Optical transparency method for quickly acquiring three-dimensional data of large-volume sample

A three-dimensional data, large-volume technology, applied in the field of biomedical optical imaging, can solve the problems of affecting three-dimensional reconstruction, non-uniform refractive index, large sample deformation, etc., to reduce the number of slices, accelerate efficiency, low viscosity and concentration. Effect

Pending Publication Date: 2021-08-31
HUST SUZHOU INST FOR BRAINMATICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Organic reagents have high transparency efficiency, but have the disadvantages of large sample deformation and poor fluorescence retention. Hydrophilic reagents have the characteristics of low deformation and high fluorescence retention, but their transparency efficiency is low, which is difficult for large-volume samples such as macaque brain. are completely transparent and are therefore only commonly used for 3D imaging of small volume samples
For example, the imaging device and method disclosed in Chinese patent 20161031568

Method used

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  • Optical transparency method for quickly acquiring three-dimensional data of large-volume sample
  • Optical transparency method for quickly acquiring three-dimensional data of large-volume sample
  • Optical transparency method for quickly acquiring three-dimensional data of large-volume sample

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] The light transparent solution in Example 1 is an aqueous solution containing 30wt% fructose, 20wt% urea, and 20v / v% dimethyl sulfoxide. The biological sample tissue is the brain of an adult rhesus monkey, and the acquisition of three-dimensional data is completed according to the following steps:

[0071] S1. Firstly, the brain of an adult macaque was labeled with AAV-GFP virus. After a few weeks of virus expression, it was anesthetized and perfused. The perfusion solution was firstly 0.01M phosphate buffer solution and then 4% paraformaldehyde solution. His brain was used as a biological sample tissue. Soak the biological sample tissue in 4% paraformaldehyde solution and continue to fix it for a period of time until the sample hardens.

[0072] S2. Perform pretreatment on the obtained biological sample tissue, that is, immerse the biological sample tissue in a light-transparent solution for at least 24 hours. During this process, the light-transparent solution first ...

Embodiment 2

[0084] The light transparent solution in Example 2 is an aqueous solution containing 30wt% fructose, 20wt% urea, and 20v / v% dimethyl sulfoxide. The biological sample tissue is the liver tissue of an adult domestic pig, and the acquisition of three-dimensional data is completed according to the following steps:

[0085] S1, first obtain the liver tissue of an adult domestic pig as a biological sample tissue, soak it in 4% paraformaldehyde solution and fix it for about 3 months until the sample becomes hard.

[0086] S2, performing pretreatment on the biological sample tissue, that is, immersing the biological sample tissue in a light-transparent solution for at least 24 hours, so as to first dehydrate and make the surface transparent.

[0087] S3, performing agarose embedding on the pretreated biological sample tissue.

[0088] S4, the embedded biological sample tissue is soaked in the transparent solution again for not less than 24 hours.

[0089] S5, pasting the embedded bi...

Embodiment 3

[0098] The light transparent solution in Example 3 is an aqueous solution containing 30 wt% fructose and 20 wt% urea. The biological sample tissue is the brain of an adult mouse, and the acquisition of three-dimensional data is completed according to the following steps:

[0099] S1, an adult Thy1-GFP-M mouse was first anesthetized and perfused. The perfusion solution was firstly 0.01M phosphate buffer solution and then 4% paraformaldehyde solution, and its brain was taken as a biological sample tissue. The biological sample tissue was soaked in 4% paraformaldehyde solution and then fixed for 24 hours.

[0100] S2, performing pretreatment on the biological sample tissue, that is, immersing the biological sample tissue in a light-transparent solution for at least 24 hours, so as to first dehydrate and make the surface transparent.

[0101] S3, performing agarose embedding on the pretreated biological sample tissue.

[0102] S4, the embedded biological sample tissue is soaked ...

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Abstract

The invention discloses a method for rapidly acquiring three-dimensional data of a large-volume biological sample. The method comprises the following steps: S1, a first immersion step; s2, an embedding step; s3, a second immersion step; s4, a sample installation step; s5, an optical imaging step; and s6, a slicing step; wherein the S5 and S6 are circularly and alternately carried out until a three-dimensional image of the biological sample tissue is obtained; the light transparent solution comprises the following raw materials in percentage by mass: 20-40% of sugar or alcohol or sugar-alcohol compound, 10 to 30% of urea, 0-30% of a penetration enhancer and the balance of water. According to the invention, the optical transparent solution and the microsection tomography technology are combined for use; optical imaging and microsection are alternately carried out, and meanwhile, the optical transparent solution is used for replacing liquid between tissues of a biological sample in real time; therefore, the refractive index of the biological sample tissue is quickly balanced, the depth of single imaging is expanded, and the slicing frequency of the whole sample is remarkably reduced. Therefore, the applicable sample range of light transparency and the continuous acquisition efficiency of three-dimensional data are greatly improved.

Description

technical field [0001] The invention relates to the technical field of biomedical optical imaging, in particular to an optically transparent method for rapidly acquiring three-dimensional data of a large-volume sample. Background technique [0002] The brain is one of the most important and complex organs in humans. Its structure is complex and neurons are abundant. But at present, we know very little about the function and structure of the brain. Brain science research will help scientists to better decipher brain function and brain diseases, and brain research can promote the development of artificial intelligence. Whole-brain three-dimensional imaging technology is often used in the study of neural circuits. A neural circuit is an intricate series of circuits in which neurons are interconnected by synaptic structures. These circuits perform the higher functions of the brain, controlling behavior and emotion, and the study of neural circuits is considered fundamental t...

Claims

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Application Information

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IPC IPC(8): G01N1/30G01N1/36G01N1/28G01N21/84
CPCG01N1/30G01N1/36G01N1/286G01N21/84G01N2001/2873
Inventor 杨孝全张云飞李向宁龚辉
Owner HUST SUZHOU INST FOR BRAINMATICS
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