Super-resolution imaging method based on fluorescence resonance energy transfer

A fluorescence resonance energy and super-resolution imaging technology, applied in the field of super-resolution imaging, can solve the problems of complex imaging software and high cost

Active Publication Date: 2012-12-19
SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Therefore, the technical problem to be solved by the present invention is to provide a method based on fluorescence resonance energy transfer (FRET) for the defects that the existing super-resolution imaging technology requires a large amount of modification to the fluorescence microscope, the cost is very expensive, and the imaging software is also very complicated. ) super-resolution imaging method

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  • Super-resolution imaging method based on fluorescence resonance energy transfer
  • Super-resolution imaging method based on fluorescence resonance energy transfer

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1 Super-resolution imaging of Cy3-Cy5 probe-labeled Hela cell microtubules

[0042] Construction of Cy3-Cy5 fluorescent probes with high FRET efficiency: Fluorescent nanospheres were prepared by inverse microemulsion method.

[0043] Experimental materials: sodium silicate solution (water glass) (the water glass modulus is 3.1, Baume degree is 40), hydrochloric acid (concentration is 1mol / L), sodium lauryl sulfate SDS, toluene, n-amyl alcohol , deionized water (W), fluorescent dye Cy3-Cy5.

[0044] The specific steps of this technique include:

[0045] Osmotic reaction: take 0.1g surfactant SDS (S), 0.2g fluorescent dye (donor Cy3 and acceptor Cy5, donor acceptor molar ratio 1:2), 0.2ml toluene and 5ml water glass solution prepared in advance Put it into a beaker, and mix it uniformly by ultrasonic (50W ultrasonic power, 1 minute mixing time) to obtain a colostrum, add n-amyl alcohol dropwise until the system is suddenly transparent, and obtain microemulsion A...

Embodiment 2

[0055] Example 2 Super-resolution imaging of Hela cell microtubules labeled with Atto488-Atto540 probe

[0056] Construction of Atto488-Atto540 Fluorescent Probe with High FRET Efficiency

[0057] Experimental materials: sodium silicate solution (water glass) (the water glass modulus is 3.2, Baume degree is 40), hydrochloric acid (concentration is 1mol / L), sodium lauryl sulfate SDS, toluene, n-amyl alcohol , deionized water (W), fluorescent dyes Atto488-Atto540.

[0058] Take 0.1g of surfactant SDS (S), 0.2g of fluorescent dye (donor Atto488 and acceptor Atto540, donor-acceptor molar ratio 1:2), 0.2ml of toluene and 5ml of water glass solution prepared in advance into a beaker In the process, ultrasonically mix evenly (ultrasonic power 50W, mixing time 1 minute) to obtain a primary emulsion, add n-amyl alcohol dropwise until the system is suddenly transparent, and obtain microemulsion A.

[0059] Polymerization reaction: replace the water glass solution with 1mol / L HCl solut...

Embodiment 3

[0067] Example 3 Super-resolution imaging of Hela cell microtubules labeled with Atto550-Atto647N probe

[0068] Construct Atto550-Atto647N fluorescent probe with high FRET efficiency:

[0069] Experimental materials: sodium silicate solution (water glass) (the water glass modulus is 3.3, Baume degree is 40), hydrochloric acid (concentration is 1mol / L), sodium lauryl sulfate SDS, toluene, n-pentanol , deionized water (W), fluorescent dye Atto550-Atto647N.

[0070] Osmotic reaction: take 0.1g surfactant SDS (S), 0.2g fluorescent dye (donor Atto550 and acceptor Atto647N, donor acceptor molar ratio 1:2), 0.2ml toluene and 5ml water glass solution prepared in advance Put it into a beaker, and mix it uniformly by ultrasonic (50W ultrasonic power, 1 minute mixing time) to obtain a colostrum, add n-amyl alcohol dropwise until the system is suddenly transparent, and obtain microemulsion A.

[0071] Polymerization reaction: replace the water glass solution with 1mol / L HCl solution, a...

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Abstract

The invention discloses a super-resolution imaging method based on fluorescence resonance energy transfer (FRET). The super-resolution imaging method comprises the following steps: 1) marking a sample to be detected with a fluorescent probe having high FRET efficiency, wherein the fluorescent probe having high FRET efficiency has a marked FRET molecule pair, the FRET molecule pair includes a first fluorophore (a donor) and a second fluorophore (a receptor), and the first fluorophore can exert FRET on the second fluorophore; and 2) carrying out laser scanning confocal microscope imaging at an excitation light threshold with an excitation light intensity capable of allowing the FRET molecule pair in the step 1) to generate FRET. A super resolution technology based on saturated FRET in the invention can realize super resolution imaging of a biological sample on an ordinary laser confocal microscope, and the method provided by the invention has high resolution.

Description

technical field [0001] The invention belongs to the technical field of super-resolution imaging, in particular to a super-resolution imaging method based on fluorescence resonance energy transfer (FRET, Fluorescence Resonance Energy Transfer). Background technique [0002] Many important research achievements in modern life science rely on the progress of microscopic imaging technology. However, it has been believed for a long time that the limit resolution that optical microscopy can achieve is about 200 nanometers, which is half the wavelength of light. Until the early 1990s, with the emergence of a series of new technologies in the field of optics, the optical diffraction limit was broken and the resolution of optical microscopes was increased to tens of nanometers. [0003] The basis of super-resolution imaging technology mainly relies on various nonlinear optical effects. In 1991, a research group at Cornell University realized the multiphoton fluorescence imaging tech...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6458G01N21/6428G01N2021/6441G02B21/00G02B21/0076
Inventor 樊春海黄庆程亚陈建芳邓素辉梁乐
Owner SHANGHAI INST OF APPLIED PHYSICS - CHINESE ACAD OF SCI
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