Rapid tissue transparent processing method based on water-soluble reagent and application thereof

A processing method and water-soluble technology, applied in the field of biomedical optical imaging, can solve the problems of limiting the application of light-transparency methods, slow transparent speed, limited transparent effect, etc., and achieve the effect of fast transparent speed, improved imaging depth, and short transparent time.

Active Publication Date: 2019-04-16
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, methods based on water-soluble reagents are slow to clear, often taking weeks or even months to implement tissue clearing, and...

Method used

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  • Rapid tissue transparent processing method based on water-soluble reagent and application thereof
  • Rapid tissue transparent processing method based on water-soluble reagent and application thereof
  • Rapid tissue transparent processing method based on water-soluble reagent and application thereof

Examples

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Effect test

Embodiment 1

[0042] The biological tissue of the embodiment of the present invention is taken from the brain tissue of a C57 mouse, and its 2mm slices are processed through the transparent treatment method of the embodiment 1 of the present invention, which specifically includes the following steps:

[0043]First, soak the thin slices in the tissue swelling agent (10% m-xylylenediamine, 5% sorbitol) for 4-6 hours; then soak the thin slices in the tissue shrinking agent (m-xylylenediamine 30%) %, sorbitol 20%, phosphate buffer saline 0.03M), processing time 2-3h; finally the section is soaked in the refractive index matching agent (m-xylylenediamine 40%, sorbitol 40%), processing time 1- 2h;

[0044] Figure 2a-2b Visual images taken before and after light-transparent treatment of isolated mouse 2mm brain slices are given. in Figure 2a For the situation before the transparent treatment, due to the turbidity of the tissue, the lines below the tissue are completely covered; Figure 2b Fo...

Embodiment 2

[0046] The biological tissue of the embodiment of the present invention is taken from the brain tissue of Thy-1EGFP and Cx3cr1-EGFP mice, and its 2mm slices are processed through the transparent processing method of the embodiment 1 of the present invention, which specifically includes the following steps:

[0047] First, soak the thin slices in the tissue swelling agent (10% m-xylylenediamine, 5% sorbitol) for 4-6 hours; then soak the thin slices in the tissue shrinking agent (m-xylylenediamine 30%) %, sorbitol 20%, phosphate buffer saline 0.05M), processing time 2-3h; finally the section is soaked in the refractive index matching agent (m-xylylenediamine 40%, sorbitol 40%), processing time 1- 2h; immerse the transparent 2mm brain slice in the refractive index matching agent for confocal fluorescence imaging.

[0048] Figures 3a-3c Fluorescence images of cortical pyramidal neurons and microglia taken before and after the light-transparent treatment of the mouse brain slices...

Embodiment 3

[0050] The biological tissue of the embodiment of the present invention is taken from various organs of C57 mice, and the whole organ is processed through the transparent treatment method of the embodiment 3 of the present invention, which specifically includes the following steps:

[0051] First, soak various organs in the tissue swelling treatment agent (m-xylylenediamine 20%, sorbitol 10%). The soaking time is respectively according to the tissue type: mouse whole brain 24-36h, mouse heart 12-24h, Liver 24-36h, kidney 12-24h, lung 12-24h, spleen 12-24h, stomach 6-12h; then soak each organ in tissue contraction agent (40% m-xylylenediamine, 30% sorbitol, phosphoric acid Salt buffer 0.01M), the soaking time according to the tissue type is: mouse whole brain 8-12h, mouse heart 4-8h, liver 8-12h, kidney 8-12h, lung 8-12h, spleen 4-8h, Stomach 4-8h; Finally, each organ is soaked in the refractive index matching agent (m-xylylenediamine 50%, sorbitol 40%), mouse whole brain 8-12h...

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Abstract

The invention provides a rapid tissue transparent processing method based on water-soluble reagent and an application thereof. The processing method comprises the following steps: placing a tissue fixed through paraformaldehyde into a tissue expansion treating agent, sufficiently soaking to loosen and expand the tissue, and removing lipid; placing the tissue sufficiently soaked by the tissue expansion treating agent into a tissue contraction agent so that the tissue can be contracted to an original state from the expansion state, and acquiring an initial transparent effect; placing the tissuesufficiently treated in the tissue contraction agent into a refractive index matching reagent to soak, and finally obtaining the highly transparent tissue with relatively uniform refractive index. Thetissue treated through the method disclosed by the invention is highly transparent, and can be used for tissue entire three-dimensional fluorescence imaging, thereby realizing three-dimensional high-resolution imagining on the whole brain neural network and an organ vascular network structure.

Description

technical field [0001] The invention belongs to the technical field of biomedical optical imaging, and in particular relates to a treatment method for rapid tissue clearing based on water-soluble reagents and an application thereof. Background technique [0002] The overall high-resolution imaging of biological tissues can provide complete and fine three-dimensional tissue structure information for life science research, so as to better understand the relationship between tissue network structure and function. Obtaining three-dimensional structures based on traditional tissue slicing techniques is time-consuming and labor-intensive, and easily leads to loss of structural information, making image reconstruction difficult. In recent years, the combination of various fluorescent labeling technologies and microscopic imaging technologies has provided a new idea for obtaining three-dimensional structure information of tissues. However, the high scattering characteristics of bio...

Claims

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Application Information

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IPC IPC(8): G01N1/28G01N1/30G01N21/64
CPCG01N1/28G01N1/30G01N21/6458
Inventor 朱朱京谭俞婷婷
Owner HUAZHONG UNIV OF SCI & TECH
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