Transparentizing agents, transparentizing agent combinations, kits containing same, and methods of transparentizing bone tissue for non-diagnostic purposes

Abstract

The application relates to the technical field of biology, in particular to a transparent agent, a transparent agent combination product containing the same, a kit and a bone tissue transparent method for non-diagnostic purposes. The transparent agent comprises an acetic acid aqueous solution and an acetic acid-ethylenediaminetetraacetic acid buffer with a specific formula; the transparent agent reacts strongly with inorganic calcium salt (calcium carbonate) in a bone sample, helps to form loose and dense bone tissue, promotes the penetration of subsequent other transparent agents, and helps to enhance the transparent effect; the transparent agent is combined with other transparent agents, which not only makes the skull optically transparent, but also softens the skull to avoid ultrasonic signal attenuation, realizes high-resolution optical imaging, and improves the ultrasonic positioning imaging effect.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to clearing agents, clearing agent combination products containing the same, kits, and methods for clearing bone tissue for non-diagnostic purposes. Background Technology

[0002] High-resolution neurovascular imaging provides an important research tool for elucidating the structure and function of cerebral blood vessels and nerves. Optical imaging techniques can be used for high spatiotemporal resolution monitoring of changes in the structure and function of cerebral blood vessels. For example, optical coherence tomography (OCT) can acquire three-dimensional structural information of cortical vessels; laser speckle contrast imaging can monitor the dynamic blood flow response of cortical microvessels; hyperspectral imaging can analyze blood oxygenation and permeability of cortical vessels; and two-photon imaging can be used to acquire fine structural and flow velocity information of cortical vessels. However, the depth of optical imaging of biological tissues is relatively limited.

[0003] For deeper cerebral blood vessels, ultra-high resolution ultrasound localization imaging has demonstrated unique advantages. Ultrasound localization microscopy is an ultrasound localization imaging method that uses ultrafast ultrasound imaging technology to record spatially discrete microbubble scintillation. It can perform ultra-high resolution imaging of microvessels on the order of 10 μm, providing information on the structure and function of microvessels.

[0004] However, the cloudy skull tissue not only severely restricts the resolution and depth of optical imaging, but also acts as a multi-layered acoustic barrier, which can attenuate ultrasound energy by tens of decibels and distort the wavefront through reflection, refraction, scattering, absorption and mode conversion, thus seriously reducing the image quality of optical imaging and ultrasound positioning imaging.

[0005] Currently, tissue light-transparency technology offers a novel approach to fundamentally addressing the problem of high scattering in biological tissues. It involves introducing highly permeable, high-refractive-index chemical reagents into the tissue to improve the refractive index matching of its components, thereby reducing scattering and making the tissue transparent to light. However, traditional light-transparency agents are primarily designed for optical imaging and cannot effectively reduce the attenuation of ultrasound by the skull. There is still a lack of a light-transparency agent that can be used for both enhanced ultrasound and optical imaging. Summary of the Invention

[0006] Based on this, one or more embodiments of this application provide a clearing agent, a combination product of clearing agents containing the same, a kit, and a method for clearing bone tissue for non-diagnostic purposes. This includes the following technical solutions:

[0007] One or more embodiments of this application provide a clearing agent comprising an aqueous acetic acid solution and an acetic acid-ethylenediaminetetraacetic acid buffer solution; the pH value of the acetic acid-ethylenediaminetetraacetic acid buffer solution is 5.0 to 6.0; based on an aqueous acetic acid solution with a mass fraction of 36%, the volume ratio of the aqueous acetic acid solution to the acetic acid-ethylenediaminetetraacetic acid buffer solution is 5:(0.5 to 2.5).

[0008] One or more embodiments of this application provide a clearing agent combination product, the clearing agent combination product including a first clearing agent and other clearing agents; the first clearing agent is the clearing agent.

[0009] In some embodiments of this application, the other transparent reagent includes one or more of the following: a second transparent reagent, a third transparent reagent, and a fourth transparent reagent as defined below:

[0010] The second transparent reagent includes ethylenediaminetetraacetic acid or its salt;

[0011] The third transparent reagent includes collagenase;

[0012] The fourth clearing agent includes sodium dodecylbenzenesulfonate.

[0013] In some embodiments of this application, the clearing agent combination product includes the first clearing agent, the second clearing agent, and the third clearing agent; or includes the first clearing agent, the second clearing agent, the third clearing agent, and the fourth clearing agent.

[0014] In some embodiments of this application, the second transparent reagent is ethylenediaminetetraacetic acid or a salt solution thereof, with a pH value of 7.0 to 7.5.

[0015] In some embodiments of this application, the third transparent reagent is a collagenase solution with a collagenase mass fraction of 0.8% to 1.2%.

[0016] In some embodiments of this application, the fourth transparent reagent is an aqueous solution of sodium dodecylbenzenesulfonate with a mass fraction of 15% to 20%.

[0017] One or more embodiments of this application provide a kit comprising the aforementioned clearing agent combination product.

[0018] One or more embodiments of this application provide a method for making bone tissue transparent for non-diagnostic purposes, the method comprising the following steps:

[0019] Using the aforementioned clearing agent combination product or the aforementioned kit, bone samples are treated sequentially with the first clearing agent, the second clearing agent, and the third clearing agent; or, bone samples are treated sequentially with the first clearing agent, the second clearing agent, the third clearing agent, and the fourth clearing agent.

[0020] In some embodiments of this application, the bone sample is of the skull type; and / or the species source of the bone sample is mouse.

[0021] Compared with traditional technologies, this application has the following advantages:

[0022] This application provides a specific formulation of a clearing agent comprising an aqueous acetic acid solution and an acetic acid-ethylenediaminetetraacetic acid buffer solution. This clearing agent reacts strongly with inorganic calcium salts (calcium carbonate) in bone samples, which helps to form loose and dense bone tissue, promotes the penetration of subsequent clearing agents, and helps to enhance the clearing effect. When this clearing agent is used in combination with other clearing agents, it can not only make the skull optically transparent, but also soften the skull to avoid ultrasound signal attenuation, achieving high-resolution optical imaging while also improving the ultrasound positioning imaging effect. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments and examples of this application, and to more completely understand this application and its beneficial effects, the accompanying drawings used in the description of the embodiments or examples will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of this application. Those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0024] Figure 1 The procedure for making mouse skull tissue transparent.

[0025] Figure 2 The bubble characteristics are those of the first transparent reagent acting on the surface of the mouse skull.

[0026] Figure 3 This image shows the imaging characteristics of the skull before and after transparency and the cerebral blood vessels in the cortex of mice after craniotomy under optical coherence tomography.

[0027] Figure 4 This image shows the imaging features of mouse cortical neurons before and after skull transparency under a two-photon microscope.

[0028] Figure 5 Imaging features of blood vessels in the sagittal plane of the brain of mice before and after skull transparency under ultra-high resolution ultrasound localization microscopy and after craniotomy.

[0029] Figure 6 The results of ultrasound imaging of the mouse skull after treatment with 36% acetic acid as the first clearing agent and subsequent clearing steps.

[0030] Figure 7 The results of ultrasound imaging of the brain after the mouse skull was treated with a first clearing agent consisting of a 1:1 mixture of 36% acetic acid and acetic acid-EDTA, followed by a subsequent clearing step.

[0031] Figure 8 Visual images of isolated mouse skull tissue placed on graph paper under different treatment methods. Detailed Implementation

[0032] The present application will be further described in detail below with reference to the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. For example, features described or illustrated as part of one embodiment can be combined in a suitable manner in another embodiment to produce new embodiments. Furthermore, numerous details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the specification of this application is for descriptive purposes only and is not intended to be limiting of the application.

[0034] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0035] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0036] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0037] The terms “combinations thereof,” “any combination thereof,” and “any combination thereof” as used in this application include all suitable combinations of any two or more of the listed items.

[0038] In this application, the term "suitable" as used in "suitable combination", "suitable method", "any suitable method", etc., refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0039] In this application, terms such as "preferred," "better," "more suitable," and "ideal" are merely used to describe implementation methods or embodiments that achieve better results, and should be understood not to limit the scope of protection of this application.

[0040] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0041] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0042] In this application, where the method flow involves multiple steps, unless otherwise explicitly stated in this application, there is no strict order restriction on the execution of these steps; they can be executed in an order other than that described. Moreover, any step may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily completed at the same time, but can be executed at different times. Their execution order is not necessarily sequential, but can be performed alternately, concurrently, or in turn with other steps or parts of the sub-steps or stages of other steps.

[0043] In this application, the terms "first aspect," "second aspect," "third aspect," "fourth aspect," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or quantity, nor should they be construed as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first," "second," "third," "fourth," etc., serve only as a non-exhaustive enumeration and should be understood not to constitute a closed limitation on quantity.

[0044] In this application, the technical features or solutions described using open-ended language include both closed-ended technical features or solutions composed of the listed content and open-ended technical features or solutions that include the listed content. In this application, when numerical intervals (i.e., numerical ranges) are involved, unless otherwise specified, the selectable numerical distribution within the aforementioned numerical interval is considered continuous, and includes the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval only refers to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this application, this is equivalent to directly listing every integer; for example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed in this application should be understood to include any and all sub-ranges included therein.

[0045] A first aspect of this application provides a clearing agent comprising an aqueous acetic acid solution and an acetic acid-ethylenediaminetetraacetic acid buffer solution; the pH value of the acetic acid-ethylenediaminetetraacetic acid buffer solution is 5.0~6.0, for example, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0; the aqueous acetic acid solution is calculated as 36% by mass, and the aqueous acetic acid solution and the acetic acid-ethylenediaminetetraacetic acid buffer solution are... The volume ratio of diaminetetraacetic acid buffer is 5:(0.5~2.5), for example 5:0.5, 5:0.6, 5:0.7, 5:0.8, 5:0.9, 5:1, 5:1.1, 5:1.2, 5:1.3, 5:1.4, 5:1.5, 5:1.6, 5:1.7, 5:1.8, 5:1.9, 5:2, 5:2.1, 5:2.2, 5:2.3, 5:2.4, 5:2.5.

[0046] A second aspect of this application provides a transparent agent combination product, the transparent agent combination product including a first transparent reagent and other transparent reagents; the first transparent reagent is the transparent agent.

[0047] In some embodiments of this application, the other transparent reagent includes one or more of the following: a second transparent reagent, a third transparent reagent, and a fourth transparent reagent as defined below:

[0048] The second transparent reagent includes ethylenediaminetetraacetic acid or its salt;

[0049] The third transparent reagent includes collagenase;

[0050] The fourth clearing agent includes sodium dodecylbenzenesulfonate.

[0051] In some embodiments of this application, the clearing agent combination product includes the first clearing agent, the second clearing agent, and the third clearing agent; or includes the first clearing agent, the second clearing agent, the third clearing agent, and the fourth clearing agent.

[0052] In some embodiments of this application, the second transparent reagent is ethylenediaminetetraacetic acid or a salt solution thereof, with a pH value of 7.0 to 7.5, for example 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5.

[0053] In some embodiments of this application, the third transparent reagent is a collagenase solution with a collagenase mass fraction of 0.8% to 1.2%, for example, 0.8%, 0.9%, 1.0%, 1.1%, or 1.2%.

[0054] In some embodiments of this application, the fourth transparent reagent is an aqueous solution of sodium dodecylbenzenesulfonate with a mass fraction of 15% to 20%, for example, 15%, 16%, 17%, 18%, 19%, or 20%.

[0055] A third aspect of this application provides a kit comprising the aforementioned clearing agent combination product.

[0056] A fourth aspect of this application provides a method for making bone tissue transparent for non-diagnostic purposes, the method comprising the following steps:

[0057] Using the aforementioned clearing agent combination product or the aforementioned kit, bone samples are treated sequentially with the first clearing agent, the second clearing agent, and the third clearing agent; or, bone samples are treated sequentially with the first clearing agent, the second clearing agent, the third clearing agent, and the fourth clearing agent.

[0058] In some embodiments of this application, the bone sample is of the skull type; and / or the species source of the bone sample is mouse.

[0059] The implementation schemes of this application will be described in detail below with reference to some specific embodiments. It should be understood that these embodiments are only used to illustrate this application and are not intended to limit the scope of this application. For experimental methods in the following embodiments where conditions are not specified, please refer to the guidelines given in this application first, or follow the experimental manual or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0060] In the following examples, the measurement parameters of the raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0061] Example 1

[0062] The transparent reagents provided in this embodiment mainly include reagents No. 1, No. 2, No. 3, and No. 4. The preparation method is as follows:

[0063] Reagent 1 (First Clearing Reagent): 36% acetic acid (A112880, Aladdin, China) and acetic acid-ethylenediaminetetraacetic acid / EDTA buffer (A301488, Aladdin, China) were mixed at a volume ratio of 5:1;

[0064] The pH of the acetic acid-EDTA buffer solution is 5.5; 36% acetic acid refers to an aqueous solution of acetic acid with a mass fraction of 36%. If using acetic acid aqueous solutions with other mass fractions, the volume equivalent to a 36% acetic acid aqueous solution can be calculated according to the mass fraction, or the solution can be diluted / adjusted to a 36% acetic acid aqueous solution before preparation according to the stated volume ratio. The principle is to utilize a biosafe, weakly acidic reagent to react with calcium salts in the skull, achieving a dual effect of skull osteoporosis and decalcification.

[0065] Reagent No. 2 (Second Clearing Reagent): EDTA decalcification solution (BL616B, Biosharp), pH 7.2. It chelates with calcium salts in the skull, causing calcium salt precipitation, softening the skull, and enhancing ultrasound penetration into the skull.

[0066] Reagent No. 3 (Third Clearing Reagent): 1% collagenase (10269638001, Roche) solution. Dissociates cranial collagen, further enhancing the clearing effect.

[0067] Reagent No. 4 (Fourth Transparent Reagent): A 17.5% sodium dodecylbenzenesulfonate aqueous solution.

[0068] The transparency steps are as follows (e.g.) Figure 1 (as shown)

[0069] Step 1: Scrape the hair off the head of 10-week-old adult male Balb / c mice, cut off the head skin, clean the mucous membrane on the surface of the skull and dry the skull surface;

[0070] In the implementation of step 1, the specific procedure is as follows: after anesthetizing the mouse, the hair on its head is shaved off, the skin on the head is cut off with ophthalmic scissors, the mucous membrane on the surface of the skull is cleaned with a moist cotton ball, and the surface of the skull is dried with compressed air.

[0071] Step 2: Construct a transparent reagent groove;

[0072] In step 2, the prepared fluid dental cement is applied to the surface of the mouse skull (except for the two parietal bones and the sagittal suture between them), and the edges are piled with semi-solid dental cement to form a bowl-shaped groove to hold the transparent reagent.

[0073] Step 3: Temporarily seal the sagittal suture with quick-drying glue;

[0074] Apply a thin layer of 502 glue to the sagittal suture between the two parietal bones to prevent leakage of reagent No. 1.

[0075] Step 4: Reagent No. 1 is applied to the surface of the skull;

[0076] Add 0.5 mL of reagent No. 1 to the groove and let it sit for 5 minutes. During this time, a large number of air bubbles will appear on the surface of the skull. The result is as follows. Figure 2 As shown.

[0077] Step 5: Clean the gel-like product from the surface of the skull;

[0078] Gently scrape away the gel-like reaction substance on the surface of the skull with a micro-surgical blade, then wipe with 75% ethanol and blow dry the skull surface.

[0079] Step 6: Reagent No. 2 is applied to the surface of the skull;

[0080] Add 0.5 mL of reagent No. 2 and allow it to react for 10 min.

[0081] Steps 7-8: Clean the chelated calcium salt crystals that have precipitated on the surface of the skull;

[0082] Wipe off reagent No. 2 and blow dry the skull surface. Gently scrape off the white product on the skull surface with a microblade.

[0083] Step 9: Reagent No. 3 is applied to the surface of the skull;

[0084] Add 0.5 mL of reagent No. 3 and allow it to react for 5 min.

[0085] Step 10-1: Wipe off reagent No. 3, add 0.5 mL of reagent No. 4, and perform optical imaging after 3 min;

[0086] Step 10-2: Wipe off reagent No. 3, apply ultrasound coupling agent, and perform ultrasound imaging.

[0087] The optical imaging steps are as follows:

[0088] (1) Using optical coherence tomography (OCT), images of cerebral blood vessels were taken before and after transparency, and compared with the results of craniotomy after removal of the skull. Figure 3The results showed that before transparency, only some large cortical vessels were visible, resulting in poor image contrast. After transparency, both large and small cortical vessels were clearly distinguishable, and their morphology was intact. While craniotomy can meet the requirements for high-resolution imaging of cortical vessels, compared to transparency, some vessels disappear due to skull removal, leading to information loss. Therefore, this transparency process not only meets the requirements for high-resolution imaging of cortical vessels but also completely preserves cortical vascular information.

[0089] (2) Use two-photon microscopy to distinguish and photograph information of cortical neurons before and after transparency. Figure 4 The results showed that before the skull was made transparent, only some neurons on the surface of the cortex were faintly visible, with an imaging depth of about 300 μm. After the skull was made transparent, the imaging depth was greatly improved, reaching 700 μm, and the cell bodies of cortical neurons and even the deeper parts were clearly distinguishable.

[0090] The steps of ultrasound imaging are as follows:

[0091] Images of the sagittal vascular plane of the mouse brain were captured using a high-resolution ultrasound localization microscope before, after, and after craniotomy. Figure 5 The results showed that before transparency, only some superficial blood vessels could be effectively distinguished, while after transparency, all blood vessels in the sagittal plane could be effectively distinguished.

[0092] Example 2

[0093] The transparent reagent provided in this embodiment mainly includes reagent No. 1. The preparation method is as follows:

[0094] Reagent 1 (First Clearing Reagent): 36% acetic acid (A112880, Aladdin, China) and acetic acid-ethylenediaminetetraacetic acid / EDTA (A301488, Aladdin, China) are mixed at a volume ratio of 5:2. The pH of the acetic acid-EDTA buffer is 5.5.

[0095] The steps to make it transparent are as follows:

[0096] Step 1: Scrape the hair off the head of 10-week-old adult male Balb / c mice, cut off the head skin, clean the mucous membrane on the surface of the skull and dry the skull surface;

[0097] In the implementation of step 1, the specific procedure is as follows: after anesthetizing the mouse, the hair on its head is shaved off, the skin on the head is cut off with ophthalmic scissors, the mucous membrane on the surface of the skull is cleaned with a moist cotton ball, and the surface of the skull is dried with compressed air.

[0098] Step 2: Construct a transparent reagent groove;

[0099] In step 2, the prepared fluid dental cement is applied to the surface of the mouse skull (except for the two parietal bones and the sagittal suture between them), and the edges are piled with semi-solid dental cement to form a bowl-shaped groove to hold the transparent reagent.

[0100] Step 3: Temporarily seal the sagittal suture with quick-drying glue;

[0101] Apply a thin layer of 502 glue to the sagittal suture between the two parietal bones to prevent leakage of reagent No. 1.

[0102] Step 4: Reagent No. 1 is applied to the surface of the skull;

[0103] Add 0.5 mL of reagent No. 1 to the groove and let it sit for 5 minutes. During this time, a large number of air bubbles will appear on the surface of the skull. The result is as follows. Figure 2 As shown.

[0104] Comparative Example 1

[0105] The transparent reagent provided in this comparative example mainly includes reagent No. 1. The preparation method is as follows:

[0106] Reagent No. 1 (First Transparent Reagent): 36% Acetic Acid (A112880, Aladdin, China).

[0107] The steps to make it transparent are as follows:

[0108] Step 1: Scrape the hair off the head of 10-week-old adult male Balb / c mice, cut off the head skin, clean the mucous membrane on the surface of the skull and dry the skull surface;

[0109] In the implementation of step 1, the specific procedure is as follows: after anesthetizing the mouse, the hair on its head is shaved off, the skin on the head is cut off with ophthalmic scissors, the mucous membrane on the surface of the skull is cleaned with a moist cotton ball, and the surface of the skull is dried with compressed air.

[0110] Step 2: Construct a transparent reagent groove;

[0111] In step 2, the prepared fluid dental cement is applied to the surface of the mouse skull (except for the two parietal bones and the sagittal suture between them), and the edges are piled with semi-solid dental cement to form a bowl-shaped groove to hold the transparent reagent.

[0112] Step 3: Temporarily seal the sagittal suture with quick-drying glue;

[0113] Apply a thin layer of 502 glue to the sagittal suture between the two parietal bones to prevent leakage of reagent No. 1.

[0114] Step 4: Reagent No. 1 is applied to the surface of the skull;

[0115] Add 0.5 mL of reagent No. 1 to the groove and let it sit for 5 minutes. During this time, a small number of air bubbles will appear on the surface of the skull. The results are as follows. Figure 2 As shown.

[0116] Step 5: Clean the gel-like product from the surface of the skull;

[0117] Gently scrape away the gel-like reaction substance on the surface of the skull with a micro-surgical blade, then wipe with 75% ethanol and blow dry the skull surface.

[0118] Step 6: Reagent No. 2 is applied to the surface of the skull;

[0119] Add 0.5 mL of reagent No. 2 and allow it to react for 10 min.

[0120] Steps 7-8: Clean the chelated calcium salt crystals that have precipitated on the surface of the skull;

[0121] Wipe off reagent No. 2 and blow dry the skull surface. Gently scrape off the white product on the skull surface with a microblade.

[0122] Step 9: Reagent No. 3 is applied to the surface of the skull;

[0123] Add 0.5 mL of reagent No. 3 and allow it to react for 5 min.

[0124] Step 10-1: Wipe off reagent No. 3, apply ultrasound coupling agent, and perform ultrasound imaging.

[0125] The steps of ultrasound imaging are as follows:

[0126] Images of sagittal blood vessels in the mouse brain before and after transparency were captured using a high-resolution ultrasound localization microscope. Figure 2 and Figure 6 The results showed that the reaction strength of 36% acetic acid alone with calcium salts on the skull surface was lower than that with the addition of some acetic acid-EDTA buffer, resulting in insufficient understanding of skull osteoporosis. This led to a slightly weaker effect of subsequent reagents 2 and 3, and lower ultrasound imaging quality. Figure 5 The ultrasound imaging quality shown.

[0127] Comparative Example 2

[0128] The transparent reagent provided in this comparative example mainly includes reagent No. 1. The preparation method is as follows:

[0129] Reagent 1 (First Clearing Reagent): 36% acetic acid (A112880, Aladdin, China) and acetic acid-ethylenediaminetetraacetic acid / EDTA (A301488, Aladdin, China) are mixed at a volume ratio of 5:3. The pH of the acetic acid-EDTA buffer is 5.5.

[0130] The steps to make it transparent are as follows:

[0131] Step 1: Scrape the hair off the head of 10-week-old adult male Balb / c mice, cut off the head skin, clean the mucous membrane on the surface of the skull and dry the skull surface;

[0132] In the implementation of step 1, the specific procedure is as follows: after anesthetizing the mouse, the hair on its head is shaved off, the skin on the head is cut off with ophthalmic scissors, the mucous membrane on the surface of the skull is cleaned with a moist cotton ball, and the surface of the skull is dried with compressed air.

[0133] Step 2: Construct a transparent reagent groove;

[0134] In step 2, the prepared fluid dental cement is applied to the surface of the mouse skull (except for the two parietal bones and the sagittal suture between them), and the edges are piled with semi-solid dental cement to form a bowl-shaped groove to hold the transparent reagent.

[0135] Step 3: Temporarily seal the sagittal suture with quick-drying glue;

[0136] Apply a thin layer of 502 glue to the sagittal suture between the two parietal bones to prevent leakage of reagent No. 1.

[0137] Step 4: Reagent No. 1 is applied to the surface of the skull;

[0138] Add 0.5 mL of reagent No. 1 to the groove and let it sit for 5 minutes. During this time, a small number of air bubbles will appear on the surface of the skull. The results are as follows. Figure 2 As shown.

[0139] Comparative Example 3

[0140] The transparent reagent provided in this comparative example mainly includes reagent No. 1. The preparation method is as follows:

[0141] Reagent 1 (First Clearing Reagent): 36% acetic acid (A112880, Aladdin, China) and acetic acid-ethylenediaminetetraacetic acid / EDTA (A301488, Aladdin, China) are mixed at a volume ratio of 1:1. The pH of the acetic acid-EDTA buffer is 5.5.

[0142] The steps to make it transparent are as follows:

[0143] Step 1: Scrape the hair off the head of 10-week-old adult male Balb / c mice, cut off the head skin, clean the mucous membrane on the surface of the skull and dry the skull surface;

[0144] In the implementation of step 1, the specific procedure is as follows: after anesthetizing the mouse, the hair on its head is shaved off, the skin on the head is cut off with ophthalmic scissors, the mucous membrane on the surface of the skull is cleaned with a moist cotton ball, and the surface of the skull is dried with compressed air.

[0145] Step 2: Construct a transparent reagent groove;

[0146] In step 2, the prepared fluid dental cement is applied to the surface of the mouse skull (except for the two parietal bones and the sagittal suture between them), and the edges are piled with semi-solid dental cement to form a bowl-shaped groove to hold the transparent reagent.

[0147] Step 3: Temporarily seal the sagittal suture with quick-drying glue;

[0148] Apply a thin layer of 502 glue to the sagittal suture between the two parietal bones to prevent leakage of reagent No. 1.

[0149] Step 4: Reagent No. 1 is applied to the surface of the skull;

[0150] Add 0.5 mL of reagent No. 1 to the groove and let it sit for 5 minutes. During this time, a small number of air bubbles will appear on the surface of the skull. The results are as follows. Figure 2 As shown.

[0151] Step 5: Clean the gel-like product from the surface of the skull;

[0152] Gently scrape away the gel-like reaction substance on the surface of the skull with a micro-surgical blade, then wipe with 75% ethanol and blow dry the skull surface.

[0153] Step 6: Reagent No. 2 is applied to the surface of the skull;

[0154] Add 0.5 mL of reagent No. 2 and allow it to react for 10 min.

[0155] Steps 7-8: Clean the chelated calcium salt crystals that have precipitated on the surface of the skull;

[0156] Wipe off reagent No. 2 and blow dry the skull surface. Gently scrape off the white product on the skull surface with a microblade.

[0157] Step 9: Reagent No. 3 is applied to the surface of the skull;

[0158] Add 0.5 mL of reagent No. 3 and allow it to react for 5 min.

[0159] Step 10-2: Wipe off reagent No. 3, apply ultrasound coupling agent, and perform ultrasound imaging.

[0160] The steps of ultrasound imaging are as follows:

[0161] Images of sagittal blood vessels in the mouse brain before and after transparency were captured using a high-resolution ultrasound localization microscope. Figure 2 and Figure 7 The results showed that the reaction intensity of reagent No. 1, which was a 1:1 volume mixture of 36% acetic acid and acetic acid-EDTA, was significantly lower than that of other groups when the skull surface was treated. This resulted in a severe deficiency in the degree of skull osteoporosis, which weakened the effect of subsequent reagents No. 2 and No. 3. Therefore, their improvement on brain ultrasound imaging was not significant.

[0162] Comparative Example 4

[0163] This comparative example is the comparative example of Example 1. The only difference from Example 1 is that reagent 1 was not used; only reagents 2, 3, and 4 were used for mouse skull clearing treatment. All other aspects are the same as in Example 1. Mouse skulls that were not cleared and mouse skulls treated with reagents 1, 2, 3, and 4 were used as controls.

[0164] The results are as follows Figure 8 As shown: The leftmost mouse skull, without any transparency treatment, appears cloudy, with background letters faintly visible and squares completely indistinguishable; the middle skull, treated only with reagents 2, 3, and 4, shows increased transparency, with background letters visible, but some squares at the edges are still indistinguishable; the rightmost skull, treated with reagents 1, 2, 3, and 4, clearly shows both background letters and squares. Therefore, this comparative example demonstrates that without reagent 1, the effect of skull transparency cannot be significantly enhanced.

[0165] In summary, this application, based on the principle of light and sound transparency, eliminates the need to grind the skull. By using a transparent agent, it enables the skull to be transparent to both light and sound simultaneously, thereby combining various optical and ultrasound imaging technologies to support high-resolution visualization studies of cerebral blood vessels and nerves.

[0166] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0167] The above-described embodiments are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. It should be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided by the present invention through logical analysis, reasoning, or limited experimentation are all within the protection scope of the appended claims. Therefore, the protection scope of this invention patent should be determined by the content of the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A transparent agent combination product, characterized in that, The clearing agent combination product includes a first clearing agent and other clearing agents; the first clearing agent includes an aqueous acetic acid solution and an acetic acid-ethylenediaminetetraacetic acid buffer solution; the pH value of the acetic acid-ethylenediaminetetraacetic acid buffer solution is 5.0~6.0; based on an aqueous acetic acid solution with a mass fraction of 36%, the volume ratio of the aqueous acetic acid solution to the acetic acid-ethylenediaminetetraacetic acid buffer solution is 5:(1~2). The other transparent reagents are the second transparent reagent, the third transparent reagent, and the fourth transparent reagent; The second transparent reagent is a solution of ethylenediaminetetraacetic acid or its salt, with a pH of 7.0-7.5; The third transparent reagent is a collagenase solution with a collagenase mass fraction of 0.8% to 1.2%; The fourth transparent reagent is an aqueous solution of sodium dodecylbenzenesulfonate with a mass fraction of 15% to 20%.

2. A reagent kit, characterized in that, The kit comprises the clearing agent combination product of claim 1.

3. A method for translucent bone tissue for non-diagnostic purposes, characterized in that, The method includes the following steps: The bone sample is treated sequentially with the first clearing reagent, the second clearing reagent, and the third clearing reagent; or, the bone sample is treated sequentially with the first clearing reagent, the second clearing reagent, the third clearing reagent, and the fourth clearing reagent. The first transparent reagent comprises an aqueous acetic acid solution and an acetic acid-ethylenediaminetetraacetic acid buffer solution; the pH value of the acetic acid-ethylenediaminetetraacetic acid buffer solution is 5.0~6.0; based on an aqueous acetic acid solution with a mass fraction of 36%, the volume ratio of the aqueous acetic acid solution to the acetic acid-ethylenediaminetetraacetic acid buffer solution is 5:(1~2); The second transparent reagent is a solution of ethylenediaminetetraacetic acid or its salt, with a pH of 7.0-7.5; The third transparent reagent is a collagenase solution with a collagenase mass fraction of 0.8% to 1.2%; The fourth transparent reagent is an aqueous solution of sodium dodecylbenzenesulfonate with a mass fraction of 15% to 20%.

4. The method for non-diagnostic bone tissue transparency according to claim 3, characterized in that, The bone sample was a skull.

5. The method for non-diagnostic bone tissue transparency according to claim 3, characterized in that, The bone samples were derived from mice.

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