Method and device for two-photon fluorescence stimulated emission differential super-resolution microscopy

Abstract

The invention discloses a method for two-photon fluorescence stimulated emission differential super-resolution microscopy. The method includes the steps that (1) after being collimated, pulsed laser beams are converted into linear polarized light and polarization modulation is conducted on the linear polarized light, so that radial polarized light is obtained; (2) the radial polarized light is converted into circular polarized light and projected onto a sample to be tested, two-photon stimulated emission is conducted, fluorescence is collected, and therefore first signal light intensity I1 is obtained; (3) polarization modulation is conducted on the linear polarized light obtained in the step (1) and the linear polarized light is converted into tangential polarized light; (4) the tangential polarized light is converted into circular polarized light and projected onto the sample to be tested, two-photon stimulated emission is conducted, fluorescence is collected, and therefore second signal light intensity I2 is obtained; (5) effective signal light intensity I is calculated according to a formula I=I1-gammaI2 , so that super-resolution imaging is achieved. The invention further discloses a device for two-photon fluorescence stimulated emission differential super-resolution microscopy. The device is simple, free of light division, low in light power, capable of weakening the photobleaching effect, higher in resolution and larger in 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 full width at half maximum (FWHM: Full Width at HalfMaximum) as where λ is t...

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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