Method for optical clearing of biological tissues

A biological tissue, aldehyde compound technology, applied in fluorescence/phosphorescence, material analysis by optical means, material excitation analysis, etc., can solve the problems of poor repeatability, slow transparency, and low degree of transparency. The effect of the original morphology, maintaining the original morphology, and cheap and easy availability of reagents

Inactive Publication Date: 2017-04-05
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Aiming at the above defects or improvement needs of the prior art, the present invention provides a method for rapidly transparentizing biological tissue, the purpose of which is to use a strong polar aprotic solvent for degreasing treatment, and then select a specific refractive index matching reagent , match the refractive index of the biological tissue after degreasing to achieve the maximum degree of transparency, thereby solving the technical problems of slow transparent speed, low degree of transparent and poor repeatability in the existing biological tissue transparent methods

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  • Method for optical clearing of biological tissues
  • Method for optical clearing of biological tissues

Examples

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Embodiment 1

[0070] (1) The 3000 μm mouse brain block was fixed by chemical fixation method, and fixed with 4% paraformaldehyde at 4°C for 24 hours;

[0071] (2) Rinse the fixed mouse brain block with PBS buffer solution for 12 hours, and then carry out immunohistochemical fluorescent labeling. First, use 20% DMSO and 1% triton X-100 to punch holes for 12 hours, and then add serum Incubate for 12 hours, then add primary antibody and incubate for 48 hours, rinse for 1 hour / 3 times, then add secondary antibody for 8 hours, rinse for 1 hour / 3 times;

[0072] (3) Dehydrate the immunohistochemically labeled mouse brain blocks with tetrahydrofuran, and dehydrate according to 50% tetrahydrofuran / 2 hours+75% tetrahydrofuran / 2 hours+95% tetrahydrofuran / 2 hours+100% tetrahydrofuran / 2 hours+100 % THF / 12 hour program for gradient dehydration;

[0073] (4) Put the dehydrated mouse brain block into a strong polar aprotic solvent 1,4-dioxane, and place it at room temperature in a dark environment for im...

Embodiment 2

[0078] (1) The legs of mice were fixed by chemical fixation means, and fixed with 4% paraformaldehyde at 4°C for 24 hours;

[0079] (2) Dehydrate the fixed mouse legs with ethanol, dehydration according to 50% ethanol / 2 hours+75% ethanol / 2 hours+95% ethanol / 2 hours+100% ethanol / 2 hours+100% ethanol / 12 hour program for gradient dehydration;

[0080] (4) Put the dehydrated mouse legs into the strong polar aprotic solvent 1,3-dimethyl-2-imidazolidinone for 48 hours at room temperature to avoid light and degrease until the biological tissue is translucent;

[0081] (5) Put the degreased mouse legs into the refractive index matching reagent for transparent treatment. The matching reagent o-dichlorobenzene (refractive index 1.55-1.552) is immersed in the dark for 24 hours at room temperature until the biological tissue is transparent ;

[0082] (6) Take pictures of the cleared mouse legs.

[0083] Figure 3 is a comparison diagram of the effects before and after the transparency ...

Embodiment 3

[0085] (1) The whole brain of the mouse was fixed by chemical fixation method, and fixed with 4% paraformaldehyde at 4°C for 24 hours;

[0086] (2) The whole mouse brain was dehydrated with tetrahydrofuran, dehydration according to 50% ethanol / 2 hours+75% ethanol / 2 hours+95% ethanol / 2 hours+100% ethanol / 2 hours+100% ethanol / 12 hours The procedure for gradient dehydration;

[0087] (4) Put the whole brain of the dehydrated mouse into a strong polar aprotic solvent for degreasing treatment. The degreasing reagent N-methylpyrrolidone (NMP) is placed at room temperature and immersed in the dark for 48 hours until the biological tissue is translucent. ;

[0088] (5) Put the degreased mouse whole brain into the refractive index matching reagent 3-phenoxybenzyl alcohol (refractive index 1.592-1.594) for transparent treatment, and immerse it in the dark at room temperature for 12 hours until the biological tissue Transparent;

[0089] (6) Take pictures of the cleared mouse whole br...

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Abstract

The invention discloses a method for rapid optical clearing of biological tissues. The method includes: employs a strong polar aprotic solvent to conduct degreasing, wherein the strong polar aprotic solvent can be methyl methacrylate, hexamethylphosphoric triamide, N-methyl pyrrolidone, 1, 3-dimethyl-2-imidazolidinone, 1, 4-dioxane or 2, 5-dimethylfuran, then selecting a specific refractive index matching reagent with a refractive index of 1.52-1.59 to conduct clearing treatment, thus realizing rapid clearing of biological tissues. The degreasing and clearing treatment totally need only 18h to realize high clearing of mouse brain tissues, the method not only can realize brain tissue clearing, but also can achieve clearing of various organs, can well maintain the original morphology of biological tissues, and has high clearing degree and repeatability, and good fluorescence retaining effect, the soma and nerve fiber have strong fluorescence signal, and the application range is wide.

Description

technical field [0001] The invention belongs to the field of biological imaging, and more specifically relates to a method for light-transparent biological tissue. Background technique [0002] Obtaining detailed molecular-level structural information from complete biological systems is a great cross-field challenge, which can promote technological progress in the entire field of science and technology in the future, especially the study of the relationship between brain structure and function from the complete system organization function. In general, many valuable statistical information of inter-system relations will help us comprehensively analyze the structure of the complete system instead of simply reconstructing the information of a single organization. It is still very difficult to study the molecular structure information of a single biological tissue in a complete biological system. At present, there are many methods involving tomographic imaging and 3D structure ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 曾绍群周宏福刚亚栋陈瑞希刘秀丽朱丹
Owner HUAZHONG UNIV OF SCI & TECH
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