A single-molecule positioning microscopic imaging method, optical component and imaging system

Abstract

The invention discloses a single-molecule positioning micro-imaging method, an optical assembly and an imaging system. The method comprises the following steps: establishing an optical module with a double helix point dispersion function and a deformed multi-value pure phase grating multi-order imaging property; after fluorescence beams emitted by to-be-tested molecules of multiple sample surfacesare emitted through the optical module, carrying out imaging at different positions of the same detection surface, so as to respectively generate double helix images; determining transverse positionsof the to-be-tested molecules according to positions of the centers of double helix sidelobes in the double helix images on the imaging surfaces; and determining the axial positions of the to-be-tested molecules according to the middle points of the double helix sidelobes in the double helix images and a rotation angle of a connection line between the two sidelobes. The molecular information of multiple layers in the sample can be imaged at different positions of the same detection surface in a double helix manner, so that the axial positioning range and resolution ratio of a double helix point spread function can be improved without scanning, and the problem of large field depth detection in single molecule positioning and tracing techniques in living cells is solved.

Description

technical field [0001] The invention relates to the technical field of super-resolution microscopic imaging, in particular to a single-molecule positioning microscopic imaging method, an optical component and an imaging system. Background technique [0002] Today, with the rapid development of life science and technology, in order to further understand and study the interaction between living organisms and the mechanism of disease, people urgently need to obtain more accurate structural information inside cells. However, due to the existence of the optical diffraction limit, the resolution of conventional optical microscopes can only reach about 200nm, which is difficult to meet the needs of modern biomedicine. In recent years, single-molecule localization super-resolution fluorescence microscopy has emerged, such as photosensitive localization microscopy (PLM), stochastic optical reconstruction microscopy (STORM), fluorescence photosensitive localization microscopy (FPLM), ...

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Problems solved by technology

However, in the process of 3D scanning, different levels of information will affect each other, resulting in background noise and fluorescence bleaching, reducing resolution

Some researchers propose to use deformable gratings to detect multi-layered samples, but since the energy of deformed gratings is mainly distributed in the 0th order and ±1st order of diffraction, this method can only simultaneously image nine different layers in the cell at most. Therefore, Inability to maintain high axial resolution while achieving large axial detection range

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