Structure for optical observation, method for forming same, method for observing sample, and mounting liquid for microscopic observation

Abstract

A structure for optical observation according to the present disclosure is obtained by sequentially arranging a liquid immersion objective lens, an immersion liquid, a light-transmitting sample placement member, and a sample that has been processed with a mounting liquid in the stated order on the optical axis of the liquid immersion objective lens. In the visible light region, the refractive index difference and the Abbe number difference between the immersion liquid and the mounting liquid are both 5% or less.

Description

Structures for optical observation and their manufacturing methods, sample observation methods, and mounting solutions for microscope observation

[0001] The present invention relates to a structure for optical observation and a method for producing the same, a method for observing a sample using the structure for optical observation, and a mounting liquid for microscopic observation using an immersion objective lens.

[0002] When observing biological tissues for pathological analysis, it is common to prepare stained tissue sections and mount them on glass slides for observation. Most biological tissue observations are performed macroscopically using air-immersion objectives with low magnification and low NA (numerical aperture). However, in some cases, it is necessary to observe small structures within biological tissues with higher resolution. In such cases, oil-immersion objectives are often used. Oil-immersion objectives have a high numerical aperture (NA), making them attractive lenses that allow even very small objects or factors to be observed with high resolution.

[0003] Typically, biological tissue sections are immersed in PBS(-) or 50% glycerin-containing PBS(-) (hereafter referred to as "Gly50-PBS") and then mounted for observation. While various mounting media are commercially available, most of them have low refractive indices. When using an immersion objective lens, such as an oil immersion objective, to observe tissue sections in such conditions, the refractive index of the sample deviates from that of the optical system, resulting in noticeable spherical aberrations, especially at the depths of the specimen, which adversely affect the image quality. Therefore, research and development of mounting media for use with immersion objective lenses, such as oil immersion objective lenses, is ongoing.

[0004] For example, Non-Patent Document 1 discloses that thiodiethanol diluted to 97 (w / v)% with water (hereinafter referred to as "TDE97") has a refractive index close to the refractive index of the immersion liquid, 1.518 (@546 nm, 23°C). When TDE97 is used in place of the medium solution for fixed cultured cells, spherical aberration is suppressed, allowing for the acquisition of very high-resolution images.

[0005] Furthermore, Non-Patent Document 2 discloses a high refractive index mounting medium (SeeDBS2). It is disclosed that super-resolution imaging at a depth of approximately 100 μm was performed using SeeDBS2 and an oil immersion objective. Furthermore, Non-Patent Documents 3 and 4 disclose CUBIC-R+ and PuClear, respectively, as high refractive index clearing reagents.

[0006] Staudt, T., et al. 2,2'-thiodiethanol: a new water soluble mounting medium for high resolution optical microscopy. Microsc Res Tech. 2007 Jan;70(1):1-9.Ke, MT, et al. Super-Resolution Mapping of Neuronal Circuitry With an Index-Optimized Clearing Agent. Cell Rep. 2016 Mar 22;14(11):2718-32.Tainaka, K. et al. Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents. Cell Rep. 2018 Aug 21;24(8):2196-2210.e9.Xu, F. et al. High-throughput mapping of a whole rhesus monkey brain at micrometer resolution. Nat Biotechnol. 2021 Dec;39(12) 1521-1528

[0007] Using the mounting media and clearing reagents disclosed in Non-Patent Documents 1-4, it is believed that it is practically possible to obtain high-resolution images without spherical aberration when observing with an immersion objective. However, when using these mounting media or clearing reagents, for example, when performing multicolor imaging with multi-wavelength excitation, there is a possibility that the images of each color may not show the correct relative localization (possibility of axial chromatic aberration), especially at deep locations. Because the color shift of such axial chromatic aberration is not constant with depth but increases proportionally, it cannot be addressed by simply shifting the image. More complex image analysis techniques are required, but this may result in artifacts.

[0008] As such, it is currently extremely difficult to analyze the colocalization of proteins deep within a sample from the overlap of multicolor signals, and further improvements are needed.

[0009] An object of one aspect of the present invention is to provide a structure for optical observation that can reduce spherical aberration and / or axial chromatic aberration during microscope observation using an immersion objective lens.

[0010] In order to solve the above problems, the present invention includes the following aspects: <1> A structure for optical observation, comprising an immersion objective lens, an immersion liquid, a translucent sample placement member, and a sample treated with a mounting liquid, arranged in this order on the optical axis of the immersion objective lens, wherein the difference in refractive index and Abbe number between the immersion liquid and the mounting liquid are all within a range of 5% or less in the visible light range. <2> A structure for optical observation, comprising an immersion objective lens, an immersion liquid, a translucent sample placement member, and a sample treated with a mounting liquid, arranged in this order on the optical axis of the immersion objective lens, wherein the Abbe number of the mounting liquid is within a range of 38 to 47, and the refractive index of the mounting liquid is within a range of 1.35 to 1.52 for light with a wavelength of 561 nm. <3> A mounting solution for microscopic observation using an immersion objective lens, the mounting solution having a refractive index of 1.35 or more and 1.52 or less for light with a wavelength of 561 nm, and containing water and at least two or more components other than water, one of which is selected from the group consisting of sugars, sugar alcohols, and glycol-based solvents.

[0011] According to one aspect of the present invention, it is possible to reduce spherical aberration or axial chromatic aberration in microscopic observation using an immersion objective lens.

[0012] 1 is an Abbe diagram in which each material is plotted.

[0033] FIG. 1 is a schematic diagram showing the relationship between the refractive index of an immersion liquid and a mounting liquid and axial chromatic aberration.

[0034] FIG. 2 is a diagram showing the measurement results of the refractive index of a mounting liquid at each wavelength.

[0035] FIG. 3 is a diagram showing the apparatus used to measure the refractive index.

[0036] FIG. 4 is a schematic diagram of the experimental protocol used to verify the amount of spherical aberration / axial chromatic aberration using beads.

[0037] FIG. 5 is a schematic diagram of the experimental protocol used to verify the amount of spherical aberration / axial chromatic aberration using beads.

[0038] FIG. 6 is a diagram showing the results of verification of the amount of axial chromatic aberration using beads.

[0039] FIG. 7 is a diagram showing the results of verification of the amount of axial chromatic aberration using beads.

[0039] FIG. 8 is a diagram showing the results of verification of the axial chromatic aberration of Chromatie.

[0039] FIG. 9 is a diagram showing the results of verification of the axial chromatic aberration of SeeDBS2.

[0039] FIG. 10 is a diagram showing the results of verification of the axial chromatic aberration of CUBIC-R+.

[0039] FIG. 11 is a diagram showing the results of verification of the axial chromatic aberration of TDE97.

[0039] FIG. 12 is a diagram showing the results of verification of the axial chromatic aberration of PuClear.

[0039] FIG. 13 is a diagram showing the results of observation of transparency and fluorescence bleaching by various mounting liquid treatments.

[0039] FIG. 14 is a diagram showing the results of observation of deep tissue using Gly50-PBS. FIG. 1 is a diagram showing the results of observation of deep tissue using Chromatie. FIG. 2 is a diagram showing the results of observation of deep tissue using various mounting solution treatments. FIG. 3 is a diagram showing the results of verification of ProLong (registered trademark) Glass. FIG. 4 is a diagram showing the results of verification of Chromatie. FIG. 5 is a schematic diagram of an experimental protocol used to verify the amount of axial chromatic aberration used in immunohistochemical staining. FIG. 6 is a schematic diagram of an experimental protocol used to verify the amount of axial chromatic aberration used in immunohistochemical staining. FIG. 7 is a diagram showing the results of verification regarding axial chromatic aberration of Chromatie. FIG. 8 is a diagram showing the results of verification regarding axial chromatic aberration of SeeDBS2. FIG. 9 is a diagram showing the results of verification regarding axial chromatic aberration of CUBIC-R+. FIG. 10 is a schematic diagram of a structure 10 for optical observation according to one embodiment of the present invention. FIG. 11 is a diagram showing the results of verification regarding axial chromatic aberration of Chromatie. FIG. 12 is a diagram showing the results of verification regarding axial chromatic aberration of Chromatie.

[0013] 1. Structure for Optical Observation A structure 10 for optical observation according to one embodiment of the present invention will be described with reference to Fig. 25. Fig. 25 shows a schematic diagram of the structure 10 for optical observation. The structure 10 for optical observation is configured by arranging an immersion objective lens 2, an immersion liquid 1, a light-transmitting sample placement member 3, and a sample treated with a mounting liquid 4, in this order, on the optical axis of the immersion objective lens 2. The direction of the optical axis of the immersion objective lens 2 is referred to as the z-direction.

[0014] (Immersion Objective Lens 2) The immersion objective lens 2 can be an immersion objective lens used in a microscope. Examples of microscopes include optical microscopes such as confocal laser microscopes, light sheet microscopes, multiphoton excitation microscopes (e.g., one-photon excitation type or two-photon excitation type), STED microscopes, RESOLFT microscopes, SIM microscopes, PALM / STORM microscopes, Bessel beam microscopes, and light lattice sheet microscopes; high-resolution image acquisition in combination with image processing (deconvolution); and the like. Examples of high-resolution image acquisition include high-resolution image acquisition by applying three-dimensional super-resolution microscopy techniques (e.g., STED, 3D PALM, FPALM, 3D STORM, and SIM).

[0015] 25 shows an example of a so-called inverted microscope in which the immersion objective lens 2 faces upward, but the optical observation structure 10 can also be applied to a microscope in which the arrangement shown in Fig. 25 is reversed (for example, a microscope in which a sample held in a hanging drop is observed from above). However, it is more preferable to apply the optical observation structure 10 to an inverted microscope.

[0016] Examples of immersion objective lenses include oil immersion objective lenses, silicone immersion objective lenses, and glycerin immersion objective lenses. The numerical aperture (NA) of the immersion objective lens is not particularly limited, but a typical example is 1.3 or more, and preferably 1.4 or more. The upper limit of the numerical aperture (NA) is, for example, 1.5 or less.

[0017] (Immersion liquid 1) Immersion liquid 1 is placed on the tip of the immersion objective lens 2. The rear surface 3b of the sample placement member 3 is placed on the immersion liquid 1. This allows the immersion objective lens 2 and the sample placement member 3 to come into contact with each other via the immersion liquid 1. The rear surface 3b is the surface of the sample placement member 3 that faces away from the sample placement surface 3a (described later).

[0018] The immersion liquid 1 may be a commercially available immersion liquid. Examples of immersion liquids include immersion oil Type-F (manufactured by Olympus; refractive index 1.5180, Abbe number 41.0), Type F (manufactured by Nikon; refractive index 1.5180, Abbe number 41.0), Immersol 518F (manufactured by Carl Zeiss; refractive index 1.5180, Abbe number 45.0), Type-F (manufactured by Leica; refractive index 1.5180, Abbe number 46.0), Type B (manufactured by Cargile; refractive index 1.5180, Abbe number 42.6), ibidi immersion liquid (refractive index 1.5180, Abbe number 46.0), silicone oil (manufactured by Olympus; refractive index 1.4060, Abbe number 52.0), and silicone oil (manufactured by Nikon; refractive index 1.4060, Abbe number 52.2). The refractive index (N) and Abbe number (ν) in this paragraph are values ​​at 23° C. and 546 nm, and are nominal values ​​of each manufacturer.

[0019] (Translucent Sample Placement Member 3) The translucent sample placement member 3 is preferably made of glass (mainly composed of borosilicate glass). Examples of the sample placement member 3 include a cover glass, a glass plate, and a glass Petri dish. The sample placement surface 3a and the rear surface 3b correspond to the two rear-facing flat surfaces of the cover glass, the two rear-facing flat surfaces of the glass plate, and the bottom surface of the glass Petri dish.

[0020] The distance between the sample placement surface 3a and the back surface 3b of the sample placement member 3 (corresponding to the thickness of the sample placement member 3) is, for example, preferably in the range of 150 μm to 190 μm, or in the range of 165 μm to 175 μm. Note that, if the difference in the refractive index and the difference in the Abbe number between the immersion liquid 1 and the mounting liquid 4 are within a predetermined range described below, even if spherical aberration and / or axial chromatic aberration occur at the interface with the sample placement member 3, these aberrations will not substantially change in the depth direction regardless of the thickness of the sample placement member 3.

[0021] The sample mounted with the mounting liquid 4 may be in direct contact with the sample mounting surface 3a, or the mounting liquid 4 may be in direct contact with the sample mounting surface 3a (i.e., the sample may be spaced a predetermined distance from the sample mounting surface 3a via the mounting liquid 4). Alternatively, the sample may be indirectly in contact with the sample mounting surface 3a via the mounting liquid 4. The sample mounted with the mounting liquid 4 may be encapsulated to prevent the mounting liquid 4 or the sample from drying, or to keep the sample in a predetermined position. For example, the sample mounted with the mounting liquid 4 may be encapsulated by placing a member other than the sample mounting member 3 on the sample mounted with the mounting liquid 4 and bonding the sample mounting member 3 to the member with an adhesive or the like. For example, in FIG. 5 , a cover slip is the sample mounting member 3, and the sample mounting surface of the cover slip corresponds to the sample mounting surface 3a. A slide placed opposite the cover slip corresponds to the member described above, and the sample is sandwiched and sealed between the cover slip and the slide slip.

[0022] (Mounting Liquid 4) In the visible light region, the difference in refractive index and the difference in Abbe number between the immersion liquid 1 and the mounting liquid 4 are both within a range of 5% or less. By having the difference within the above numerical range, spherical aberration and / or axial chromatic aberration can be reduced, enabling high-resolution observation deep into the sample. Furthermore, multicolor imaging without color shift is possible even with multi-wavelength excitation. The difference in refractive index and the difference in Abbe number are both 5% or less, which further reduces spherical aberration and / or axial chromatic aberration. The difference in Abbe number is preferably 4% or less or 3% or less, more preferably 2% or less, even more preferably 1% or less, and particularly preferably 0%. The difference in refractive index is preferably 4% or less or 3% or less, more preferably 2% or less, even more preferably 1% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less, and particularly preferably 0%. Mounting liquid 4 is also included in one aspect of the present invention.

[0023] In the visible light region, the difference in refractive index between the immersion liquid 1 and the mounting liquid 4 being a predetermined % or less means that the difference in refractive index between them at one or more wavelengths in the visible light region (for example, selected from wavelengths of 560 to 600 nm) is a predetermined % or less, preferably means that the difference in refractive index between them at each of two or more wavelengths (for example, one selected from wavelengths of 400 to 500 nm and another selected from wavelengths of 630 to 700 nm) is a predetermined % or less, and more preferably means that the difference in refractive index between them at each of three or more wavelengths (for example, one selected from wavelengths of 400 to 500 nm, one selected from wavelengths of 560 to 600 nm, and one selected from wavelengths of 630 to 700 nm) is a predetermined % or less.

[0024] The Abbe number (ν Ｄ ) can be calculated using the following formula: Ｄ = (N Ｄ −1) / (N Ｆ -N Ｃ ) In the formula, N Ｃ , N Ｄ and N Ｆare the refractive indices for the Fraunhofer C line (wavelength 656 nm), D line (wavelength 589 nm), and F line (wavelength 486 nm), respectively. The Abbe number can be measured using, for example, a known Abbe refractometer.

[0025] <Abbe number of mounting liquid 4> The Abbe number of mounting liquid 4 may preferably be 38 or greater, and more preferably 41 or greater or 42 or greater. The Abbe number of mounting liquid 4 may preferably be 47 or less, and more preferably 46 or less or 45 or less. If the Abbe number of mounting liquid 4 is within this range, it is easy to match it with the Abbe number of commercially available immersion liquids. The Abbe number of mounting liquid 4 is measured at a temperature of 24.8°C or greater and 25.2 or less.

[0026] <Refractive Index of Mounting Liquid 4> The refractive index of the mounting liquid 4 is preferably 1.32 or more and 1.52 or less, more preferably 1.35 or more and 1.52 or less, even more preferably 1.50 or more and 1.52 or less, and even more preferably 1.51 or more and 1.52 or less, for light with a wavelength of 561 nm, in order to reduce the difference from the refractive index of a typical cover glass and enable observation of samples of various shapes, etc. Furthermore, the refractive index of the mounting liquid 4 may be preferably 1.45 or more or 1.46 or more, and may be more preferably 1.47 or more, 1.48 or more, 1.49 or more, 1.50 or more, 1.505 or more, or 1.51 or more.

[0027] <Combination of Abbe number and refractive index of mounting liquid 4> The mounting liquid 4 may preferably have an Abbe number in the range of 38 to 47 and a refractive index in the range of 1.32 to 1.52. The Abbe number of this mounting liquid 4 may preferably be, for example, 41 or more, or 42 or more. The mounting liquid 4 may also preferably have an Abbe number of, for example, 46 or less, or 45 or less. The refractive index of this mounting liquid 4 is more preferably 1.35 to 1.51. The refractive index of this mounting liquid 4 may also preferably be 1.45 or more, or 1.46 or more, and more preferably 1.47 or more, 1.48 or more, or 1.49 or more.

[0028] The preferred ranges of the Abbe number and refractive index of the immersion liquid 1 are the same as the preferred ranges of the Abbe number and refractive index of the mounting liquid 4 .

[0029] <Composition of Mounting Solution 4> The mounting solution 4 preferably contains water; at least one component with a low refractive index and a high Abbe number (refractive index: 1.5 or less, Abbe number: 55 or more) selected from the group consisting of sugars, sugar alcohols, and glycols (Group B in FIG. 1); and at least one component with a high refractive index (refractive index: 1.518 or more) selected from the group consisting of nonionic organic iodine compounds and thiodiethanol (Group A in FIG. 1). By including these components, a mounting solution 4 with the desired Abbe number and refractive index can be obtained. Furthermore, by including these components, a sample can be made transparent, enabling the acquisition of three-dimensional images deep within the sample.

[0030] A preferred embodiment of the mounting liquid 4 is a mounting liquid having a refractive index of 1.35 to 1.52 for light with a wavelength of 561 nm; containing water and at least two or more components other than water, one of which is selected from the group consisting of sugars, sugar alcohols, and glycol-based solvents. The refractive index of the mounting liquid is approximately the same as that of the immersion liquid, thereby reducing spherical aberration and / or axial chromatic aberration during microscopic observation using an immersion objective lens. This allows for high-resolution observation even deep within a sample, and enables multicolor imaging without color shift even with multi-wavelength excitation.

[0031] The water content in the mounting liquid 4 is preferably 10% by weight or more, more preferably 15% by weight or more, from the viewpoint of the solubility of the raw materials, and is preferably 20% by weight or less, more preferably 10% by weight or less, from the viewpoint of achieving a high refractive index.

[0032] At least one low refractive index component selected from the group consisting of sugars, sugar alcohols, and glycols tends to have a low refractive index and a high Abbe number. Examples of sugars or sugar alcohols include fructose, mannose, sucrose, sorbitol, xylitol, erythritol, malbit, lactitol, glycerol, glucose, psicose, allose, tagatose, and isomers thereof. Examples of glycols include monoethylene glycol (MEG), polyethylene glycol (PEG), monopropylene glycol (MPG), polypropylene glycol (PPG), butylene glycol (BG), butanetriol (BT), and the like. The low refractive index component may be one type or two or more types.

[0033] The low refractive index component also has the effect of maintaining the fluorescence that labels the sample. Maintaining this fluorescence allows the mounting solution to be used as a storage solution for samples in high-resolution imaging. From the standpoint of maintaining fluorescence, the content of the low refractive index component in the mounting solution 4 is preferably 10 wt % or more, more preferably 20 wt % or more, and even more preferably 30 wt % or more. Furthermore, from the standpoint of viscosity or prevention of solidification, the content of the low refractive index component in the mounting solution 4 is preferably 40 wt % or less, more preferably 30 wt % or less, and even more preferably 20 wt % or less.

[0034] At least one high refractive index component selected from the group consisting of nonionic organic iodine compounds and thiodiethanol tends to have a high refractive index and a low Abbe number. Nonionic organic iodine compounds are compounds used as iodine-based nonionic contrast agents. Examples of nonionic organic iodine compounds include iopamidol, iomeprol, ioxilan, ioversol, iohexol, iopromide, iotrolan, and iodisanol. Two or more types of nonionic organic iodine compounds may be contained in the mounting solution 4.

[0035] When a nonionic organic iodine compound is used as a component of the mounting liquid 4, it is preferable to use thiodiethanol as a component of the mounting liquid 4. Among high refractive index materials, thiodiethanol has a relatively high Abbe number, and therefore can also be used for refractive index adjustment.

[0036] The content of the high refractive index component in the mounting liquid 4 is preferably 10% by weight or more, more preferably 20% by weight or more, and even more preferably 30% by weight or more, from the viewpoint of improving the refractive index, and is preferably 70% by weight or less, more preferably 50% by weight or less, and even more preferably 40% by weight or less, from the viewpoint of suppressing a decrease in the Abbe number or solidification.

[0037] The content ratio of the high refractive index component and the low refractive index component (high refractive index component:low refractive index component) in the mounting liquid 4 can be determined, for example, using an Abbe diagram. Figure 1 shows an Abbe diagram in which the high refractive index component and the low refractive index component are plotted.

[0038] An example of a method for determining the content ratio of the high-refractive-index component and the low-refractive-index component in the mounting liquid 4 (high-refractive-index component:low-refractive-index component) will be described using FIG. 1 . First, the target Abbe number and refractive index (in the case of FIG. 1 , the Abbe number and refractive index of the immersion liquid (Imm. Oil)) are plotted. If the mounting liquid 4 contains water, water is plotted, and an Abbe curve (the curve in FIG. 1 ) connecting the plot of water and the plot of the immersion liquid is drawn. Then, the content ratio of the high-refractive-index component and the low-refractive-index component is determined so as to match a point on the curve that is higher in refractive index than the target Abbe number and refractive index (Imm. Oil) (for example, the intersection of the line segment connecting the plot of the selected high-refractive-index component and the plot of the low-refractive-index component in FIG. 1 ), and the two components are mixed. Since the refractive index and Abbe number of the mixed solution are roughly proportional to the content ratio of the high-refractive-index component and the low-refractive-index component, the division ratio at the intersection of the line segment connecting the plot of the selected high-refractive-index component and the plot of the low-refractive-index component with the curve roughly corresponds to the content ratio of the two components. Finally, the amount of water required to adjust the Abbe number and refractive index is calculated and added to prepare a mounting solution having the desired Abbe number and refractive index.

[0039] The content ratio of the high refractive index component to the low refractive index component in the mounting liquid 4 (high refractive index component:low refractive index component) may be 10-90:90-10, 20-80:80-20, 30-70:70-30, or 40-60:60-40.

[0040] In addition to water, the low refractive index component, and the high refractive index component, other components may be contained in the mounting liquid 4. Examples of other components include dimethyl sulfoxide (DMSO), triethanolamine (TEA), formamide (FA), urea, etc. (Group C in FIG. 1 ).

[0041] The use of urea as another component improves the pourability, inhibits precipitation of low-refractive index components or high-refractive index components, and inhibits drying of the mounting solution. The urea content in the mounting solution 4 may be, for example, 15% by weight or more. In one example, the addition of urea is carried out so as not to substantially affect the refractive index, Abbe number, spherical aberration, and axial chromatic aberration. In another example, the addition of urea does not affect the transparency of the sample. In another example, when performing in situ hybridization, it may be preferable for the mounting solution 4 not to contain urea.

[0042] Examples of specific compositions of mounting solutions are as follows: (1) A mounting solution consisting of 5% to 50% by weight of a sugar or sugar alcohol, 2% to 50% by weight of thiodiethanol, 5% to 60% by weight of a non-ionic organic iodide, and 2% to 30% by weight of water. (2) A mounting solution consisting of 10% to 40% by weight of a sugar or sugar alcohol, 5% to 40% by weight of thiodiethanol, 10% to 50% by weight of a non-ionic organic iodide, and 5% to 20% by weight of water. (3) A mounting solution consisting of 0% to 20% by weight of urea, 5% to 35% by weight of a sugar or sugar alcohol, a total of 30% to 70% by weight of thiodiethanol and non-ionic organic iodide, and 2% to 30% by weight of water. (4) A mounting solution comprising 0% to 16% by weight of urea, 10% to 35% by weight of a sugar or sugar alcohol, a total amount of 45% to 70% by weight of thiodiethanol and a non-ionic organic iodide, and 5% to 20% by weight of water. (5) A mounting solution comprising 10% to 20% by weight of a sugar or sugar alcohol, a total amount of 45% to 70% by weight of thiodiethanol and a non-ionic organic iodide, and 5% to 20% by weight of water. This mounting solution may contain 16% or less by weight of urea.

[0043] In any of the mounting solutions (1) to (5), the total of all components is 100% by weight, and these mounting solutions satisfy the above-mentioned values ​​of the <Abbe number of mounting solution 4> and the <refractive index of mounting solution 4>.

[0044] In the mounting solutions (3), (4), and (5) above, the ratio (weight ratio) of thiodiethanol to nonionic organic iodide is, for example, within the range of 3:1 to 3:15.

[0045] (Sample) Examples of samples to be observed using the optical observation structure 10 according to one embodiment of the present invention include biological materials. The type of biological material is not particularly limited, but plant-derived materials or animal-derived materials are preferred, with animal-derived materials such as fish, amphibians, reptiles, birds, or mammals (mammals) being more preferred, and mammal-derived materials being particularly preferred. The type of mammal is also not particularly limited, but examples include laboratory animals such as primates other than humans, such as mice, rats, rabbits, guinea pigs, and marmosets; pet animals such as dogs, cats, and ferrets; livestock such as pigs, cows, and horses; and humans.

[0046] The biological material may be an individual (excluding a living human individual), or may be an organ, tissue, or cell obtained from a plant or animal individual. The cell may also be a cultured cell.

[0047] Furthermore, the biological material may be a material that has been fixed with paraformaldehyde or the like for microscopic observation, or may be an unfixed material. Fixation treatment can be carried out, for example, by a conventionally known method.

[0048] Furthermore, the biological material may specifically be, for example, biological tissue injected with a fluorescent chemical substance, biological tissue stained with a fluorescent chemical substance, biological tissue into which cells expressing a fluorescent protein have been transplanted, or biological tissue of a genetically modified animal that expresses a fluorescent protein.

[0049] The thickness of the sample may be 1 μm or more, 10 μm or more, 30 μm or more, 50 μm or more, 100 μm or more, 200 μm or more, 500 μm or more, or 1 mm or more. The thickness of the sample may also be 10 mm or less, 5 mm or less, or 2 mm or less. In conventional techniques, spherical aberration or axial chromatic aberration occurs during optical observation, particularly at deep portions of the sample (e.g., at a depth of 50 μm or 100 μm from the sample surface). The optical observation structure 10 according to one aspect of the present invention reduces spherical aberration or axial chromatic aberration even at deep portions of the sample, enabling multicolor imaging of the deep portions.

[0050] Examples of a "sample treated with a mounting solution" include a sample immersed in a mounting solution or a sample encapsulated in a mounting solution.

[0051] The structure for optical observation and the mounting solution according to one aspect of the present invention can be used for pathological diagnosis or pathological testing, as described below.

[0052] [2. Sample Observation Method] A sample observation method according to one aspect of the present invention (hereinafter sometimes referred to as "the present observation method") is a method for observing a sample using the above-described structure for optical observation, and includes a step of observing the sample through an immersion objective lens. In the present observation method, the descriptions of each component (e.g., "structure for optical observation," "immersion objective lens," and "sample") are those described in [1. Structure for Optical Observation].

[0053] This observation method allows multicolor imaging to a depth of approximately 50 to 200 μm when using a high-resolution microscope or super-resolution microscope. This depth is limited by the working distance (WD) of the objective lens, so adjusting the WD of the objective lens makes it possible to observe even deeper areas.

[0054] Furthermore, by using the above-described optical observation structure, this observation method can reduce spherical aberration or axial chromatic aberration in multicolor imaging or three-dimensional imaging of deep inside a sample, particularly in three-dimensional multicolor imaging of deep inside a sample combined with a sample transparency technique. For example, this observation method allows a cultured cell mass or the like in a medium to be observed as is by replacing it with a mounting solution.

[0055] A method for assisting pathological diagnosis using this observation method is also included in one aspect of the present invention. By appropriately adjusting the refractive index of the biological sample, not only aberration but also diffuse reflection can be reduced, improving resolution. It has been confirmed that using the above-described optical observation structure, a biological sample 4 μm thick can be observed with significantly higher resolution than with conventional mounting media (not shown). Furthermore, in conventional pathological diagnosis, a thickness of approximately 5 μm is recommended because it is not possible to confirm the penetration of staining agents into the tissue. The method for assisting pathological diagnosis according to one aspect of the present invention allows for high-resolution observation of thick sections (e.g., 30 μm or more, 50 μm or more, 100 μm or more, etc.) of biological material, thereby improving the accuracy of pathological diagnosis. The type of multicolor imaging used in the method for assisting pathological diagnosis is not particularly limited, but examples include fluorescent in situ hybridization (FISH) methods such as break-apart FISH for examining translocations and amplification FISH for examining amplified genes, fluorescent immunohistochemistry (IHC) using antibodies, and other molecular-specific fluorescent dye staining methods.

[0056] [3. Method for Preparing a Structure for Optical Observation] A method for preparing a structure for optical observation according to one aspect of the present invention (hereinafter sometimes referred to as "this preparation method") is a method that uses the above-mentioned mounting liquid. In this preparation method, the descriptions of each component (e.g., "structure for optical observation" and "mounting liquid") are incorporated by reference from [1. Structure for Optical Observation] and [2. Sample Observation Method].

[0057] For example, the refractive index and Abbe number of the immersion liquid to be used are examined, and a mounting liquid with a similar refractive index and Abbe number is prepared. After preparing the mounting liquid, an immersion objective lens, the immersion liquid, a translucent sample placement member, and a sample treated with the mounting liquid are arranged in this order on the optical axis of the immersion objective lens, thereby creating a structure for optical observation. In a more specific example, the immersion liquid is placed on the tip of the immersion objective lens. Separately, a sample for optical observation is prepared and placed on a translucent sample placement member, after which the mounting liquid is applied to the sample. Next, the sample placement member with the sample placed on it is set in the microscope. Next, the distance between the immersion objective lens and the sample placement member is adjusted to create the above-mentioned structure for optical observation.

[0058] [Summary] The present invention includes the following aspects. <1> A structure for optical observation, comprising an immersion objective lens, an immersion liquid, a translucent sample placement member, and a sample treated with a mounting liquid, arranged in this order on the optical axis of the immersion objective lens, wherein the difference in refractive index and Abbe number between the immersion liquid and the mounting liquid are all within 5% or less in the visible light region. <2> A structure for optical observation, comprising an immersion objective lens, an immersion liquid, a translucent sample placement member, and a sample treated with a mounting liquid, arranged in this order on the optical axis of the immersion objective lens, wherein the Abbe number of the mounting liquid is within the range of 38 to 47, and wherein the refractive index of the mounting liquid is within the range of 1.35 to 1.52 for light with a wavelength of 561 nm. <3> The structure for optical observation according to <1>, wherein the refractive index of the mounting liquid is within the range of 1.35 to 1.52 for light with a wavelength of 561 nm. <4> A method for observing a sample using the structure for optical observation described in any one of <1> to <3>, comprising a step of observing the sample through the immersion objective lens. <5> The observation method described in <4>, in which the observation of the sample is multicolor imaging. <6> A mounting liquid for microscope observation using an immersion objective lens, to be used in the structure for optical observation described in <1>, the mounting liquid having an Abbe number in the range of 38 to 47. <7> The mounting liquid described in <6>, having a refractive index in the range of 1.35 to 1.52 for light with a wavelength of 561 nm. <8> The mounting liquid described in <6>, comprising water, at least one component selected from the group consisting of sugars, sugar alcohols, and glycols, and at least one component selected from the group consisting of non-ionic organic iodine compounds and thiodiethanol. <9> A mounting solution for microscopic observation using an immersion objective lens, the mounting solution having a refractive index of 1.35 or more and 1.52 or less for light with a wavelength of 561 nm, and containing water and at least two or more components other than water, one of which is selected from the group consisting of sugars, sugar alcohols, and glycol-based solvents. <10> The mounting solution according to <9>, having an Abbe number of 38 or more and 47 or less.<11> A method for creating a structure for optical observation according to <1> or <2>, wherein the mounting liquid according to any one of <6> to <10> is used as the mounting liquid. <12> The mounting liquid according to any one of <6>, <7>, <9>, and <10>, further comprising at least one component selected from the group consisting of a non-ionic organic iodine compound and thiodiethanol. <13> The mounting liquid according to <12>, which comprises a non-ionic organic iodine compound and does not contain thiodiethanol. <14> The structure for optical observation according to any one of <1> to <3>, wherein the mounting liquid further comprises at least one component selected from the group consisting of a non-ionic organic iodine compound and thiodiethanol. <15> The structure for optical observation according to <14>, wherein the mounting liquid comprises a non-ionic organic iodine compound and does not contain thiodiethanol. <16> A method for observing a sample using the structure for optical observation according to <14> or <15>, comprising a step of observing the sample through the immersion objective lens. <17> The observation method according to <16>, wherein the observation of the sample is multicolor imaging.

[0059] The following examples are provided to further explain the embodiments of the present invention. It goes without saying that the present invention is not limited to the following examples, and various modifications are possible in detail. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed herein are also included in the technical scope of the present invention. Furthermore, all of the documents cited in this specification are incorporated by reference.

[0060] [1. Consideration of Mounting Solutions] The refractive index (ND) / Abbe number (νD) of the following materials that could be candidates for mounting solutions was plotted on an Abbe diagram. The Abbe diagram with each material plotted is shown in Figure 1. Based on Figure 1, the materials were classified as follows:

[0061] (Group A: High refractive index materials) Aqueous solutions of iodine-based nonionic contrast agents such as iopamidol, iomeprol, ioxilan, ioversol, iohexol, iopromide, iotrolan, and iodisanol; organic solvents such as 2,2'-thiodiethanol (TDE), dichlorobenzene, trichlorobenzene, bromonaphthalene, benzyl alcohol, benzyl benzoate, benzoic acid esters, butylbenzyl phthalate, diphenylamine, and diphenyl ether; silicone oils such as polysiloxane and polydimethylsiloxane.

[0062] (Group B: Low-dispersion materials (low refractive index materials)) Sugar or sugar alcohol solutions containing fructose, mannose, sucrose, sorbitol, xylitol, erythritol, malbit, lactitol, glycerol, glucose, psicose, allose, tagatose, and isomers thereof; glycol solutions such as monoethylene glycol (MEG), polyethylene glycol (PEG), monopropylene glycol (MPG), polypropylene glycol (PPG), butylene glycol (BG), and butanetriol (BT); mineral oils such as liquid paraffin (mineral oil).

[0063] (Group C: Other materials) Dimethyl sulfoxide (DMSO), triethanolamine (TEA), formamide (FA), urea, etc.

[0064] High-refractive-index materials in Group A tend to have low Abbe numbers, while low-refractive-index materials in Group B tend to have high Abbe numbers. The refractive index of the mounting solution is determined by the blending ratio of the materials in Groups A and B. Therefore, by connecting the selected materials with an Abbe curve (or a straight line), the blending ratio can be roughly determined by the division ratio of the line segments. Here, the immersion solution was used as the target, and two or more materials (plus water) were combined to achieve the desired refractive index / Abbe number. To achieve the final adjustment with water, an Abbe curve (dotted line in Figure 1) connecting the water and the target was first drawn, and the refractive index of the two mixed solutions was adjusted to lie along the extension of this curve (the high-refractive-index side). Furthermore, rather than simply matching the refractive index, the mounting solution was prepared with the following three points in mind:

[0065] (1) High Refractive Index When preparing a mounting liquid with a high refractive index of 1.5 or more, it is essential to select a material from Group A.

[0066] (2) Maintenance of fluorescence: Addition of sugar, sugar alcohol, or glycol (liquid) is effective in maintaining the fluorescence of the labeled sample. Therefore, when preparing a mounting solution, it is preferable to include a large amount of group B materials.

[0067] (3) Dryness or moisture absorption: Iodine-based nonionic contrast agents and sugars that are powder-based at room temperature (such as fructose and sorbitol) tend to dry out quickly even when dissolved in a small amount of water, which can lead to high refractive index fluctuations or solidification. If these are selected as components of the mounting solution, adding an appropriate amount of moisturizing urea can suppress refractive index fluctuations and further improve the miscibility of materials (especially powders).

[0068] With these three points in mind, mounting solutions were prepared that matched the refractive index of each manufacturer's immersion oil. Tables 1 to 3 show the compositions of mounting solutions that matched the refractive index of Olympus Type-F. Table 4 shows the compositions of mounting solutions that matched the refractive index of Nikon Type-F. Table 5 shows the compositions of mounting solutions that matched the refractive index of Carl Zeiss Immersol 518F and Leica Type-F. Table 6 shows the compositions of mounting solutions that matched the refractive index of Olympus silicone oil. In the examples, immersion oil may also be referred to as "immersion liquid."

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075] In the following examples, unless otherwise specified, verification was performed using the M47 (U:3M) mounting solution (referred to as Chromatie) in Table 1.

[0076] [2. Refractive index of mounting liquid in a specific wavelength range] In order to substantially completely eliminate spherical aberration and axial chromatic aberration, the refractive index and Abbe number of the immersion liquid and the sample must be substantially perfectly matched. Figure 2 is a schematic diagram showing the relationship between the refractive index of the immersion liquid and the mounting liquid and the axial chromatic aberration. As shown on the left side of Figure 2, even if the refractive index of the immersion liquid and the mounting liquid match only at a specific wavelength, axial chromatic aberration (ca) will occur due to differences in the degree of refraction at the glass interface for other wavelengths. As shown on the right side of Figure 2, if the refractive index of the immersion liquid and the mounting liquid match within a certain wavelength range (e.g., 400 nm to 700 nm) and the Abbe number ν Ｄ Therefore, no axial chromatic aberration occurs.

[0077] The refractive index of each mounting solution was measured in a specific wavelength range. SeeDB2S, CUBIC-R+, TDE97, PuClear, and Chromatie were measured using the 546 nm (e-line) refractive index (N ｅ = 1.5166 ± 0.0001), and the refractive index at each wavelength was measured (measurement temperature: 25°C). The measurement results are shown in Figure 3. The multi-wavelength Abbe refractometer DR-M2 (manufactured by Atago) used to measure the refractive index is shown in Figure 4.

[0078] Details of SeeDB2S are described in Non-Patent Document 2, CUBIC-R+ in Non-Patent Document 3, TDE97 in Non-Patent Document 1, and PuClear in Non-Patent Document 4. RapiClear (registered trademark) 1.52 is a product of Sunjin Lab Co., Ltd.

[0079] The vertical axis in Fig. 3 indicates the difference in refractive index between the immersion liquid and the mounting liquid. The numbers in parentheses in Fig. 3 indicate the refractive index of the immersion liquid, and the numbers in brackets [ ] indicate the Abbe number (ν Ｄ As shown in Figure 3, it was confirmed that the refractive index at wavelengths other than 546 nm for all mounting solutions except Chromatie did not match the refractive index of the immersion liquid.

[0080] [3. Verification of the Amount of Axial Chromatic Aberration in Vitro] Multicolor beads (TetraSpeck TMFirst, the refractive index (N) of the immersion liquid at 561 nm was measured to match the wavelength of the laser light installed in the confocal microscope (Olympus FV3000) used. ５６１ The refractive index and Abbe number of each mounting solution were adjusted again to match the refractive index (RI) of the known mounting solution (measurement temperature: 28°C).

[0081]

[0082] Next, a device for measuring chromatic aberration was fabricated. Multicolor beads (TetraSpeck® beads, particle size 1.0 μm, Thermo Fisher Scientific) were scattered on a glass slide and a cover glass (thickness: 0.17 mm, both manufactured by Matsunami Glass Co., Ltd.) and then dried and fixed. These two glasses were stacked with a 0.1 mm thick spacer in between, and various mounting solutions with adjusted refractive indices were injected into the gap. The periphery of the cover glass was sealed with nail polish. The cover glass was placed on an inverted microscope stage with the objective lens facing the objective lens, and excitation was performed sequentially with four wavelengths (405, 488, 561, and 640 nm) (Figure 5). Images of the beads on the surface of the cover glass (Base) and those directly below the glass slide (Deep) were taken at 0.2 μm intervals (Z direction), covering 31 images (6 μm in total). The acquired XY images were converted to XZ images, and MIP images were generated (Figure 6).

[0083] First, we compared MIP images of beads injected into the device with various mounting solutions (Figure 7). As shown on the right side of Figure 7, solutions with a lower refractive index than the immersion solution (Gly50-PBS (50% glycerol-containing PBS(-)) or PBS(-)) exhibited significant spherical aberration, resulting in an elongated focal depth and reduced fluorescence intensity. In contrast, five mounting solutions (Chromatie, SeeDB2S, CUBIC-R+, TDE97, and PuClear) whose refractive index at 561 nm was adjusted to match the refractive index of the immersion solution showed no elongation or reduction in fluorescence intensity from Base (0 μm depth) to Deep (approximately 90 μm depth) (Figure 7, top left). Furthermore, when images excited at 488 nm and 640 nm were overlaid, solutions with low Abbe numbers (SeeDB2S, CUBIC-R+, and PuClear) showed noticeable color shifts that could be seen even on the image (Figure 7, bottom left).

[0084] To investigate this more specifically, we used ImageJ to obtain a vertical line profile for each bead. Then, the difference in peak positions after Gaussian fitting, Δ Ｚ (X λ１ -X λ２ ) was defined as chromatic aberration and analyzed (Figure 8). The objective lens used in analyzing each mounting solution was the ultra-low chromatic aberration objective PLAPON60XOSC (60x / NA1.40, Olympus). The microscope conditions used in this test are shown in Table 8.

[0085]

[0086] The analysis results for each mounting solution are shown in Figures 9 to 13. In Figures 9 to 13, the vertical axis represents chromatic aberration (Δ Ｚ ) and the horizontal axis indicates the depth of the sample. The numbers in parentheses indicate the number of measurement points.

[0087] Although the magnitude of axial chromatic aberration differs depending on the wavelength used, it was confirmed that the amount of chromatic aberration increased in proportion to the depth of the sample in all solutions with low Abbe numbers (Figures 10, 11, and 13). Furthermore, a mounting solution with a slightly higher Abbe number (TDE97) exhibited chromatic aberration with color shift in the opposite direction to the other mounting solutions (Figure 12). On the other hand, Chromatie, whose refractive index in the visible light range perfectly matches that of the immersion liquid, showed almost no change in the depth direction, confirming the absence of axial chromatic aberration (Figure 9) (where the number in parentheses indicates the number of measurement points).

[0088] [4. Confirmation of Fluorescence Fading] Coronal sections (50 μm thick, PFA-fixed) of brain tissue from Tg mice expressing Thy1-ChR2-EYFP were used to confirm the fluorescence fading of various mounting solutions. Female Tg mice, 117 weeks old, were used. In addition to five mounting solutions (Chromatie, SeeDB2S, CUBIC-R+, TDE97, and PuClear), commercially available ProLong® Glass and the commonly used Gly50-PBS were also used as solution samples. First, the section samples were attached to a cover glass, coated with various solutions, and mounted. The results after one day of mounting are shown in Figure 14. The microscope conditions used in this study are listed in Table 9.

[0089]

[0090] As shown in Figure 14, the four solutions other than TDE97 did not significantly affect fluorescence fading. On the other hand, TDE97 caused significant fading and tissue shrinkage.

[0091] [5. Deep Observation of Actual Tissue] Deep observations were performed on mouse brain tissue sections. Frozen brain tissue sections (t: 100 μm) from Thy1-YFP-H mice (74-week-old, male mice) were perfusion-fixed with PBS containing 4% PFA. The frozen brain tissue sections were then stained with DAPI and propidium iodide (PI) and observed. The results of observations using Gly50-PBS as the mounting solution are shown in Figure 15, and the results of observations using Chromatie are shown in Figure 16. The microscope conditions used in this study are listed in Table 10.

[0092]

[0093] The Z axis in Figures 15 and 16 indicates the depth of the sample. As shown in Figure 16, when Chromatie was used as the mounting solution, spherical aberration was suppressed, allowing bright, high-resolution imaging deep into the tissue. On the other hand, as shown in Figure 15, when Gly50-PBS was used as the mounting solution, neither DAPI nor PI staining was observed beyond a certain depth. Small molecular structures such as nucleoli could not be clearly observed even at a small depth due to poor resolution.

[0094] Next, deep sections of mouse tongue tissue were observed. Frozen sections (50 μm thick) of tongue tissue from CD11c-YFP mice (21-week-old, male mice) were perfused with PBS containing 4% PFA. Then, the sections were stained with DAPI and propidium iodide (PI), and the frozen sections of brain tissue were observed. Gly50-PBS or Chromatie (New Solution) was used as the mounting medium. The observation results are shown in Figure 17. The microscope conditions used in this study are listed in Table 11.

[0095]

[0096] As shown in FIG. 17, when Chromatie (New Solution) was used as the mounting solution, a high-resolution image was obtained in which dendrites could be observed.

[0097] The mounting solution used was either the commercially available mounting medium ProLong® Glass (Thermo Fisher Scientific) or Chromatie, and images of deep tissue were compared. Brain tissue cryosections (t: 100 μm) from wild-type mice (C57BL / 6J, 47 weeks old, female) were used as samples. The brain tissue cryosections were perfusion-fixed with PBS containing 4% PFA. They were then stained with DAPI and propidium iodide (PI) and observed. The macrozoom microscope conditions are shown in Table 12, and the confocal laser scanning microscope conditions are shown in Table 13.

[0098]

[0099]

[0100] Figure 18 shows the observation results using ProLong® Glass, and Figure 19 shows the observation results using Chromatie. The left side of each figure shows the results of observation using a macro zoom microscope, and the right side shows the results of observation using a confocal laser scanning microscope. The Z axis in Figures 18 and 19 indicates the depth of the sample. Observation using a macro zoom microscope revealed no significant difference between ProLong® Glass and Chromatie. Meanwhile, observation using a confocal laser scanning microscope revealed that fluorescence from deep areas could be detected with Chromatie, whereas fluorescence from deep areas could not be detected with ProLong® Glass. It is believed that spherical aberration prevented fluorescence from being detected in observations using ProLong® Glass.

[0101] 6. Verification of Axial Chromatic Aberration in Actual Tissue Next, we verified the amount of axial chromatic aberration using actual mouse brain tissue sections. The mouse brain tissue sections were coronal sections (cutting thickness: 50 and 100 μm) from mice (C57BL / 6J). Targeting presynaptic terminals, immunohistochemical staining (IHC) was performed using an anti-Synaptophysin 1 mouse monoclonal antibody conjugated with Oyster550 dye (Synaptophysin 1-Oyster550) as the primary antibody. Further secondary antibody staining was performed with anti-mouse Alexa488 antibody (F(ab')2 fragments) to create a two-color colocalization model sample. The model sample is shown in Figure 20.

[0102] The stained tissue section was attached to a cover glass, various mounting solutions (approximately 20 μL) were dropped onto it, and a slide glass was placed on top, then inverted and sealed. The cover glass was set on the inverted microscope stage so that the surface was facing the objective lens, and excited sequentially at two wavelengths (488, 561 nm). 451 images (total 90 μm) were taken at a depth of -10 to 80 μm in 0.2 μm steps (Z direction), and the acquired XY images were converted into XZ section images. Several synapse-like structures were picked out from the images, and a line profile was taken in the vertical direction, as with the beads. After Gaussian fitting, the difference in peak position Δ Ｚ (X ５６１ -X λ２ ) were collected (Figure 21). Here, Chromatie, SeeDB2S, and CUBIC-R+ were used as representative solutions, and values ​​were collected and analyzed at different depths within the tissue (0-5 μm, 40-45 μm, and 70-75 μm). The microscope conditions used in this study are shown in Table 14.

[0103]

[0104] The analysis results of each mounting solution are shown in Figures 22 to 24. In Figures 22 to 24, the vertical axis represents chromatic aberration (Δ Ｚ ) and the horizontal axis indicates the depth of the sample. The numbers in parentheses indicate the number of measurement points.

[0105] As shown in Figures 23 and 24, the SeeDB2S and CUBIC-R+, which have low Abbe numbers, showed an increase in chromatic aberration in proportion to the depth of the tissue. On the other hand, as shown in Figure 22, the Chromatie showed almost no change in chromatic aberration even deep in the tissue.

[0106] [7. Verification of Amount of Axial Chromatic Aberration In Vitro] Regarding [3. Verification of Amount of Axial Chromatic Aberration In Vitro], we verified whether axial chromatic aberration would occur even if the confocal microscope used was changed when a mounting liquid whose refractive index in the visible light region matched the refractive index of the immersion liquid was used. The confocal microscope used was Ti2AX with N-SPARC (manufactured by Nikon Corporation), and the mounting liquid used was M55 (hereinafter sometimes referred to as Chromatie / M55) in Table 4. The refractive index at 561 nm of the immersion liquid for this confocal microscope (Nikon Type F in Table 4) (N ５６１ The refractive index (N) of each mounting solution was adjusted again to match the refractive index (N) of the Chromatie / M55 at 561 nm after preparation. ５６１ ) is 1.5161, and the Abbe number (ν D ) was 38.7.

[0107] Next, a device for measuring the amount of chromatic aberration was fabricated in the same manner as in [3. Verification of the amount of axial chromatic aberration in vitro], and observation was performed using a microscope. The microscope conditions used in this test are shown in Table 15, and the analysis results are shown in Figures 26 and 27. The vertical axis of Figure 27 represents the amount of axial chromatic aberration (Δ Ｚ ) and the horizontal axis indicates the depth of the sample. The numbers in parentheses indicate the number of measurement points.

[0108]

[0109] As shown in Figures 26 and 27, regardless of the type of confocal microscope used, Chromatie / M55, whose refractive index in the visible light range is perfectly matched to the refractive index of the immersion liquid, shows almost no change in the depth direction, and it was confirmed that no axial chromatic aberration occurs.

[0110] 8. Summary Table 16 is a summary of the amount of axial chromatic aberration obtained with each mounting solution.

[0111]

[0112] As shown in Table 16, even if the refractive index of the mounting fluid at a specific wavelength (561 nm) matches that of the immersion fluid, differences in Abbe number result in axial chromatic aberration. This phenomenon occurs not only in in vitro systems using beads, but also in brain tissue, and the amount of chromatic aberration is nearly identical. Furthermore, even with the TDE97, which has a slightly higher Abbe number, the amount of chromatic aberration does indeed increase with depth. At first glance, the TDE97 image appears to have almost no color shift. However, this is simply because the objective lens itself also has axial chromatic aberration, which coincidentally runs in the opposite direction to the slope of the line, resulting in a color shift within ±0.1 μm.

[0113] In contrast to these known mounting solutions, Chromatie, whose refractive index and Abbe number match those of the immersion solution, shows almost no change in chromatic aberration from the glass surface (depth 0) to the depths of the sample. While there is a slight degree of chromatic aberration between 640 and 405 nm, this is due to axial chromatic aberration inherent in the objective lens itself, and therefore is a problem that can be resolved by improving the objective lens. Furthermore, this deviation can be offset by shifting the entire XZ image, making it possible to virtually eliminate axial chromatic aberration in Chromatie.

[0114] In this way, the newly developed Chromatie can achieve high-resolution fluorescence imaging without color shift deep within a sample, and it is highly likely that it will be possible to visualize microscopic objects or factors that have not been visible until now using an optical microscope.

[0115] The present invention can reduce spherical aberration or axial chromatic aberration during microscopic observation using an immersion objective lens, particularly during high-resolution observation, and can therefore be used in, for example, high-resolution observation for basic research or pathological examination.

[0116] 1 Immersion liquid (immersion liquid) 2 Immersion objective lens 3 Sample placement member 3a Sample placement surface 3b Back surface 4 Sample and mounting liquid

Claims

1. an immersion objective lens, an immersion liquid, a light-transmitting sample placement member, and a sample treated with a mounting liquid are arranged in this order on the optical axis of the immersion objective lens; a difference in refractive index and an Abbe number between the immersion liquid and the mounting liquid are both within a range of 5% or less in a visible light region; the Abbe number of the mounting liquid is within the range of 38 to 47, The refractive index of the mounting liquid is in the range of 1.35 to 1.52 for light having a wavelength of 561 nm. Structure for optical observation.

2. an immersion objective lens, an immersion liquid, a light-transmitting sample placement member, and a sample treated with a mounting liquid are arranged in this order on the optical axis of the immersion objective lens; the Abbe number of the mounting liquid is within the range of 38 to 47, The refractive index of the mounting liquid is in the range of 1.35 to 1.52 for light having a wavelength of 561 nm. Structure for optical observation.

3. 3. A method for observing a sample using the structure for optical observation according to claim 1, comprising the step of observing the sample through the immersion objective lens.

4. The observation method according to claim 3 , wherein the observation of the sample is multi-color imaging.

5. A mounting liquid for microscope observation using an immersion objective lens, which is used in the optical observation structure according to claim 1, comprising: The Abbe number is within the range of 38 to 47, A mounting solution having a refractive index of 1.35 or more and 1.52 or less for light with a wavelength of 561 nm.

6. Water, At least one component selected from the group consisting of sugars, sugar alcohols, and glycols; 6. The mounting solution according to claim 5, comprising at least one component selected from the group consisting of non-ionic organic iodine compounds and thiodiethanol.

7. A mounting solution for use in microscopic observation using an immersion objective lens, comprising: The refractive index for light with a wavelength of 561 nm is in the range of 1.35 to 1.52, Contains water and at least two or more components other than water, one of the components is selected from the group consisting of sugars, sugar alcohols, and glycol solvents; A mounting solution having an Abbe number in the range of 38 to 47.

8. A method for producing a structure for optical observation according to claim 1 or 2, comprising the steps of: A method for producing a mounting liquid, comprising using the mounting liquid according to any one of claims 5 to 7 as the mounting liquid.