WO2024160125A1 - Degreasing and decolorizing composition for non-deformation tissue clearing and use thereof - Google Patents

Abstract

A degreasing and decolorizing composition for tissue clearing, comprising: a betaine-based surfactant containing a fatty acid side chain; and an anionic surfactant containing a cholic acid side chain. A tissue clearing method, comprising: treating a tissue sample by using the degreasing and decolorizing composition for tissue clearing.

Description

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

The invention relates to the field of tissue clearing, and in particular to a degreasing and decolorizing composition for non-transformable tissue clearing and application thereof.

Background Art

Optical clearing refers to the process of treating tissues to reduce the inhomogeneity of the refractive index within the tissues, thereby allowing light to penetrate thick tissues for imaging and enhancing the imaging effect. Tissue clearing technology makes it possible to image and reconstruct the complete three-dimensional structure of animal tissues and organs, thereby promoting a systematic understanding of how complex biological systems are constructed.

The essence of tissue transparency is to remove or match the components with uneven refractive index in the tissue, thereby reducing the refraction and reflection of light when penetrating 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 transparency 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 transparency.

Tissue clearing technology can be roughly divided into three categories according to the principle: aqueous clearing technology, organic solvent clearing technology and gel clearing technology. Aqueous clearing technology, such as CUBIC-X, ScaleA2, ScaleS and other technologies, mainly uses high concentration urea (4M) in combination with non-ionic surfactants (e.g., TritonX-100) for clearing. Urea, as a transmembrane protein denaturant, can form hydrogen bonds with the hydrophobic part of the protein structure to change the protein structure, thereby making the protein that is insoluble or has a low degree of solvation highly solvated, or called hyperhydration. The hyperhydrated protein component will tend to be consistent with the refractive index of the solvent. Combining the degreasing effects of urea and surfactants, the overall tissue can achieve better transparency after a longer immersion time. However, using this method, protein hyperhydration will lead to irreversible denaturation of the protein and thus the loss of endogenous fluorescence signals, and the loosening of the fibrin structure will lead to severe swelling and deformation of the tissue as a whole.

Organic solvent clearing techniques, such as iDisco and BABB, use water-insoluble organic reagents to Degreasing, dehydration and refractive index matching. Generally, organic solvents denature proteins more strongly than aqueous reagents, and it is difficult to maintain the stability of intrinsic fluorescence, and it will cause more severe tissue shrinkage. Most organic solvent clearing reagents are toxic and require safety precautions.

Gel clearing techniques, such as CLARITY, PACT, SHIELD, etc., use hydrogel grids such as polyacrylamide or epoxy resin to coat tissues, restrict the movement of biomacromolecules, and then use high concentrations of ionic strong detergents (e.g., SDS) to bind to lipids, and usually use electric fields to accelerate the negatively charged lipid groups from the tissue. Since SDS is also a relatively strong protein denaturant, similar to the effect of aqueous clearing, gel clearing technology will also cause loss of fluorescent signals and expansion and deformation of tissues, but the speed is faster than most aqueous clearing methods after using electric fields.

Since transparency requires relatively large changes in the properties of various tissue components, almost all existing transparency methods 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 are not sufficient to maintain intrinsic fluorescence, so weak signals cannot be detected more effectively. The maintenance of macroscopic morphology and the maintenance of intrinsic fluorescence are both essentially the 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.

The water-based transparent ScaleS technology uses low-concentration detergents and high-concentration urea and high-concentration sugars to shrink the tissue to its original size after it expands. This is because the protein superhydration stretching force caused by urea and the osmotic pressure caused by high-concentration sugars reach a balance. However, this method still does not maintain the morphology of cells in principle at the microscopic level, and there is a process in the treatment process where high-concentration urea causes the tissue to expand to about twice its size. In addition, this method has limited transparency because it does not effectively remove lipids.

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

In view of the above problems in the prior art, the main purpose of the present invention is to provide a novel defatting 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 being able to achieve tissue clearing efficiently and quickly.

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

Moreover, since there is no or almost no tissue deformation during the tissue treatment process using the tissue clearing and decolorizing composition of the present invention, the animal can be perfused transcardially, thereby further accelerating the uniformity and speed of clearing. In addition, since there is no or almost no tissue deformation, the tissue clearing and decolorizing composition of the present invention can also be used for clearing tissue sections on glass slides, so as to be applied to high signal-to-noise ratio super-resolution imaging and large-scale rapid clearing imaging.

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

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.

In some embodiments, in the degreasing and decolorizing composition for tissue clearing according to the present invention, the anionic surfactant containing bile acid side chains may contain side chains derived from bile acid compounds. Preferably, the bile acid compounds may be selected from one or more of bile 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.

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

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.

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

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 may be the same or different.

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.

In some embodiments, the tissue clearing method according to the present invention further comprises treating the tissue with the defatting and decolorizing composition. The treated tissue samples were maintained in an aqueous refractive index matching fluid.

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 transparent degreasing and decolorizing composition according to the present invention or the tissue transparent method using the same, an excellent degreasing and decolorizing effect can be obtained, while not changing the tissue morphology, thereby protecting the endogenous fluorescence. The degreasing efficiency of the tissue transparent degreasing and decolorizing composition of the present invention is much higher than 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 several weeks to less than two days. In addition, due to the non-deformation characteristics of the tissue transparent 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.

The tissue clearing degreasing and decolorizing composition of the present invention or the tissue clearing method using the same can be applied to in vitro tissues, in vivo organs, thick slices, thin slices, and whole organs, but is not limited thereto. In particular, due to the advantage of being able to perform perfusion degreasing, it has great application potential in large animals (e.g., non-human primates) and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

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

Figure 1 shows schematic diagrams of the chemical structures of several different types of degreasing molecules, among which Class 1 is urea, such as urea; Class 2 is fatty acid chain anionic surfactants, such as dodecyl sulfate ion; Class 3 is non-ionic surfactants, such as Triton X-100; Class 4 is bile acid chain zwitterionic surfactants, such as 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS), such as lauryl amide propyl hydroxysulfonate (LHSB); Class 5 is bile acid anionic surfactants, such as deoxycholate ion; Class 6 is fatty acid chain zwitterionic surfactants (betaine surfactants).

Figure 2 shows the effects of several different types of delipidation molecules on tissue size, where 1 to 6 are shown in Figure 1. 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 other types all cause tissue expansion to varying degrees.

FIG. 3 shows the results of measuring the degreasing ability of several different types of degreasing molecules, wherein 1 to 6 are respectively shown in FIG. 1 .

FIG. 4 is a diagram showing the use of the degreasing and degreasing method according to an embodiment of the present invention compared with the prior art transparent method. A graph showing the results of the degreasing effect of the color composition on tissue clearing.

5 is a graph showing the results of tissue deformation, degreasing efficiency, and fluorescent signal retention for tissue clearing using a degreasing and decolorizing composition according to an embodiment of the present invention compared to a clearing method of the prior art.

Figure 6 shows photos of the results of whole-organ transparentization of mice using the defatting and decolorizing composition according to an embodiment of the present invention. The upper figure shows, from left to right, photos of the mouse brain before defatting and decolorizing, after defatting and decolorizing, and after refractive index matching; the lower figure shows photos of the morphology of the mouse heart, liver, spleen, lung, kidney, and brain after transparentization.

Figure 7 shows the results of imaging a 200 μm thick Thy1-YFP mouse brain slice that was cleared. Among them, a) only refractive index matching was performed 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; d) maximum brightness projection images of two viewing angles of three-dimensional imaging, the left side is after clearing treatment, and the right side is before clearing treatment; e) bright field image after complete clearing treatment; f) bright field image with only refractive index matching; g) bright field image of a 100 μm mouse brain slice without any clearing treatment.

DETAILED DESCRIPTION

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.

Since transparency requires relatively large changes in the properties of various tissue components, almost all transparency methods in the prior art cannot maintain tissue scale and ensure tissue morphology, that is, it is impossible to eliminate the influence of the transparency method itself on the organ tissue structure. In addition, various transparency methods in the prior art are not sufficient to maintain intrinsic fluorescence, so weak signals cannot be detected 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.

The inventors conducted in-depth studies on the degreasing ability, effects on tissue morphology, and effects on endogenous fluorescent proteins of various surfactants and found that by compounding betaine surfactants and bile acid surfactants, not only can degreasing and decolorization be achieved that is significantly superior to the methods of the prior art, 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, tissue deformation will not be caused.

Figure 1 shows the chemical structures of several different types of degreasing molecules, of which one type is urea, e.g. urea; Class 2 is fatty acid chain anionic surfactants, such as dodecyl sulfate ion; Class 3 is nonionic surfactants, such as Triton X-100; Class 4 is bile acid chain zwitterionic surfactants, such as 3-[3-(cholamidopropyl)dimethylamino]propanesulfonic acid inner salt (CHAPS); Class 5 is bile acid anionic surfactants, such as deoxycholate ion; Class 6 is fatty acid chain zwitterionic surfactants (betaine surfactants), such as lauryl amide propyl hydroxysulfobetaine (LHSB). Figure 2 shows the effects of several different types of delipidated molecules on tissue size, where 1 to 6 are Class 1 to 6 shown in Figure 1. It can be seen that Class 5 bile acid anionic surfactants, including N,N-diisopropylethanolamine-bile salt (DIDC), do not cause tissue deformation; Class 6 betaine surfactants also do not cause tissue deformation, while other classes all cause tissue expansion to varying degrees.

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

Betaine surfactants containing fatty acid side chains have rapid tissue degreasing effects, properties that do not cause tissue deformation, good intrinsic fluorescence maintenance ability, high chemical stability and extremely high solubility, and solubilization of other functional decolorizing agents. In some embodiments, the betaine surfactants containing fatty acid side chains can be selected from one or more of laurylamidopropyl hydroxysulfobetaine LHSB, dodecyl dimethyl hydroxypropyl sulfobetaine, and tetradecyl dimethyl hydroxypropyl sulfobetaine, but are not limited thereto.

In some embodiments, the composition may include about 0% w/v to about 50% w/v of a betaine surfactant containing a fatty acid side chain. In some embodiments, the composition may include 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.

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 of rapid degreasing and rapid decolorization, 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.

In some embodiments, the composition may include about 1% w/v to 30% w/v of anionic surfactants containing bile acid side chains. Preferably, the content of anionic surfactants containing bile acid side chains 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.

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.

In some embodiments, the degreasing and decolorizing composition for tissue clearing according to the present invention may further include a fluidity enhancer. In some embodiments, the fluidity enhancer may be selected from thiourea compounds and urea, and preferably, the thiourea compounds include thiourea. By further adding a fluidity enhancer to the degreasing and decolorizing composition, cell membrane fluidity, lipid solubility, and tissue morphology can be increased in the early stages of degreasing. In a preferred embodiment, the degreasing and decolorizing composition comprises 5% w/v to 25% w/v of a fluidity enhancer.

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).

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.

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

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 may be the same or different.

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.

In a particularly preferred embodiment, the first degreasing and decolorizing composition is a combination for rapid pre-degreasing and decolorizing. In a particularly preferred embodiment, the second degreasing and decolorizing composition is a composition for final degreasing and decolorizing, which comprises about 12% w/v sodium deoxycholate, about 10% w/v N,N-diisopropylethanolamine, about 25% w/v lauramidopropylhydroxysulfonate betaine 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 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 betaine and about 16% w/v thiourea is used for final degreasing and decolorizing, thereby removing the remaining lipids and pigments to the maximum extent.

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.

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

1) Obtaining a tissue sample to be cleared;

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;

3) performing a second degreasing and decolorizing of the tissue sample that has undergone 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;

4) The tissue sample that has undergone the second defatting and decolorization is washed and transferred to an aqueous refractive index matching solution for storage.

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

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

Hereinafter, the present invention will be described in more detail by 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.

Example

Materials and methods

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

Reagents, materials, and instruments:

1) Determination of phospholipid concentration: phospholipid detection kit (MAK122, Sigma-Aldrich); Nanodrop3000 micro-volume spectrophotometer (ThermoFisher); EnVision multi-function microplate reader (Perkin).

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

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), lauroamidopropyl hydroxysulfobetaine (LHSB, Greensense), 3-[3- (Cholamidopropyl) 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).

Example 1. Preparation of degreasing and decolorizing composition

FIG1 shows schematic diagrams of the chemical structures of several different types of degreasing molecules, 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 fatty acid chain zwitterionic surfactants (betaine surfactants), such as laurylamidopropyl hydroxysulfobetaine (LHSB); type 5 is bile acid anionic surfactants, such as deoxycholate ion; type 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 types of degreasing molecules and their effects on tissue size.

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

Furthermore, the degreasing ability of different degreasing agents was evaluated by measuring the highest phospholipid concentration (c(PL)) of brain tissue slices immersed in different degreasing agents for 2 hours and 32 hours. Referring to FIG3 , it can be seen that 5 types of bile acid anionic surfactants or ethanolamine-bile salts and 6 types of betaine surfactants 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.

Sodium deoxycholate, N,N-diisopropylethanolamine and lauramide propyl hydroxysulfonate betaine and optional thiourea are mixed in a solution to prepare the degreasing and decolorizing composition of the present invention. Specifically, a degreasing and decolorizing composition comprising 12% w/v sodium deoxycholate, 10% w/v N,N-diisopropylethanolamine and 3% w/v lauramide propyl hydroxysulfonate betaine is prepared as a rapid pre-degreasing and decolorizing reagent, which has the ability to quickly degrease the tissue in the early stage and will reach saturation faster. In addition, a degreasing and decolorizing composition containing 12% w/v sodium deoxycholate, 10% w/v N,N-diisopropylethanolamine, 25% w/v lauramide propyl hydroxysulfonate betaine and 16% w/v thiourea is also prepared as a final degreasing and decolorizing reagent, which can remove the remaining lipids and pigments to the maximum extent.

Example 2. Passive degreasing, decolorization and clearing of thick sections

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, and the specific steps were as follows:

1- Obtain mouse brain slices with a thickness of 300 μm by vibratome or cryosectioning, and place the fixed tissue slices in a rapid pre-defatting and decolorizing reagent at 37°C for 12 h.

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

3- Transfer the tissue sections to the final defatting and decolorizing reagent at 37°C for 12h.

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

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

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

Example 3. Degreasing, decolorization and transparentization on slides

By using the rapid pre-degreasing and decolorizing reagent and the final degreasing and decolorizing reagent prepared in Example 1, the tissue sections attached to the slide are degreased, decolorized and transparentized, and the specific steps are as follows:

1- Obtain 50 μm thick mouse brain slices by vibratome or cryosectioning, attach the tissue slices to a slide and place the slide in PBS to fully wash off the embedding agent 2-3 times.

2- Place the slide in a staining jar, add rapid pre-decolorization reagent, and incubate at 37℃ for 1 hour.

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

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

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

Example 4: Whole-organ clearing by transcardiac perfusion in vivo

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, and the specific steps were as follows:

1-Perfuse the mouse transcardially with pre-chilled heparin-saline solution until the blood is fully drained.

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

3-Perfuse the mouse transcardially with 15 ml of rapid pre-defatting and decolorizing reagent, and establish a circulation to recycle the fluid and continue perfusing for 8 hours.

4-Replace with new 15ml rapid pre-degreasing and decolorizing reagent and circulate the solution for 8h.

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

6-Replace with new 15 ml final defatting and decolorizing reagent and circulate the solution for 8 hours.

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

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

Figure 6 shows a photo of the results of using a defatting and decolorizing composition according to an embodiment of the present invention to make the whole organ of a mouse transparent. The upper figure shows, from left to right, photos of the mouse brain before defatting and decolorizing, after defatting and decolorizing, and after refractive index matching; the lower figure shows photos of the morphology of the heart, liver, spleen, lung, kidney, and brain of the mouse after transparency. Among them, after defatting and decolorizing by perfusion, refractive index matching is performed using a refractive index matching agent to achieve transparency of the whole organ.

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

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.

The mouse brain tissue, which was post-fixed for 1 day and dehydrated with 30% sucrose for 2 days, was cut into 300 μm slices using a freezing microtome and weighed immediately. 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 slices was added for tissue clearing. 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 agent (24% w/v urea, 10% w/v glycerol, 40% w/v sorbitol, 0.2% w/v TritonX-100, 15% w/v DMSO) for tissue clearing. The mouse brain slices treated with the rapid degreasing and decolorizing agent of the present invention, CUBIC-X degreasing agent or ScaleS clearing agent were sampled, and the relative area changes, supernatant phospholipid content and relative fluorescence changes of the three groups of brain slices were measured respectively. For fluorescence measurement, thy1-YFP mice were used, and the green fluorescence intensity of the fluorescently labeled cells was measured.

Figure 4 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 prior art. It can be seen that the degreasing rate using the rapid pre-degreasing and decolorizing reagent of Example 1 is significantly faster than the degreasing and decolorizing reagent in the classic aqueous clearing methods CUBIC and ScaleS. Figure 5 is a graph showing the results of tissue deformation, degreasing efficiency and fluorescence signal retention using the degreasing and decolorizing composition according to an embodiment of the present invention for tissue clearing compared with the clearing method of the prior art. It can be seen that compared with the degreasing and decolorizing reagent in the classic aqueous clearing methods CUBIC and ScaleS, the use of the rapid pre-degreasing and decolorizing reagent of Example 1 does not cause brain slice tissue deformation during the degreasing and decolorizing process (left), the degreasing efficiency is higher (middle), and the GFP fluorescence signal is better protected (right).

Example 6: Effect of degreasing and decolorizing composition on imaging effect

The improvement of imaging effect by using the defatting and decolorizing agent of the present invention for tissue clearing was further tested. Tissue clearing was performed using the rapid pre-defatting and decolorizing reagent and the final defatting and decolorizing reagent prepared in Example 1. Figure 7 shows a photograph of the imaging results of a 200-micron-thick Thy1-YFP mouse brain slice that was cleared. a, b, c, and d in Figure 7 were taken using a rotating disk confocal microscope, where a, b, and c were taken using a 10x air mirror, and d was taken using a 20x air mirror, and both took 488/520 green fluorescence channels; e, f, and g in Figure 7 were taken using a wide-field microscope bright field light source and a camera.

In FIG7 , 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 refractive index matching only; g) bright field image of 100 micron mouse brain slice without any transparent treatment. As can be seen from FIG7 , compared with tissue slices that are not transparentized at all or only 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 the rapid pre-degreasing and decolorizing agent, further using the final degreasing and decolorizing agent after the rapid pre-degreasing and decolorizing agent treatment achieves a better imaging effect.

Claims (11)

A degreasing and decolorizing composition for tissue clearing, the composition comprising: Betaine surfactants containing fatty acid side chains; and Anionic surfactant containing bile acid side chains. The degreasing and decolorizing composition for tissue clearing according to claim 1, wherein 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, tetradecylsulfobetaine and lauryldimethylsulfobetaine. The degreasing and decolorizing composition for tissue clearing according to claim 1 or 2, wherein the composition comprises 0% w/v to 50% w/v of the betaine surfactant containing a fatty acid side chain. The degreasing and decolorizing composition for tissue clearing according to any one of claims 1 to 3, wherein the composition comprises 1% w/v to 50% w/v of the betaine surfactant containing a fatty acid side chain. The degreasing and decolorizing composition for tissue clearing according to any one of claims 1 to 4, wherein the anionic surfactant containing bile acid side chains comprises side chains derived from bile acid compounds, preferably, the bile acid compounds are selected from one or more of bile acid, lithocholic acid, deoxycholic acid and chenodeoxycholic acid; Preferably, the anionic surfactant containing bile acid side chains further forms an ammonium salt complex with an amine compound; More preferably, 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. The degreasing and decolorizing composition for tissue clearing according to any one of claims 1 to 5, wherein the composition comprises 0.5% w/v to 35% w/v of the anionic surfactant containing a bile acid side chain. The degreasing and decolorizing composition for tissue clearing according to any one of claims 1 to 6, wherein the anionic surfactant containing a bile acid side chain further forms an ammonium salt complex with an amine compound; Preferably, 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, and the concentration of the amine compound is 1% w/v to 20% w/v. The degreasing and decolorizing composition for tissue clearing according to any one of claims 1 to 7, wherein the composition further comprises a fluidity enhancer; preferably, the fluidity enhancer is selected from thiourea compounds and urea. Preferably, the thiourea compound comprises thiourea; and even more preferably, the composition comprises 5% w/v to 25% w/v of the fluidity enhancer. A tissue clearing method, comprising treating a tissue sample with the defatting and decolorizing composition for tissue clearing according to any one of claims 1 to 8. The tissue clearing method according to claim 9, wherein the method comprises: treating the tissue sample with a first defatting and decolorizing composition; and optionally transferring the tissue sample treated with the first defatting and decolorizing composition to a second defatting and decolorizing composition, Wherein, the first degreasing and decolorizing composition and the second degreasing and decolorizing composition are the same or different and are the degreasing and decolorizing composition for tissue clearing according to any one of claims 1 to 9; Preferably, the second degreasing and decolorizing composition is different from the first degreasing and decolorizing composition; more preferably, the first degreasing and decolorizing composition does not contain a fluidity enhancer; and even more preferably, the second degreasing and decolorizing composition contains a higher content of betaine surfactants containing fatty acid side chains than the first degreasing and decolorizing composition. The tissue clearing method according to claim 9 or 10, wherein the method further comprises maintaining the tissue sample treated with the defatting and decolorizing composition in an aqueous refractive index matching liquid.