WO2021238893A1

WO2021238893A1 - Efficient and effective tissue clearing agents and their compositions

Info

Publication number: WO2021238893A1
Application number: PCT/CN2021/095690
Authority: WO
Inventors: Ho Ko; Hei Ming LAI; Yumi TANG
Original assignee: The Chinese University Of Hong Kong
Priority date: 2020-05-25
Filing date: 2021-05-25
Publication date: 2021-12-02

Abstract

Provided are compositions and methods for tissue clearing. Many types of tissues can be cleared effectively in a short amount of time by mixing the tissue with a homogenizing agent, a high refractive index chemical with high water-octanol partition coefficient, a high refractive index chemical with low water-octanol partition coefficient, and, optionally, a Lewis acid and/or Lewis base.

Description

EFFICIENT AND EFFECTIVE TISSUE CLEARING AGENTS AND THEIR COMPOSITIONS

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/029,582, filed May 25, 2020, which is hereby incorporated by reference in its entirety including any tables, figures, or drawings.

BACKGROUND OF THE INVENTION

Histology is the study of tissue microanatomy, and there exists many approaches to visualize tissues in 3D. Tissue clearing is one approach that has the unique advantage of leaving the tissue intact by optically sectioning a transparent sample. Tissue clearing is a recently emerged field in which physicochemical processes are used to turn naturally opaque tissues visibly more transparent.

Tissue clearing allows for 3D imaging of a tissue. Instead of serially cutting a tissue into thin sections and reconstructing the 3D image using computational methods, tissue clearing techniques directly turn tissues transparent, which allow imaging deep within the tissue. With the use of microscopes capable of imaging a selective plane of depth (i.e. optically sectioning the tissue) , a 3D image can be rapidly acquired, with no cutting artifacts or sample destruction in serial-sectioning methods.

Recently, there has been a surge of interest in developing novel tissue clearing agents and techniques. Invariably, these techniques alter the physicochemical properties of the tissue to achieve optical homogeneity. Thus, the overall process of tissue clearing can be viewed as tissue refractive index (RI) homogenization. A large number of techniques has been developed, and can be classified into aqueous-based simple RI homogenization, delipidation-assisted RI homogenization, and organic solvent-based RI homogenization. The latter two categories each has their own strengths, but can cause substantial tissue damage and has problems when applied to postmortem human tissues.

Accordingly, a solution to the challenging problem of limiting tissue damage while producing rapid and effective tissue clearing is needed.

BRIEF SUMMARY OF THE INVENTION

The subject invention pertains to a tissue clearing composition and associated method that is capable of turning a non-osseous, non-collagenous tissue visibly transparent. The tissue can be rodent or human and up to a 5 mm-thick slice. The method is a single step immersion, allowing for fast process that be can completed in as little as 2 hours for a 5 mm-thick sample. The method avoids distortion of sample, which is common for other tissue clearing methods, preserves tissue structure and molecular properties, is compatible with a large number of staining and molecular labelling methods and reagents, applicable to wide range of samples (those with prolonged fixation, clinical samples recovered from paraffin blocks) , compatible with most of the aqueous-based delipidation-facilitated tissue clearing methods, low cost, and easy to prepare and handle.

In certain embodiments, the tissue clearing composition comprises 4 to 5 compounds. The composition alters the optical properties of the tissues without damaging the physical components of the tissues. The now-transparent tissues can be visualized in 3D under a laser microscope, without physically sectioning the tissue. Embodiments of the subject invention are particularly useful for large tissues or in situations where 3D reconstruction of tissue morphology is required.

The subject invention can be helpful from bench-based research on animal tissues to clinical applications in diagnostic pathology.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. Depiction of brain tissue clearing capabilities of OPTIClear v2a and OPTIClear2b compared to OPTIClear and a control solution, performed on 2 mm-thick fixed mouse brain tissues. OPTIClear v2a and OPTIClear v2b are composed of a combination of one or more of 1-allylimidazole, 1- (3-aminopropyl) imidazole, 2, 2’-thiodiethanol, 2- (methylthio) ethanol, 3, 6-dithiaoctane-1, 8-diol, and iopromide. For OPTIClear2a, the ratio of 1-allylimidazole, 1- (3-aminopropyl) imidazole, 2, 2’-thiodiethanol, 2- (methylthio) ethanol, 3, 6-dithiaoctane-1, 8-diol, iopromide is 10: 10: 25: 0: 0: 32 in terms of percentages. For OPTIClear2b, the ratio of 1-allylimidazole, 1- (3-aminopropyl) imidazole, 2, 2’-thiodiethanol, 2- (methylthio) ethanol, 3, 6-dithiaoctane-1, 8-diol, iopromide is 0: 20: 14.4: 21.6: 6: 32. For OPTIClear2c, the ratio of 1-allylimidazole, 1- (3-aminopropyl) imidazole, 2, 2’-thiodiethanol, 2- (methylthio) ethanol, 3, 6-dithiaoctane-1, 8-diol, iopromide is 0: 20: 16: 24: 0: 32.

FIGURE 2. Confocal microscopy imaging of the tissue depicted in FIGURE 1 stained for 3 molecules (somatostain, calretinin, calbindin) using an immunofluorescence technique and cleared in OPTIClear2a. The scale bar has been provided for size reference.

FIGURE 3. Confocal microscopy imaging of a 1 mm-thick 4%formaldehyde-fixed brain tissue stained for anpep RNA transcript molecules using fluorescent in situ hybridization technique (shown in green) , brain vessel endothelium using fluorescently-labelled Lycopersicon esculentum lectin (shown in red) , and cell nuclei using 4′, 6-diamidino-2-phenylindole stain (shown in blue) . The stained tissue has been cleared in OPTIClear2b prior to imaging. The scale bar has been provided for size reference.

DETAILED DISCLOSURE OF THE INVENTION

Selected Definitions

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 20 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20, as well as all intervening decimal values between the aforementioned integers such as, for example, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to sub-ranges, “nested sub-ranges” that extend from either end point of the range are specifically contemplated. For example, a nested sub-range of an exemplary range of 1 to 50 may comprise 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

As used herein a “reduction” means a negative alteration, and an “increase” means a positive alteration, wherein the negative or positive alteration is at least 0.001%, 0.01%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or 100%.

The transitional term “comprising, ” which is synonymous with “including, ” or “containing, ” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. By contrast, the transitional phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel characteristic (s) ” of the claimed invention. Use of the term “comprising” contemplates other embodiments that “consist” or “consist essentially of” the recited component (s) .

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms “a, ” “and” and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term “about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01%of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

As used herein “tissue clearing, ” is the process of turning naturally opaque tissues visibly more transparent.

Tissue Clearing Composition

The subject invention is a tissue clearing composition comprising one or more homogenizing agents, one or more high refractive index chemicals with high water-octanol partition coefficients, and one or more high refractive index chemicals with low water-octanol partition coefficients. In certain embodiments, the tissue clearing composition further comprises one or more Lewis acids and/or Lewis bases. A chemical with high water-octanol partition coefficient is defined as d as log [ (solute concentration in octanol) / (solute concentration in water) ] > 0.0, after dissolving the chemical in a 1: 1 octanol: water mixture and allowing the two phases of solvent to separate. A chemical with low water-octanol partition coefficient is defined as d as log [ (solute concentration in octanol) / (solute concentration in water) ] ≤ 0.0, after dissolving the chemical in a 1: 1 octanol: water mixture and allowing the two phases of solvent to separate.

In certain embodiments, the homogenizing agent can be a denaturant of proteins, a denaturant of nucleic acids, a denaturant of proteins and nucleic acids, a chaotropic agent, a hydrotrope, a solubilization agent, and/or an emollient. Exemplary homogenizing agents that can be used in the subject invention are of N-methylglucamine, N-ethylglucamine, urea, thiourea, guanidine, guanidinium chloride, lithium perchlorate, ethylenediamine, triethanolamine, N-butylethanolamine, triethylamine, tetraethylammonium, imidazole, 1-methylimidazole, 1-ethylimidazole, 1- (3-aminopropylimidazole) , 1-allylimidazole, antipyrine, hydantoin, 1-phenyl-3- (phenylamino) pyrrolidine-2, 5-dione, ethylene carbonate, propylene carbonate, formamide, N-methylacetamide, 2-chloro-N- (hydroxymethyl) acetamide, N-dimethylacetamide, and/or derivatives thereof. The concentration of the homogenizing agent can be about 5%w/v to about 60%w/v, about 7.5%w/v to about 55%w/v, or about 10%to about 50%w/v.

In certain embodiments, the high refractive index chemical with high water-octanol partition coefficient or the high refractive index chemical with low water-octanol partition coefficient has a refractive index higher than water at about 25℃ to 60℃. In certain embodiments both the high refractive index chemical with high water-octanol partition coefficient and the high refractive index chemical with low water-octanol partition coefficient have a refractive index higher than water at about 25℃ to 60℃.

In certain embodiments, the high refractive index chemical with high water-octanol partition coefficient or the high refractive index chemical with low water-octanol partition coefficient has a refractive index of about 1.40 to about 1.50 at about 25℃ to 60℃. In certain embodiments, both the high refractive index chemical with high water-octanol partition coefficient and the high refractive index chemical with low water-octanol partition coefficient have a refractive index of about 1.40 to about 1.50 at about 25℃ to 60℃.

In certain embodiments, the high refractive index chemical with low water-octanol partition coefficient can be iohexol, iopromide, diatrizoic acid, metrizoic acid, iodamide, iotalamic acid, ioxitalamic acid, ioglicic acid, acetrizoic acid, iocarmic acid, methiodal, diodone, metrizamide, ioxaglic acid, iopamidol, iotrolan, ioversol, iopentol, iodixanol, iomeprol, iobitridol, ioxilan, iodoxamic acid, iotroxic acid, ioglycamic acid, adipiodone, iobenzamic acid, iopanoic acid, iocetamic acid, sodium iopodate, tyropanoic acid, lipiodol, iopydol, iofendylate, propyliodone, and/or derivatives thereof. The concentration of the high refractive index chemical with low water-octanol partition coefficient can be about 5%to about 70%w/v, about 7.5%to about 60%w/v, or about 10%to about 50%w/v. In some embodiments, the high refractive index chemical with high water-octanol partition coefficient is miscible with water.

In certain embodiments, the high refractive index chemical with high water-octanol partition coefficient can be 2, 2’-thiodiethanol, propylene glycol, ethylene glycol, glycerol, propylene carbonate, 1-thioglycerol, 2- (methylthio) ethanol, 2, 2’-sulfonyldiethanol, 2- (methylsulfonyl) ethanol, 3, 5’-dithia-1, 7-heptanediol, 3, 3’-thiodipropanol, 3, 6’-dithia-1, 8-octanediol, (methylthio) acetaldehyde dimethyl acetal, methyl (methylsulfinyl) methyl sulfide, and derivatives thereof. The concentration of the high refractive index chemical with high water-octanol partition coefficient can be about 5%to about 70%w/v, about 7.5%to about 60%w/v, or about 10%to about 50%w/v.

In certain embodiments, the tissue clearing composition further comprises one or more Lewis acids and/or Lewis bases. The Lewis acids and/or Lewis bases can be boric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, periodic acid, perchloric acid, sulfuric acid, nitric acid, methanesulfonic acid, ethylenediaminetetraacetic acid, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, potassium carbonate, potassium bicarbonate, lithium carbonate, tris (hydroxymethyl) aminomethane, 2, 2-bis (hydroxymethyl) -2, 2’, 2”-nitrilotriethanol, 3-morpholinopropane-1-sulfonic acid, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, 2- (N-morpholino) ethanesulfonic acid, and/or derivatives thereof, at concentrations of about 0.1mM to about 10M or about 0.1mM to about 1M as required to achieve pH of about 6.0 to about 9.0.

In certain embodiments, the tissue clearing composition can further comprise one or more excipients including liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, or combinations thereof. The liquid vehicle can be solvents, dispersion media, and diluents. The diluents can be aqueous media, such as, for example, water, acid solutions, and buffered solutions. Exemplary isotonic agents are sodium chloride, potassium chloride, sodium lactate, calcium chloride, and glucose.

In certain embodiments, the tissue clearing composition can have a refractive index of about 1.40 to about 1.55 at 25℃. Furthermore, the tissue clearing composition can have a pH in the range of about 5 to about 10, about 5 to about 9, about 5.5 to about 8.5, about 6 to about 8, or about 7 to about 10.

Tissue Clearing

The tissue clearing compositions of the subject invention can be used to change the refractive index of any type of tissue, particularly tissues from mammals, such as, for example, human, llama, goat, mouse, rat, mouse, horse, monkey, or tissues from birds, such as, for example, chickens. In preferred embodiments, the types of tissues that can be cleared are non-osseous tissues, including muscle, epithelial, blood, connective, cartilage, adipose, and nervous. Additionally, osseous (bone) tissues can be cleared. Other types of tissues that can be cleared include, for example, columnar epithelium, stratified epithelium, cuboidal epithelium, smooth muscle, skeletal muscle, cardiac muscle, and loose connective. The tissue can be from a variety of organs including, for example, brain, lung, liver, bladder, renal (kidney) , ureter, urethra, heart, stomach, intestines, salivary glands, larynx, esophagus, gallbladder, mesentery, pancreas, nerve, eye, ear, tongue, osseous, and skin. Furthermore, the tissue sample can be pathological or a tumor. The tissue sample can be fresh, archived, or retrieved from a paraffin wax-embedded tissue.

In certain embodiments, the tissue can be at least 0.1 mm, 0.25 mm, 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, or greater in width. In preferred embodiments, the tissue can be at least 2 mm to about 5 mm thick.

In preferred embodiments, the tissue clearing composition shows improved tissue clearing capacity for non-osseous tissues or organs relative to corresponding compositions. Corresponding compositions can be osseous or connective tissues that can be rich in collagen.

Prior to incubating the tissue sample, the homogenizing agent, the high refractive index chemical with low water-octanol partition coefficient, the high refractive index chemical with high water-octanol partition coefficient, and, optionally, a Lewis acid and/or Lewis base can be mixed to form the tissue clearing composition.

Before or after the tissue sample is incubated in the tissue clearing composition, one or more of the following steps, can be performed in any order:

(i) staining the tissue sample, preferably using one or more fluorescent dyes;

(ii) imaging one or more proteins expressed in the tissue sample, preferably fluorescently dyed proteins;

(iii) performing immunohistochemistry, fluorescent histochemistry, fluorescent in situ hybridization or both on the tissue sample; and/or

(iv) characterizing the tissue sample using transmission electron microscopy and routine pathology.

Methods of tissue staining, tissue visualization, histochemistry, and characterization of the tissue are well-known in the art, particularly methods using fluorescence. Practitioners of the art can perform a variety of modifications in order to effectively and efficiently stain; visualize; hybridize; characterize; and/or perform immunofluorescence, histochemistry, lipophilic tracing, fluorescent in situ hybridization or other related methods.

To clear a tissue, the tissue sample is incubated in a tissue clearing composition of the subject invention. The tissue sample can incubated for a period of time ranging from about 0.1 to about 48 hours or about 0.1 to about 24 hours at a temperature ranging from about 37℃ to about 55℃. In preferred embodiments, the tissue can be cleared in less than about 2 hours.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1-PREFERRED TISSUE CLEARING COMPOSITIONS

One exemplary tissue clearing composition comprises the homogenizing agent 1-allyimidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, and the high refractive index chemical with high water-octanol partition coefficient 2- (methylthio) ethanol. The concentration of 1-allylimidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, the concentration of 2- (methylthio) ethanol is about 25 w/v %.

A second exemplary tissue clearing composition comprises the homogenizing agent 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, and the high refractive index chemical with high water-octanol partition coefficient 2- (methylthio) ethanol, and the Lewis acid hydrochloric acid. The concentration of 1- (3-aminopropyl) imidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, the concentration of 2- (methylthio) ethanol is about 25 w/v %, and the concentration of hydrochloric acid is about 1.676 M.

A third exemplary tissue clearing composition comprises the homogenizing agents 1-allylimidazole and 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, and the high refractive index chemical with high water-octanol partition coefficient 2- (methylthio) ethanol. The concentration of 1-allylimidazole is about 10 v/v %, the concentration of 1- (3-aminopropyl) imidazole is about 10 v/v %, the concentration of iopromide is about 32 w/v %, and the concentration of 2- (methylthio) ethanol is about 25 w/v%.

A fourth exemplary tissue clearing composition comprises the homogenizing agent 1-allyimidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, and the high refractive index chemical with high water-octanol partition coefficient 3, 6-dithia-octane-1, 8-diol. The concentration of 1-allylimidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, and the concentration of 3, 6-dithia-octane-1, 8-diol is about 25 w/v %.

A fifth exemplary tissue clearing composition comprises the homogenizing agent 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, and the high refractive index chemical with high water-octanol partition coefficient 3, 6-dithia-octane-1, 8-diol, and the Lewis acid hydrochloric acid. The concentration of 1- (3-aminopropyl) imidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, the concentration of 3, 6-dithia-octane-1, 8-diol is about 25 w/v %, and the concentration of hydrochloric acid is about 1.676 M.

A sixth exemplary tissue clearing composition comprises the homogenizing agents 1-allylimidazole and 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, and the high refractive index chemical with high water-octanol partition coefficient 3, 6-dithia-octane-1, 8-diol. The concentration of 1-allylimidazole is about 10 v/v %, the concentration of 1- (3-aminopropyl) imidazole is about 10 v/v %, the concentration of iopromide is about 32 w/v %, and the concentration of 3, 6-dithia-octane-1, 8-diol is about 25 w/v %.

A seventh exemplary tissue clearing composition comprises the homogenizing agent 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, and the high refractive index chemicals with high water-octanol partition coefficients 2- (methylthio) ethanol and 2, 2’-thiodiethanol. The concentration of 1- (3-aminopropyl) imidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, the concentration of 2- (methylthio) ethanol is about 24 v/v %, and the concentration of 2, 2’-thiodiethanol is about 16 v/v %.

An eighth exemplary tissue clearing composition comprises the homogenizing agent 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, the high refractive index chemicals with high water-octanol partition coefficients 2- (methylthio) ethanol and 2, 2’-thiodiethanol, and the Lewis acid hydrochloric acid. The concentration of 1- (3-aminopropyl) imidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, the concentration of 2- (methylthio) ethanol is about 24 v/v %, the concentration of 2, 2’-thiodiethanol is about 16 v/v %, and the concentration of hydrochloric acid is about 1.676 M.

An ninth exemplary tissue clearing composition comprises the homogenizing agent 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, and the high refractive index chemicals with high water-octanol partition coefficients 2- (methylthio) ethanol, 2, 2’-thiodiethanol, and 3, 6-dithia-octane-1, 8-diol. The concentration of 1- (3-aminopropyl) imidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, the concentration of 2- (methylthio) ethanol is about 21.6 v/v %, the concentration of 2, 2’-thiodiethanol is about 14.4 v/v %, and the concentration of 3, 6-dithia-octane-1, 8-diol is about 6 w/v %.

An tenth exemplary tissue clearing composition comprises the homogenizing agent 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, the high refractive index chemicals with high water-octanol partition coefficients 2- (methylthio) ethanol, 2, 2’-thiodiethanol, and 3, 6-dithia-octane-1, 8-diol, and the Lewis acid hydrochloric acid. The concentration of 1- (3-aminopropyl) imidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, the concentration of 2- (methylthio) ethanol is about 21.6 v/v %, the concentration of 2, 2’-thiodiethanol is about 14.4 v/v %, the concentration of 3, 6-dithia-octane-1, 8-diol is about 6 w/v %, and the concentration of the hydrochloric acid is about 1.676 M.

An eleventh exemplary tissue clearing composition comprises the homogenizing agent 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, and the high refractive index chemicals with high water-octanol partition coefficients 2- (methylthio) ethanol, 2, 2’-thiodiethanol, and 3, 6-dithia-octane-1, 8-diol. The concentration of 1- (3-aminopropyl) imidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, the concentration of 2- (methylthio) ethanol is about 20.4 v/v %, the concentration of 2, 2’-thiodiethanol is about 13.6 v/v %, and the concentration of 3, 6-dithia-octane-1, 8-diol is about 6 w/v %.

A twelfth exemplary tissue clearing composition comprises the homogenizing agent 1- (3-aminopropyl) imidazole, the high refractive index chemical with low water-octanol partition coefficient iopromide, the high refractive index chemicals with high water-octanol partition coefficients 2- (methylthio) ethanol, 2, 2’-thiodiethanol, and 3, 6-dithia-octane-1, 8-diol, and the Lewis acid hydrochloric acid. The concentration of 1- (3-aminopropyl) imidazole is about 20 v/v %, the concentration of iopromide is about 32 w/v %, the concentration of 2- (methylthio) ethanol is about 20.4 v/v %, the concentration of 2, 2’-thiodiethanol is about 13.6 v/v %, the concentration of 3, 6-dithia-octane-1, 8-diol is about 6 w/v %, and the concentration of the hydrochloric acid is about 1.676 M.

EXAMPLE 2-METHODS OF USING THE TISSUE CLEARING COMPOSITIONS

Prior to incubating the tissue sample, 10 v/v %1-allylimidazole, 10 v/v %1- (3-aminopropyl) imidazole, 32 w/v %iopromide, 25 v/v %2, 2’-thiodiethanol are mixed to form the tissue clearing composition. The tissue sample, preferably brain and kidney tissues from human or rodents, is incubated in 10 v/v %1-allylimidazole, 10 v/v %1- (3-aminopropyl) imidazole, 32 w/v %iopromide, and 25 v/v %2, 2’-thiodiethanol for 1 hour at about 37℃.

A second incubation condition is as follows: prior to incubating the tissue sample, 10 v/v %1-allylimidazole, 10 v/v %1- (3-aminopropyl) imidazole, 32 w/v %iopromide, 25 v/v %2, 2’-thiodiethanolare mixed to form the tissue clearing composition. The tissue sample, preferably brain and kidney tissues from human or rodents, is incubated in 10 v/v %1-allylimidazole, 10 v/v %1- (3-aminopropyl) imidazole, 32 w/v %iopromide, 25 v/v %2, 2’-thiodiethanol for a period of time ranging from about 0.1 to about 24 hours at a temperature ranging from about 37℃ to about 55℃ .

A third incubation condition is as follows: prior to incubating a 2 mm-thick 4%paraformaldehyde-fixed mouse brain tissue sample, 1-allylimidazole 1- (3-aminopropyl) imidazole, iopromide, 2- (methylthio) ethanol, and 2, 2’-thiodiethanol are mixed. The 2 mm-thick 4%paraformaldehyde-fixed mouse brain tissue sample is incubated in a mixture of 20 v/v % 1- (3-aminopropyl) imidazole, 32%w/v iopromide, 21.6 v/v %2- (methylthio) ethanol, 14.4%v/v 2, 2’-thiodiethanol, and 6 %w/v 3, 6-dithia-1, 8-octanediol for about 1 hour at about37℃.

As demonstrated in Figure 1, the compositions of the subject invention improved tissue clearance compared to OPTIClear and the control. OPTIClear is composed of N-methylglucamine, 2, 2'-thiodiethanol, iohexol, and hydrochloric acid. OPTIClear2 is composed of 1-allylimidazole, 1- (3-aminopropyl) imidazole, 2, 2’-thiodiethanol, 2- (methylthio) ethanol, 3, 6-dithiaoctane-1, 8-diol, iopromide, with versions 2a and 2b possessing different ratios of the aforementioned compounds. For OPTIClear2a, the ratio of 1-allylimidazole, 1- (3-aminopropyl) imidazole, 2, 2’-thiodiethanol, 2- (methylthio) ethanol, 3, 6-dithiaoctane-1, 8-diol, iopromide is 10: 10: 25: 0: 0: 32 in terms of percentages. For OPITClear2b, the ratio of 1-allylimidazole, 1- (3-aminopropyl) imidazole, 2, 2’-thiodiethanol, 2- (methylthio) ethanol, 3, 6-dithiaoctane-1, 8-diol, iopromide is 0: 20: 14.4: 21.6: 6: 32. For OPTIClear2c, the ratio of 1-allylimidazole, 1- (3-aminopropyl) imidazole, 2, 2’-thiodiethanol, 2- (methylthio) ethanol, 3, 6-dithiaoctane-1, 8-diol, iopromide is 0: 20: 16: 24: 0: 32.

The result is a much improved tissue clearing efficacy at an accelerated speed. The 2 mm mouse brain tissue used in Figure 1 can be cleared in less than 2 hours using OPTIClear v2 compared to 6 hours with OPTIClear. Furthermore, OPTIClear v2 can clear 5 mm-thick brain tissue. OPTIClear cannot clear 5 mm-thick brain tissue. In addition, OPTIClear v2 has a much wider application to nearly all tissue types, except for bone and collagenous tissues, while OPTIClear is mostly applicable to neural tissues.

EXAMPLE 3-THERMO-IMMUNOHISTOCHEMISTRY WITH OPTIMIZED KINETICS (ThICK) STAINING PROTOCOL USING OPTIClear2 OR OPTIClear

Fresh brain tissues were obtained and stored from C57BL/6 and Thy1-GCaMP6f transgenic adult mice of at least 2 months old were used. Formaldehyde-fixed and SHIELD-protected brain tissues were harvested as previously described in Park, Y. -G. et al. Protection of tissue physicochemical properties using polyfunctional crosslinkers. Nat Biotechnol 37, 73–83 (2019) , which is hereby incorporated by reference. SHIELD protection is preferably for samples labeled with fluorescent proteins. After adequate washings with PBST, tissues were stored at 4℃ in 1× PBS until use. Tissues < 300 μm-thick were permeabilized for 1 day in PBST at 37℃, while larger samples were treated with 4%w/v SDS in 0.2 M borate buffer, pH 8.5 at 37℃ until optically transparent. The permeabilized sample was then washed thoroughly in PBST at 37℃ for 3 times (1 hour each) . This is essential as any residual SDS will precipitate with guanidinium chloride (GnCl) used in the next step. The washed sample was then equilibrated in roughly five-times the tissue volume of PBST with 1 M GnCl at 55℃ for 30 minutes, after which 10 μl of SPEAR reaction mixture per 100 μl staining buffer was added to the staining solution and incubated at 55℃ for 16 –72 hours, depending on the sample thickness. The staining duration can be increased by 8 hours for every 200 μm staining depth, although it is likely that optimization of antibody concentration and staining duration will be required for individual antibody-antigen pairs. After incubating at 55℃, the sample was cooled to room temperature and incubated further for 1 hour. The sample was then briefly washed in PBST to remove any residual GnCl and incubated in OPTIClear2 for 2 hours or OPTIClear for 6 hours at 37℃. The optically cleared sample can then be imaged.

EXAMPLE 4-CLEARNING LARGE SCALE HUMAN PONS SECTION USING OPTIClear2 OR OPTIClear

A human postmortem brainstem sample was fixed in 10%neutral buffered formalin for 3 weeks before washing and storage in PBS at 4℃. A 5 mm-thick transverse section of the pons was then cut. The pons slice was then sectioned sagittally and cut posterior to the medial lemniscus to obtain a subdivision containing the locus coeruleus. The sample was then permeabilized in 4%w/v SDS in 0.2 M borate buffer, pH 8.5 at 55℃ for 24 hours and washed three times in PBST, 2 hours each. Meanwhile, a Tyrosine hydroxylase (TH) synergistically protected poly epoxide-crosslinked Fab-complexed antibody reagents catalyzed by pyridine (SPEAR ^py) (with AlexaFluor 594) were prepared from 30 μl rabbit anti-TH antibody (AB152, Millipore) with 16 hours of incubation at 13℃ for 24 hours, as described below.

To create a SPEAR ^py, Primary antibodies were reconstituted in 1× PBS with 0.1%w/v sodium azide to 1 μg/μl if lyophilized. The constituents of the storage buffer were reviewed for presence of any additives (except BSA) containing primary amine groups. If the storage buffer contains > 0.1 M Tris, the antibodies were buffer exchanged to 1× PBS using ultracentrifugal filters with molecular weight cut-off of 50 kDa (Amicon Ultra-0.5 centrifugal filter unit, Cat. no. UFC505008, Millipore, Burlington, MA) . Purified antibodies in serum are preferred, as non-specific IgGs would consume the Fab fragments. 2 μl of 0.05 μg/μl antibody complexed with 2 μl of 1 μg/μl Fab fragment performed as satisfactorily as 2 μl of 1 μg/μl antibody, although using a larger amount of antibody may help to further boost signal.

SPEARs were freshly synthesized 1 day prior to staining. 2 μl of the primary antibody and 1 μl of the corresponding Fab fragment at 2 μg/μl were thoroughly mixed and incubated at room temperature for 10 minutes to form the Ab-Fab complex. During this time, 200 μl of P3PE was pipetted into a 1.5 ml Eppendorf tube using cut 1000-μl tips and the reverse pipetting technique. 800 μl of distilled water was then added and the tube was tightly capped and vigorously vortexed for 1 minute where the mixture would become a homogeneous milky emulsion. The tube was then centrifuged at 15,000 × g for 3 minutes at room temperature and allowed to sit at room temperature for not longer than an hour. To form the IgG-Fab complex, 1 μl of 1 M sodium carbonate pH 10 buffer followed by 4 μl of water and optional 0.5 μl of 10%v/v pyridine: water were then added and thoroughly mixed. This is followed by 1 μl of the freshly prepared P3PE supernatant, and the tube was rigorously vortexed. Using a thermocycler, the 10 μl reaction mixture was then reacted at 13℃ for 16 hours followed by cooling to 4℃ and kept for not more than 24 hours until further use.

The washed human postmortem brainstem was then placed in 3ml fresh PBST with 300 μl TH SPEARs ^py reaction mixture and ThICK-stained at 55℃ for 24 hours. After ThICK staining, the sample was cooled to 4℃ overnight, washed in PBST briefly for 1 hour at RT, and incubated in 20 ml OPTIClear at 37℃ overnight.

The stained and cleared sample was then imaged using an in-house custom built two-photon microscope in tiled Z-stack mode (total acquisition field-of-view of 1773 × 2754 ×1084 μm ³) using an Olympus XLPLN10XSVMP (10×, NA 0.6, WD 8 mm) objective. The Z-stacks were then imported into Zen Blue software (ZEN 3.3, Carl Zeiss, Oberkochen, Germany) . Gaussian blurred Z-stacks were then generated from each tile and used to correct shading inhomogeneity. The adjusted images were then background subtracted. Stitching was performed using the ImageJ plugin BigStitcher. The stitched image was imported into Imaris (v9, Bitplane) and cells were segmented with local background contrast option and filtered based on volume and sphericity parameters, followed by manual refinements. To quantify cell distance from the nearest tissue surface, a surface was generated encompassing all voxels outside of the tissue. A new channel with a linear gradient of voxel intensity that scales with the distance from the above generated surface was created using distance transformation in MATLAB (R2018b, MathWorks) . The mean intensities of the distance transformation channel for the segmented cell surfaces were thus their distance from the nearest tissue surface.

For comparison, a 1.5 mm-thick human pons samples that also contained the locus coeruleus was fixed in 10%neutral-buffered formalin for 3 weeks, permeabilized in 4%w/v SDS in 0.2 M borate buffer, pH 8.5 at 55℃ for 24 hours and washed three times in PBST, 2 hours each. 10 μl of Rabbit anti-TH antibody was then added every day to the immunostaining PBST solution to a total of 100 μl, and the tissue was then incubated for an additional 4 days at 37℃. The sample was then washed in PBST overnight for 1 day, and AlexaFluor 594-labelled donkey anti-rabbit secondary antibody (Invitrogen, R37119) ) was applied in a similar regimen. The sample was then washed and cleared in OPTIClear overnight. Imaging was performed with a Carl Zeiss LSM 780 confocal microscope using a 10× objective (Carl Zeiss Plan-Apochromat 10×/NA 0.45 M27) with an imaging depth of 1,500 μm (i.e. full-thickness imaging) . Stitched was performed alongside acquisition in Zen Black software (ZEN 2.3, Carl Zeiss) .

EXEMPLARY EMBODIMENTS

Embodiment 1. A tissue clearing composition comprising a homogenizing agent, a high refractive index chemical with high water-octanol partition coefficient, a high refractive index chemical with low water-octanol partition coefficient, and, optionally, a Lewis acid and/or Lewis base.

Embodiment 2. The tissue clearing composition of Embodiment 1, wherein the homogenizing agent is a denaturant of proteins, denaturant of nucleic acids, a denaturant of proteins and nucleic acids, a chaotropic agent, a hydrotrope, a solubilization agent, or an emollient.

Embodiment 3. The tissue clearing composition of Embodiment 1, wherein the homogenizing agent is N-methylglucamine, N-ethylglucamine, urea, thiourea, guanidine, guanidinium chloride, lithium perchlorate, ethylenediamine, triethanolamine, N-butylethanolamine, triethylamine, tetraethylammonium, imidazole, 1-methylimidazole, 1-ethylimidazole, 1- (3-aminopropylimidazole) , 1-allylimidazole, antipyrine, hydantoin, 1-phenyl-3- (phenylamino) pyrrolidine-2, 5-dione, ethylene carbonate, propylene carbonate, formamide, N-methylacetamide, 2-chloro-N- (hydroxymethyl) acetamide, N-dimethylacetamide, or a derivative of any thereof.

Embodiment 4. The tissue clearing composition Embodiment 1, wherein the concentration of the homogenizing agent is about 5 to about 60 w/v %or about 10 to about 50 w/v %.

Embodiment 5. The tissue clearing composition of Embodiment 1, wherein the high refractive index chemical with high water-octanol partition coefficient, the high refractive index chemical with low water-octanol partition coefficient, or both the high refractive index chemical with high water-octanol partition coefficient and the high refractive index chemical with low water-octanol partition coefficient have a refractive index higher than water at about 25℃ to 60℃.

Embodiment 6. The tissue clearing composition of Embodiment 1, wherein the high refractive index chemical with high water-octanol partition coefficient, the high refractive index chemical with low water-octanol partition coefficient, or both the high refractive index chemical with high water-octanol partition coefficient and the high refractive index chemical with low water-octanol partition coefficient have a refractive index of about 1.40 to about 1.50 at about 25℃ to about 60℃.

Embodiment 7. The tissue clearing composition of Embodiment 1, wherein the high refractive index chemical with low water-octanol partition coefficient is iohexol, iopromide, diatrizoic acid, metrizoic acid, iodamide, iotalamic acid, ioxitalamic acid, ioglicic acid, acetrizoic acid, iocarmic acid, methiodal, diodone, metrizamide, ioxaglic acid, iopamidol, iotrolan, ioversol, iopentol, iodixanol, iomeprol, iobitridol, ioxilan, iodoxamic acid, iotroxic acid, ioglycamic acid, adipiodone, iobenzamic acid, iopanoic acid, iocetamic acid, sodium iopodate, tyropanoic acid, lipiodol, iopydol, iofendylate, propyliodone, or a derivative of any thereof.

Embodiment 8. The tissue clearing composition of Embodiment 1, wherein the concentration of the high refractive index chemical with low water-octanol partition coefficient is about 5 to about 70 w/v %or about 10 to about 50 w/v %.

Embodiment 9. The tissue clearing composition of Embodiment 1, wherein the high refractive index chemical with high water-octanol partition coefficient is miscible with water.

Embodiment 10. The tissue clearing composition of Embodiment 1, wherein the high refractive index chemical with high water-octanol partition coefficient is 2, 2’-thiodiethanol, propylene glycol, ethylene glycol, glycerol, propylene carbonate, 1-thioglycerol, 2- (methylthio) ethanol, 2, 2’-sulfonyldiethanol, 2- (methylsulfonyl) ethanol, 3, 5’-dithia-1, 7-heptanediol, 3, 3’-thiodipropanol, 3, 6’-dithia-1, 8-octanediol, (methylthio) acetaldehyde dimethyl acetal, methyl (methylsulfinyl) methyl sulfide, or a derivative of any thereof.

Embodiment 11. The tissue clearing composition of Embodiment 1, wherein the concentration of the high refractive index chemical with high water-octanol partition coefficient is about 5 to about 70 w/v %or about 10 to about 50 w/v %.

Embodiment 12. The tissue clearing composition of Embodiment 1, the Lewis acid and/or the Lewis base is boric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, periodic acid, perchloric acid, sulfuric acid, nitric acid, methanesulfonic acid, ethylenediaminetetraacetic acid, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, potassium carbonate, potassium bicarbonate, lithium carbonate, tris (hydroxymethyl) aminomethane, 2, 2-bis (hydroxymethyl) -2, 2’, 2”-nitrilotriethanol, 3-morpholinopropane-1-sulfonic acid, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, 2- (N-morpholino) ethanesulfonic acid, or a derivative of any thereof.

Embodiment 13. The tissue clearing composition of Embodiment 1, further comprising one or more excipients selected from liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and combinations thereof.

Embodiment 14. The tissue clearing composition of Embodiment 13, wherein the liquid vehicle is a solvent, dispersion medium, and/or diluent.

Embodiment 15. The tissue clearing composition of Embodiment 14, wherein the diluent is an aqueous medium.

Embodiment 16. The tissue clearing composition of Embodiment 15, wherein the aqueous medium is water, an acid solution, or a buffered solution.

Embodiment 17. The tissue clearing composition of Embodiment 13, wherein the isotonic agents are any one or more of sodium chloride, potassium chloride, sodium lactate, calcium chloride, and glucose.

Embodiment 18. The tissue clearing composition of Embodiment 1, wherein the tissue clearing composition has a refractive index of about 1.40 to about 1.55 at 25℃.

Embodiment 19. The tissue clearing composition of Embodiment 1, wherein the tissue clearing composition has a pH of about 5 to about 10, about 5 to about 9, about 5.5 to about 8.5, or about 6 to about 8.

Embodiment 20. The tissue clearing composition of Embodiment 1, wherein the tissue clearing composition shows improved tissue clearing capacity for non-osseous tissues or organs relative to corresponding compositions.

Embodiment 21. A method of clearing tissues, comprising incubating a tissue sample in the tissue clearing composition of any one of Embodiments 1-20.

Embodiment 22. The method of Embodiment 21, wherein the tissue sample is incubated for about 0.1 to about 24 hours at a temperature of about 37℃ to about 55℃.

Embodiment 23. The method of Embodiment 21, wherein the tissue sample is a mammalian tissue sample.

Embodiment 24. The method of Embodiment 23, wherein the mammalian tissue sample is a human tissue sample.

Embodiment 25. The method of Embodiment 21, wherein the tissue sample is from non-osseous tissue or organ.

Embodiment 26. The method of Embodiment 21, wherein the tissue sample is a renal tissue sample.

Embodiment 27. The method of Embodiment 21, wherein the tissue sample is a pathological tissue sample.

Embodiment 28. The method of Embodiment 21, wherein the tissue sample is a tumor tissue sample.

Embodiment 29. The method of Embodiment 21, wherein the tissue sample is fresh, archived, or retrieved from a paraffin wax-embedded tissue.

Embodiment 30. The method of Embodiment 21, further comprising, prior to incubating the tissue sample, mixing the tissue clearing composition of any one of Embodiments 1-20.

Embodiment 31. The method of Embodiment 21, further comprising, before incubating the tissue sample, one or more of the following steps, in any order:

(i) staining the tissue sample using one or more fluorescent dyes;

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

(iii) performing immunohistochemistry, fluorescent histochemistry, fluorescent in situ hybridization or any combination thereof on the tissue sample; and/or

Embodiment 32. The method of Embodiment 21, further comprising, after incubating the tissue sample, one or more of the following steps, in any order:

(i) staining the tissue sample using one or more fluorescent dyes;

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

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

Claims

A tissue clearing composition comprising a homogenizing agent, a high refractive index chemical with high water-octanol partition coefficient, a high refractive index chemical with low water-octanol partition coefficient, and, optionally, a Lewis acid and/or Lewis base.
The tissue clearing composition of claim 1, wherein the homogenizing agent is a denaturant of proteins, denaturant of nucleic acids, a denaturant of proteins and nucleic acids, a chaotropic agent, a hydrotrope, a solubilization agent, or an emollient.
The tissue clearing composition of claim 1, wherein the homogenizing agent is N-methylglucamine, N-ethylglucamine, urea, thiourea, guanidine, guanidinium chloride, lithium perchlorate, ethylenediamine, triethanolamine, N-butylethanolamine, triethylamine, tetraethylammonium, imidazole, 1-methylimidazole, 1-ethylimidazole, 1- (3-aminopropylimidazole) , 1-allylimidazole, antipyrine, hydantoin, 1-phenyl-3-(phenylamino) pyrrolidine-2, 5-dione, ethylene carbonate, propylene carbonate, formamide, N-methylacetamide, 2-chloro-N- (hydroxymethyl) acetamide, N-dimethylacetamide, or a derivative of any thereof.
The tissue clearing composition claim 1, wherein the concentration of the homogenizing agent is about 5 to about 60 w/v %or about 10 to about 50 w/v %.
The tissue clearing composition of claim 1, wherein the high refractive index chemical with high water-octanol partition coefficient, the high refractive index chemical with low water-octanol partition coefficient, or both the high refractive index chemical with high water-octanol partition coefficient and the high refractive index chemical with low water-octanol partition coefficient have a refractive index higher than water at about 25℃ to 60℃.
The tissue clearing composition of claim 1, wherein the high refractive index chemical with high water-octanol partition coefficient, the high refractive index chemical with low water-octanol partition coefficient, or both the high refractive index chemical with high water-octanol partition coefficient and the high refractive index chemical with low water-octanol partition coefficient have a refractive index of about 1.40 to about 1.50 at about 25℃ to about 60℃.
The tissue clearing composition of claim 1, wherein the high refractive index chemical with low water-octanol partition coefficient is iohexol, iopromide, diatrizoic acid, metrizoic acid, iodamide, iotalamic acid, ioxitalamic acid, ioglicic acid, acetrizoic acid, iocarmic acid, methiodal, diodone, metrizamide, ioxaglic acid, iopamidol, iotrolan, ioversol, iopentol, iodixanol, iomeprol, iobitridol, ioxilan, iodoxamic acid, iotroxic acid, ioglycamic acid, adipiodone, iobenzamic acid, iopanoic acid, iocetamic acid, sodium iopodate, tyropanoic acid, lipiodol, iopydol, iofendylate, propyliodone, or a derivative of any thereof.
The tissue clearing composition of claim 1, wherein the concentration of the high refractive index chemical with low water-octanol partition coefficient is about 5 to about 70 w/v %or about 10 to about 50 w/v %.
The tissue clearing composition of claim 1, wherein the high refractive index chemical with high water-octanol partition coefficient is miscible with water.
The tissue clearing composition of claim 1, wherein the high refractive index chemical with high water-octanol partition coefficient is 2, 2’-thiodiethanol, propylene glycol, ethylene glycol, glycerol, propylene carbonate, 1-thioglycerol, 2- (methylthio) ethanol, 2, 2’-sulfonyldiethanol, 2- (methylsulfonyl) ethanol, 3, 5’-dithia-1, 7-heptanediol, 3, 3’-thiodipropanol, 3, 6’-dithia-1, 8-octanediol, (methylthio) acetaldehyde dimethyl acetal, methyl (methylsulfinyl) methyl sulfide, or a derivative of any thereof.
The tissue clearing composition of claim 1, wherein the concentration of the high refractive index chemical with high water-octanol partition coefficient is about 5 to about 70 w/v %or about 10 to about 50 w/v %.
The tissue clearing composition of claim 1, the Lewis acid and/or the Lewis base is boric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, periodic acid, perchloric acid, sulfuric acid, nitric acid, methanesulfonic acid, ethylenediaminetetraacetic acid, sodium carbonate, sodium bicarbonate, sodium hydroxide, potassium hydroxide, lithium hydroxide, cesium hydroxide, potassium carbonate, potassium bicarbonate, lithium carbonate, tris (hydroxymethyl) aminomethane, 2, 2-bis (hydroxymethyl) -2, 2’ , 2”-nitrilotriethanol, 3-morpholinopropane-1-sulfonic acid, 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid, 2- (N-morpholino) ethanesulfonic acid, or a derivative of any thereof.
The tissue clearing composition of claim 1, further comprising one or more excipients selected from liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and combinations thereof.
The tissue clearing composition of claim 13, wherein the liquid vehicle is a solvent, dispersion medium, and/or diluent.
The tissue clearing composition of claim 14, wherein the diluent is an aqueous medium.
The tissue clearing composition of claim 15, wherein the aqueous medium is water, an acid solution, or a buffered solution.
The tissue clearing composition of claim 13, wherein the isotonic agents are any one or more of sodium chloride, potassium chloride, sodium lactate, calcium chloride, and glucose.
The tissue clearing composition of claim 1, wherein the tissue clearing composition has a refractive index of about 1.40 to about 1.55 at 25℃.
The tissue clearing composition of claim 1, wherein the tissue clearing composition has a pH of about 5 to about 10, about 5 to about 9, about 5.5 to about 8.5, or about 6 to about 8.
The tissue clearing composition of claim 1, wherein the tissue clearing composition shows improved tissue clearing capacity for non-osseous tissues or organs relative to corresponding compositions.
A method of clearing tissues, comprising incubating a tissue sample in the tissue clearing composition of any one of claims 1-20.
The method of claim 21, wherein the tissue sample is incubated for about 0.1 to about 24 hours at a temperature of about 37℃ to about 55℃.
The method of claim 21, wherein the tissue sample is a mammalian tissue sample.
The method of claim 23, wherein the mammalian tissue sample is a human tissue sample.
The method of claim 21, wherein the tissue sample is from non-osseous tissue or organ.
The method of claim 21, wherein the tissue sample is a renal tissue sample.
The method of claim 21, wherein the tissue sample is a pathological tissue sample.
The method of claim 21, wherein the tissue sample is a tumor tissue sample.
The method of claim 21, wherein the tissue sample is fresh, archived, or retrieved from a paraffin wax-embedded tissue.
The method of claim 21, further comprising, prior to incubating the tissue sample, mixing the tissue clearing composition of any one of claims 1-20.
The method of claim 21, further comprising, before incubating the tissue sample, one or more of the following steps, in any order:

(i) staining the tissue sample using one or more fluorescent dyes;

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

(iii) performing immunohistochemistry, fluorescent histochemistry, fluorescent in situ hybridization or any combination thereof on the tissue sample; and/or

(iv) characterizing the tissue sample using transmission electron microscopy and routine pathology.
The method of claim 21, further comprising, after incubating the tissue sample, one or more of the following steps, in any order:

(i) staining the tissue sample using one or more fluorescent dyes;

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

(iii) performing immunohistochemistry, fluorescent histochemistry, fluorescent in situ hybridization or any combination thereof on the tissue sample; and/or

(iv) characterizing the tissue sample using transmission electron microscopy and routine pathology.