A biological thick tissue section transparentizing reagent, a preparation method and application thereof

Abstract

The present disclosure belongs to the technical field of biological tissue optical imaging, and provides a biological thick tissue section transparentization reagent, a preparation method and application thereof. The reagent comprises a lipase solution, an organic compound and an amide compound, and has a pH of 7-9; the thickness of the biological thick tissue section is 50-400 μm. The preparation method comprises: mixing the organic compound and the amide compound with the lipase solution to prepare a mixed reagent; and adjusting the pH of the mixed reagent to 7-9 to obtain the reagent. The use method of the reagent comprises: adding the reagent dropwise on a biological thick tissue section sample or immersing the biological thick tissue section sample in the reagent, and the transparentization is completed after 2-3 min, and then imaging can be performed. The transparentization reagent can quickly realize the transparentization of a thick tissue sample, improve the imaging depth, and has a simple formula, is easy to obtain, and is non-toxic and harmless. Meanwhile, the reagent can maintain the stability of a fluorescent protein, and maintain that fluorescence is not weakened or quenched.

Description

Technical Field

[0001] This disclosure relates to the field of optical imaging technology for biological tissues, and in particular to a reagent for clearing thick biological tissue sections, its preparation method, and its application. Background Technology

[0002] Tissue section imaging is currently the most common detection technique in biological research; however, this method is limited to two-dimensional imaging. For some thick tissue sections, three-dimensional imaging is required. The acquisition of three-dimensional imaging data currently relies mainly on layer-by-layer sample cutting imaging and the rapidly developing tissue transparency imaging. Tissue transparency imaging is widely used in three-dimensional tissue imaging, and its main principle lies in reducing light absorption and scattering during the imaging process.

[0003] However, current tissue clearing methods generally require complex clearing steps and long processing times, reducing the efficiency of 3D imaging data acquisition. For example, the DISCO series of organic solvent-based clearing methods, currently the most widely used in tissue clearing imaging, requires dehydration with gradient concentrations of reagents and refractive index matching with dibenzyl ether organic solvent, with a clearing time of at least 3 days. Among water-soluble clearing methods, the CUBIC technique requires dehydration and decolorization at 37°C and a refractive index matching process, with a clearing time of over one week. The latest improved version, liver-CUBIC, also requires 3-4 days of clearing processing. It is evident that the above tissue clearing methods are mainly for intact tissues and organs. For acquiring imaging data from thick tissue sections, there are currently no more convenient and rapid processing reagents.

[0004] Therefore, in order to improve the efficiency of acquiring three-dimensional imaging data of thick biological tissue sections, there is an urgent need to develop a simple and faster clearing reagent. Summary of the Invention

[0005] This disclosure provides a biological thick tissue section clearing reagent, its preparation method, and its application, in order to at least solve the above-mentioned technical problems existing in the prior art.

[0006] According to a first aspect of this disclosure, a biological thick tissue section clearing reagent is provided, comprising a lipase solution, an organic compound, and an amide compound, wherein the pH of the biological thick tissue section clearing reagent is 7-9; and the thickness of the biological thick tissue section is 50-400 μm.

[0007] According to one possible implementation of this disclosure, at least the following beneficial effects are achieved:

[0008] This disclosed biological thick tissue section clearing reagent achieves delipidation and permeation of biological tissues through a lipase solution. Furthermore, due to the presence of high-refractive-index organic compounds and amide compounds, this high-refractive-index clearing reagent can replace low-refractive-index components in biological tissues, such as water and fat. By rapidly penetrating and homogenizing the refractive index of the tissue sample, this reagent reduces light absorption and scattering during imaging, enabling its numerous applications in biological thick tissue section imaging studies. It plays a crucial role in studying the distribution of tissue structures at single-cell resolution. In addition, by maintaining the pH within the range of 7–9, this clearing reagent can preserve the stability of fluorescent proteins, preventing weakening or quenching of their fluorescence.

[0009] Therefore, the clearing reagent disclosed herein can rapidly achieve the clearing of thick biological tissue samples (clearing can be completed in about 3 minutes, and microscopic imaging can be performed without any other operations), improving the imaging depth of thick biological tissue sections. Furthermore, the formulation is simple, readily available, non-toxic, and harmless. Simultaneously, this clearing reagent is compatible with endogenously fluorescently labeled transgenic tissue samples, and multicolor labeled tissue microenvironment spatial information can be obtained through microscopic optical imaging.

[0010] In one embodiment, the lipase solution is prepared by dissolving a lipase in a solvent, wherein the lipase is selected from at least one of pancreatic lipase, triglyceride hydrolase, and phospholipase.

[0011] Specifically, the solvent is selected from water.

[0012] Specifically, the specific activity of the lipase is 100–500 U / mg.

[0013] Specifically, the lipase solution is a saturated solution with a high concentration of lipase, and its main function is to degrease and permeate, accelerating the penetration of the clearing agent into thick biological tissues.

[0014] In one embodiment, the organic compound is selected from at least one of imidazole, antipyrine, and urea.

[0015] In one embodiment, the organic compound has a mass-to-volume ratio of 5% to 65%.

[0016] In one embodiment, the amide compound is selected from at least one of formamide, nicotinamide, and N-methylnicotinamide.

[0017] In one embodiment, the amide compound has a mass-to-volume ratio of 20-60%.

[0018] According to a second aspect of this disclosure, a method for preparing the above-mentioned biological thick tissue section clearing reagent is provided, comprising the following steps:

[0019] S1: Prepare a lipase solution by mixing organic compounds and amide compounds with the lipase solution to form a mixed reagent;

[0020] S2: Adjust the pH of the mixed reagent to 7-9 to obtain the biological thick tissue section clearing reagent.

[0021] In one embodiment, the method for preparing the lipase solution in step S1 includes the following steps: dissolving a lipase with a specific activity of 100-500 U / mg in a solvent to obtain the lipase solution.

[0022] Specifically, the solvent is water.

[0023] In one embodiment, step S1 involves dissolving 5-65% by mass / volume of the organic compound and 20-60% by mass / volume of the amide compound in the lipase solution to prepare the mixed reagent.

[0024] In one embodiment, step S2 involves adjusting the pH of the mixed reagent to 7-9 by adding an alkaline substance to obtain the biological thick tissue section clearing reagent.

[0025] In one embodiment, the alkaline substance includes either sodium hydroxide or sodium bicarbonate.

[0026] Specifically, the pH of the mixed reagent prepared in step S1 is around 5-6, which is slightly acidic. This is very unfavorable for fluorescent proteins that are not alkali-resistant, and may lead to weakening or quenching of sample fluorescence. In contrast, fluorescent proteins can exist stably in a neutral pH (7-8) environment, which is the most reliable for maintaining fluorescence. Therefore, adjusting the pH of the mixed reagent to 7-9 is more appropriate.

[0027] According to a third aspect of this disclosure, a method for using the above-mentioned biological thick tissue section clearing agent is provided, comprising the following steps:

[0028] The biological thick tissue section clearing reagent is dropped onto the biological thick tissue section sample, or the biological thick tissue section sample is immersed in the biological thick tissue section clearing reagent. After 2-3 minutes, the clearing is completed, and the cleared biological thick tissue section sample can be imaged.

[0029] In one possible embodiment, prior to treatment with the biological thick tissue sectioning reagent, the step includes fixing and sectioning the biological tissue to obtain biological thick tissue section samples with a thickness of 50 to 400 μm.

[0030] In one embodiment, the biological thick tissue section clearing agent can also be used as a mounting medium. After the biological thick tissue section sample is treated with it, it can be directly mounted without the need for additional mounting medium for imaging.

[0031] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this disclosure, nor is it intended to limit the scope of this disclosure. Other features of this disclosure will become readily apparent from the following description. Attached Figure Description

[0032] The above and other objects, features, and advantages of this disclosure will become readily apparent from the following detailed description of exemplary embodiments, taken in conjunction with the accompanying drawings. Several embodiments of this disclosure are illustrated in the drawings by way of example and not limitation, in which:

[0033] In the accompanying drawings, the same or corresponding reference numerals indicate the same or corresponding parts.

[0034] Figure 1 A schematic diagram of the process for clearing thick tissue using a biological thick tissue sectioning agent is shown in the experimental examples of this disclosure;

[0035] Figure 2 The image shows the effect of transparency on thick tissue sections of the brain, liver, and kidney of adult mice in the experimental examples disclosed in this publication; the image was taken with a camera and the background grid size is 1.6mm × 1.6mm.

[0036] Figure 3 The following are fluorescence micrographs of adult mouse liver sections after treatment with biological thick tissue section clearing agent 2 in the experimental examples of this disclosure; where a is the cleared section and b is the uncleared section. Detailed Implementation

[0037] To make the objectives, features, and advantages of this disclosure more apparent and understandable, the technical solutions in the embodiments of this disclosure will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this disclosure, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this disclosure without creative effort are within the scope of protection of this disclosure.

[0038] Most existing tissue transparency technologies are designed for transparentizing intact tissues and organs, involving complex procedures and lengthy operation times. For example, the CUBIC series of technologies requires several days of degreasing treatment (including continuous solution changes and soaking with 50% and 100% degreasing solutions), followed by 6 hours of PBS washing, and finally several days of refractive index matching (including solution changes and soaking with 50% and 100% refractive index matching reagents). The total time generally exceeds one week.

[0039] This disclosure presents a clearing reagent primarily for clearing thick biological tissue sections, which differs from the objectives of existing tissue clearing techniques. Furthermore, the most prominent features of this clearing reagent are its ultra-fast and simple operation; only one reagent needs to be prepared and used to treat the thick biological tissue section sample, achieving clearing within 3 minutes without any further steps, after which imaging can proceed. Currently, no other clearing technique can achieve sample clearing in such a short time without changing the reagent.

[0040] The following examples illustrate this in detail.

[0041] Example 1

[0042] In this embodiment, biological thick tissue section clearing reagent 1 was prepared. The specific process is as follows:

[0043] (1) Add about 100g of a mixture of pancreatic lipase, triglyceride hydrolase and phospholipase with a specific activity of 300U / mg to 50mL of ultrapure water, dissolve and mix well, centrifuge at 4000g for 10min, take the supernatant and filter it with 200 mesh gauze to remove the undissolved part, prepare a saturated lipase solution, and store at 4℃.

[0044] (2) Weigh 25-35% by volume of imidazole and 20-40% by volume of nicotinamide, dilute to volume with the saturated lipase solution prepared in step (1) and dissolve in 50 mL of the system to prepare a mixed reagent.

[0045] (3) Adjust the pH of the mixed reagent prepared in step (2) to 8-9 with NaOH to obtain biological thick tissue section clearing reagent 1, and store it at 4℃.

[0046] Example 2

[0047] In this embodiment, biological thick tissue section clearing reagent 2 was prepared. The specific process is as follows:

[0048] (1) Add about 160g of a mixture of triglyceride hydrolase and phospholipase with a specific activity of 500U / mg to 80mL of ultrapure water, dissolve and mix well, centrifuge at 4000g for 10min, take the supernatant and filter it with 200 mesh gauze to remove the undissolved part, and prepare a saturated lipase solution, and store at 4℃.

[0049] (2) Weigh 30-50% by volume of urea and 30-60% by volume of nicotinamide, dilute to volume with the saturated lipase solution prepared in step (1) and dissolve in 50 mL of the system to prepare a mixed reagent.

[0050] (3) Adjust the pH of the mixed reagent prepared in step (2) to 8-9 with NaOH to obtain biological thick tissue section clearing reagent 2, and store at 4℃.

[0051] Example 3

[0052] In this embodiment, biological thick tissue section clearing reagent 3 was prepared. The specific process is as follows:

[0053] (1) Add about 100g of a mixture of triglyceride hydrolase with a specific activity of 100U / mg to 50mL of ultrapure water, dissolve and mix well, centrifuge at 4000g for 10min, take the supernatant and filter it with 200 mesh gauze to remove the undissolved part, and prepare a saturated lipase solution, and store at 4℃.

[0054] (2) Weigh 5-25% antipyrine, 15-25% imidazole and 20-40% N-methylnicotinamide, and dilute to 50 mL with the saturated lipase solution prepared in step (1) to make a mixed reagent.

[0055] (3) Adjust the pH of the mixed reagent prepared in step (2) to 7-9 with NaOH to obtain biological thick tissue section clearing reagent 3, and store it at 4℃.

[0056] Test case

[0057] This experimental example uses the biological thick tissue section clearing reagent prepared in the previous example to perform a clearing test on a specific biological thick tissue section:

[0058] The overall process for transparentization using the transparentizing reagent prepared in the examples is as follows: Figure 1 First, prepare a biological thick tissue section clearing reagent. Then, slice the tissue sample to be tested that has been fixed in paraformaldehyde (PFA). After slicing, add the clearing reagent to the tissue sample slice. After 2-3 minutes, the tissue sample slice can be imaged under a microscope.

[0059] 1. Clearing test of thick tissue sections from different organs of mice using biological thick tissue section clearing reagent 1 prepared in Example 1:

[0060] (1) Take fresh brain, liver and kidney of adult mice, fix the organs with 4% PFA for 24h, and then wash the fixed samples with 1×PBS at room temperature.

[0061] (2) After the sample is cleaned, the three organs are sliced ​​to a thickness of 200 μm by vibration slicing to obtain thick tissue slices of brain, liver and kidney.

[0062] (3) After the biological thick tissue section clearing reagent 1 is brought back to room temperature or heated to 37°C in a water bath, it is dropped onto the thick tissue sections of the brain, liver and kidney respectively. After waiting for 3 minutes, the clearing process is completed. At this time, the section samples can be directly mounted and imaged.

[0063] The transparency effects of three thick tissue sections are shown in the following figures. Figure 2 As shown. Figure 2 The results showed that, compared to the untreated tissue sections, approximately 3 minutes after adding the biological thick tissue section clearing reagent 1, the brain, liver, and kidney sections exhibited significantly higher levels of transparency, with the background mesh clearly visible. This indicates that the clearing reagent prepared in Example 1 has a rapid and effective clearing effect on thick tissue sections.

[0064] 2. Clearing test of thick tissue sections of mouse liver using biological thick tissue section clearing reagent 2 prepared in Example 2:

[0065] To examine the effect of biological thick tissue section clearing reagent 2 on the preservation of tissue sample fluorescence, confocal optical microscopy was performed on the cleared mouse liver.

[0066] (1) Take fresh livers from adult mice, fix them with 4% PFA for 24 hours, and then wash the fixed samples with 1×PBS at room temperature.

[0067] (2) After the sample is cleaned, the liver is sliced ​​to a thickness of 400 μm by vibrating the slice to obtain a thick tissue slice of the liver.

[0068] (3) After the biological thick tissue section clearing reagent 2 is brought back to room temperature or heated to 37°C in a water bath, it is dropped onto the thick tissue section of the liver. After waiting for 3 minutes, the clearing process is completed. At this time, the section sample can be directly mounted and optically microscopically imaged.

[0069] Imaging was performed using a 10x water microscope on an LSM780 NLO confocal microscope (Zeiss, Germany) with an excitation wavelength of 633 nm. Fluorescence microscopy was used to image liver slices, both before and after rapid clearing, under the same imaging conditions. The results are as follows: Figure 3 As shown. Figure 3The results show that both the opaque (b) and rapidly transparent (a) liver sections exhibit relatively good preservation of mCherry fluorescence. In terms of imaging depth, the opaque liver sections have an imaging depth of less than 50 μm, while the rapidly transparent liver sections have an imaging depth of approximately 400 μm. Therefore, the biological thick tissue section transparentizing reagent 2 prepared in Example 2 not only maintains the fluorescence of fluorescent proteins without weakening or quenching, but also increases the imaging depth of liver section samples by approximately eight times.

[0070] In summary, the biological thick tissue section clearing reagent prepared in this disclosure has an ultra-fast clearing speed and a good clearing effect; it can also maintain the stability of fluorescent proteins and prevent the fluorescence emitted by the fluorescent proteins from being weakened or quenched; it is compatible with transgenic tissue samples with endogenous fluorescent labels, and spatial information of the microenvironment of multicolor labeled tissues can be obtained through microscopic optical imaging, which has strong practicality.

[0071] It should be understood that the various forms of processes shown above can be used to rearrange, add, or delete steps. For example, the steps described in this disclosure can be executed in parallel, sequentially, or in different orders, as long as the desired result of the technical solution of this disclosure can be achieved, and this is not limited herein.

[0072] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this disclosure, "a plurality of" means two or more, unless otherwise explicitly specified.

[0073] The above description is merely a specific embodiment of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this disclosure should be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A clearing agent for thick biological tissue sections, characterized in that, The biological thick tissue section clearing reagent is composed of lipase solution, organic compounds and amide compounds; the pH of the biological thick tissue section clearing reagent is 7-9, which is used to maintain the stability of fluorescent proteins and prevent fluorescence from being weakened or quenched; the thickness of the biological thick tissue section is 50-400 μm. The lipase solution is a saturated lipase solution, and the specific activity of the lipase in the saturated lipase solution is 100~500U / mg. The lipase is selected from at least one of pancreatic lipase, triglyceride hydrolase, and phospholipase. The transparentizing time of the biological thick tissue section clearing reagent on the biological thick tissue section is 2~3min.

2. The biological thick tissue section clearing reagent according to claim 1, characterized in that, The organic compound is selected from at least one of imidazole, antipyrine, and urea.

3. The biological thick tissue section clearing reagent according to claim 1, characterized in that, The organic compound has a mass-to-volume ratio of 5-65%.

4. The biological thick tissue section clearing reagent according to claim 1, characterized in that, The amide compound is selected from at least one of formamide, nicotinamide, and N-methylnicotinamide.

5. The biological thick tissue section clearing reagent according to claim 1, characterized in that, The mass-to-volume ratio of the amide compounds is 20-60%.

6. A method for preparing the biological thick tissue section clearing reagent according to any one of claims 1 to 5, characterized in that, Includes the following steps: S1: Prepare a lipase solution by mixing an organic compound and an amide compound with the lipase solution to form a mixed reagent; S2: Adjust the pH of the mixed reagent to 7-9 to obtain the biological thick tissue section clearing reagent.

7. The preparation method according to claim 6, characterized in that, Step S1 involves dissolving 5-65% by mass / volume of the organic compound and 20-60% by mass / volume of the amide compound in the lipase solution to prepare the mixed reagent.

8. The preparation method according to claim 6, characterized in that, Step S2 involves adjusting the pH of the mixed reagent to 7-9 by adding an alkaline substance to obtain the biological thick tissue section clearing reagent.

9. The preparation method according to claim 8, characterized in that, The alkaline substance includes either sodium hydroxide or sodium bicarbonate.

10. The method of using the biological thick tissue section clearing reagent according to any one of claims 1 to 5, characterized in that, Includes the following steps: The biological thick tissue section clearing reagent is dropped onto the biological thick tissue section sample, or the biological thick tissue section sample is immersed in the biological thick tissue section clearing reagent. After 2-3 minutes, the clearing is completed, and the cleared biological thick tissue section sample can be imaged.

11. The method of use according to claim 10, characterized in that, Prior to the transparentization process of the biological thick tissue sections, the procedure includes fixing and slicing the biological tissue to obtain biological thick tissue section samples with a thickness of 50 to 400 μm.

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