A Two-Photon Fluorescence Stimulated Emission Differential Super-resolution Microscopy Method and Device

Abstract

The invention discloses a two-photon fluorescence stimulated emission differential super-resolution microscopy method, comprising the steps of: 1) converting a pulsed laser beam into linearly polarized light after being collimated, and then performing polarization modulation on the linearly polarized light to obtain a diameter 2) convert radially polarized light into circularly polarized light and project it on the sample to be measured, perform two-photon excitation, collect fluorescence to obtain the first signal light intensity I 1 ; 3) carry out polarization modulation to the linearly polarized light obtained in step 1), and convert it into tangentially polarized light; 4) convert tangentially polarized light into circularly polarized light and project it on the sample to be measured, perform two-photon excitation, and collect fluorescence Get the second signal light intensity I 2 ; 5) according to the formula I=I 1 -γI 2 Calculate the effective signal intensity I to realize super-resolution imaging. The invention also discloses a two-photon fluorescence stimulated emission differential super-resolution microscopic device. The device of the invention is simple, does not need light splitting, uses lower optical power, weakens photobleaching effect, and has higher resolution and greater imaging depth.

Description

technical field [0001] The invention relates to the field of super-resolution, in particular to a two-photon fluorescence stimulated emission differential super-resolution microscopy method and device capable of exceeding the diffraction limit in the far field and realizing super-resolution. Background technique [0002] Due to the existence of the optical diffraction limit, the traditional far-field optical microscopy method has a limit to the resolution that can be achieved. This limit is determined by Abbe's diffraction limit theory. After the beam is focused by the microscope objective lens, a blurred spot is formed on the focal plane. The resolution of an optical microscope is defined as the minimum distance between two spots of equal brightness that can be distinguished. Therefore, the size of the spot determines the limit resolution of the microscope. The size of the spot is represented by the full width at half maximum (FWHM: FullWidthatHalfMaximum) as where λ is ...

AI Technical Summary

Problems solved by technology

The traditional fluorescence optical microscope adopts single-photon excitation method, and uses short-wavelength excitation light to excite fluorescence. The sample has a strong scattering effect on short-wavelength excitation light, and the intensity of excitation light decays exponentially with the increase of depth, thus limiting the imaging depth of the microscope.

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