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

A two-photon fluorescence and stimulated emission technology, which is applied in the field of super-resolution, can solve the problems of strong scattering and limited imaging depth of the microscope, and achieve the effect of weakening scattering, high resolution and simple device

CN104062750BInactive Publication Date: 2016-07-06ZHEJIANG UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2016-07-06
Estimated Expiration
Not applicable · inactive patent

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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.
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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 ...

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Embodiment Construction

[0042] The present invention will be described in detail below in conjunction with the embodiments and accompanying drawings, but the present invention is not limited thereto.

[0043] Such as figure 1 As shown, the fluorescence stimulated emission differential super-resolution microscopy device includes: femtosecond pulsed laser 1, single-mode fiber 2, collimator lens 3, polarizer 4, liquid crystal polarization converter 5, dichroic mirror 6, scanning Galvanometer system 7, scanning lens 8, field lens 9, 1 / 4 wave plate 10, microscope objective lens 11, sample stage 12, optical filter 13, focusing lens 14, pinhole 15, detector 16, controller 17.

[0044] Single-mode optical fiber 2, collimator lens 3, polarizer 4, liquid crystal polarization converter 5, dichroic mirror 6 are located on the optical axis of the outgoing beam of femtosecond pulse laser 1 in sequence, and the light transmission axis of polarizer 4 Parallel to the vertical direction, the scanning galvanometer sys...