Method and kit for cleaning in-vitro tissue surface after magnetic resonance imaging

Abstract

The invention discloses a kit for cleaning the surface of in vitro tissue after magnetic resonance imaging, which comprises a reagent A, a reagent B, a reagent C and a reagent D. The reagent A is a mixture of n-hexane and n-pentane; the reagent B is a mixture of n-hexane, n-pentane and an alcohol substance; the reagent C is an alcohol substance; the reagent D is a mixture of an alcohol substance and a phosphate buffer solution. The invention further discloses a method for cleaning the surface of the in-vitro tissue after magnetic resonance imaging. The method comprises the step of sequentially rinsing an in-vitro biological tissue sample after magnetic resonance imaging by using the reagent A, the reagent B, the reagent C and the reagent D in the kit. According to the invention, n-hexane is introduced into the kit to remove perfluoropolyether on the tissue surface, and the technical problems of incomplete cleaning of fluorocarbon oil on the tissue surface, slow cleaning and complex process in the prior art are solved at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of magnetic resonance imaging, and in particular relates to a method and a kit for cleaning the surface of ex vivo tissue after magnetic resonance imaging. Background Art

[0002] MRI (magnetic resonance imaging) technology has a very broad application prospect in ex vivo biological tissues. It not only provides high-resolution, multi-parameter, 3D reconstructible imaging capabilities, but also expands new research fields and applications by combining with other advanced technologies. However, when MRI is used to image ex vivo biological tissues, in order to reduce background signals and improve image resolution, it is often necessary to immerse the biological tissues in fluorine oil (perfluoropolyether) for imaging. Fluorine oil is a colorless and transparent liquid composed of three elements: fluorine, carbon, and oxygen. It has the characteristics of safety, non-toxicity, thermal stability, high temperature resistance, non-flammability, and chemical stability. The high chemical inertness makes common chemical cleaning agents almost ineffective against fluorine oil; the high temperature resistance makes it impossible to remove fluorine oil by heating; the good viscosity-temperature property increases the difficulty of cleaning; the low surface tension makes it difficult to be wetted and emulsified by water and other common cleaning agents; the non-stickiness to water and oil makes it difficult to clean it even with water-based or oil-based cleaning agents.

[0003] The shortcomings of existing methods for cleaning the surface of ex vivo biological tissues after MRI mainly include the following aspects: incomplete cleaning leads to residual contaminants; the use of certain chemical cleaning agents may damage the structure of biological tissues, affecting their subsequent research or use; strong cleaning may cause changes in the microstructure of biological tissues, thereby affecting the accuracy of experimental results; different types of biological tissues may require different cleaning agents and methods, which makes the standardized process complicated and difficult to achieve; the cleaned biological tissues may require drying or other post-processing steps, which increases the complexity of the entire cleaning process.

[0004] In summary, a large amount of fluorinated oil often adheres to the surface of ex vivo biological tissue after magnetic resonance imaging. Fluorinated oil is difficult to clean due to its high chemical inertness, thermal stability, non-flammability, viscosity-temperature properties, and special application requirements, which has adverse effects on subsequent biological tissue processing such as embedding and slicing. Therefore, how to quickly clean the fluorinated oil on the surface of ex vivo biological tissue after MRI is a technical problem in the field of nuclear magnetic resonance, and technicians in this field need to try different cleaning methods or cleaning kits. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention proposes a method and a kit for cleaning the surface of ex vivo tissue after magnetic resonance imaging, which can solve the technical problems of the prior art in that fluorine oil cleaning of the tissue surface is incomplete, slow and has a complicated process.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a kit for cleaning the surface of an ex vivo tissue after magnetic resonance imaging, the kit comprising reagent A, reagent B, reagent C and reagent D, wherein:

[0008] The reagent A is a mixture of n-hexane and n-pentane;

[0009] The reagent B is a mixture of n-hexane, n-pentane and alcohols;

[0010] The reagent C is an alcohol substance;

[0011] The reagent D is a mixture of alcohol substances and phosphate buffer.

[0012] In one or more embodiments, the phosphate buffer may be a potassium phosphate buffer or a sodium phosphate buffer.

[0013] In one or more embodiments, the alcohol material may be anhydrous ethanol or anhydrous methanol, preferably anhydrous ethanol.

[0014] In one or more embodiments, the mass ratio of n-hexane to n-pentane in the reagent A is 9:1.

[0015] In one or more embodiments, the mass ratio of n-hexane, n-pentane and alcohol in the reagent B is 45:5:50.

[0016] In one or more embodiments, the mass ratio of the alcohol substance to the phosphate buffer in the reagent D is 1:1.

[0017] In a second aspect, the present invention provides a method for cleaning the surface of ex vivo tissue after magnetic resonance imaging, the method comprising the steps of using reagent A, reagent B, reagent C and reagent D in the kit described in any one of the items of the present invention to rinse the ex vivo biological tissue sample after magnetic resonance imaging in sequence.

[0018] In one or more embodiments, the method comprises the following steps:

[0019] (1) taking out the in vitro biological tissue sample after magnetic resonance imaging from the sample bottle filled with fluorine oil, and washing the fluorine oil on the surface of the sample with phosphate buffer;

[0020] (2) using reagent A, reagent B, reagent C and reagent D in the kit to sequentially rinse the ex vivo biological tissue sample after magnetic resonance imaging;

[0021] (3) Take out the rinsed biological tissue and place it in phosphate buffer and store it under low temperature conditions.

[0022] In one or more embodiments, when rinsing the ex vivo biological tissue sample in step (2), the amount of reagent A, reagent B, reagent C and reagent D used is 10 times the volume of the ex vivo biological tissue.

[0023] In one or more embodiments, when rinsing the ex vivo biological tissue sample in step (2), the ex vivo biological tissue sample is placed in a sample bottle containing reagent A, reagent B, reagent C and reagent D in sequence, and then the sample bottle caps are closed and the sample bottles are placed in a constant temperature oscillator in sequence for rinsing.

[0024] In one or more embodiments, the rinsing time in the constant temperature oscillator in step (2) is 30 minutes each time.

[0025] The present invention introduces n-hexane into the kit and uses it in combination with other reagents to remove perfluoropolyether on the surface of ex vivo tissues, while solving the technical problems of incomplete cleaning of fluorinated oil on the surface of ex vivo tissues, slow cleaning, and complicated processes in the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a flow chart of the cleaning method described in the present invention.

[0027] Figure 2 It is a comparison chart of the surface of the ex vivo tissue before and after cleaning in Example 1.

[0028] Figure 3 It is a comparison chart of the surface of the ex vivo tissue before and after cleaning in Example 2. DETAILED DESCRIPTION

[0029] It should be understood that the tissue, ex vivo tissue, biological tissue, ex vivo biological tissue, biological ex vivo tissue, etc. mentioned in the present invention all have the same meaning. The tissue needs to be immersed in fluorinated oil (perfluoropolyether) during MRI imaging, and the fluorinated oil on the surface of the tissue needs to be removed when the tissue is subsequently sectioned or immunized as a whole, otherwise it will affect the final result.

[0030] The shortcomings of existing methods for cleaning the surface of ex vivo biological tissues after MRI mainly include the following aspects: incomplete cleaning leads to residual contaminants; the use of certain chemical cleaning agents may damage the structure of biological tissues, affecting their subsequent research or use; strong cleaning may cause changes in the microstructure of biological tissues, thereby affecting the accuracy of experimental results; different types of biological tissues may require different cleaning agents and methods, which makes the standardized process complicated and difficult to achieve; the cleaned biological tissues may require drying or other post-processing steps, which increases the complexity of the entire cleaning process.

[0031] The above technical problems will have a great impact on the subsequent biological tissue sectioning or overall immunostaining, which will in turn affect the subsequent microscopic imaging of the biological tissue, and ultimately make it difficult to obtain the structural information of the biological tissue or the obtained structural information is not accurate enough.

[0032] In view of the existing problems, the inventors have found that by combining n-hexane with other chemical substances, the fluorinated oil on the surface of ex vivo tissue can be cleaned more quickly and thoroughly. Therefore, the present invention provides a kit and method for cleaning the surface of ex vivo tissue after magnetic resonance imaging.

[0033] In the present invention, the kit comprises reagent A, reagent B, reagent C and reagent D, wherein: reagent A is a mixture of n-hexane and n-pentane; reagent B is a mixture of n-hexane, n-pentane and alcohol substances; reagent C is an alcohol substance; and reagent D is a mixture of alcohol substances and phosphate buffer.

[0034] The inventors found that in the above kit, the alcohol substance can be anhydrous ethanol or anhydrous methanol, both of which can fully clean the surface of the ex vivo tissue. However, the use of methanol is likely to cause damage to the tissue surface, so anhydrous ethanol is preferably used.

[0035] It should be understood that for different ex vivo tissues, when using the kit of the present invention for cleaning, the amount of each component in the kit can be adjusted according to actual needs. Preferably, in the kit of the present invention, the mass ratio of n-hexane to n-pentane in the reagent A is 9:1; the mass ratio of n-hexane, n-pentane and alcohol in the reagent B is 45:5:50; the mass ratio of alcohol to phosphate buffer in the reagent D is 1:1.

[0036] In addition, the present invention also provides a method for cleaning the surface of ex vivo tissue after magnetic resonance imaging, the method comprising the steps of using reagent A, reagent B, reagent C and reagent D in the kit of the present invention to rinse the ex vivo biological tissue sample after magnetic resonance imaging in sequence.

[0037] In one or more embodiments, the method is as follows Figure 1 As shown, the following steps are included:

[0038] (1) taking out the in vitro biological tissue sample after magnetic resonance imaging from the sample bottle filled with fluorine oil, and washing the fluorine oil on the surface of the sample with phosphate buffer;

[0039] (2) using reagent A, reagent B, reagent C and reagent D in the kit to sequentially rinse the ex vivo biological tissue sample after magnetic resonance imaging;

[0040] (3) Take out the rinsed biological tissue and place it in phosphate buffer and store it under low temperature conditions.

[0041] It should be understood that for the above-mentioned method, for different ex vivo tissues, the specific parameters of each step can be adjusted according to actual needs during the specific cleaning or rinsing process. For example, according to the size of the ex vivo tissue, an appropriate amount of phosphate buffer can be used to rinse its surface in step (1); for example, according to actual needs, the amount of each reagent added in the kit and the rinsing time of each reagent for the ex vivo biological tissue sample can be adjusted in step (2).

[0042] It should be understood that the phosphate buffer used in the method is a reagent commonly used in the art, which can be a mixture of sodium dihydrogen phosphate and disodium hydrogen phosphate, or a mixture of potassium dihydrogen phosphate and dipotassium hydrogen phosphate. Those skilled in the art can adjust the content of each component in the mixture according to actual needs or the actual required buffer pH. Preferably, the pH value of the phosphate buffer in the present invention is 5.7-8.0.

[0043] Preferably, when rinsing the ex vivo biological tissue sample in step (2), the amount of reagent A, reagent B, reagent C and reagent D used is 10 times the volume of the ex vivo biological tissue.

[0044] Preferably, when rinsing the ex vivo biological tissue sample in step (2), the ex vivo biological tissue sample is placed in a sample bottle containing reagent A, reagent B, reagent C and reagent D in sequence, and then the sample bottle caps are closed and the sample bottles are placed in a constant temperature oscillator in sequence for rinsing.

[0045] Preferably, each rinsing time in the constant temperature oscillator in step (2) is 30 minutes.

[0046] Example

[0047] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0048] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0049] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0050] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0051] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0052] Embodiment 1:

[0053] A kit for rapid cleaning of the surface of in vitro biological tissue after magnetic resonance imaging, the kit comprising reagents A, B, C and D.

[0054] Reagent A includes n-hexane and n-pentane, and the mass ratio of the two is 9:1.

[0055] The components of reagent B are a mixture of alkanes and alcohols, wherein the alkanes are n-hexane and n-pentane, and the alcohol is anhydrous ethanol; the mass ratio of the components is n-hexane: n-pentane: anhydrous ethanol = 45:5:50.

[0056] The ingredients in reagent C are alcohols, wherein the alcohol is anhydrous ethanol.

[0057] The ingredients in reagent D are a mixture of alcohols and buffers. The alcohol is anhydrous ethanol, and the buffer is sodium phosphate buffer. The mass ratio of anhydrous ethanol to sodium phosphate buffer is 1:1.

[0058] The above kit is used to clean the surface of biological tissue in vitro after magnetic resonance imaging, and the specific method includes the following steps:

[0059] S1: First, the in vitro biological tissue sample after magnetic resonance imaging is taken out from the sample bottle filled with fluorine oil.

[0060] (1) Use sodium phosphate buffer to rinse the fluorine oil on the surface of the sample, then place the biological sample in a new sample bottle, add 10 times the volume of tissue reagent A to the sample bottle, then cover the sample bottle cap, place the sample bottle in a constant temperature oscillator, and rinse for 30 minutes.

[0061] (2) Pour out reagent A from the sample bottle, add 10 times the volume of reagent B into the sample bottle, then cover the sample bottle, place the sample bottle in a constant temperature oscillator, and rinse for 30 minutes.

[0062] (3) Pour out reagent B from the sample bottle, add 10 times the volume of reagent C to the sample bottle, then cover the sample bottle, place the sample bottle in a constant temperature oscillator, and rinse for 30 minutes.

[0063] (4) Pour out the reagent C in the sample bottle, add 10 times the volume of the tissue into the sample bottle, then cover the sample bottle cap, place the sample bottle in a constant temperature oscillator, and rinse for 30 minutes.

[0064] S2: Take out the rinsed biological tissue and put it into sodium phosphate buffer and store it under low temperature conditions;

[0065] S3: Biological tissues can be sectioned or immunostained as a whole and used for confocal imaging, two-photon imaging, or light-sheet fluorescence microscopy to obtain structural information.

[0066] Figure 2 The kit and method described in this embodiment are used to clean the brain tissue of Wuzhishan miniature pigs after MRI imaging. After 2 hours, the tissue photos before and after cleaning are compared. It can be clearly seen that the surface of the biological tissue before cleaning is covered with fluorinated oil that is difficult to remove. After cleaning, there is no fluorinated oil residue in the biological tissue, and the biological tissue is not damaged in any way. Therefore, it has no effect on subsequent research or other treatments.

[0067] Embodiment 2:

[0068] A kit for rapid cleaning of the surface of in vitro biological tissue after magnetic resonance imaging, the kit comprising reagents A, B, C and D.

[0069] Reagent A includes n-hexane and n-pentane, and the mass ratio of the two is 9:1.

[0070] The components of reagent B are a mixture of alkanes and alcohols, wherein the alkanes are n-hexane and n-pentane, and the alcohol is anhydrous methanol; the mass ratio of the components is n-hexane: n-pentane: anhydrous methanol = 45:5:50.

[0071] The ingredients in reagent C are alcohols, wherein the alcohol is anhydrous methanol.

[0072] The components of reagent D are a mixture of alcohol and buffer. The alcohol is anhydrous methanol, and the buffer is potassium phosphate buffer. The mass ratio of anhydrous methanol to potassium phosphate buffer is 1:1.

[0073] The above kit is used to clean the surface of biological tissue in vitro after magnetic resonance imaging, and the specific method includes the following steps:

[0074] S1: First, the in vitro biological tissue sample after magnetic resonance imaging is taken out from the sample bottle filled with fluorine oil.

[0075] (1) Use potassium phosphate buffer to rinse the fluorine oil on the surface of the sample, then place the biological sample in a new sample bottle, add 10 times the volume of tissue reagent A to the sample bottle, then cover the sample bottle cap, place the sample bottle in a constant temperature oscillator, and rinse for 30 minutes.

[0076] (2) Pour out reagent A from the sample bottle, add 10 times the volume of reagent B into the sample bottle, then cover the sample bottle, place the sample bottle in a constant temperature oscillator, and rinse for 30 minutes.

[0077] (3) Pour out reagent B from the sample bottle, add 10 times the volume of reagent C to the sample bottle, then cover the sample bottle, place the sample bottle in a constant temperature oscillator, and rinse for 30 minutes.

[0078] (4) Pour out the reagent C in the sample bottle, add 10 times the volume of the tissue into the sample bottle, then cover the sample bottle cap, place the sample bottle in a constant temperature oscillator, and rinse for 30 minutes.

[0079] S2: Take out the rinsed biological tissue and put it into potassium phosphate buffer and store it under low temperature conditions;

[0080] S3: Biological tissues can be sectioned or immunostained as a whole and used for confocal imaging, two-photon imaging, or light-sheet fluorescence microscopy to obtain structural information.

[0081] Figure 3 In order to use the kit and method described in this embodiment to clean the kidney tissue of the Wuzhishan miniature pig after MRI imaging, the tissue photos before and after cleaning were compared 2 hours later. It can be clearly seen that the surface of the biological tissue before cleaning was covered with fluorinated oil that was difficult to remove. After cleaning, there was no fluorinated oil residue on the biological tissue. The surface of the biological tissue was slightly damaged, but it had little impact on subsequent research or other treatments.

[0082] In summary, the kit and method described in the present invention can completely remove the fluorinated oil on the surface of biological tissue within 2 hours, solving the technical problems of the prior art in which the fluorinated oil on the tissue surface is not thoroughly cleaned, and the cleaning process is slow and complicated.

[0083] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A kit for cleaning the surface of ex vivo tissue after magnetic resonance imaging, characterized in that: The kit comprises reagent A, reagent B, reagent C and reagent D, wherein: The reagent A is a mixture of n-hexane and n-pentane; The reagent B is a mixture of n-hexane, n-pentane and alcohols; The reagent C is an alcohol substance; The reagent D is a mixture of alcohol substances and phosphate buffer.

2. The kit according to claim 1, characterized in that The alcohol substance is anhydrous ethanol or anhydrous methanol.

3. The kit according to claim 1, characterized in that The mass ratio of n-hexane to n-pentane in the reagent A is 9:

1.

4. The kit according to claim 1, characterized in that The mass ratio of n-hexane, n-pentane and alcohol in the reagent B is 45:5:

50.

5. The kit according to claim 1, characterized in that The mass ratio of the alcohol substance to the phosphate buffer in the reagent D is 1:

1.

6. A method for cleaning the surface of ex vivo tissue after magnetic resonance imaging, characterized in that: The method comprises the steps of using reagent A, reagent B, reagent C and reagent D in the kit according to any one of claims 1 to 5 to sequentially rinse the ex vivo biological tissue sample after magnetic resonance imaging.

7. The method according to claim 6, characterized in that The method comprises the following steps: (1) taking out the in vitro biological tissue sample after magnetic resonance imaging from the sample bottle filled with fluorine oil, and washing the fluorine oil on the surface of the sample with phosphate buffer; (2) using reagent A, reagent B, reagent C and reagent D in the kit to sequentially rinse the ex vivo biological tissue sample after magnetic resonance imaging; (3) Take out the rinsed biological tissue and place it in phosphate buffer and store it under low temperature conditions.

8. The method according to claim 7, characterized in that When rinsing the ex vivo biological tissue sample in step (2), the amount of reagent A, reagent B, reagent C and reagent D used is 10 times the volume of the ex vivo biological tissue.

9. The method according to claim 7, characterized in that When rinsing the ex vivo biological tissue sample in step (2), the ex vivo biological tissue sample is placed in a sample bottle containing reagent A, reagent B, reagent C and reagent D in sequence, then the sample bottle caps are closed, and the sample bottles are placed in a constant temperature oscillator in sequence for rinsing.

10. The method according to claim 9, characterized in that In the step (2), each rinsing time in the constant temperature oscillator is 30 minutes.

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