Compositions and Methods for Tissue Transparency

Abstract

Investigations of fine tissue structures, such as those in non-neural, non-bony tissues or organs, are best carried out in intact tissues. Compositions and methods for rendering tissues transparent for subsequent three-dimensional analysis are described herein. The inventive compositions as tissue clearing compositions consist of four core components: (1) homogenizing agents such as N-methylglucamine, urea, thiourea, guanidine, guanidine chloride, lithium perchlorate, ethylenediamine and its derivatives; (2) water-soluble regulators such as iohexol, sodium thiosulfate, polyethylene glycol and its derivatives; (3) lipid-soluble regulators such as 2,2'-thiodiethanol, propylene glycol and its derivatives; and (4) borate compounds such as boric acid, tetraboric acid, disodium tetraborate and its derivatives. The disclosed tissue clearing compositions are particularly suitable for non-neural, non-bony tissues or organs. These tissues or organs can be fresh, archived or recovered from paraffin-embedded tissues.

Description

Field of the Invention

[0001] The disclosed invention generally relates to the field of tissue clearing, and more particularly to the field of biological tissue analysis for making biological tissues transparent using tissue clearing compositions. Background of the Invention

[0003] Although biological specimens are inherently three-dimensional, the obscuring effect of light scattering hinders high-resolution deep tissue imaging. One way to visualize thick tissue is to serially section it into thin slices and reconstruct a three-dimensional image from these slices using computational methods. However, this method is not only laborious but also has limitations in cases where the true three-dimensional nature of the tissue cannot be determined from thin slices. Investigation of fine tissue structures, such as those in non-neural, non-bony tissues or organs, is best performed in intact tissue.

[0004] To preserve the true three-dimensional structure of tissues, there has been a surge of interest in developing tissue clearing agents and techniques. Tissue clearing techniques directly render tissues transparent, allowing imaging deep within the tissue. By using a microscope capable of imaging selective planes in depth (i.e., optically sectioning the tissue), three-dimensional (3D) images can be rapidly obtained without the cutting artifacts or sample destruction of the serial sectioning method. A good optical clearing method facilitates deep tissue bioimaging by reducing in situ light scattering while preserving tissue integrity for accurate signal reconstruction.

[0005] Tissue clearing techniques often alter the physicochemical properties of tissues. Currently available compositions for tissue clearing can cause significant swelling, resulting in structural deformation. In some cases, due to the limited clearing efficacy of the clearing agents, they are only suitable for very small samples. Existing clearing agents also require a long time to clear tissues and multiple steps of tissue processing before optical clearing. In addition, they are incompatible with archived tissues that have been fixed with formalin for a long time.

[0006] The overall process of tissue clearing can be considered as tissue refractive index (RI) homogenization (i.e., homogenizing or making the refractive indices of tissues more equal). Currently available methods can be classified into: (1) simple water-based RI homogenization, (2) degreasing-assisted RI homogenization, and (3) organic solvent-based RI homogenization (Table 1). The latter two categories each have their advantages, but they cause significant tissue damage and are generally not suitable for high-resolution imaging studies where the most fine structures are to be studied in detail.

[0007] Table 1. Currently available tissue clearing methods

[0008]

[0009] In contrast, although the Class (1) methods cause the least damage, their tissue clearing effect is not as good as the other two methods. This creates a need for improved water-based tissue clearing methods that result in improved tissue transparency and maximum structural retention. Preferably, these methods are applicable to human tissue and are compatible not only with all existing chemical staining methods and electron microscopy, but also with future diagnostic and research uses.

[0010] In biomedical research, there is an increasing need for 3D imaging and analysis. 3D imaging helps to understand the biological structure and function of organs during development and pathogenesis. Clearing of stained tissue results in improved image quality.

[0011] One object of the present invention is to provide a tissue clearing composition having improved tissue clearing ability, particularly for non-neural, non-bone tissues or organs.

[0012] A further object of the present invention is to provide a kit for clearing tissue.

[0013] A further object of the present invention is to provide improved methods for clearing tissue, particularly methods for clearing human tissue. SUMMARY OF THE INVENTION

[0015] Compositions and methods for clearing tissue are disclosed herein and are useful for, e.g., subsequent 3D analysis. In some forms, the disclosed tissue clearing compositions are composed of four core components:

[0016] (1) Homogenizing agents (such as N-methylglucamine, urea, thiourea, guanidine, guanidine chloride, lithium perchlorate, ethylenediamine, and their derivatives);

[0017] (2) Water-soluble RI regulators for the cytoplasm (such as iohexol, sodium thiosulfate, polyethylene glycol, and their derivatives);

[0018] (3) Lipid-soluble RI regulators for the membrane (such as 2,2'-thiodiethanol (TDE), propylene glycol, and their derivatives); and

[0019] (4) Borate compounds in the form of hydrogen or metal borates (such as boric acid, tetraboric acid, disodium tetraborate, and their derivatives).

[0020] In a preferred form, the disclosed tissue clearing compositions do not contain strong detergents or strong denaturants, which allows for the retention of lipid membranes for lipophilic tracing and subsequent imaging. In some forms, the disclosed methods may involve a single-step incubation of the tissue in the disclosed tissue clearing composition.

[0021] Different forms of tissue clearing compositions are particularly suitable for different tissue and source types, such as compositions specifically for non-neural, non-bone tissues or organs, non-neural, non-bone pathological tissues or organs, or non-neural, non-bone human tissues or organs. Some specific tissue clearing compositions are particularly useful for tissues recovered from archival sources (such as those archived for up to about 50 years) and recently fixed tissues (such as those fixed within about 3 weeks to about 3 months, which is typical for human tissues).

[0022] In some forms, the disclosed tissue clearing compositions can contain N-methylglucamine as a homogenizing agent, iohexol as a water-soluble RI regulator, 2,2'-thiodiethanol as a lipid-soluble RI regulator, and boric acid as a borate compound. In some forms, the concentrations of N-methylglucamine, iohexol, and 2,2'-thiodiethanol each range from about 10 to about 50 w / v%, and the molar ratio of N-methylglucamine to boric acid is between about 0.5 and about 2. In some forms, the tissue clearing composition can comprise about 20% w / v of N-methylglucamine, about 32% w / v of iohexol, about 25% w / v of 2,2'-thiodiethanol, and boric acid with a molar ratio of about 1 to N-methylglucamine.

[0023] Preferably, the tissue clearing composition has improved tissue clearing ability for non-neural, non-bone tissues and organs relative to the corresponding composition without a borate compound, relative to the corresponding composition in which the borate compound is replaced with an organic or inorganic acid, or relative to both.

[0024] Preferably, the tissue clearing composition has improved tissue clearing ability for non-neural, non-bone tissues or organs relative to neural tissues or organs (such as the brain).

[0025] Since it can be applicable to any specific application, the concentrations of the four core components of the tissue clearing composition can vary. In some forms, the composition is suitable for solid general applications. In some forms, the composition is suitable for fresh tissues. In some forms, the composition is suitable for long-term fixed tissues. In some forms, the composition is suitable for in vivo clearing applications.

[0026] The disclosed tissue clearing compositions can contain additional components, which for example enable the composition to be used or adjusted for the specific tissue and source types to be applied. In some forms, the disclosed tissue clearing compositions are compatible with further processing methods such as those used in histology and electron microscopy studies, other tissue clearing methods, different tissue staining methods (such as immunohistochemistry, chemical staining, transgenic cell labeling methods, imaging probes, in situ tissue chemistry, and virus tracing methods), or combinations thereof.

[0027] Additional advantages of the disclosed methods and compositions will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the subject matter claimed.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the description, serve to explain the principles of the disclosed methods and compositions.

[0030] Figure 1 An exemplary protocol for processing formalin-fixed, paraffin-embedded kidney or tumor tissue is illustrated, where the disclosed tissue clearing composition, OPTIClear B (20 w / v% N-methylglucamine, 25 w / v% 2,2'-thiodiethanol, 32 w / v% iohexol, and 6.335% w / v boric acid) is used as an example. DETAILED DESCRIPTION OF THE INVENTION

[0032] Compositions and methods for tissue clearing are disclosed herein that are useful for, e.g., subsequent 3D analysis. Investigation of fine tissue architecture, such as that in non-neural, non-bone tissues or organs, is best performed in intact tissue. The disclosed compositions and methods eliminate the need for tissue sectioning, making the process faster (e.g., 12 - 15 times faster) than conventional histological studies. The transparency obtained by using the disclosed compositions and methods can improve the ability to observe cellular structures and signal detection sensitivity, where cellular structures include, e.g., fluorescent and non-fluorescent cellular structures. The disclosed compositions and methods also allow 3D observation of tissues in any orientation (whole or virtual sections) and avoid other problems associated with conventional 3D imaging techniques, such as slide loss in conventional histology.

[0033] It has been found that by adjusting the RI of different parts of cells and tissues (such as the aqueous part, the lipid / hydrophobic part, the protein part, the cytoplasm, and the nucleus) to match (be the same or similar), relative transparency or translucency of the whole or entire tissue can be obtained. It has been found that by using at least one reagent that modulates the RI of the aqueous part of the tissue and at least one reagent that modulates the RI of the lipid / hydrophobic part of the tissue, the RI of these different tissue parts can be made the same or their values can be made closer. Such adjustment of the RI of different tissue parts results in less refractive distortion, thereby increasing transparency or translucency. Generally, the modulators are selected to partition or divide the tissue components to be modulated. This can typically be achieved by using, for example, relatively hydrophilic reagents for modulating the RI of the aqueous part of the tissue and relatively hydrophobic reagents for modulating the RI of the lipid / hydrophobic part of the tissue. Preferably, the modulators are selected to adjust the RI of their target tissue parts to the RI of other tissue parts.

[0034] It has also been found that since the modulators for modulating the RI of tissue parts cannot effectively physically approach some biological macromolecules in the tissue (such as some undenatured proteins), and since such unmodulated undenatured proteins can affect the RI of the tissue part in which they are located, for some tissues, it is useful to use homogenizing agents that make some or all of the problematic biological macromolecules more accessible to the modulators. Such homogenizing agents allow for a more complete adjustment or matching of the RI of different tissue parts.

[0035] It has further been found that the addition of a borate compound such as boric acid that can react with one or more components of the tissue clearing composition can improve tissue transparency, as compared to the corresponding tissue clearing composition without the borate compound, as compared to the corresponding tissue clearing composition in which the borate compound is replaced with an organic or inorganic acid, or as compared to both.

[0036] Based on the fundamental findings discussed above, it is recognized that since different tissues contain different components, the refractive indices of tissue parts in different tissues can be different. Therefore, it is recognized that the results of the disclosed compositions and methods can be improved by selecting reagents, their concentrations / formulations, or combinations thereof that can adjust the RI of the target tissue parts to the correct degree based on the specific properties of the target tissue components. This feature of selecting reagents and the concentrations / formulations of the reagents to adjust or match to a given target tissue can generally be simplified by noting the RI of a given part of the target tissue and selecting reagents and reagent concentrations / formulations to adjust the RI of different parts of the tissue to the same or similar RI values. In this way, the present discovery adjusts or matches the formulation of the tissue clearing composition to a variety of different tissues by following the clear principles discovered and developed.

[0037] In some forms, the disclosed tissue clearing compositions are characterized by low viscosity, low osmotic pressure, low chemical concentration, or combinations thereof. These properties translate into easier handling, faster tissue clearing times, single-step methods, better tissue retention, lower production costs, or combinations thereof.

[0038] In preferred forms, the disclosed tissue clearing compositions are free of strong detergents or strong denaturants, thereby allowing retention of lipid membranes for lipophilic tracing and subsequent imaging. In some forms, the disclosed tissue clearing compositions are free of detergents or denaturants. In some forms, the disclosed methods can involve a single-step incubation of tissue in the disclosed tissue clearing compositions.

[0039] In some forms, the disclosed tissue clearing compositions exhibit improved clearing ability in non-neural, non-bone tissues or organs, which is difficult to achieve with other methods and other compositions, optionally allowing visualization of structures at as low as 300 μm in about 3 hours. In some forms, the disclosed tissue clearing compositions can be used to clear archived and / or formalin-fixed, paraffin-embedded (FFPE) tissues. In some forms, the disclosed tissue clearing compositions can be used to clear biopsy tissues from a clinical setting to facilitate pathological diagnosis. In some forms, long-term storage after tissue clearing is feasible.

[0040] I. Definitions

[0041] As used herein, the term "tissue clearing" refers to a process that has the effect of adjusting, matching, or homogenizing the refractive index (RI) of a tissue, thereby generally resulting in increased tissue transparency. The transparency of a tissue can be quantitatively determined by light absorption spectrophotometry, such as by measuring light transmission through the tissue or by confocal microscopy.

[0042] As used herein, the term "homogenization" refers to the act of making a composition (such as a solution, tissue, or tissue part) uniform throughout by mixing unlike elements or characteristics. For example, in the context of tissue RI, homogenization results in a more uniform or matched RI throughout the tissue.

[0043] As used herein in the context of tissue RI, the term "adjustment" refers to the act of making the RI of different tissue parts more uniform throughout. For example, in the context of tissue RI, adjustment results in a more uniform or matched RI throughout the tissue. In the context of a reagent used to adjust tissue RI, adjustment refers to the selection of the reagent and the ratio of the reagent to achieve the adjustment of tissue RI.

[0044] As used herein in the context of tissue RI, the term "matching" refers to the act of making the RIs of different tissue parts more uniform with each other. For example, in the context of tissue RI, matching results in a more uniform or matched RI throughout the tissue. In the context of a reagent used to match tissue RI, matching refers to the selection of the reagent and the ratio of the reagent to achieve the matching of tissue RI.

[0045] As used herein, the term "homogenizing agent" refers to a compound or composition that increases the homogeneity of a mixture that is difficult to mix, such as tissue.

[0046] As used herein, the terms "water-soluble regulator" and "water-soluble RI regulator" refer to a compound or composition that can selectively regulate the RI of the aqueous compartments of tissue, such as cytoplasm, cytosol, extracellular compartment, tissue fluid, blood, plasma, and lymph.

[0047] As used herein, the term "water-soluble" with respect to a component refers to the ability of the component to dissolve in water.

[0048] As used herein, the terms "lipid-soluble regulator" and "lipid-soluble RI regulator" refer to a compound or composition that can selectively regulate the RI of the lipid-rich, membrane, or fat compartments of tissue.

[0049] As used herein, the term "lipid-soluble" with respect to a component refers to the ability of the component to dissolve in fat, oil, lipid, and nonpolar solvents.

[0050] As used herein, the term "refractive index regulator" or "RI regulator" refers to a compound or composition that selectively regulates the RI of the lipid-rich or aqueous compartments of tissue.

[0051] As used herein, the term "refractive index" or "RI" refers to the ratio of the speed of radiation, such as electromagnetic radiation or light, in one medium, such as air, glass, or vacuum, to the speed in another medium.

[0052] As used herein, the term "archived tissue" refers to tissue that has been retained for short-term or long-term storage. The tissue can be retained by heat fixation, immersion in a fixing solution, perfusion, freezing, formalin fixation and paraffin embedding, or any other chemical or other available method.

[0053] The term "denaturant" refers to a reagent that can cause the denaturation of biological macromolecules such as proteins and / or nucleic acids. Denaturation is the process by which a protein or nucleic acid loses its quaternary, tertiary, and / or secondary structure that exists in its native state. Denatured proteins can exhibit a wide range of characteristics, from conformational changes and solubility loss to aggregation due to the exposure of hydrophobic groups. In some forms, denaturants can include chaotropic agents such as urea, guanidine chloride, guanidine, and lithium perchlorate.

[0054] The term "chaotropic agent" refers to a molecule in an aqueous solution that can disrupt the hydrogen bond network between water molecules, i.e., exhibit chaotropic activity. It affects the stability of the native state of other molecules in the solution, mainly macromolecules (such as proteins and nucleic acids), by weakening the hydrophobic interaction. For example, a chaotropic agent can reduce the amount of order in the protein structure formed by water molecules, including the hydration layers around bulk and hydrophobic amino acids, and can cause its denaturation.

[0055] In some forms, chaotropic agents can disrupt the structure of macromolecules such as proteins and nucleic acids (e.g., DNA and RNA) and cause their denaturation. Chaotropic agents increase the entropy of the system by interfering with intermolecular interactions mediated by non-covalent forces such as hydrogen bonds, van der Waals forces, and hydrophobic interactions. The structure and function of macromolecules depend on the net effect of these forces, so an increase in chaotropic agents in a biological system will denature the macromolecules. Tertiary protein folding depends on the hydrophobic forces of amino acids throughout the protein sequence. Due to the disorder of water molecules adjacent to the protein, chaotropic agents can reduce the net hydrophobic interaction in the hydrophobic region. This dissolves the hydrophobic region in the solution, thus denaturing the protein. This also directly applies to the hydrophobic region in the lipid bilayer; if the critical concentration of the chaotropic agent (in the hydrophobic region of the bilayer) is reached, membrane integrity may be compromised and the cell will lyse.

[0056] Chaotropic salts that dissociate in solution exert chaotropic effects via different mechanisms. Chaotropic compounds such as ethanol interfere with the above-mentioned non-covalent intramolecular forces, and chaotropic salts can have chaotropic properties by shielding charges and preventing the stabilization of salt bridges. Hydrogen bonds are stronger in non-polar media, so salts that increase the chemical polarity of the solvent will also destabilize hydrogen bonds. Mechanistically, this is because there are not enough water molecules to effectively solvate the ions. This leads to an ion-dipole interaction between the salt and the hydrogen bond species, which is more favorable than the normal hydrogen bond. Exemplary chaotropic agents include n-butanol, ethanol, guanidine chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea.

[0057] The term "miscible" means forming a homogeneous mixture when mixed together. In some forms, the term refers to the ability to mix in any proportion without phase separation.

[0058] The term "solid organ" refers to an internal organ that has a fixed tissue coherence and is neither hollow (such as gastrointestinal organs) nor liquid (such as blood). Exemplary solid organs include the heart, kidney, liver, lung, and pancreas.

[0059] The term "derivative" refers to a compound / portion that has a structure similar to that of a parent compound / portion but is different from it in one or more components, functional groups, atoms, etc. Derivatives can be formed from the parent compound / portion by chemical reactions. The differences between a derivative and a parent compound / portion can include, but are not limited to, replacing one or more functional groups with one or more different functional groups or introducing or removing substituents of one or more hydrogen atoms. In some forms, a derivative can also differ from the parent compound / portion in terms of its protonation state. In some forms, a derivative can be derived from the parent compound / portion via an acid-base reaction. Preferably, the derivative retains the biological activity of the parent compound / portion, such as at least 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, and 60% of the biological activity of the parent compound / portion. In some forms, the derivative has higher activity compared to the parent compound / portion.

[0060] The term "organic acid" refers to an organic compound that has acidic properties. The most common organic acids are carboxylic acids, and their acidity is related to their carboxyl group -COOH.

[0061] II. Compositions

[0062] Compositions are provided herein for making tissues transparent for, for example, subsequent 3D analysis. The disclosed compounds can include additional components, for example, to make the compositions useful for or adapted for a particular tissue and source type to be applied.

[0063] The physical basis of opacity lies in the bending of light when passing through the boundary between two media with different RIs, resulting in a perceived boundary. Adjusting the RI of one or both media to make the two RIs close to or equal to each other can eliminate the bending of light and avoid the perceived boundary.

[0064] In tissues, tissue compartments have different properties and thus different RIs. This is the origin of tissue opacity. For example, compared to the RI of the extracellular space (~1.37), lipid membranes / compartments in tissues generally have a higher RI (~1.45); due to refraction, the interface between them causes light bending and scattering. By selecting chemical substances with specific optical properties that preferentially dissolve in specific compartments, the refractive indices of these compartments can be adjusted accordingly so that they match each other, resulting in optical homogeneity despite physical heterogeneity - the structure does not change, but they appear the same optically, that is, transparency.

[0065] Thus, the use of a lipid-soluble modifier plus a water-soluble RI modifier can result in optical homogenization of two major physical compartments within the tissue. In some forms, the water-soluble modifier may not be physically close enough to the protein, resulting in some physical heterogeneity that hinders optical homogenization. This explains why all current tissue clearing formulations use denaturants to achieve better clearing effects (e.g., urea in ScaleA2 and ScaleS; formamide in Clear T ; SDS in CLARITY TM ). However, it is reasonable to assume that if physical homogeneity is sufficient to achieve optical homogeneity, denaturation may not be necessary. Non-denaturation or partially controlled denaturation of proteins will help avoid side effects such as tissue and antigen damage, tissue swelling, and incompatibility with lipophilic tracers that typically involve denaturation.

[0066] In most forms, the disclosed tissue clearing compositions utilize the optical properties of various chemicals to reduce light loss through the tissue sample, thereby improving light recovery efficiency. Further, the disclosed tissue clearing compositions utilize homogenizing agents to improve the physical homogeneity of the tissue sample. The disclosed tissue clearing compositions also utilize borate compounds to enhance the tissue clearing efficacy of the tissue clearing compositions, especially for non-neural, non-bone tissues or organs.

[0067] In some forms, the disclosed tissue clearing compositions consist of four core components: (1) a homogenizing agent, (2) a water-soluble modifier, (3) a lipid-soluble modifier, and (4) a borate compound. In some forms, the water-soluble modifier is a water-soluble RI modifier. In some forms, the lipid-soluble modifier is a lipid-soluble RI modifier.

[0068] In some forms, the tissue clearing composition has a refractive index of about 1.4 to about 1.5 at 25 °C, such as about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, and about 1.50.

[0069] In some forms, the tissue clearing composition has improved tissue clearing ability for non-neural, non-bone tissues or organs relative to the corresponding composition without the borate compound, relative to the corresponding composition with an organic or inorganic acid replacing the borate compound, or relative to both. Such comparisons can be made by comparing the opacity of two parallel groups of tissue samples: one group treated with the tissue clearing composition and the other group treated with the corresponding composition without the borate compound or with an organic or inorganic acid replacing the borate compound. The opacity of the samples can be measured by light transmission.

[0070] In some forms, the tissue clearing composition has a reduced ability to clear neural tissue or organs relative to the corresponding composition without the borate compound, relative to the corresponding composition with an organic acid or an inorganic acid replacing the borate compound, or relative to both. Such comparisons can be made by comparing the opacities of two parallel groups of tissue samples: one group treated with the disclosed tissue clearing composition and the other group treated with the corresponding composition without the borate compound or with an organic acid or an inorganic acid replacing the borate compound. The opacity of the samples can be measured by light transmission.

[0071] In some forms, the tissue clearing composition exhibits improved tissue clearing ability for non-neural, non-bony tissues or organs relative to neural tissue or organs. Such comparisons can be made by comparing the opacity of the treated non-neural, non-bony tissues or organs with the opacity of the treated neural tissue or organs. For example, a comparison can be made between a treated kidney sample and a treated brain sample. The opacity of the samples can be measured by light transmission.

[0072] In some forms, the non-neural, non-bony tissues or organs are non-neural, non-bony solid organs such as the heart, kidney, liver, lung, and pancreas. In some forms, the solid organ is the kidney. In some forms, the neural tissue or organ is the brain.

[0073] In some forms, the non-neural, non-bony tissues or organs are non-neural, non-bony pathological tissues or organs such as tumor tissue. In some forms, the organic acid can be acetic acid, succinic acid, maleic acid, malic acid, glutamic acid, aspartic acid, lactic acid, formic acid, citric acid, oxalic acid, uric acid, or derivatives thereof. In some forms, the inorganic acid can be hydrochloric acid, sulfuric acid, nitric acid, or phosphoric acid.

[0074] A. Homogenizing agent

[0075] Homogenization is any of several processes used to make a mixture of two immiscible liquids the same or similar throughout. This is typically achieved by transforming one liquid into a state consisting of extremely small particles uniformly distributed throughout the other liquid. A homogenizing agent is a product that improves the homogeneity of a mixture that is difficult to mix. This promotes true homogenization of water-soluble and water-insoluble reagents with tissue components to achieve better optical homogeneity.

[0076] In some forms, the homogenizing agent is a denaturing agent for proteins, nucleic acids, or combinations thereof. Denaturation of these biopolymers, especially proteins, can promote physical homogenization because the higher-order structures of these biopolymers are disrupted. This is partly due to the fact that RI regulators, especially water-soluble RI regulators, can closely approach the biopolymers after denaturation, including regions that were inaccessible to the solvent before denaturation, thereby achieving optical homogenization at the molecular scale and resulting in more effective tissue clearing.

[0077] In a preferred form, the homogenizing agent is not a strong denaturant of proteins, nucleic acids, or combinations thereof that can cause aggregation and precipitation of denatured biological macromolecules.

[0078] In some forms, the denaturant is a chaotropic agent. Using a chaotropic agent as a denaturant has the advantage of avoiding aggregation of denatured biological macromolecules, especially proteins, because chaotropic agents can reduce the net hydrophobic effect of the hydrophobic regions of biological macromolecules exposed to the solvent. This helps dissolve the hydrophobic regions of denatured biological macromolecules.

[0079] Preferably, the concentration of the chaotropic agent is below the critical concentration of the lipid bilayer such that it cannot dissolve the hydrophobic region of the lipid bilayer or disrupt the membrane integrity of the cell.

[0080] Exemplary homogenizing agents include, but are not limited to, N-methylglucamine, urea, thiourea, guanidine, guanidine hydrochloride, lithium perchlorate, ethylenediamine, triethanolamine, triethylamine, tetraethylammonium, and derivatives thereof. However, as will be understood by those skilled in the art, there are many other reagents or methods known to those skilled in the art that can be used to homogenize a mixture. Such reagents and methods can be used in conjunction with the disclosed compositions and methods.

[0081] In some forms of the tissue clearing composition, N-methylglucamine can be used as a homogenizing agent. In other forms, urea can be used as a homogenizing agent. In certain forms, ethylenediamine can be used as a homogenizing agent.

[0082] The final concentration of the homogenizing agent in the tissue clearing composition can vary. However, higher concentrations of the homogenizing agent may compromise tissue integrity or fluorescence protein signal intensity. In some forms, the concentration range of urea in the tissue clearing composition can be from about 5 to about 60 w / v% or from about 10 to about 50 w / v%, such as from about 10 to about 24 w / v%. Preferably, the concentration of urea is about 10 w / v%. In some forms, the concentration range of N-methylglucamine in the tissue clearing composition can be from about 10 to about 50 w / t%. Preferably, the concentration of N-methylglucamine is about 20 w / t%.

[0083] B. Modulator

[0084] In some forms, the modulator is a refractive index modulator.

[0085] The RI difference between two opposing media causes the light path to bend and can be eliminated by adjusting the RI of each medium to match each other. In the tissue context, modulating the RI of different cellular compartments (such as the nucleus, cytoplasm, and membrane) by selectively dissolving chemicals in the cellular compartments can result in a minimally destructive and highly efficient tissue clearing effect.

[0086] In some forms, the water-soluble modifier, the lipid-soluble modifier, or both have an RI higher than that of water at 25 °C.

[0087] In some forms, the water-soluble modifier, the lipid-soluble modifier, or both have an RI of from about 1.40 to about 1.50 at 25 °C, such as, for example, about 1.40, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, and 1.50.

[0088] In some forms, at 25 °C, the RI of the water-soluble modifier is about 10%, about 8%, about 5%, or about 2% or within that of the RI of the lipid-soluble modifier.

[0089] The water-soluble RI modifier selectively modulates the RI of the aqueous compartments of tissues, such as the RI of cytoplasm, cytosol, extracellular compartments, interstitial fluid, blood, plasma, and lymph. Water-soluble RI modifiers suitable for the disclosed tissue clearing compositions include, but are not necessarily limited to, reagents such as iohexol, sodium thiosulfate, polyethylene glycol, and its derivatives. In other forms, the water-soluble modifier may include metrizamide, iodixanol, sodium diatrizoate, sodium iodide, and their derivatives. In some forms, the concentration of the water-soluble modifier is between about 5 and about 60 w / v% or between about 10 and about 50 w / v%.

[0090] The lipid-soluble RI modifier selectively modulates the RI of the lipid-rich, membrane, or fatty compartments of tissues.

[0091] In some forms, the lipid-soluble RI modifier may be miscible with water. Lipid-soluble RI modifiers suitable for the disclosed tissue clearing compositions include, but are not necessarily limited to, reagents such as 2,2'-thiodiethanol (TDE), propylene glycol, and their derivatives. In other forms, the lipid-soluble RI modifier may include glycerol, ethylene glycol, sodium dodecyl sulfate, trimethylamine, triethanolamine, triethanolamine-boric acid (1:1) adduct, and their derivatives. In some forms, the concentration of the lipid-soluble modifier is between about 5 and about 70 w / v% or between about 10 and about 50 w / v%.

[0092] Various assays can be used to determine the suitability of a particular RI modifier. In some forms, the assay involves incubating tissue homogenates in various concentrations of the modifier in the presence of a homogenizing agent, such as N-methylglucamine or urea. Preferably, the assay includes both water-soluble and lipid-soluble modifiers to obtain a desired reduction in homogenate opacity. Homogenate opacity can be measured using a spectrophotometer in the ultraviolet, visible, and near-infrared and far-infrared light ranges.

[0093] C. Borate Compounds

[0094] The tissue clearing composition further includes a borate compound. In some forms, the borate compound is a hydrogen or metal borate in anhydrous or hydrated form. Exemplary hydrogen borates include boric acid (H 3 BO 3 ), metaboric acid (H 3 B 3 O 6 ), and tetraboric acid (H 2 B 4 O 7 ). Exemplary metal borates contain a borate oxyanion selected from one of the following: metaborate (e.g., BO 2 - ), diborate (e.g., B 2 O 5 4- ), triborate (e.g., B 3 O 7 5- ), tetraborate (e.g., B 4 O 7 2- , B 4 O 5 (OH) 4 2- , B 4 O 9 6- and combinations thereof) and hydroxyborate (e.g., B(OH) 4 - ). In some forms, the borate compound is boric acid or tetraboric acid. In some forms, the borate compound is disodium tetraborate, such as anhydrous disodium tetraborate (i.e., Na 2 B 4 O 7 ), disodium tetraborate pentahydrate (i.e., Na 2 B 4 O 7 ·5H 2 O), disodium tetraborate decahydrate (i.e., Na 2 B 4 O 7 ·10H 2 O), disodium octaborate tetrahydrate (i.e., Na 2 B 4 O 5 (OH) 4 ·8H 2 O) and combinations thereof.

[0095] In some forms, the borate compound can generate the tetrahydroxyborate anion B(OH) 4 -, which can form covalent or non-covalent conjugates with other components of the tissue clearing agent, preferably the homogenizing agent. Covalent conjugates can be produced by dehydration reactions, resulting in borate esters. Exemplary covalent conjugates include borate esters. Non-covalent conjugates can be produced via hydrogen bond interactions. Further, the tetrahydroxyborate anion can also form covalent and non-covalent bonds with reactive chemical groups from proteins, cells, tissues, or combinations thereof from a tissue sample upon physical contact.

[0096] The following equations illustrate examples of covalent and non-covalent bonds formed between the tetrahydroxyborate anion and hydroxyl groups from other chemical entities.

[0097]

[0098] The use of borate compounds in the composition can modulate the ionic strength of the medium in which the composition is dissolved or suspended. Additionally, borates are weak bases, so borate solutions can be used as alkaline buffer solutions. The alkaline pH can facilitate the coupling reaction between the borate compound and reactive amines from the homogenizing agent and / or proteins, cells, or tissues from the tissue sample.

[0099] Boric acid can react with N-methylglucosamine to form a covalent cyclic adduct as shown in the following equation. This adduct can be converted to a zwitterion after protonation of the amine group. The zwitterion provides strong hydration through electrostatic interactions with water molecules.

[0100]

[0101] In some forms, the molar ratio of the homogenizing agent to the borate compound is between about 0.5 and about 2. Preferably, the molar ratio of the homogenizing agent to the borate compound is about 1.

[0102] D. Excipients

[0103] The compositions disclosed herein can additionally contain one or more excipients, as used herein, which include any and all solvents, dispersion media, diluents, or other liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc., applicable to a particular composition, and combinations thereof. Unless any conventional excipient medium is incompatible with the substance or its derivatives, such as by producing any undesired biological effects or interacting in a detrimental manner with any other component of the pharmaceutical composition, its use is considered to be within the scope of this disclosure.

[0104] In some forms, the liquid carrier comprises an aqueous medium selected from water, acid solutions, and buffer solutions. Suitable acid solutions include hydrochloric acid, nitric acid, phosphoric acid, and sulfuric acid. Suitable buffer solutions include phosphate buffered saline (pH 6.8 - 7.6) and TrisHCl buffer (pH 6.8 - 7.6). The aqueous medium may contain sodium azide, such as 0.01 - 0.05 w / v%.

[0105] In some forms, the pH of the tissue clearing composition ranges from about 5 to about 9, about 5.5 to about 8.5, about 6 to about 8, or about 7 to about 10. In some forms, the pH of the composition can be adjusted to the desired range or value by titrating one or more acid solutions. In some forms, no pH adjustment is required.

[0106] In some forms, the composition contains one or more osmotic agents, such as sodium chloride (e.g., 142 - 148 mM), potassium chloride (e.g., 3 - 7 mM), sodium lactate (e.g., 28 - 32 mM), calcium chloride (e.g., 1.2 - 2.4 mM), and glucose (e.g., 5 - 7 mM).

[0107] E. Specific Composition

[0108] In some forms, the homogenizing agent may account for about 5 to about 60%, about 5 to about 59%, about 5 to about 58%, about 5 to about 57%, about 5 to about 56%, about 5 to about 55%, about 5 to about 54%, about 5 to about 53%, about 5 to about 52%, about 5 to about 51%, about 5 to about 50%, about 5 to about 49%, about 5 to about 48%, about 5 to about 47%, about 5 to about 46%, about 5 to about 45%, about 5 to about 44%, about 5 to about 43%, about 5 to about 42%, about 5 to about 41%, about 5 to about 40%, about 5 to about 39%, about 5 to about 38%, about 5 to about 37%, about 5 to about 36%, about 5 to about 35%, about 5 to about 34%, about 5 to about 33%, about 5 to about 32%, about 5 to about 31%, about 5 to about 30%, about 5 to about 29%, about 5 to about 28%, about 5 to about 27%, about 5 to about 26%, about 5 to about 25%, about 5 to about 24%, about 5 to about 23%, about 5 to about 22%, about 5 to about 21%, about 5 to about 20%, about 5 to about 19%, about 5 to about 18%, about 5 to about 17%, about 5 to about 16%, about 5 to about 15%, about 5 to about 14%, about 5 to about 13%, about 5 to about 12%, about 5 to about 11%, about 5 to about 10%, about 5 to about 9%, about 5 to about 8%, about 5 to about 7%, about 5 to about 6%, about 6 to about 50%, about 7 to about 50%, about 8 to about 50%, about 9 to about 50%, about 10 to about 50%, about 11 to about 50%, about 12 to about 50%, about 13 to about 50%, about 14 to about 50%, about 15 to about 50%, about 16 to about 50%, about 17 to about 50%, about 18 to about 50%, about 19 to about 50%, about 20 to about 50%, about 21 to about 50%, about 22 to about 50%, about 23 to about 50%, about 24 to about 50%, about 25 to about 50%, about 26 to about 50%, about 27 to about 50%, about 28 to about 50%, about 29 to about 50%, about 30 to about 50%, about 31 to about 50%, about 32 to about 50%, about 33 to about 50%, about 34 to about 50%, about 35 to about 50%, about 36 to about 50%, about 37 to about 50%, about 38 to about 50%, about 39 to about 50%, about 40 to about 50%, about 41 to about 50%, about 42 to about 50%, about 43 to about 50%, about 44 to about 50%, about 45 to about 50%, about 46 to about 50%, about 47 to about 50%, about 48 to about 50%, about 49 to about 50%, about 6 to about 48%, about 6 to about 46%, about 7 to about 46%, about 7 to about 44%, about 8 to about 44%, about 8 to about 42%, about 9 to about 42%, about 9 to about 40%, about 10 to about 40%, about 10 to about 38%from about 11 to about 38%, from about 11 to about 36%, from about 12 to about 36%, from about 12 to about 34%, from about 13 to about 34%, from about 13 to about 32%, from about 14 to about 32%, from about 14 to about 30%, from about 15 to about 30%, from about 15 to about 28%, from about 16 to about 28%, from about 16 to about 26%, from about 17 to about 26%, from about 17 to about 24%, from about 18 to about 24%, from about 18 to about 22%, from about 19 to about 22%, from about 19 to about 20%, from about 10 to about 20%, from about 10 to about 19%, from about 11 to about 19%, from about 11 to about 20%, about 18%, from about 12 to about 18%, from about 12 to about 17%, from about 13 to about 17%, from about 13 to about 16%, from about 14 to about 16%, from about 14 to about 15%, from about 15 to about 25%, from about 15 to about 24%, from about 16 to about 24%, from about 16 to about 23%, from about 17 to about 23%, from about 17 to about 22%, from about 18 to about 22%, from about 18 to about 21%, from about 19 to about 21%, from about 19% to about 20%, about 60%, about 59%, about 58%, about 57%, about 56%, about 55%, about 54%, about 53%, about 52%, about 51%, about 50%, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6% or about 5%. In some forms, the foregoing percentage values refer to w / v%.

[0109] In some forms, the water-soluble modifier may account for about 5 to about 60%, about 5 to about 59%, about 5 to about 58%, about 5 to about 57%, about 5 to about 56%, about 5 to about 55%, about 5 to about 54%, about 5 to about 53%, about 5 to about 52%, about 5 to about 51%, about 5 to about 50%, about 5 to about 49%, about 5 to about 48%, about 5 to about 47%, about 5 to about 46%, about 5 to about 45%, about 5 to about 44%, about 5 to about 43%, about 5 to about 42%, about 5 to about 41%, about 5 to about 40%, about 5 to about 39%, about 5 to about 38%, about 5 to about 37%, about 5 to about 36%, about 5 to about 35%, about 5 to about 34%, about 5 to about 33%, about 5 to about 32%, about 5 to about 31%, about 5 to about 30%, about 5 to about 29%, about 5 to about 28%, about 5 to about 27%, about 5 to about 26%, about 5 to about 25%, about 5 to about 24%, about 5 to about 23%, about 5 to about 22%, about 5 to about 21%, about 5 to about 20%, about 5 to about 19%, about 5 to about 18%, about 5 to about 17%, about 5 to about 16%, about 5 to about 15%, about 5 to about 14%, about 5 to about 13%, about 5 to about 12%, about 5 to about 11%, about 5 to about 10%, about 5 to about 9%, about 5 to about 8%, about 5 to about 7%, about 5 to about 6%, about 6 to about 50%, about 7 to about 50%, about 8 to about 50%, about 9 to about 50%, about 10 to about 50%, about 11 to about 50%, about 12 to about 50%, about 13 to about 50%, about 14 to about 50%, about 15 to about 50%, about 16 to about 50%, about 17 to about 50%, about 18 to about 50%, about 19 to about 50%, about 20 to about 50%, about 21 to about 50%, about 22 to about 50%, about 23 to about 50%, about 24 to about 50%, about 25 to about 50%, about 26 to about 50%, about 27 to about 50%, about 28 to about 50%, about 29 to about 50%, about 30 to about 50%, about 31 to about 50%, about 32 to about 50%, about 33 to about 50%, about 34 to about 50%, about 35 to about 50%, about 36 to about 50%, about 37 to about 50%, about 38 to about 50%, about 39 to about 50%, about 40 to about 50%, about 41 to about 50%, about 42 to about 50%, about 43 to about 50%, about 44 to about 50%, about 45 to about 50%, about 46 to about 50%, about 47 to about 50%, about 48 to about 50%, about 49 to about 50%, about 6 to about 48%, about 6 to about 46%, about 7 to about 46%, about 7 to about 44%, about 8 to about 44%, about 8 to about 42%, about 9 to about 42%, about 9 to about 40%, about 10 to about 40%, about 10 to about 38%From about 11 to about 38%, from about 11 to about 36%, from about 12 to about 36%, from about 12 to about 34%, from about 13 to about 34%, from about 13 to about 32%, from about 14 to about 32%, from about 14 to about 30%, from about 15 to about 30%, from about 15 to about 28%, from about 16 to about 28%, from about 16 to about 26%, from about 17 to about 26%, from about 17 to about 24%, from about 18 to about 24%, from about 18 to about 22%, from about 19 to about 22%, from about 19 to about 20%, from about 10 to about 20%, from about 10 to about 19%, from about 11 to about 19%, from about 11 to about 20%, about 18%, from about 12 to about 18%, from about 12 to about 17%, from about 13 to about 17%, from about 13 to about 16%, from about 14 to about 16%, from about 14 to about 15%, from about 15 to about 25%, from about 15 to about 24%, from about 16 to about 24%, from about 16 to about 23%, from about 17 to about 23%, from about 17 to about 22%, from about 18 to about 22%, from about 18 to about 21%, from about 19 to about 21%, from about 19% to about 20%, about 60%, about 59%, about 58%, about 57%, about 56%, about 55%, about 54%, about 53%, about 52%, about 51%, about 50%, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6% or about 5%. In some forms, the foregoing percentage values refer to w / v%.

[0110] In some forms, the lipid-soluble modifier may account for about 5 to about 70%, about 5 to about 69%, about 5 to about 68%, about 5 to about 67%, about 5 to about 66%, about 5 to about 65%, about 5 to about 64%, about 5 to about 63%, about 5 to about 62%, about 5 to about 61%, 5 to about 60%, about 5 to about 59%, about 5 to about 58%, about 5 to about 57%, about 5 to about 56%, about 5 to about 55%, about 5 to about 54%, about 5 to about 53%, about 5 to about 52%, about 5 to about 51%, about 5 to about 50%, about 5 to about 49%, about 5 to about 48%, about 5 to about 47%, about 5 to about 46%, about 5 to about 45%, about 5 to about 44%, about 5 to about 43%, about 5 to about 42%, about 5 to about 41%, about 5 to about 40%, about 5 to about 39%, about 5 to about 38%, about 5 to about 37%, about 5 to about 36%, about 5 to about 35%, about 5 to about 34%, about 5 to about 33%, about 5 to about 32%, about 5 to about 31%, about 5 to about 30%, about 5 to about 29%, about 5 to about 28%, about 5 to about 27%, about 5 to about 26%, about 5 to about 25%, about 5 to about 24%, about 5 to about 23%, about 5 to about 22%, about 5 to about 21%, about 5 to about 20%, about 5 to about 19%, about 5 to about 18%, about 5 to about 17%, about 5 to about 16%, about 5 to about 15%, about 5 to about 14%, about 5 to about 13%, about 5 to about 12%, about 5 to about 11%, about 5 to about 10%, about 5 to about 9%, about 5 to about 8%, about 5 to about 7%, about 5 to about 6%, about 6 to about 60%, about 7 to about 60%, about 8 to about 60%, about 9 to about 60%, about 10 to about 60%, about 11 to about 60%, about 12 to about 60%, about 13 to about 60%, about 14 to about 60%, about 15 to about 60%, about 16 to about 60%, about 17 to about 60%, about 18 to about 60%, about 19 to about 60%, about 20 to about 60%, about 21 to about 60%, about 22 to about 60%, about 23 to about 60%, about 24 to about 60%, about 25 to about 60%, about 26 to about 60%, about 27 to about 60%, about 28 to about 60%, about 29 to about 60%, about 30 to about 60%, about 31 to about 60%, about 32 to about 60%, about 33 to about 60%, about 34 to about 60%, about 35 to about 60%, about 36 to about 60%, about 37 to about 60%, about 38 to about 60%, about 39 to about 60%, about 40 to about 60%, about 41 to about 60%, about 42 to about 60%, about 43 to about 60%, about 44 to about 60%, about 45 to about 60%, about 46 to about 60%, about 47 to about 60%, about 48 to about 60%, about 49 to about 60%, about 16 to about 44%, about 16 to about 43%, about 17 to about 43%, about 17 to about 42%, about 18 to about 42%, about 18 to about 41%, about 19 to about 41%,From about 19 to about 40%, from about 20 to about 40%, from about 20 to about 39%, from about 21 to about 39%, from about 21 to about 38%, from about 22 to about 38%, from about 22 to about 37%, from about 23 to about 37%, from about 23 to about 36%, from about 24 to about 36%, from about 24 to about 35%, from about 25 to about 35%, from about 25 to about 34%, from about 26 to about 34%, from about 26 to about 33%, from about 27 to about 33%, from about 27 to about 32%, from about 28 to about 32%, from about 28 to about 31%, from about 29 to about 31%, from about 29 to about 30%, from about 25 to about 35%, from about 25 to about 34%, from about 26 to about 34%, from about 26 to about 33%, from about 27 to about 33%, from about 27 to about 32%, from about 28 to about 32%, from about 28 to about 31%, from about 29 to about 31%, from about 29 to about 30%, from about 20 to about 30%, from about 20 to about 29%, from about 21 to about 29%, from about 21 to about 28%, from about 22 to about 28%, from about 22 to about 27%, from about 23 to about 27%, from about 23 to about 26%, from about 24 to about 26%, from about 24 to about 25%, about 70%, about 69%, about 68%, about 67%, about 66%, about 65%, about 64%, about 63%, about 62%, about 61%, about 60%, about 59%, about 58%, about 57%, about 56%, about 55%, about 54%, about 53%, about 52%, about 51%, about 50%, about 49%, about 48%, about 47%, about 46%, about 45%, about 44%, about 43%, about 42%, about 41%, about 40%, about 39%, about 38%, about 37%, about 36%, about 35%, about 34%, about 33%, about 32%, about 31%, about 30%, about 29%, about 28%, about 27%, about 26%, about 25%, about 24%, about 23%, about 22%, about 21%, about 20%, about 19%, about 18%, about 17%, about 16%, about 15%, about 14%, about 13%, about 12%, about 11%, about 10%, about 9%, about 8%, about 7%, about 6% or about 5%. In some forms, the foregoing percentage values refer to w / v%.

[0111] In some forms, the molar ratio of the homogenizing agent to the borate compound is between about 0.5 and about 2, between about 0.5 and about 1.9, between about 0.5 and about 1.8, between about 0.5 and about 1.7, between about 0.5 and about 1.6, between about 0.5 and about 1.5, between about 0.5 and about 1.4, between about 0.5 and about 1.3, between about 0.5 and about 1.2, between about 0.5 and about 1.1, between about 0.5 and about 1, between about 0.6 and about 2, between about 0.6 and about 1.9, between about 0.6 and about 1.8, between about 0.6 and about 1.7, between about 0.6 and about 1.6, between about 0.6 and about 1.5, between about 0.6 and about 1.4, between about 0.6 and about 1.3, between about 0.6 and about 1.2, between about 0.6 and about 1.1, between about 0.6 and about 1, between about 0.7 and about 2, between about 0.7 and about 1.9, between about 0.7 and about 1.8, between about 0.7 and about 1.7, between about 0.7 and about 1.6, between about 0.7 and about 1.5, between about 0.7 and about 1.4, between about 0.7 and about 1.3, between about 0.7 and about 1.2, between about 0.7 and about 1.1, between about 0.7 and about 1, between about 0.8 and about 2, between about 0.8 and about 1.9, between about 0.8 and about 1.8, between about 0.8 and about 1.7, between about 0.8 and about 1.6, between about 0.8 and about 1.5, between about 0.8 and about 1.4, between about 0.8 and about 1.3, between about 0.8 and about 1.2, between about 0.8 and about 1.1, between about 0.8 and about 1, between about 0.9 and about 2, between about 0.9 and about 1.9, between about 0.9 and about 1.8, between about 0.9 and about 1.7, between about 0.9 and about 1.6, between about 0.9 and about 1.5, between about 0.9 and about 1.4, between about 0.9 and about 1.3, between about 0.9 and about 1.2, between about 0.9 and about 1.1, between about 0.9 and about 1, between about 1 and about 2, between about 1 and about 1.9, between about 1 and about 1.8, between about 1 and about 1.7, between about 1 and about 1.6, between about 1 and about 1.5, between about 1 and about 1.4, between about 1 and about 1.3, between about 1 and about 1.2, between about 1 and about 1.1, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, or about 2.

[0112] In some forms, the disclosed tissue clearing compositions include N-methylglucamine, iohexol, and 2,2'-thiodiethanol, and the concentration range of each of these components is from about 10 to about 50 w / v%. Preferably, the tissue clearing composition may consist of about 20 w / v% N-methylglucamine, about 32 w / v% iohexol, and about 25 w / v% thiodiethanol.

[0113] In some forms, the disclosed tissue clearing compositions include N-methylglucamine, iohexol, and propylene glycol. The concentration range of N-methylglucamine and iohexol is each from about 10 to about 50 w / v%, and the concentration range of propylene glycol is from about 10 to about 60 w / v%. Preferably, the tissue clearing composition may consist of about 20 w / v% N-methylglucamine, about 32 w / v% iohexol, and about 35 w / v% propylene glycol.

[0114] In some forms, the disclosed tissue clearing compositions include urea, iohexol, and 2,2'-thiodiethanol. The concentration range of urea is from about 5 to about 50 w / v%, and the concentration range of iohexol and 2,2'-thiodiethanol is each from about 10 to about 50 w / v%. Preferably, the tissue clearing composition may consist of about 10 w / v% urea, about 32 w / v% iohexol, and about 25 w / v% 2,2'-thiodiethanol.

[0115] In some forms, the disclosed tissue clearing compositions include N-methylglucamine, iohexol, 2,2'-thiodiethanol, and boric acid. The concentration range of N-methylglucamine, iohexol, and 2,2'-thiodiethanol is each from about 10 to about 50 w / v%, and the molar ratio of N-methylglucamine to boric acid is between about 0.5 and about 2. Preferably, the tissue clearing composition may consist of about 20 w / v% N-methylglucamine, about 32 w / v% iohexol, and about 25 w / v% thiodiethanol, and the molar ratio of N-methylglucamine to boric acid is about 1.

[0116] In some forms, the disclosed tissue clearing compositions include N-methylglucamine, iohexol, propylene glycol, and boric acid. The concentration range of N-methylglucamine and iohexol is each from about 10 to about 50 w / v%, the concentration range of propylene glycol is from about 10 to about 60 w / v%, and the molar ratio of N-methylglucamine to boric acid is between about 0.5 and about 2. Preferably, the tissue clearing composition may consist of about 20 w / v% N-methylglucamine, about 32 w / v% iohexol, and about 35 w / v% propylene glycol, and the molar ratio of N-methylglucamine to boric acid is about 1.

[0117] In some forms, the disclosed tissue clearing compositions comprise urea, iohexol, 2,2'-thiodiethanol, and boric acid, wherein the concentration of urea ranges from about 10 to about 50 w / v%, and the molar ratio of urea to boric acid is from 0.5 to 2. Preferably, the tissue clearing composition can consist of about 10 w / v% urea, about 32 w / v% iohexol, and about 25 w / v% 2,2'-thiodiethanol, and the molar ratio of urea to boric acid is about 1.

[0118] However, as can be understood by those skilled in the art, the components, the concentration ranges and sub-ranges of any such components can vary depending on the specific application of the tissue clearing composition.

[0119] In some forms, the composition is suitable for robust conventional applications. In some forms, the composition is suitable for fresh tissue. In some forms, the composition is suitable for long-term fixed tissue. In some forms, the composition is suitable for in vivo clearing applications.

[0120] In some forms, the disclosed tissue clearing compositions are compatible with further processing methods such as those used in histological and electron microscopy studies, other tissue clearing methods, different tissue staining methods (such as immunohistochemistry, chemical staining, transgenic cell labeling methods, imaging probes, in situ tissue chemistry, and virus tracing methods), or combinations thereof.

[0121] Different forms of the tissue clearing composition can be formulated and used for different tissue and source types. In some forms, the composition is suitable for clearing non-neural, non-bone tissues or organs. In some forms, the non-neural, non-bone tissues or organs are non-neural, non-bone solid organs such as the heart, kidney, liver, lung, and pancreas. In some forms, the non-neural, non-bone solid organ is the kidney. In some forms, the non-neural, non-bone tissues or organs are non-neural, non-bone pathological tissues or organs such as tumor tissues.

[0122] In some forms, the composition is suitable for clearing plant tissues, animal tissues or organs, or both. In some forms, the composition is suitable for clearing mammalian tissues or organs. In some forms, the composition is suitable for clearing human tissues or organs.

[0123] In some forms, the composition is suitable for clearing tissues recovered from archival sources. In some forms, the composition is suitable for clearing tissues archived for any time between about 3 months and about 50 years. In some forms, the composition is suitable for clearing recently fixed tissues, such as tissues fixed for any time between about 3 weeks and about 3 months.

[0124] In some forms, the tissue to be made transparent can be archived tissue, where the archived tissue has been stored for at least 3 weeks, 4 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 2 months, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 3 months, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 4 months, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 5 months, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 6 months, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 7 months, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 8 months, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 9 months, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 10 months, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 11 months, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 12 months, 1 year, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 2 years, 30 months, 36 months, 3 years, 42 months, 48 months, 4 years, 54 months, 60 months, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 22 years, 24 years, 25 years, 26 years, 28 years, 30 years, 35 years, 40 years, or 50 years.

[0125] However, as will be understood by those skilled in the art, the time range for tissue fixation can vary before applying the disclosed tissue clearing composition and is thus not limited to the time ranges identified above.

[0126] It should also be understood that the uses of the compositions disclosed herein are not limited to the tissues, sources, or types of sources identified above and can thus vary.

[0127] III. Kits

[0128] The disclosed tissue clearing compositions and other materials can be packaged together in any suitable combination as kits for performing or assisting in performing the disclosed methods. A kit is useful if the kit components in a given kit are designed and adapted to be used together in the disclosed methods.

[0129] The disclosed compositions can include additional components, e.g., to make the composition useful for or adapted for a particular tissue and type of source to be applied.

[0130] In some forms, the tissue clearing composition disclosed in the kit consists of four core components: (1) a homogenizing agent, (2) a water-soluble regulator, (3) a lipid-soluble regulator, and (4) a borate compound.

[0131] In some forms, the tissue clearing kit may include a composition having about 20 w / v% N-methylglucamine, about 32 w / v% iohexol, and about 25 w / v% thiodiglycol, with a molar ratio of N-methylglucamine to boric acid of about 1. In some forms, the kit may include a composition having about 20 w / v% N-methylglucamine, about 32 w / v% iohexol, and about 35 w / v% propylene glycol, with a molar ratio of N-methylglucamine to boric acid of about 1. In some forms, the kit may include a composition having about 10 w / v% urea, about 32 w / v% iohexol, and about 25 w / v% 2,2'-thiodiglycol, with a molar ratio of urea to boric acid of about 1.

[0132] However, as will be understood by those skilled in the art, the components in a particular kit, the concentration ranges and sub-ranges of any such components, may vary depending on the specific application of the tissue clearing composition.

[0133] The different forms of the tissue clearing composition in the kit can be formulated and used for different tissue and source types. In some forms, the composition is suitable for clearing non-neural, non-bone tissues or organs. In some forms, the non-neural, non-bone tissues or organs are non-neural, non-bone solid organs, such as the heart, kidney, liver, lung, and pancreas. In some forms, the non-neural, non-bone solid organ is the kidney. In some forms, the non-neural, non-bone tissues or organs are non-neural, non-bone pathological tissues or organs, such as tumor tissues.

[0134] In some forms, the composition is suitable for clearing plant tissues, animal tissues or organs, or both. In some forms, the composition is suitable for clearing mammalian tissues or organs. In some forms, the composition is suitable for clearing human tissues or organs.

[0135] In some forms, the composition is suitable for clearing tissues recovered from archival sources. In some forms, the composition is suitable for clearing tissues archived at any time between about 3 months and about 50 years. In some forms, the composition is suitable for clearing recently fixed tissues, such as tissues fixed at any time between about 3 weeks and about 3 months.

[0136] In some forms, the disclosed kit may further include a liquid carrier. In some forms, the liquid carrier is an aqueous solution. In some forms, the aqueous solution is a concentrate, such as a 5x, 10x, or 20x concentrate. In some forms, the aqueous solution contains one or more of the following: sodium azide (optionally at a concentration of about 10% w / v); a surfactant, such as Triton X-100 or sodium dodecyl sulfate (optionally at a concentration between about 4 and about 8 w / v%); and a buffer, such as a borate compound (e.g., boric acid, optionally at a concentration of 0.05 - 0.5 M, and optionally with a buffer pH between about 8 and about 9) or a buffer of phosphate-buffered saline.

[0137] In some forms, the disclosed kit may further include a 10% w / v sodium azide solution, a 10x P concentrate, phosphate-buffered saline with 0.1% Triton X-100 and 0.01% sodium azide, 4% w / v sodium dodecyl sulfate in 0.2 M sodium borate buffer at pH 8.5, or 8% w / v sodium dodecyl sulfate in phosphate-buffered saline at pH 7.4.

[0138] IV. Method of Use

[0139] Also disclosed is a method of rendering a tissue sample transparent using the tissue clearing composition. The method includes incubating the tissue sample with the tissue clearing composition. The incubation time range can be from about 3 hours to about 24 hours. The incubation temperature range can be from about 37°C to about 55°C.

[0140] The method may further include the following steps, prior to incubating the tissue sample: (a) selecting a homogenizing agent, a water-soluble regulator, a lipid-soluble regulator, and a borate compound, and (b) mixing the homogenizing agent, the water-soluble regulator, the lipid-soluble regulator, and the borate compound to form the tissue clearing composition.

[0141] The method can be combined with one or more detection and / or characterization steps before or after incubating the tissue sample, such as:

[0142] (i) staining the tissue sample with one or more fluorescent dyes;

[0143] (ii) imaging one or more fluorescent proteins expressed in the tissue sample;

[0144] (iii) performing immunohistochemistry, fluorescent histochemistry, or both on the tissue sample; and

[0145] (iv) characterizing the tissue sample using a transmission electron microscope.

[0146] In some forms, the method can be used in conjunction with other tissue clearing methods.

[0147] The disclosed method of tissue clearing can be used in 3D histology. Histology is the study of the microscopic anatomy of tissues, and there are many methods to visualize tissues in 3D, one of which is tissue clearing. Tissue clearing has unique advantages in that it can preserve the integrity of the tissue so that subsequent optical sectioning of the cleared sample can be performed, as opposed to other methods for 3D histology that require physically sectioning the sample into many thin slices followed by computer reconstruction.

[0148] A. RI homogenization using a tissue clearing composition

[0149] The present disclosure also includes methods for clearing biological materials of a subject. In some forms, the method can include a single step of incubating a tissue sample in a suitable tissue clearing composition as described herein at a specific temperature for a sufficient time. The incubation can be carried out for a period of about 3 hours to about 24 hours in a temperature range of about 37°C to about 55°C. Preferably, the sample is incubated in the tissue clearing composition at about 37°C for about 6 hours.

[0150] In some forms, the tissue sample is incubated in a tissue clearing composition comprising N-methylglucamine, iohexol, 2,2'-thiodiethanol, and boric acid, wherein the concentration range of each of N-methylglucamine, iohexol, and 2,2'-thiodiethanol is about 10 to about 50 w / v% and the molar ratio of N-methylglucamine to boric acid is between about 0.5 and about 2. Preferably, the tissue sample is incubated in a tissue clearing composition containing about 20 w / v% of N-methylglucamine, about 32 w / v% of iohexol, and about 25 w / v% of thiodiethanol, and the molar ratio of N-methylglucamine to boric acid is about 1.

[0151] In other forms, the tissue sample is incubated in a tissue clearing composition comprising N-methylglucamine, iohexol, propylene glycol, and boric acid, wherein the concentration range of each of N-methylglucamine and iohexol is about 10 to about 50 w / v%, the concentration range of propylene glycol is about 10 to about 60 w / v%, and the molar ratio of N-methylglucamine to boric acid is between about 0.5 and about 2. Preferably, the tissue sample is incubated in a tissue clearing composition containing about 20 w / v% of N-methylglucamine, about 32 w / v% of iohexol, and about 35 w / v% of propylene glycol, and the molar ratio of N-methylglucamine to boric acid is about 1.

[0152] In other forms, the tissue sample is incubated in a tissue clearing composition comprising urea, iohexol, 2,2'-thiodiethanol, and boric acid, where the urea concentration ranges from about 5 to about 50 w / v%, the concentrations of iohexol and 2,2'-thiodiethanol each range from about 10 to about 50 w / v%, and the molar ratio of urea to boric acid is between 0.5 and 2. Preferably, the tissue sample is incubated in a tissue clearing composition containing about 10 w / v% urea, about 32 w / v% iohexol, and about 25 w / v% 2,2'-thiodiethanol, and the molar ratio of urea to boric acid is about 1.

[0153] However, as can be understood by those skilled in the art, the tissue sample can be incubated in a composition, where the components of the composition, the concentration ranges and sub-ranges of any such components, vary depending on the tissue and the specific application. The tissue sample can be tissue or an organ of a plant or an animal, preferably an animal tissue or organ such as an insect, fish, amphibian, bird, and mammal; more preferably, a tissue or organ of a mammal. Mammals can include, but are not limited to: laboratory animals such as mice, rats, rabbits, guinea pigs, and primates; pet animals such as dogs and cats; farm animals such as cows, horses, and sheep; and humans. Preferably, the tissue or organ is derived from a human or a mouse. Most preferably, the tissue or organ is of human origin.

[0154] In some forms, the tissue sample is from a non-neural, non-bone tissue or organ. In some forms, the tissue sample is from a non-neural, non-bone solid organ such as the heart, kidney, liver, lung, and pancreas. Preferably, the non-neural, non-bone solid organ is the kidney. In some forms, the tissue sample is a kidney tissue sample. In some forms, the tissue sample is a pathological tissue sample such as a tumor tissue sample. In some forms, the tissue sample is not from a neural tissue or organ. In some forms, the tissue sample is not a brain tissue sample.

[0155] The tissue sample can be fresh, archived, or recovered from paraffin-embedded tissue.

[0156] B. Tissue Staining and Processing Using the Tissue Clearing Composition

[0157] The tissue sample can be pre-labeled with an imaging tracer of a dye, fluorescent protein, or antibody so that after the sample tissue is cleared and made transparent, the imaging tracer can be traced under a microscope (preferably a confocal microscope). Figure 1 An exemplary protocol for tissue staining and processing is illustrated.

[0158] In some forms, animal tissues (such as human, rodent, and mouse tissues) that have been chemically fixed with formalin or formalin-fixed paraffin-embedded (FFPE) can be recovered and subjected to various processing steps, such as rehydration through a series of organic solvents and washing methods, in preparation for subsequent "SDS treatment". The SDS treatment involves partial defatting of the prepared tissue for subsequent labeling. This can be achieved by immersing the prepared tissue in 4% or 8% SDS in a specific buffer and then incubating at a specific temperature to allow tissue permeabilization and partial defatting.

[0159] In some forms, lipophilic dye tracing is performed on the recovered archived tissue. DiI and CM DiI dyes can be directly applied to non-SDS-treated kidney tissues (such as human or rodent kidney tissues) that have been fixed with formalin for ≥1 year. Subsequently, the tissue can be made transparent using a tissue clearing composition and visualized in 3D using various imaging methods, such as differential interference contrast, confocal microscopy, light sheet microscopy, supermicroscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, structured illumination microscopy, ground state depletion microscopy, stimulated emission depletion microscopy, scanning electron microscopy, transmission electron microscopy, wide-field fluorescence microscopy, conventional transmitted light microscopy, dissecting microscopy, spectrophotometry, fluorescence plate assay, and fluorescence chip assay, etc.

[0160] In other forms, chemical staining is performed on the recovered archived tissue. These chemical stains can include but are not limited to Dil staining, DAPI staining, or tomato lectin staining. Chemical staining can also include using a fluorophore-labeled antibody (such as a specific antibody for AQP2) to detect specific biomolecules in the tissue.

[0161] Dil staining uses Dil, which is a lipophilic tracer for lipids. DAPI staining uses DAPI, which is a nucleic acid stain. Tomato lectin staining can be used to perform lectin histochemistry to detect glycosylation; it targets various tubule types.

[0162] In other forms, immunostaining is performed on the recovered archived tissue using any suitable antibody, such as an antibody targeting AQP2, which can be used to visualize the distal convoluted tubules in the kidney. In some forms, the antibodies can be applied in a sequential manner at high dilutions to enable them to further penetrate into the tissue. In other forms, in addition to a binding kinetics controller, the antibodies can be applied at low dilutions in a single step to facilitate their penetration into the tissue. In some forms, the immunostaining technique can be applied with or without signal amplification techniques. In some forms, the immunostaining technique can be combined with other preparation processes and subsequent methods to complete.

[0163] C. Applications

[0164] In some forms, the disclosed methods are applicable to plants. Preferably, the disclosed methods are applicable to tissues or organs of animals, such as insects, fish, amphibians, birds, and mammals; more preferably, tissues or organs of mammals. Mammals can include, but are not limited to: laboratory animals, such as mice, rats, rabbits, guinea pigs, and primates; pet animals, such as dogs and cats; farm animals, such as cows, horses, sheep; and humans. Preferably, the tissues or organs are of human origin. In some forms, the tissue samples are from non-neural, non-bone tissues or organs. In some forms, the tissue samples are from non-neural, non-bone solid organs, such as the heart, kidney, liver, lung, and pancreas. Preferably, the non-neural, non-bone solid organ is the kidney. In some forms, the tissue sample is a kidney tissue sample. In some forms, the tissue sample is a pathological tissue sample, such as a tumor tissue sample. In some forms, the tissue sample is not from neural tissue or organ. In some forms, the tissue sample is not a brain tissue sample.

[0165] In some forms, the disclosed methods are applicable to clinical pathology research and tests for routine clinical use to improve the diagnosis of patients' diseases (such as cancer and kidney diseases).

[0166] The disclosed compositions and methods can be further understood by the following numbered paragraphs.

[0167] 1. A tissue clearing composition comprising a homogenizing agent, a water solubility regulator, a lipid solubility regulator, and a borate compound.

[0168] 2. The tissue clearing composition of paragraph 1, wherein the borate compound is a hydroborate or metal borate in anhydrous or hydrated form.

[0169] 3. The tissue clearing composition of paragraph 1 or 2, wherein the borate compound is a hydroborate selected from the group consisting of boric acid (H 3 BO 3 ), metaboric acid (H 3 B 3 O 6 ), and tetraboric acid (H 2 B 4 O 7 ).

[0170] 4. The tissue clearing composition of any one of paragraphs 1 or 2, wherein the borate compound is a metal borate having a boron-containing oxyanion selected from the group consisting of metaborate (BO 2 - ), diborate (B 2 O 5 4- ), triborate (B 3 O 7 5-)、 tetraborate (B 4 O 7 2- ), B 4 O 5 (OH) 4 2- ), B 4 O 9 6- ), or a combination thereof) and hydroxyboric acid (B(OH) 4 - ).

[0171] 5. An optically clear tissue clearing composition according to any one of paragraphs 1 - 4, wherein the borate compound is selected from boric acid, tetraboric acid, disodium tetraborate and derivatives thereof.

[0172] 6. An optically clear tissue clearing composition according to any one of paragraphs 1 - 5, wherein the molar ratio of the homogenizing agent to the borate compound is from about 0.5 to about 2.

[0173] 7. An optically clear tissue clearing composition according to any one of paragraphs 1 - 6, wherein the molar ratio of the homogenizing agent to the borate compound is about 1.

[0174] 8. An optically clear tissue clearing composition according to any one of paragraphs 1 - 7, wherein the homogenizing agent is a denaturing agent for proteins, nucleic acids or a combination thereof.

[0175] 9. An optically clear tissue clearing composition according to any one of paragraphs 1 - 8, wherein the homogenizing agent is a chaotropic agent.

[0176] 10. An optically clear tissue clearing composition according to any one of paragraphs 1 - 9, wherein the homogenizing agent is selected from N - methylglucamine, urea, thiourea, guanidine, guanidine hydrochloride, lithium perchlorate, ethylenediamine, triethanolamine, triethylamine, tetraethylammonium and derivatives thereof.

[0177] 11. An optically clear tissue clearing composition according to any one of paragraphs 1 - 10, wherein the concentration of the homogenizing agent is from about 5 w / v% to about 60 w / v% or from about 10 w / v% to about 50 w / v%.

[0178] 12. An optically clear tissue clearing composition according to any one of paragraphs 1 - 11, wherein the water - soluble regulator and the lipid - soluble regulator are refractive index regulators.

[0179] 13. An optically clear tissue clearing composition according to any one of paragraphs 1 - 12, wherein the refractive index of the water - soluble regulator, the lipid - soluble regulator or both at 25 °C is higher than the refractive index of water.

[0180] 14. An optically clear tissue clearing composition according to any one of paragraphs 1 - 13, wherein the refractive index of the water - soluble regulator, the lipid - soluble regulator or both at 25 °C is from about 1.40 to about 1.50.

[0181] 15. An optically clear tissue composition according to any one of paragraphs 1-14, wherein the refractive index of the water-soluble modifier at 25 °C is 10% or less of the refractive index of the lipid-soluble modifier.

[0182] 16. An optically clear tissue composition according to any one of paragraphs 1-15, wherein the water-soluble modifier is selected from iohexol, sodium thiosulfate, polyethylene glycol, ethylene carbonate, and derivatives thereof.

[0183] 17. An optically clear tissue composition according to any one of paragraphs 1-16, wherein the concentration of the water-soluble modifier is from about 5 w / v% to about 60 w / v%, or from about 10 w / v% to about 50 w / v%.

[0184] 18. An optically clear tissue composition according to any one of paragraphs 1-17, wherein the lipid-soluble modifier is miscible with water.

[0185] 19. An optically clear tissue composition according to any one of paragraphs 1-18, wherein the lipid-soluble modifier is selected from 2,2'-thiodiethanol, propylene glycol, ethylene carbonate, and derivatives thereof.

[0186] 20. An optically clear tissue composition according to any one of paragraphs 1-19, wherein the concentration of the lipid-soluble modifier is from about 5 w / v% to about 70 w / v%, or from about 10 w / v% to about 50 w / v%.

[0187] 21. The optically clear tissue composition of paragraph 1, wherein

[0188] (a) the homogenizing agent is selected from N-methylglucamine, urea, thiourea, guanidine, guanidine hydrochloride, lithium perchlorate, ethylenediamine, and derivatives thereof;

[0189] (b) the water-soluble modifier is selected from iohexol, sodium thiosulfate, polyethylene glycol, and derivatives thereof;

[0190] (c) the lipid-soluble modifier is selected from 2,2'-thiodiethanol, propylene glycol, and derivatives thereof; and

[0191] (d) the borate compound is selected from boric acid, tetraboric acid, disodium tetraborate, and derivatives thereof.

[0192] 22. The optically clear tissue composition of paragraph 21, wherein the homogenizing agent is N-methylglucamine, the water-soluble modifier is iohexol, the lipid-soluble modifier is 2,2'-thiodiethanol, and the borate compound is boric acid.

[0193] 23. The tissue clearing composition of paragraph 22, wherein the concentration ranges of N-methylglucamine, iohexol, and 2,2'-thiodiethanol are each about 10 w / v% to about 50 w / v%, and the molar ratio of N-methylglucamine to boric acid is about 0.5 to about 2.

[0194] 24. The tissue clearing composition of paragraph 23, wherein the concentration of N-methylglucamine is about 20 w / v%, the concentration of iohexol is about 32 w / v%, the concentration of thiodiethanol is about 25 w / v%, and the molar ratio of N-methylglucamine to boric acid is about 1.

[0195] 25. The tissue clearing composition of paragraph 21, wherein the homogenizing agent is N-methylglucamine, the water-soluble regulator is iohexol, the lipid-soluble regulator is propylene glycol, and the borate compound is boric acid.

[0196] 26. The tissue clearing composition of paragraph 25, wherein the concentration ranges of N-methylglucamine and iohexol are each about 10% to about 50%, the concentration range of propylene glycol is about 10 w / v% to about 60 w / v%, and the molar ratio of N-methylglucamine to boric acid is about 0.5 to about 2.

[0197] 27. The tissue clearing composition of paragraph 26, wherein the concentration of N-methylglucamine is about 20 w / v%, the concentration of iohexol is about 32 w / v%, the concentration of propylene glycol is about 35 w / v%, and the molar ratio of N-methylglucamine to boric acid is about 1.

[0198] 28. The tissue clearing composition of paragraph 21, wherein the homogenizing agent is urea, the water-soluble regulator is iohexol, the lipid-soluble regulator is 2,2'-thiodiethanol, and the borate compound is boric acid.

[0199] 29. The tissue clearing composition of paragraph 28, wherein the concentration range of urea is about 5 w / v% to about 50 w / v%, the concentration ranges of iohexol and 2,2'-thiodiethanol are each about 10 w / v% to about 50 w / v%, and the molar ratio of urea to boric acid is about 0.5 to about 2.

[0200] 30. The tissue clearing composition of paragraph 29, wherein the concentration of urea is about 10 w / v%, the concentration of iohexol is about 32 w / v%, the concentration of 2,2'-thiodiethanol is about 25 w / v%, and the molar ratio of urea to boric acid is about 1.

[0201] 31. The tissue clearing composition of any one of paragraphs 1-30, further comprising one or more excipients selected from the group consisting of: liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and combinations thereof.

[0202] 32. The tissue clearing composition of paragraph 31, wherein the liquid carrier is selected from solvents, dispersion media, and diluents.

[0203] 33. The tissue clearing composition of paragraph 32, wherein the diluent comprises an aqueous medium selected from water, acid solutions, and buffer solutions.

[0204] 34. The tissue clearing composition of any one of paragraphs 31 - 33, wherein the isotonic agent comprises sodium chloride, potassium chloride, sodium lactate, calcium chloride, and glucose.

[0205] 35. The tissue clearing composition of any one of paragraphs 1 - 34, wherein the tissue clearing composition has a refractive index of about 1.4 to about 1.5 at 25 °C.

[0206] 36. The tissue clearing composition of any one of paragraphs 1 - 35, wherein the tissue clearing composition has a pH of about 5 to about 9, about 5.5 to about 8.5, about 6 to about 8, or about 7 to about 10.

[0207] 37. The tissue clearing composition of any one of paragraphs 1 - 36, wherein the tissue clearing composition shows improved tissue clearing ability for non - neural, non - bone tissues or organs compared to the corresponding composition without the borate compound, compared to the corresponding composition in which the borate compound is replaced by an organic or inorganic acid, or compared to both.

[0208] 38. A method for clearing tissue, which comprises incubating a tissue sample in the tissue clearing composition of any one of paragraphs 1 - 37.

[0209] 39. The method of paragraph 38, wherein the homogenizing agent is N - methylglucamine, the water - soluble regulator is iohexol, the lipid - soluble regulator is 2,2'-thiodiethanol, and the borate compound is boric acid.

[0210] 40. The method of paragraph 39, wherein the concentration ranges of N - methylglucamine, iohexol, and 2,2'-thiodiethanol are each about 10 w / v% to about 50 w / v%, and the molar ratio of N - methylglucamine to boric acid is about 0.5 to about 2.

[0211] 41. The method of paragraph 40, wherein the concentration of N - methylglucamine is about 20 w / v%, the concentration of iohexol is about 32 w / v%, the concentration of thiodiethanol is about 25 w / v%, and the molar ratio of N - methylglucamine to boric acid is about 1.

[0212] 42. The method of any one of paragraphs 38 - 41, wherein the tissue sample is incubated for a period of about 3 to about 24 hours at a temperature of about 37 to about 55 °C.

[0213] The method of any one of paragraphs 38 - 42, wherein the tissue sample is a mammalian tissue sample.

[0214] The method of any one of paragraphs 38 - 43, wherein the tissue sample is a human tissue sample.

[0215] The method of any one of paragraphs 38 - 44, wherein the tissue sample is from a non - neural, non - bone tissue or organ.

[0216] The method of any one of paragraphs 38 - 45, wherein the tissue sample is from a non - neural, non - bone solid organ.

[0217] The method of any one of paragraphs 38 - 46, wherein the tissue sample is a kidney tissue sample.

[0218] The method of any one of paragraphs 38 - 47, wherein the tissue sample is a pathological tissue sample.

[0219] The method of any one of paragraphs 38 - 48, wherein the tissue sample is a tumor tissue sample.

[0220] The method of any one of paragraphs 38 - 49, wherein the tissue sample is not a brain tissue sample.

[0221] The method of any one of paragraphs 38 - 50, wherein the tissue sample is fresh, archived, or recovered from paraffin - embedded tissue.

[0222] The method of any one of paragraphs 38 - 51, further comprising, before incubating the tissue sample:

[0223] (a) selecting a homogenizing agent, a water - soluble regulator, a lipid - soluble regulator, and a borate compound; and

[0224] (b) mixing the homogenizing agent, the water - soluble regulator, the lipid - soluble regulator, and the borate compound to form the tissue clearing composition.

[0225] The method of any one of paragraphs 38 - 52, further comprising, before or after incubating the tissue sample, performing one or more of the following steps in any order:

[0226] (i) staining the tissue sample with one or more fluorescent dyes;

[0227] (ii) imaging one or more fluorescent proteins expressed in the tissue sample;

[0228] (iii) performing immunohistochemistry, fluorescence histochemistry, or both on the tissue sample; and

[0229] (iv) Characterize the tissue sample using a transmission electron microscope. Examples

[0230] Example 1. Acid screening

[0231] OPTIClear A (20 w / v% N-methylglucamine, 25 w / v% 2,2'-thiodiethanol, and 32 w / v% iohexol) requires hydrochloric acid titration to obtain a neutral pH. A group of organic and inorganic acids were screened to replace hydrochloric acid to improve the tissue clearing ability of the composition for non-neural, non-bone tissues or organs with or without prior paraffin embedding (e.g., formalin-fixed kidney tissues from rats and / or mice).

[0232] A series of common acids, including organic acids (such as acetic acid, succinic acid, maleic acid, malic acid, and glutamic acid) and inorganic acids (such as sulfuric acid, nitric acid, and phosphoric acid), were tested at a concentration with a molar ratio of 1:1 to N-methylglucamine. Interestingly, compared with OPTIClear A, the organic acids tested always gave worse tissue clearing results, indicating that the choice of acid is important. Further, other common inorganic acids (such as sulfuric acid, nitric acid, and phosphoric acid) also failed to produce satisfactory tissue clearing.

[0233] Driven by these negative results, further screening of acids was carried out to identify acids that would not produce separated anions that might hinder the penetration of N-methylglucamine into tissues once neutralized. Boric acid was identified as showing improved tissue clearing for kidney tissues compared to all other acids tested.

[0234] Boric acid can react with vicinal diols in N-methylglucamine to form cyclic borate esters. Although a coordination covalent bond may form between electron-deficient boric acid and N-methylglucamine, 11 11B NMR confirmed that the formation of cyclic borate esters is more reasonable.

[0235] The tissue clearing composition supplemented with boric acid is designated as OPTIClear B (20 w / v% N-methylglucamine, 25 w / v% 2,2'-thiodiethanol, 32 w / v% iohexol, and 6.335% w / v boric acid).

[0236] Example 2. Tissue clearing with OPTIClear B

[0237] Like OPTIClear A, OPTIClear B is detergent-free, non-toxic, and has a low concentration of iohexol, thus quenching less of the xanthene fluorophore. Compared with OPTIClear A, OPTIClear B is easier to prepare because boric acid can be directly added to the solid mixture without titration.

[0238] OPTIClear B can clear non-neural, non-bony tissues of 2-5 mm, which ranges from formalin-fixed, non-paraffin-embedded mouse kidneys, livers, spleens, and intestines to implanted mouse skin tumors and human kidney samples recovered from paraffin-embedded blocks, without causing significant tissue swelling, shrinkage, or deformation.

[0239] Example 3. Three-dimensional imaging using OPTIClear B

[0240] OPTIClear B is compatible with a range of fluorescent tissue labeling reagents such as lipophilic tracers, fluorophore-conjugated antibodies, fluorophore-conjugated lectins, fluorescent proteins, and nucleic acid stains. Combinations of these different stains are used to visualize different structures in formalin-fixed, non-paraffin-embedded tissue blocks. The stained tissue is then made transparent by immersion in OPTIClear B. Confocal microscopy is used to generate 3D images of kidney and tumor structures with unprecedented detail. Staining is performed before the tissue clearing step using OPTIClear B.

[0241] For example, 3D projection images of whole 2-mm-thick mouse kidney sections stained with the chemical stains DAPI (for nuclear DNA), AQP2 (for distal convoluted tubules), and tomato lectin (for various tubule types) demonstrate the compatibility of OPTIClear B with these staining methods. Formalin-fixed kidney sections are stained with the above-mentioned stains and then cleared in OPTIClear B. 3D projection images can then be generated by confocal microscopy imaging of the cleared tissue.

[0242] In another example, 3D color-coded projection images and 3D renderings of mouse kidneys with 1-mm-thick transverse sections that have been perfusion-stained with the lipophilic tracer dye DiI were generated. DiI was injected into the heart of sacrificed mice and then perfusion-fixed with formalin. The mouse kidneys were dissected and cut into 1-mm-thick sections, immersed in OPTIClear B, and imaged using confocal microscopy. The imaging results showed that the thin cell membranes of the endothelium remained intact after tissue clearing with OPTIClear B.

[0243] In another embodiment, as described in the previous embodiments, multiple classes of fluorescent stains were applied, including DAPI (nucleic acid stain), anti-laminin antibody (immunofluorescence), DiI (lipid lipophilic tracer), and Dylight 694-conjugated tomato lectin (lectin histochemistry for detecting glycosylation). The multi-colored stained tissue was then immersed in OPTIClear B and imaged using a confocal microscope. This process generated 3D images of mouse glomeruli from mouse kidney tissue. The imaging results showed that all stained targets were well-preserved in the mouse kidney tissue after tissue clearing.

[0244] In another embodiment, as described in the previous embodiments, multiple classes of fluorescent stains were applied, including DAPI (nucleic acid stain), GFP (fluorescent protein), DiI (lipophilic tracer for lipids), and anti-CD31 antibody (immunofluorescence). The multi-colored stained tissue was then immersed in OPTIClear B and imaged using a confocal microscope. This process generated 3D images of mouse tumor implants. Again, the imaging results showed that all stained targets were well-preserved in the mouse tumor tissue after tissue clearing.

[0245] It should be understood that the disclosed methods and compositions are not limited to the specific methodologies, protocols, and reagents described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims.

Claims

1. A tissue clearing composition comprising a homogenizing agent, a water-soluble regulator, a lipid-soluble regulator, and boric acid, wherein the homogenizing agent is N-methylglucamine, the water-soluble regulator is iohexol, and the lipid-soluble regulator is propylene glycol; wherein the concentration ranges of N-methylglucamine and iohexol are each 10 w / v% to 50 w / v%, the concentration range of propylene glycol is 10 w / v% to 60 w / v%, and the molar ratio of N-methylglucamine to boric acid is 0.5 to 2.

2. The tissue clearing composition according to claim 1, wherein the molar ratio of the homogenizing agent to the boric acid is 1.

3. The tissue clearing composition according to claim 1, wherein the concentration of the homogenizing agent is 5 w / v% to 60 w / v%.

4. The tissue clearing composition according to claim 3, wherein the concentration of the homogenizing agent is 10 w / v% to 50 w / v%.

5. The tissue clearing composition according to any one of claims 1-4, wherein the water-soluble regulator and the lipid-soluble regulator are refractive index regulators.

6. The tissue clearing composition according to any one of claims 1-4, wherein the refractive index of the water-soluble regulator, the lipid-soluble regulator, or both at 25 °C is higher than the refractive index of water.

7. The tissue clearing composition according to claim 6, wherein the refractive index of the water-soluble regulator, the lipid-soluble regulator, or both at 25 °C is 1.40 to 1.

50.

8. The tissue clearing composition according to any one of claims 1-4, wherein the refractive index of the water-soluble regulator at 25 °C is 10% of the refractive index of the lipid-soluble regulator.

9. The tissue clearing composition according to any one of claims 1-4, wherein the refractive index of the water-soluble regulator at 25 °C is within 10% of the refractive index of the lipid-soluble regulator.

10. The tissue clearing composition according to any one of claims 1-4, wherein the lipid-soluble regulator is miscible with water.

11. The tissue clearing composition according to any one of claims 1-4, wherein the concentration of N-methylglucamine is 20 w / v%, the concentration of iohexol is 32 w / v%, the concentration of propylene glycol is 35 w / v%, and the molar ratio of N-methylglucamine to boric acid is 1.

12. The tissue clearing composition according to any one of claims 1-4, further comprising one or more excipients selected from the following: liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, and combinations thereof.

13. The tissue clearing composition according to claim 12, wherein the liquid carrier is selected from solvents and diluents.

14. The tissue clearing composition according to claim 12, wherein the liquid carrier is selected from dispersion media.

15. The tissue clearing composition according to claim 13, wherein the diluent comprises an aqueous medium selected from water, acid solutions, and buffer solutions.

16. The tissue clearing composition according to claim 12, wherein the isotonic agent comprises sodium chloride, potassium chloride, sodium lactate, calcium chloride, and glucose.

17. The tissue clearing composition according to any one of claims 1-4, wherein the tissue clearing composition has a refractive index of 1.4 to 1.5 at 25 °C.

18. The tissue clearing composition according to any one of claims 1-4, wherein the tissue clearing composition has a pH of 5 to 9.

19. The tissue clearing composition according to claim 18, wherein the tissue clearing composition has a pH of 5.5 to 8.

5.

20. The tissue clearing composition according to any one of claims 1-4, wherein the tissue clearing composition has a pH of 6 to 8.

21. The tissue clearing composition according to any one of claims 1-4, wherein the tissue clearing composition has a pH of 7 to 10.

22. The tissue clearing composition according to any one of claims 1-4, wherein the tissue clearing composition exhibits improved tissue clearing ability for non-neural, non-bone tissues or organs.

23. Use of the tissue clearing composition according to any one of claims 1-22 in the preparation of a medicament for clearing tissue, said use comprising incubating a tissue sample in the tissue clearing composition.

24. The use according to claim 23, wherein the tissue sample is incubated for a period of 3 to 24 hours at a temperature of 37 °C to 55 °C.

25. The use according to claim 23 or 24, wherein the tissue sample is a mammalian tissue sample.

26. The use according to claim 23 or 24, wherein the tissue sample is a human tissue sample.

27. The use according to claim 23 or 24, wherein the tissue sample is from a non-neural, non-bone tissue or organ.

28. The use according to claim 23 or 24, wherein the tissue sample is from a non-neural, non-bone solid organ.

29. The use according to claim 23 or 24, wherein the tissue sample is a kidney tissue sample.

30. The use according to claim 23 or 24, wherein the tissue sample is a pathological tissue sample.

31. The use according to claim 23 or 24, wherein the tissue sample is a tumor tissue sample.

32. The use according to claim 23 or 24, wherein the tissue sample is not a brain tissue sample.

33. The use according to claim 23 or 24, wherein the tissue sample is fresh, archived, or recovered from paraffin-embedded tissue.

34. The use according to claim 23 or 24, which further comprises, prior to incubating the tissue sample: (a) selecting a homogenizing agent, a water-soluble regulator, a lipid-soluble regulator, and boric acid; and (b) mixing the homogenizing agent, the water-soluble regulator, the lipid-soluble regulator, and boric acid to form the tissue clearing composition.

35. The use according to claim 23 or 24, which further comprises, before or after incubating the tissue sample, in any order, one or more of the following steps: (i) staining the tissue sample with one or more fluorescent dyes; (ii) Imaging one or more fluorescent proteins expressed in the tissue sample; (iii) Performing immunohistochemistry, fluorescence histochemistry, or both on the tissue sample; and (iv) Characterizing the tissue sample using a transmission electron microscope.

Images