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Up-conversion super-resolution imaging nanoprobe and its preparation method and application

A super-resolution imaging and nanoprobe technology, applied in the field of fluorescence microscopy and immunofluorescence, can solve the problems of easy photolysis and photobleaching, long-term observation of unfavorable biological samples, poor photochemical stability, etc. No photobleaching, good photochemical stability, and large anti-Stokes shift

Active Publication Date: 2021-01-15
SOUTH CHINA NORMAL UNIVERSITY
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  • Description
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Problems solved by technology

At present, most of the fluorescent groups commonly used in immunofluorescence experiments are organic fluorescent dyes. Organic fluorescent dyes not only have poor photochemical stability, but also are prone to photolysis and photobleaching, which is very unfavorable for long-term preservation of biological samples (cells or tissue slices). Observed

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  • Up-conversion super-resolution imaging nanoprobe and its preparation method and application
  • Up-conversion super-resolution imaging nanoprobe and its preparation method and application
  • Up-conversion super-resolution imaging nanoprobe and its preparation method and application

Examples

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Embodiment 1

[0049] see figure 1 In this example, the synthesis, carboxyl modification and optical loss methods of the up-conversion super-resolution imaging nanoprobe are mainly explained. Specifically, the following steps are included:

[0050] 1. The method (solvothermal method) of synthesizing up-conversion nanoparticle, concrete steps are as follows:

[0051] (1) Synthesis of super-resolution upconversion nanoparticle core structure:

[0052] In the oleic acid / 1-octadecene (v:v=8:15, unit: mL) system, add 5ml of 0.2mol / L (n=1mmol) Ln(CH3CO2)3(Ln=Y:Yb:Tm =74:18:8) solution. Expose and heat to 150°C, and stir for 40 minutes. After cooling down to room temperature, 10ml 0.4mol / L NH4-methanol solution and 2.5ml 1mol / L NaOH-methanol solution were added dropwise, and the water bath was kept at 40°C and stirred for 2h. Then the water bath was removed, and the system was heated to 100°C and vacuumed for 30 minutes to completely remove methanol. After the vacuuming was completed, the temp...

Embodiment 2

[0063] Applying the up-conversion super-resolution imaging nanoprobe described in Example 1 to fluorescence super-resolution imaging, the following imaging method can be used: the near-infrared depleted light is modulated into a hollow beam with a spatial phase modulation plate, and collimated with the excitation beam. After yoke focusing, rare earth-doped upconversion nanoparticles and their labeling are applied to cell immunofluorescence imaging. Based on this microscopic imaging method, a super-resolution optical microscopic imaging system consisting of an excitation light generation module, a loss light generation module, a dichroic mirror, a multiphoton microscopic scanning module and a photodetection module is built on the basis of existing technologies , to obtain low-cost, low-complexity, high-resolution, simple and effective real-time dynamic three-dimensional images.

[0064] This embodiment specifically describes a super-resolution optical microscopy imaging system ...

Embodiment 3

[0072] In this example, the specific immunolabeling method of the up-conversion super-resolution imaging nanoprobe modified by the primary antibody is illustrated: for example, the primary antibody protein (anti-desmin protein primary antibody , desmin protein is a modification of cytoskeleton protein) for cytoskeleton-specific immunolabeling, and then super-resolution imaging of cytoskeleton:

[0073] (1) Carboxylated up-conversion super-resolution imaging nanoprobes are modified with primary antibodies, and the specific steps are as follows:

[0074] Take 1ml of 1mg / ml UCNPs-COOH, add 20μl of 0.3mg / μl NHS and 20μl of 0.2mg / μl EDC to it and stir at room temperature for two hours. Collect by centrifugation at 15,000rpm for 30min, and dissolve with 1ml deionized water. The pH was adjusted to 7.2-7.5, and 100 μl of the primary antibody of the antibody was added to prepare the primary antibody-modified up-conversion super-resolution imaging nanoprobe.

[0075] (2) The primary a...

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Abstract

The invention discloses an upconversion super-resolution imaging nanoprobe as well as a preparation method and application thereof. The upconversion super-resolution imaging nanoprobe has a core@shellstructure. The preparation method comprises the following steps: preparing rare-earth upconversion nano-particles with controllable particle size and excellent dispersion property by adopting a solvothermal method; performing surface modification by polyacrylic acid; and finally performing protein modification. The upconversion super-resolution imaging nanoprobe is applied to immunofluorescent labeling of a subcellular structure of cells, so that fluorescent super-resolution imaging of the subcellular structure is realized. Compared with the traditional fluorescein-based super-resolution optical imaging method, the method disclosed by the invention is a fluorescence zero-bleaching and zero-flicker super-resolution microscopic imaging method based on the upconversion super-resolution imaging nanoprobe, and biological samples can be observed for a long time.

Description

technical field [0001] The invention belongs to the technical fields of fluorescence microscopic imaging and immunofluorescence, and in particular relates to an up-conversion super-resolution imaging nanoprobe, and a preparation method and application of the probe. Background technique [0002] According to the German scientist Abbe, the diffraction resolution limit was deduced for the first time based on the diffraction theory, that is, the distance between two points that can be optically resolved is about half of the wavelength of the incident light. Therefore, the resolution of optical fluorescence imaging technology has been limited to around 200nm. This obviously cannot satisfy scientists' observation and research on submicroscopic structures. Therefore, in recent years, a variety of fluorescence imaging techniques that break the optical diffraction limit have been proposed and used. These fluorescence imaging techniques that break the optical diffraction limit are ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C09K11/85C09K11/02B82Y20/00B82Y40/00G01N21/64G01N33/533
Inventor 詹求强周超黄冰如
Owner SOUTH CHINA NORMAL UNIVERSITY
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