Composition for preparing biological material with excellent optical transparency and use thereof

Abstract

To provide a rarefaction technique facilitating processing and suitable for imaging with high throughput and at low magnification.SOLUTION: The present invention relates to a rarefaction technique using a solution including at least one of a compound having lipid removal ability, a compound having biological pigment decolorization ability, a compound having decalcification ability, a compound having refractive index adjustment ability, and a compound having tissue swelling ability. The rarefaction technique facilitates processing and is suitable for imaging with high throughput and at low magnification.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a composition for preparing a biological material having excellent optical transparency and use thereof. [Background technology]

[0002] In recent years, in order to perform advanced observation of intracellular gene expression, intracellular localization, and cell morphology, pretreatment (clearing treatment) has been performed using clearing agents to increase the optical transparency of tissues, and various clearing agents and pretreatment methods have been developed.

[0003] For example, Non-Patent Documents 1 and 2 disclose a clearing treatment technique using benzyl benzoate / benzyl alcohol (BABB method). The BABB method is a classical tissue clearing method using an organic solvent, and is used as a method for preparing cleared samples to be applied to sheet illumination macro zoom microscopes.

[0004] Non-Patent Document 3 discloses a technique (SeeDB method) for adjusting the refractive index using fructose. The main purpose is to minimize the changes in biological tissues that occur during processing, so the molecular level structure, membrane structure, and axon shape are almost completely maintained. Furthermore, by using a two-photon microscope and an optimized lens, observation at the whole brain level is possible.

[0005] Non-Patent Document 4 and Patent Document 1 disclose a technique (the Scale method) that successfully minimizes the loss of fluorescent signals by using a water-soluble reagent. This technique also has the main purpose of minimizing changes in biological tissues that occur during processing, and is intended for use in the observation of nerve axons in neuroscience and the detection of amyloid plaques in Alzheimer's disease tissues.

[0006] Non-Patent Document 5 discloses a technology (CLARITY method) for achieving transparency by a physicochemical method using an electrophoresis device. The CLARITY method achieves transparency by physicochemically treating brain tissue solidified with an acrylamide polymer using an electrophoresis device to remove lipid components. A high degree of transparency is achieved in about two weeks while maintaining the molecular-level structure within the tissue. It is applicable to both fluorescent protein labeling and immunostaining.

[0007] Non-Patent Document 6 discloses a technique (PACT method, PARS method) that overcomes the difficulty of the physicochemical method using an electrophoresis apparatus in the CLARITY method and performs transparency by perfusion of an SDS (surfactant) solution used in the CLARITY method.

[0008] Non-patent document 7 discloses a technology (SWITCH method) that overcomes the difficulty of the physicochemical method using an electrophoresis device in the CLARITY method, and performs clarification by treating the SDS (surfactant) solution used in the CLARITY method at high temperatures. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2012 / 161143 [Non-patent literature]

[0010] [Non-Patent Document 1] Hans-Ulrich Dodt et al. "Ultramicroscopy: three-dimensional visualization of neural networks in the whole mouse brain" Nature Methods vol.4 no.4: 331-336 (2007) [Non-Patent Document 2] Klaus Becker et al."Chemical Clearing and Dehydration of GFP Expressing Mouse Brains"PLoS ONE vol.7 issue 3: e33916 (2012) [Non-Patent Document 3] Meng-Tsen Ke et al. "SeeDB: a simple and morphology-preserving optical clearing agent for neural circuit reconstruction" advance online publication (published online 23 June 2013) [Non-Patent Document 4] Hiroshi Hama et al. "Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain" Nature Neuroscience vol.14 no.11: 1481-1490 (2011) [Non-Patent Document 5] Kwanghun Chung et al."Structural and molecular interrogation of intact biological systems"Nature vol.497: 332-339 (2013) [Non-Patent Document 6] Bin Yang et al. "Single-CellPhenotyping within Transparent Intact Tissue through Whole-Body Clearing"Cell vol.158: 945-958 (2014) [Non-Patent Document 7] Evan Murray et al. "Simple,Scalable Proteomic Imaging for High-Dimensional Profiling of IntactSystems" Cell vol.163: 1500-1514 (2015) Summary of the Invention [Problem to be solved by the invention]

[0011] However, in the BABB method described in Non-Patent Documents 1 and 2, the fluorescent protein is rapidly extinguished due to the dehydration and organic solvent treatment, making it impossible to detect the signal in a short period of time. In addition, organic solvents generally pose safety concerns.

[0012] The SeeDB method described in Non-Patent Document 3 allows observation at the whole brain level, but requires several days for scanning. In addition, since it is assumed to be used with a two-photon microscope, the transparency of the processed tissue (especially the adult brain) is not high enough to be adapted to a microscope with a sheet illumination system.

[0013] The Scale method described in Non-Patent Document 4 is intended for use with confocal and two-photon microscopes, so the transparency of the processed tissues (especially adult brains) is not high enough to be compatible with microscopes using sheet illumination systems.

[0014] The CLARITY method described in Non-Patent Document 5, and the PACT and PARS methods described in Non-Patent Document 6, are not suitable for application to multiple samples because they involve complicated steps and require dedicated devices.

[0015] The SWITCH method described in Non-Patent Document 7 requires high-temperature treatment using a surfactant, which results in severe fading of the fluorescent protein.

[0016] The present invention has been made to solve the above problems, and a main object of the present invention is to provide a clearing technique which is easy to process and suitable for high-throughput, low-magnification imaging. [Means for solving the problem]

[0017] In order to solve the above problems, the present inventors conducted a large-scale screening based on various unique viewpoints, and as a result, discovered several compounds capable of making biological materials transparent, thereby completing the present invention.

[0018] That is, the composition according to the present invention is a composition for preparing a biological material having excellent optical transparency, and comprises at least one of the following (A) to (E): (A) Compound with high lipid removal ability (B) Compounds showing higher decolorizing activity than N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine (C) EDTA in combination with acids and bases (D) A compound that is soluble in water at 60 wt% or more and has a refractive index in an aqueous solution at at least one concentration in the range of 60 to 80 wt% in the range of 1.47 to 1.50. (E) Compounds exhibiting higher swelling activity than urea The solution is characterized in that it contains:

[0019] The method of the present invention is a method for preparing a biological material with excellent optical transparency, and is characterized by including a step of infiltrating the biological material with a solution containing at least one of the above (A) to (E).

[0020] The biomaterial with excellent optical transparency according to the present invention is characterized in that it is infiltrated with a solution containing at least one of the above (A) to (E). Effect of the Invention

[0021] The present invention is a technology based on chemical processing, and does not require the use of special devices or complicated procedures. Furthermore, the present invention can realize a high level of transparency to a level that enables high-throughput analysis of multiple samples by a simple process. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a diagram showing the correlation between delipidation activity and compound category in an example of the present invention. [Diagram 2] FIG. 1 is a graph showing the correlation between delipidation activity and pH in an example of the present invention. [Diagram 3] FIG. 1 shows the efficiency of clearing and defatting of a mouse hemibrain in an example of the present invention. [Figure 4] FIG. 1 is a diagram showing the correlation between transparency and phospholipid content in mouse hemibrain in an example of the present invention. [Diagram 5] FIG. 1 is a diagram showing a comparison of the transparency of human brain white matter by 10 wt% #0070+10 wt% chemical in an example of the present invention. [Figure 6] FIG. 1 is a diagram showing a comparison of the transparency of human brain white matter by 10 wt% #0070+10 wt% surfactant in an example of the present invention. [Figure 7] FIG. 1 is a diagram showing the correlation between EGFP fluorescence intensity and pH in an example of the present invention. [Figure 8] FIG. 1 is a diagram showing a comparison of the transparency of a mouse hemibrain caused by basic chemicals in an example of the present invention. [Figure 9] FIG. 13 is a graph showing the efficiency of mouse kidney clearing and defatting by 10 wt% #0414+10 wt% chemical in an example of the present invention. [Figure 10] FIG. 1 shows the quenching efficiency of EGFP by the #0414 cocktail in an example of the present invention. [Figure 11] FIG. 1 shows the transparency of a mouse whole brain with 10 wt% #0414+10 wt% Triton X-100 in an example of the present invention. [Figure 12] FIG. 1 shows the depigmentation efficiency of mouse spleen in an example of the present invention. [Figure 13] FIG. 1 shows the transparency of mouse femurs and quenching of GFP fluorescence by an EDTA cocktail in an example of the present invention. [Figure 14]FIG. 2 is a diagram showing a solubilization test of a hydroxyapatite suspension in an example of the present invention. [Figure 15] FIG. 2 is a graph showing the pH dependency of solubilization of a hydroxyapatite suspension with an EDTA+#0414 cocktail in an example of the present invention. [Figure 16] FIG. 2 is a graph showing the temperature dependence and EDTA concentration dependence of solubilization of a hydroxyapatite suspension with an EDTA+#0414 cocktail in an example of the present invention. [Figure 17] FIG. 2 is a diagram showing a comparison of demineralization activity using 10 wt % EDTA and a basic chemical in an embodiment of the present invention. [Figure 18] FIG. 1 shows the transparency of mouse kidneys using various refractive index adjusting agents in an example of the present invention. [Figure 19] FIG. 1 shows the transparency of mouse lungs using various refractive index adjusting agents in an example of the present invention. [Figure 20] FIG. 1 shows top compounds in swelling screening using gelatin in an example of the present invention. [Figure 21] FIG. 13 shows swelling of the brain after delipidation treatment with swelling activity reagent #1352 in an example of the present invention. [Figure 22] FIG. 1 shows a swollen brain made transparent using a mixed solution of #0640+#1352 in an example of the present invention. [Diagram 23] FIG. 1 shows clearing of tissues including mouse liver and human brain white matter in an example of the present invention. [Figure 24] FIG. 1 shows the clearing of mouse brain tissue in an example of the present invention. [Diagram 25] FIG. 13 is a diagram showing refractive index adjustment of a degreased tissue in an embodiment of the present invention. [Figure 26] FIG. 1 shows transparency of mouse organs and the whole body in an example of the present invention. [Figure 27] FIG. 1 shows the clearing of human tissue in an embodiment of the present invention. [Figure 28] FIG. 1 shows the clearing of human brain tissue in an embodiment of the present invention. [Figure 29] FIG. 1 shows the transparency of a marmoset brain in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Hereinafter, an embodiment of the present invention will be described in detail.

[0024] [1. Composition] The present invention provides a composition for preparing a biological material having excellent optical transparency. The composition according to the present invention is characterized in that it is a solution containing at least one (one, two, three, four, or five) of the following (A) to (E).

[0025] As used herein, the term "light transmittance" refers to the degree to which transmitted light passes through the incident light, or the degree to which fluorescence due to irradiation with excitation light passes through and scattering is low, and the expression "excellent light transmittance" refers, for example, to a high ratio of light emitted from the observation target (i.e., transmitted light) to light incident on the observation target. Light transmittance can be expressed as a percentage, for example, with the ratio of transmitted light to incident light being 0% for a light-blocking substance (e.g., thick black plastic or metal) and 100% for a transparent liquid such as water. In one embodiment of a biological material having excellent light transmittance, the ratio of transmitted light to incident light is 40% or more, preferably 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, or 99% or more.

[0026] The type of biological material to which the present invention is applied is not particularly limited, but is preferably a plant-derived material or an animal-derived material, more preferably an animal-derived material such as fish, amphibians, reptiles, birds, or mammals (mammals), and particularly preferably a mammal-derived material. The type of mammal is not particularly limited, but may include laboratory animals such as mice, rats, rabbits, guinea pigs, and primates other than humans (marmosets, etc.); pet animals such as dogs and cats; livestock such as cows and horses; and humans.

[0027] The biological material may be an individual (excluding a living human individual), or may be an organ, tissue, body fluid (e.g., blood, saliva, serum, plasma, urine, synovial fluid, cerebrospinal fluid, etc.) or cell obtained from an individual of a multicellular organism. The organism may be an adult or a fetus. Since the composition according to the present invention has an excellent transparency processing ability, the transparency processing can be applied even if the biological material is a tissue or organ derived from a multicellular animal (e.g., the whole or a part of a bone, brain, heart, lung, liver, spleen, or kidney), or an individual of a multicellular animal other than a human (e.g., an embryo, an adult, etc.). The biological material may be a material fixed with paraformaldehyde or the like, or may not be fixed.

[0028] Specifically, the biological material may 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 from a genetically modified animal expressing a fluorescent protein.

[0029] By using the present invention, the optical transparency of biological materials can be significantly increased, enabling imaging with high throughput. Loss of signal from fluorescent proteins and fluorescent substances is minimized. -Multiple samples (tens to hundreds) can be processed in parallel at the same time. -High degree of transparency is achieved so that it can be used in combination with sheet illumination microscopes. The effect on the structure of the tissue area can be minimized. - Effectively achieves clearing even for samples that are large and comprised of a variety of tissues, such as adult samples.

[0030] Furthermore, the clearing treatment using the composition according to the present invention is reversible to a certain extent. Specifically, the clearing state of the biological material after the clearing treatment can be released by simply immersing it in a balanced salt solution, and the material can be used for storage, immunohistochemical assay, biochemical assay, and the like. Specific examples of the balanced salt solution include phosphate-buffered balanced salt solutions such as PBS and HBSS; Tris-hydrochloride-buffered balanced salt solutions (TBS); artificial cerebrospinal fluid (ACSF); MEM, DMEM, and Ham's F-12, and other basal media for cell culture. Since the antigenicity of proteins and the like is preserved without change before and after the clearing treatment, analysis using normal tissue staining and immunostaining techniques is possible.

[0031] ((A) Compound having degreasing and removing ability) The compound (A) that can be contained in the composition according to the present invention is a compound having lipid removing ability. In one example, the compound (A) has high lipid removing ability.

[0032] In one embodiment, the compound (A) is a compound whose mixed solution has an OD600 of 0.336 or less when measured by the method described in the Examples. In another embodiment, the compound (A) is at least one of polyether, anionic surfactant, cationic surfactant, amphoteric surfactant, amine, aminoalcohol, polyhydric alcohol, surfactant-like aromatic ether, and pyridine. In terms of high tissue penetration, amine and polyalcohol are preferred, and amine is more preferred.

[0033] The amine may be any of primary amines, secondary amines, tertiary amines, and quaternary amines. The number of amine groups contained in the compound may be one, two, three, four, or more. The hydrocarbon portion of the amine may be linear, branched, or cyclic. The hydrocarbon portion of the amine may be saturated or unsaturated. It may also have an aromatic ring such as a benzene ring. The number of carbon atoms in the hydrocarbon portion of the amine is not particularly limited, and may be, for example, 1 to 20, 1 to 15, 1 to 10, or 2 to 8.

[0034] More specific examples of the compound (A) include the compounds listed in Table 1 and their analogs. In one example, #0070 (1,3-bis(aminomethyl)cyclohexane) in Table 1 and its analogs are preferred. Examples of analogs of #0070 include bis(aminoalkyl)cycloalkyl. The number of carbon atoms of the alkyl in the aminoalkyl group is not particularly limited, but may be, for example, 1 to 8, and in a preferred example, 1. The number of carbon atoms of the alkyl in the cycloalkyl portion is not particularly limited, but may be, for example, 3 to 8, and in a preferred example, 6. The position at which the two aminoalkyl groups are bonded is not particularly limited, and may be 1,2-, 1,3-, or 1,4-. Among the compounds listed in Table 1 and their analogs, #0070 (1,3-bis(aminomethyl)cyclohexane) in Table 1 is preferred from the viewpoints of low cost and low odor.

[0035] In addition, the compound (A) may be one type or multiple types. In one embodiment, the compound (A) may be a combination of multiple types of polyethers, anionic surfactants, cationic surfactants, amphoteric surfactants, amines, amino alcohols, polyhydric alcohols, surfactant-like aromatic ethers, and pyridines. In one example, it may be preferable to combine an amine with a surfactant. Preferred combinations of an amine with a surfactant include, for example, a combination of #0070 and #0631, and a combination of #0070 and SDS, and among them, a combination of #0070 and #0631 is more preferable.

[0036] Furthermore, when the biological material contains a fluorescent protein, it is preferable to use a compound with a pH of 5 to 12, preferably 7 to 11. Among them, from the viewpoint of transparency, #0389 and #0414 are preferable, and #0414 is more preferable. #0414 may be further combined with other compounds, and preferable combinations include #0414 and Triton X-100, #0414 and Tween-40, #0414 and #1051, #0414 and #0694, and #0414 and sucrose, among which #0414 and Triton X-100 is more preferable.

[0037] The concentration of the compound (A) in the composition is not particularly limited, but is preferably 5 to 60 wt%, more preferably 10 to 40 wt%, and even more preferably 15 to 30 wt% (total value when two or more types are included). In one example, when an amine and a surfactant are combined, the amine can be 10 to 20 wt%, and the surfactant can be 5 to 10 wt%.

[0038] ((B) Compounds capable of decolorizing biological pigments) The compound (B) that can be contained in the composition according to the present invention is a compound having a biological pigment depigmenting ability. In one example, the compound (B) has a high biological pigment depigmenting ability.

[0039] In one embodiment, the compound (B) is a compound that exhibits a bleaching activity higher than that of an aminoalcohol (N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine). The bleaching activity can be measured, for example, by the method described in the Examples.

[0040] The compound of (B), in another embodiment, is, for example, at least one of an alkylimidazole, a phosphine, a surfactant, an amine, a pyridine, an aminoalcohol, an alcohol, a benzylamide, and a pyrazolone.

[0041] More specific examples of the compound (B) include the compounds and analogs thereof listed in Table 2. In one example, the compound (B) is preferably one that retains the fluorescence of the fluorescent protein, and examples of such compounds include #0441, #0470, #0484, #0635, #0651, #0938, #1283, and analogs thereof listed in Table 2.

[0042] From the viewpoint of retaining the fluorescence of the fluorescent protein and exhibiting high bleaching activity, #0651, #0938, and analogs thereof are preferred, and #0938 and analogs thereof are more preferred.

[0043] Analogs of #0938 include compounds included in alkylimidazoles. The alkyl group may be linear, branched, or cyclic. The alkyl group may be saturated or unsaturated. The number of carbon atoms in the alkyl group is not particularly limited, and may be, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. The bonding position of the alkyl group is not particularly limited, and is preferably the 1-position (1-alkylimidazole). From the viewpoint of low cost, 1-methylimidazole is preferred.

[0044] The compound (B) may be of one type or of multiple types.

[0045] The concentration of the compound (B) in the composition is not particularly limited, but is, for example, preferably 5 to 40 wt %, more preferably 5 to 30 wt %, and even more preferably 10 to 20 wt % (the total value when two or more types are included).

[0046] In one example, the compound (B) has a high affinity for heme and may be able to remove heme from hemoglobin without destroying the protein.

[0047] ((C) Compounds with demineralizing ability) The compound (C) that can be contained in the composition according to the present invention is a compound having demineralizing ability. In one example, the compound (C) has high demineralizing ability.

[0048] In one embodiment, the compound (C) is a combination of EDTA (ethylenediaminetetraacetic acid), an acid, and a base. By combining EDTA, an acid, and a base, demineralization can be performed efficiently. In one example, the loss of the signal of the fluorescent protein is minimized.

[0049] Examples of the acid include halogenated monocarboxylic acids, hydroxymonocarboxylic acids, aliphatic monocarboxylic acids, alkylaminomonocarboxylic acids, acetylaminomonocarboxylic acids, other monocarboxylic acids, dicarboxylic acids, malic acid, thiodicarboxylic acids, tartaric acid, malonic acid, other dicarboxylic acids, polycarboxylic acids, sugars, tetrafluoroborates, phosphonic acids, sulfonic acids, hydrocarbons, acid salts of amines, and acid salts of aromatic amines. In one example, the acid preferably has a pH of 2.0 or higher from the viewpoint of tissue damage.

[0050] More specific examples of the acid include the compounds listed in Table 4. Among the compounds listed in Table 4, preferred acids include compounds having a score value of 0.9 or less in the Examples. Among the compounds listed in Table 4, more preferred acids include #1165 and #1300. The acid may be one type or multiple types.

[0051] As the base, in one example, a compound having degreasing and removing ability is preferable. Specific examples of the base include the bases having degreasing and removing ability described in (A) above (amines, amino alcohols, and pyridines), among which amino alcohols are preferable, and #0414 and its analogs are more preferable. An example of an analog of #0414 is N-alkyl dialcoholamine. The alkyl group in the N-alkyl dialcoholamine may be linear, branched, or cyclic. The alkyl group in the N-alkyl dialcoholamine may be saturated or unsaturated. The number of carbon atoms in the alkyl group in the N-alkyl dialcoholamine is not particularly limited, but may be, for example, 1 to 8, and in a preferred example, 4. The number of carbon atoms in the two alcohol moieties is not particularly limited, and may be the same or different, but may be, for example, 1 to 8, and in a preferred example, 2. The base may be one type or multiple types.

[0052] As the base, in another example, imidazole analogues are preferred, and #1352 is more preferred. In addition, heterocyclic amines such as #1278 (4-methylmorpholine) and #0370 (2-amino-6-methylpyridine) are also preferred.

[0053] The pH of the solution containing (C) is preferably 6 to 11, more preferably 7 to 9, and even more preferably around 8.

[0054] The EDTA concentration in the solution containing (C) is not particularly limited, but is preferably 5 to 25 wt%, more preferably 7.5 to 20 wt%, and even more preferably around 10 wt%. The acid concentration in the solution containing (C) is not particularly limited, but is preferably 5 to 25 wt%, more preferably 7.5 to 20 wt%, and even more preferably around 10 wt%. The base concentration in the solution containing (C) is not particularly limited, and may be such that the pH of the solution becomes the target pH. In one example of a method for preparing a solution containing (C), a base is added to a solution containing EDTA and an acid.

[0055] ((D) Compound having refractive index adjusting ability) The compound (D) that can be contained in the composition according to the present invention is a compound having a refractive index adjusting ability. In one example, the compound (D) has a high refractive index adjusting ability.

[0056] In one embodiment, the compound (D) is a compound that is soluble in water at 60 wt % or more and has a refractive index in an aqueous solution at at least one concentration in the range of 60 to 80 wt % within the range of 1.47 to 1.50.

[0057] The compound of (D), in another embodiment, is, for example, at least one of benzylamine, pyridine, amine, benzylaminoalcohol, hydrazine, and thiourea.

[0058] More specific examples of the compound (D) include the compounds listed in Table 3 and analogs thereof. From the viewpoint of suppressing shrinkage, the compound (D) is preferably a compound that does not contain a salt. Examples of such compounds include #0370, #0389, #0587, #0609, #0640, #1102, #1283, and analogs thereof listed in Table 3. Among these compounds, from the viewpoint of achieving higher transparency, #0587, #0640, #1102, #1283, and analogs thereof are preferred, and #0587 and analogs thereof are more preferred.

[0059] Furthermore, among the compounds listed in Table 3 and their analogues, #0640, #0450, #1283, #0864, ​​#1052, #0788, #0389, and analogues thereof are preferred from the viewpoint of maintaining the fluorescence of the fluorescent protein.

[0060] Furthermore, from the viewpoint that higher transparency can be achieved, a combination of #0640 and #1283, a combination of #0640 and nicotinamide (#0855), and a combination of Histodenz and #1283 are also preferable.

[0061] The compound (D) may be of one type or of multiple types.

[0062] The concentration of the compound (D) in the composition is preferably, for example, a concentration that results in a refractive index of 1.47 to 1.53, and specifically, for example, is preferably 50 to 90 wt%, more preferably 60 to 85 wt%, and even more preferably 70 to 80 wt% (total value when two or more types are included). In the specific combinations listed above, for example, the former is preferably 40 to 50 wt%, and the latter is preferably 25 to 35 wt%.

[0063] ((E) Compounds Having Tissue Swelling Ability) The compound (E) that can be contained in the composition according to the present invention is a compound having tissue swelling ability. In one example, the compound (E) has high tissue swelling ability.

[0064] In one embodiment, the compound (E) is a compound that exhibits a swelling activity higher than that of urea. The swelling activity can be measured, for example, by the method described in the Examples.

[0065] Compounds of (E) include, for example, the compounds shown in FIG. 20, imidazole derivatives and antipyrine analogues.

[0066] The compound (E) may be of one type or of multiple types.

[0067] The concentration of the compound (E) in the composition is not particularly limited, but is, for example, preferably 5 to 40 wt %, more preferably 5 to 30 wt %, and even more preferably 5 to 25 wt % (the total value when two or more types are included).

[0068] In one example, the compound (B) can expand the volume of a biological material 4 to 10 times while maintaining the shape of the material.

[0069] (combination) A single composition may contain a plurality of components (A) to (E).

[0070] In one example, the composition preferably essentially contains at least one of (A) and (E). When the biological material is rich in blood or a vital pigment such as myoglobin, the composition preferably contains (B). When the biological material is bone tissue, the composition preferably contains (C).

[0071] (solvent) The type of solvent used in the composition according to the present invention is not particularly limited as long as it can dissolve the above-mentioned active ingredients, but it is preferable to use water as the main solvent, and it is particularly preferable to use only water as the solvent.In the present invention, "using water as the main solvent" refers to the proportion of water in the volume of the total solvent used being the largest compared to other solvents, and preferably refers to the use of water in an amount of more than 50% and not more than 100% of the total volume of the total solvent used.In addition, the composition according to the present invention prepared using water as the main solvent is called a reagent as an aqueous solution.

[0072] When water is used as the main solvent, for example, dimethyl sulfoxide (DMSO) may be mixed with water for use in the fixed specimen. For example, mixing DMSO with the fixed specimen is expected to improve the permeability of the reagent into the biological material and promote the clearing treatment of tissues having a keratinous surface.

[0073] The main advantages of using water as a solvent are: 1) The active ingredient of the composition of the present invention has excellent solubility in water, which makes the composition of the present invention easy to prepare and inexpensive; 2) Compared to using an organic solvent as the main solvent, the clearing process does not involve dehydration of the target biological material, which makes it possible to prevent the problem of shrinkage of the biological material; 3) Compared to using organic solvents as the main solvent, the possibility of damaging fluorescent proteins is significantly reduced, making it possible to observe fluorescent proteins in biological materials that have been cleared; 4) It is not limited to fixed materials, but can be applied to the clearing process of live materials; 5) The clearing process is reversible, and the cleared biological specimen can be restored to its pre-clearing state if necessary; 6) Compared with the case where an organic solvent is used as the main solvent, the handling is safer.

[0074] The composition according to the present invention may be a buffer solution capable of maintaining a suitable pH for the biological material to be cleared. Furthermore, the osmotic pressure of the composition according to the present invention may be adjusted to such an extent that the biological material to be cleared is not deformed and the active ingredient is sufficiently permeated into the biological material.

[0075] (Further Optional Ingredients) In the composition according to the present invention, the solution may contain further optional components as necessary, such as a pH adjuster and an osmotic pressure adjuster.

[0076] [2. Method] The present invention provides a method for preparing a biological material having excellent optical transparency. The method according to the present invention is characterized by including a step (infiltration step) of infiltrating a biological material with a solution containing at least one (one, two, three, four, or five) of the above-mentioned (A) to (E). The solution is as described above.

[0077] In one embodiment, the method according to the present invention may include a step of infiltrating a biological material with a solution (first solution) containing at least one of (A) to (E) (first infiltration step) and a step of infiltrating a biological material with a solution (second solution) containing at least another one of (A) to (E) (second infiltration step), and may further include at least one (one, two, three, four, or five) of a step of infiltrating a biological material with a solution (third solution) containing at least another one of (A) to (E) (third infiltration step), a step of infiltrating a biological material with a solution (fourth solution) containing at least another one of (A) to (E) (fourth infiltration step), and a step of infiltrating a biological material with a solution (fifth solution) containing at least another one of (A) to (E) (fifth infiltration step).

[0078] (Infiltration process) In the infiltration step, at least one of degreasing with a compound (A), decolorization with a compound (B), demineralization with a compound (C), refractive index adjustment with a compound (D), and swelling with a compound (E) is performed. That is, the method according to the present invention includes at least one (one, two, three, four, or five) of a degreasing step with a compound (A), a decolorization step with a compound (B), a demineralization step with a compound (C), a refractive index adjustment step with a compound (D), and a swelling step with a compound (E). When multiple steps are included, the steps may be performed separately, or two or more may be performed simultaneously.

[0079] The biological material subjected to the delipidation process has excellent light transmittance because light scattering substances (mainly lipids) are removed. Therefore, the delipidation process is a particularly effective process for making transparent biological materials that contain a lot of lipids (for example, organs or tissues that contain a lot of lipids). The delipidation process may be carried out, for example, at 25 to 45°C for about 1 to 10 days.

[0080] The biological material subjected to the decolorization step has excellent light transmittance due to decolorization of the biological pigment (mainly hemoglobin in blood). Therefore, the decolorization step is particularly effective in making blood transparent and in making biological materials containing a large amount of blood (for example, organs or tissues containing a large amount of blood) transparent. The decolorization step may be carried out, for example, at 25 to 45°C for about 1 to 5 days.

[0081] In the biological material subjected to the demineralization process, calcium-rich tissues (such as bones and teeth) are demineralized, and the biological material exhibits excellent light transmittance. Therefore, the demineralization process is a particularly effective process for making calcium-rich biological materials (such as organs or tissues that contain a lot of calcium) transparent. The demineralization process may be carried out, for example, at 25 to 45°C for about 3 to 10 days.

[0082] The biological material subjected to the refractive index adjustment process exhibits excellent light transmittance because the refractive index of the liquid component is close to that of the biological molecules such as proteins. Therefore, this process is effective for all biological materials. The refractive index adjustment process may be performed, for example, at 25 to 37°C for about 1 to 4 days.

[0083] The biological material subjected to the swelling step exhibits excellent light transmittance due to swelling. In addition, the optical resolution is also improved. Therefore, this step is effective for all biological materials. The swelling step may be carried out, for example, at 10 to 35° C. for about 1 to 7 days.

[0084] The order of each step is not particularly limited, but when all five steps are performed, they are preferably performed in the following order: (1) → (2) → (3). (1) Degreasing process, bleaching process (2) Decalcification process, swelling process (3) Refractive index adjustment process In (1), the degreasing step and the decolorizing step may be carried out separately or simultaneously. In one example, the compound (A) used in the degreasing step and the compound (B) used in the decolorizing step are partially or entirely the same and can be carried out simultaneously. Alternatively, the compound (A) and the compound (B) may be mixed and carried out simultaneously. In addition, when they are carried out separately, either one may be carried out first, but it is preferable to carry out the degreasing step first. In (2), the order of the demineralizing step and the swelling step is not particularly limited, and either one may be carried out first.

[0085] It is also preferable to follow the above sequence even if at least one of the five steps is not performed.

[0086] (Further optional steps) In the method according to the present invention, a step of washing the biological material (washing step) may be carried out at any timing. The washing step may be carried out, for example, before the infiltration step is started, between each infiltration step, and / or after the infiltration step is completed. Washing may be carried out, for example, using PBS or distilled water. Furthermore, washing may be carried out, for example, 1 to 5 times for each washing step.

[0087] In the method according to the present invention, the cleared biological material may be stored at any time, for example, at room temperature or in a low temperature environment (cleared sample storage step). In this case, the biological material is preferably stored immersed in the above-mentioned solution or a balanced salt solution such as PBS to prevent drying.

[0088] The method according to the present invention may be combined with a transparentization method other than that of the present invention. For example, the degreasing step and the decolorization step may be performed by the method according to the present invention, and the refractive index adjustment may be performed by a transparentization method other than that of the present invention (for example, the SeeDB method).

[0089] The biological material that has been made transparent is subjected to a step of observing the biological material with, for example, an optical microscope (observation step). The biological material to be subjected to the observation step may be subjected to a visualization step such as staining or marking before the transparentizing treatment of the present invention, or after the transparentizing treatment and before the observation step, as necessary. For example, when a fluorescent protein is used in the visualization step, a fluorescent protein gene is introduced into a living biological material before the transparentizing treatment to express the fluorescent protein. In addition, when a fluorescent chemical substance (excluding fluorescent proteins) is injected into the biological material or a biological material is stained with a fluorescent chemical substance as the visualization step, it is preferable to perform the visualization step before the transparentizing treatment, but it can also be performed after the transparentizing treatment. Furthermore, staining with a chemical substance other than a fluorescent chemical substance can also be performed as the visualization step.

[0090] The observation step can be performed using any type of optical microscope. For example, the observation step can be performed using a sheet illumination microscope, a two-photon laser microscope, and / or a confocal microscope. The method of the present invention can be particularly suitably applied to high-throughput analysis of multiple samples at low magnification.

[0091] [3. Kit] The present invention also provides a kit comprising a composition for preparing a biological material having excellent optical transparency.

[0092] As used herein, the term "kit" refers to a package that includes a container (e.g., a bottle, a plate, a tube, a dish, etc.) that contains a particular material (e.g., a component). The kit preferably includes instructions for using each material. When used herein in the context of a kit, "included" refers to a state in which the material is contained in any of the individual containers that constitute the kit. The kit of the present invention may also be a package in which multiple different compositions are packaged together, and the above-mentioned composition may be contained in multiple different containers when in solution form. The kit of the present invention may include multiple components mixed in the same container or in separate containers. The "instructions" may be written or printed on paper or other media, or may be attached to electronic media such as magnetic tape, computer-readable disk or tape, CD-ROM, etc., and describe procedures for realizing the use of the kit. The kit of the present invention may also include containers that contain diluents, solvents, washing solutions, or other reagents. In addition, the kit of the present invention may include instruments and reagents necessary for carrying out procedures for realizing the use of the kit.

[0093] In one example, the kit may include a plurality of the above-mentioned compositions. For example, when the degreasing step and the decolorizing step are performed separately, the kit may include a composition containing (A) and a composition containing (B). In one embodiment, the kit includes at least two (two, three, four, or five) of a composition containing (A), a composition containing (B), a composition containing (C), a composition containing (D), and a composition containing (E). In another embodiment, the kit includes at least a composition containing at least two (two, three, four, or five) of (A) to (E) and a composition containing at least another one (one, two, three, four, or five) of (A) to (E).

[0094] The following examples are provided to further explain the embodiments of the present invention. Of course, the present invention is not limited to the following examples, and various aspects of the details are possible. Furthermore, the present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims, and embodiments obtained by appropriately combining the technical means disclosed are also included in the technical scope of the present invention. This specification also incorporates the contents described in the specification and drawings of Japanese Patent Application No. 2016-092025, which is the basis of the priority of this application. Furthermore, all publications and patents cited in this specification are incorporated herein by reference as they are. EXAMPLES

[0095] 1. Search for a clearing agent We developed an experimental method to evaluate, in a high-throughput manner, a library of 1,619 water-soluble compounds to select favorable compounds for each of the five parameters required for transparency: (A) lipid removal ability, (B) biological pigment decolorization ability, (C) decalcification ability, (D) refractive index adjustment ability, and (E) tissue swelling ability.

[0096] (A. Lipid removal ability) Using the fixed brain tissue solubilization assay method described in Susaki et al., Cell, 157, 726-739, 2014, we screened a library of 1,619 water-soluble compounds for compounds with lipid removal ability.

[0097] Specifically, the excised mouse brain was sheared using a utility knife and further crushed by ultrasonication. An equal volume of 8% PFA solution was added to these suspensions and fixed at 4°C for 1 hour. The supernatant was removed by centrifugation, washed three times with PBS, and PBS was added to each brain to make 2 mL of the suspension. A fixed brain tissue suspension was obtained by adding 130 μL of 10 wt% chemicals to 20 μL of the suspension diluted 1 / 3.5, shaking at 37°C overnight, and then measuring the absorbance (turbidity) of these mixed solutions at 600 nm. Chemical solutions that reduce the turbidity of the fixed brain suspension are expected to reduce light scattering by solubilizing lipids in the brain tissue, which are scattering factors. The top 50 chemicals that contribute to reducing turbidity were classified according to their chemical properties, and it was shown that surfactants, amino alcohols, aliphatic amines, and pyridines were effective (Table 1, Figure 1). In addition, the decrease in turbidity of the fixed brain suspension correlated with the pH of the solution, and the more basic the solution, the more pronounced the decrease in turbidity (Figure 2).

[0098] Next, we investigated the lipid removal efficiency of a series of compounds for mammalian tissues. A mouse half brain was treated with 10 wt% chemical solution at 37°C for 3 days, washed with PBS, and the refractive index was adjusted with CUBIC-2A (22.5 wt% sucrose, 22.5 wt% antipyrine, 25 wt% urea, 10 wt% triethanolamine). After measuring the transmittance of the brain tissue, it was washed with PBS, the tissue was crushed, and the lipid amount (phospholipid amount and cholesterol amount) of the suspension was quantified (Figure 3). As a result, a high correlation was observed between the amount of residual phospholipid and the transmittance of the tissue (Figure 4). In other words, the more the amount of phospholipid is reduced, the higher the transparency of the tissue is. This result directly demonstrated that the removal of lipids contributes to transparency. Importantly, compared to surfactants such as #1035, #1455, #1460 and Triton X-100, basic chemicals such as amino alcohols and aliphatic amines, as well as some neutral chemicals such as #1051, were found to be more effective at degreasing and clearing the tissue. This indicates that the basic chemicals and some neutral chemicals mentioned above are superior in terms of their ability to penetrate into tissues compared to surfactants. Of the basic chemicals obtained, #0070 (1,3-bis(aminomethyl)cyclohexane) was selected from the standpoints of price and the odor of the reagent.

[0099] We investigated combinations that synergistically contribute to delipidation so as to maximize the delipidation activity based on #0070 (Figure 5). Small pieces of human brain white matter were treated with a cocktail of 10wt% #0070 + 10wt% chemicals at 37°C for 4 days, washed with PBS, and then the refractive index was adjusted using CUBIC-2A. As a result, we found that a combination with SDS, a surfactant, was the most excellent in terms of achieving high transparency without damaging the tissue.

[0100] Furthermore, to search for the optimal surfactant, various 10wt% #0070 + 10wt% surfactant cocktails were prepared and their effects on human brain white matter transparency were compared (Figure 6). As a result, the cocktail of 10wt% #0070 + 10wt% #0631 was found to be the optimal combination.

[0101] It was shown that the fluorescence of EGFP, a representative fluorescent protein, is quenched at pH 12 or higher at 37°C (Figure 7). Therefore, we limited the use of basic chemicals with a pH of 11 to 12 to compare the transparency of mouse hemibrains (Figure 8). Mouse hemibrains were treated with 10 wt% chemical solutions at 37°C for 3 days, washed with PBS, and then the refractive index was adjusted using CUBIC-2A. Based on these results, #0389 and #0414 were selected, but after further investigation, it was found that #0414 was superior in terms of organ transparency, and therefore #0414 (N-butyldiethanolamine) was selected as a highly efficient degreasing chemical.

[0102] Next, we investigated combinations that synergistically contribute to delipidation so as to maximize the delipidation activity based on #0414 (Figure 9). Mouse kidneys were treated with a cocktail of 10wt% #0414 + 10wt% chemicals at 37°C for one day, washed with PBS, and then the refractive index was adjusted with CUBIC-2A. As a result, it was shown that the combination with Triton X-100 was optimal for both delipidation activity and transparency. Furthermore, it was shown that the cocktail of #0414 + Triton X-100 did not induce EGFP quenching at various mixing ratios (Figure 10). Mouse whole brains were treated with a cocktail of 10wt% #0414 + 10wt% Triton X-100 at 37°C for three days, washed with PBS, and then the refractive index was adjusted with CUBIC-2A. As a result, a dramatic improvement in transparency and delipidation efficiency was confirmed compared to the conventional CUBIC-1 reagent (Figure 11).

[0103] (B. Biopigment decolorization ability) We developed a high-throughput screening system that evaluates decolorization ability by mixing various compound solutions with coagulated bovine blood and measuring the absorbance of heme recovered in the supernatant. Using this system, we screened compounds with biological pigment decolorization ability from a library of 1,619 water-soluble compounds.

[0104] Specifically, an equal amount of 8% PFA solution was mixed with bovine blood and reacted overnight at 4°C. The mixture was centrifuged at 1500×g for 5 minutes, the supernatant containing PFA was discarded, and the initial volume of PBS was added to prepare a coagulated blood sample. 50 μL of the coagulated blood sample was added to 150 μL of various 10 wt% aqueous compound solutions and reacted overnight at 37°C. After centrifuging the reaction solution at 1500×g for 5 minutes, 100 μL of the supernatant was added to a plate and the absorbance was measured. Since samples suspended without being dissolved in water show high absorbance values ​​despite a weak decolorizing effect, OD720 was measured as an index of scattering and OD420 as an index of heme elution, and OD420-OD700 was defined as the decolorizing activity of each chemical, and a group of decolorizing activities higher than amino alcohol (N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine) that has already been shown to exhibit decolorizing activity was identified (Table 2). Through this screening, surfactants such as alkylimidazoles and pyridinium salts, as well as bases such as alkylamines, were identified as compounds with significant decolorizing activity.

[0105] In order to examine the decolorization efficiency of a series of compounds for mammalian tissues, mouse spleen was treated overnight at 37°C with various 10 wt% chemical solutions that do not induce EGFP quenching, and then the OD420 of the supernatant was measured and the organs were photographed (Figure 12). As a result, #0938 (1-ethylimidazole) showed the highest decolorization activity for mouse spleen. Since this reagent is expensive at 176 yen per gram, we decided to use 1-methylimidazole (25.8 yen per gram) as an alternative with equivalent decolorization activity. In hemoglobin, heme is coordinated to oxygen and histidine and bound to them. Since 1-alkylimidazole has a skeleton similar to histidine, it is expected that it has a high affinity for heme and therefore showed strong decolorization activity. On the other hand, almost no decolorization activity was observed with imidazole in which the nitrogen atom at position 1 is not alkylated, which suggests that the alkylation of the nitrogen atom at position 1 improves the affinity for heme.

[0106] (C. Demineralization ability) In order to comprehensively screen compounds with demineralizing activity from a library of 1619 water-soluble compounds, a screening system was constructed using hydroxyapatite, the main component of bone tissue. Hydroxyapatite (200mg / mL) was suspended in 40v / v% glycerol and mixed with various compound aqueous solutions, and the turbidity was measured. Specifically, 20μL of 200mg / mL hydroxyapatite 40v / v% glycerol suspension was dispensed into a 96-well plate reader, and 130μL of each of the various reagents was added and shaken and stirred overnight at 37℃. The demineralizing activity was then evaluated by measuring OD600. The score value of each compound aqueous solution was calculated when the OD600 value when treated with water was set to Score 1.0. The closer the score value is to 0, the stronger the demineralizing activity of the chemical is. It is generally known that demineralization proceeds quickly with strong acids such as hydrochloric acid and nitric acid, but the fluorescent protein signal cannot be observed under these conditions. On the other hand, EDTA in the neutral range can retain the signal of the fluorescent protein, but its decalcification ability is weaker than that of strong acids, and a long decalcification operation is required. In the present invention, a solution that shows dramatically improved decalcification activity in combination with EDTA in the neutral range was developed. As a result of screening, the score of most compounds was 0 when the pH was below 2.0. Therefore, a list of compounds with a pH of 2.0 or more sorted by the characteristics of their structural formula is shown in Table 4. Among these compound groups, compounds with low scores are shown in a box.

[0107] In order to verify whether these compounds are effective for decalcification, a cocktail was prepared by neutralizing a mixed solution of 10wt% EDTA + 10wt% decalcification candidate compound to pH 7-8 with #0070 solution, which has been found to be useful as a degreasing reagent. After treating mouse femurs with EDTA-Na solution adjusted to pH 7-8 with NaOH or EDTA-decalcification candidate compound-#0070 solution, the change in translucency of the femurs was measured (the experiment was performed independently twice). As a result, it was found that EDTA-decalcification candidate compound-#0070 solution showed dramatically higher decalcification activity than the conventional EDTA-Na solution (Figure 13). In addition, no quenching of GFP was observed at all. The results of Figure 13 showed that EDTA-#1165-#0070 solution and EDTA-#1300-#0070 solution were the most effective decalcification reagents.

[0108] Next, we attempted to optimize the basic chemicals used for pH adjustment. Since the experimental system using femoral bones has a large experimental error, we conducted an evaluation system using hydroxyapatite to optimize the demineralization activity of EDTA in more detail. Specifically, 20 μL of 60 or 200 mg / mL hydroxyapatite 40 v / v% glycerol suspension was dispensed into a 96-well plate reader, and 130 μL of each reagent was added and shaken and stirred overnight at 37°C. The demineralization activity was then evaluated by measuring OD600. For 10 wt% EDTA, a cocktail neutralized to about pH 8 using basic amines such as #0070, #0390, #0414, #1027, #1439, and #1456, which have been found to be useful as degreasing reagents, was used, and 10 wt% EDTA-#0414 showed the most effective demineralization effect (Figure 14).

[0109] When the pH dependency of demineralization activity of a cocktail in which 10 wt% EDTA was neutralized with #0414 (EDTA-#0414) and a cocktail in which 10 wt% #0414 was neutralized with EDTA (#0414-EDTA) was examined, it was confirmed that the demineralization activity was more pronounced at around pH 8 than the conventional EDTA-NaOH solution (Fig. 15). When the temperature dependency and EDTA concentration dependency were examined, it was found that the higher the temperature, the stronger the demineralization activity, and the maximum demineralization activity was observed at an EDTA concentration of approximately 10 wt% (Fig. 16). When mouse femurs were made transparent by dissolving the surrounding tissue with sodium hydroxide under these conditions, demineralization proceeded efficiently.

[0110] Furthermore, a comprehensive search for basic chemicals was performed using an evaluation system using hydroxyapatite. Specifically, 20 μL of 150 mg / mL hydroxyapatite 40 v / v% glycerol suspension was dispensed into a 96-well plate reader, and 130 μL of each of the various reagents was added and shaken and stirred overnight at 37°C. The decalcification activity was then evaluated by measuring OD600. Decalcification was performed using a cocktail of 10 wt% EDTA neutralized to about pH 8 using a basic chemical with a pH of 11 or higher (Figure 17). As a result of comparing the relative decalcification activity, imidazole analogues had a higher decalcification activity than other categories, and the neutralization cocktail using #1352 (imidazole) showed the highest decalcification. In addition, neutralization cocktails using the heterocyclic amine #1278 (4-methylmorpholine) and the pyridine skeleton #0370 (2-amino-6-methylpyridine) showed high demineralization activity.

[0111] (D.Refractive index adjustment ability) It is generally known that the refractive index of an aqueous solution increases depending on the concentration of the solute, and the refractive index per unit weight varies depending on each reagent. Therefore, the properties required for preparing a high refractive index aqueous solution are (1) extremely high water solubility, and (2) a solvent with a high refractive index even in a low-concentration aqueous solution. In order to comprehensively search for reagents that meet the criteria of (2) from a library of 1,619 water-soluble compounds, the refractive index at 589 nm of various 10 wt% aqueous solutions of the compounds was measured, and compounds with high refractive indexes were screened. Furthermore, in order to select compounds with high water solubility from among them, 60-80 wt% aqueous solutions were prepared, and a group of compounds with refractive indices of 1.47-1.50 was identified (Table 3).

[0112] From the viewpoint of suppressing shrinkage, the effect of adjusting the refractive index was compared among these compound groups for seven compounds that did not contain salt. Specifically, the kidneys of 8-week-old male mice were shaken in a 10wt% #0414 + 10wt% Triton X-100 mixed solution at 37°C for 4 days, and then washed with PBS. The kidneys were further shaken overnight at 37°C in a 1 / 2-fold diluted high refractive index solution, and then further shaken overnight at 37°C in the original high refractive index solution, after which the transmission images and tissue translucency were measured (Figure 18). For comparison, CUBIC-2A solution, 70wt% Histodenz solution, and 70wt% sucrose solution were used. As a result, the transparency of #0587, #0640, #1102, and #1283 was higher in both the appearance and translucency of the organs than the high-concentration Histodenz solution and high-concentration sucrose solution that have already been reported.

[0113] Next, in order to search for a highly refractive index modifier using a cocktail of these chemicals, various 46wt% / 30wt% mixed solutions were prepared. After shaking for 4 days in a mixed solution of #0414 + 10wt% Triton X-100 at 37°C, the tissue was washed with PBS. The tissue was further shaken overnight at 37°C in a 1 / 2-fold diluted high refractive index solution, and then shaken overnight in the original high refractive index solution at 37°C. The transmitted image and tissue transparency were then measured (Figure 19). As a result, all of the tissues achieved good transparency, but 46wt% #0640 + 30wt% #1283, 46wt% #0640 + 30wt% nicotinamide (#0855), and 46wt% Histodenz + 30wt% #1283 achieved the highest transparency.

[0114] (E. Tissue swelling ability) It is generally known that tissue transparency can be increased by inducing swelling in tissues. We confirmed that the swelling and shrinkage behavior of the brain and that of gelatin show a high correlation, and established a screening system using gelatin. A 9% gelatin aqueous solution and a 0.8% paraformaldehyde solution were mixed, and the mixed solution was dispensed in 200 μL on a 96-well plate and incubated at 4 °C for 16 hours. 170 μL of a 10 wt% compound aqueous solution from a library of 1619 water-soluble compounds was dispensed onto the solidified gelatin plate and incubated at room temperature for 72 hours. The gelatin plate was then washed with pure water about three times, wiped clean, and the absorbance at 975 nm, one of the absorption wavelengths of water, was measured. From the library of 1619 water-soluble compounds, a group of compounds with swelling activity exceeding that of urea was screened, with urea's swelling activity set as Score 1. As a result, water-soluble compounds with swelling activity were identified (Figure 20).

[0115] To verify whether the identified compounds actually have swelling activity on mouse brains, we used #1352, one of the compounds mentioned above, to shake a delipidated brain using CUBIC-1 for 3 days with a change of solution every 12 hours. As a result, we observed high brain swelling (Figure 21). To verify whether the swollen brains actually show high transparency, we shaked the brains swollen with #1352 in a 55wt% #0640 + 5wt% #1352 mixed solution, which has high refractive index adjustment ability, and succeeded in producing brain samples that remained swollen and had high transparency (Figure 22).

[0116] [2. Example of use of clearing reagent] (1. Clearing tissues including mouse liver and human brain white matter) Tissues including mouse liver and human brain white matter can be cleared, for example, by the following procedure. (1) Mouse liver that has been perfusion-fixed with 4% paraformaldehyde solution or human brain tissue that has been formalin-fixed is immersed in PBS. (2) Immerse in permeabilization solution (Sol. PE: 10 wt% 1,3-bis(aminomethyl)cyclohexane, 10 wt% sodium dodecylbenzenesulfonate or sodium dodecyl sulfate) at 37°C for about 2 to 4 days. (3) Wash with PBS several times, for several hours each time. (4) Immerse in clearing solution (Sol. CL: 22.5 wt% sucrose, 22.5 wt% antipyrine, 10 wt% triethanolamine, 25% urea) at 50°C for about 2 to 4 days. (5) If necessary, wash again with PBS and store.

[0117] The results of actually performing the above procedure are shown in Figure 23.

[0118] (2. Decolorization of Mouse Spleen) Mouse spleen destaining can be carried out, for example, by the following procedure. (1) Spleen tissue that has been perfused and fixed in 4% paraformaldehyde solution is immersed in PBS. (2) Immerse in 10 wt% #0441 (benzethonium chloride), #0484 (1-(3-aminopropyl)imidazole), #0651 (decyldimethyl(3-sulfopropyl)ammonium hydroxide inner salt), #0938 (1-ethylimidazole), or #1283 (N-methylnicotinamide) at 37°C for 2-4 days. As controls, also prepare specimens by immersing in #10 (N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine), CUBIC-1, or PBS at 37°C for 2-4 days. (3) Photograph the decolorized spleen. Quantify the heme dissolved in the supernatant by measuring the absorbance at 420 nm.

[0119] Figure 12 shows the results of actually performing the above procedure.

[0120] (3. Clearing mouse brain tissue) Mouse brain tissue can be made transparent, for example, by the following procedure. (1) Mouse brain tissue that has been perfused and fixed in 4% paraformaldehyde solution is immersed in PBS. The brain is then cut into hemibrains. (2) Immerse in brain delipidation solution (10 wt% N-butyldiethanolamine, 10 wt% Triton X-100) at 37°C for about 2 to 4 days. (3) Wash with PBS several times, for several hours each time. (4) Immerse in clearing solution (Sol. CL: 22.5 wt% sucrose, 22.5 wt% antipyrine, 10 wt% triethanolamine, 25% urea) at 37°C for about one day. (5) If necessary, wash again with PBS and store.

[0121] The results of actually performing the above procedure are shown in Figure 24.

[0122] (4. Adjustment of refractive index of degreased tissue) The refractive index of the degreased tissue can be adjusted, for example, by the following procedure. (1) Mouse brain tissue that has been perfusion-fixed in 4% paraformaldehyde solution is post-fixed in 4% paraformaldehyde solution and then immersed in PBS. Cut into hemibrains. (2) The dissected brain tissue is immersed in CUBIC-1 at 37°C for approximately 3 to 5 days. (3) Immerse in PBS for about a day. (4) Immerse in 70 wt% #0389 (N-benzylethanolamine), 60 wt% #0640 (antipyrine), or 70 wt% #0788 (N,N-dimethylbenzamide) at room temperature for approximately one day.

[0123] The results of actually performing the above procedure are shown in Figure 25.

[0124] (5. Clearing various mouse organs) Various mouse organs (brain, heart, lungs, liver, and kidneys) can be made transparent, for example, by the following procedure. (1) Mouse organs that have been perfusion-fixed in 4% paraformaldehyde solution are removed and post-fixed in 4% paraformaldehyde solution at 4°C overnight. (2) Wash with PBS several times, for several hours each time. (3) Immerse in Permeabilization CUBIC-L solution (Sol. PE CUBIC-L: 10 wt% N-butyldiethanolamine, 10 wt% Triton X-100) at 37°C for 2 to 4 days. (4) Wash with PBS several times, for several hours each time. (5) Immerse overnight at room temperature in Clearing CUBIC-R solution (Sol. CL CUBIC-R: 46 wt% antipyrine, 30 wt% nicotinamide) diluted 1 / 2 with distilled water. (6) Immerse in undiluted solution Sol. CL CUBIC-R for 2 days.

[0125] The actual results of following the above procedure are shown on the left in Figure 26.

[0126] (6. Making the whole mouse body transparent) The whole body of a mouse can be made transparent, for example, by the following procedure. (1) After perfusion fixation with 4% paraformaldehyde solution, perfuse 100 to 200 mL of Sol. PE CUBIC-L throughout the body. (2) Immerse in Sol. PE CUBIC-L at 37°C for 7 days. During this time, change the liquid every two days. (3) Wash with PBS several times, for several hours each time, and then soak in PBS at room temperature overnight. (4) Immerse overnight at room temperature in Sol. CL CUBIC-R diluted 1 / 2 with distilled water. (5) Immerse in the original solution Sol. CL CUBIC-R for 2 days.

[0127] The actual results of following the above procedure are shown in the center of Figure 26.

[0128] (7. Making the mouse spine transparent) The mouse spine can be made transparent, for example, by the following procedure. (1) Mouse vertebrae that have been perfusion-fixed in 4% paraformaldehyde solution are removed and post-fixed in 4% paraformaldehyde solution at 4°C overnight. (2) Wash with PBS several times, for several hours each time. (3) Immerse in Sol. PE CUBIC-L at 37°C for 3 days. (4) Wash with PBS several times, for several hours each time. (5) Immerse in decalcification CUBIC-B solution (Sol. DC CUBIC-B: 10 wt% EDTA, 15 wt% imidazole) at 37°C for 6 days. (6) Wash with PBS several times, for several hours each time. (7) Immerse in Sol. PE CUBIC-L at 37°C for 3 days. (8) Wash with PBS several times, for several hours each time. (9) Immerse overnight at room temperature in Sol. CL CUBIC-R diluted 1 / 2 with distilled water. (10) Immerse in undiluted solution Sol. CL CUBIC-R for 2 days.

[0129] The actual results of following the above procedure are shown on the right side of Figure 26.

[0130] (8. Clearing human tissue) (1) Formalin-fixed human tissue is immersed in PBS, washed several times for several hours each, and then immersed in PBS overnight at room temperature. (2) Immerse in Sol. PE at 37°C for about 5 to 10 days. (3) Wash with PBS several times, for several hours each time, and then soak in PBS at room temperature overnight. (4) Immerse overnight at room temperature in Sol. CL CUBIC-R diluted 1 / 2 with distilled water. (5) Immerse in the original solution Sol. CL CUBIC-R for 2 days.

[0131] The results of actually performing the above procedure are shown in Figure 27.

[0132] (9. Clearing Human Brain Tissue) (1) Formalin-fixed human tissue is immersed in PBS, washed several times for several hours each, and then immersed in PBS overnight at room temperature. (2) Immerse in Sol. PE CUBIC-HBL solution (Sol. PE CUBIC-HBL: 10 wt% N-butyldiethanolamine, 10 wt% Triton X-100, 15 wt% imidazole) at 37°C for about 5 to 10 days. (3) Wash with PBS several times, for several hours each time, and then soak in PBS at room temperature overnight. (4) Immerse overnight at room temperature in Sol. CL CUBIC-R diluted 1 / 2 with distilled water. (5) Immerse in the original solution Sol. CL CUBIC-R for 2 days.

[0133] The results of actually performing the above procedure are shown in Figure 28.

[0134] (10. Making the Marmoset Brain Transparent) (1) Marmoset brains were perfusion-fixed in 4% paraformaldehyde solution, then removed and post-fixed in 4% paraformaldehyde solution at 4°C overnight. (2) Wash with PBS several times, for several hours each time. (3) Immerse in Sol. PE CUBIC-L at 37°C for about 21 days. During this time, change the liquid every two days. (4) Immerse in PBS containing 0.01% sodium azide (hereafter referred to as AzidoPBS) at room temperature for one week. During this time, change the solution every day. (5) Immerse in PBS containing 10 μg / mL propidium iodide and 1.5 M NaCl at 37° C. for 12 days. (6) Immerse in AzidoPBS for 2 days and wash. During this time, change the solution once every half day. (7) Immerse overnight at room temperature in Sol. CL CUBIC-R diluted 1 / 2 with distilled water. (8) Immerse in undiluted solution Sol. CL CUBIC-R for 4 days. (9) Immerse in AzidoPBS and wash for 2 days, changing the solution once every half day. (10) Immerse in a 10 wt% imidazole aqueous solution at room temperature for two days. During this time, change the solution once every half day. (11) Immerse in Sol. CL CUBIC-R containing 1 wt% #0414 diluted 1 / 2 with distilled water (hereinafter, Sol. CL CUBIC-R2) at room temperature for 4 days. (12) Immerse in Sol. CL CUBIC-R2 at room temperature for 7 days, changing the solution once during the period. (13) Immerse in AzidoPBS for 2 days and wash. During this time, change the liquid once every half day. (14) Immerse in Sol. PE CUBIC-L at room temperature for 3 days. Then, soak at 37°C for 2 weeks, and then soak at 45°C for a further week. During this process, change the liquid once every two days. (15) Immerse in AzidoPBS for 2 days and wash. During this time, change the solution once every half day. (16) Immerse in PBS containing 10 μg / mL propidium iodide and 1.5 M NaCl at 37°C for 7 days. (17) Immerse in AzidoPBS for 2 days and wash. During this time, change the solution once every half day. (18) Immerse in 10 wt% imidazole aqueous solution at room temperature for 2 days. During this time, change the solution once every half day. (19) Immerse in Sol. CL CUBIC-R2 diluted 1 / 2 with distilled water at room temperature for 3 days. (20) Immerse in Sol. CL CUBIC-R2 at room temperature for 7 days, changing the solution once during the period.

[0135] The results of actually performing the above procedure are shown in Figure 29.

[0136] [Table 1]

[0137] TIFF0007669054000002.tif235169

[0138] TIFF0007669054000003.tif249169

[0139] TIFF0007669054000004.tif251169

[0140] [Table 2]

[0141] TIFF0007669054000006.tif248169

[0142] TIFF0007669054000007.tif248169

[0143] TIFF0007669054000008.tif245169

[0144] TIFF0007669054000009.tif246169

[0145] TIFF0007669054000010.tif245169

[0146] [Table 3]

[0147] TIFF0007669054000012.tif244169

[0148] TIFF0007669054000013.tif252169

[0149] [Table 4]

[0150] TIFF0007669054000015.tif237169

[0151] TIFF0007669054000016.tif212169 [Industrial Applicability]

[0152] The present invention can be used, for example, for high-throughput monitoring of tissue gene expression, cell localization, and cell morphology at the whole tissue level.

Claims

1. A composition for adjusting the refractive index in the preparation of biological materials with excellent optical transparency, characterized in that the composition is a solution containing N-benzylethanolamine, N-methylnicotinamide, N,N-dimethylbenzamide, or a combination of 2,3-dimethyl-1-phenyl-5-pyrazolone and nicotinamide.

2. A method for adjusting the refractive index in preparing a biological material with excellent optical transparency, comprising a step of infiltrating the biological material with a solution containing N-benzylethanolamine, N-methylnicotinamide, N,N-dimethylbenzamide, or a combination of 2,3-dimethyl-1-phenyl-5-pyrazolone and nicotinamide.

3. A biological material having a controlled refractive index and excellent optical transparency, characterized in that it is infiltrated with a solution containing N-benzylethanolamine, N-methylnicotinamide, N,N-dimethylbenzamide, or a combination of 2,3-dimethyl-1-phenyl-5-pyrazolone and nicotinamide.

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