 14 will be described. Fig. 7 shows a support 14A according to this modified example. As shown in Fig. 7, the support 14A includes a pair of thin films 26 that face each other via a spacer 25. The inner surface of one of the pair of thin films 26 provides a support surface 14a that supports the observation object 2. When the observation object 2 is observed using the support 14A, an emulsion solution containing the observation object 2 and the solution 3 is enclosed between the pair of thin films 26 and observed.

[0063] When observing the observation object 2, the support surface 14a of the support 14A is placed at a non-focal position Pn, which is different from the focal position Pf, on the optical path of the laser light L1, and the laser light L1 is irradiated onto the observation object 2 inside the support 14A. As a result, the laser light L1 passes through the observation object 2 and an image 2i of the observation object 2 is formed on the light-receiving surface 15a, which is placed on the rear side in the optical axis direction.

[0064] Fig. 8 shows a support 14B according to another modified example. As shown in Fig. 8, the support 14B has a pair of thin films 31 and a flow path 32 formed between the pair of thin films 31. When observing the observation object 2, the support 14B is placed at a non-focal position Pn different from the focal position Pf, and an emulsion solution containing the observation object 2 and a solution 3 is supplied to the flow path 32. Then, the laser light L1 is irradiated onto the solution 3 in the support 14B, and an enlarged image of the observation object 2 contained in the solution 3 is observed on the light-receiving surface 15a.

[0065] 8 , the control device 17 continuously or periodically acquires images of the observation object 2 from the first image sensor 15 to generate a moving image including a plurality of frames 36 in which the observation object 2 is captured. The control device 17 performs image analysis processing on the generated moving image to acquire information such as the particle size distribution (e.g., particle size distribution) or shape distribution (e.g., circularity distribution) of the observation object 2 contained in the solution 3. The flow rate of the solution 3 flowing through the flow path 32 is set according to the frame rate of the moving image. For example, the solution 3 may flow through the flow path 32 at a flow rate that allows the same observation object 2 to be imaged twice.

[0066] Next, an observation device according to another embodiment will be described. Fig. 9 is a diagram schematically showing an observation device 1A according to another embodiment. The observation device 1A differs from the observation device 1 shown in Fig. 1 in that the observation device 1A acquires an image of the observation target 2 while moving the support 14. In the following explanation, differences from the observation device 1 will be mainly described, and overlapping explanations will be omitted.

[0067] As shown in Fig. 9, the observation device 1A includes a drive device 30 that moves the support 14. The drive device 30 includes an actuator such as a pneumatic or hydraulic cylinder, a motor, or a piezoelectric element, and is connected to the support 14. The operation of the drive device 30 is controlled by, for example, the control device 17. The drive device 30 moves the support 14 in a direction intersecting the optical axis direction. More specifically, the drive device 30 moves the support 14 so as to cross the laser light L1 along a plane that is perpendicular to the optical axis Z of the objective lens 23 and includes the non-focal position Pn.

[0068] The control device 17 continuously or periodically observes the observation object 2 while moving the support 14. When the laser light L1 is irradiated while moving the support 14, the angle of incidence of the light changes depending on the position of the observation object 2. For example, as shown in FIG. 10( a), when the observation object 2 is located to the right of the optical axis Z, light traveling from the upper left to the lower right is incident on the observation object 2. Therefore, an image 2i of the observation object 2 observed from the upper left at an angle is received on the light receiving surface 15a. Furthermore, as shown in FIG. 10( b), when the observation object 2 is located on the optical axis Z, light traveling from directly above to directly below is incident on the observation object 2. Therefore, an image 2i of the observation object 2 observed from directly above is received on the light receiving surface 15a. As shown in FIG. 10( c), when the observation object 2 is located to the left of the optical axis Z, light traveling from the upper right to the lower left is incident on the observation object 2. Therefore, the light receiving surface 15a receives an image 2i of the object 2 observed obliquely from the upper left.

[0069] The first imaging element 15 periodically captures an image 2i of the observation object 2 observed on the light receiving surface 15a when the laser light L1 is irradiated onto the observation object 2 while moving the support 14, and outputs a plurality of images 40. The plurality of images 40 are tilted images in which the angles of incidence of light onto the observation object 2 are different from one another.

[0070] In one embodiment, the observation device 1A further includes an image processing device 34 that generates a three-dimensional model of the observation object 2. The image processing device 34 generates a plurality of enlarged images 42 by cutting out an area including an image 2i of the observation object 2 from a plurality of images 40. The image processing device 34 then generates a three-dimensional model of the observation object 2 by reconstructing the plurality of enlarged images 42 according to the angle of incidence of light. The three-dimensional model is a stereoscopic digital model of the observation object 2 created on a computer. The three-dimensional structure of the observation object 2 can be observed by rendering an image from any viewpoint from the three-dimensional model.

[0071] Next, a method for observing an observation object 2 according to another embodiment will be described with reference to Fig. 11. Fig. 11 is a flowchart showing the observation method according to the another embodiment. Each step shown in Fig. 11 is performed using the observation device 1A shown in Fig. 9.

[0072] 11, steps ST11 to ST15 are the same as steps ST1 to ST5 shown in FIG. 4, and therefore will not be described further. In this observation method, while irradiating the laser light L1 onto the observation object 2 placed at the non-focal position Pn, the drive device 30 is driven to move the support 14 in a direction intersecting (perpendicular to) the optical axis Z (step ST16). When the observation object 2 supported on the support 14 moves in accordance with the drive of the drive device 30, the angle of incidence of the laser light L1 with respect to the observation object 2 changes depending on the position of the observation object 2. As a result, an image 2i of the observation object 2 corresponding to the angle of incidence of the laser light L1 is received on the light receiving surface 15a of the first image sensor 15.

[0073] The first image sensor 15 outputs an image corresponding to the intensity of the light received by the light receiving surface 15 a (step ST17). The observation device 1A periodically outputs images of the observation object 2 while moving the support 14 in a direction intersecting the optical axis Z until the movement exceeds a predetermined range, thereby generating a plurality of images 40 in which the angles of incidence of the laser light L1 on the observation object 2 are different from one another (step ST18).

[0074] After the predetermined movement range has been exceeded, the image processing device 34 cuts out an area including the image 2i of the observation object 2 from the plurality of images 40 to generate a plurality of enlarged images 42, and reconstructs these plurality of enlarged images 42 to reconstruct a three-dimensional model of the observation object 2 (step ST19). The user can observe the three-dimensional structure of the observation object 2 by rendering an image from any viewpoint from the three-dimensional model.

[0075] As described above, in the observation method shown in FIG. 11, a three-dimensional model is generated from a plurality of images 40 with different angles of incidence, thereby making it possible to analyze the three-dimensional structure of the observation object 2 in detail.

[0076] Below, we will explain experimental examples and comparative experimental examples of the observation device 1. It should be noted that the present invention is not limited to the following experimental examples.

[0077] In the experimental example and comparative experimental example, an image of the observation object 2 was generated using the observation device 1 shown in FIG. 1 . In the experimental example and comparative experimental example, laser light with a wavelength of 532 nm was used as the laser light L1. A silicon nitride thin film was used as the support 14. In the experimental example, a sunscreen containing fine particles of an ultraviolet reflector (titanium oxide or zinc oxide) with a diameter of 100 nm was used as the observation object 2. In the comparative experimental example, polystyrene beads with a diameter of 1 μm were used as the observation object 2.

[0078] FIGS. 12(a) to 12(d) show images of the observation object 2 (polystyrene beads) generated in the comparative experimental example. FIG. 12(a) is an image of the polystyrene beads (output from the second image sensor 16) generated using white light L2. FIG. 12(b) is an image showing the left portion of the polystyrene beads (output from the first image sensor 15) generated using laser light L1. FIG. 12(c) is an image showing the central portion of the polystyrene beads generated using laser light L1. FIG. 12(d) is an image showing the right portion of the polystyrene beads generated using laser light L1. Because the polyethylene beads have a diameter larger than the wavelength of the laser light L1, as shown in FIG. 12(a), when white light L2 was used, the polystyrene beads could be observed with high contrast. On the other hand, as shown in FIGS. 12(b) to 12(d), when the polystyrene beads were observed using laser light L1, diffraction of the laser light L1 occurred, resulting in an image in which interference fringes appeared around the polystyrene beads. From these results, it was confirmed that when an observation object 2 having a size larger than the wavelength of the laser light L1 is observed using the laser light L1, an image with low resolution is generated.

[0079] On the other hand, Figures 13(a) and (b) show images of the observation object 2 (UV reflector contained in a sunscreen) generated in the experimental example. Figure 13(a) is an image of the UV reflector generated using white light L2 (output from the second image sensor 16). Figure 13(b) is an image of the UV reflector generated using laser light L1 (output from the first image sensor 15). Because the UV reflector has a diameter smaller than the wavelength of the laser light L1, as shown in Figure 13(a), when white light L2 was used, only image noise was observed, and no particulate UV reflector was observed. On the other hand, when laser light L1 was used, the particulate UV reflector or its aggregates were observed with high contrast. These results confirmed that by observing a nanometer-scale observation object 2 using the observation device 1, the observation object 2 can be optically observed with high resolution exceeding the diffraction limit.

[0080] The observation devices 1, 1A and observation methods according to various embodiments have been described above, but the invention is not limited to the above-described embodiments and various modifications can be made within the scope that does not change the gist of the invention.

[0081] 1 , the support body 14, the first image pickup element 15, and the second image pickup element 16 are arranged on the optical axis Z of the objective lens 23, but as long as the support body 14, the first image pickup element 15, and the second image pickup element 16 are arranged on the optical paths of the laser light L1 and the white light L2, they do not have to be arranged on the optical axis Z of the objective lens 23. For example, the laser light L1 and the white light L2 may be reflected by a mirror in a direction intersecting the optical axis Z, and the reflected laser light L1 and the white light L2 may be output to the support body 14, the first image pickup element 15, or the second image pickup element 16.

[0082] Furthermore, although the observation device 1 is equipped with a laser light source 11 that outputs laser light L1, it may also be equipped with a light source that outputs non-laser light instead of the laser light source 11. For example, the observation device 1 may be equipped with a mercury lamp or LED light source that outputs light having a wavelength of 340 nm to 550 nm. If light having a wavelength longer than the size of the observation object 2 that is placed at the non-focal position Pn is irradiated onto the observation object 2, it is possible to observe the observation object 2 with high resolution by utilizing the attenuation of light due to Rayleigh scattering.

[0083] Furthermore, in the above embodiment, the observation object 2 (support surface 14 a) is placed at the non-focal position Pn behind the focal position Pf, but in one embodiment, the observation object 2 (support surface 14 a) may be placed in front of the focal position Pf. Even in this case, Rayleigh scattering occurs, so the observation object 2 can be observed with high resolution.

[0084] The various embodiments described above can be combined to the extent that no contradiction occurs.

[0085] The present disclosure includes the following contents.

[0086] [1] An observation method comprising the steps of: placing an object to be observed at a non-focal position on an optical path that is different from a focal position of a focusing optical system; irradiating the object to be observed placed at the non-focal position with light having a wavelength longer than the size of the object to be observed via the focusing optical system; and receiving the light irradiated to the object to be observed and attenuated by Rayleigh scattering on a light-receiving surface to generate an image of the object to be observed.

[0087] [2] The observation method according to [1], wherein the light irradiated onto the object to be observed is visible light.

[0088] [3] The observation method according to [1] or [2], wherein the light irradiated onto the object to be observed is laser light having a wavelength of 340 nm to 550 nm.

[0089] [4] The observation method according to any one of [1] to [3], wherein the non-focal position is located closer to the light receiving surface than the focal position.

[0090] [5] The observation method according to any one of [1] to [4], wherein the optical path length between the focal position and the non-focal position is 0.1 μm or more.

[0091] [6] The observation method according to any one of [1] to [5], wherein a ratio of an optical path length between the focal position and the light receiving surface to an optical path length between the focal position and the non-focal position is 100 or more and 10,000 or less.

[0092] [7] The observation method according to any one of [1] to [6], further comprising the steps of: moving the object to be observed in a direction intersecting the optical path; and periodically capturing images of the object to be observed that are received on the light-receiving surface while moving the object to be observed, to generate a plurality of images in which the angles of incidence of the light on the object to be observed are different from one another.

[0093] [8] The observation method according to [7], further comprising the step of reconstructing the plurality of images to generate a three-dimensional model of the object to be observed.

[0094] [9] An observation device comprising: a light source that outputs light having a wavelength longer than the size of an object to be observed; a focusing optical system that is arranged on an optical path of the light and focuses the light at a focal position; a support having a support surface that supports the object to be observed, the support surface being arranged at a non-focal position on the optical path that is different from the focal position; and an image sensor that receives, at a light-receiving surface, transmitted light that has been attenuated by Rayleigh scattered light generated by irradiating the object to be observed with the light, and generates an image of the object to be observed.

[0095]

[10] The observation device according to [9], wherein the light output from the light source is visible light.

[0096]

[11] The observation device according to [9], wherein the light output from the light source is laser light having a wavelength of 340 nm to 550 nm.

[0097]

[12] The observation device according to [9] or

[10] , wherein the non-focal position is located closer to the light receiving surface than the focal position.

[0098]

[13] The observation device according to any one of [9] to

[12] , wherein the optical path length between the focal position and the non-focal position is 0.1 μm or more.

[0099]

[14] The observation device according to any one of [9] to

[13] , wherein a ratio of an optical path length between the focal position and the light receiving surface to an optical path length between the focal position and the non-focal position is 100 or more and 10,000 or less.

[0100]

[15] The observation device according to any one of [9] to

[14] , wherein the center line average roughness of the support is 100 nm or less.

[0101]

[16] The observation device according to any one of [9] to

[15] , wherein the support has a flow path for a solution containing the object to be observed.

[0102]

[17] The observation device according to any one of [9] to

[16] , further comprising a drive device that moves the support in a direction intersecting the optical path, wherein the image sensor periodically captures an image of the object of observation received on the light receiving surface while moving the support, thereby generating a plurality of images in which the angles of incidence of the light on the object of observation are different from one another.

[0103]

[18] The observation device according to

[17] , further comprising an image processing device that reconstructs the plurality of images to generate a three-dimensional model of the observation object.

[0104] 1, 1A...observation device, 2...object to be observed, 2i...image of object to be observed, 13...light-collecting optical system, 14, 14A, 14B...support, 14a...support surface, 15a, 16a...light-receiving surface, 30...drive device, 32...flow path, 34...image processing device, 40...plurality of images, D1, D2...optical path length, L1...laser light, Pf...focal position, Pn...non-focal position, Z...optical axis

Claims

1. An observation method comprising the steps of: placing an object to be observed at a non-focal position on an optical path that is different from the focal position of a focusing optical system; irradiating the object to be observed placed at the non-focal position with light having a wavelength longer than the size of the object to be observed via the focusing optical system; and receiving the light irradiated to the object to be observed and attenuated by Rayleigh scattering on a light-receiving surface to generate an image of the object to be observed.

2. The observation method according to claim 1, wherein the light irradiated onto the object to be observed is visible light.

3. The observation method according to claim 1, wherein the light irradiated onto the object to be observed is laser light having a wavelength of 340 nm to 550 nm.

4. The observation method according to claim 1, wherein the non-focus position is located closer to the light receiving surface than the focus position.

5. The observation method according to claim 1, wherein the optical path length between the focal position and the non-focal position is 0.1 μm or more.

6. An observation method according to claim 1, wherein the ratio of the optical path length between the focal position and the light receiving surface to the optical path length between the focal position and the non-focal position is 100 or more and 10,000 or less.

7. The observation method according to claim 1, further comprising the steps of: moving the object to be observed in a direction intersecting the optical path; and periodically capturing images of the object to be observed that are received on the light-receiving surface while moving the object to be observed, thereby generating a plurality of images in which the angles of incidence of the light on the object to be observed are different from one another.

8. The observation method according to claim 7, further comprising the step of reconstructing the plurality of images to generate a three-dimensional model of the observed object.

9. An observation device comprising: a light source that outputs light having a wavelength longer than the size of an object to be observed; a focusing optical system that is arranged on the optical path of the light and focuses the light at a focal position; a support having a support surface that supports the object to be observed, the support surface being arranged at a non-focal position on the optical path that is different from the focal position; and an image sensor that receives, on a light-receiving surface, transmitted light that has been attenuated by Rayleigh scattered light generated by irradiating the object to be observed with the light, and generates an image of the object to be observed.

10. The observation device according to claim 9, wherein the light output from the light source is visible light.

11. The observation device according to claim 9, wherein the light output from the light source is laser light having a wavelength of 340 nm to 550 nm.

12. The observation device according to claim 9, wherein the non-focus position is located closer to the light receiving surface than the focus position.

13. The observation device according to claim 9, wherein the optical path length between the focal position and the non-focal position is 0.1 μm or more.

14. An observation device according to claim 9, wherein the ratio of the optical path length between the focal position and the light receiving surface to the optical path length between the focal position and the non-focal position is 100 or more and 10,000 or less.

15. The observation device according to claim 9, wherein the center line average roughness of the support is 100 nm or less.

16. The observation device according to claim 9, wherein the support has a flow path for a solution containing the object to be observed.

17. An observation device according to claim 9, further comprising a drive device that moves the support in a direction intersecting the optical path, wherein the imaging element periodically captures images of the object of observation received on the light receiving surface while moving the support, thereby generating a plurality of images in which the angles of incidence of the light on the object of observation are different from one another.

18. The observation device according to claim 17, further comprising an image processing device that reconstructs the plurality of images to generate a three-dimensional model of the observed object.

Images