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A method of tomographic imaging

A tomography and imaging technology, which is applied in the field of biological fluorescence microscopy imaging, can solve the problems of requirements, limitations, and high sample transparency of light sheet lighting technology, and achieve shortened imaging time, fast imaging speed, and good tomographic effect Effect

Active Publication Date: 2020-06-26
HUAZHONG UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The light sheet illumination imaging technology only illuminates the imaging focal plane and does not illuminate other parts. When collecting data, the surface imaging mode can be used. Compared with the point imaging method, it is faster and the fluorescence quenching is smaller, but most of the light sheet The tomographic capability of lighting imaging technology can only reach a few microns, and the light sheet lighting technology has high requirements for sample transparency, which is greatly restricted when imaging large samples, especially opaque large samples

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Examples

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Effect test

Embodiment 1

[0088] Chemical tomography of the whole brain of Thy1-EGFP-M mice, including the following steps:

[0089] (1) The sample is fixed.

[0090] The whole brain of Thy1-EGFP-M mice was fixed by means of chemical fixation to obtain fixed mouse brain biological tissue. Specific steps are as follows:

[0091] After the heart was perfused at 4°C, the dissected whole brain of the mouse was soaked in 4% PFA solution for about 12 hours. Use 40ml of PBS solution, rinse for four hours each time.

[0092] (2) Sample dehydration.

[0093] The fixed mouse brain tissue was replaced with ethanol, so that the biological tissue was dehydrated, and the dehydrated EGFP-labeled mouse whole brain was obtained. The specific steps are:

[0094] At 4°C, dehydrate the fixed mouse whole brain sequentially through 20 ml of gradient ethanol double-distilled aqueous solution for 2 hours. The concentration gradient of ethanol double distilled water solution is 50%, 75%, 95%, 100%, 100% ethanol according...

Embodiment 2

[0105] A method for chemical tomography of the mouse whole brain overexpressing the pH-sensitive fluorescent protein pHuji, comprising the following steps:

[0106] (1) The sample is fixed.

[0107] The whole brain of mice overexpressing pHuji was fixed by chemical fixation means to obtain fixed pHuji-labeled biological tissues. The specific steps are as follows: at 4 degrees Celsius, after perfusing the heart, the dissected whole brain of the mouse was soaked in a 4% PFA solution for about 12 hours, the amount of PFA solution was 20ml per mouse, and then rinsed three times with PBS solution. Each animal uses 40ml of PBS solution each time, and rinses for four hours each time.

[0108] (2) Sample dehydration.

[0109]The fixed pHuji-labeled mouse whole brain was replaced with ethanol, so that the biological tissue was dehydrated, and the dehydrated pHuji-labeled mouse whole brain was obtained. The specific steps are as follows: at 4°C, soak the fixed pHuji-labeled mouse who...

Embodiment 3

[0120] A chemical tomography method for the whole brain of mice overexpressing EYFP, comprising the following steps:

[0121] (1) The sample is fixed.

[0122] The whole brain of the overexpressing EYFP mouse was fixed by chemical fixation means to obtain the fixed mouse brain biological tissue. The specific steps are as follows: at 4 degrees Celsius, after perfusing the heart, the dissected whole brain of the mouse was soaked in a 4% PFA solution for about 12 hours, the amount of PFA solution was 20ml per mouse, and then rinsed three times with PBS solution. Each animal uses 40ml of PBS solution each time, and rinses for four hours each time.

[0123] (2) Sample dehydration.

[0124] The fixed mouse brain tissue was replaced with ethanol, so that the biological tissue was dehydrated, and the dehydrated EGFP-labeled mouse whole brain was obtained. The specific steps are as follows: at 4 degrees Celsius, the fixed mouse whole brain is sequentially passed through 20 ml of gra...

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Abstract

The invention discloses a tomographic imaging method, which comprises the following steps: activating the surface fluorescence of a biological tissue sample marked with a protein label or a fluorescent dye that does not emit fluorescence or only emits specific fluorescence, to obtain an activated surface biological tissue sample; Fluorescence excitation and imaging of the obtained surface biological tissue sample to obtain a fluorescence image of the surface; cutting off the surface; after cutting off the surface, a new non-activated surface is exposed, and the new surface is repeatedly activated, imaged and The subtraction step is repeated tomographic imaging in this way until a two-dimensional image of each layer of the biological tissue sample is obtained, and the two-dimensional images are superimposed to obtain a complete three-dimensional image of the biological tissue sample, so as to obtain a complete three-dimensional image of the biological tissue sample. 3D structural information. The imaging method proposed by the invention has good chromatographic ability, high resolution, fast imaging speed, small fluorescence destructive quenching, wide application range and good imaging effect.

Description

technical field [0001] The invention belongs to the field of biological fluorescence microscopic imaging, and in particular relates to a chemical tomographic imaging method, and more specifically, designs an imaging method which utilizes chemical means to realize rapid tomographic capability. Background technique [0002] Fluorescence microscopy imaging technology is an indispensable technology in current life science research. With the development of molecular biology and the development of fluorescent labeling technology, imaging methods for fluorescent biological samples are also developing. [0003] In the research of life sciences, it is of great significance to obtain continuous fine structure information of biological samples in a large range. Early ordinary fluorescence microscopes used wide-field illumination imaging to image biological samples, and could only obtain two-dimensional images for thin-section samples. In order to obtain the three-dimensional fine stru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6428G01N21/6486G01N2021/6432G01N21/6456
Inventor 曾绍群骆清铭熊汗青郭文炎吕晓华
Owner HUAZHONG UNIV OF SCI & TECH
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