WO2021215438A1

WO2021215438A1 - Optical microscope and sample substrate holder

Info

Publication number: WO2021215438A1
Application number: PCT/JP2021/016047
Authority: WO
Inventors: 暁藤芳; 啓太石田
Original assignee: 国立大学法人東京工業大学
Priority date: 2020-04-20
Filing date: 2021-04-20
Publication date: 2021-10-28
Also published as: JP2023078490A

Abstract

This optical microscope comprises: a light source which emits irradiation light so as to be incident on a sample; a sample substrate which has a mounting surface on which the sample is mounted; a first optical element which irradiates the sample with the irradiation light and on which detection light from the sample is incident; a second optical element which has a recessed part that covers the sample formed therein, emits the irradiation light from the first optical element to the sample, and emits the incident detection light from the sample to the first optical element; and an imaging device which captures an image of the sample using the detection light emitted from the first optical element. The second optical element has a first incidence/emission surface, and a second incidence/emission surface which includes the recessed part and through which the irradiation light incident on the first incidence/emission surface is emitted to the sample and the detection light incident from the sample is emitted to the first incidence/emission surface, and the space formed by the recessed part and the mounting surface can be filled with a liquid.

Description

Optical microscope and sample substrate holder

The present disclosure relates to an optical microscope and a sample substrate holder.

There is an increasing need for an optical microscope that can image samples in nanoscale units. For example, by using an optical microscope that can be observed on a nanoscale, an object such as a cell nucleus contained in a sample can be observed. When observing an object contained in a sample, it is necessary not only to observe the sample containing the object in a plane but also to observe in the thickness direction of the sample, so-called depth observation.

Patent Document 1 describes a reflective microscope using an objective lens composed of two concave mirrors, a plane mirror and a convex mirror. The objective lens of the reflective microscope described in Patent Document 1 prevents a decrease in MTF and produces a high-resolution image by making the radius of curvature of the reflector and the distance between the reflectors have a specific relationship. Can be obtained.

Non-Patent Document 1 describes a cryo-optical microscope that employs a low-temperature objective mirror having a high NA of 0.93 and a wide field of view of 36 μm. The cryo-optical microscope described in Non-Patent Document 1 can clearly image minute objects such as mammalian cells by adopting a low-temperature objective mirror having a high NA and a wide field of view.

Japanese Unexamined Patent Publication No. 04-000407

However, the reflective microscope described in Patent Document 1 obtains a clear image when the positions of the plurality of optical elements are not sufficiently adjusted according to the focal lengths of the plurality of optical elements including two concave mirrors, a plane mirror and a convex mirror. Therefore, it is desirable to adjust the positions of a plurality of optical elements with high accuracy. In general, it is not easy to adjust the positions of a plurality of optical elements with high precision, and it is not easy to obtain a clear image by using the reflective microscope described in Patent Document 1.

Further, in Non-Patent Document 1, since a planar interface exists on the surface of the sample arranged apart from the low temperature objective mirror, spherical aberration occurs due to the refraction of the irradiation light incident on the sample and is included in the sample. The object may be distorted and imaged.

The present disclosure has been made to solve the above-mentioned problems, and an object of the present disclosure is to provide an optical microscope capable of acquiring an image with less distortion of a sample.

In order to achieve the above object, the optical microscope according to the present disclosure is mounted on a light source that emits irradiation light incident on the sample, a sample substrate having a mounting surface on which the sample is mounted, and a mounting surface. Along with irradiating the sample with irradiation light, a first optical element in which detection light is incident from the irradiated sample and a recess covering the sample are formed, and the irradiation light incident from the first optical element is emitted to the sample. At the same time, it has a second optical element that emits detection light incident from the sample to the first optical element, and an imaging device that captures an image of the sample formed by the detection light emitted from the first optical element. The two optical elements have a spherical shape, and the irradiation light is incident from the first optical element, and at least a part of the first inlet / output surface that emits the detected light to the first optical element and the wall portion of the recess. It has a second inlet / output surface that emits the irradiation light incident on the first inlet / output surface to the sample and emits the detection light incident from the sample to the first inlet / outlet surface, and has a recess and a mounting surface. The space formed by the optics can be filled with liquid.

In the optical microscope according to the present disclosure, the second optical element has a bottom surface on which the outer edge is connected to the first entrance / exit surface and the inner edge is connected to the second entrance / exit surface so that the second optical element can be arranged in close contact with the mounting surface. It is preferable to have more.

In the optical microscope according to the present disclosure, it is preferable that the second optical element is arranged so that the center of the spherical surface forming the first entrance / exit surface coincides with the focal point of the first optical element.

In the optical microscope according to the present disclosure, it is preferable that the second entrance / exit surface has a spherical shape whose center coincides with the center of the spherical surface forming the first entrance / exit surface.

In the optical microscope according to the present disclosure, it is preferable that the first optical element, the second optical element, and the sample substrate can be moved independently.

In the optical microscope according to the present disclosure, it is preferable that the second optical element and the sample substrate are integrally held.

In the optical microscope according to the present disclosure, it is preferable that the first optical element is composed of one or a plurality of optical lenses.

In the optical microscope according to the present disclosure, the first optical element has a planar shape, the first transmitting surface that emits the detected light to the image pickup apparatus while the irradiation light is incident from the light source, and the spherical shape. It has a first reflecting surface that is arranged to face the first transmitting surface, reflects the irradiation light incident from the first transmitting surface, and reflects the detected light to the first transmitting surface, and has a spherical shape. In addition, the second reflecting surface is arranged so that the inner edge is in contact with the outer edge of the first transmitting surface, reflects the irradiation light incident from the first reflecting surface, and reflects the detected light to the first reflecting surface, and a spherical shape. It has a shape, the inner edge is in contact with the outer edge of the first reflecting surface, and is arranged so as to face the second reflecting surface. It is preferable to have a second transmitting surface that emits the detection light emitted from the emitting surface to the second reflecting surface.

In the optical microscope according to the present disclosure, it is preferable that the first optical element is arranged so that the center of the second transmission surface coincides with the focal point of the first optical element.

The sample substrate holder according to the present disclosure is formed with a sample substrate having a mounting surface on which the sample is placed and a recess covering the sample, emits irradiation light to the sample, and emits detection light incident from the sample. The second optical element has a second optical element and a holding member for holding the sample substrate, and the second optical element has a spherical shape, and a first input / output surface that incidents irradiation light and emits detection light. A second inlet / output surface that includes at least a part of the wall portion of the recess and emits irradiation light incident on the first inlet / outlet surface to the sample and emits detection light incident from the sample to the first inlet / outlet surface. The space formed by the recess and the mounting surface can be filled with liquid.

The optical microscope according to the present disclosure can acquire an image of a sample with little distortion.

It is a figure which shows an example of the optical microscope which concerns on 1st Embodiment, (a) is the figure which shows the structure of the optical microscope, (b) is the optical path of the irradiation light which emits a light source 11 and is incident on a sample 3. It is a figure which shows, and (c) is the figure which shows the optical path of the detection light which emits a sample and is incident on an image pickup apparatus. (A) is a cross-sectional view of the second optical element shown in FIG. 1, and (b) is a cross-sectional view of a modified example of the second optical element shown in FIG. It is a figure which shows an example of the structure of the optical microscope which concerns on 2nd Embodiment. It is a figure which shows the optical path of the irradiation light of the optical microscope shown in FIG. It is a figure which shows the optical path of the detection light of the optical microscope shown in FIG. It is sectional drawing of the 1st optical element 13'and the 2nd optical element 14 shown in FIG. FIG. 3 is a view (No. 1) for explaining the holding member shown in FIG. 3, FIG. 3A is a perspective view of a second optical element and a ring member holding the second optical element shown in FIG. 3, and FIG. 3B is shown in FIG. Is a cross-sectional view of the second optical element and the ring member holding the second optical element shown in FIG. FIG. 2 is a view (No. 2) for explaining the holding member shown in FIG. 3, and is a cross-sectional view showing a state in which an intermediate member is coupled to the ring member shown in FIG. 7. 3 is a view for explaining the holding member shown in FIG. 3, FIG. 3A is a cross-sectional view of an installation member included in the holding member shown in FIG. 3, and FIG. 3B is a second optical element and a sample substrate. And is a cross-sectional view of a holding member. It is a figure which shows the optical simulation result of the optical microscope which concerns on the comparative example which the 2nd optical element 14 is not arranged, (a) shows the optical path of the irradiation light and the detection light between the 1st optical element 13', and a sample 3. It is a figure, (b) is a figure which shows the light intensity ratio at a depth Z with respect to the depth Z = 0, (c) is a figure which shows a simulation image, (d) is a figure which shows the intensity distribution. .. It is a figure which shows the optical simulation result of the optical microscope which concerns on 2nd Embodiment, (a) shows the optical path of irradiation light and detection light, (b) shows the light intensity ratio at depth Z with respect to depth Z = 0. It is a figure which shows, (c) is a figure which shows a simulation image, (d) is a figure which shows the intensity distribution. It is a photographed image when beads having a diameter of 6 μm were placed in a sample and irradiated with fluorescence. It is sectional drawing of the sample substrate holder which the optical microscope which concerns on 3rd Embodiment has.

Hereinafter, the optical microscope according to one aspect of the present disclosure will be described with reference to the drawings. However, it should be noted that the technical scope of the present disclosure is not limited to those embodiments, but extends to the inventions described in the claims and their equivalents. In the following description and figures, components having the same functional configuration are designated by the same reference numerals, so that duplicate description will be omitted.

FIG. 1 is a diagram showing an optical microscope according to the first embodiment, FIG. 1 (a) is a diagram showing a configuration of an optical microscope, and FIG. 1 (b) is a diagram in which a light source 11 is emitted and is incident on a sample 3. It is a figure which shows the optical path of the irradiation light, and FIG. 1C is a figure which shows the optical path of the detection light which emits a sample and is incident on an image pickup apparatus.

The optical microscope 1 includes a light source 11, a beam splitter 12, a first optical element 13, a second optical element 14, a condenser lens 15, and an image pickup device 16, and has a mounting surface 21 of a sample substrate 2. The sample 3 placed on the image is imaged. The optical microscope 1 may further include a holding member 17 that integrally holds the sample substrate 2 and the second optical element 14.

The light source 11 is, for example, a laser light source, and generates beam-shaped irradiation light which is, for example, infrared rays, visible light, and ultraviolet rays, and emits the generated irradiation light to the beam splitter 12. The beam splitter 12 is, for example, a half mirror, and reflects the irradiation light incident from the light source 11 on the first optical element 13. The first optical element 13 is an objective lens composed of one or a plurality of lenses. The irradiation light that has passed through the first optical element 13 is incident on the sample 3 mounted on the mounting surface 21 via the second optical element 14.

The detection light emitted from the sample 3 to which the irradiation light is incident is incident on the beam splitter 12 via the objective lens which is the second optical element 14 and the first optical element 13. The detection light incident from the beam splitter 12 passes through the beam splitter 12 and is incident on the condenser lens 15.

The condenser lens 15 collects the incident detection light and emits it to the image pickup apparatus 16. Instead of the condenser lens 15, the detection light incident from the beam splitter 12 may be focused, for example, by combining a concave mirror and a plane mirror. The detection light incident from the beam splitter 12 may be focused by combining an optical lens, a concave mirror and a plane mirror.

The image pickup device 16 is a storage device that stores an image pickup surface in which image pickup elements such as CCD (Charge Coupled Device) and CMOS (Complementary Metal Oxide Semiconductor) are arranged in an array, and image data indicating an image formed on the image pickup surface. Have. The image pickup apparatus 16 captures an image of a sample formed by the detection light emitted from the condenser lens 15, and stores image data indicating the captured image.

The holding member 17 may be arranged on the transfer device 4. The transfer device 4 moves the holding member in the x-axis, y-axis, and z-axis directions shown in the figure, for example, by electronic control of a personal computer. The plane extending along the x-axis and the y-axis is a plane extending in a direction parallel to the extending direction of the mounting surface 21. The z-axis direction is a direction parallel to the normal direction of the mounting surface 21, and is a depth direction of the sample 3. The transfer device 4 may be integrated with the optical microscope 1.

FIG. 2A is a cross-sectional view of the second optical element 14, and FIG. 5B is a cross-sectional view of a modified example of the second optical element 14.

The second optical element 14 is made of glass, for example, and has a recess 141, a first entrance / exit surface 142, a second entrance / exit surface 143, a bottom portion 144, and a side portion 145. The recess 141 covers the sample 3 and is filled with a liquid submerged in the sample 3. The liquid filled in the recess 141 preferably has a small difference in refractive index from that of sample 3. For example, when the sample 3 has a refractive index of about 1.4 to 1.7, the liquid is preferably ethanol having a refractive index of 1.36 rather than water having a refractive index of 1.33. Further, the liquid is more preferably an optical oil having a refractive index of 1.8 or less, which is also called immersion oil. The difference between the refractive index of the liquid and the refractive index of the sample is preferably 0.10 or less, and more preferably 0.05 or less. Various liquids can be used, and can be appropriately selected so that the difference in refractive index from the sample becomes small.

When the mounting surface 21 is in close contact with the bottom 144 while the liquid is filled, for example, the mounting surface 21 is fixed to the bottom 144 by the surface tension of the filled liquid, and the liquid can be sealed in the recess 141. Here, the close contact is, for example, a state in which the liquid filled by the surface lift of the liquid is not washed away, for example, a state in which the distance between the mounting surface 21 and the bottom portion 144 is 1 mm or less. The distance between the bottom 144 to which the second optical element 14 and the sample substrate 2 can be brought into close contact with the mounting surface 21 is appropriately determined depending on the size of the second optical element 14 and the sample substrate 2 and the type of liquid to be filled. Will be done.

The first input / output surface 142 has a spherical shape, and emits the irradiation light incident from the first optical element to the sample 3 and emits the detection light incident from the sample 3 to the first optical element 13. The second inlet / outlet surface 143 has a spherical shape including at least a part of the wall portion of the recess 141, emits the irradiation light incident on the first inlet / outlet surface 142 to the sample 3, and detects the incident light from the sample 3. Light is emitted to the first entrance / exit surface 142. The second optical element 14 is a meniscus lens in which the first entrance / exit surface 142, which is one of the curved surfaces of the lens, is convex, and the second entrance / exit surface 143, which is the other, is concave. The bottom 144 has an inner edge connected to the peripheral edge of the recess 141 and an outer edge connected to the first entrance / exit surface 142 and can be brought into close contact with the mounting surface 21. The side portion 145 extends vertically from the peripheral edge of the first entrance / exit surface 142 toward the mounting surface 21 and connects to the bottom portion 144.

As shown in FIG. 2B, the optical microscope according to the embodiment does not have a side portion 145 and has a hemispherical first entrance / exit surface 142'instead of the first entrance / exit surface 142. The two optical elements 14'may be used instead of the second optical element 14.

The objective lens, the second optical element 14, and the sample substrate 2, which are the first optical elements 13, can be moved independently of each other. The second optical element 14 and the sample substrate 2 may be integrally held by the holding member 17. Easy to adjust.

The first entrance / exit surface 142, which is one of the curved surfaces of the lens, is convex, and the second entrance / exit surface 143, which is the other, is concave. The following effects are produced by filling with.
(1) Since the lens is spherical with respect to the incident direction, it is vertically incident and is not affected by the refractive index.
(2) Since a liquid having a refractive index almost equal to that of the sample is used, it is not affected by the refractive index at the sample interface. Therefore, the image is not distorted.
(3) The objective lens can be used without being immersed in a liquid, and the liquid can be exchanged only on the meniscus lens side without being affected by the objective lens, and the objective lens can be easily changed.
(4) Only the liquid filled in the recesses of the second optical element 14 and the second optical element 14 is arranged in the optical path between the sample 3 and the first optical element 13 which is an objective lens, and it depends on the difference in the refractive index of the substance. It is possible to suppress the occurrence of image distortion.

FIG. 3 is a diagram showing an optical microscope according to the second embodiment.

The optical microscope 1'has a light source 11, a beam splitter 12, a first optical element 13', a second optical element 14, a condenser lens 15, and an image pickup device 16, and mounts the sample substrate 2. The sample 3 placed on the surface 21 is imaged. The optical microscope 1 ′ further includes a holding member 17 that integrally holds the sample substrate 2 and the second optical element 14.

FIG. 4 is a diagram showing an optical path of irradiation light emitted from the light source 11 and incident on the sample 3.

The light source 11 is, for example, a laser light source, and generates beam-shaped irradiation light which is, for example, infrared rays, visible light, and ultraviolet rays, and emits the generated irradiation light to the beam splitter 12. The beam splitter 12 reflects the irradiation light incident from the light source 11 on the first optical element 13'. The first optical element 13'is an integrally molded (Single Component) objective mirror integrally molded with quartz glass and provided with two reflecting mirrors. The irradiation light that has passed through the first optical element 13'is incident on the sample 3 mounted on the mounting surface 21 via the second optical element 14.

FIG. 5 is a diagram showing an optical path of the detection light that emits the sample 3 and is incident on the image pickup apparatus 16.

The detection light emitted from the sample 3 to which the irradiation light is incident is incident on the beam splitter 12 via the second optical element 14 and the first optical element 13'. The detection light incident from the beam splitter 12 passes through the beam splitter 12 and is incident on the condenser lens 15.

The condenser lens 15 collects the incident detection light and emits it to the image pickup apparatus 16. The image pickup apparatus 16 captures an image of a sample formed by the detection light emitted from the condenser lens 15, and stores image data indicating the captured image.

The optical microscope 1'may have a holding member 17. The holding member 17 may be arranged on the transfer device 4.

FIG. 6 is a cross-sectional view of the first optical element 13'and the second optical element 14 shown in FIG.

The base material of the first optical element 13'is, for example, quartz glass, and the accommodating portion 131, the first transmission surface 132, the first reflection surface 133, the second reflection surface 134, and the second transmission surface 135 are provided. Have. The accommodating portion 131 is a substantially hemispherical recess capable of accommodating at least a part of the second optical element 14. The first transmission surface 132 has a circular planar shape, and the irradiation light is incident from the light source 11 and the detection light is emitted to the image pickup apparatus 16.

The first reflecting surface 133 has a spherical shape and is arranged so as to face the first transmitting surface 132, reflects the irradiation light incident from the first transmitting surface 132, and is incident from the second reflecting surface 134. The detection light is reflected on the first transmission surface 132. The first reflective surface 133 is formed by coating a metal film formed of a highly reflective material such as aluminum, gold, and silver on glass as a base material. The first reflecting surface 133 may have an aspherical shape as long as it is convex toward the first transmitting surface 132 with respect to the inner edge of the second transmitting surface 135.

The second reflecting surface 134 has a spherical shape and is arranged so that the inner edge is in contact with the outer edge of the first transmitting surface 132, reflects the irradiation light incident from the first reflecting surface 133, and the second transmitting surface. The detection light incident from 135 is reflected on the first reflecting surface 133. The second reflective surface 134 is formed by coating a metal film formed of a highly reflective material such as aluminum, gold, and silver on glass as a base material.

The second transmission surface 135 has a spherical shape forming a part of the accommodating portion 131, and is arranged so that the inner edge is in contact with the outer edge of the first reflection surface 133 and is opposed to the second reflection surface 134. The second transmission surface 135 emits the irradiation light incident from the second reflection surface 134 as a spherical wave to the first inlet / output surface 142 of the second optical element 14, and is also emitted from the first inlet / outlet surface 142 as a spherical wave. The detected light is emitted to the second reflecting surface 134.

It is preferable that the center of the second transmission surface 135 is arranged so as to coincide with the focal point of the first optical element 13'. Further, it is preferable that the focal point of the first optical element 13'is aligned with the center of the spherical surface forming the first entrance / exit surface 142 of the second optical element 14. By aligning the focal point of the first optical element 13'with the center of the spherical surface of the first entrance / exit surface 142, the irradiation light incident on the first entrance / exit surface 142 from the second transmission surface 135 as a spherical wave is the second. 1 It is possible to make the entrance / exit surface 142 perpendicular to the entrance / exit surface 142. Since the irradiation light is incident perpendicularly to the first entrance / exit surface 142, spherical aberration is caused by the refraction of the irradiation light when it is incident on the first entrance / exit surface 142, and the image in the thickness direction of the sample is prevented from being distorted. can.

Similar to the first embodiment, the recess 141 of the second optical element 14 covers the sample 3 and is filled with the liquid submerged in the sample 3.

The sample 3 is housed in the recess 141 of the second optical element 14, the recess 141 is filled with a transparent liquid, and the combination of the first optical element 13'and the second optical element 14 produces the following effects.
(1) The first entrance / exit surface 142 of the second optical element 14 is spherical with respect to the incident direction of the irradiation light, and is arranged so that the focal point and the center of the first optical element 13'are aligned with each other. It is incident and is not affected by the refractive index.
(2) Since the sample 3 is submerged in the recess 141 by a liquid having a small difference in the refractive index from the sample 3, the influence of the refractive index can be minimized when the irradiation light is incident on the sample 3, so that the sample 3 can be used. Distortion of the image of the contained object is suppressed.
(3) Since the first optical element 13'is used without being immersed in a liquid for immersion and a dry optical element can be adopted, an optical element having desired optical characteristics is selected according to the type of sample 3. It can be used as an optical element 13'.
(4) Since the first optical element 13'is integrally molded, there is no possibility that the positional relationship between the first reflecting surface 133 and the second reflecting surface 134 deviates from the desired positional relationship and spherical aberration occurs.

The first optical element 13', the second optical element 14, and the sample substrate 2 can be moved independently of each other. The second optical element 14 and the sample substrate 2 may be integrally held by the holding member 17. Easy to adjust.

FIG. 7 is a diagram (No. 1) for explaining the holding member 17, FIG. 8 is a diagram (No. 2) for explaining the holding member, and FIG. 9 is a diagram (No. 2) for explaining the holding member. 3). FIG. 7A is a perspective view of a ring member holding the second optical element 14 and the second optical element 14, and FIG. 7B is a ring member holding the second optical element 14 and the second optical element 14. It is a cross-sectional view of. FIG. 8 is a cross-sectional view showing a state in which an intermediate member is coupled to the ring member shown in FIG. 7. FIG. 9A is a cross-sectional view of the installation member included in the holding member 17, and FIG. 9B is a cross-sectional view of the second optical element 14, the sample substrate 2, and the holding member 17.

The holding member 17 has a ring member 171, an intermediate member 172, and an installation member 173. The ring member 171 extends perpendicularly to the lower surface 1712 from between the outer circumference and the inner circumference of the cylindrical element installation portion 1714 having the annular upper surface 1711 and lower surface 1712 and the cylindrical side surface 1713 and the lower surface 1712 of the element installation portion. It has a cylindrical joint 1715 to be used. The holding member 17 integrally holds the second optical element 14 and the sample substrate 2. Further, the second optical element 14, the sample substrate 2, and the holding member 17 form a sample substrate holder 18.

A notch 1716 in which the second optical element 14 is arranged is formed in the central portion of the upper surface 1711 of the ring member 171. A gap 1717 is formed between the side surface of the cutout portion 1716 and the side portion 145 of the second optical element 14. The second optical element 14 is fixed by filling the gap 1717 with an adhesive member such as an adhesive resin. In the joint portion 1715, an internal threaded portion 1718 screwed with the intermediate member 172 is formed on the inner surface surface, and an external threaded portion 1719 screwed with the installation member 173 is formed on the outer surface surface.

The intermediate member 172 includes a cylindrical support main body portion 1725 having an annular upper surface 1721, an annular lower surface 1722, an outer surface 1723, and an inner side surface 1724, and a cylindrical support lower portion extending vertically from the inner circumference of the lower surface 1722. It has 1726 and. The outer peripheral radius of the support lower portion 1726 is smaller than the outer peripheral radius of the support main body 1725. The outer surface 1723 of the support main body is formed with a main body screw portion 1727 that is screwed with the internal screw portion 1718. The intermediate member 172 is formed so as to support the sample substrate 2 via the first buffer ring 1728 and to be detachably attached to the ring member 171 by screwing the main body threaded portion 1727 to the internal threaded portion 1718.

The installation member 173 has a cylindrical storage portion 1733 in which the first opening 1731 and the second opening 1732 are formed. In the storage portion 1733, a storage screw portion 1734 that is screwed with the external screw portion 1719 of the ring member 171 is formed on the first opening 1731 side of the inner side surface of the cylinder. The inner diameter of the storage portion 1733 on the first opening 1731 side is larger than the inner diameter on the second opening 1732 side. Since the inner diameters of the first opening 1731 side and the second opening 1732 side are different, a step portion 1735 is formed on the inner side surface of the storage portion 1733. The installation member 173 is connected to the bottom surface of the cylinder on the side of the second opening 1732 of the storage portion 1733, and has a cylindrical hem portion 1736 having an outer peripheral radius larger than the outer peripheral radius of the storage portion 1733. The hem portion 1736 is arranged so that the central axis coincides with the storage portion 1733.

The ring member 171 and the intermediate member 172 are inserted into the first opening 1731 of the installation member 173 in a screwed state, and the external screw portion 1719 of the ring member 171 and the storage screw portion 1734 of the installation member 173 are screwed together. The second buffer ring 1737 is arranged at the step portion 1735. By arranging the second buffer ring 1737 on the step portion 1735, it is possible to prevent the force from being conducted to the sample substrate 2 and the second optical element 14 even when a force is applied from the outside, and the sample substrate 2 and the second 2 The close contact state of the optical element 14 is maintained.

FIG. 10 is a diagram showing an optical simulation result of an optical microscope according to a comparative example in which the second optical element 14 is not arranged. (A) is a diagram showing an optical path of irradiation light and detection light between the first optical element 13'and sample 3, and (b) is a light intensity ratio EE1st (z) at a depth Z with respect to a depth Z = 0. ) / EE1st (z = 0), (c) is a diagram showing a simulation image, and (d) is a diagram showing an intensity distribution.

Energy in a circle (EE: Encircled Energy) represents the light intensity in a circle with a radius r, with the center of the light spot as zero. EE1st is r = first anechoic ring radius, and the first anechoic ring radius is the radius where the intensity is zero at the beginning of the Airy function.

In the optical simulation shown in FIG. 10, Zemax was used as the optical simulation software. The light intensity ratio EE1st (z) / EE1st (z = 0) decreases to 0.8 at a depth of 12 μm from the mounting surface. As shown in (c), the image at a depth of 12 μm from the mounting surface is greatly distorted. The intensity distribution shown in (d) is gradually lower than the sample position. This is because the incident irradiation light on the sample is refracted at the interface, which causes spherical aberration and distorts the image of the object inside the sample.

FIG. 11 is a diagram showing an example of the simulation result of the optical microscope 1 ′ according to the second embodiment. The recess 141 of the second optical element 14 is filled with a liquid. (A) shows the optical path of the irradiation light and the detection light between the first optical element 13', the second optical element 14, and the sample 3, and (b) is the light intensity ratio at the depth Z to the depth Z = 0. (C) is a diagram showing a simulation image, and (d) is a diagram showing an intensity distribution.

In the optical simulation shown in FIG. 11, the light intensity ratio EE1st (z) / EE1st (z = 0) decreases to 0.8 at a depth of 300 μm from the mounting surface 21 of the sample substrate 2. As shown in (c), the image at a depth of 300 μm is not so distorted. The intensity distribution shown in (d) is sharply lower than the sample position. Depth observation of about 300 μm is possible from the mounting surface 21. This is because the liquid filled in the recess 141 eliminates refraction at the sample interface.

FIG. 12 is an image taken when beads having a diameter of 6 μm are placed in a sample and irradiated with fluorescence, (a) is an image taken by a confocal microscope, and (b) is an image taken by an optical microscope according to a second embodiment. It is an image.

The image in the focal plane xy direction parallel to the mounting surface is almost the same as the image taken by the confocal microscope and the microscope of the second embodiment, but the image in the optical axis (z) direction is distorted by the image taken by the confocal microscope. On the other hand, it can be seen that the microscopic image of the second embodiment is clearly good.

FIG. 13 is a cross-sectional view of a sample substrate holder included in the optical microscope according to the third embodiment.

The optical microscope according to the third embodiment is different from the optical microscope 1'according to the second embodiment in that the sample substrate holder 18'is provided in place of the sample substrate holder 18. The components and functions of the components of the optical microscope according to the third embodiment other than the sample substrate holder 18'are the same as the components and functions of the optical microscope 1'with the same reference numerals. Is omitted.

The sample substrate holder 18'is different from the sample substrate holder 18 in that it has the fixing member 100 and the support member 110 instead of the holding member 17. Further, the sample substrate holder 18 ′ integrally holds the first optical element 13 ′ and the second optical element 14, and the sample substrate 2 on which the sample 3 is placed is placed on the first optical element 13 ′ and the second optical element. It differs from the sample substrate holder 18 in that it can move relative to 14.

In the fixing member 100, the first optical element 13'and the second optical element 14 are aligned so that the focal point of the first optical element 13'and the center of the spherical surface forming the first entrance / exit surface 142 of the second optical element 14 coincide with each other. To fix.

The fixing member 100 has a disk shape and has an upper surface 101 on which the first optical element 13'and the second optical element 14 are arranged, and a lower surface 102 on the opposite side of the upper surface 101. The upper surface 101 has a first notch 103 for installing the first optical element 13'and a second notch 104 for installing the second optical element 14 inside the first notch 103. The inner diameter of the disk of the fixing member 100 is a diameter that allows the support member 110 that supports the sample substrate 2 to be inserted and moved in the x-axis, y-axis, and z-axis directions.

The support member 110 has a columnar upper surface 111 on which the sample substrate 2 is arranged and a lower surface 112 facing the transfer device, and the sample substrate 2 is movably supported by the transfer device 4. The upper surface 111 of the support member 110 has a notch 113 into which the sample substrate 2 is fitted. The hole 114 penetrates from the upper surface 111 to the lower surface 112 along the central axis of the support member 110 so as to insert a pin (not shown) into the notch 113 from the lower surface 112 and remove the sample substrate 2 to be aligned. It is formed. The support member 110 may be formed with a slit instead of the hole 114. Further, the support member 110 further has a hem member 115 on the lower surface side for stabilizing the installation on the transfer device 4.

In the optical microscope according to the third embodiment, since the sample substrate 2 can be moved relative to the first optical element 13'and the second optical element 14, the range is wider than that of the optical microscope 1'according to the second embodiment. Can move the sample 3 in the x-axis and y-axis directions. In the optical microscope 1 ′ according to the second embodiment, when the sample substrate 2 is moved by about 0.5 mm in the x-axis and the y-direction, the positions between the first optical element 13 ′ and the second optical element 14 shift and the sample 3 The clear image of is not captured. On the other hand, in the optical microscope according to the third embodiment, the sample substrate 2 can be moved in the x-axis and the y-direction to capture a clear image of the sample 3 over a field of view of about 8 mm □.

Further, in the optical microscope described, the light source emits beam-shaped irradiation light, but the optical microscope according to the embodiment may be an optical sheet microscope in which the light source emits sheet-shaped irradiation light. Further, the light source may generate light from a light bulb, a fluorescent lamp, or an LED and use it as irradiation light.

The irradiation light may be applied to the sample from the surface opposite to the mounting surface of the sample substrate.

It should be understood that one of ordinary skill in the art can make various changes, replacements and amendments to this without departing from the spirit and scope of this disclosure.

Claims

A light source that emits irradiation light incident on the sample,
A sample substrate having a mounting surface on which the sample is mounted, and
A first optical element that irradiates the sample placed on the above-mentioned mounting surface with the irradiation light and incidents detection light from the sample irradiated with the irradiation light.
With a second optical element in which a recess covering the sample is formed, the irradiation light incident from the first optical element is emitted to the sample, and the detection light incident from the sample is emitted to the first optical element. ,
It has an imaging device that captures an image of the sample formed by the detection light emitted from the first optical element.
The second optical element is
A first input / output surface having a spherical shape, in which the irradiation light is incident from the first optical element and the detection light is emitted to the first optical element.
The irradiation light that includes at least a part of the wall portion of the recess and is incident on the first inlet / outlet surface is emitted to the sample, and the detection light incident from the sample is emitted to the first inlet / outlet surface. Has a second entrance / exit surface,
An optical microscope characterized in that the space formed by the recess and the above-mentioned mounting surface can be filled with a liquid.
The second optical element further has a bottom surface in which an outer edge is connected to the first entrance / exit surface and an inner edge is connected to the second entrance / exit surface so that the second optical element can be arranged in close contact with the above-mentioned mounting surface. Item 1. The optical microscope according to item 1.
The optical microscope according to claim 1 or 2, wherein the second optical element is arranged so that the center of a spherical surface forming the first entrance / exit surface coincides with the focal point of the first optical element.
The optical microscope according to any one of claims 1 to 3, wherein the second entrance / exit surface has a spherical shape in which the center of the spherical surface forming the first entrance / exit surface coincides with the center.
The optical microscope according to any one of claims 1 to 4, wherein the first optical element, the second optical element, and the sample substrate can be moved independently.
The optical microscope according to any one of claims 1 to 4, further comprising a holding member that integrally holds the second optical element and the sample substrate.
The optical microscope according to any one of claims 1 to 6, wherein the first optical element is composed of one or a plurality of optical lenses.
The first optical element is
A first transmission surface having a planar shape, in which the irradiation light is incident from the light source, and the detection light is emitted to the image pickup apparatus.
A first that has a spherical shape and is arranged so as to face the first transmission surface, reflects the irradiation light incident from the first transmission surface, and reflects the detection light to the first transmission surface. Reflective surface and
It has a spherical shape and is arranged so that the inner edge is in contact with the outer edge of the first transmitting surface, reflects the irradiation light incident from the first reflecting surface, and transmits the detected light to the first reflecting surface. The second reflecting surface that reflects,
It has a spherical shape, its inner edge is in contact with the outer edge of the first reflecting surface and is arranged so as to face the second reflecting surface, and the irradiation light incident from the second reflecting surface is emitted from the first input / exit surface. A second transmission surface that emits the detection light emitted from the first entrance / exit surface to the second reflection surface, and a second transmission surface that emits the detection light to the second reflection surface.
The optical microscope according to any one of claims 1 to 6.
The optical microscope according to claim 8, wherein the first optical element is arranged so that the center of the second transmission surface coincides with the focal point of the first optical element.
A sample substrate having a mounting surface on which the sample is mounted, and
A second optical element, which is formed with a recess covering the sample, emits irradiation light to the sample, and emits detection light incident from the sample.
It has a holding member for holding the sample substrate, and has
The second optical element is
A first inlet / output surface having a spherical shape, in which the irradiation light is incident, and the detection light is emitted.
A second portion that includes at least a part of the wall portion of the recess, emits the irradiation light incident on the first inlet / outlet surface to the sample, and emits the detected light incident from the sample to the first inlet / outlet surface. Has an entrance / exit surface,
A sample substrate holder characterized in that the space formed by the recess and the above-mentioned mounting surface can be filled with a liquid.