Fluorescent three-dimensional nano-resolution imaging method with axial selectivity excitation and device thereof

Abstract

The invention is applicable to three-dimensional imaging of samples such as cells and the like, and provides a fluorescent three-dimensional nano-resolution imaging method with axial selectivity excitation and a device thereof. The method comprises the following steps of: generating exciting light and extinguishing light; light split, splitting the exciting light into first exciting light and second exciting light, and splitting the extinguishing light into first extinguishing light and second extinguishing light; and adjusting the phase difference and the intensity ratio between the first exciting light and the second exciting light, and adjusting the phase difference and the intensity ratio between the first extinguishing light and the second extinguishing light, and leading the first exciting light and the second exciting light as well as the first extinguishing light and the second extinguishing light to be coherent at a thin layer or more thin layers along the axial direction of the sample to form axial modulation illumination and realize axial selectivity excitation. Most fluorescence labeling objects in a fluorescent state can be distributed inside one or more thin layer(s) in a centralized way along the axial direction by the axial selectivity excitation, the background noise caused by thick sample imaging can be greatly inhibited, and the image information acquisition rate is improved.

Description

technical field [0001] The invention belongs to optical microscopic imaging technology, in particular to a fluorescent three-dimensional nano-resolution imaging method and device with axial selective excitation. Background technique [0002] As we all know, nano-resolution fluorescence imaging can visually display the spatial distribution of labeled molecules in the labeled object with nanometer spatial resolution, and can be used to study the interaction process between labeled molecules, especially for the study of DNA in cells , The interaction and movement rules between RNA and protein molecules are important tools for using cells as "test tubes" to study the interaction rules of major biological macromolecules in situ. In recent years, a class of fluorescent marker localization microscopy techniques using the switching effect of the fluorescent marker itself has emerged to perform nano-resolution imaging through time-division multiplexing, centroid localization, and ima...

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Problems solved by technology

[0004] The purpose of the embodiments of the present invention is to provide an efficient three-dimensional nano-resolution imaging method and device for fluorescence with axial selective excitation, aiming to solve the problem of low efficiency of existing molecular positioning microscopy imaging

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