Degreasing and decolorizing composition for transparentizing non-deformable tissue and its application

Abstract

The present invention relates to a non-deformable degreasing and decolorizing composition for tissue clearing and its use. The present invention provides a tissue clearing degreasing and decolorizing composition comprising: a betaine surfactant containing fatty acid side chains; and an anionic surfactant containing bile acid side chains. Furthermore, the present invention provides a tissue clearing method comprising treating a tissue sample with the tissue clearing degreasing and decolorizing composition. The tissue clearing degreasing and decolorizing composition of the present invention has minimal impact on protein structure, does not cause macroscopic tissue deformation, and effectively protects against endogenous fluorescence, thereby enabling efficient and rapid tissue clearing.

Description

Technical Field

[0001] The present invention relates to the field of tissue clearing, in particular to a degreasing and decolorizing composition for non-deformable tissue clearing and application thereof. Background Art

[0002] Optical clearing involves treating tissue to reduce refractive index heterogeneity, allowing light to penetrate thick tissue for imaging and enhancing imaging quality. Tissue clearing technology enables the imaging and reconstruction of the complete three-dimensional structure of animal tissues and organs, thereby promoting a systematic understanding of the structure of complex biological systems.

[0003] The essence of tissue clearing is to remove or match the components with uneven refractive index in the tissue, thereby reducing the refraction and reflection of light when it penetrates the refractive index interface. From a macroscopic perspective, the tissue becomes transparent. Components with higher refractive index in the tissue include insoluble fibrin bundles (1.52), lipid membranes and lipid-membrane-rich organelles (1.4-1.5), cytoplasm (1.4), etc., while components with low refractive index are mainly extracellular fluid (1.33-1.37). Therefore, how to match these components with uneven refractive index to the same level is the key to the development of various tissue clearing technologies. Regardless of the refractive index matching scheme, the lipid components in the tissue must be removed to ensure the transparency of the tissue, so the use of defatting and decolorization reagents is the core step of tissue clearing.

[0004] Tissue clearing techniques can be broadly categorized based on their principles: aqueous, organic, and gel-based. Aqueous clearing techniques, such as CUBIC-X, ScaleA2, and ScaleS, primarily utilize high-concentration urea (4M) in combination with a nonionic surfactant (e.g., Triton X-100). Urea, a transmembrane protein denaturant, can form hydrogen bonds with hydrophobic regions of proteins, thereby altering their structure. This can lead to hyperhydration of otherwise insoluble or poorly solvated proteins. Hyperhydrated protein components tend to align more closely with the refractive index of the solvent. Combined with the degreasing effects of urea and surfactants, the overall tissue can achieve relatively good transparency after extended immersion. However, with this method, protein hyperhydration can lead to irreversible denaturation, resulting in a loss of intrinsic fluorescence signal, and the loosening of the fibrin structure can cause severe swelling and deformation of the tissue.

[0005] Organic solvent clearing techniques, such as iDisco and BABB, utilize water-insoluble organic reagents for degreasing, dehydration, and refractive index matching. Organic solvents typically denature proteins more strongly than aqueous reagents, making it difficult to maintain stable intrinsic fluorescence and causing significant tissue shrinkage. Most organic solvent clearing reagents are toxic and require safety precautions.

[0006] Gel clearing techniques, such as CLARITY, PACT, and SHIELD, use hydrogel grids such as polyacrylamide or epoxy resin to coat the tissue, restricting the movement of biomacromolecules within. High concentrations of strong ionic detergents (e.g., SDS) are then used to bind to lipids, and an electric field is typically used to accelerate the removal of negatively charged lipid clusters from the tissue. Because SDS is also a relatively strong protein denaturant, similar to aqueous clearing, gel clearing techniques can also result in loss of fluorescent signal and swelling and deformation of the tissue. However, the use of an electric field accelerates the process faster than most aqueous clearing methods.

[0007] Since clearing requires relatively large changes in the properties of various tissue components, almost all existing clearing methods are unable to maintain tissue scale and ensure tissue morphology, that is, they cannot eliminate the influence of the clearing method itself on the organ tissue structure. In addition, various clearing methods are not sufficient to maintain intrinsic fluorescence, which makes it impossible to detect weak signals more effectively. The maintenance of macroscopic morphology and the maintenance of intrinsic fluorescence are both essentially the protection of protein structure. The high concentration of urea commonly used in existing aqueous clearing methods, the organic solvent clearing methods and the high concentration of strong anionic detergents in the gel clearing methods are all factors that lead to protein denaturation.

[0008] The ScaleS water-based clearing technique uses a low concentration of detergent, high concentrations of urea, and high concentrations of sugars to expand tissue and then shrink it back to its original size. This is because the protein superhydration and stretching force caused by urea and the osmotic pressure caused by the high sugar concentrations reach a balance. However, this method still does not fundamentally maintain cell morphology at the microscopic level, and the high concentration of urea causes the tissue to expand to approximately twice its size during the treatment process. Furthermore, since this method does not effectively remove lipids, transparency is limited.

[0009] Therefore, it is necessary to develop a fast and efficient defatting and decolorizing formula for tissue transparentization that has no morphological effect on the tissue and maintains the activity of biological macromolecules in the tissue. Summary of the Invention

[0010] In response to the above-mentioned problems in the prior art, the main purpose of the present invention is to provide a novel degreasing and decolorizing composition for tissue clearing, which has little effect on protein structure, does not cause macroscopic tissue deformation, and has a good protective effect on endogenous fluorescence, thereby enabling efficient and rapid tissue clearing.

[0011] Another object of the present invention is to provide a method for tissue clearing using the defatting and decolorizing composition for tissue clearing according to the present invention, which can avoid deformation of the tissue during the defatting and decolorizing process while maintaining endogenous fluorescent signals and protein activity.

[0012] Furthermore, because the tissue clearing and decolorizing composition of the present invention causes little or no tissue deformation during tissue processing, animals can be transcardially perfused, further accelerating the uniformity and speed of clearing. Furthermore, because it causes little or no tissue deformation, the tissue clearing and decolorizing composition of the present invention can also be used to clear tissue sections on glass slides, enabling applications such as high-signal-to-noise ratio super-resolution imaging and large-scale rapid clearing imaging.

[0013] Therefore, in one aspect, the present invention provides a degreasing and decolorizing composition for tissue clearing, comprising: a betaine surfactant containing a fatty acid side chain; and an anionic surfactant containing a bile acid side chain.

[0014] In some embodiments, in the degreasing and decolorizing composition for tissue clearing according to the present invention, the betaine surfactant containing a fatty acid side chain can be selected from one or more of laurylamidopropyl hydroxysulfobetaine LHSB, dodecyldimethylhydroxypropylsulfobetaine and tetradecyldimethylhydroxypropylsulfobetaine.

[0015] In some embodiments, in the tissue clearing and decolorizing composition according to the present invention, the anionic surfactant containing bile acid side chains may include side chains derived from bile acid compounds. Preferably, the bile acid compound may be selected from one or more of cholic acid, lithocholic acid, deoxycholic acid, and chenodeoxycholic acid. In some specific embodiments, the anionic surfactant containing bile acid side chains may further form an ammonium salt complex with an amine compound.

[0016] In some embodiments, the defatting and decolorizing composition for tissue clearing according to the present invention may further comprise a fluidity enhancer.

[0017] In another aspect, the present invention provides a method for tissue clearing, comprising treating a tissue sample with the defatting and decolorizing composition for tissue clearing according to the present invention.

[0018] In some embodiments, the tissue clearing method according to the present invention comprises: treating the tissue sample with a first defatting and decolorizing composition; and

[0019] transferring the tissue sample treated with the first defatting and decolorizing composition to a second defatting and decolorizing composition,

[0020] The first degreasing and decolorizing composition and the second degreasing and decolorizing composition may be the same or different.

[0021] In some specific embodiments, the first degreasing and decolorizing composition is different from the second degreasing and decolorizing composition. In some preferred embodiments, the first degreasing and decolorizing composition does not contain a fluidity enhancer. In some preferred embodiments, the second degreasing and decolorizing composition contains a higher content of a betaine surfactant containing a fatty acid side chain than the first degreasing and decolorizing composition.

[0022] In some embodiments, the tissue clearing method according to the present invention further comprises placing the tissue sample treated with the defatting and decolorizing composition in an aqueous refractive index matching liquid.

[0023] In the present invention, a new aqueous transparent degreasing and decolorizing formula is provided by compounding betaine surfactants, bile acid surfactants and amines. By using the tissue clearing degreasing and decolorizing composition according to the present invention or the tissue clearing method using the same, an excellent degreasing and decolorizing effect can be obtained while not changing the tissue morphology, thereby protecting the intrinsic fluorescence. The degreasing efficiency of the tissue clearing degreasing and decolorizing composition of the present invention is much higher than that of the aqueous degreasing method commonly used in the prior art (e.g., CUBIC-X), and due to its characteristic of not changing the tissue morphology, it can be used for transcardial perfusion, which can greatly accelerate the degreasing process, for example, shortening the degreasing process of up to several weeks to less than two days. In addition, due to the non-deformation characteristics of the tissue clearing degreasing and decolorizing composition of the present invention, it can also be applied to the transparentization of tissue sections on glass slides, thereby being applied to high signal-to-noise ratio super-resolution imaging and large-scale rapid transparent imaging.

[0024] The tissue clearing degreasing and decolorizing composition of the present invention, or the tissue clearing method using the same, can be applied to, but is not limited to, ex vivo tissues, in vivo organs, thick sections, thin sections, and whole organs. In particular, due to the advantage of perfusion degreasing, it has great potential for application in large animals (e.g., non-human primates). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The drawings described herein are for illustrative purposes only of selected embodiments of the present invention and not all possible embodiments, and are not intended to limit the scope of the present invention.

[0026] Figure 1 Schematic diagrams of the chemical structures of several different types of degreasing molecules are shown, among which type 1 is urea, such as urea; type 2 is fatty acid chain anionic surfactants, such as dodecyl sulfate ion; type 3 is nonionic surfactants, such as Triton X-100; type 4 is bile acid chain zwitterionic surfactants, such as 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS), such as laurylamidopropyl hydroxysulfobetaine (LHSB); type 5 is bile acid anionic surfactants, such as deoxycholate ion; type 6 is fatty acid chain zwitterionic surfactants (betaine surfactants).

[0027] Figure 2 The effects of several different types of delipidated molecules on tissue size are shown, with 1 to 6 being Figure 1 As shown in the figure, it can be seen that 5 types of bile acid anionic surfactants, including N,N-diisopropylethanolamine-bile salt (DIDC), do not cause tissue deformation; 6 types of betaine surfactants also do not cause tissue deformation, while the other types all cause varying degrees of tissue expansion.

[0028] Figure 3 The results of the determination of the degreasing ability of several different types of degreasing molecules are shown, among which 1 to 6 are respectively as follows Figure 1 shown.

[0029] Figure 4 This is a graph showing the results of the degreasing effect of tissue clearing using the degreasing and decolorizing composition according to an embodiment of the present invention, compared with the clearing method of the related art.

[0030] Figure 5 Graphs showing the results of tissue deformation, degreasing efficiency, and fluorescent signal retention in tissue clearing using a degreasing and decolorizing composition according to an embodiment of the present invention compared to a conventional clearing method.

[0031] Figure 6 Photographs showing the results of whole-organ clearing of mice using a degreasing and decolorizing composition according to an embodiment of the present invention. The top image, from left to right, shows the mouse brain before, after, and after refractive index matching. The bottom image shows the morphology of the cleared mouse heart, liver, spleen, lungs, kidneys, and brain.

[0032] Figure 7Photographs showing the imaging results of 200-μm-thick Thy1-YFP mouse brain sections that had been cleared. (a) Refractive index matching only without destaining agent treatment; b) Treatment with a rapid pre-destaining reagent and refractive index matching; c) Treatment with a rapid pre-destaining reagent and a final destaining reagent and refractive index matching; d) Maximum intensity projection images of two viewing angles of 3D imaging, with clearing treatment on the left and before clearing on the right; e) Brightfield image after complete clearing; f) Brightfield image with refractive index matching only; g) Brightfield image of a 100-μm mouse brain section without any clearing treatment. DETAILED DESCRIPTION

[0033] Hereinafter, the embodiments of the present invention will be described in more detail to help understand the present invention. It should be understood that the description of these embodiments is only for illustrative purposes and is not intended to limit the scope of protection claimed by the present invention in any way.

[0034] Since transparency requires relatively large changes in the properties of various tissue components, almost all transparency methods in the prior art are unable to maintain tissue scale and ensure tissue morphology, that is, they cannot eliminate the influence of the transparency method itself on the organ tissue structure. In addition, various transparency methods in the prior art are insufficient for maintaining intrinsic fluorescence, which makes it impossible to detect weak signals more effectively. The maintenance of macroscopic morphology and the maintenance of intrinsic fluorescence are both essentially protection of protein structure. High-concentration urea commonly used in existing aqueous transparency methods, organic solvent transparency methods, and high-concentration strong anionic detergents in gel transparency methods are all factors that lead to protein denaturation.

[0035] After in-depth research on the degreasing ability, effects on tissue morphology, and endogenous fluorescent proteins of various surfactants, the inventors found that by compounding betaine surfactants and bile acid surfactants, not only can degreasing and decolorization methods that are significantly superior to those of the prior art be achieved, but also, especially when urea and urea analogs or reagents containing amide bonds with superhydration effects and strong ionic surfactants such as sodium dodecyl sulfate (SDS) are excluded, no tissue deformation will be caused.

[0036] Figure 1Schematic diagrams of the chemical structures of several different types of degreasing molecules are shown, among which type 1 is urea, such as urea; type 2 is fatty acid chain anionic surfactants, such as dodecyl sulfate ion; type 3 is nonionic surfactants, such as Triton X-100; type 4 is bile acid chain zwitterionic surfactants, such as 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS); type 5 is bile acid anionic surfactants, such as deoxycholate ion; type 6 is fatty acid chain zwitterionic surfactants (betaine surfactants), such as laurylamidopropyl hydroxysulfobetaine (LHSB). Figure 2 The effects of several different types of delipidation molecules on tissue size are shown, with 1 to 6 being Figure 1 As shown in Figures 1 to 6, it can be seen that the bile acid anionic surfactants of type 5, including N,N-diisopropylethanolamine-bile salt (DIDC), do not cause tissue deformation; the betaine surfactants of type 6 also do not cause tissue deformation, while the other types all cause varying degrees of tissue expansion.

[0037] Therefore, in one aspect, the present invention provides a degreasing and decolorizing composition for tissue clearing, comprising: a betaine surfactant containing a fatty acid side chain; and an anionic surfactant containing a bile acid side chain.

[0038] Betaine surfactants containing fatty acid side chains have rapid tissue degreasing effects, do not cause tissue deformation, have good intrinsic fluorescence maintenance capabilities, are highly chemically stable and extremely high in solubility, and can solubilize other functional decolorizing agents. In some embodiments, the betaine surfactant containing fatty acid side chains can be selected from one or more of lauramidopropyl hydroxysulfobetaine LHSB, dodecyldimethylhydroxypropylsulfobetaine, and tetradecyldimethylhydroxypropylsulfobetaine, but are not limited thereto.

[0039] In some embodiments, the composition may comprise from about 1% w / v to about 50% w / v of a betaine surfactant containing a fatty acid side chain. Preferably, the content of the betaine surfactant containing a fatty acid side chain is from about 1% w / v to about 50% w / v, more preferably from about 3% w / v to about 30% w / v, and most preferably from about 3% w / v to about 25% w / v.

[0040] In some embodiments, the anionic surfactant containing bile acid side chains may include side chains derived from bile acid compounds. Preferably, the bile acid compound may include one or more of bile acid, lithocholic acid, deoxycholic acid or chenodeoxycholic acid, but is not limited thereto. In some specific embodiments, the anionic surfactant containing bile acid side chains may further form an ammonium salt complex with an amine compound. In particular, by mixing an alkaline amine decolorant (e.g., N,N-diisopropylethanolamine) with a bile acid compound, a slightly alkaline amine-cholate complex is obtained, which has the ability to quickly degrease and quickly decolorize, and does not introduce other invalid ions, such as sodium ions in sodium deoxycholate. Preferably, the amine compound is selected from one or more of N,N-diisopropylethanolamine, N-butyldiethanolamine, N,N',N,N'-tetra-hydroxypropylethylaminetetraacetic acid (Quadrol), 1-(3-aminopropyl)imidazole, triethanolamine and N-methyldiethanolamine, but is not limited thereto.

[0041] In some embodiments, the composition may comprise about 1% w / v to 30% w / v of an anionic surfactant containing a bile acid side chain. Preferably, the content of the anionic surfactant containing a bile acid side chain is about 1% w / v to about 30% w / v, more preferably about 5% w / v to about 25% w / v, and most preferably about 15% w / v to about 25% w / v.

[0042] In some embodiments, the anionic surfactant containing bile acid side chains can further form an ammonium salt complex with an amine compound. Preferably, the amine compound is selected from one or more of N,N-diisopropylethanolamine, N-butyldiethanolamine, Quadrol, 1-(3-aminopropyl)imidazole, triethanolamine and N-methyldiethanolamine, but is not limited thereto. In some specific embodiments, based on the degreasing and decolorizing composition for tissue clearing, the concentration of the anionic surfactant containing bile acid side chains is 1% w / v to 15% w / v, preferably 3% w / v to 15% w / v. In some specific embodiments, based on the degreasing and decolorizing composition for tissue clearing, the concentration of the amine compound is 1% w / v to 20% w / v, preferably 3% w / v to 12% w / v.

[0043] In some embodiments, the degreasing and decolorizing composition for tissue clearing according to the present invention may further comprise a fluidity enhancer. In some embodiments, the fluidity enhancer may be selected from thiourea compounds and urea, preferably, the thiourea compound comprises thiourea. Adding a fluidity enhancer to the degreasing and decolorizing composition can increase cell membrane fluidity, enhance lipid solubility, and help maintain tissue morphology during the initial stages of degreasing. In a preferred embodiment, the degreasing and decolorizing composition comprises 5% w / v to 25% w / v of the fluidity enhancer.

[0044] In some embodiments, the degreasing and decolorizing composition according to the present invention does not contain urea or urea analogs. In some embodiments, the degreasing and decolorizing composition according to the present invention does not contain strong ionic surfactants such as sodium dodecyl sulfate (SDS).

[0045] In another aspect, the present invention provides a method for tissue clearing, comprising treating a tissue sample with the defatting and decolorizing composition for tissue clearing according to the present invention.

[0046] In some embodiments, the tissue clearing method according to the present invention comprises: treating the tissue sample with a first defatting and decolorizing composition; and

[0047] transferring the tissue sample treated with the first defatting and decolorizing composition to a second defatting and decolorizing composition,

[0048] The first degreasing and decolorizing composition and the second degreasing and decolorizing composition may be the same or different.

[0049] In some specific embodiments, the first degreasing and decolorizing composition is different from the second degreasing and decolorizing composition. In some preferred embodiments, the first degreasing and decolorizing composition does not contain a fluidity enhancer. In some preferred embodiments, the second degreasing and decolorizing composition contains a higher content of a betaine surfactant containing a fatty acid side chain than the first degreasing and decolorizing composition.

[0050] In a particularly preferred embodiment, the first degreasing and decolorizing composition is a composition for rapid pre-degreasing and decolorizing, comprising about 12% w / v sodium deoxycholate, about 10% w / v N,N-diisopropylethanolamine, and about 3% w / v lauramidopropyl hydroxysulfobetaine. In a particularly preferred embodiment, the second degreasing and decolorizing composition is a composition for final degreasing and decolorizing, comprising about 12% w / v sodium deoxycholate, about 10% w / v N,N-diisopropylethanolamine, about 25% w / v lauramidopropyl hydroxysulfobetaine, and about 16% w / v thiourea. In certain preferred embodiments, a first degreasing and decolorizing composition comprising about 12% w / v sodium deoxycholate, about 10% w / v N,N-diisopropylethanolamine, and about 3% w / v lauramidopropyl hydroxysulfonate can be used for rapid pre-degreasing and decolorizing, which can quickly degrease the tissue in the initial stage and reach saturation relatively quickly; and after the rapid pre-degreasing and decolorizing with the first degreasing and decolorizing composition, a second degreasing and decolorizing composition comprising about 12% w / v sodium deoxycholate, about 10% w / v N,N-diisopropylethanolamine, about 25% w / v lauramidopropyl hydroxysulfonate and about 16% w / v thiourea is used for final degreasing and decolorizing, thereby maximally removing the remaining lipids and pigments.

[0051] In some embodiments, the tissue clearing method according to the present invention further comprises placing the tissue sample treated with the defatting and decolorizing composition in an aqueous refractive index matching liquid.

[0052] In some embodiments, the tissue clearing method may include the following steps:

[0053] 1) Obtaining a tissue sample to be cleared;

[0054] 2) performing at least one first degreasing and decolorizing step on the obtained tissue sample using a first degreasing and decolorizing composition; preferably, the first degreasing and decolorizing step is performed at about 34° C. to 45° C. for about 8 to 24 hours;

[0055] 3) performing a second degreasing and decolorizing of the tissue sample after the first degreasing and decolorizing using a second degreasing and decolorizing composition; preferably, the second degreasing and decolorizing is performed at 34° C. to 45° C. for about 8 to 24 hours;

[0056] 4) Wash the tissue sample after the second defatting and decolorization, and transfer it into an aqueous refractive index matching solution for storage.

[0057] In some embodiments, the first defatting and decolorizing can be performed at least once, for example, once, twice, or three times or more.

[0058] In some embodiments, the tissue sample includes, but is not limited to, ex vivo tissue, in vivo organ, thick slice, thin slice, or whole organ. In some embodiments, the tissue sample is obtained from a mammal, such as, but not limited to, a cow, horse, pig, dog, sheep, rat, mouse, or non-human primate.

[0059] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention. Those skilled in the art can make various modifications and variations without departing from the scope and spirit of the present invention, and the modifications and variations also fall within the scope of the present invention.

[0060] Example

[0061] Materials and methods

[0062] Experimental Animals: Adult male or female C57BL / 6N mice aged 8-16 weeks were used. Mice were anesthetized with an overdose of sodium pentobarbital (>100 mg / kg) before perfusion.

[0063] Reagents, materials, and instruments:

[0064] 1) Determination of phospholipid concentration: Phospholipid detection kit (MAK122, Sigma-Aldrich); Nanodrop 3000 micro-volume spectrophotometer (ThermoFisher); EnVision multi-function microplate reader (Perkin).

[0065] 2) Tissue size bright field imaging, fluorescence imaging, etc.: fully automatic digital slide scanning system Axio Z1 (Zeiss); spinning disk confocal microscope Dragonfly (Andor).

[0066] 3) Main reagents: deoxycholic acid (D2510, Sigma-Aldrich), sodium deoxycholate (264103, Sigma-Aldrich), N,N-diisopropylethanolamine (D838068, Macklin), N-butyldiethanolamine (N802391, Macklin), 1-(3-aminopropyl)imidazole (272264, Sigma-Aldrich), lauramidopropyl hydroxysulfobetaine (LHSB, Greensense), 3-[3- (Choleamidopropyl) dimethylamino] propanesulfonic acid inner salt (CHAPS-RO, Sigma-Aldrich), sodium dodecyl sulfate (V900859, Sigma-Aldrich), sodium dodecylbenzenesulfonate (PHR1305, Sigma-Aldrich), TritonX-100 (T8787, Sigma-Aldrich), urea (U5128, Sigma-Aldrich), thiourea (T7875, Sigma-Aldrich).

[0067] Example 1. Preparation of degreasing and decolorizing composition

[0068] Figure 1 Schematic diagrams of the chemical structures of several different degreasing molecules are shown. Among them, category 1 is urea, such as urea; category 2 is fatty acid chain anionic surfactants, such as lauryl sulfate ion; category 3 is nonionic surfactants, such as Triton X-100; category 4 is fatty acid chain zwitterionic surfactants (betaine surfactants), such as laurylamidopropyl hydroxysulfobetaine (LHSB); category 5 is bile acid anionic surfactants, such as deoxycholate ion; and category 6 is bile acid chain zwitterionic surfactants, such as 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS). The inventors tested the degreasing ability of these degreasing molecules and their effects on tissue size.

[0069] After post-fixing the mouse brain for 4 hours, the mouse brain was cut into 100 μm slices using a vibrating microtome, and temporary slides were prepared with PBS and photographed with a bright field camera to extract the brain slice area; the brain slices were immersed in the corresponding reagent at 37°C for 24 hours, and temporary slides were prepared with the corresponding reagent and photographed with a bright field camera to extract the brain slice area and obtain the relative area. Figure 2 , which shows the effect of degreasing and decolorizing the tissue size after using the above-mentioned different types of degreasing molecules. 1 to 6 are Figure 1 As shown in Figures 1 to 6. It can be seen that the bile acid anionic surfactants of type 5, including ethanolamine-bile salt (DIDC), do not cause tissue deformation; the betaine surfactants of type 6 also do not cause tissue deformation, while the other types all cause varying degrees of tissue expansion.

[0070] Furthermore, the degreasing ability of different degreasing agents was evaluated by measuring the maximum phospholipid concentration (c(PL)) of brain tissue sections after immersion in different degreasing agents for 2 hours and 32 hours. Figure 3 It can be seen that bile acid anionic surfactants or ethanolamine-bile salts of type 5 and betaine surfactants of type 6 have the highest degreasing ability and degreasing rate. Therefore, in order to achieve the best degreasing effect, the inventors compounded these two types of degreasing agents.

[0071] The degreasing and decolorizing composition of the present invention is prepared by mixing sodium deoxycholate, N,N-diisopropylethanolamine, lauramidopropylhydroxysulfobetaine, and optionally thiourea in a solution. Specifically, a degreasing and decolorizing composition comprising 12% w / v sodium deoxycholate, 10% w / v N,N-diisopropylethanolamine, and 3% w / v lauramidopropylhydroxysulfobetaine was prepared as a rapid pre-degreasing and decolorizing reagent, which rapidly degreases tissues in the initial stage and reaches saturation more quickly. Furthermore, a degreasing and decolorizing composition comprising 12% w / v sodium deoxycholate, 10% w / v N,N-diisopropylethanolamine, 25% w / v lauramidopropylhydroxysulfobetaine, and 16% w / v thiourea was prepared as a final degreasing and decolorizing reagent, which maximizes the removal of residual lipids and pigments.

[0072] Example 2. Passive defatting, decolorization and clearing of thick sections

[0073] By using the rapid pre-degreasing and decolorizing reagent and the final degreasing and decolorizing reagent prepared in Example 1, the thick tissue sections were passively degreased, decolorized, and transparentized. The specific steps are as follows:

[0074] 1- Obtain 300 μm thick mouse brain slices using a vibratome or cryosectioner. Place the fixed tissue slices in a rapid pre-defat decolorizing reagent at 37°C for 12 h.

[0075] 2- Place in new rapid pre-degreasing and decolorizing reagent and continue treatment at 37℃ for 12 hours.

[0076] 3- Transfer the tissue sections to the final defatting and decolorizing reagent at 37°C for 12 hours.

[0077] 4- Place in new final defatting and decolorizing reagent and continue treatment for 12 hours.

[0078] 5- Rinse three times with PBS at room temperature, 10 minutes each time.

[0079] 6- Transfer to aqueous refractive index matching solution and mount the slides using aqueous refractive index matching solution.

[0080] Example 3. Degreasing, decolorization and transparentization on slides

[0081] The tissue sections attached to the slides were degreased, decolorized, and transparentized using the rapid pre-decolorization reagent and the final decolorization reagent prepared in Example 1. The specific steps are as follows:

[0082] 1- Obtain 50 μm thick mouse brain slices by vibratome or cryosectioning, attach the tissue slices to glass slides and place the slides in PBS to thoroughly wash off the embedding medium 2-3 times.

[0083] 2- Place the slides in a staining jar, add rapid pre-decolorization reagent, and incubate at 37°C for 1 hour.

[0084] 3-Replace the reagent in the staining jar with the final decolorizing reagent and incubate at 37°C for 1 hour.

[0085] 4- Wash the slides thoroughly in PBS 2-3 times to remove the decolorizing agent.

[0086] 5- Remove excess PBS and mount the slides with aqueous refractive index matching solution.

[0087] Example 4: In vivo whole-organ clearing by transcardial perfusion

[0088] By using the rapid pre-defatting and decolorizing reagent and the final defatting and decolorizing reagent prepared in Example 1, mice were subjected to whole-organ clearing by transcardial perfusion. The specific steps are as follows:

[0089] 1- Perfuse the mouse transcardially with ice-cold heparin-saline solution until the blood is fully drained.

[0090] 2- Mice were transcardially perfused with 4% paraformaldehyde (containing 0.01 M phosphate buffer) for 15 min.

[0091] 3-Perfuse the mouse transcardially with 15 ml of rapid pre-delipidation and decolorization reagent, and establish a circulation to recycle the fluid and continue perfusing for 8 hours.

[0092] 4-Replace with new 15ml rapid pre-degreasing and decolorizing reagent and circulate the perfusion for 8h.

[0093] 5-Replace 15 ml of the final degreasing and decolorizing reagent and circulate the perfusion for 8 hours.

[0094] 6-Replace with new 15ml of final defatting and decolorizing reagent and circulate the perfusion for 8h.

[0095] 7- Remove the organs and rinse with PBS to remove the decolorizing agent.

[0096] 8- Immerse the organ in aqueous refractive index matching fluid.

[0097] Figure 6 Photographs show the results of whole-organ clearing of mice using a degreasing and decolorizing composition according to an embodiment of the present invention. The top image shows, from left to right, photographs of a mouse brain before, after, and after refractive index matching. The bottom image shows the morphology of the mouse heart, liver, spleen, lungs, kidneys, and brain after clearing. After degreasing and decolorizing by perfusion, refractive index matching was performed using a refractive index matching agent, achieving whole-organ clearing.

[0098] Example 5: Degreasing effect of the rapid degreasing and decolorizing composition

[0099] We further compared the rapid degreasing and decolorizing agent of the present invention with the commonly used degreasing and decolorizing reagents and methods in the prior art in terms of degreasing rate, tissue deformation, and protection of fluorescent signals.

[0100] Mouse brain tissue, post-fixed for one day and dehydrated with 30% sucrose for two days, was cut into 300-μm sections using a freezing microtome and weighed immediately. Tissue clearing was performed by adding the rapid pre-defatting and decolorizing reagent prepared in Example 1 (containing 12% w / v sodium deoxycholate, 10% w / v N,N-diisopropylethanolamine, and 3% w / v lauramidopropyl hydroxysulfobetaine) equivalent to 20 times the mass of the mouse brain sections. In addition, the same mouse brain slices were treated with CUBIC-X degreasing agent (25% w / v urea, 5% w / v Quadrol (N,N',N'-tetra-hydroxypropylethylaminetetraacetic acid), 15% w / v TritonX-100) or ScaleS clearing reagent (24% w / v urea, 10% w / v glycerol, 40% w / v sorbitol, 0.2% w / v TritonX-100, 15% w / v DMSO). At different time points, the mouse brain slices treated with the rapid degreasing and color removal agent of the present invention, CUBIC-X degreasing agent or ScaleS clearing reagent were sampled, and the relative area changes, supernatant phospholipid content and relative fluorescence changes of the three groups of brain slices were measured. For fluorescence measurement, thy1-YFP mice were used, and the green fluorescence intensity of the fluorescently labeled cells was measured.

[0101] Figure 4 This graph shows the results of tissue clearing using a degreasing and decolorizing composition according to an embodiment of the present invention, compared to prior art clearing methods. As can be seen, the rapid pre-degreasing and decolorizing reagent of Example 1 achieves significantly faster degreasing rates than the degreasing and decolorizing reagents used in the classic aqueous clearing methods CUBIC and ScaleS. Figure 5 Graphs showing tissue deformation, degreasing efficiency, and fluorescence signal preservation during tissue clearing using a degreasing and decolorizing composition according to embodiments of the present invention, compared to prior art clearing methods. As can be seen, compared to the degreasing and decolorizing reagents used in the classic aqueous clearing methods CUBIC and ScaleS, the rapid pre-degreasing and decolorizing reagent of Example 1 does not cause tissue deformation in brain slices during the degreasing and decolorizing process (left), exhibits higher degreasing efficiency (center), and better preserves the GFP fluorescence signal (right).

[0102] Example 6: Effect of the degreasing and decolorizing composition on imaging effects

[0103] The improvement in imaging effect of tissue clearing using the degreasing and decolorizing agent of the present invention was further tested. Tissue clearing was performed using the rapid pre-degreasing and decolorizing reagent and the final degreasing and decolorizing reagent prepared in Example 1. Figure 7 Photographs showing the results of imaging cleared 200-μm-thick Thy1-YFP mouse brain sections. Figure 7Images a, b, c, and d were taken using a spinning disk confocal microscope. Images a, b, and c were taken using a 10x air objective, and images d were taken using a 20x air objective. Images were taken using the 488 / 520 green fluorescence channel. Figure 7 Images e, f, and g were captured using a wide-field microscope with bright-field illumination and a camera.

[0104] exist Figure 7 Among them, a) only refractive index matching without degreasing and decolorizing agent treatment; b) treatment with rapid pre-degreasing and decolorizing agent and refractive index matching; c) treatment with rapid pre-degreasing and decolorizing agent and final degreasing and decolorizing agent and refractive index matching (complete transparent treatment); d) maximum brightness projection images of two viewing angles of three-dimensional imaging, the left side is after transparent treatment, and the right side is before transparent treatment; e) bright field image after complete transparent treatment; f) bright field image with only refractive index matching; g) bright field image of 100 micron mouse brain slice without any transparent treatment. Figure 7 It can be seen that compared with tissue sections that are not subjected to any transparent treatment or are only subjected to a refractive index matching treatment, the imaging effect after treatment with the degreasing and decolorizing agent of the present invention is significantly improved; in addition, compared with only using a rapid pre-degreasing and decolorizing reagent, further using a final degreasing and decolorizing reagent after treatment with a rapid pre-degreasing and decolorizing reagent achieves a better imaging effect.

Claims

1. A degreasing and decolorizing composition for transparentizing non-deformable tissue, comprising: Betaine surfactants containing fatty acid side chains; and Anionic surfactants containing bile acid side chains, in, The betaine surfactant containing a fatty acid side chain is selected from one or more of laurylamidopropyl hydroxysulfobetaine, dodecyldimethylhydroxypropylsulfobetaine, tetradecyldimethylhydroxypropylsulfobetaine, N,N-dimethyl-N-dodecylglycine betaine and lauryldimethylsulfobetaine. Wherein, the anionic surfactant containing bile acid side chains comprises side chains derived from bile acid compounds, Wherein, the degreasing and decolorizing composition for tissue clearing comprises 1% w / v to 50% w / v of the betaine surfactant containing a fatty acid side chain, and Wherein, the degreasing and decolorizing composition for tissue clearing contains 0.5% w / v to 35% w / v of the anionic surfactant containing bile acid side chains.

2. The degreasing and decolorizing composition for transparentizing non-deformable tissue according to claim 1, wherein The bile acid compound is selected from one or more of cholic acid, lithocholic acid, deoxycholic acid and chenodeoxycholic acid.

3. The degreasing and decolorizing composition for transparentizing non-deformable tissue according to claim 1 or 2, wherein The anionic surfactant containing bile acid side chains further forms an ammonium salt complex with an amine compound.

4. The degreasing and decolorizing composition for transparentizing non-deformable tissue according to claim 3, wherein The amine compound is selected from one or more of N,N-diisopropylethanolamine, N-butyldiethanolamine, N,N',N,N'-tetra-hydroxypropylethylaminetetraacetic acid, 1-(3-aminopropyl)imidazole, triethanolamine and N-methyldiethanolamine.

5. The degreasing and decolorizing composition for transparentizing non-deformable tissue according to claim 1 or 2, wherein The anionic surfactant containing bile acid side chains further forms an ammonium salt complex with the amine compound; Based on the tissue clearing and decolorizing composition, the concentration of the anionic surfactant containing a bile acid side chain is 1% w / v to 15% w / v, and the concentration of the amine compound is 1% w / v to 20% w / v.

6. The degreasing and decolorizing composition for transparentizing non-deformable tissue according to claim 1 or 2, wherein The composition also includes a flow enhancer.

7. The degreasing and decolorizing composition for transparentizing non-deformable tissue according to claim 6, wherein The fluidity enhancer is selected from thiourea compounds and urea.

8. The degreasing and decolorizing composition for transparentizing non-deformable tissue according to claim 7, wherein The thiourea compound includes thiourea.

9. The degreasing and decolorizing composition for transparentizing non-deformable tissue according to claim 8, wherein The composition comprises 5% w / v to 25% w / v of the flowability enhancer. 10 . A tissue clearing method, comprising treating a tissue sample with the non-deformable tissue clearing defatting and decolorizing composition according to claim 1 .

11. The tissue clearing method according to claim 10, wherein: The method comprises: treating the tissue sample with a first defatting and decolorizing composition; and transferring the tissue sample treated with the first defatting and decolorizing composition to a second defatting and decolorizing composition, The first degreasing and decolorizing composition and the second degreasing and decolorizing composition are the same or different, and the first degreasing and decolorizing composition and the second degreasing and decolorizing composition are each the degreasing and decolorizing composition for transparentizing amorphous tissue according to any one of claims 1 to 9.

12. The tissue clearing method according to claim 11, wherein: The second degreasing and decolorizing composition is different from the first degreasing and decolorizing composition.

13. The tissue clearing method according to claim 11, wherein: The second degreasing and decolorizing composition contains a higher content of a betaine-based surfactant containing a fatty acid side chain than the first degreasing and decolorizing composition.

14. The tissue clearing method according to claim 10, wherein: The method further comprises maintaining the tissue sample treated with the defatting and decolorizing composition in an aqueous refractive index matching fluid.

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