A tissue clearing reagent for stimulated raman scattering imaging and preparation method and application thereof

Abstract

The present application belongs to the technical field of biomedical imaging, and particularly relates to a transparent reagent for stimulated Raman scattering imaging and a preparation method and application thereof. The reagent components include 20-60% urea by mass percentage, 0.5-5% cyclodextrin compounds by mass percentage, and the rest is deuterated dimethyl sulfoxide; and the reagent is obtained by ultrasonic mixing and filtering of microparticles. The reagent can realize rapid transparentization of a millimeter-thick tissue sample in a short time by introducing a temperature regulation and a cyclodextrin-mediated cholesterol depletion mechanism, and significantly improves the transparentization efficiency, thereby significantly improving the imaging depth and contrast of the stimulated Raman scattering microscope, and realizing label-free and real-time three-dimensional histological imaging. The present application not only breaks through the limitation of traditional transparentization relying on lipid removal leading to loss of lipid signal in stimulated Raman imaging, but also solves the technical problem of lack of suitable transparentization scheme for stimulated Raman scattering imaging in large-volume tissues.

Description

Technical Field

[0001] This invention belongs to the field of biomedical imaging technology, specifically relating to a tissue clearing reagent for stimulated Raman scattering imaging, its preparation method, and its application. Background Technology

[0002] Tissue transparency technology is a rapidly developing tissue processing method in recent years. Its core objective is to achieve optical transparency of tissues while maintaining their structural integrity by adjusting the tissue's refractive index and reducing light scattering and absorption. With the help of transparency processing, optical microscopy can overcome traditional scattering barriers, enabling deep three-dimensional imaging of samples with thicknesses ranging from millimeters to centimeters. This technology has been widely applied in fields such as neuroscience, developmental biology, and oncology, and has driven the rapid development of large-volume biological tissue imaging.

[0003] Currently, common methods for achieving organizational transparency mainly include the following categories:

[0004] 1. Organic solvent-based method

[0005] Represented by BABB (benzyl alcohol / benzyl benzoate), 3DISCO, and their modified formulations, clearing is achieved by progressive dehydration and defatting followed by infiltration of tissues with high-refractive-index organic solvents. These methods are relatively fast and produce significant clearing effects, but they often cause tissue shrinkage and loss of fluorescence signals. Furthermore, organic solvents frequently exhibit strong background signals in the infrared and Raman spectral regions, severely interfering with optical imaging that relies on intrinsic molecular vibrational signals.

[0006] 2. Water-soluble reagent type method

[0007] Represented by CUBIC, this method utilizes water-soluble chemical reagents such as amino alcohols and urea to achieve good tissue transparency while preserving fluorescence signals as much as possible. This type of method has good biocompatibility and a wide range of applications. However, its multi-step processing (including degreasing and refractive index matching) often takes several days, which is not conducive to rapid application.

[0008] 3. Hydrogel Embedding Method

[0009] Represented by CLARITY, this method achieves transparency by cross-linking tissue with a hydrogel network and then performing electrophoresis or chemical degreasing. While this method offers advantages in preserving tissue structure and fluorescence signals, it is complex to operate and has a long processing time (typically several weeks), making it difficult to apply to rapid imaging scenarios.

[0010] 4. New rapid transparency strategy

[0011] Some recently proposed methods (such as SeeDB, ScaleS, Fast-Clear, etc.) attempt to simplify the steps and improve the transparency efficiency, but most of them are still focused on the field of fluorescence microscopy and still have shortcomings in the speed and uniformity of transparency of large-volume tissues, and their compatibility with molecular vibration optical imaging technology is also limited.

[0012] Although the above methods have made some progress in scientific research, existing transparency technologies generally suffer from the following problems:

[0013] 1. Processing time is too long: Most methods take several hours to several days to complete, which is not conducive to rapid detection or clinical application;

[0014] 2. Complex operation: The process is cumbersome and relies on a variety of chemical reagents, which increases the risk of experimental errors and sample loss;

[0015] 3. Spectral signal interference: Many transparentizing reagents produce strong background in the infrared and Raman spectral regions, which seriously affects imaging methods that rely on intrinsic molecular vibrational signals;

[0016] 4. Insufficient transparency of large-volume tissues: For tissues in the millimeter range or larger, existing methods are unable to achieve uniform transparency within a limited time, resulting in limited imaging depth;

[0017] 5. Loss of lipid signal: Most clearing methods rely on lipid removal as the core step to achieve transparency, but lipid molecules are an important source of contrast in stimulated Raman scattering imaging. Lipid removal not only disrupts the biochemical integrity of tissues but also severely weakens the label-free imaging capabilities based on lipids, limiting the application value of molecular vibrational imaging in pathological diagnosis and metabolic research.

[0018] Stimulated Raman scattering (SRS) is a nonlinear Raman microscopy technique that detects molecular vibrations. It enables label-free imaging of the spatial distribution and in-situ content of molecules such as lipids, proteins, nucleic acids, and carbohydrates in biological samples. Lipid signals primarily originate from the CH2 vibration peak of fatty acid chains, sensitively reflecting cell membrane, myelin sheath, and lipid metabolism processes. Protein signals mainly originate from the CH3 vibration peak and amide peak, revealing the distribution of the cell nucleus and cytoplasm, as well as pathological changes. Together, these two components constitute the main contrast of SRS imaging, used to present tissue morphology, metabolic state, and pathological features. Leveraging its label-free imaging advantage, SRS has demonstrated unique advantages in areas such as dynamic lipid metabolism research, tumor boundary identification, and rapid clinical pathological diagnosis.

[0019] Compared to spontaneous Raman imaging, stimulated Raman scattering (SRS) imaging offers advantages such as high spatiotemporal resolution (~300 nm in the xy direction, ~1 μm in the z direction), high video imaging speed, and optical slicing capabilities. However, due to the non-uniform light scattering in biological tissues, the effective imaging depth of conventional SRS imaging is typically limited to 100–300 μm, insufficient for three-dimensional imaging of large tissues at the millimeter or even centimeter scale. More importantly, existing commercially available tissue clearing methods are designed for fluorescence imaging and are not suitable for modalities that use SRS as the imaging contrast mechanism. There are two main technical incompatibilities: first, current tissue clearing methods generally rely on lipid removal to enhance the clearing effect; however, this directly weakens or even eliminates the lipid signal, which is the core contrast in SRS; second, traditional clearing reagents generate strong Raman background interference in the CH stretching vibration region of the Raman spectrum. In summary, current traditional clearing methods are difficult to integrate with SRS imaging; simultaneously, the lack of effective tissue clearing strategies also limits the application of SRS in three-dimensional imaging of large tissues.

[0020] Therefore, there is an urgent need to develop a rapid, simple, low-interference tissue transparency method that can preserve lipid signals. This method should not only meet the transparency requirements of conventional fluorescence microscopy, but also fully leverage the advantages of stimulated Raman scattering imaging in label-free three-dimensional chemical imaging, thereby enabling integrated and rapid analysis of the morphology and chemical composition of large-volume tissues. Summary of the Invention

[0021] The purpose of this invention is to provide a tissue clearing reagent with fast clearing speed, capable of preserving endogenous signals such as tissue lipids, and particularly suitable for stimulated Raman scattering imaging, as well as its preparation method and application.

[0022] The tissue clearing agent for stimulated Raman scattering imaging provided by this invention comprises, by mass percentage:

[0023] Urea: 30–50%;

[0024] Cyclodextrin compounds: 0.5–5%, preferably 1–2%; the cyclodextrin compounds are selected from β-cyclodextrin or methyl-β-cyclodextrin;

[0025] Deuterated dimethyl sulfoxide (DMSO-d6): Balance, as solvent;

[0026] All three conditions are met at 100%.

[0027] The urea reduces light scattering by adjusting the difference in tissue refractive index, while cyclodextrin compounds enhance transparency by binding to lipids and partially removing cholesterol. Deuterated dimethyl sulfoxide not only has good solubility and permeability, but also has a low signal background in the Raman spectral region, which helps to maintain the signal purity of stimulated Raman scattering imaging.

[0028] The preparation method of the tissue clearing agent in this invention includes the following specific steps:

[0029] (1) Weigh out solid urea and powdered cyclodextrin compounds and place them in a clean centrifuge tube;

[0030] (2) Add deuterated dimethyl sulfoxide solution to form the desired mixed solution;

[0031] (3) Place the mixed solution in an ultrasonic cleaner at room temperature (20–25℃) and sonicate for 5–20 minutes until the urea and cyclodextrin compounds are completely dissolved to obtain a homogeneous, clear, and transparent solution.

[0032] (4) The transparent solution is filtered through a filter membrane to remove the particles, and the resulting solution is the tissue transparentizing reagent.

[0033] In this invention, the filter membrane can be a 0.22μm filter membrane.

[0034] This invention also provides the application of the above-mentioned tissue clearing agent in stimulated Raman scattering imaging, the specific steps of which are as follows:

[0035] (1) Cut fresh or fixed biological tissue samples to form tissue sections;

[0036] (2) Preheat the prepared tissue clearing reagent at 25–65°C, preferably 60°C;

[0037] (3) Place the tissue sections into the preheated tissue clearing reagent and let them stand at a constant temperature for clearing.

[0038] (4) Take out the cleared tissue section, place it on a glass slide, and then add tissue clearing reagent for mounting.

[0039] (5) Place the mounted tissue sections under a stimulated Raman scattering microscope for three-dimensional volume imaging for subsequent analysis.

[0040] In this invention, the tissue clearing agent is preheated using a constant-temperature metal bath.

[0041] The tissue transparency method of the present invention is applicable to a variety of mammalian tissues, and the tissue sections include, but are not limited to:

[0042] Fresh or fixed mouse brain slices (0.3–2 mm thick);

[0043] Fixed sections of mouse heart, kidney, lung, spleen, liver and other tissues (0.3–1 mm thick);

[0044] Tissue sections of brain tumor biopsy samples from patients (thickness 0.3–1 mm).

[0045] All raw materials are commercially available chemical reagents, such as urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl β-cyclodextrin (Adamas, 19185A), and β-cyclodextrin (Adamas, 81437A).

[0046] Beneficial effects

[0047] The transparentizing reagent and method of the present invention have the following advantages:

[0048] 1. Speed: It can achieve transparency of tissues with a thickness of millimeters in a very short time (minutes), which is significantly faster than traditional methods;

[0049] 2. Preservation of lipid signals: By avoiding strong degreasing steps, the lipid contrast in the tissue is preserved, thus ensuring the core advantages of stimulated Raman scattering imaging;

[0050] 3. Low background interference: Deuterated dimethyl sulfoxide has a low signal in the Raman spectral region and does not introduce significant background, thus ensuring the authenticity of the vibrational signals of intrinsic molecules;

[0051] 4. Enhanced imaging depth: In stimulated Raman scattering imaging, the tissue imaging depth can be extended from the conventional 100–300 μm to the millimeter level;

[0052] 5. Dual applicability: This method is applicable to both conventional fluorescence imaging and stimulated Raman scattering imaging, combining broad applicability with specificity.

[0053] The reagents of this invention, through the introduction of temperature regulation and cyclodextrin-mediated cholesterol consumption mechanisms, can rapidly achieve transparentization of tissue samples with a thickness of millimeters within a short time, significantly improving transparentization efficiency. This, in turn, significantly enhances the imaging depth and contrast of stimulated Raman scattering microscopy, enabling label-free, near real-time three-dimensional histological imaging. The application of this invention not only overcomes the limitations of traditional transparentization methods that rely on delipidemia leading to lipid signal loss, but also fills the technical gap in the lack of suitable transparentization schemes for large-volume tissues in stimulated Raman scattering imaging, providing a novel solution for label-free three-dimensional histological research. Attached Figure Description

[0054] Figure 1 A one-step, rapid organizational transparency process applicable to SRS.

[0055] Figure 2 The background signal of the clearing reagents at imaging wavenumbers is shown in (a) for spontaneous Raman spectra of dimethyl sulfoxide (cyan), urea (pink), deuterated dimethyl sulfoxide (purple), and mouse brain tissue (orange). (b) is the spontaneous Raman spectrum of a mouse brain slice (blue) and a 50% urea solution containing 1% β-cyclodextrin (green) or 1% methyl β-cyclodextrin (orange).

[0056] Figure 3 The graph illustrates how temperature and β-cyclodextrin-induced cholesterol consumption induce lipid phase transitions. (a) shows the spontaneous Raman spectra of a 100 μm thick mouse brain slice before and after incubation in PBS at 60 °C for 10 minutes. Spectra have been calculated based on 2888 cm⁻¹. -1 The peak was normalized. 2888cm -1 The peak broadening at this point corresponds to the transition of lipids from solid to liquid state. (b) Stimulated Raman imaging hyperspectral analysis of myelin-rich white matter regions in mouse cerebellum tissue before and after transparency, normalized to 2850 cm⁻¹. -1 2888cm -1 The intensity of the area decreases, which corresponds to the consumption of cholesterol.

[0057] Figure 4 The images show photographs of mouse brain tissue before and after transparency under the preferred scheme, as well as stimulated Raman volumetric imaging after transparency.

[0058] Figure 5 This is a stimulated Raman volumetric imaging image of other organs and tissues in mice after transparency under the preferred scheme.

[0059] Figure 6 The images show photographs of fresh mouse brain tissue before and after transparency, and stimulated Raman volumetric imaging after transparency, under the preferred scheme.

[0060] Figure 7 The images show the human tumor biopsy samples before and after transparency, and the stimulated Raman volumetric imaging after transparency, under the preferred scheme.

[0061] Figure 8 The attached figure is for Example 1.

[0062] Figure 9 The attached figure is for Example 2.

[0063] Figure 10 The attached figure is for Example 3.

[0064] Figure 11 The attached figure is for Example 4.

[0065] Figure 12 The attached figure is for Example 5.

[0066] Figure 13 The attached figure is for Example 6.

[0067] Figure 14 The attached figure is for Example 7.

[0068] Figure 15 The attached figure is for Example 8.

[0069] Figure 16 The attached figure is for Example 9.

[0070] Figure 17 The attached figure is for Example 10.

[0071] Figure 18 The attached figure is for Example 11.

[0072] Figure 19 The attached figure is for Example 12.

[0073] Figure 20 The attached figure is for Example 13. Detailed Implementation

[0074] Example 1

[0075] Composition and preparation of transparentizing reagents

[0076] The clearing agent used in this embodiment consists of urea, β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0077] Urea: 50wt%;

[0078] β-Cyclodextrin: 1 wt%;

[0079] The balance is deuterated dimethyl sulfoxide;

[0080] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), β-cyclodextrin (Adamas, 81437A).

[0081] The preparation method is as follows:

[0082] 1. Weigh 5g of solid urea and 0.1g of β-cyclodextrin powder into a 10mL centrifuge tube;

[0083] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0084] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0085] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0086] The resulting transparentizing reagent can be used directly without further dilution.

[0087] Tissue transparency and stimulated Raman scattering imaging methods

[0088] 1. Select 6–20 week old C57BL / 6J mice, take brain tissue and fix it with 4% paraformaldehyde, then section it and use a vibratory microtome to cut the tissue into sections with a thickness of 2000 μm.

[0089] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 60°C;

[0090] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 35 minutes, the tissue samples will become completely transparent;

[0091] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0092] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0093] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0094] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have shown that this method can achieve complete transparency of brain tissue slices up to 2 mm thick and enable high-resolution imaging of deep structures.

[0095] Equipment used

[0096] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0097] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0098] Example 2

[0099] Composition and preparation of transparentizing reagents

[0100] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0101] Urea: 30wt%;

[0102] Methyl-β-cyclodextrin: 0.5 wt%;

[0103] The balance is deuterated dimethyl sulfoxide;

[0104] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0105] The preparation method is as follows:

[0106] 1. Weigh 3g of solid urea and 0.05g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0107] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0108] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0109] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0110] The resulting transparentizing reagent can be used directly without further dilution.

[0111] Tissue transparency and stimulated Raman scattering imaging methods

[0112] 1. Select 6–20 week old C57BL / 6J mice, take brain tissue and fix it with 4% paraformaldehyde, then section it and use a vibratory microtome to cut the tissue into sections with a thickness of 500 μm.

[0113] 2. Immerse the tissue sections directly in the pre-prepared clearing reagent at room temperature (25°C) (no additional buffer or dilution step required);

[0114] 3. After 7 minutes, the tissue sample became completely transparent;

[0115] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0116] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0117] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0118] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have shown that this method can achieve complete transparency of brain tissue slices up to 0.5 mm thick and enable high-resolution imaging of deep structures.

[0119] Equipment used

[0120] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0121] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0122] Example 3

[0123] Composition and preparation of transparentizing reagents

[0124] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0125] Urea: 50wt%;

[0126] Methyl-β-cyclodextrin: 5 wt%;

[0127] The balance is deuterated dimethyl sulfoxide;

[0128] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0129] The preparation method is as follows:

[0130] 1. Weigh 5g of solid urea and 0.5g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0131] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0132] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0133] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0134] The resulting transparentizing reagent can be used directly without further dilution.

[0135] Tissue transparency and stimulated Raman scattering imaging methods

[0136] 1. Select 6–20 week old C57BL / 6J mice, take brain tissue and fix it with 4% paraformaldehyde, then section it and use a vibratory microtome to cut the tissue into sections with a thickness of 700 μm.

[0137] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 37°C;

[0138] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 8 minutes, the tissue samples will become completely transparent;

[0139] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0140] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0141] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0142] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have demonstrated that this method can achieve complete transparency of brain tissue slices up to 0.7 mm thick and enable high-resolution imaging of deep structures.

[0143] Equipment used

[0144] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0145] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0146] Example 4

[0147] Composition and preparation of transparentizing reagents

[0148] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0149] Urea: 30wt%;

[0150] Methyl-β-cyclodextrin: 5 wt%;

[0151] The balance is deuterated dimethyl sulfoxide;

[0152] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0153] The preparation method is as follows:

[0154] 1. Weigh 3g of solid urea and 0.5g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0155] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0156] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0157] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0158] The resulting transparentizing reagent can be used directly without further dilution.

[0159] Tissue transparency and stimulated Raman scattering imaging methods

[0160] 1. Select 6–20 week old C57BL / 6J mice, take kidney tissue and fix it with 4% paraformaldehyde, then section it and use a vibratory microtome to cut the tissue into sections with a thickness of 700 μm.

[0161] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 42°C;

[0162] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 10 minutes, the tissue samples will become completely transparent;

[0163] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0164] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0165] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0166] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have demonstrated that this method can achieve complete transparency of brain tissue slices up to 0.7 mm thick and enable high-resolution imaging of deep structures.

[0167] Equipment used

[0168] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0169] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0170] Example 5

[0171] Composition and preparation of transparentizing reagents

[0172] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0173] Urea: 30wt%;

[0174] Methyl-β-cyclodextrin: 1 wt%;

[0175] The balance is deuterated dimethyl sulfoxide;

[0176] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0177] The preparation method is as follows:

[0178] 1. Weigh 3g of solid urea and 0.1g of methyl-β-cyclodextrin powder into a 10mL centrifuge tube;

[0179] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0180] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0181] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0182] The resulting transparentizing reagent can be used directly without further dilution.

[0183] Tissue transparency and stimulated Raman scattering imaging methods

[0184] 1. Select 6–20 week old C57BL / 6J mice, take brain tissue and fix it with 4% paraformaldehyde, then cut the tissue into sections with a thickness of 300 μm using a vibratory microtome;

[0185] 2. Immerse the tissue sections directly in the pre-prepared clearing reagent at room temperature (25°C) (no additional buffer or dilution step required);

[0186] 3. After 5 minutes, the tissue sample becomes completely transparent;

[0187] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0188] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0189] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0190] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have shown that this method can achieve complete transparency of brain tissue slices up to 0.3 mm thick and enable high-resolution imaging of deep structures.

[0191] Equipment used

[0192] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0193] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0194] Example 6

[0195] Composition and preparation of transparentizing reagents

[0196] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0197] Urea: 50wt%;

[0198] Methyl-β-cyclodextrin: 2 wt%;

[0199] The balance is deuterated dimethyl sulfoxide;

[0200] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0201] The preparation method is as follows:

[0202] 1. Weigh 5g of solid urea and 0.2g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0203] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0204] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0205] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0206] The resulting transparentizing reagent can be used directly without further dilution.

[0207] Tissue transparency and stimulated Raman scattering imaging methods

[0208] 1. Take a tissue section (1mm thick) from the patient's brain tumor biopsy sample;

[0209] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 60°C;

[0210] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 9 minutes, the tissue samples will become completely transparent;

[0211] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0212] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0213] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0214] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have shown that this method can achieve complete transparency of brain tissue slices up to 1 mm thick and enable high-resolution imaging of deep structures.

[0215] Equipment used

[0216] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0217] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0218] Example 7

[0219] Composition and preparation of transparentizing reagents

[0220] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0221] Urea: 50wt%;

[0222] Methyl-β-cyclodextrin: 5 wt%;

[0223] The balance is deuterated dimethyl sulfoxide;

[0224] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0225] The preparation method is as follows:

[0226] 1. Weigh 5g of solid urea and 0.5g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0227] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0228] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0229] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0230] The resulting transparentizing reagent can be used directly without further dilution.

[0231] Tissue transparency and stimulated Raman scattering imaging methods

[0232] 1. Take a tissue section (1mm thick) from the patient's brain tumor biopsy sample;

[0233] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 60°C;

[0234] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 8 minutes, the tissue samples will become completely transparent;

[0235] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0236] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0237] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0238] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have shown that this method can achieve complete transparency of brain tissue slices up to 1 mm thick and enable high-resolution imaging of deep structures.

[0239] Equipment used

[0240] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0241] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0242] Example 8

[0243] Composition and preparation of transparentizing reagents

[0244] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0245] Urea: 50wt%;

[0246] Methyl-β-cyclodextrin: 3 wt%;

[0247] The balance is deuterated dimethyl sulfoxide;

[0248] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0249] The preparation method is as follows:

[0250] 1. Weigh 5g of solid urea and 0.3g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0251] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0252] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0253] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0254] The resulting transparentizing reagent can be used directly without further dilution.

[0255] Tissue transparency and stimulated Raman scattering imaging methods

[0256] 1. Select 6–20 week old C57BL / 6J mice, take brain tissue and fix it with 4% paraformaldehyde, then section it and use a vibratory microtome to cut the tissue into sections with a thickness of 1000 μm.

[0257] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 42°C;

[0258] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 10 minutes, the tissue samples will become completely transparent;

[0259] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0260] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0261] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0262] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have shown that this method can achieve complete transparency of brain tissue slices up to 1 mm thick and enable high-resolution imaging of deep structures.

[0263] Equipment used

[0264] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0265] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0266] Example 9

[0267] Composition and preparation of transparentizing reagents

[0268] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0269] Urea: 40wt%;

[0270] Methyl-β-cyclodextrin: 5 wt%;

[0271] The balance is deuterated dimethyl sulfoxide;

[0272] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0273] The preparation method is as follows:

[0274] 1. Weigh 4g of solid urea and 0.5g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0275] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0276] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0277] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0278] The resulting transparentizing reagent can be used directly without further dilution.

[0279] Tissue transparency and stimulated Raman scattering imaging methods

[0280] 1. Select 6–20 week old C57BL / 6J mice, take brain tissue and fix it with 4% paraformaldehyde, then section it and use a vibratory microtome to cut the tissue into sections with a thickness of 1000 μm.

[0281] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 42°C;

[0282] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 10 minutes, the tissue samples will become completely transparent;

[0283] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0284] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0285] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0286] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have shown that this method can achieve complete transparency of brain tissue slices up to 1 mm thick and enable high-resolution imaging of deep structures.

[0287] Equipment used

[0288] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0289] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0290] Example 10

[0291] Composition and preparation of transparentizing reagents

[0292] The clearing agent used in this embodiment consists of urea, β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0293] Urea: 50wt%;

[0294] β-Cyclodextrin: 1 wt%;

[0295] The balance is deuterated dimethyl sulfoxide;

[0296] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), β-cyclodextrin (Adamas, 81437A).

[0297] The preparation method is as follows:

[0298] 1. Weigh 5g of solid urea and 0.1g of β-cyclodextrin powder into a 10mL centrifuge tube;

[0299] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0300] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0301] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0302] The resulting transparentizing reagent can be used directly without further dilution.

[0303] Tissue transparency and stimulated Raman scattering imaging methods

[0304] 1. Select 6–20 week old C57BL / 6J mice, take brain tissue and fix it with 4% paraformaldehyde, then section it and use a vibratory microtome to cut the tissue into sections with a thickness of 1500 μm.

[0305] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 60°C;

[0306] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 20 minutes, the tissue samples will become completely transparent;

[0307] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0308] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0309] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0310] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have demonstrated that this method can achieve complete transparency of brain tissue slices up to 1.5 mm thick and enable high-resolution imaging of deep structures.

[0311] Equipment used

[0312] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0313] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0314] Example 11

[0315] Composition and preparation of transparentizing reagents

[0316] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0317] Urea: 50wt%;

[0318] Methyl-β-cyclodextrin: 1 wt%;

[0319] The balance is deuterated dimethyl sulfoxide;

[0320] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0321] The preparation method is as follows:

[0322] 1. Weigh 5g of solid urea and 0.1g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0323] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0324] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0325] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0326] The resulting transparentizing reagent can be used directly without further dilution.

[0327] Tissue transparency and stimulated Raman scattering imaging methods

[0328] 1. Select 6–20 week old C57BL / 6J mice, take fresh brain tissue, and use a vibratory microtome to cut the tissue into sections with a thickness of 500 μm;

[0329] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 60°C;

[0330] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 10 minutes, the tissue samples will become completely transparent;

[0331] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0332] 4. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0333] 5. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.) and collect samples at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals are used for three-dimensional volume imaging;

[0334] 6. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have shown that this method can achieve complete transparency of brain tissue slices up to 0.5 mm thick and enable high-resolution imaging of deep structures.

[0335] Equipment used

[0336] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0337] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0338] Example 12

[0339] Composition and preparation of transparentizing reagents

[0340] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0341] Urea: 50wt%;

[0342] Methyl-β-cyclodextrin: 1 wt%;

[0343] The balance is deuterated dimethyl sulfoxide;

[0344] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0345] The preparation method is as follows:

[0346] 1. Weigh 5g of solid urea and 0.1g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0347] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0348] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, yielding a clear and transparent homogeneous solution;

[0349] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0350] The resulting transparentizing reagent can be used directly without further dilution.

[0351] Tissue transparency and confocal fluorescence imaging and stimulated Raman scattering imaging methods

[0352] 1. Select 6–20 week old C57BL / 6J mice, take fresh brain tissue, and use a vibratory microtome to cut the tissue into sections with a thickness of 1000 μm;

[0353] 2. Soak tissue sections in 1% Tween solution for 10 min, then wash with PBS for 5 min × 3 times; then block in 5% BSA solution at room temperature for 1 h, then wash with PBS for 5 min × 3 times; then add 5 μg / mL tomato lectin solution specifically labeled for vascular endothelial cells and incubate in the dark for 1 h, then wash with 0.1% Triton X-100 solution for 5 min × 3 times; finally wash with PBS for 5 min × 3 times to complete the fluorescent labeling of vascular endothelial cells;

[0354] 3. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 60°C;

[0355] 4. Place the fluorescently labeled tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 10 minutes, the tissue samples will become completely transparent;

[0356] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing;

[0357] 5. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0358] 6. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.), for example, at 2850 cm⁻¹. -1 (lipids) and 2930cm -1 (Protein) signals, while using confocal microscopy to perform three-dimensional volumetric imaging of the fluorescence channel;

[0359] 7. Subsequent image reconstruction yields a three-dimensional chemical image of the tissue. Experiments have shown that this method can achieve complete transparency of brain tissue slices up to 1 mm thick and enable high-resolution imaging of deep structures.

[0360] Equipment used

[0361] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0362] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0363] Example 13

[0364] Composition and preparation of transparentizing reagents

[0365] The clearing agent used in this embodiment is composed of urea, methyl-β-cyclodextrin, and deuterated dimethyl sulfoxide (DMSO-d6). The specific ratio is as follows:

[0366] Urea: 50wt%;

[0367] Methyl-β-cyclodextrin: 1 wt%;

[0368] The balance is deuterated dimethyl sulfoxide;

[0369] Raw material sources: urea (Sangon Biotech, A610148-0500), deuterated dimethyl sulfoxide (Adamas, 38114L), methyl-β-cyclodextrin (Adamas, 19185A).

[0370] The preparation method is as follows:

[0371] 1. Weigh 5g of solid urea and 0.1g of methyl-β-cyclodextrin powder and place them in a 10mL centrifuge tube;

[0372] 2. Add 4.9 mL of deuterated dimethyl sulfoxide solution to the tube;

[0373] 3. Place the mixture in an ultrasonic cleaner and sonicate at room temperature for 10 minutes until completely dissolved, obtaining a clear and transparent homogeneous solution;

[0374] 4. Optional step: Filter the obtained solution through a 0.22μm filter membrane to remove insoluble impurities.

[0375] The resulting transparentizing reagent can be used directly without further dilution.

[0376] Tissue transparency and stimulated Raman scattering imaging methods

[0377] 1. Select 6–20 week old C57BL / 6J mice, take brain tissue and fix it with 4% paraformaldehyde, then cut the tissue into sections with a thickness of 300 μm using a vibratory microtome;

[0378] 2. Place the tissue before clearing on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.) and collect 2850 cm⁻¹ samples from different regions. -1 (Lipid) signal, used for subsequent comparison

[0379] 2. Place the centrifuge tube containing the pre-prepared clearing reagent in a constant temperature metal bath and heat it to 60°C;

[0380] 3. Place the tissue sections directly into centrifuge tubes for immersion (no additional buffer or dilution step required); after 10 minutes, the tissue samples will become completely transparent;

[0381] In this process, the clearing agent promotes lipid phase transition through a positive feedback mechanism of cholesterol consumption and increased membrane disorder, thereby achieving rapid tissue clearing. At the same time, a longer immersion time is used to test the lipid retention rate.

[0382] 5. Remove the cleared tissue section, place it on a glass slide, and add a small amount of clearing reagent for mounting to maintain the clear effect;

[0383] 6. Place the mounted tissue sample on a stimulated Raman scattering microscope (built-in system or commercial system, such as Leica SP8-CARS, Invenio, etc.) and collect samples at 2850 cm⁻¹. -1 (Lipid) signal, calculation of lipid retention rate;

[0384] Equipment used

[0385] Temperature control equipment: constant temperature metal bath, drying oven, constant temperature shaker or incubator are all acceptable;

[0386] Imaging equipment: Stimulated Raman scattering microscope, including pump light and Stokes light source, modulator, lock-in amplifier and high numerical aperture objective lens, etc.

[0387] Summary of technical effects

[0388] Through Examples 1–13, the clearing reagent of this invention can achieve rapid clearing of tissues with a thickness of millimeters within 2–10 minutes, and even for tissues with a thickness doubled to 2 mm, clearing can be completed within 40 minutes. Compared with traditional clearing methods that rely on degreasing, this invention avoids the loss of lipid signals caused by excessive degreasing, and can effectively preserve key endogenous molecular signals such as lipids and proteins, thereby significantly improving the contrast and imaging depth of stimulated Raman scattering imaging. Experimental results show that the method of this invention can achieve complete clearing of mouse brain slices with a thickness of up to 2 mm, and obtain clear three-dimensional chemical imaging under stimulated Raman scattering microscopy, verifying its application value in rapid, label-free histological imaging.

[0389] Verification Description of Embodiments of the Invention

[0390] 1. Design and validation of lipid signal preservation

[0391] Stimulated Raman scattering imaging typically relies on the CH2 vibration signal (approximately 2850 cm⁻¹) generated by the abundant fatty acid alkane chains in the cell membrane and intracellular lipid droplets. -1 Traditional transparency methods often achieve transparency through a strong degreasing process. While this improves tissue transparency, it also severely damages lipid signals, resulting in a significant reduction in the contrast of stimulated Raman scattering imaging.

[0392] The transparency method of this invention avoids excessive extraction of long fatty acid chains, selectively removing only free cholesterol and some membrane cholesterol from the tissue, thereby preserving most lipid signals while maintaining overall tissue transparency. Experiments show that cholesterol consumption does not significantly affect the 2850 cm⁻¹ area. -1The CH2 peak intensity at the location. The final imaging verification results showed that the tissue sample after one-step rapid clearing treatment retained more than 95% of the lipid signal (after one hour of clearing reagent treatment), ensuring the intrinsic contrast on which stimulated Raman scattering imaging depends.

[0393] 2. Raman background removal

[0394] To ensure high-quality molecular vibrational signals under stimulated Raman scattering microscopy, deuterated dimethyl sulfoxide (DMSO-d6) was selected as the solvent for the clearing agent in this invention. Compared with ordinary DMSO, DMSO-d6 exhibits higher molecular vibrational signals in the CH region (2800–3000 cm⁻¹). -1 It produces almost no interference, thus effectively eliminating background signals that may be introduced by conventional clearing agents.

[0395] Further experiments showed that adding 1% β-cyclodextrin or methyl-β-cyclodextrin to the solution did not produce a significant background signal in the CH2 and CH3 regions. Figure 2 As shown, this reagent system is in CH2(2850cm) -1 ) and CH3 (2930cm -1 The signal-to-noise ratio (S / B) in the region remained above 80, indicating that the transparency system did not interfere with the detection of lipid or protein signals.

[0396] 3. Positive feedback mechanism enables rapid transparency

[0397] The binding and consumption of cholesterol in tissues by β-cyclodextrin and its derivatives has been widely confirmed. This invention utilizes this property, combined with heating conditions to induce lipid phase transition, establishing a "positive feedback" mechanism to accelerate the transparentization process.

[0398] Raman spectroscopy results show that 2888 cm⁻¹ -1 The vibrational peak at 2888 cm⁻¹ can serve as a marker of the conformational order of alkane chains: when alkane chains transition from a condensed state to a gel-like phase, this peak broadens and is accompanied by an intensity decrease. When the tissue is heated alone, the peak is at 2888 cm⁻¹. -1 The peak broadens and reaches 2888cm. -1 / 2850cm -1 The intensity ratio decreased from 1.08 to 1.05; after completing the full one-step transparency process, the ratio further decreased from 0.998 to 0.914.

[0399] These results confirm that:

[0400] The reduction of cholesterol induced a transition in membrane lipid conformation from a condensed state to a disordered state;

[0401] Phase change enhances membrane fluidity and significantly increases the permeation rate of urea and deuterated dimethyl sulfoxide in tissues;

[0402] This process creates a positive feedback mechanism of "cholesterol consumption – membrane disorder – accelerated solvent penetration", enabling tissues to achieve uniform transparency with a thickness of millimeters within minutes.

[0403] In summary, this invention, through its three core designs of lipid signal preservation, background elimination, and positive feedback acceleration mechanism, overcomes the limitations of existing transparency techniques in stimulated Raman scattering imaging, achieving rapid, low-interference, and label-free three-dimensional histological imaging.

[0404] To verify the effectiveness of the transparentizing reagent and method described in this invention, the following experimental methods were used to test the transparentizing efficiency, signal fidelity, and imaging performance:

[0405] 1. Organizational Transparency Efficiency Testing

[0406] Mouse brain tissue sections were selected and treated using the reagent of this invention and conventional clearing methods (such as DISCO, FOCM, etc.). Tissue transparency was compared before and after treatment using transmitted light imaging, and the required time was recorded. The results showed that the reagent of this invention could achieve uniform clearing of tissue sections less than 1 mm thick at 60°C within 2–10 minutes, and tissue sections 1–2 mm thick at 60°C within approximately 30 minutes, while conventional methods typically require several hours to several days.

[0407] 2. Validation of lipid signal retention

[0408] Stimulated Raman scattering microscopy at 2850 cm⁻¹ -1 (CH2 vibration peak) signal intensity was collected. The peak intensity changes in the same region before and after transparency were compared to calculate the lipid signal retention rate. Experimental results show that after transparency treatment according to this invention, the lipid signal retention rate is greater than 95%, while the lipid retention rate of traditional lipid-reducing transparency methods is far below 50%.

[0409] 3. Raman Background Assessment

[0410] Confocal Raman spectroscopy was used to perform spectral analysis on the clearing agent itself and the treated tissue samples. The spectra were analyzed at 2800–3000 cm⁻¹. -1 Within this region, the transparent system of this invention exhibits almost no background interference, and the signal-to-noise ratio of the CH2 / CH3 signal is greater than 80. In contrast, ordinary DMSO or other organic solvent systems show significant interference peaks in this region.

[0411] 4. Verification of Imaging Depth and Quality

[0412] Three-dimensional imaging of mouse brain slices, both untreated and treated with the method of this invention, was performed using stimulated Raman scattering (SRS) microscopy with the same technical parameters. The results showed that the effective imaging depth of the untreated samples was typically only 100–300 μm (with a signal-to-noise ratio (SNR) ≥ 2 as the effective imaging standard); while samples treated with the method of this invention could achieve complete three-dimensional imaging in tissues with a thickness of 1–2 mm, with an SNR greater than 2, and significantly improved image clarity and contrast. The improved SNR and image quality validate the advantages of this invention in enhancing SRS imaging depth and quality.

[0413] 5. Validation of the positive feedback mechanism

[0414] Raman spectroscopy was used to monitor the tissue at 2888 cm⁻¹ during the heating and transparentization process. -1 Peak width and 2888cm -1 / 2850cm -1 Intensity ratio change. The results showed that the ratio decreased from 0.998 to 0.914 during the transparency process, confirming that β-cyclodextrin-mediated cholesterol consumption induced lipid phase transition, accelerated solvent penetration, and formed a "positive feedback" mechanism, thereby achieving rapid transparency.

[0415] Comparative Analysis Table of Representative Technologies

[0416]

[0417] 1. Ertürk A, Becker K, N, Mauch CP, Hojer CD, Egen JG, Hellal F, Bradke F, Sheng M, Dodt HU. Three-dimensional imaging of solvent-cleared organs using 3DISCO. Nat Methods. 2012;9(7):703-709.

[0418] 2.Susaki EA,Tainaka K,Perrin D,Kishino F,Tawara T,Watanabe TM,Yokoyama C,Onoe H,Eguchi M,Yamaguchi S,Abe T,Kiyonari H,Shimizu Y,Miyawaki A,Yokota H,Ueda HR.Whole-brain imaging with single-cell resolution usingchemical cocktails and computational analysis.Cell.2014;157(3):726-739.

[0419] 3.Tomer R,Ye L,Hsueh B,Deisseroth K.Advanced CLARITY for rapid andhigh-resolution imaging of intact tissues.Nat Protoc.2014;9(7):1682-1697.

[0420] 4.Perbellini F,Liu AK,Watson SA,Bardi I,Terracciano CM.Free-of-acrylamide SDS-based tissue clearing(FASTClear):a novel protocol of tissueclearing for three-dimensional visualization of human braintissues.Neuropathol Appl Neurobiol.2017;43(4):346-351.

[0421] 5.Cai R,Pan C,Ghasemigharagoz A,Todorov MI, B, Zhao S, BhatiaHS, Parra-Damas A, Mrowka L, Theodorou D, Rempfler M, Radoshevich L, Alberti S, Menze B, Krammer T, Plesnila N, Dichgans M, Herms J, Bechmann I, Steinke H, et al. Panoptic imaging of transparent mice reveals whole-body neuronal projections and skull-meninges connections. Nat Neurosci.2019;22(2):317-327.

[0422] 6. Li J, Lin P, Tan Y, Cheng JX. Volumetric stimulated Raman scattering imaging of cleared tissues towards three-dimensional chemicalhistopathology. Biomed Opt Express. 2019; 10(8): 4329-4339.

[0423] 7. Wei M, Shi L, Shen Y, Zhao Z, Guzman A, Kaufman LJ, Wei L, MinW. Volumetric chemical imaging by clearing-enhanced stimulated Ramanscattering microscopy. Proc Natl Acad Sci US A. 2019; 116(14): 6608-6617.

[0424] Summary table of transparency capabilities in the method of this invention

[0425]

[0426]

[0427] SBR: signal to background.

Claims

1. A tissue clearing agent for stimulated Raman scattering imaging, characterized in that, Its components, by mass percentage, include: Urea: 30–50%; Cyclodextrin compounds: 0.5–5%, preferably 1–2%; the cyclodextrin compounds are selected from β-cyclodextrin or methyl-β-cyclodextrin; Deuterated dimethyl sulfoxide (DMSO-d6): Balance, as solvent; All three conditions are met at 100%.

2. A method for preparing the tissue clearing agent as described in claim 1, characterized in that, The specific steps are as follows: (1) Weigh out solid urea and powdered cyclodextrin compounds and place them in a clean centrifuge tube; (2) Add deuterated dimethyl sulfoxide solution to form the required mixed solution; (3) Place the mixed solution in an ultrasonic cleaner at room temperature (20–25℃) and sonicate for 5–20 minutes until the urea and cyclodextrin compounds are completely dissolved to obtain a homogeneous, clear, and transparent solution. (4) The transparent solution is filtered through a filter membrane to remove the particles, and the resulting solution is the tissue transparentizing reagent.

3. The preparation method according to claim 2, characterized in that, The filter membrane used is a 0.22 μm filter membrane.

4. The application of the tissue clearing agent as described in claim 1 in stimulated Raman scattering imaging, characterized in that, The specific steps are as follows: (1) Cut fresh or fixed biological tissue samples to form tissue sections; (2) Preheat the prepared tissue clearing reagent and maintain it at 25–65 °C; (3) Place the tissue sections into the preheated tissue clearing reagent and let them stand at a constant temperature for clearing; (4) Take out the cleared tissue section, place it on a glass slide, and then add tissue clearing reagent for mounting. (5) Place the mounted tissue sections under a stimulated Raman scattering microscope for three-dimensional volume imaging for subsequent analysis.

5. The application as described in claim 4, characterized in that, The tissue-clearing reagent is preheated using a constant-temperature metal bath.

6. The application as described in claim 4 or 5, characterized in that, The biological tissue samples are 0.3–2 mm thick mouse brain slices, 0.3–1 mm thick fixed mouse heart, kidney, lung, spleen, and liver tissue slices, or 0.3–1 mm thick biopsy tissue slices of patient brain tumors.

Images