Method for realizing super-resolution dipole orientation analysis

A dipole and super-resolution technology, applied in the field of super-resolution imaging, can solve the problems of limited observation capability of living cells and low temporal resolution, and achieve the effects of anti-noise and stability, improving accuracy and improving effectiveness

Active Publication Date: 2016-12-07
PEKING UNIV
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Different wavelengths have strict requirements on fluorescent markers, which restricts the application. At the

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  • Method for realizing super-resolution dipole orientation analysis
  • Method for realizing super-resolution dipole orientation analysis
  • Method for realizing super-resolution dipole orientation analysis

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Embodiment Construction

[0028] The present invention will be described in detail below in conjunction with the accompanying drawings and embodiments.

[0029] The invention provides a method for realizing super-resolution dipole orientation analysis. The method is based on the fact that fluorescent proteins have dipole characteristics, and then uses fluorescent proteins to mark target proteins. Under the condition that the two proteins form a rigid structure, by exciting to study fluorescent protein dipole orientations, allowing analysis of structural changes in the target protein. The present invention specifically comprises the following steps:

[0030] 1) if figure 1As shown, a micro-imaging system is set, and the micro-imaging system adds a half-wave plate on the basis of a wide-field fluorescence microscope (epi-fluorescence microscope), which includes a laser light source 1, a half-wave plate 2, a collimator lens 3, two Chromatic mirror 4, objective lens 5, reflecting mirror 6, converging len...

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Abstract

The invention relates to a method for realizing super-resolution dipole orientation analysis. The method comprises the following steps: a microscopic imaging system is arranged, wherein the system comprises a laser light source, a half-wave plate, a collimating lens, a dichroscope, an object lens, a reflector, a convergent lens, and an EMCCD imaging device; light splitting difference correction for different polarized light due to diffraction, background noise, and the dichroscope as well as Poisson sampling property factors of EMCCD acquisition signals are considered, a fluorescent polarization modulation microscopic imaging process is modeled, and a super-resolution intensity signal is obtained; after counter-demodulation, polarization modulation properties of singles in a single pixel are used, least square fitting is carried out for the super-resolution intensity signal, average dipole orientation in a single pixel is extracted, and signals with super-diffraction limiting resolution are realized; a direction consistency factor OUF is defined for evaluating validity of average dipole direction after reconstruction; and two visualization methods of super-resolution signals are used, dimensions for distinguishing different molecules are added from the aspect of an angle resolution, and fine understanding of protein compositions is realized.

Description

technical field [0001] The invention relates to a super-resolution imaging method, in particular to a method for realizing super-resolution dipole orientation analysis. Background technique [0002] Fluorescent labeling microscopy is widely used to observe the subcellular structure of biological samples. Fluorescence has four characteristics: intensity (intensity, reflecting fluorescence density), wavelength (wavelength, absorption and emission spectrum), time (time, fluorescence decay lifetime) and polarization (polarization, caused by dipole orientation). The dipole orientation of fluorescent molecules can reflect the spatial orientation of the target protein, and then study the structure and dynamics of the target protein in living cells. [0003] Fluorescence polarization microscopy (FPM) measures the orientation of dipoles by analyzing the polarization properties to study the orientation of the target protein. FPM is already a relatively mature optical imaging system,...

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Application Information

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IPC IPC(8): G01N21/64
CPCG01N21/6458G01N2201/06113
Inventor 陈龙张昊杨旭三王淼妍张奇伟高军涛席鹏
Owner PEKING UNIV
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