Prism, and device and method for observing living body using the same

Abstract

To provide a prism that can obtain information on a deep part of a living body with high accuracy.SOLUTION: A prism 100 of the present invention is a prism used for observation of a specific portion 20 of a living body, is a solid body having a first surface 101 and a second surface 102 causing light to enter and exit, and a third surface 103 reflecting the light entering from one of the first surface 101 and the second surface 102 toward the other, and is formed of material having a low refractive index of 90% or more and 110% or less of the refractive index of the specific portion 20.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a prism, and an observation device and an observation method for a living body using the prism. [Background technology]

[0002] In recent years, two-photon microscopes that use infrared lasers as the light source have become widespread, making it possible to observe the inside of living organisms with high spatial resolution. Infrared lasers have excellent penetration into living tissue, and when used in two-photon microscopes, they emit excitation light only at the focal plane, making it possible to obtain high spatial resolution microscopic images of areas up to about 1 mm deep from the tissue surface with minimal invasiveness.

[0003] To observe brain activity, synapses, and other microstructures, microscope images of deeper areas are required, and a method for observing deeper areas using a prism has been developed (Non-Patent Document 1). By implanting a prism into the deeper area, irradiating laser light through the prism and detecting the reflected light, it is possible to obtain information at a depth of more than 1 mm. This method has been used to obtain information on the deep areas of the prefrontal cortex, which is an important area involved in, for example, fearful experiences and PTSD. However, the images obtained as information on the deep areas do not have sufficient brightness and spatial resolution, and it is difficult to grasp information on the deep areas with high accuracy based on these images. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Cellular resolution optical access to brain regions in fissures: Imaging medial prefrontal cortex and grid cells in entorhinal cortex, PNAS, December 30, 2014, vol. 111, no. 52, 18739-18744 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made in consideration of the above circumstances, and has an object to provide a prism that makes it possible to obtain information about deep inside a living body with high accuracy, and a living body observation device and observation method that use the same. [Means for solving the problem]

[0006] The inventor focused on the refractive index difference between the observed part of the living body and the prism, and thought that by reducing the refractive index difference, an image with high brightness and spatial resolution could be obtained. Based on this, the inventor succeeded in fabricating a prism made of a material with a refractive index close to that of the living body as a result of repeated ingenuity, and completed the present invention. In order to solve the above problems, the present invention employs the following means.

[0007] (1) A prism according to one embodiment of the present invention is a prism used for observing a specific part of a living body, which is a solid body having a first surface and a second surface through which light enters and leaves, and a third surface that reflects the light that enters from one of the first and second surfaces toward the other surface, and is made of a material having a low refractive index that is 95% or more and 105% or less of the refractive index of the specific part.

[0008] (2) In the prism described in (1) above, the low refractive index is preferably 1.4 or less.

[0009] (3) In the prism described in either (1) or (2) above, it is preferable that the material contains a fluorine compound as a main component.

[0010] (4) In the prism described in any one of (1) to (3) above, a metal film is preferably formed on the third surface.

[0011] (5) In the prism described in any one of (1) to (4) above, it is preferable that the three-dimensional object is a triangular prism having the first surface, the second surface, and the third surface as side surfaces.

[0012] (6) In the prism described in (5) above, it is preferable that the base of the triangular prism is a right-angled isosceles triangle.

[0013] (7) In the prism described in (6) above, it is preferable that the lengths of the two sides forming the right angle of the right-angled isosceles triangle are 0.5 mm or more and 8.0 mm or less.

[0014] (8) A living body observation device according to one aspect of the present invention includes the prism according to any one of (1) to (7) above, and a multiphoton microscope.

[0015] (9) A method for observing a living organism according to one embodiment of the present invention is a method for observing a living organism using a prism described in any one of (1) to (7) above, comprising a first step of placing the prism at a specific portion of the living organism, a second step of using a multiphoton microscope to irradiate the observation portion within the living organism with light via the prism, and a third step of detecting light emitted from the excited observation portion via the prism.

[0016] (10) In the first step of the method for observing a living organism described in (9) above, it is preferable to install the prism so that the first surface faces the objective lens of the multiphoton microscope and the second surface faces the observation portion. Effect of the Invention

[0017] According to the present invention, it is possible to provide a prism that makes it possible to obtain information about a deep part of a living body with high accuracy, and a living body observation device and observation method that use the prism. [Brief description of the drawings]

[0018] [Figure 1]1A is a perspective view of a prism according to an embodiment of the present invention, and FIGS. 1B and 1C are plan views of the prism of the same embodiment, illustrating the incidence and emission of light. [Diagram 2] 5A and 5B are diagrams illustrating a method for manufacturing a prism according to the embodiment. [Diagram 3] 10 is a diagram showing a schematic diagram of a state in which a living body is observed using the living body observation device according to the embodiment. FIG. [Figure 4] 13(a) and 13(b) are plan views of a prism according to a first modified example of the same embodiment. [Diagram 5] FIG. 11 is a perspective view of a prism according to a second modified example of the embodiment. [Figure 6] FIG. 11 is a diagram showing a schematic view of a living body being observed using a living body observation device according to Modification 2. [Figure 7] 1A and 1B are diagrams illustrating an implementation procedure in the first embodiment of the present invention. [Figure 8] 1 is a photograph of a prism according to Example 1. [Figure 9] This is a photograph of the prism immersed in gel. [Figure 10] 13 is an image of the inside of a gel obtained by observation in a comparative example. [Figure 11] 1 shows an image of the inside of a gel obtained by observation in Example 1. [Figure 12] 4(a) to 4(c) are enlarged images of the inside of the gel obtained by observation in the comparative example, at positions at depths of 0 μm, 300 μm, and 600 μm from the surface of the gel. [Figure 13] 1(a) to 1(c) are enlarged images of the inside of the gel obtained by the observation in Example 1, at positions at depths of 0 μm, 300 μm, and 600 μm from the surface of the gel. [Figure 14] 13(a) and 13(b) are graphs comparing the full width at half maximum (FWHM) obtained from the distribution of the spread of fluorescence obtained at depths of 0 μm, 300 μm, and 600 μm in Example 1 and the comparative example. [Figure 15]13(a) and 13(b) are graphs comparing the average intensity and maximum intensity of fluorescence obtained at depths of 0 μm, 300 μm, and 600 μm in Example 1 and the comparative example. [Figure 16] 13 is an in vivo image obtained in Example 2 of the present invention. [Figure 17] 17 is a high-magnification image of a portion of FIG. 16. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Hereinafter, a prism according to an embodiment of the present invention and a living body observation device and observation method using the prism will be described in detail with reference to the drawings. Note that the drawings used in the following description may show characteristic parts in an enlarged scale for the sake of convenience in order to make the characteristics easier to understand, and the dimensional ratios of each component may not necessarily be the same as in reality. In addition, the materials, dimensions, etc. exemplified in the following description are merely examples, and the present invention is not limited to them, and can be appropriately modified and implemented within the scope of the present invention.

[0020] (prism) Fig. 1(a) is a perspective view of a prism 100 according to one embodiment of the present invention. Fig. 1(b) and (c) are plan views of the prism 100 as viewed from a direction (here, the Y direction) perpendicular to the direction in which light is incident and the direction in which light is emitted, and are diagrams for explaining the incidence and emission of light into and from the prism 100.

[0021] The prism 100 is an optical element used for observing the inside of a living body, etc., and is mainly made of a material that transmits light, and has the function of changing the path of light.

[0022] Prism 100 is a solid having first surface 101 and second surface 102 through which light enters and leaves, and third surface 103 that reflects light entering from one of first surface 101 and second surface 102 toward the other surface. First surface 101, second surface 102, and third surface 103 are all assumed to be flat within the range of manufacturing precision. The shape of prism 100 is not particularly limited, but here, a case where it is a triangular prism is shown as an example.

[0023] As shown in Fig. 1(b), light incident from the first surface 101 is reflected by the third surface 103 and emitted from the second surface 102. Conversely, light incident from the second surface 102 is reflected by the third surface 103 and emitted from the first surface 101, as shown in Fig. 1(c).

[0024] Prism 100 is mainly composed of a material (approximately 90% or more) having a refractive index similar to that of the specific portion to be observed, specifically, 95% or more and 105% or less, preferably 98% or more and 102% or less, and most preferably 100% of the refractive index of the specific portion. The material constituting prism 100 is preferably a material that is easy to mold into a predetermined shape such as a triangular prism.

[0025] For example, when observing the brain of a living organism, the refractive index of the brain is about 1.33, so the refractive index of the prism 100 is preferably 1.3 or more and 1.4 or less, and preferably about the same as 1.33. Examples of such low refractive index materials include magnesium fluoride, fluorine-based resins, photocrosslinkable resins such as BIO-133 (manufactured by MY POLYMER), and fluorine compounds containing perfluorobutenyl vinyl ether, 2,2-bis(trifluoromethyl)-1,3-dioxole, tetrafluoroethylene, Cytop (registered trademark) (manufactured by AGC Inc.), Teflon (registered trademark) AF resin (manufactured by DuPont) as the main component (about 90% or more). In particular, BIO-133 is preferred because it is an easy-to-form material.

[0026] In this embodiment, the term "living body" refers to a part or the whole body of an organism, and is preferably in a living state or a state close to that. The type of organism is not particularly limited, and examples thereof include vertebrates such as mammals including humans, invertebrates, and plants.

[0027] As shown in FIG. 1(a), a metal film (mirror, reflective film) 100B for increasing the reflectance of incident light may be formed on the third surface of the prism 100. That is, the prism 100 may be composed of a base material 100A containing the above-mentioned low refractive index material as a main component and having the above-mentioned three-dimensional structure, and a metal film 100B formed on the third surface 103. Examples of the metal film 100B include films made of aluminum, silver, platinum, etc. The thinner the metal film, the more preferable it is. The thickness of the metal film may be, for example, 100 nm or more and 10 μm or less from the viewpoint of practical use or actual manufacturing.

[0028] (How to make a prism) The method for producing the above-mentioned prism 100 is not particularly limited, but may be, for example, the following method.

[0029] A metal such as aluminum is vacuum-deposited on a glass substrate. The metal film after deposition is irradiated with a UV laser from the side opposite to the deposition surface, and the metal film is cut out to a desired size. It is preferable to subject the deposition surface of the glass substrate to oxygen plasma treatment before deposition.

[0030] A mold for molding the raw material of a prism is prepared or fabricated, and the raw material is filled (stored) in the recess of the mold. Fig. 2(a) is a diagram showing a state in which a metal film (not shown) and raw material 100C of a substrate are stored in recess 40A of mold 40 placed in pressure device 46. Pressure device 46 is equipped with a pressure means such as screw 45, and is configured to apply pressure to the surface of mold 40. Here, a case is illustrated in which the internal space of recess 40A is a triangular prism.

[0031] 2(b) is a perspective view of the mold 40 in the middle of assembly. Here, as an example, the mold 40 is used in which the recess 40A is formed from four members (metal plates or the like) 41-44, and the members are pressed and fixed with screws 45 so that there are no gaps between them except in the recess 40A, but other molds may be used as long as the desired recess 40A is formed.

[0032] Here, the inner wall surfaces (side wall surfaces and bottom wall surface) of the recess 40A are configured so as to obtain a triangular prism. First, a prepared metal film (not shown) is placed on the portion (inclined surface) of the inner wall surface of the recess 40A where the third surface (reflective surface) of the prism is to be formed. In order to prevent the metal film from sticking to the inner wall surface of the recess 40A, it is preferable to spray-coat it with Teflon (registered trademark). Next, a low refractive index material that will be the raw material for the base material of the prism is filled into the recess 40A.

[0033] Check that there are no bubbles in the filled raw material, and remove any bubbles that are present. If necessary, cover the recess 40A with a cover glass (glass plate) to seal the raw material in the recess 40A. In this state, apply 0.05 to 1 W / cm to the raw material in the recess 40A. 2 The cover glass is irradiated with UV light at an intensity of about 3 to 20 minutes. It is preferable to perform oxygen plasma treatment on the portion of the cover glass facing the raw material in the recess 40A before irradiating it with UV light. The raw material is hardened in the shape of the recess 40A by irradiation with UV light, and the base material of the prism is formed, and the third surface is bonded to the metal film. Finally, the prism composed of the base material and the metal film is taken out from inside the recess 40A.

[0034] (Living Body Observation Apparatus and Observation Method) 3 is a diagram showing a schematic diagram of a state in which the inside of a specific part 20 of a living organism is observed using a living organism observation device 10. The living organism observation device 10 mainly includes a prism 100 and a multiphoton microscope 200. A known multiphoton microscope can be used as the multiphoton microscope 200. Here, only an objective lens 201 of the multiphoton microscope 200 is specifically illustrated, and other parts are not illustrated.

[0035] The method for observing a living body using the prism 100 mainly includes the following first, second and third steps.

[0036] (first step) A prism 100 is inserted (implanted) into a specific portion (hereinafter, sometimes simply referred to as a living body) 20 including an observation portion (portion to be observed) 20a of a living body. The prism 100 is installed near the observation portion 20a so that the first surface 101 faces the objective lens 201 and the second surface 102 faces the observation portion 20a. The prism 100 is preferably hard enough to maintain its original shape (here, a triangular prism shape) even when inserted into the living body. Next, the multiphoton microscope is installed so that the objective lens 201 faces the first surface 101. As a result, a light propagation path is formed between the objective lens 201 and the observation portion 20a via the prism 100, and an image of the observation portion can be detected. The objective lens 201 may be directly in contact with the first surface 101 of the prism, or may be indirectly in contact with the first surface 101 of the prism via air or liquid such as water.

[0037] It is preferable that the first surface 101 is perpendicular to the optical axis 201a of the objective lens 201. When the prism 100 is installed, a portion on the first surface 101 side is exposed to the outside of the living body 20, and a push-in portion (tip portion) 104 on the opposite side to the first surface 101 is pushed in the vicinity of the observation portion 20a inside the living body 20. It is preferable that the push-in portion 104 has a pointed (tapered) shape as shown in FIG. 3 so that it can be pushed in the vicinity of the observation portion 20a. The position where the push-in portion 104 is pushed in may be a recessed portion that is originally present in the vicinity of the observation portion 20a, a flat portion, or a portion where a new cut is made.

[0038] (Second process) Using a multiphoton microscope 200, light S1 is irradiated onto an observation portion 20a of a living body via a prism 100. More specifically, light S1 irradiated from an objective lens 201 is made incident on a first surface 101 of the prism. The incident light S1 is reflected inwardly by a third surface 103 of the prism, and the reflected light S2 is emitted from a second surface 102 toward the observation portion 20a. The light S1 irradiated onto the prism 100 is not particularly limited, but may be any light that transmits through a living body, and is preferably a laser in the infrared range (for example, with a wavelength of 0.78 μm or more and 2 μm or less, preferably 0.78 μm or more and 1 μm or less) with excellent transmittance.

[0039] (Third step) The light S3 emitted from the excited observation portion 20a by the light S2 emitted from the second surface 102 is detected via the prism 100. More specifically, when the light S2 emitted from the second surface 102 of the prism is irradiated onto the observation portion 20a, the fluorescent dye molecules in the ground state contained in the observation portion 20a absorb the irradiated light S2, and emit light (fluorescence) S3 when returning to the ground state after being excited once. Of the emitted light S3, the light S3 incident from the second surface 102 of the prism is reflected inward by the third surface 103, and the reflected light S4 reaches the objective lens 201. By detecting the light S4 that reaches the objective lens 201, it is possible to observe inside the living body and obtain various information inside the living body.

[0040] As shown in FIG. 1(a), the prism 100 is preferably a triangular prism having a first surface 101, a second surface 102, and a third surface 103 as side surfaces, that is, a triangular prism composed of rectangular side surfaces (the first surface 101, the second surface 102, and the third surface 103) and two triangular base surfaces 105 and 106. In this case, the direction of light incident on the prism 100 can be bent according to the angle of incidence. The shape of the base surfaces 105 and 106 of the triangular prism is not particularly limited, but a right-angled isosceles triangle is more preferable because the direction of light perpendicularly incident on the first surface 101 can be bent accurately to a direction perpendicular to the first surface 101. In this case, in the right-angled isosceles triangle, the lengths L1 and L2 of the two sides (sides other than the hypotenuse) forming a right angle are preferably 0.5 mm or more and 8.0 mm or less, respectively, and more preferably 1.0 mm or more and 7.5 mm or less. The length L3 of the side connecting the two bottom surfaces 105, 106 is not particularly limited, but is preferably, for example, 0.5 mm or more and 8.0 mm or less, and more preferably 1.0 mm or more and 7.5 mm or less.

[0041] It should be noted that the lengths L2 and L3 are preferably larger since they can capture more light, but it is preferable to determine the lengths L2 and L3 while taking into consideration the extent to which the installation of the prism 100 does not cause too much damage to the specific part 20 of the living body.

[0042] The size of the prism 100 is not particularly limited, and can be determined taking into consideration the position (distance from the surface) and size of the observation portion 20a, the allowable size of the press, the lens diameter and lens area of ​​the objective lens 201, etc.

[0043] From the viewpoint of increasing the accuracy of the reflection direction of the incident light, it is preferable to reflect the light in the vicinity of a central portion 103c, which is less affected by the edge of the third surface 103. Therefore, it is preferable that, when the prism 100 is installed, this central portion 103c is located at a position at approximately the same depth as (the center of) the observation portion 20a.

[0044] When the prism 100 is pressed into the inside of the living body 20 so that the first surface 101 is approximately aligned with the surface 20a of the living body, it is preferable that the length L2 of the side of the second surface facing the observation portion 20a be approximately twice the depth D of the observation portion 20a. For example, when a portion about 1 mm deep from the surface 20a of the living body is the observation portion 20a (its center), the length L2 may be set to approximately 2 mm.

[0045] It is also possible that when the prism 100 is pressed in, the first surface 101 may be located outside the surface 20a of the living body, that is, a part of the first surface 101 may protrude outside the surface 20a of the living body. In such a case, it is preferable to set the length of the pressed-in portion to be at least twice the depth of the observation portion 20a.

[0046] From the viewpoint of obtaining information (image) of the observation portion 20a with high accuracy, it is preferable that as much light irradiated from the objective lens 201 is made incident on the first surface 101 and is made to exit toward the observation portion 20a via the prism 100. From the same viewpoint, it is preferable that as much light generated in the observation portion 20a and made to exit from the first surface 101 via the prism 100 is made incident on the objective lens 201. Therefore, it is preferable that the objective lens 201 is included in the first surface 101 when the objective lens 201 and the first surface 101 are overlapped in a plan view from the direction of the optical axis 201a of the objective lens (Z direction).

[0047] The prism 100 is made of a material having a refractive index similar to that of at least the portion through which the light S2 and S3 pass, that is, the observation portion 20a in contact with the second surface 102. The refractive index similar to that of the observation portion 20a means a low refractive index of 90% or more and 110% or less, preferably 95% or more and 105% or less, and most preferably 100% of the refractive index of the observation portion 20a. Since the difference in refractive index between the prism 100 and the observation portion 20a is small, it is possible to reduce the amount of light emitted from the observation portion 20a that is reflected in a direction deviating from the path toward the objective lens 201 at the interface with the second surface 102. Therefore, the image detected by the objective lens 201 is clearer with significantly improved spatial resolution and brightness than when a prism such as glass having a refractive index higher than that of a living body is used.

[0048] As described above, the prism 100 of this embodiment has the first surface 101 and the second surface 102 through which light is incident and exits, and the third surface that reflects light incident from one of the first surface 101 and the second surface 102 toward the other. By installing this prism 100 in the vicinity of the observation portion 20a so that the first surface 101 faces the objective lens 201 and the second surface 102 faces the observation portion 20a, a light propagation path is formed connecting the objective lens 201 and the observation portion 20a via the prism 100, and an image of the observation portion 20a can be detected.

[0049] A prism used for general in vivo observation is made of the same material as the lens in the optical path of the microscope, such as borosilicate crown glass, and has a high refractive index. In contrast, the refractive index of the prism 100 of this embodiment is approximately the same as that of the observation portion 20a, and the refractive index difference between the prism 100 and the observation portion 20a is small, so that the occurrence of spherical aberration and unnecessary reflection due to the refractive index difference can be reduced. As a result, the light that maintains the state when emitted from the observation portion 20a can reach the objective lens 201, and the image of the observation portion 20a to be detected can be made clear, making it possible to obtain information on the deep part of the living body with high accuracy.

[0050] The prism 100 of this embodiment can be used for various purposes by combining it with other components. For example, a gradient index type (GRIN lens) of a predetermined length can be bonded to the first surface 101 of the prism 100 to adjust the focal length. In addition, the prism 100 having a predetermined electrode on its surface can be pushed into a specific part of a living body to observe the electric potential of the specific part. In addition, the prism 100 having a microdialysis membrane on its surface can be pushed into a specific part of a living body to collect molecules present in the specific part.

[0051] (Variation 1) 4(a) and (b) are plan views of a prism 110 according to a first modified example of the above embodiment. The same reference numerals are used to designate parts corresponding to the prism 100 of the above embodiment. Fig. 4(a) is a plan view of the prism 110 as viewed from the second surface 102 side facing the observation portion. Fig. 4(b) is a plan view of the prism 110 as viewed from the bottom surface 105 side not involved in the incidence and emission of light.

[0052] The prism 110 is a part located deepest from the surface of the living body when the prism 110 is inserted into a specific part of the living body, and the end 104 that is not in contact with the first surface 101 is rounded. The degree of roundness is not particularly limited, but for example, -4 That's it, 10 -3 The rounding may be within the following range. The rest of the configuration is the same as that of prism 100, and at least the same effect as prism 100 of the same embodiment is achieved. Prism 110 has rounded end 104, which reduces damage to a living body caused by pushing prism 110 in, compared to when end 104 is sharp.

[0053] (Variation 2) FIG. 5 is a perspective view of a prism 120 according to the second modification of the embodiment. The same reference numerals are used to indicate parts corresponding to the prism 100 of the embodiment. The prism 120 has a trapezoidal column with the first surface 101, the second surface 102, the third surface 103, and the newly added fourth surface 107 as side surfaces, and the bottom surfaces 105 and 106 as trapezoidal columns. Compared to the prism 100, the prism 120 has a shape in which the second surface 102 extends to the side opposite the pressed-in portion 104, and the first surface 101 is away from the third surface 103, in other words, a shape in which a square column made of the same material as the prism 100 is further joined to the first surface of the prism 100. The angle between the second surface 102 and the third surface 103 is preferably 45 degrees, but may be less than 45 degrees. The other configurations are the same as those of the prism 100, and at least the same effects as the prism 100 of the embodiment are achieved.

[0054] 6 is a schematic diagram showing a state in which the inside of a specific part 20a of a living body is observed using a living body observation device 10 in which the prism 100 is replaced with a prism 120. The prism 120 has a second surface 102 that extends further than the prism 100, and is longer in the depth direction (here, the Z direction), so that the prism 120 can be pushed to a deeper position from the surface 20b. This makes it possible to observe a part located deeper.

[0055] In this case, the distance from the first surface 101 facing the objective lens 201 to the third surface 103 facing the observation portion 20a increases, but the angle of incidence of light incident from the first surface 101 and the second surface 102 to the third surface 103 is the same as in the above embodiment. In addition, since the entire prism 120 except for the third surface 103 has a refractive index similar to that of the observation portion 20a, unnecessary reflection due to the refractive index difference between the prism 120 and the observation portion 20a and within the prism 120 can be reduced.

[0056] Therefore, even when observing a deep position that cannot be reached by the triangular prism 100, the prism 120 can detect a clear image with significantly improved spatial resolution and brightness, similar to the case of observing a shallow position using the prism 100. Furthermore, even when the prism 120 is pressed into a position deeper than the surface 20b of the living body, the area of ​​the first surface 101 is constant and is not enlarged in a direction parallel to the surface 20b of the living body. Therefore, the groove or wound formed by pressing the prism 120 does not expand beyond the area of ​​the third surface 103, and damage to the living body can be minimized. EXAMPLES

[0057] The effects of the present invention will be more clearly understood by the following examples. Note that the present invention is not limited to the following examples, and can be modified as appropriate without departing from the spirit and scope of the present invention.

[0058] Example 1 The living body observation method of the above embodiment was carried out using a structure produced by imitating an observation part of a living body. Figures 7(a) and (b) are explanatory diagrams thereof. Figure 7(a) is a cross-sectional view of the structure 31 cut along a plane (here, XZ plane) including the direction in which light is emitted and received when the prism 34 is pressed into the structure 31. Figure 7(b) is a plan view of the prism 34 and the structure 31 in the same state, viewed from the direction in which light is emitted and received (here, Z direction). A living body observation device was prepared according to the following procedure, and the inside of the structure 31 was observed.

[0059] First, a structure 31 was placed in a container 30. The structure 31 used was composed of an agarose gel 32 with a refractive index of 1.33 and YG beads 33 dispersed in the gel 32. The YG beads 33 correspond to the fluorescent dye molecules dispersed in the observation area of ​​the living body.

[0060] Next, a triangular prism 34 was pressed into the surface 31a of the structure 31 in the same manner as in the above embodiment, and its orientation was adjusted. The prism 34 was made of BIO-133 with a refractive index of 1.33, and was composed of a triangular prism base material 34A with a first surface, a second surface, and a third surface as side surfaces, and an aluminum film 34B formed on the third surface. The shape of the bottom surface of the prism 34 was a right-angled isosceles triangle, and the lengths of the two sides forming the right angle were each 2 mm. The thickness of the aluminum film 34B was 300 nm.

[0061] Fig. 8 is a photograph of the prism 34 used here in the atmosphere. Fig. 9 is a photograph of the prism 34 immersed in the gel 32. In the photograph of Fig. 9, only the metal film 34B of the prism 34 is visible, and the portion of the base material 34A that was visible in the photograph of Fig. 8 has become transparent and is no longer visible. This is because the refractive index of the base material 34A of the prism is the same as that of the gel 32, and reflection due to the difference in refractive index at the interface between the base material 34A and the gel 32 is reduced, allowing the back side of the prism 34 to be seen almost as it is.

[0062] Next, water 35 was placed in the container 30 so as to cover the surface 31a of the structure 31 and the first face 34a of the prism.

[0063] Next, the objective lens 36 of the two-photon microscope was brought into contact with the water 32 at a position facing the first surface 34a of the prism, and light was irradiated into the gel 33 via the prism 34, as in the above-described embodiment. Here, configurations other than the objective lens 36 of the two-photon microscope are not shown in the figure.

[0064] (Comparative Example) An observation device was prepared in the same manner as in Example 1, except that a base material 34A of the prism was made of glass having a refractive index of 1.55, and the inside of the structure was observed.

[0065] 10 and 11 are images of the inside of the gel obtained by observation in the comparative example and in Example 1, respectively. In the image of the comparative example using a glass prism, the distribution of the YG beads can be barely confirmed, but the fluorescence from the YG beads is hardly confirmed. In the comparative example, it is considered that a part of the fluorescence emitted by the YG beads is lost due to reflection due to the refractive index difference at the interface between the gel and the prism, and only the remaining small amount of fluorescence can be detected. On the other hand, in the image of Example 1 using a prism made of BIO-133 with a low refractive index, the brightness and spatial resolution are high, and the fluorescence from the distributed YG beads can be clearly confirmed. In Example 1, it is considered that all of the fluorescence emitted by the YG beads can be detected because there is no reflection due to the refractive index difference at the interface between the gel and the prism.

[0066] Figures 12(a)-(c) are enlarged images of the focal plane (xy) and the optical axis plane (xz) perpendicular to the focal plane at depths of 0 μm, 300 μm, and 600 μm from the gel surface, respectively, among images obtained in the observation of the comparative example. Comparing the three images, it can be seen that the deeper the image, the darker it becomes, and the more difficult it is to see the fluorescence from the YG beads. Furthermore, in the image on the optical axis plane, the fluorescent image from the YG beads appears to be spread in the direction of the optical axis.

[0067] 13(a)-(c) are magnified images of the focal plane (xy) and the optical axis plane (xz) perpendicular thereto at depths of 0 μm, 300 μm, and 600 μm from the gel surface, respectively, among the images obtained by the observation in Example 1. Comparing the three images, it can be seen that the images tend to become slightly darker as they get deeper, but are not as dark as in the comparative example. It can also be seen that the spread of the fluorescent image of the YG beads in the optical axis direction is smaller than in the comparative example. From these results, it can be seen that the observation method of the present invention using a low refractive index prism greatly improves the brightness and spatial resolution compared to observation using a glass prism, regardless of the depth from the surface, and a clearer image of the inside of the body can be obtained.

[0068] 14(a) and (b) are graphs comparing the full width at half maximum (FWHM) obtained from the spatial distribution of the fluorescent image obtained at depths of 0 μm, 300 μm, and 600 μm in Example 1 and Comparative Example. The full width at half maximum in FIG. 14(a) is the full width at half maximum (axial FWHM) obtained from the distribution of the spread in the depth direction (Z direction in FIG. 7). The full width at half maximum in FIG. 14(b) is the full width at half maximum (lateral FWHM) obtained from the distribution of the spread in the direction perpendicular to the light propagation path (Y direction in FIG. 7). In any direction and depth, the full width at half maximum in Example 1 is smaller than the full width at half maximum in Comparative Example, so that it can be seen that the low refractive index prism used in Example 1 has a superior light collecting ability compared to the prism in Comparative Example.

[0069] 15(a) and (b) are graphs comparing the intensity (brightness) of the fluorescence obtained at depths of 0 μm, 300 μm, and 600 μm in Example 1 and Comparative Example. The intensities in FIG. 15(a) and (b) are the average intensity and maximum intensity of the fluorescence obtained at each depth, respectively. Since the intensity in Example 1 exceeds the intensity in the Comparative Example at each depth, it is understood that the low refractive index prism used in Example 1 has superior light-collecting properties compared to the prism in the Comparative Example, realizing high spatial resolution observation and enabling detection of images with high brightness.

[0070] Example 2 The inside of a mouse brain, which is a living organism, was observed using the living organism observation method of the above embodiment. As shown in Fig. 3, the pushing-in part (tip part) of the pointed prism was pushed in so that the intersection line between the second surface and the third surface of the stage was approximately parallel to the midline of the brain. In this state, a two-photon microscope was used to obtain images of multiple cross sections on the side opposite the second surface of the prism.

[0071] FIG. 16 is an image of one cross section (2.0 mm×2.0 mm) obtained. A clear image was obtained in a depth range of 2.0 mm from the brain surface (deep brain). In particular, a brighter image was obtained in a depth range of about 1.4 mm from the brain surface. FIG. 17 is an image of a part of the image of FIG. 16 taken at a high magnification near a depth of 1.4 mm. The dendrites branching out in a complex manner from the nerve cells can be confirmed. The contact structure (synapse) between the dendrites can also be confirmed. The limit of the depth that can be observed with conventional technology is about 1.0 mm from the brain surface, but it can be seen that by applying the present invention, it is possible to observe deeper regions while maintaining high spatial resolution. The resolution of the image obtained by the present invention can be further improved by further adding super-resolution imaging technology. [Explanation of symbols]

[0072] 100, 110, 120... Prism 100A...Base material 100B...Metal film 100C...Raw material 101...Front page 102...Second side 103...Third side 104... Push-in part 105, 106... Bottom 107...Fourth side 200...Multiphoton microscope 201 Objective lens 201a...Optical axis 10. Biological observation device 20. Living body 20a Observation part 20b: Biological surface D Depth L1, L2, L3...length S1, S2, S3, S4...Light 30...container 31...Structure 32. Gel 33...YG beads 34 Prism 34A...Base material 34B Aluminum film 34a...Front page 34b...Second side 35...water 36 Objective lens 40, 41, 42, 43, 44: Mold components 45···Screw 46 Pressurizing device

Claims

1. A prism for use in observing a specific part of a living body, comprising: a first surface and a second surface through which light is incident and exits; a third surface that reflects the light incident from one of the first surface and the second surface toward the other surface, A prism comprising a material having a low refractive index of 95% or more and 105% or less of the refractive index of the specific portion.

2. 2. The prism according to claim 1, wherein the low refractive index is 1.4 or less.

3. 3. The prism according to claim 1, wherein the material contains a fluorine compound as a main component.

4. 3. The prism according to claim 1, wherein a metal film is formed on the third surface.

5. 3. The prism according to claim 1, wherein the solid body is a triangular prism having the first surface, the second surface, and the third surface as side surfaces.

6. 6. The prism according to claim 5, wherein the base of the triangular prism is a right-angled isosceles triangle.

7. 7. The prism according to claim 6, wherein the lengths of the two sides forming the right angle of the right-angled isosceles triangle are 0.5 mm or more and 8.0 mm or less.

8. A prism according to claim 1 or 2; A living body observation device comprising: a multiphoton microscope.

9. A method for observing a living body using the prism according to claim 1 or 2, comprising: a first step of placing the prism on a specific part of the living body; a second step of irradiating the observation area in the living body with light via the prism using a multiphoton microscope; and a third step of detecting light emitted from the excited observation portion via the prism.

10. In the first step, 10. The method for observing a living organism according to claim 9, wherein the prism is disposed so that the first surface faces an objective lens of the multiphoton microscope and the second surface faces the observation portion.

Images