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WO2021210637A1 - Lighting device for microscope, microscope, and observation method - Google Patents

Lighting device for microscope, microscope, and observation method Download PDF

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Publication number
WO2021210637A1
WO2021210637A1 PCT/JP2021/015550 JP2021015550W WO2021210637A1 WO 2021210637 A1 WO2021210637 A1 WO 2021210637A1 JP 2021015550 W JP2021015550 W JP 2021015550W WO 2021210637 A1 WO2021210637 A1 WO 2021210637A1
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WO
WIPO (PCT)
Prior art keywords
light
guide plate
light guide
microscope
end portion
Prior art date
Application number
PCT/JP2021/015550
Other languages
French (fr)
Japanese (ja)
Inventor
小野寺　宏
Original Assignee
国立大学法人東京大学
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Filing date
Publication date
Application filed by 国立大学法人東京大学 filed Critical 国立大学法人東京大学
Publication of WO2021210637A1 publication Critical patent/WO2021210637A1/en

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements

Definitions

  • the present invention relates to a microscope illumination device, a microscope, and an observation method.
  • a multi-photon microscope such as a two-photon microscope and a three-photon microscope may be used for observing the inside of a living tissue (see, for example, Patent Document 1).
  • a multi-photon microscope one fluorescent molecule is excited by a plurality of photons at the same time.
  • the multiphoton microscope uses near-infrared pulsed laser light having a wavelength that is multiple times the normal excitation wavelength. Since a long wavelength laser beam is used, scattering of excitation light in living tissue is suppressed. Further, in multiphoton excitation, excitation of fluorescent molecules existing other than the focal plane is unlikely to occur. Therefore, according to the multi-photon microscope, it is possible to obtain a high-resolution observation image.
  • the price of a multiphoton microscope is as high as about 100 million yen, and the multiphoton microscope has problems that it is difficult to operate and it takes time to take a picture.
  • an optical sheet microscope may be used for observing the inside of living tissue (see, for example, Patent Document 2).
  • a sheet-shaped excitation light focused by a cylindrical lens and an objective lens is irradiated from the side of the sample to obtain an optical cross-sectional image of the sample.
  • the sample if the sample has an opaque portion, the excitation light cannot advance beyond the opaque portion. Therefore, when observing the sample with the optical sheet microscope, the sample is rotated with respect to the excitation light, and it takes time to observe the sample with the optical sheet microscope. Also, since the sample is rotated, the resolution may decrease. Further, in the optical sheet microscope, the excitation light can be scattered inside the sample, so that the transparency of the sample is preferably high.
  • One of the objects of the present invention is to provide an inexpensive microscope illumination device, a microscope, and an observation method capable of observing a cross section of an observation target not limited to a living tissue.
  • a light guide plate having a light source for irradiating light, a light incident end portion where light is incident inside, and a light emitting end portion where light is emitted from the inside in a sheet shape is provided.
  • a microscope lighting device for illuminating an observation target with sheet-like light emitted from a light emitting end portion is provided.
  • a hole may be provided in the light guide plate, the light incident end may be located on the outer periphery of the light guide plate, and the light emitting end may be located on the inner circumference of the hole in the light guide plate. ..
  • the above-mentioned illumination device for a microscope may include a plurality of light sources that inject light into a light guide plate.
  • the light guide plate may be provided with a transparent substrate.
  • the transparent substrate may contain silicon oxide.
  • the transparent substrate may contain a resin.
  • the light guide plate may include a reflecting portion facing the light guide plate.
  • the above-mentioned illumination device for a microscope may further include an observation target moving device that moves the observation target in the direction perpendicular to the surface of the light guide plate.
  • the above-mentioned illumination device for a microscope may further include a light guide plate moving device that moves the light guide plate in a direction perpendicular to the surface of the light guide plate.
  • a light guide plate having a light source for irradiating light, a light incident end portion where light is incident inside, and a light emitting end portion where light is emitted from the inside in a sheet shape.
  • a microscope comprising a lens for observing an observation object irradiated with sheet-like light emitted from a light emitting end portion of a light guide plate.
  • the light guide plate may be provided with a hole, the light incident end may be located on the outer circumference of the light guide plate, and the light emitting end may be located on the inner circumference of the hole in the light guide plate.
  • the above microscope may be provided with a plurality of light sources that inject light into the light guide plate.
  • the light guide plate may be provided with a transparent substrate.
  • the transparent substrate may contain silicon oxide.
  • the transparent substrate may contain a resin.
  • the light guide plate may include a reflecting portion facing the light guide plate.
  • the light guide plate may be arranged in the direction perpendicular to the optical axis of the lens.
  • the above microscope may further include an observation target moving device that moves the observation target in the direction perpendicular to the surface of the light guide plate.
  • the above-mentioned microscope may further include an imaging device that photographs the observation object through the lens each time the observation object moves.
  • the microscope described above may further include a light guide plate moving device that moves the light guide plate in a direction perpendicular to the surface of the light guide plate.
  • the above-mentioned microscope may further include an imaging device that photographs an observation target through a lens each time the light guide plate moves.
  • the light incident end portion of the light guide plate having a light incident end portion where light is incident inside and a light emitting end portion where light is emitted from the inside in a sheet shape is irradiated with light.
  • an observation method including the process of irradiating the observation target with sheet-shaped light emitted from the light emitting end of the light guide plate, and observing the observation target irradiated with the sheet-shaped light. Will be done.
  • the light guide plate may be provided with a hole, the light incident end may be located on the outer circumference of the light guide plate, and the light emitting end may be located on the inner circumference of the hole in the light guide plate.
  • light may be incident on the light guide plate from a plurality of light sources.
  • the light guide plate may include a transparent substrate.
  • the transparent substrate may contain silicon oxide.
  • the transparent substrate may contain a resin.
  • the light guide plate may include a reflective portion facing the light guide plate.
  • the observation target may be observed using a lens
  • the light guide plate may be arranged in the direction perpendicular to the optical axis of the lens.
  • the above observation method may further include moving the observation target in the direction perpendicular to the surface of the light guide plate.
  • the above observation method may further include photographing the observation target each time the observation target moves.
  • the above observation method may further include moving the light guide plate in the direction perpendicular to the surface of the light guide plate.
  • the above observation method may further include photographing the observation target each time the light guide plate moves.
  • the present invention it is possible to provide an inexpensive microscope illumination device, a microscope, and an observation method capable of observing a cross section of an observation target.
  • FIG. 1 is a schematic side view of a microscope lighting device according to an embodiment.
  • FIG. 2 is a schematic top view of the microscope lighting device according to the embodiment.
  • FIG. 3 is a schematic side view of the microscope lighting device according to the embodiment.
  • FIG. 4 is a schematic side view of the illumination device for a microscope according to the embodiment.
  • FIG. 5A is an image of observing a mouse stomach specimen according to Example 1.
  • FIG. 5B is an image of observing a mouse stomach specimen according to a comparative example.
  • FIG. 6 is an image of observing a mouse stomach specimen according to Example 2.
  • FIG. 7A is an image of observing a mouse stomach specimen according to Example 2.
  • FIG. 7B is an image of observing a mouse stomach specimen according to a comparative example.
  • FIG. 8 is an image of observing a mouse stomach specimen according to Example 3.
  • FIG. 9 is an image of observing a mouse stomach specimen according to a comparative example.
  • FIG. 10 is an image of observing a mouse stomach specimen according to a comparative example.
  • FIG. 11 is an image of observing a mouse lung specimen according to Example 4.
  • FIG. 12 is an image of observing a mouse lung specimen according to Example 4.
  • FIG. 13 is an image of observing a mouse lung specimen according to Example 4.
  • FIG. 14 is an image of observing a mouse lung specimen according to Example 4.
  • FIG. 15 is an image of observing a sample of a mouse lung according to Example 4.
  • FIG. 16 is an image of observing a sample of a mouse lung according to Example 4.
  • FIG. 17 is an image of observing a mouse lung specimen according to Example 5.
  • the microscope lighting device includes a light source 10 that irradiates light, a light incident end portion 21 that incidents light inside, and a light emitting end that emits light from the inside in a sheet shape.
  • a light guide plate 20 having a portion 22 is provided.
  • the microscope illumination device according to the embodiment is used in combination with a microscope.
  • the microscope illumination device according to the embodiment is used to irradiate an observation target observed with a microscope with sheet-like light emitted from a light emitting end portion 32.
  • the microscope combined with the microscope illumination device according to the embodiment can be a general-purpose optical microscope.
  • the microscope includes a lens 40 for observing an observation object.
  • Examples of the light source 10 include a light emitting diode (LED) and a laser. Examples of LEDs include chip LEDs and power LEDs.
  • the light source 10 irradiates light toward the light incident end portion 21 of the light guide plate 20.
  • the light source 10 shows a form including a cover, but the light source 10 may not have a cover.
  • an LED without a cover may be brought into close contact with the light incident end 21 of the light guide plate 20.
  • a power supply and an electronic circuit are connected to the light source 10. Examples of electronic circuits include constant current circuits and laser excitation circuits.
  • a light source lens 11 may be arranged between the light source 10 and the light guide plate 20 so that the light emitted by the light source 10 is diffused in the light guide plate 20. Further, optionally, a cooling device 12 such as a fan for cooling the light source 10 may be provided.
  • the light source 10 is arranged so as to face the light incident end portion 21 of the light guide plate 20 by using, for example, a bracket. Alternatively, the light emitted by the light source 10 may be guided to the light incident end portion 21 of the light guide plate 20 via an optical fiber.
  • the number of the light sources 10 may be one or a plurality.
  • the light source 10 is arranged so as to irradiate the observation target 30 from two opposite sides or from all sides, but is not particularly limited.
  • the number of light incident end portions 21 of the light guide plate 20 may be one or a plurality.
  • the number of light emitting end portions 22 of the light guide plate 20 may be one or a plurality.
  • the light source 10 may be arranged on each side forming the polygon, or the light source 10 may be arranged on at least a part of the sides forming the polygon.
  • the shape of the light guide plate 20 is circular, the light sources 10 may be arranged at predetermined intervals around the circular shape.
  • the light guide plate 20 is arranged in a direction perpendicular to the optical axis of the lens 40 of the microscope for observing the observation target 30, for example.
  • the light guide plate 20 includes, for example, a transparent substrate.
  • the light guide plate 20 is a transparent substrate.
  • the transparent substrate is made of a material that is transparent to light.
  • the material of the transparent substrate may be silicon oxide such as silicon dioxide.
  • the transparent substrate may be glass.
  • the material of the transparent substrate may be a resin such as acrylic, polyethylene terephthalate (PET), polycarbonate, and polyvinyl chloride.
  • the material of the transparent substrate may be a monorefringent material or a birefringent material.
  • the lower surface, side surface, and upper surface of the light guide plate 20 which is a transparent substrate are, for example, smooth.
  • the light guide plate 20 may be provided with a hole 23.
  • the observation target 30 may be arranged in the hole 23 of the light guide plate 20.
  • the light incident end portion 21 is located on the outer periphery of the light guide plate 20.
  • the light emitting end portion 22 is located on the inner circumference of the hole 23 of the light guide plate 20.
  • the hole 23 is provided in the light guide plate 20 by laser processing or the like.
  • the shape of the light guide plate 20 is a square such as a square, a polygon, or a circle, but is not particularly limited.
  • the length of one side of the light guide plate 20 when it is square, or the diameter of the light guide plate 20 when it is circular is, for example, 5 mm or more, 10 mm or more, 15 mm or more, or 20 mm or more, but is not particularly limited.
  • the length of one side of the light guide plate 20 when it is square, or the diameter of the light guide plate 20 when it is circular is, for example, 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less, or 10 mm or less. There is no particular limitation.
  • the thickness of the light guide plate 20 which is a transparent substrate is, for example, 1 ⁇ m or more, 4 ⁇ m or more, 5 ⁇ m or more, 10 ⁇ m or more, 15 ⁇ m or more, 20 ⁇ m or more, 25 ⁇ m or more, or 30 ⁇ m or more, but is not particularly limited.
  • the thickness of the light guide plate 20 which is a transparent substrate is, for example, 500 ⁇ m or less, 400 ⁇ m or less, 300 ⁇ m or less, 200 ⁇ m or less, 150 ⁇ m or less, 100 ⁇ m or less, 90 ⁇ m or less, 80 ⁇ m or less, 70 ⁇ m or less, 60 ⁇ m or less, 50 ⁇ m or less, 40 ⁇ m or less.
  • the thickness of the sheet-shaped light emitted from the light guide plate 20 is close to the thickness of the light guide plate 20 which is a transparent substrate. Therefore, by selecting the thickness of the light guide plate 20 which is a transparent substrate, it is possible to adjust the thickness of the sheet-like light emitted from the light guide plate 20.
  • the thickness of the light guide plate 20 may be selected according to the purpose of observation.
  • the diameter of the hole 23 of the light guide plate 20 is, for example, 0.5 mm or more, 1 mm or more, 2 mm or more, or 3 mm or more, but is not particularly limited.
  • the diameter of the hole 23 of the light guide plate 20 is, for example, 20 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, or 5 mm or less, but is not particularly limited.
  • the inner peripheral side surface of the hole 23 of the light guide plate 20 serving as the light emitting end portion 22 is smooth so that the emitted sheet-like light is not diffused.
  • the light guide plate 20 shown in FIG. 1 is held by, for example, holding devices 51 and 52.
  • the holding devices 51 and 52 hold the light guide plate 20 by sandwiching the light guide plate 20, for example.
  • the holding device 51 arranged under the light guide plate 20 is provided with a hole 53 so that the hole 23 provided in the light guide plate 20 is exposed.
  • the holding device 52 arranged on the upper side of the light guide plate 20 is provided with a hole 54 so that the hole 23 provided in the light guide plate 20 is exposed.
  • the upper surface of the holding device 51 in contact with the lower surface of the light guide plate 20 is smooth.
  • the lower surface of the holding device 52 in contact with the upper surface of the light guide plate 20 is smooth.
  • the holding devices 51 and 52 are made of, for example, a rigid material.
  • Examples of the materials of the holding devices 51 and 52 include metals and resins. At least a part of each shape of the holding devices 51 and 52 may be the same as the shape of the light guide plate 20. Therefore, the shapes of the holding devices 51 and 52 are square, polygonal, or circular, such as a square, but are not particularly limited.
  • a support substrate 60 for supporting the observation target 30 is arranged below the light guide plate 20.
  • the observation target 30 is arranged on the support substrate 60 and in the hole 23 of the light guide plate 20.
  • a liquid may be arranged between the light emitting end 22 of the light guide plate 20 and the observation target 30 to reduce the difference in refractive index between the inside and the outside of the light emitting end 22 of the light guide plate 20.
  • the observation target 30 may be arranged in the transparent container 70 which can be arranged on the support substrate 60 and which can be inserted into the hole 23 of the light guide plate 20.
  • the liquid may be arranged between the light emitting end portion 22 of the light guide plate 20 and the transparent container 70. Further, the liquid may be put in the transparent container 70.
  • the support substrate 60 When observing the observation target 30 from below the support substrate 60, the support substrate 60 is transparent.
  • the material of the support substrate 60 and the transparent container 70 may be silicon oxide such as silicon dioxide.
  • the support substrate 60 and the transparent container 70 may be made of glass.
  • the material of the support substrate 60 and the transparent container 70 may be a resin such as acrylic, polyethylene terephthalate (PET), polycarbonate, and polyvinyl chloride.
  • PET polyethylene terephthalate
  • the material of the support substrate 60 and the transparent container 70 may be a monorefractive material or a birefringent material.
  • the lower surface, side surfaces, and upper surface of the support substrate 60 are, for example, smooth.
  • the lower surface and sides of the transparent container 70 are, for example, smooth.
  • the support substrate 60 may be provided with an opening 62.
  • the diameter of the opening 62 may be larger than the diameter of the lens 40, which will be described later, for example. As a result, the lens 40 can enter the opening 60
  • the observation target 30 is at least partially transparent.
  • the observation target 30 include biological samples of humans, non-human animals, and plants.
  • biological samples include, but are not limited to, organs, bones, at least a part of organs such as connective tissue, and cells.
  • the biological sample may be alive.
  • the observation target 30 is, for example, transparentized before observation.
  • the observation target 30 is a biological sample, the observation target 30 is subjected to a clearing treatment with, for example, a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organic iodine compound.
  • the observation target 30 may be dyed with a fluorescent reagent excited by the light emitted by the light source 10.
  • staining include, but are not limited to, tissue staining and immunostaining.
  • the observation target 30 is not limited to the biological sample, and may be an artificial object such as a resin molded product.
  • the method of making the observation target 30 transparent is not limited to the above method.
  • the observation target 30 may be made transparent by the Clarity method, the CUBIC method, the Scale method, and the FocusClear (registered trademark).
  • the thickness of the sheet-like light emitted from the light emitting end portion 22 is substantially the same as the thickness of the transparent substrate forming the light guide plate 20.
  • the sheet-shaped light emitted from the light emitting end portion 22 irradiates the observation target 30 while keeping the thickness substantially constant.
  • the observation target 30 irradiated with the sheet-shaped light is magnified and observed through the lens 40 of the microscope.
  • the lens 40 is, for example, an objective lens.
  • the lens 40 is arranged on the skeleton 41.
  • an imaging device 42 for photographing the observation target 30 observed through the lens 40 is arranged in the frame 41 of the microscope.
  • the photographing device 42 includes, for example, a solid-state image sensor such as a CCD image sensor or a CMOS image sensor. Any optical system may be arranged between the lens 40 and the photographing device 42.
  • a fluorescence observation filter is arranged in the frame 41 of the microscope.
  • An eyepiece for observation may be provided on the skeleton 41.
  • the observation target 30 When the observation target 30 is transparent, even if the number of light sources 10 is one, the sheet-shaped light can pass through the cross section of the observation target 30.
  • the light source 10 need only emit light for a short period of time while the photographing device 42 takes a picture.
  • the observation target 30 has an opaque portion, a part of the sheet-like light may not advance beyond the opaque portion.
  • a plurality of light sources 10 are provided, it is possible to irradiate a portion where the light emitted from one light source 10 cannot reach with the light emitted from another light source 10.
  • the light source 10 may emit light only for a short time while the photographing device 42 takes a picture. If the observation target 30 has no opaque portion, the number of light sources 10 may be one. According to the illumination device for a microscope according to the embodiment, since it is possible to shorten the irradiation time of light on the observation target 30, the phototoxicity to the observation target 30 is low, and the observation target 30 is deteriorated by light. It can be suppressed. In addition, it is possible to suppress photobleaching of fluorescent molecules in the observation target 30. Further, since it is not necessary to rotate the observation target 30, it is possible to observe the observation target 30 with a high resolution.
  • the observation target 30 is a living biological sample
  • light may be emitted from the light source 10 at predetermined intervals to observe the time change of the biological sample.
  • the microscope lighting device may further include an observation target moving device 61 that moves the observation target 30 in the direction perpendicular to the surface of the light guide plate 20.
  • the observation target moving device 61 moves, for example, the support substrate 60 in the direction perpendicular to the surface of the light guide plate 20, that is, in the optical axis direction of the lens 40 of the microscope, and the observation target 30 with respect to the light guide plate 20.
  • the observation target moving device 61 moves the observation target 30 by the same distance as the thickness of the sheet-shaped light, for example.
  • an actuator can be used as the observation target moving device 61.
  • the light source 10 emits light each time the observation target moving device 61 moves the observation target 30, and irradiates the observation target 30 with sheet-shaped light. However, the light source 10 may continuously emit light.
  • the imaging device 42 of the microscope photographs the observation target 30 through the lens 40 each time the observation target moving device 61 moves the observation target 30. This makes it possible to obtain a three-dimensional image of the observation target 30.
  • the microscope lighting device may further include a light guide plate moving device 55 that moves the light guide plate 20 in a direction perpendicular to the surface of the light guide plate 20.
  • the light guide plate moving device 55 moves the light guide plate 20 in the optical axis direction of the lens 40 of the microscope to move the light guide plate 20 with respect to the observation target 30.
  • the light guide plate moving device 55 moves the light guide plate 20 by the same distance as the thickness of the sheet-shaped light, for example.
  • an actuator can be used for the light guide plate moving device 55.
  • the light source 10 emits light each time the light guide plate moving device 55 moves the light guide plate 20, and irradiates the observation target 30 with sheet-shaped light. However, the light source 10 may continuously emit light.
  • the photographing device 42 photographs the observation target 30 via the lens 40 each time the light guide plate moving device 55 moves the light guide plate 20. This makes it possible to obtain a three-dimensional image of the observation target 30.
  • the microscope 40 a lens included in a general optical microscope can be used.
  • the photographing device 42 a photographing device 42 included in a general optical microscope can be used. Therefore, the microscope according to the embodiment can be manufactured by arranging the illumination device for a microscope including the light source 10 and the light guide plate 20 on the stage of a general optical microscope. Therefore, the microscope according to the embodiment can be manufactured at a lower cost than the multiphoton microscope, which costs about 100 million yen, and the conventional optical sheet microscope and confocal microscope, which cost about 30 million yen. Needless to say, a microscope incorporating the microscope lighting device according to the embodiment from the beginning may be manufactured.
  • the optical system that generates sheet-shaped light is the light guide plate 20, and the light source 10 irradiates the light guide plate 20 with light without complicated adjustment of the optical system. Then, it is possible to generate sheet-like light. Therefore, the microscope lighting device according to the embodiment is easy to operate. Further, since the light source 10 may be an LED having a low power consumption such as 20 W or less, such as an LED having a power consumption of 1 W or an LED having a power consumption of 3 W, the device can be miniaturized, and the light source 10 can be driven by a battery. Is also possible. Therefore, the microscope lighting device according to the embodiment is easy to carry, and is also easy to use, for example, outdoors.
  • the microscope lighting device and the microscope according to the embodiment it is possible to continuously take a cross-sectional image of the living tissue in a short time, so that the cancer existing in the living tissue can be reliably detected. Can be possible.
  • the present invention has been described by embodiment as described above, the descriptions and drawings that form part of this disclosure should not be understood to limit the invention.
  • This disclosure should reveal to those skilled in the art various alternative embodiments, examples and operational techniques.
  • the light guide plate may be the reflecting portions 24 and 25 facing each other.
  • the reflecting portions 24 and 25 are made of a material capable of reflecting light.
  • a metal such as aluminum can be used as the material of the reflecting portions 24 and 25, a metal such as aluminum can be used.
  • the reflecting portions 24 and 25 are arranged in parallel.
  • the space between the reflecting portions 24 and 25 is, for example, a cavity.
  • the reflecting portions 24 and 25 may be provided with holes 23.
  • the observation target 30 may be arranged in the holes 23 of the reflection portions 24 and 25.
  • the light incident end portion 21 is located on the outer periphery of the reflecting portions 24 and 25.
  • the light emitting end portion 22 is located on the inner circumference of the holes 23 of the reflecting portions 24 and 25.
  • the distance between the reflecting portions 24 and 25 is, for example, 1 ⁇ m or more, 4 ⁇ m or more, 5 ⁇ m or more, 10 ⁇ m or more, 15 ⁇ m or more, 20 ⁇ m or more, or 25 ⁇ m or more, but is not particularly limited.
  • the intervals between the reflecting portions 24 and 25 are, for example, 500 ⁇ m or less, 400 ⁇ m or less, 300 ⁇ m or less, 200 ⁇ m or less, 150 ⁇ m or less, 100 ⁇ m or less, 90 ⁇ m or less, 80 ⁇ m or less, 70 ⁇ m or less, 60 ⁇ m or less, 50 ⁇ m or less, 40 ⁇ m or less, 30 ⁇ m.
  • the following, or 15 ⁇ m or less but is not particularly limited.
  • the light emitted by the light source 10 travels while being reflected between the reflecting portions 24 and 25, and is emitted in a sheet shape from the light emitting end portion 22.
  • the thickness of the sheet-shaped light emitted from the light emitting end portion 22 is close to the distance between the reflecting portions 24 and 25. Therefore, it is possible to adjust the thickness of the sheet-like light emitted from the light emitting end portion 22 by selecting the intervals between the reflecting portions 24 and 25.
  • the number of the light sources 10 is not particularly limited even if the light guide plates are the reflecting portions 24 and 25 facing each other, and the number of the light sources 10 may be one or a plurality.
  • the shapes of the reflecting portions 24 and 25 are not particularly limited, and may be, for example, a square such as a square, a polygon, or a circle.
  • Example 1 Two quadrangular transparent glass substrates having a thickness of 140 ⁇ m were prepared as light guide plates.
  • the two light guide plates were opposed to each other with a lateral spacing, and two blue LEDs were arranged as light sources at the center of the outer sides of the two light guide plates.
  • the illumination device for a microscope according to Example 1 was produced.
  • the bottom surface between the two light guide plates was closed with a cover glass, and the space between the two light guide plates was filled with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organic iodine compound.
  • Mouse stomach specimens were obtained from transgenic mice in which the green fluorescent protein (GFP) excited by blue light is expressed in the cell nucleus.
  • GFP green fluorescent protein
  • the microscope lighting device according to Example 1 was placed on the stage of a general microscope (Keyence). A power of 0.25 W was supplied to each of the two LEDs, blue light was emitted from the two LEDs, and a sheet-shaped blue light was irradiated to the mouse stomach specimen from the transparent glass substrate. A mouse stomach specimen was observed using the GFP filter attached to the microscope. As a result, as shown in FIG. 5A, an observation image having a high resolution equivalent to that of a conventional expensive optical sheet microscope was obtained. On the other hand, when the same specimen is observed by irradiating it from below with a mercury lamp of a normal microscope without using the illumination device for a microscope according to Example 1, the cell structure is as shown in FIG. 5 (b). I could't observe it at all.
  • Example 2 A rectangular glass transparent substrate having a thickness of 50 ⁇ m and having a hole having a diameter of 3 mm in the center was prepared as a light guide plate. A blue LED was arranged as a light source at the center of each of the four outer sides of the light guide plate. As a result, a microscope lighting device including four LEDs according to Example 2 was produced. The bottom surface of the hole of the light guide plate was closed with a cover glass, and the hole of the light guide plate was filled with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organic iodine compound. Further, a 2 mm thick mouse stomach specimen similar to that in Example 1 was placed in the hole of the light guide plate.
  • the microscope lighting device according to Example 2 was placed on the stage of a general microscope (Keyence). A power of 0.25 W was supplied to the LED, blue light was emitted from the LED, and a sheet-shaped blue light was irradiated to the stomach specimen of the mouse from the transparent glass substrate. A mouse stomach specimen was observed using the GFP filter attached to the microscope. As a result, as shown in FIG. 6, an observation image having a high resolution equivalent to that of a conventional expensive optical sheet microscope was obtained. Even when the magnification was increased, a high-resolution observation image was obtained as shown in FIG. 7 (a). On the other hand, when the same specimen is observed by irradiating it from below with a mercury lamp of a normal microscope without using the microscope illumination device according to Example 2, the cell structure is as shown in FIG. 7 (b). I could't observe it at all.
  • Example 3 The illumination device for a microscope according to Example 3 having the same configuration as that of Example 2 except that the light guide plate is a transparent glass substrate having a thickness of 30 ⁇ m was produced.
  • a 2 mm thick mouse stomach specimen similar to Example 2 was observed in the same manner as in Example 2.
  • FIG. 8 an observation image having a high resolution equivalent to that of a conventional expensive optical sheet microscope was obtained.
  • the same specimen is irradiated from below with a mercury lamp of a normal microscope without using the microscope illumination device according to Example 3, the cell structure is completely observed as shown in FIG. I could't.
  • FIG. 8 When the same specimen was observed with an expensive confocal microscope, cell nuclei surrounding the gastric glands were observed, as shown in FIG. Since the same structure was observed in the image shown in FIG. 8, it was shown that an image equal to or higher than that of an expensive confocal microscope can be obtained by using the microscope illumination device according to the third embodiment.
  • Example 4 A rectangular glass transparent substrate having a thickness of 15 ⁇ m and having a hole having a diameter of 3 mm in the center was prepared as a light guide plate.
  • a block of mouse lung that had been previously cleared with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organoiodine compound was prepared.
  • the thickness of the block was 1 mm.
  • the nuclei of mouse lung cells were stained with DAPI (4', 6-diamidino-2-phenylindole) or SYBR Gold® (registered trademark, Thermo Fisher).
  • the inner wall of the blood vessel of the mouse lung was stained with DyLight594 (funakoshi), which is a tomato lectin. Stained mouse lungs were placed in a clear case.
  • the perimeter of the mouse lung in a clear case was filled with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organoiodine compound.
  • a transparent case containing mouse lungs was placed in a hole in a square glass transparent substrate having a thickness of 15 ⁇ m.
  • An LED was placed as a light source at the center of each of the three outer sides of the light guide plate.
  • the three LEDs were an LED emitting 405 nm light, an LED emitting 475 nm light, and an LED emitting 594 nm light, respectively.
  • Light at 405 nm can be used as excitation light for DAPI.
  • the light of 475 nm can be used as the excitation light of SYBR Gold.
  • the light of 594 nm can be used as the excitation light of DyLight594.
  • Example 5 A quadrangular transparent glass substrate having a thickness of 10 ⁇ m and having a hole having a diameter of 3 mm in the center was prepared as a light guide plate.
  • a block of mouse lung that had been previously cleared with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organoiodine compound was prepared.
  • the thickness of the block was 1 mm.
  • the nuclei of mouse lung cells were stained with DAPI (4', 6-diamidino-2-phenylindole). Stained mouse lungs were placed in a clear case.
  • the perimeter of the mouse lung in a clear case was filled with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organoiodine compound.
  • a transparent case containing mouse lungs was placed in a hole in a square glass transparent substrate having a thickness of 10 ⁇ m.
  • An LED that emits light of 405 nm was arranged as a light source on the outside of the light guide plate. Light at 405 nm can be used as excitation light for DAPI.
  • Mouse lungs were observed with a microscope (Keyence, Biozero) equipped with a 10x objective lens (Nikon, Planfluol). As shown in FIG. 17, alveoli were observed.
  • the 10 ⁇ m-thick sheet-shaped light emitted from the 10 ⁇ m-thick glass transparent substrate is equivalent to the thickness of the sheet-shaped light used in a conventional optical sheet microscope. It was shown that by using a thin light guide plate, the internal structure of the unsliced mouse lung block can be observed with high resolution.
  • the illumination device for a microscope according to the embodiment is not particularly limited, but can be used in industries related to clinical pathology, basic medicine, pharmacy, science, agriculture, and the like.
  • the microscope illumination device according to the embodiment can be used for new drug screening and side effect screening.
  • the microscope illuminator according to the embodiment is available for observing at least some of the flora and fauna.
  • the microscope illuminator according to the embodiment can be used for observing cultivated plants.

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Abstract

A lighting device for a microscope provided with a light source 10 for radiating light, and a light guide plate 20 having a light-incident end part 21 into which light enters and a light-emitting end part 22 from the interior of which light is emitted in a sheet-like manner, wherein an observation subject is illuminated with the sheet-like light emitted from the light-emitting end part 32.

Description

顕微鏡用照明装置、顕微鏡、及び観察方法Microscope illuminator, microscope, and observation method
  本発明は、顕微鏡用照明装置、顕微鏡、及び観察方法に関する。 The present invention relates to a microscope illumination device, a microscope, and an observation method.
　生体組織内部の観察に、２光子顕微鏡及び３光子顕微鏡等の多光子顕微鏡が用いられることがある（例えば、特許文献１参照。）。多光子顕微鏡においては、１個の蛍光分子を、同時に複数個の光子で励起する。多光子顕微鏡は、通常の励起波長の複数倍の波長を有する近赤外パルスレーザー光を用いる。長波長のレーザー光を用いるため、生体組織内における励起光の散乱は抑制される。また、多光子励起において、焦点面以外に存在する蛍光分子の励起が生じにくい。そのため、多光子顕微鏡によれば、高い解像度の観察画像を得ることが可能である。しかし、多光子顕微鏡の価格は約１億円と高く、また多光子顕微鏡は操作が難しい、撮影に時間がかかるという問題がある。 A multi-photon microscope such as a two-photon microscope and a three-photon microscope may be used for observing the inside of a living tissue (see, for example, Patent Document 1). In a multi-photon microscope, one fluorescent molecule is excited by a plurality of photons at the same time. The multiphoton microscope uses near-infrared pulsed laser light having a wavelength that is multiple times the normal excitation wavelength. Since a long wavelength laser beam is used, scattering of excitation light in living tissue is suppressed. Further, in multiphoton excitation, excitation of fluorescent molecules existing other than the focal plane is unlikely to occur. Therefore, according to the multi-photon microscope, it is possible to obtain a high-resolution observation image. However, the price of a multiphoton microscope is as high as about 100 million yen, and the multiphoton microscope has problems that it is difficult to operate and it takes time to take a picture.
　また、生体組織内部の観察に、光シート顕微鏡が用いられることがある（例えば、特許文献２参照。）。従来の光シート顕微鏡は、シリンドリカルレンズと対物レンズで絞られたシート状の励起光を試料の側方から照射して、試料の光学断面像を得る。光シート顕微鏡では、試料に不透明な部分があると、励起光は不透明な部分の先に進めない。そのため、光シート顕微鏡で試料を観察する際には、励起光に対して試料を回転させており、光シート顕微鏡による試料の観察は時間がかかる。また、試料を回転させるため、解像度が低下し得る。さらに、光シート顕微鏡では、励起光が試料内部で散乱し得るため、試料の透明度が高いことが好ましい。そのため、生体組織を２，２´－チオジエタノールと非イオン性有機ヨウ素化合物を含む水溶性溶媒中に浸漬して、生体組織を透明化する方法が提案されている（例えば、特許文献３参照。）。従来の光シート顕微鏡の価格は約３千万円であり、多光子顕微鏡より安価であるものの、高価である。また、従来の光シート顕微鏡も、操作が難しい、撮影に時間がかかるという問題がある。 In addition, an optical sheet microscope may be used for observing the inside of living tissue (see, for example, Patent Document 2). In a conventional optical sheet microscope, a sheet-shaped excitation light focused by a cylindrical lens and an objective lens is irradiated from the side of the sample to obtain an optical cross-sectional image of the sample. In an optical sheet microscope, if the sample has an opaque portion, the excitation light cannot advance beyond the opaque portion. Therefore, when observing the sample with the optical sheet microscope, the sample is rotated with respect to the excitation light, and it takes time to observe the sample with the optical sheet microscope. Also, since the sample is rotated, the resolution may decrease. Further, in the optical sheet microscope, the excitation light can be scattered inside the sample, so that the transparency of the sample is preferably high. Therefore, a method of immersing the living tissue in a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organic iodine compound to make the living tissue transparent has been proposed (see, for example, Patent Document 3). ). The price of a conventional optical sheet microscope is about 30 million yen, which is cheaper than a multiphoton microscope, but is expensive. Further, the conventional optical sheet microscope also has problems that it is difficult to operate and it takes time to take a picture.
特許第６２５４０９６号公報Japanese Patent No. 6254096 特許第６６３４０２４号公報Japanese Patent No. 6634024 特許第６３２５４６１号公報Japanese Patent No. 6325461
　本発明は、生体組織に限られない観察対象の断面を観察可能な安価な顕微鏡用照明装置、顕微鏡、及び観察方法を提供することを目的の一つとする。 One of the objects of the present invention is to provide an inexpensive microscope illumination device, a microscope, and an observation method capable of observing a cross section of an observation target not limited to a living tissue.
　本発明の態様によれば、光を照射する光源と、光が内部に入射する光入射端部と、光が内部からシート状で出射する光出射端部と、を有する導光板と、を備え、光出射端部から出射したシート状の光で、観察対象を照射するための、顕微鏡用照明装置が提供される。 According to the aspect of the present invention, a light guide plate having a light source for irradiating light, a light incident end portion where light is incident inside, and a light emitting end portion where light is emitted from the inside in a sheet shape is provided. , A microscope lighting device for illuminating an observation target with sheet-like light emitted from a light emitting end portion is provided.
　上記の顕微鏡用照明装置において、導光板に孔が設けられており、光入射端部が導光板の外周に位置し、光出射端部が導光板の孔の内周に位置していてもよい。 In the above-mentioned illumination device for a microscope, a hole may be provided in the light guide plate, the light incident end may be located on the outer periphery of the light guide plate, and the light emitting end may be located on the inner circumference of the hole in the light guide plate. ..
　上記の顕微鏡用照明装置が、導光板に光を入射する光源を複数備えていてもよい。 The above-mentioned illumination device for a microscope may include a plurality of light sources that inject light into a light guide plate.
　上記の顕微鏡用照明装置において、導光板が透明基板を備えていてもよい。 In the above-mentioned illumination device for a microscope, the light guide plate may be provided with a transparent substrate.
　上記の顕微鏡用照明装置において、透明基板が酸化ケイ素を含んでいてもよい。 In the above microscope lighting device, the transparent substrate may contain silicon oxide.
　上記の顕微鏡用照明装置において、透明基板が樹脂を含んでいてもよい。 In the above microscope lighting device, the transparent substrate may contain a resin.
　上記の顕微鏡用照明装置において、導光板が、対向する反射部を備えていてもよい。 In the above-mentioned illumination device for a microscope, the light guide plate may include a reflecting portion facing the light guide plate.
　上記の顕微鏡用照明装置が、観察対象を導光板の表面に対して垂直方向に移動させる観察対象移動装置をさらに備えていてもよい。 The above-mentioned illumination device for a microscope may further include an observation target moving device that moves the observation target in the direction perpendicular to the surface of the light guide plate.
　上記の顕微鏡用照明装置が、導光板を導光板の表面に対して垂直方向に移動させる導光板移動装置をさらに備えていてもよい。 The above-mentioned illumination device for a microscope may further include a light guide plate moving device that moves the light guide plate in a direction perpendicular to the surface of the light guide plate.
　また、本発明の態様によれば、光を照射する光源と、光が内部に入射する光入射端部と、光が内部からシート状で出射する光出射端部と、を有する導光板と、導光板の光出射端部から出射したシート状の光を照射された観察対象を観察するためのレンズと、を備える、顕微鏡が提供される。 Further, according to the aspect of the present invention, a light guide plate having a light source for irradiating light, a light incident end portion where light is incident inside, and a light emitting end portion where light is emitted from the inside in a sheet shape. Provided is a microscope comprising a lens for observing an observation object irradiated with sheet-like light emitted from a light emitting end portion of a light guide plate.
　上記の顕微鏡において、導光板に孔が設けられており、光入射端部が導光板の外周に位置し、光出射端部が導光板の孔の内周に位置していてもよい。 In the above microscope, the light guide plate may be provided with a hole, the light incident end may be located on the outer circumference of the light guide plate, and the light emitting end may be located on the inner circumference of the hole in the light guide plate.
　上記の顕微鏡が、導光板に光を入射する光源を複数備えていてもよい。 The above microscope may be provided with a plurality of light sources that inject light into the light guide plate.
　上記の顕微鏡において、導光板が透明基板を備えていてもよい。 In the above microscope, the light guide plate may be provided with a transparent substrate.
　上記の顕微鏡において、透明基板が酸化ケイ素を含んでいてもよい。 In the above microscope, the transparent substrate may contain silicon oxide.
　上記の顕微鏡において、透明基板が樹脂を含んでいてもよい。 In the above microscope, the transparent substrate may contain a resin.
　上記の顕微鏡において、導光板が、対向する反射部を備えていてもよい。 In the above microscope, the light guide plate may include a reflecting portion facing the light guide plate.
　上記の顕微鏡において、導光板が、レンズの光軸に対して垂直方向に配置されていてもよい。 In the above microscope, the light guide plate may be arranged in the direction perpendicular to the optical axis of the lens.
　上記の顕微鏡が、観察対象を導光板の表面に対して垂直方向に移動させる観察対象移動装置をさらに備えていてもよい。 The above microscope may further include an observation target moving device that moves the observation target in the direction perpendicular to the surface of the light guide plate.
　上記の顕微鏡が、観察対象が移動するごとにレンズを介して観察対象を撮影する撮影装置をさらに備えていてもよい。 The above-mentioned microscope may further include an imaging device that photographs the observation object through the lens each time the observation object moves.
　上記の顕微鏡が、導光板を導光板の表面に対して垂直方向に移動させる導光板移動装置をさらに備えていてもよい。 The microscope described above may further include a light guide plate moving device that moves the light guide plate in a direction perpendicular to the surface of the light guide plate.
　上記の顕微鏡が、導光板が移動するごとにレンズを介して観察対象を撮影する撮影装置をさらに備えていてもよい。 The above-mentioned microscope may further include an imaging device that photographs an observation target through a lens each time the light guide plate moves.
　また、本発明の態様によれば、光が内部に入射する光入射端部と、光が内部からシート状で出射する光出射端部と、を有する導光板の光入射端部に光を照射することと、導光板の光出射端部から出射したシート状の光を観察対象に照射することと、シート状の光を照射された観察対象を観察することと、を含む、観察方法が提供される。 Further, according to the aspect of the present invention, the light incident end portion of the light guide plate having a light incident end portion where light is incident inside and a light emitting end portion where light is emitted from the inside in a sheet shape is irradiated with light. Provided is an observation method including the process of irradiating the observation target with sheet-shaped light emitted from the light emitting end of the light guide plate, and observing the observation target irradiated with the sheet-shaped light. Will be done.
　上記の観察方法において、導光板に孔が設けられており、光入射端部が導光板の外周に位置し、光出射端部が導光板の孔の内周に位置していてもよい。 In the above observation method, the light guide plate may be provided with a hole, the light incident end may be located on the outer circumference of the light guide plate, and the light emitting end may be located on the inner circumference of the hole in the light guide plate.
　上記の観察方法において、複数の光源から導光板に光を入射してもよい。 In the above observation method, light may be incident on the light guide plate from a plurality of light sources.
　上記の観察方法において、導光板が透明基板を備えていてもよい。 In the above observation method, the light guide plate may include a transparent substrate.
　上記の観察方法において、透明基板が酸化ケイ素を含んでいてもよい。 In the above observation method, the transparent substrate may contain silicon oxide.
　上記の観察方法において、透明基板が樹脂を含んでいてもよい。 In the above observation method, the transparent substrate may contain a resin.
　上記の観察方法において、導光板が、対向する反射部を備えていてもよい。 In the above observation method, the light guide plate may include a reflective portion facing the light guide plate.
　上記の観察方法において、レンズを用いて観察対象が観察され、導光板が、レンズの光軸に対して垂直方向に配置されていてもよい。 In the above observation method, the observation target may be observed using a lens, and the light guide plate may be arranged in the direction perpendicular to the optical axis of the lens.
　上記の観察方法が、観察対象を導光板の表面に対して垂直方向に移動させることをさらに含んでいてもよい。 The above observation method may further include moving the observation target in the direction perpendicular to the surface of the light guide plate.
　上記の観察方法が、観察対象が移動するごとに観察対象を撮影することをさらに含んでいてもよい。 The above observation method may further include photographing the observation target each time the observation target moves.
　上記の観察方法が、導光板を導光板の表面に対して垂直方向に移動させることをさらに含んでいてもよい。 The above observation method may further include moving the light guide plate in the direction perpendicular to the surface of the light guide plate.
　上記の観察方法が、導光板が移動するごとに観察対象を撮影することをさらに含んでいてもよい。 The above observation method may further include photographing the observation target each time the light guide plate moves.
  本発明によれば、観察対象の断面を観察可能な安価な顕微鏡用照明装置、顕微鏡、及び観察方法を提供可能である。 According to the present invention, it is possible to provide an inexpensive microscope illumination device, a microscope, and an observation method capable of observing a cross section of an observation target.
図１は、実施形態に係る顕微鏡用照明装置の模式的側面図である。FIG. 1 is a schematic side view of a microscope lighting device according to an embodiment. 図２は、実施形態に係る顕微鏡用照明装置の模式的上面図である。FIG. 2 is a schematic top view of the microscope lighting device according to the embodiment. 図３は、実施形態に係る顕微鏡用照明装置の模式的側面図である。FIG. 3 is a schematic side view of the microscope lighting device according to the embodiment. 図４は、実施形態に係る顕微鏡用照明装置の模式的側面図である。FIG. 4 is a schematic side view of the illumination device for a microscope according to the embodiment. 図５（ａ）は、実施例１に係るマウスの胃の標本を観察した画像である。図５（ｂ）は、比較例に係るマウスの胃の標本を観察した画像である。FIG. 5A is an image of observing a mouse stomach specimen according to Example 1. FIG. 5B is an image of observing a mouse stomach specimen according to a comparative example. 図６は、実施例２に係るマウスの胃の標本を観察した画像である。FIG. 6 is an image of observing a mouse stomach specimen according to Example 2. 図７（ａ）は、実施例２に係るマウスの胃の標本を観察した画像である。図７（ｂ）は、比較例に係るマウスの胃の標本を観察した画像である。FIG. 7A is an image of observing a mouse stomach specimen according to Example 2. FIG. 7B is an image of observing a mouse stomach specimen according to a comparative example. 図８は、実施例３に係るマウスの胃の標本を観察した画像である。FIG. 8 is an image of observing a mouse stomach specimen according to Example 3. 図９は、比較例に係るマウスの胃の標本を観察した画像である。FIG. 9 is an image of observing a mouse stomach specimen according to a comparative example. 図１０は、比較例に係るマウスの胃の標本を観察した画像である。FIG. 10 is an image of observing a mouse stomach specimen according to a comparative example. 図１１は、実施例４に係るマウスの肺の標本を観察した画像である。FIG. 11 is an image of observing a mouse lung specimen according to Example 4. 図１２は、実施例４に係るマウスの肺の標本を観察した画像である。FIG. 12 is an image of observing a mouse lung specimen according to Example 4. 図１３は、実施例４に係るマウスの肺の標本を観察した画像である。FIG. 13 is an image of observing a mouse lung specimen according to Example 4. 図１４は、実施例４に係るマウスの肺の標本を観察した画像である。FIG. 14 is an image of observing a mouse lung specimen according to Example 4. 図１５は、実施例４に係るマウスの肺の標本を観察した画像である。FIG. 15 is an image of observing a sample of a mouse lung according to Example 4. 図１６は、実施例４に係るマウスの肺の標本を観察した画像である。FIG. 16 is an image of observing a sample of a mouse lung according to Example 4. 図１７は、実施例５に係るマウスの肺の標本を観察した画像である。FIG. 17 is an image of observing a mouse lung specimen according to Example 5.
  以下、本発明の実施形態について図面を参照して説明する。以下の図面の記載において、同一又は類似の部分には同一又は類似の符号で表している。ただし、図面は模式的なものである。したがって、具体的な寸法等は以下の説明を照らし合わせて判断するべきものである。また、図面相互間においても互いの寸法の関係や比率が異なる部分が含まれていることはもちろんである。 Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the description of the drawings below, the same or similar parts are represented by the same or similar reference numerals. However, the drawings are schematic. Therefore, the specific dimensions and the like should be determined in light of the following explanations. In addition, it goes without saying that the drawings include parts having different dimensional relationships and ratios from each other.
　実施形態に係る顕微鏡用照明装置は、図１に示すように、光を照射する光源１０と、光が内部に入射する光入射端部２１と、光が内部からシート状で出射する光出射端部２２と、を有する導光板２０と、を備える。実施形態に係る顕微鏡用照明装置は、顕微鏡と組み合わされて使用される。実施形態に係る顕微鏡用照明装置は、光出射端部３２から出射したシート状の光で、顕微鏡で観察される観察対象を照射するために使用される。実施形態に係る顕微鏡用照明装置と組み合わされる顕微鏡は、汎用的な光学顕微鏡であり得る。顕微鏡は、観察対象を観察するためのレンズ４０を備える。 As shown in FIG. 1, the microscope lighting device according to the embodiment includes a light source 10 that irradiates light, a light incident end portion 21 that incidents light inside, and a light emitting end that emits light from the inside in a sheet shape. A light guide plate 20 having a portion 22 is provided. The microscope illumination device according to the embodiment is used in combination with a microscope. The microscope illumination device according to the embodiment is used to irradiate an observation target observed with a microscope with sheet-like light emitted from a light emitting end portion 32. The microscope combined with the microscope illumination device according to the embodiment can be a general-purpose optical microscope. The microscope includes a lens 40 for observing an observation object.
　光源１０の例としては、発光ダイオード（ＬＥＤ）及びレーザーが挙げられる。ＬＥＤの例としては、チップＬＥＤ及びパワーＬＥＤが挙げられる。光源１０は、導光板２０の光入射端部２１に向けて光を照射する。図１において、光源１０はカバーを備える形態を示しているが、光源１０はカバーを備えていなくてもよい。例えば、カバーのないＬＥＤを導光板２０の光入射端部２１に密着させてもよい。光源１０には、例えば、電源と電子回路が接続される。電子回路の例としては、定電流回路及びレーザー励起回路が挙げられる。光源１０と導光板２０の間に、光源１０が発した光が導光板２０内に拡散するよう、光源用レンズ１１を配置してもよい。また、任意で、光源１０を冷却するファン等の冷却装置１２を設けてもよい。 Examples of the light source 10 include a light emitting diode (LED) and a laser. Examples of LEDs include chip LEDs and power LEDs. The light source 10 irradiates light toward the light incident end portion 21 of the light guide plate 20. In FIG. 1, the light source 10 shows a form including a cover, but the light source 10 may not have a cover. For example, an LED without a cover may be brought into close contact with the light incident end 21 of the light guide plate 20. For example, a power supply and an electronic circuit are connected to the light source 10. Examples of electronic circuits include constant current circuits and laser excitation circuits. A light source lens 11 may be arranged between the light source 10 and the light guide plate 20 so that the light emitted by the light source 10 is diffused in the light guide plate 20. Further, optionally, a cooling device 12 such as a fan for cooling the light source 10 may be provided.
　光源１０は、例えば、ブラケットを用いて、導光板２０の光入射端部２１に対向するように配置される。あるいは、光源１０が発した光を、光ファイバを介して導光板２０の光入射端部２１に導いてもよい。光源１０の数は１つであってもよいし、複数であってもよい。例えば、光源１０は観察対象３０を対向する二方から、あるいは四方から照射するように配置されるが、特に限定されない。また、導光板２０の光入射端部２１の数は１つであってもよいし、複数であってもよい。導光板２０の光出射端部２２の数は１つであってもよいし、複数であってもよい。例えば、導光板２０の形状が多角形である場合、多角形をなす各辺に光源１０を配置してもよいし、多角形をなす辺の少なくとも一部に光源１０を配置してもよい。導光板２０の形状が円形である場合、円形をなす周に所定の間隔をおいて光源１０を配置してもよい。 The light source 10 is arranged so as to face the light incident end portion 21 of the light guide plate 20 by using, for example, a bracket. Alternatively, the light emitted by the light source 10 may be guided to the light incident end portion 21 of the light guide plate 20 via an optical fiber. The number of the light sources 10 may be one or a plurality. For example, the light source 10 is arranged so as to irradiate the observation target 30 from two opposite sides or from all sides, but is not particularly limited. Further, the number of light incident end portions 21 of the light guide plate 20 may be one or a plurality. The number of light emitting end portions 22 of the light guide plate 20 may be one or a plurality. For example, when the shape of the light guide plate 20 is a polygon, the light source 10 may be arranged on each side forming the polygon, or the light source 10 may be arranged on at least a part of the sides forming the polygon. When the shape of the light guide plate 20 is circular, the light sources 10 may be arranged at predetermined intervals around the circular shape.
　導光板２０は、例えば、観察対象３０を観察するための顕微鏡のレンズ４０の光軸に対して垂直方向に配置される。導光板２０は、例えば、透明基板を備える。あるいは、導光板２０は、透明基板である。透明基板は、光に対して透明な材料からなる。透明基板の材料は、二酸化ケイ素等の酸化ケイ素であってもよい。透明基板はガラスであってもよい。透明基板の材料は、アクリル、ポリエチレンテレフタレート（ＰＥＴ）、ポリカーボネート、及びポリ塩化ビニル等の樹脂であってもよい。透明基板の材料は、単屈折性材料であってもよいし、複屈折性材料であってもよい。透明基板である導光板２０の下表面、側面、及び上表面は、例えば、平滑である。 The light guide plate 20 is arranged in a direction perpendicular to the optical axis of the lens 40 of the microscope for observing the observation target 30, for example. The light guide plate 20 includes, for example, a transparent substrate. Alternatively, the light guide plate 20 is a transparent substrate. The transparent substrate is made of a material that is transparent to light. The material of the transparent substrate may be silicon oxide such as silicon dioxide. The transparent substrate may be glass. The material of the transparent substrate may be a resin such as acrylic, polyethylene terephthalate (PET), polycarbonate, and polyvinyl chloride. The material of the transparent substrate may be a monorefringent material or a birefringent material. The lower surface, side surface, and upper surface of the light guide plate 20 which is a transparent substrate are, for example, smooth.
　図２に示すように、導光板２０に、孔２３が設けられていてもよい。導光板２０の孔２３の中に、観察対象３０を配置してもよい。この場合、光入射端部２１は、導光板２０の外周に位置する。光出射端部２２は、導光板２０の孔２３の内周に位置する。孔２３は、レーザー加工等により、導光板２０に設けられる。 As shown in FIG. 2, the light guide plate 20 may be provided with a hole 23. The observation target 30 may be arranged in the hole 23 of the light guide plate 20. In this case, the light incident end portion 21 is located on the outer periphery of the light guide plate 20. The light emitting end portion 22 is located on the inner circumference of the hole 23 of the light guide plate 20. The hole 23 is provided in the light guide plate 20 by laser processing or the like.
　導光板２０の形状は、正方形等の四方形、多角形、又は円形であるが、特に限定されない。正方形である場合の導光板２０の一辺の長さ、又は円形である場合の導光板２０の直径は、例えば、５ｍｍ以上、１０ｍｍ以上、１５ｍｍ以上、あるいは２０ｍｍ以上であるが、特に限定されない。また、正方形である場合の導光板２０の一辺の長さ、又は円形である場合の導光板２０の直径は、例えば、５０ｍｍ以下、４０ｍｍ以下、３０ｍｍ以下、２０ｍｍ以下、あるいは１０ｍｍ以下であるが、特に限定されない。 The shape of the light guide plate 20 is a square such as a square, a polygon, or a circle, but is not particularly limited. The length of one side of the light guide plate 20 when it is square, or the diameter of the light guide plate 20 when it is circular is, for example, 5 mm or more, 10 mm or more, 15 mm or more, or 20 mm or more, but is not particularly limited. The length of one side of the light guide plate 20 when it is square, or the diameter of the light guide plate 20 when it is circular is, for example, 50 mm or less, 40 mm or less, 30 mm or less, 20 mm or less, or 10 mm or less. There is no particular limitation.
　透明基板である導光板２０の厚みは、例えば、１μｍ以上、４μｍ以上、５μｍ以上、１０μｍ以上、１５μｍ以上、２０μｍ以上、２５μｍ以上、あるいは３０μｍ以上であるが、特に限定されない。また、透明基板である導光板２０の厚みは、例えば、５００μｍ以下、４００μｍ以下、３００μｍ以下、２００μｍ以下、１５０μｍ以下、１００μｍ以下、９０μｍ以下、８０μｍ以下、７０μｍ以下、６０μｍ以下、５０μｍ以下、４０μｍ以下、３０μｍ以下、あるいは１５μｍ以下であるが、特に限定されない。導光板２０から出射するシート状の光の厚みは、透明基板である導光板２０の厚みに近似する。したがって、透明基板である導光板２０の厚みを選択することにより、導光板２０から出射するシート状の光の厚みを調整することが可能である。導光板２０の厚みが薄いと、観察対象３０の像の解像度が高くなる傾向にある。また、導光板２０の厚みが厚いと、観察対象３０の三次元構造の把握が容易になる傾向にある。したがって、観察目的に応じて、導光板２０の厚みを選択してもよい。 The thickness of the light guide plate 20 which is a transparent substrate is, for example, 1 μm or more, 4 μm or more, 5 μm or more, 10 μm or more, 15 μm or more, 20 μm or more, 25 μm or more, or 30 μm or more, but is not particularly limited. The thickness of the light guide plate 20 which is a transparent substrate is, for example, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 40 μm or less. , 30 μm or less, or 15 μm or less, but is not particularly limited. The thickness of the sheet-shaped light emitted from the light guide plate 20 is close to the thickness of the light guide plate 20 which is a transparent substrate. Therefore, by selecting the thickness of the light guide plate 20 which is a transparent substrate, it is possible to adjust the thickness of the sheet-like light emitted from the light guide plate 20. When the light guide plate 20 is thin, the resolution of the image of the observation target 30 tends to be high. Further, when the light guide plate 20 is thick, the three-dimensional structure of the observation target 30 tends to be easily grasped. Therefore, the thickness of the light guide plate 20 may be selected according to the purpose of observation.
　導光板２０の孔２３の直径は、例えば、０．５ｍｍ以上、１ｍｍ以上、２ｍｍ以上、あるいは３ｍｍ以上であるが、特に限定されない。また、導光板２０の孔２３の直径は、例えば、２０ｍｍ以下、１０ｍｍ以下、９ｍｍ以下、８ｍｍ以下、７ｍｍ以下、６ｍｍ以下、あるいは５ｍｍ以下であるが、特に限定されない。 The diameter of the hole 23 of the light guide plate 20 is, for example, 0.5 mm or more, 1 mm or more, 2 mm or more, or 3 mm or more, but is not particularly limited. The diameter of the hole 23 of the light guide plate 20 is, for example, 20 mm or less, 10 mm or less, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less, or 5 mm or less, but is not particularly limited.
　光出射端部２２となる導光板２０の孔２３の内周の側面は、出射するシート状の光が拡散しないように、滑らかであることが好ましい。 It is preferable that the inner peripheral side surface of the hole 23 of the light guide plate 20 serving as the light emitting end portion 22 is smooth so that the emitted sheet-like light is not diffused.
　図１に示す導光板２０は、例えば、保持器具５１、５２で保持される。保持器具５１、５２は、例えば、導光板２０を挟んで、導光板２０を保持する。導光板２０の下側に配置される保持器具５１には、導光板２０に設けられた孔２３が露出するように、孔５３が設けられている。導光板２０の上側に配置される保持器具５２には、導光板２０に設けられた孔２３が露出するように、孔５４が設けられている。導光板２０の下表面に接する保持器具５１の上表面は、平滑である。導光板２０の上表面に接する保持器具５２の下表面は平滑である。保持器具５１、５２は、例えば、剛性のある材料からなる。保持器具５１、５２の材料の例としては、金属及び樹脂が挙げられる。保持器具５１、５２のそれぞれの形状の少なくとも一部は、導光板２０の形状と同じであってもよい。したがって、保持器具５１、５２のそれぞれの形状は、正方形等の四方形、多角形、又は円形であるが、特に限定されない。 The light guide plate 20 shown in FIG. 1 is held by, for example, holding devices 51 and 52. The holding devices 51 and 52 hold the light guide plate 20 by sandwiching the light guide plate 20, for example. The holding device 51 arranged under the light guide plate 20 is provided with a hole 53 so that the hole 23 provided in the light guide plate 20 is exposed. The holding device 52 arranged on the upper side of the light guide plate 20 is provided with a hole 54 so that the hole 23 provided in the light guide plate 20 is exposed. The upper surface of the holding device 51 in contact with the lower surface of the light guide plate 20 is smooth. The lower surface of the holding device 52 in contact with the upper surface of the light guide plate 20 is smooth. The holding devices 51 and 52 are made of, for example, a rigid material. Examples of the materials of the holding devices 51 and 52 include metals and resins. At least a part of each shape of the holding devices 51 and 52 may be the same as the shape of the light guide plate 20. Therefore, the shapes of the holding devices 51 and 52 are square, polygonal, or circular, such as a square, but are not particularly limited.
　導光板２０の下方には、観察対象３０を支持するための支持基板６０が配置される。例えば、支持基板６０上、かつ導光板２０の孔２３の中に観察対象３０が配置される。導光板２０の光出射端部２２と観察対象３０の間に、液体を配置して、導光板２０の光出射端部２２の中と外の間の屈折率差を低減してもよい。観察対象３０は、支持基板６０上に配置可能であり、かつ導光板２０の孔２３の中に挿入可能な透明容器７０の中に配置されてもよい。この場合、導光板２０の光出射端部２２と透明容器７０の間に液体を配置してもよい。また、透明容器７０内に液体を入れてもよい。 A support substrate 60 for supporting the observation target 30 is arranged below the light guide plate 20. For example, the observation target 30 is arranged on the support substrate 60 and in the hole 23 of the light guide plate 20. A liquid may be arranged between the light emitting end 22 of the light guide plate 20 and the observation target 30 to reduce the difference in refractive index between the inside and the outside of the light emitting end 22 of the light guide plate 20. The observation target 30 may be arranged in the transparent container 70 which can be arranged on the support substrate 60 and which can be inserted into the hole 23 of the light guide plate 20. In this case, the liquid may be arranged between the light emitting end portion 22 of the light guide plate 20 and the transparent container 70. Further, the liquid may be put in the transparent container 70.
　支持基板６０の下方から観察対象３０を観察する場合、支持基板６０は透明である。支持基板６０及び透明容器７０の材料は、二酸化ケイ素等の酸化ケイ素であってもよい。支持基板６０及び透明容器７０はガラスであってもよい。支持基板６０及び透明容器７０の材料は、アクリル、ポリエチレンテレフタレート（ＰＥＴ）、ポリカーボネート、及びポリ塩化ビニル等の樹脂であってもよい。支持基板６０及び透明容器７０の材料は、単屈折性材料であってもよいし、複屈折性材料であってもよい。支持基板６０の下表面、側面、及び上表面は、例えば、平滑である。透明容器７０の下表面及び側面は、例えば、平滑である。あるいは、支持基板６０に開口６２を設けてもよい。開口６２の径は、例えば、後述するレンズ４０の径より大きくてもよい。これにより、レンズ４０が開口６０の中に入ることが可能であり、レンズ４０を観察対象３０に近づけることが可能である。 When observing the observation target 30 from below the support substrate 60, the support substrate 60 is transparent. The material of the support substrate 60 and the transparent container 70 may be silicon oxide such as silicon dioxide. The support substrate 60 and the transparent container 70 may be made of glass. The material of the support substrate 60 and the transparent container 70 may be a resin such as acrylic, polyethylene terephthalate (PET), polycarbonate, and polyvinyl chloride. The material of the support substrate 60 and the transparent container 70 may be a monorefractive material or a birefringent material. The lower surface, side surfaces, and upper surface of the support substrate 60 are, for example, smooth. The lower surface and sides of the transparent container 70 are, for example, smooth. Alternatively, the support substrate 60 may be provided with an opening 62. The diameter of the opening 62 may be larger than the diameter of the lens 40, which will be described later, for example. As a result, the lens 40 can enter the opening 60, and the lens 40 can be brought closer to the observation target 30.
　観察対象３０は、少なくとも部分的に透明である。観察対象３０の例としては、ヒト、非ヒト動物、及び植物の生体試料が挙げられる。生体試料の例としては、臓器、骨、結合組織等の器官の少なくとも一部、及び細胞が挙げられるが、特に限定されない。生体試料は生きていてもよい。観察対象３０は、例えば、観察の前に、透明化処理される。観察対象３０が生体試料である場合、観察対象３０は、例えば、２，２´－チオジエタノールと非イオン性有機ヨウ素化合物を含む水溶性溶媒で、透明化処理される。また、観察対象３０は、光源１０が照射した光で励起される蛍光試薬で染色されていてもよい。染色の例としては、組織染色及び免疫染色が挙げられるが、特に限定されない。なお、観察対象３０は、生体試料に限られず、樹脂成型物等の人工物であってもよい。 The observation target 30 is at least partially transparent. Examples of the observation target 30 include biological samples of humans, non-human animals, and plants. Examples of biological samples include, but are not limited to, organs, bones, at least a part of organs such as connective tissue, and cells. The biological sample may be alive. The observation target 30 is, for example, transparentized before observation. When the observation target 30 is a biological sample, the observation target 30 is subjected to a clearing treatment with, for example, a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organic iodine compound. Further, the observation target 30 may be dyed with a fluorescent reagent excited by the light emitted by the light source 10. Examples of staining include, but are not limited to, tissue staining and immunostaining. The observation target 30 is not limited to the biological sample, and may be an artificial object such as a resin molded product.
　なお、観察対象３０を透明化する方法は上記方法に限定されない。例えば、Ｃｌａｒｉｔｙ法、ＣＵＢＩＣ法、Ｓｃａｌｅ法、及びＦｏｃｕｓＣｌｅａｒ（登録商標）により、観察対象３０を透明化してもよい。 The method of making the observation target 30 transparent is not limited to the above method. For example, the observation target 30 may be made transparent by the Clarity method, the CUBIC method, the Scale method, and the FocusClear (registered trademark).
　光源１０から発せられ、光入射端部２１から導光板２０の内部に入射した光は、導光板２０の下表面と上表面で反射しながら、導光板２０内部を進行し、光出射端部２２から出射する。光出射端部２２から出射したシート状の光の厚みは、導光板２０をなす透明基板の厚みとほぼ同じである。光出射端部２２から出射したシート状の光は、ほぼ厚みを一定に保ったまま、観察対象３０を照射する。 The light emitted from the light source 10 and incident on the inside of the light guide plate 20 from the light incident end 21 travels inside the light guide plate 20 while being reflected by the lower surface and the upper surface of the light guide plate 20, and the light emitting end 22 Emit from. The thickness of the sheet-like light emitted from the light emitting end portion 22 is substantially the same as the thickness of the transparent substrate forming the light guide plate 20. The sheet-shaped light emitted from the light emitting end portion 22 irradiates the observation target 30 while keeping the thickness substantially constant.
　シート状の光を照射された観察対象３０は、顕微鏡のレンズ４０を介して拡大観察される。レンズ４０は、例えば、対物レンズである。レンズ４０は、躯体４１に配置される。顕微鏡の躯体４１内には、例えば、レンズ４０を介して観察される観察対象３０を撮影する撮影装置４２が配置される。撮影装置４２は、例えば、ＣＣＤイメージセンサ又はＣＭＯＳイメージセンサ等の固体撮像素子を備える。レンズ４０と撮影装置４２の間には、任意の光学系が配置され得る。例えば、顕微鏡の躯体４１内に、蛍光観察用フィルタが配置される。躯体４１に、観察用接眼レンズが設けられていてもよい。 The observation target 30 irradiated with the sheet-shaped light is magnified and observed through the lens 40 of the microscope. The lens 40 is, for example, an objective lens. The lens 40 is arranged on the skeleton 41. In the frame 41 of the microscope, for example, an imaging device 42 for photographing the observation target 30 observed through the lens 40 is arranged. The photographing device 42 includes, for example, a solid-state image sensor such as a CCD image sensor or a CMOS image sensor. Any optical system may be arranged between the lens 40 and the photographing device 42. For example, a fluorescence observation filter is arranged in the frame 41 of the microscope. An eyepiece for observation may be provided on the skeleton 41.
　観察対象３０が透明である場合、光源１０の数が１つでも、シート状の光は観察対象３０の断面を通過可能である。光源１０は、撮影装置４２が撮影する間の短い時間のみ、光を発すればよい。観察対象３０に不透明な部分がある場合、シート状の光の一部が、不透明な部分の先に進めない場合がある。これに対し、光源１０を複数備えれば、ある光源１０から発せられた光が到達できない部分を、別の光源１０から発せられた光で照射することが可能である。したがって、光源１０を複数備えれば、観察対象３０を回転させながら光を照射しなくともよく、光源１０は、撮影装置４２が撮影する間の短い時間のみ、光を発すればよい。観察対象３０に不透明な部分がない場合、光源１０は１つでもよい。実施形態に係る顕微鏡用照明装置によれば、観察対象３０への光の照射時間を短くすることが可能であるため、観察対象３０に対する光毒性が低く、観察対象３０が光によって劣化することを抑制することが可能である。また、観察対象３０中の蛍光分子の光退色を抑制することが可能である。さらに、観察対象３０を回転させる必要がないため、観察対象３０を高い解像度で観察することが可能である。 When the observation target 30 is transparent, even if the number of light sources 10 is one, the sheet-shaped light can pass through the cross section of the observation target 30. The light source 10 need only emit light for a short period of time while the photographing device 42 takes a picture. When the observation target 30 has an opaque portion, a part of the sheet-like light may not advance beyond the opaque portion. On the other hand, if a plurality of light sources 10 are provided, it is possible to irradiate a portion where the light emitted from one light source 10 cannot reach with the light emitted from another light source 10. Therefore, if a plurality of light sources 10 are provided, it is not necessary to irradiate the light while rotating the observation target 30, and the light source 10 may emit light only for a short time while the photographing device 42 takes a picture. If the observation target 30 has no opaque portion, the number of light sources 10 may be one. According to the illumination device for a microscope according to the embodiment, since it is possible to shorten the irradiation time of light on the observation target 30, the phototoxicity to the observation target 30 is low, and the observation target 30 is deteriorated by light. It can be suppressed. In addition, it is possible to suppress photobleaching of fluorescent molecules in the observation target 30. Further, since it is not necessary to rotate the observation target 30, it is possible to observe the observation target 30 with a high resolution.
　観察対象３０が生きている生体試料の場合、所定の間隔で光源１０から光を発して、生体試料の時間変化を観察してもよい。 When the observation target 30 is a living biological sample, light may be emitted from the light source 10 at predetermined intervals to observe the time change of the biological sample.
　実施形態に係る顕微鏡用照明装置は、観察対象３０を導光板２０の表面に対して垂直方向に移動させる観察対象移動装置６１をさらに備えていてもよい。観察対象移動装置６１は、例えば、支持基板６０を、導光板２０の表面に対して垂直方向、すなわち、顕微鏡のレンズ４０の光軸方向に移動させて、導光板２０に対して、観察対象３０を移動させる。観察対象移動装置６１は、例えば、シート状の光の厚みと同じ距離ずつ、観察対象３０を移動させる。観察対象移動装置６１には、例えば、アクチュエーターが使用可能である。 The microscope lighting device according to the embodiment may further include an observation target moving device 61 that moves the observation target 30 in the direction perpendicular to the surface of the light guide plate 20. The observation target moving device 61 moves, for example, the support substrate 60 in the direction perpendicular to the surface of the light guide plate 20, that is, in the optical axis direction of the lens 40 of the microscope, and the observation target 30 with respect to the light guide plate 20. To move. The observation target moving device 61 moves the observation target 30 by the same distance as the thickness of the sheet-shaped light, for example. For example, an actuator can be used as the observation target moving device 61.
　光源１０は、例えば、観察対象移動装置６１が観察対象３０を移動させるごとに、光を発し、観察対象３０をシート状の光で照射する。ただし、光源１０は、光を継続的に発していてもよい。顕微鏡の撮影装置４２は、観察対象移動装置６１が観察対象３０を移動させるごとに、レンズ４０を介して、観察対象３０を撮影する。これにより、観察対象３０の三次元画像を得ることが可能である。 The light source 10 emits light each time the observation target moving device 61 moves the observation target 30, and irradiates the observation target 30 with sheet-shaped light. However, the light source 10 may continuously emit light. The imaging device 42 of the microscope photographs the observation target 30 through the lens 40 each time the observation target moving device 61 moves the observation target 30. This makes it possible to obtain a three-dimensional image of the observation target 30.
　あるいは、実施形態に係る顕微鏡用照明装置は、図３に示すように、導光板２０を導光板２０の表面に対して垂直方向に移動させる導光板移動装置５５をさらに備えていてもよい。導光板移動装置５５は、例えば、導光板２０を顕微鏡のレンズ４０の光軸方向に移動させて、観察対象３０に対して導光板２０を移動させる。導光板移動装置５５は、例えば、シート状の光の厚みと同じ距離ずつ、導光板２０を移動させる。導光板移動装置５５には、例えば、アクチュエーターが使用可能である。 Alternatively, as shown in FIG. 3, the microscope lighting device according to the embodiment may further include a light guide plate moving device 55 that moves the light guide plate 20 in a direction perpendicular to the surface of the light guide plate 20. The light guide plate moving device 55, for example, moves the light guide plate 20 in the optical axis direction of the lens 40 of the microscope to move the light guide plate 20 with respect to the observation target 30. The light guide plate moving device 55 moves the light guide plate 20 by the same distance as the thickness of the sheet-shaped light, for example. For example, an actuator can be used for the light guide plate moving device 55.
　光源１０は、例えば、導光板移動装置５５が導光板２０を移動させるごとに、光を発し、観察対象３０をシート状の光で照射する。ただし、光源１０は、光を継続的に発していてもよい。撮影装置４２は、導光板移動装置５５が導光板２０を移動させるごとに、レンズ４０を介して、観察対象３０を撮影する。これにより、観察対象３０の三次元画像を得ることが可能である。 The light source 10 emits light each time the light guide plate moving device 55 moves the light guide plate 20, and irradiates the observation target 30 with sheet-shaped light. However, the light source 10 may continuously emit light. The photographing device 42 photographs the observation target 30 via the lens 40 each time the light guide plate moving device 55 moves the light guide plate 20. This makes it possible to obtain a three-dimensional image of the observation target 30.
　レンズ４０には、一般的な光学顕微鏡に含まれるレンズが使用可能である。また、撮影装置４２には、一般的な光学顕微鏡に含まれる撮影装置４２が使用可能である。したがって、光源１０と、導光板２０と、を備える顕微鏡用照明装置を一般的な光学顕微鏡のステージに配置することにより、実施形態に係る顕微鏡を作製することが可能である。そのため、約１億円する多光子顕微鏡や、約３千万円する従来の光シート顕微鏡及び共焦点顕微鏡と比較して、実施形態に係る顕微鏡は、安価に作製することが可能である。なお、実施形態に係る顕微鏡用照明装置をはじめから組み込んだ顕微鏡を製造してもよいことはもちろんである。 As the lens 40, a lens included in a general optical microscope can be used. Further, as the photographing device 42, a photographing device 42 included in a general optical microscope can be used. Therefore, the microscope according to the embodiment can be manufactured by arranging the illumination device for a microscope including the light source 10 and the light guide plate 20 on the stage of a general optical microscope. Therefore, the microscope according to the embodiment can be manufactured at a lower cost than the multiphoton microscope, which costs about 100 million yen, and the conventional optical sheet microscope and confocal microscope, which cost about 30 million yen. Needless to say, a microscope incorporating the microscope lighting device according to the embodiment from the beginning may be manufactured.
　また、実施形態に係る顕微鏡用照明装置においては、シート状の光を生成する光学系が導光板２０であり、光学系の煩雑な調整をせずとも、光源１０から導光板２０に光を照射すれば、シート状の光を生成することが可能である。したがって、実施形態に係る顕微鏡用照明装置は、操作が容易である。また、光源１０は、消費電力が１ＷのＬＥＤや３ＷのＬＥＤのように２０Ｗ以下のような低消費電力のＬＥＤでもよいため、装置の小型化が可能であり、光源１０をバッテリーで駆動することも可能である。したがって、実施形態に係る顕微鏡用照明装置は、持ち運びが容易であり、例えば屋外で使用することも容易である。 Further, in the microscope lighting device according to the embodiment, the optical system that generates sheet-shaped light is the light guide plate 20, and the light source 10 irradiates the light guide plate 20 with light without complicated adjustment of the optical system. Then, it is possible to generate sheet-like light. Therefore, the microscope lighting device according to the embodiment is easy to operate. Further, since the light source 10 may be an LED having a low power consumption such as 20 W or less, such as an LED having a power consumption of 1 W or an LED having a power consumption of 3 W, the device can be miniaturized, and the light source 10 can be driven by a battery. Is also possible. Therefore, the microscope lighting device according to the embodiment is easy to carry, and is also easy to use, for example, outdoors.
　例えば、ガンの疑いのある生体組織を顕微鏡観察する際に、生体組織の断面画像を所定の距離の間隔をおいて取得すると、取得した断面画像にガン細胞が写っていなくとも、取得した断面画像を撮影した断面どうしの間にガン細胞が存在している場合がある。この場合、生体組織がガンに侵されていることを見落とし得る。これに対し、実施形態に係る顕微鏡用照明装置と顕微鏡によれば、生体組織の断面画像を連続的に短時間で撮影することが可能であるため、生体組織に存在するガンを確実に発見することが可能になり得る。 For example, when observing a biological tissue suspected of having cancer under a microscope, if cross-sectional images of the biological tissue are acquired at intervals of a predetermined distance, the acquired cross-sectional image is obtained even if cancer cells are not shown in the acquired cross-sectional image. Cancer cells may be present between the cross-sections taken. In this case, it can be overlooked that the living tissue is affected by cancer. On the other hand, according to the microscope lighting device and the microscope according to the embodiment, it is possible to continuously take a cross-sectional image of the living tissue in a short time, so that the cancer existing in the living tissue can be reliably detected. Can be possible.
　上記のように本発明を実施形態によって記載したが、この開示の一部をなす記述及び図面はこの発明を限定するものであると理解するべきではない。この開示から当業者には様々な代替実施形態、実施例及び運用技術が明らかになるはずである。例えば、上記の実施形態では、導光板が透明基板である例を説明した。これに対し、図４に示すように、導光板は、対向する反射部２４、２５であってもよい。反射部２４、２５は、光を反射可能な材料から成る。反射部２４、２５の材料としては、アルミニウム等の金属が使用可能である。 Although the present invention has been described by embodiment as described above, the descriptions and drawings that form part of this disclosure should not be understood to limit the invention. This disclosure should reveal to those skilled in the art various alternative embodiments, examples and operational techniques. For example, in the above embodiment, an example in which the light guide plate is a transparent substrate has been described. On the other hand, as shown in FIG. 4, the light guide plate may be the reflecting portions 24 and 25 facing each other. The reflecting portions 24 and 25 are made of a material capable of reflecting light. As the material of the reflecting portions 24 and 25, a metal such as aluminum can be used.
　反射部２４、２５は、平行に配置される。反射部２４、２５の間は、例えば、空洞である。反射部２４、２５には、孔２３が設けられていてもよい。反射部２４、２５の孔２３の中に、観察対象３０を配置してもよい。この場合、光入射端部２１は、反射部２４、２５の外周に位置する。光出射端部２２は、反射部２４、２５の孔２３の内周に位置する。 The reflecting portions 24 and 25 are arranged in parallel. The space between the reflecting portions 24 and 25 is, for example, a cavity. The reflecting portions 24 and 25 may be provided with holes 23. The observation target 30 may be arranged in the holes 23 of the reflection portions 24 and 25. In this case, the light incident end portion 21 is located on the outer periphery of the reflecting portions 24 and 25. The light emitting end portion 22 is located on the inner circumference of the holes 23 of the reflecting portions 24 and 25.
　反射部２４、２５の間隔は、例えば、１μｍ以上、４μｍ以上、５μｍ以上、１０μｍ以上、１５μｍ以上、２０μｍ以上、あるいは２５μｍ以上であるが、特に限定されない。また、反射部２４、２５の間隔は、例えば、５００μｍ以下、４００μｍ以下、３００μｍ以下、２００μｍ以下、１５０μｍ以下、１００μｍ以下、９０μｍ以下、８０μｍ以下、７０μｍ以下、６０μｍ以下、５０μｍ以下、４０μｍ以下、３０μｍ以下、あるいは１５μｍ以下であるが、特に限定されない。 The distance between the reflecting portions 24 and 25 is, for example, 1 μm or more, 4 μm or more, 5 μm or more, 10 μm or more, 15 μm or more, 20 μm or more, or 25 μm or more, but is not particularly limited. The intervals between the reflecting portions 24 and 25 are, for example, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, 150 μm or less, 100 μm or less, 90 μm or less, 80 μm or less, 70 μm or less, 60 μm or less, 50 μm or less, 40 μm or less, 30 μm. The following, or 15 μm or less, but is not particularly limited.
　光源１０が発した光は、反射部２４、２５の間を反射しながら進行し、光出射端部２２からシート状に出射する。光出射端部２２から出射するシート状の光の厚みは、反射部２４、２５の間隔に近似する。したがって、反射部２４、２５の間隔を選択することにより、光出射端部２２から出射するシート状の光の厚みを調整することが可能である。導光板が対向する反射部２４、２５であっても、光源１０の数は特に限定されず、１つであってもよいし、複数であってもよい。また、反射部２４、２５のそれぞれの形状は特に限定されず、例えば、正方形等の四方形、多角形、又は円形であってもよい。 The light emitted by the light source 10 travels while being reflected between the reflecting portions 24 and 25, and is emitted in a sheet shape from the light emitting end portion 22. The thickness of the sheet-shaped light emitted from the light emitting end portion 22 is close to the distance between the reflecting portions 24 and 25. Therefore, it is possible to adjust the thickness of the sheet-like light emitted from the light emitting end portion 22 by selecting the intervals between the reflecting portions 24 and 25. The number of the light sources 10 is not particularly limited even if the light guide plates are the reflecting portions 24 and 25 facing each other, and the number of the light sources 10 may be one or a plurality. The shapes of the reflecting portions 24 and 25 are not particularly limited, and may be, for example, a square such as a square, a polygon, or a circle.
　このように、本発明はここでは記載していない様々な実施形態等を包含するということを理解すべきである。 As described above, it should be understood that the present invention includes various embodiments not described here.
　（実施例１）
　２枚の厚さ１４０μｍの四角形のガラス透明基板を導光板として用意した。２枚の導光板を横方向に間隔をおいて対向させ、２枚の導光板のそれぞれの外側の辺の中心に、２個の青色ＬＥＤを光源として配置した。これにより、実施例１に係る顕微鏡用照明装置を作製した。２枚の導光板の間の底面をカバーガラスで塞ぎ、２枚の導光板の間を２，２´－チオジエタノールと非イオン性有機ヨウ素化合物を含む水溶性溶媒で満たした。さらに、２，２´－チオジエタノールと非イオン性有機ヨウ素化合物を含む水溶性溶媒で予め透明化処理した２ｍｍ厚のマウスの胃の標本を、２枚の導光板の間に入れた。マウスの胃の標本は、青色光で励起される緑色蛍光タンパク質（ＧＦＰ）が細胞核で発現するトランスジェニックマウスより取得した。 (Example 1)
Two quadrangular transparent glass substrates having a thickness of 140 μm were prepared as light guide plates. The two light guide plates were opposed to each other with a lateral spacing, and two blue LEDs were arranged as light sources at the center of the outer sides of the two light guide plates. As a result, the illumination device for a microscope according to Example 1 was produced. The bottom surface between the two light guide plates was closed with a cover glass, and the space between the two light guide plates was filled with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organic iodine compound. Further, a 2 mm thick mouse stomach specimen pre-cleared with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organic iodine compound was placed between the two light guide plates. Mouse stomach specimens were obtained from transgenic mice in which the green fluorescent protein (GFP) excited by blue light is expressed in the cell nucleus.
　実施例１に係る顕微鏡用照明装置を一般的な顕微鏡（キーエンス）のステージに配置した。２個のＬＥＤのそれぞれに０．２５Ｗの電力を供給し、２個のＬＥＤから青色光を発して、ガラス透明基板からマウスの胃の標本にシート状の青色光を照射した。顕微鏡に付属のＧＦＰ用フィルタを用いて、マウスの胃の標本を観察した。その結果、図５（ａ）に示すように、従来の高価な光シート顕微鏡と同等の高い解像度の観察画像が得られた。一方、同じ標本を、実施例１に係る顕微鏡用照明装置を用いずに、通常の顕微鏡の水銀ランプで下方から照射して観察した場合、図５（ｂ）に示すように、細胞の構造は全く観察することができなかった。 The microscope lighting device according to Example 1 was placed on the stage of a general microscope (Keyence). A power of 0.25 W was supplied to each of the two LEDs, blue light was emitted from the two LEDs, and a sheet-shaped blue light was irradiated to the mouse stomach specimen from the transparent glass substrate. A mouse stomach specimen was observed using the GFP filter attached to the microscope. As a result, as shown in FIG. 5A, an observation image having a high resolution equivalent to that of a conventional expensive optical sheet microscope was obtained. On the other hand, when the same specimen is observed by irradiating it from below with a mercury lamp of a normal microscope without using the illumination device for a microscope according to Example 1, the cell structure is as shown in FIG. 5 (b). I couldn't observe it at all.
　（実施例２）
　厚さ５０μｍの四角形のガラス透明基板であって、中央に直径３ｍｍの孔が設けられたガラス透明基板を導光板として用意した。導光板の外側の４辺のそれぞれの中心に、青色ＬＥＤを光源として配置した。これにより、実施例２に係る、４個のＬＥＤを備える顕微鏡用照明装置を作製した。導光板の孔の底面をカバーガラスで塞ぎ、導光板の孔を２，２´－チオジエタノールと非イオン性有機ヨウ素化合物を含む水溶性溶媒で満たした。さらに、実施例１と同様の２ｍｍ厚のマウスの胃の標本を、導光板の孔に入れた。 (Example 2)
A rectangular glass transparent substrate having a thickness of 50 μm and having a hole having a diameter of 3 mm in the center was prepared as a light guide plate. A blue LED was arranged as a light source at the center of each of the four outer sides of the light guide plate. As a result, a microscope lighting device including four LEDs according to Example 2 was produced. The bottom surface of the hole of the light guide plate was closed with a cover glass, and the hole of the light guide plate was filled with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organic iodine compound. Further, a 2 mm thick mouse stomach specimen similar to that in Example 1 was placed in the hole of the light guide plate.
　実施例２に係る顕微鏡用照明装置を一般的な顕微鏡（キーエンス）のステージに配置した。ＬＥＤに０．２５Ｗの電力を供給し、ＬＥＤから青色光を発して、ガラス透明基板からマウスの胃の標本にシート状の青色光を照射した。顕微鏡に付属のＧＦＰ用フィルタを用いて、マウスの胃の標本を観察した。その結果、図６に示すように、従来の高価な光シート顕微鏡と同等の高い解像度の観察画像が得られた。倍率を上げても、図７（ａ）に示すように、高い解像度の観察画像が得られた。一方、同じ標本を、実施例２に係る顕微鏡用照明装置を用いずに、通常の顕微鏡の水銀ランプで下方から照射して観察した場合、図７（ｂ）に示すように、細胞の構造は全く観察することができなかった。 The microscope lighting device according to Example 2 was placed on the stage of a general microscope (Keyence). A power of 0.25 W was supplied to the LED, blue light was emitted from the LED, and a sheet-shaped blue light was irradiated to the stomach specimen of the mouse from the transparent glass substrate. A mouse stomach specimen was observed using the GFP filter attached to the microscope. As a result, as shown in FIG. 6, an observation image having a high resolution equivalent to that of a conventional expensive optical sheet microscope was obtained. Even when the magnification was increased, a high-resolution observation image was obtained as shown in FIG. 7 (a). On the other hand, when the same specimen is observed by irradiating it from below with a mercury lamp of a normal microscope without using the microscope illumination device according to Example 2, the cell structure is as shown in FIG. 7 (b). I couldn't observe it at all.
　（実施例３）
　導光板が厚さ３０μｍのガラス透明基板である以外は実施例２と同様の構成を有する実施例３に係る顕微鏡用照明装置を作製した。実施例２と同様の２ｍｍ厚のマウスの胃の標本を、実施例２と同様に観察した。その結果、図８に示すように、従来の高価な光シート顕微鏡と同等の高い解像度の観察画像が得られた。一方、同じ標本を、実施例３に係る顕微鏡用照明装置を用いずに、通常の顕微鏡の水銀ランプで下方から照射して観察した場合、図９に示すように、細胞の構造は全く観察することができなかった。同じ標本を高価な共焦点顕微鏡で観察したところ、図１０に示すように、胃腺を取り囲む細胞核が観察された。図８に示す画像でも、同等の構造が観察されたことから、実施例３に係る顕微鏡用照明装置を用いれば、高価な共焦点顕微鏡と同等以上の画像が得られることが示された。 (Example 3)
The illumination device for a microscope according to Example 3 having the same configuration as that of Example 2 except that the light guide plate is a transparent glass substrate having a thickness of 30 μm was produced. A 2 mm thick mouse stomach specimen similar to Example 2 was observed in the same manner as in Example 2. As a result, as shown in FIG. 8, an observation image having a high resolution equivalent to that of a conventional expensive optical sheet microscope was obtained. On the other hand, when the same specimen is irradiated from below with a mercury lamp of a normal microscope without using the microscope illumination device according to Example 3, the cell structure is completely observed as shown in FIG. I couldn't. When the same specimen was observed with an expensive confocal microscope, cell nuclei surrounding the gastric glands were observed, as shown in FIG. Since the same structure was observed in the image shown in FIG. 8, it was shown that an image equal to or higher than that of an expensive confocal microscope can be obtained by using the microscope illumination device according to the third embodiment.
　（実施例４）
　厚さ１５μｍの四角形のガラス透明基板であって、中央に直径３ｍｍの孔が設けられたガラス透明基板を導光板として用意した。 (Example 4)
A rectangular glass transparent substrate having a thickness of 15 μm and having a hole having a diameter of 3 mm in the center was prepared as a light guide plate.
　２，２´－チオジエタノールと非イオン性有機ヨウ素化合物を含む水溶性溶媒で予め透明化処理したマウス肺のブロックを用意した。ブロックの厚みは１ｍｍであった。マウス肺の細胞の核を、ＤＡＰＩ（４´，６－ｄｉａｍｉｄｉｎｏ－２－ｐｈｅｎｙｌｉｎｄｏｌｅ）又はＳＹＢＲ　Ｇｏｌｄ（登録商標、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ）で染色した。また、マウス肺の血管内壁を、トマトレクチンであるＤｙＬｉｇｈｔ５９４（フナコシ）で染色した。染色したマウス肺を、透明なケースに入れた。透明なケース内のマウス肺の周囲は、２，２´－チオジエタノールと非イオン性有機ヨウ素化合物を含む水溶性溶媒で満たした。マウス肺を入れた透明なケースを、厚さ１５μｍの四角形のガラス透明基板の孔の中に配置した。 A block of mouse lung that had been previously cleared with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organoiodine compound was prepared. The thickness of the block was 1 mm. The nuclei of mouse lung cells were stained with DAPI (4', 6-diamidino-2-phenylindole) or SYBR Gold® (registered trademark, Thermo Fisher). In addition, the inner wall of the blood vessel of the mouse lung was stained with DyLight594 (funakoshi), which is a tomato lectin. Stained mouse lungs were placed in a clear case. The perimeter of the mouse lung in a clear case was filled with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organoiodine compound. A transparent case containing mouse lungs was placed in a hole in a square glass transparent substrate having a thickness of 15 μm.
　導光板の外側の３辺のそれぞれの中心に、ＬＥＤを光源として配置した。３個のＬＥＤは、それぞれ、４０５ｎｍの光を発するＬＥＤ、４７５ｎｍの光を発するＬＥＤ、及び５９４ｎｍの光を発するＬＥＤであった。４０５ｎｍの光は、ＤＡＰＩの励起光として利用可能である。４７５ｎｍの光は、ＳＹＢＲ　Ｇｏｌｄの励起光として利用可能である。５９４ｎｍの光は、ＤｙＬｉｇｈｔ５９４の励起光として利用可能である。 An LED was placed as a light source at the center of each of the three outer sides of the light guide plate. The three LEDs were an LED emitting 405 nm light, an LED emitting 475 nm light, and an LED emitting 594 nm light, respectively. Light at 405 nm can be used as excitation light for DAPI. The light of 475 nm can be used as the excitation light of SYBR Gold. The light of 594 nm can be used as the excitation light of DyLight594.
　ＤＡＰＩとＤｙＬｉｇｈｔ５９４で染色したマウス肺に対し、ＬＥＤから４０５ｎｍの光と５９４ｎｍの光を順次発し、１０倍の対物レンズ（ニコン、プランフルオール）を装着した顕微鏡（キーエンス、Ｂｉｏｚｅｒｏ）でマウス肺を観察した。４０５ｎｍの光を照射した場合、図１１に示すように、細胞核が点々と観察された。５９４ｎｍの光を照射した場合、図１２に示すように、血管構造が観察された。図１１に示した写真と図１２に示した写真の合成写真を図１３に示す。 For mouse lungs stained with DAPI and DyLight594, 405 nm light and 594 nm light are sequentially emitted from the LED, and the mouse lungs are observed with a microscope (Keyence, Biozero) equipped with a 10x objective lens (Nikon, Planfluol). bottom. When irradiated with light of 405 nm, cell nuclei were observed in spots as shown in FIG. When irradiated with light of 594 nm, a vascular structure was observed as shown in FIG. A composite photograph of the photograph shown in FIG. 11 and the photograph shown in FIG. 12 is shown in FIG.
　ＳＹＢＲ　ＧｏｌｄとＤｙＬｉｇｈｔ５９４で染色したマウス肺に対し、ＬＥＤから４７５ｎｍの光と５９４ｎｍの光を順次発し、１０倍の対物レンズ（ニコン、プランフルオール）を装着した顕微鏡（キーエンス、Ｂｉｏｚｅｒｏ）でマウス肺を観察した。４７５ｎｍの光を照射した場合、図１４に示すように、細胞核が点々と観察された。５９４ｎｍの光を照射した場合、図１５に示すように、血管構造が観察された。図１４に示した写真と図１５に示した写真の合成写真を図１６に示す。 For mouse lungs stained with SYBR Gold and DyLight594, 475 nm light and 594 nm light are sequentially emitted from the LED, and the mouse lungs are examined with a microscope (Keyence, Biozero) equipped with a 10x objective lens (Nikon, Planfluol). Observed. When irradiated with light of 475 nm, cell nuclei were observed in spots as shown in FIG. When irradiated with light of 594 nm, a vascular structure was observed as shown in FIG. A composite photograph of the photograph shown in FIG. 14 and the photograph shown in FIG. 15 is shown in FIG.
　薄い導光板を用いることにより、薄切していないマウス肺のブロックの内部構造を高い解像度で観察できることが示された。 It was shown that by using a thin light guide plate, the internal structure of the unsliced mouse lung block can be observed with high resolution.
　（実施例５）
　厚さ１０μｍの四角形のガラス透明基板であって、中央に直径３ｍｍの孔が設けられたガラス透明基板を導光板として用意した。 (Example 5)
A quadrangular transparent glass substrate having a thickness of 10 μm and having a hole having a diameter of 3 mm in the center was prepared as a light guide plate.
　２，２´－チオジエタノールと非イオン性有機ヨウ素化合物を含む水溶性溶媒で予め透明化処理したマウス肺のブロックを用意した。ブロックの厚みは１ｍｍであった。マウス肺の細胞の核を、ＤＡＰＩ（４´，６－ｄｉａｍｉｄｉｎｏ－２－ｐｈｅｎｙｌｉｎｄｏｌｅ）で染色した。染色したマウス肺を、透明なケースに入れた。透明なケース内のマウス肺の周囲は、２，２´－チオジエタノールと非イオン性有機ヨウ素化合物を含む水溶性溶媒で満たした。マウス肺を入れた透明なケースを、厚さ１０μｍの四角形のガラス透明基板の孔の中に配置した。 A block of mouse lung that had been previously cleared with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organoiodine compound was prepared. The thickness of the block was 1 mm. The nuclei of mouse lung cells were stained with DAPI (4', 6-diamidino-2-phenylindole). Stained mouse lungs were placed in a clear case. The perimeter of the mouse lung in a clear case was filled with a water-soluble solvent containing 2,2'-thiodiethanol and a nonionic organoiodine compound. A transparent case containing mouse lungs was placed in a hole in a square glass transparent substrate having a thickness of 10 μm.
　導光板の外側に、４０５ｎｍの光を発するＬＥＤを光源として配置した。４０５ｎｍの光は、ＤＡＰＩの励起光として利用可能である。１０倍の対物レンズ（ニコン、プランフルオール）を装着した顕微鏡（キーエンス、Ｂｉｏｚｅｒｏ）でマウス肺を観察した。図１７に示すように、肺胞が観察された。 An LED that emits light of 405 nm was arranged as a light source on the outside of the light guide plate. Light at 405 nm can be used as excitation light for DAPI. Mouse lungs were observed with a microscope (Keyence, Biozero) equipped with a 10x objective lens (Nikon, Planfluol). As shown in FIG. 17, alveoli were observed.
　厚さ１０μｍのガラス透明基板から出射する厚さ１０μｍのシート状の光は、従来の光シート顕微鏡で用いられるシート状の光の厚みと同等である。薄い導光板を用いることにより、薄切していないマウス肺のブロックの内部構造を高い解像度で観察できることが示された。 The 10 μm-thick sheet-shaped light emitted from the 10 μm-thick glass transparent substrate is equivalent to the thickness of the sheet-shaped light used in a conventional optical sheet microscope. It was shown that by using a thin light guide plate, the internal structure of the unsliced mouse lung block can be observed with high resolution.
　実施形態に係る顕微鏡用照明装置は、特に限定されないが、臨床病理学、基礎医学、薬学、理学、及び農学等に関する産業に利用可能である。例えば、薬学に関する産業において、実施形態に係る顕微鏡用照明装置は、新薬スクリーニングや副作用スクリーニングに利用可能である。理学に関する産業において、実施形態に係る顕微鏡用照明装置は、動植物の少なくとも一部の観察に利用可能である。農学に関する産業において、実施形態に係る顕微鏡用照明装置は、品種改良された植物の観察に利用可能である。 The illumination device for a microscope according to the embodiment is not particularly limited, but can be used in industries related to clinical pathology, basic medicine, pharmacy, science, agriculture, and the like. For example, in the pharmaceutical industry, the microscope illumination device according to the embodiment can be used for new drug screening and side effect screening. In the science industry, the microscope illuminator according to the embodiment is available for observing at least some of the flora and fauna. In the agricultural industry, the microscope illuminator according to the embodiment can be used for observing cultivated plants.
　１０・・・光源、１１・・・レンズ、１２・・・冷却装置、２０・・・導光板、２１・・・光入射端部、２２・・・光出射端部、２３・・・孔、２４、２５・・・反射部、３０・・・観察対象、４０・・・レンズ、４１・・・躯体、４２・・・撮影装置、５１、５２・・・保持器具、５３、５４・・・孔、５５・・・導光板移動装置、６０・・・支持基板、６１・・・観察対象移動装置、７０・・・透明容器
  10 ... light source, 11 ... lens, 12 ... cooling device, 20 ... light guide plate, 21 ... light incident end, 22 ... light emitting end, 23 ... hole, 24, 25 ... Reflector, 30 ... Observation target, 40 ... Lens, 41 ... Frame, 42 ... Imaging device, 51, 52 ... Holding device, 53, 54 ... Hole, 55 ... Light source moving device, 60 ... Support substrate, 61 ... Observing object moving device, 70 ... Transparent container

Claims (14)

  1. 　光を照射する光源と、
    　前記光が内部に入射する光入射端部と、前記光が内部からシート状で出射する光出射端部と、を有する導光板と、
    　を備え、
    　前記光出射端部から出射した前記シート状の光で、観察対象を照射するための、顕微鏡用照明装置。     A light source that irradiates light and
    A light guide plate having a light incident end portion where the light is incident inside and a light emitting end portion where the light is emitted from the inside in a sheet shape.
    With
    A lighting device for a microscope for illuminating an observation target with the sheet-shaped light emitted from the light emitting end portion.
  2. 　前記導光板に孔が設けられており、前記光入射端部が前記導光板の外周に位置し、前記光出射端部が前記導光板の前記孔の内周に位置する、請求項１に記載の顕微鏡用照明装置。 The first aspect of the present invention, wherein the light guide plate is provided with a hole, the light incident end portion is located on the outer periphery of the light guide plate, and the light emitting end portion is located on the inner circumference of the hole of the light guide plate. Lighting equipment for microscopes.
  3. 　前記導光板に前記光を入射する前記光源を複数備える、請求項１又は２に記載の顕微鏡用照明装置。 The illumination device for a microscope according to claim 1 or 2, further comprising a plurality of the light sources that incident the light on the light guide plate.
  4. 　前記導光板が透明基板を備える、請求項１から３のいずれか１項に記載の顕微鏡用照明装置。 The microscope lighting device according to any one of claims 1 to 3, wherein the light guide plate includes a transparent substrate.
  5. 　前記透明基板が酸化ケイ素を含む、請求項４に記載の顕微鏡用照明装置。 The illumination device for a microscope according to claim 4, wherein the transparent substrate contains silicon oxide.
  6. 　前記透明基板が樹脂を含む、請求項４に記載の顕微鏡用照明装置。 The illumination device for a microscope according to claim 4, wherein the transparent substrate contains a resin.
  7. 　前記導光板が、対向する反射部を備える、請求項１から３のいずれか１項に記載の顕微鏡用照明装置。 The microscope lighting device according to any one of claims 1 to 3, wherein the light guide plate includes reflecting portions facing each other.
  8. 　前記観察対象を前記導光板の表面に対して垂直方向に移動させる観察対象移動装置をさらに備える、請求項１から７のいずれか１項に記載の顕微鏡用照明装置。 The illumination device for a microscope according to any one of claims 1 to 7, further comprising an observation target moving device for moving the observation target in a direction perpendicular to the surface of the light guide plate.
  9. 　前記導光板を前記導光板の表面に対して垂直方向に移動させる導光板移動装置をさらに備える、請求項１から７のいずれか１項に記載の顕微鏡用照明装置。 The illumination device for a microscope according to any one of claims 1 to 7, further comprising a light guide plate moving device for moving the light guide plate in a direction perpendicular to the surface of the light guide plate.
  10. 　光を照射する光源と、
    　前記光が内部に入射する光入射端部と、前記光が内部からシート状で出射する光出射端部と、を有する導光板と、
    　前記導光板の前記光出射端部から出射した前記シート状の光を照射された観察対象を観察するためのレンズと、
    　を備える、顕微鏡。     A light source that irradiates light and
    A light guide plate having a light incident end portion where the light is incident inside and a light emitting end portion where the light is emitted from the inside in a sheet shape.
    A lens for observing an observation target irradiated with the sheet-like light emitted from the light emitting end portion of the light guide plate.
    Equipped with a microscope.
  11. 　前記導光板が、前記レンズの光軸に対して垂直方向に配置される、請求項１０に記載の顕微鏡。 The microscope according to claim 10, wherein the light guide plate is arranged in a direction perpendicular to the optical axis of the lens.
  12. 　前記観察対象が移動するごとに前記レンズを介して前記観察対象を撮影する撮影装置をさらに備える、請求項１０又は１１に記載の顕微鏡。 The microscope according to claim 10 or 11, further comprising an imaging device that photographs the observation object through the lens each time the observation object moves.
  13. 　前記導光板が移動するごとに前記レンズを介して前記観察対象を撮影する撮影装置をさらに備える、請求項１０又は１１に記載の顕微鏡。 The microscope according to claim 10 or 11, further comprising an imaging device that photographs the observation target through the lens each time the light guide plate moves.
  14. 　光が内部に入射する光入射端部と、前記光が内部からシート状で出射する光出射端部と、を有する導光板の前記光入射端部に光を照射することと、
    　前記導光板の前記光出射端部から出射した前記シート状の光を観察対象に照射することと、
    　前記シート状の光を照射された観察対象を観察することと、
    　を含む、観察方法。
          Irradiating the light incident end portion of a light guide plate having a light incident end portion where light is incident inside and a light emitting end portion where the light is emitted from the inside in a sheet shape, and
    Irradiating the observation target with the sheet-like light emitted from the light emitting end portion of the light guide plate, and
    Observing the observation target irradiated with the sheet-shaped light and
    Observation methods, including.
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WO2024171736A1 (en) * 2023-02-17 2024-08-22 国立大学法人東京大学 Light guide plate and microscope kit

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US20120293797A1 (en) * 2009-12-17 2012-11-22 Universiteit Gent Methods and systems for optical characterisation

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US20120293797A1 (en) * 2009-12-17 2012-11-22 Universiteit Gent Methods and systems for optical characterisation

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024171736A1 (en) * 2023-02-17 2024-08-22 国立大学法人東京大学 Light guide plate and microscope kit

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