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System and method for carrying out polarization super-resolution imaging on fluorescence anisotropy

A super-resolution imaging and anisotropic technology, applied in the field of fluorescence imaging, can solve the problems of slow imaging speed and inability to apply live cell imaging

Active Publication Date: 2021-10-01
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] (3) FPM technology based on single-molecule imaging, such as Polar-dSTORM, cannot be applied to live cell imaging due to its slow imaging speed

Method used

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  • System and method for carrying out polarization super-resolution imaging on fluorescence anisotropy
  • System and method for carrying out polarization super-resolution imaging on fluorescence anisotropy
  • System and method for carrying out polarization super-resolution imaging on fluorescence anisotropy

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Embodiment 1

[0055] The OLID-SDOM imaging system provided in this embodiment excites the sample to be tested with excitation light (rotated polarized light) in different polarization directions, so that the fluorescence signal of the sample to be tested presents a cosine square modulation, and the sample to be tested that is modulated by polarization is obtained. Fluorescence image sequence.

[0056] Such as figure 1 As shown, the imaging system of OLID-SDOM is a linear dichroic system excited by polarization modulation, including excitation light source, dichroic mirror, mirror, half-wave plate, polarization compensator, beam expander system, achromatic lens, objective lens , CCD (Charge Coupled Device) camera and control unit, in which the half-wave plate is mounted on a motorized rotary stage capable of rotating at a given frequency.

[0057] Specifically, the excitation light source can be a polarized continuous wave laser, and the wavelengths of the excitation light sources in this e...

Embodiment 2

[0065] Based on the polarization-modulated fluorescence image sequence of the sample to be tested acquired by the OLID-SDOM imaging system, this embodiment provides a method for performing polarization super-resolution imaging on fluorescence anisotropy combined with optical phase-locked detection, including the following:

[0066] S1. In order to calculate the absolute angle of dipole orientation, the phase of the reference direction needs to be given first, and this process is called polarization angle calibration. Such as figure 2 As shown in b, the specific process includes:

[0067] S11. Place a standard polarizer on the focal plane of the objective lens, and its direction is parallel to the x-axis of the imaging system, wherein the z-axis of the imaging system is perpendicular to the focal plane, and the x-axis and y-axis are parallel to the focal plane and perpendicular to each other. In a Cartesian coordinate system, the x-axis of the imaging system refers to the x-a...

Embodiment 3

[0119] In this embodiment, a simulation experiment is used to verify the performance of the OLID. The simulation experiment includes two parts: fluorescence fluctuation and bleaching. Within one OLID cycle, the dipole orientations are measured with an accuracy between 5° (OUF = 0.3) and 15° (OUF = 0.15) for a typical 10% fluctuation in the membrane of an organelle. The rotation period of the half-wave plate can be improved by a factor of two (four OLID periods). Averaging the signal with a longer acquisition time yields similar performance when bleaching is ignored. On the other hand, if the magnitude of the acquisition time and the bleaching time are consistent and comparable, the measurement error will increase instead. The invention performs imaging on various target proteins marked by GFP in yeast, studies the fluorescence anisotropy of various subcellular organelles, and finds that the anisotropy is related to the function of the organelles. The present invention also o...

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Abstract

The invention relates to a system and method for carrying out polarization super-resolution imaging on fluorescence anisotropy. According to the system, an optical phase-locked detection technology and a linear dichroic system are used to obtain a polarization modulation image sequence. The method comprises the following steps of: S1, carrying out frequency domain phase-locked processing on an acquired fluorescence image sequence modulated by rotating polarized light, extracting signals with the same modulation frequency, and obtaining a sequence image IOLID which only retains background and modulation frequency information; and S2, performing demodulation, namely three-dimensional deconvolution, on the sequence image IOLID to obtain a super-resolution ac image G'ac, a reconstructed dc image G'dc and a super-resolution reconstructed image G 'at the same time, and performing phase extraction to obtain a dipole orientation distribution image. The OLID-SDOM disclosed by the invention reveals fluorescence anisotropy which is never observed before in part of samples, which shows that the OLID-SDOM has potential to observe dynamic molecular structures; and compared with a traditional microscope, the OLID-SDOM has the advantages that the spatial resolution is doubled, and biological samples are not limited during imaging.

Description

technical field [0001] The invention relates to the technical field of fluorescence imaging, in particular to a system and method for performing polarization super-resolution imaging on fluorescence anisotropy combined with optical phase-lock detection. Background technique [0002] The dipolar orientation of fluorophores attached to organelles provides information on the molecular formation, transformation, and dynamics of organelles. Due to various reasons, the application of FPM (Florescent Polarization Microscopy) in the study of subcellular structure is limited, mainly because: [0003] (1) The size of most subcellular organelles is below the diffraction limit; [0004] (2) Subcellular organelles and their proteins exhibit low polarization modulation due to the averaging of polarization signals in adjacent regions; [0005] (3) FPM technologies based on single-molecule imaging, such as Polar-dSTORM, cannot be applied to live cell imaging due to the slow imaging speed....

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N21/01
CPCG01N21/6428G01N21/6486G01N21/6456G01N21/6445G01N21/6458G01N21/01
Inventor 高军涛关美玲王淼妍张昊陈龙杨旭三席鹏张奇伟
Owner TSINGHUA UNIV
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