Sample pretreatment method for in situ correlative light-electron microscope detection of transmission electron microscopes and application thereof

A technology of sample pretreatment and transmission electron microscopy, which is applied in the preparation of test samples, measuring devices, fluorescence/phosphorescence, etc., can solve the problems of easy changes in sample shape and structure, avoid fluorescence quenching, improve the preparation process, and be easy to operate Effect

Pending Publication Date: 2020-03-06
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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Problems solved by technology

First, the fluorescent images of living cells can be observed under a fluorescent microscope. After that, the structural characteristics of the cells can be obtained under the electron microscope through sectioning, staining, etc. This method has precise positioning between the two different imaging methods And the disadvantage that the shape and structure of the sample are easy to change before and after treatment

Method used

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  • Sample pretreatment method for in situ correlative light-electron microscope detection of transmission electron microscopes and application thereof
  • Sample pretreatment method for in situ correlative light-electron microscope detection of transmission electron microscopes and application thereof
  • Sample pretreatment method for in situ correlative light-electron microscope detection of transmission electron microscopes and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Embodiment 1 adopts the detection method of transmission electron microscope

[0048] 1. Fix the HepG2 cells labeled with red fluorescent rhodan at room temperature with 3% glutaraldehyde, and then wash three times with 0.1M PBS buffer;

[0049] 2. Use gradient concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 95%, 100%) to dehydrate, and then use resin to infiltrate and embed the sample;

[0050] 3. Slice the embedded cells and load them on a marked copper grid;

[0051] 4. Fluorescence microscope is used to detect and image the sample, and obtain fluorescence pictures and bright field pictures;

[0052] 5. Use 1% osmic acid to fix, 2% uranyl acetate to stain, observe in the transmission electron microscope, and obtain the electron microscope picture of the ultrastructure of the sample;

[0053] 6. Finally, use the open software to combine the fluorescence picture and the electron microscope picture to obtain the result picture of the in situ photo-electric correla...

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Abstract

The invention provides a sample pretreatment method for in situ correlative light-electron microscope detection of transmission electron microscopes and and application thereof. According to the method, a fluorescently-labeled sample is fixed at room temperature by using glutaraldehyde, is flushed by using buffer solution, and then is directly dehydrated by using ethyl alcohol with a gradient concentration. Through going into an in situ correlative light-electron microscope detection technology, a sample pretreatment method for room-temperature in situ correlative light-electron microscope detection is innovated, so that the structural influences on the samples in the high-pressure freezing methods are avoided and the florescence of the samples is ensured not to be quenched to the greatestextent in the sample treatment process; and in the method for in situ correlative light-electron microscope detection, signals can be detected at room temperature, so that the method is convenient, rapid, relatively low in cost, easy to operate and convenient to popularize and apply, and has a huge market value.

Description

technical field [0001] The invention relates to the technical field of image detection and processing, in particular to a sample pretreatment method and application for in-situ light-electron microscope correlation detection of a transmission electron microscope. Background technique [0002] Correlative Photoelectric Microscopy (CLEM) combines the advantages of fluorescence microscopy and electron microscopy. By superimposing electron microscopy photos on fluorescent photos, information on fluorescent functional substances and high-resolution structural information on electron microscopy can be obtained in one image. Delphi in the Netherlands integrated the two optical paths for the first time in the world, pushing the correlative microscopy technology to the peak. Combining the advantages of fluorescence microscopy and electron microscopy, light-electric correlation microscopy (CLEM) can obtain overlapping images of electron micrographs and fluorescence images, which provi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N21/64
CPCG01N1/28G01N21/6402G01N21/6486
Inventor 胡西学甘雅玲郭宏博宫宁强梁兴杰
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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