CN109632756B - A real-time fluorescence radiation differential super-resolution microscopy method and device based on parallel spot scanning - Google Patents

Abstract

The invention discloses a real-time fluorescence radiation differential super-resolution microscopy method and device based on parallel spot scanning. The method divides a laser beam into S-polarized light and P-polarized light, modulates the S-polarized light into a circularly polarized solid spot, and converts the P-polarized light into a circularly polarized solid spot. The polarized light is first modulated into vortex polarized light, and then modulated into a circularly polarized hollow spot; the excitation light of the solid spot and the excitation light of the hollow spot are staggered by at least 200 nm on the object surface; the excitation light of the solid spot and the excitation light of the hollow spot are used for fluorescent samples. At the same time, two-dimensional scanning was performed to obtain a positive confocal fluorescence intensity map modulated by a solid spot and a negative confocal fluorescence intensity map modulated by a hollow spot; the two fluorescence intensity maps were shifted and matched. Due to the simultaneous scanning of two light spots, compared with the traditional method of switching the modulated light spots back and forth in the traditional fluorescence emission differential microscopy system, the sampling speed is twice as fast as the traditional one, and the super-resolution dynamic microscopy effect at the confocal scanning speed can be realized, which can significantly improve the imaging. speed.

Description

A Real-Time Fluorescence Radiation Differential Super-Resolution Microscopy Method Based on Parallel Spot Scanning with device

technical field

The invention belongs to the field of super-resolution microscopy, in particular to a method capable of simultaneously obtaining a positive confocal image modulated by a solid light spot and a negative confocal image modulated by a hollow light spot in the far field, and using a differential method to achieve the ultra-diffraction limit High-resolution super-resolution microscopy methods and apparatus.

Background technique

The development of fluorescence microscopy has greatly promoted the research in biological cytology and other fields. However, due to optical diffraction, conventional far-field optical microscopy methods have a resolution limit. According to Abbe's diffraction limit theory, the diffraction limit can be determined by the focusing of the objective lens. The full width at half maximum of the light spot is represented, that is, Δr=0.61λ/NA, where λ is the wavelength of the light wave and NA is the numerical aperture of the objective lens. In the past ten years, many researchers have made major breakthroughs in breaking the optical diffraction limit, such as stimulated radiation depletion super-resolution microscopy (Stimulation emission depletion microscopy, STED), fluorescence radiation differential super-resolution microscopy. Fluorescence emission difference microscopy (FED), structured light illumination super-resolution microscopy (SIM), photoactivated localization microscopy (PLAM) and stochastic optical reconstruction super-resolution microscopy Technology (Stochastic optical reconstruction microscopy, STORM) and so on.

Differential super-resolution microscopy of fluorescence radiation is a new type of super-resolution microscopy method recently proposed, which uses two different modes of excitation light spot excitation to generate fluorescence images on the basis of confocality, that is, one is composed of solid light spots. The positive confocal microscopic image obtained by modulation, the other is the negative confocal microscopic image obtained by modulation of the bread-shaped hollow spot, in which the center of the hollow spot is a dark spot with a size smaller than the diffraction limit. The intensity difference eliminates the edge-excited signal to achieve super-resolution, which is a type of differential imaging.

Compared with other super-resolution microscopy methods, FED can achieve lower fluorescence bleaching properties, faster imaging speed, and certain optical tomographic capabilities. However, due to the principle of FED, it requires a positive confocal image and a negative confocal image during imaging, which results in that if a super-resolution image is to be obtained, it needs to be scanned twice, which reduces the imaging speed.

SUMMARY OF THE INVENTION

The invention provides a real-time fluorescence radiation differential super-resolution microscopy method and device based on parallel spot scanning. Compared with the traditional FED method, the imaging speed of the method of the invention is increased by two times, and the ultra-high-resolution imaging speed at the confocal imaging speed is realized. Resolution imaging.

The object of the present invention is achieved through the following technical solutions: a real-time fluorescence radiation differential super-resolution microscopy method based on parallel spot scanning, comprising the following steps:

(1) The laser beam emitted by the laser is collimated and divided into S-polarized light and P-polarized light by using a polarization beam splitter (PBS);

(2) Using a quarter-wave plate to modulate the S-polarized light into a circularly polarized solid spot;

(3) Phase modulation is performed on the P polarized light to modulate into vortex polarized light;

(4) The modulated P-polarized light is further modulated into a circularly polarized hollow spot by using a quarter-wave plate;

(5) According to the circular hole diffraction limit formula, in order to ensure that the solid spot and the hollow spot will not interfere with each other, the beam deflection device is used to make the excitation light of the solid spot and the excitation light of the hollow spot staggered on the object surface by at least 200nm or more;

(6) utilize solid spot excitation light and hollow spot excitation light to carry out two-dimensional scanning to fluorescent sample simultaneously, filter out stray light and excitation light, collect fluorescence signal, obtain the positive confocal fluorescence intensity figure I ₁ ( x, y) and the negative confocal fluorescence intensity map I ₂ (x, y) obtained by the modulation of the hollow spot;

为第一信号光强I₁(x,y)的最大值，

为第二信号光强I₂(x,y)的最大值；I(x,y)为负数时，设置I(x,y)＝0。(7) The two fluorescence intensity maps are shifted and matched, and the super-resolution image I(x,y) is obtained according to the formula I(x,y)=I ₁ (x,y)-γI ₂ (x,y), where

is the maximum value of the first signal light intensity I ₁ (x, y),

is the maximum value of the second signal light intensity I ₂ (x, y); when I(x, y) is a negative number, set I(x, y)=0.

Further, in the step (1), a half-wave plate is used to adjust the splitting ratio of the two polarized lights, so that the light intensity of the S-polarized light is weaker than that of the P-polarized light.

其中ρ为光束上某点与光轴的距离，

为光束垂直光轴剖面内位置极坐标矢量与X轴的夹角。Further, in the step (3), the vortex phase plate is used to phase modulate the P-polarized light, and the modulation function is

where ρ is the distance between a point on the beam and the optical axis,

is the angle between the position polar coordinate vector and the X axis in the vertical optical axis section of the beam.

Further, in the step (5), the light beam deflecting device may use a flat mirror, a dichroic mirror, or the like.

Further, in the step (6), an avalanche photodiode (APD) or a photomultiplier tube (PMT) is used to collect the fluorescence signal.

The invention provides two real-time fluorescence radiation differential super-resolution microscopy devices based on parallel spot scanning, both of which include a laser, an excitation light modulation optical path sub-module, a stage for carrying a fluorescent sample to be measured, and a microscope for projecting light onto the stage rack and detection optical path sub-module.

The excitation light modulation optical path sub-module includes:

A beam expander used to expand the light of a point light source emitted by a laser into parallel light;

A half-wave plate used to modulate the polarization direction of the light emitted by the beam expander;

A polarizing beam splitter used to split the light emitted from the half-wave plate into P-polarized light and S-polarized light;

Vortex phase plate for 0-2π phase modulation of P-polarized light;

Quarter wave plate for modulating P-polarized and S-polarized light into circularly polarized light;

Beam splitter for combining P-polarized light and S-polarized light;

A dichroic mirror for reflecting S-polarized light and transmitting fluorescence;

A first plane mirror for staggering two circularly polarized lights on the object surface by at least 200 nm;

Scanning lens for focusing the light emitted from the beam splitter.

The microscope stand includes:

a second plane mirror for beam deflection of the light emitted from the scanning lens;

A tube mirror for collimating the light emitted by the second plane mirror;

A microscope objective used to focus the light emitted from the tube lens to the stage;

Further, the numerical aperture of the microscope objective lens is NA=1.49, the magnification is 100 times, the focal length of the tube lens is 200 mm, and the focal length of the scanning lens is 50 mm.

The detection optical path sub-module includes two schemes:

Option 1 specifically includes:

Bandpass filter used to filter out stray light in the output light of the dichroic mirror;

A doublet lens for converging the light emitted from the bandpass filter;

A semicircular plane mirror for splitting the two outgoing lights of the doublet lens;

a first 4F lens group for respectively amplifying the two signal lights emitted by the semicircular plane reflector;

A spatial filter used to spatially filter the outgoing beam of the first 4F lens group; the spatial filter can be implemented by pinhole or multimode fiber, and the size should be smaller than the diameter of an Airy disk;

The first detector used to detect the outgoing beam of the spatial filter; the first detector can be selected from a photomultiplier tube (PMT) or an avalanche photodiode (APD).

The second plan includes:

Bandpass filter used to filter out stray light in the output light of the dichroic mirror;

A doublet lens for converging the light emitted from the bandpass filter;

The second 4F lens group used to amplify the two-way outgoing light of the doublet lens;

Parallel filter fibers used to spatially filter the two paths of light emitted by the second 4F lens group; the spacing of the parallel filter fibers is 375um. In order to ensure that 60% of the energy of the Airy disk is collected, the magnification of the entire system is set to 800 times, so the distance between the two light spots on the object surface should be 470nm±50nm.

The second detector is used to detect the beam outgoing from the parallel filtered fiber; the second detector can be selected from a photomultiplier tube (PMT) or an avalanche photodiode (APD).

The principle of the invention is as follows: an innovation is made on the basis of the original fluorescence radiation differential super-resolution microscope system, the two coincident light spots are staggered by a distance greater than one Airy disk, and the detection module is improved, and a semicircular plane mirror is used. One of the staggered signals is reflected into a separate detection system, while the other is directly detected, or the staggered fluorescence signals are detected using parallel filter fibers.

其中k为波矢，a为圆孔半径，θ为孔径角，I₀为中心光强极大值，其艾里斑直径可由公式

得到，次级大的相对强度为I₂≈0.00175I₀，因此当两光斑相间大于一个艾里斑距离时，其旁瓣的影响则小于千分之二，因此两光斑需要间隔大于一个艾里斑，以保证光斑相互间不影响。According to the circular hole diffraction limit formula

where k is the wave vector, a is the radius of the circular hole, θ is the aperture angle, I ₀ is the maximum value of the central light intensity, and its Airy disk diameter can be calculated by the formula

It can be obtained that the relative intensity of the secondary maximum is I ₂ ≈ 0.00175I ₀ , so when the distance between the two light spots is greater than an Airy disk distance, the effect of the side lobes is less than two thousandths, so the two light spots need to be separated by more than one Airy distance. spot to ensure that the spots do not affect each other.

Compared with the prior art, the present invention has the following beneficial technical effects: due to the simultaneous scanning of two light spots, compared with the method of switching the modulated light spot back and forth in the traditional fluorescence emission differential microscope system, the sampling speed is twice as high as that of the traditional one, and the The super-resolution dynamic microscopy effect at the confocal scanning speed can significantly improve the imaging speed.

Description of drawings

Fig. 1 is a schematic diagram of a microscopic device scheme of the present invention;

Fig. 2 is the schematic diagram of the second scheme of the microscope device of the present invention;

3 is a schematic diagram of the spaced position of the parallel spot scanning on the object plane proposed by the present invention;

In Fig. 4, (a) is a schematic diagram of a semicircular plane mirror in the dashed-line frame of Fig. 1, and (b) is a schematic diagram of a parallel filtering optical fiber in the dashed-line frame of Fig. 2;

(a) and (b) in Fig. 5 are respectively enlarged schematic diagrams of solid light spot and hollow light spot;

FIG. 6 is a schematic diagram of differential super-resolution of solid light spots minus hollow light spots.

Detailed ways

The present invention will be described in detail below with reference to examples and accompanying drawings, but the present invention is not limited thereto.

Example 1

A real-time fluorescence radiation differential super-resolution microscopy method based on parallel spot scanning provided by this embodiment includes the following steps:

(1) The laser beam emitted by the laser is collimated and divided into S-polarized light and P-polarized light by using a polarization beam splitter (PBS);

(2) Using a quarter-wave plate to modulate the S-polarized light into a circularly polarized solid spot;

(3) Phase modulation is performed on the P polarized light to modulate into vortex polarized light;

(4) The modulated P-polarized light is further modulated into a circularly polarized hollow spot by using a quarter-wave plate;

(5) According to the circular hole diffraction limit formula, in order to ensure that the solid spot and the hollow spot will not interfere with each other, the beam deflection device is used to make the excitation light of the solid spot and the excitation light of the hollow spot staggered on the object surface by at least 200nm or more;

(6) utilize solid spot excitation light and hollow spot excitation light to carry out two-dimensional scanning to fluorescent sample simultaneously, filter out stray light and excitation light, collect fluorescence signal, obtain the positive confocal fluorescence intensity figure I ₁ ( x, y) and the negative confocal fluorescence intensity map I ₂ (x, y) obtained by the modulation of the hollow spot;

为第一信号光强I₁(x,y)的最大值，

为第二信号光强I₂(x,y)的最大值；I(x,y)为负数时，设置I(x,y)＝0。(7) The two fluorescence intensity maps are shifted and matched, and the super-resolution image I(x,y) is obtained according to the formula I(x,y)=I ₁ (x,y)-γI ₂ (x,y), where

is the maximum value of the first signal light intensity I ₁ (x, y),

is the maximum value of the second signal light intensity I ₂ (x, y); when I(x, y) is a negative number, set I(x, y)=0.

Further, in the step (1), a half-wave plate is used to adjust the splitting ratio of the two polarized lights, so that the light intensity of the S-polarized light is weaker than that of the P-polarized light.

其中ρ为光束上某点与光轴的距离，

为光束垂直光轴剖面内位置极坐标矢量与X轴的夹角。Further, in the step (3), the vortex phase plate is used to phase modulate the P-polarized light, and the modulation function is

where ρ is the distance between a point on the beam and the optical axis,

is the angle between the position polar coordinate vector and the X axis in the vertical optical axis section of the beam.

Further, in the step (5), the light beam deflecting device adopts a plane reflecting mirror, a dichroic mirror, and the like.

Further, in the step (6), an avalanche photodiode (APD) or a photomultiplier tube (PMT) is used to collect the fluorescence signal.

Example 2

As shown in FIG. 1 , a real-time fluorescence radiation differential super-resolution microscopy device based on parallel spot scanning provided in this embodiment includes a laser 14, an excitation light modulation optical path sub-module, a stage 1 for carrying a fluorescent sample to be measured, Project light to the microscope stand and detection light path sub-module of stage 1;

The excitation light modulation optical path sub-module includes:

A beam expander 12 for expanding the light of the point light source emitted by the laser 14 into parallel light;

A half-wave plate 11 for modulating the polarization direction of the light emitted from the beam expander 12;

A polarizing beam splitter 10 for dividing the light emitted from the half-wave plate 11 into P-polarized light and S-polarized light;

A vortex phase plate 9 for performing 0-2π phase modulation on P-polarized light;

A quarter-wave plate 8 for modulating P-polarized light and S-polarized light into circularly polarized light;

Beam splitter 6 for combining P-polarized light and S-polarized light;

A dichroic mirror 7 for reflecting S-polarized light and transmitting fluorescence;

The first plane mirror 13 used to stagger the two beams of circularly polarized light by at least 200nm on the object surface, and by adjusting the deflection angle of the first plane mirror 13, the combined hollow light spot and the solid light spot form a certain clip. horn.

The scanning lens 5 is used to focus the light emitted from the beam splitter 6 .

The microscope stand includes:

a second plane mirror 4 for deflecting the light emitted from the scanning lens 5;

A tube mirror 3 for collimating the light emitted from the second plane mirror 4;

The microscope objective lens 2 used for the light emitted from the tube lens 3 to converge on the stage 1;

Further, the numerical aperture of the microscope objective lens 2 is NA=1.49, the magnification is 100 times, the focal length of the tube lens 3 is 200 mm, and the focal length of the scanning lens 5 is 50 mm.

The detection optical path sub-module includes:

A bandpass filter 15 for filtering out stray light in the light emitted by the dichroic mirror 7;

A doublet lens 16 for converging the light emitted from the bandpass filter 15;

A semicircular plane mirror 17 for splitting the two outgoing lights of the doublet lens 16;

the first 4F lens group 18 for respectively amplifying the two signal lights emitted by the semicircular plane mirror 17;

A spatial filter 19 for spatially filtering the outgoing beam of the first 4F lens group 18; the spatial filter 19 can be realized by a pinhole or a multimode fiber, and the size should be smaller than an Airy disk diameter;

The first detector 20 is used to detect the beam exiting the spatial filter 19; the first detector 20 can be selected from a photomultiplier tube (PMT) or an avalanche photodiode (APD).

The apparatus also includes a computer 24 for controlling the laser 14 and the first detector 20 .

Example 3

As shown in FIG. 2 , a real-time fluorescence radiation differential super-resolution microscopy device based on parallel spot scanning provided by this embodiment includes a laser 14, an excitation light modulation optical path sub-module, a stage 1 for carrying a fluorescent sample to be measured, Project light to the microscope stand and detection light path sub-module of stage 1;

The excitation light modulation optical path sub-module includes:

A beam expander 12 for expanding the light of the point light source emitted by the laser 14 into parallel light;

A half-wave plate 11 for modulating the polarization direction of the light emitted from the beam expander 12;

A polarizing beam splitter 10 for dividing the light emitted from the half-wave plate 11 into P-polarized light and S-polarized light;

A vortex phase plate 9 for performing 0-2π phase modulation on P-polarized light;

A quarter-wave plate 8 for modulating P-polarized light and S-polarized light into circularly polarized light;

Beam splitter 6 for combining P-polarized light and S-polarized light;

A dichroic mirror 7 for reflecting S-polarized light and transmitting fluorescence;

The first plane mirror 13 for staggering the two circularly polarized lights on the object surface by at least 200 nm; the scanning lens 5 for focusing the light emitted from the beam splitter 6 .

The microscope stand includes:

a second plane mirror 4 for deflecting the light emitted from the scanning lens 5;

A tube mirror 3 for collimating the light emitted from the second plane mirror 4;

The microscope objective lens 2 used for the light emitted from the tube lens 3 to converge on the stage 1;

Further, the numerical aperture of the microscope objective lens 2 is NA=1.49, the magnification is 100 times, the focal length of the tube lens 3 is 200 mm, and the focal length of the scanning lens 5 is 50 mm.

The detection optical path sub-module includes:

A bandpass filter 15 for filtering out stray light in the light emitted by the dichroic mirror 7;

A doublet lens 16 for converging the light emitted from the bandpass filter 15;

The second 4F lens group 21 for amplifying the two-way outgoing light of the doublet lens 16;

A parallel filter fiber 22 for spatially filtering the two paths of light emitted by the second 4F lens group 21; the spacing between the parallel filter fibers 22 is 375um. In order to ensure that 60% of the Airy disk energy is collected, the magnification of the entire system is Set to 800 times, so the distance between the two light spots on the object surface should be 470nm±50nm.

The second detector 23 is used to detect the beam output from the parallel filtering fiber 22; the second detector 23 can be selected from a photomultiplier tube (PMT) or an avalanche photodiode (APD).

The apparatus also includes a computer 24 for controlling the laser 14 and the second detector 23 .

The above-mentioned specific embodiments are used to explain the present invention, rather than limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims all fall into the protection scope of the present invention.

Claims (7)

1. a real-time fluorescence radiation differential super-resolution microscopy device based on parallel spot scanning, is characterized in that, this device comprises laser, excitation light modulation optical path sub-module, the stage of carrying the fluorescent sample to be measured, the projection light to the object The microscope stand and detection light path sub-module of the stage; The excitation light modulation optical path sub-module includes: A beam expander used to expand the light of a point light source emitted by a laser into parallel light; A half-wave plate used to modulate the polarization direction of the light emitted by the beam expander; A polarizing beam splitter used to split the light emitted from the half-wave plate into P-polarized light and S-polarized light; Vortex phase plate for 0-2π phase modulation of P-polarized light; Quarter wave plate for modulating P-polarized and S-polarized light into circularly polarized light; Beam splitter for combining P-polarized light and S-polarized light; A dichroic mirror for reflecting S-polarized light and transmitting fluorescence; A first plane mirror for staggering the two beams of circularly polarized light by at least 200nm on the object surface, the first plane mirror is located on the optical path of the P-polarized light and behind the quarter-wave plate; A scanning lens for focusing the light emitted by the beam splitter; The microscope stand includes: a second plane mirror for beam deflection of the light emitted from the scanning lens; A tube mirror for collimating the light emitted by the second plane mirror; A microscope objective used to focus the light emitted from the tube lens to the stage; The detection optical path sub-module includes: Bandpass filter used to filter out stray light in the output light of the dichroic mirror; A doublet lens for converging the light emitted from the bandpass filter; A semicircular plane mirror for splitting the two outgoing lights of the doublet lens; a first 4F lens group for respectively amplifying the two signal lights emitted by the semicircular plane reflector; A spatial filter for spatially filtering the outgoing beam of the first 4F lens group; a first detector for detecting the beam exiting the spatial filter; The spatial filter is realized by using pinhole or multimode fiber, and the size should be smaller than the diameter of an Airy disk. 2. A real-time fluorescence radiation differential super-resolution microscopy device based on parallel spot scanning, characterized in that the device comprises a laser, an excitation light modulation optical path sub-module, a stage bearing the fluorescent sample to be measured, and a projection light to the object. The microscope stand and detection light path sub-module of the stage; The excitation light modulation optical path sub-module includes: A beam expander used to expand the light of a point light source emitted by a laser into parallel light; A half-wave plate used to modulate the polarization direction of the light emitted by the beam expander; A polarizing beam splitter used to split the light emitted from the half-wave plate into P-polarized light and S-polarized light; Vortex phase plate for 0-2π phase modulation of P-polarized light; Quarter wave plate for modulating P-polarized and S-polarized light into circularly polarized light; Beam splitter for combining P-polarized light and S-polarized light; A dichroic mirror for reflecting S-polarized light and transmitting fluorescence; A first plane mirror for staggering the two beams of circularly polarized light by at least 200nm on the object surface, the first plane mirror is located on the optical path of the P-polarized light and behind the quarter-wave plate; A scanning lens for focusing the light emitted by the beam splitter; The microscope stand includes: a second plane mirror for beam deflection of the light emitted from the scanning lens; A tube mirror for collimating the light emitted by the second plane mirror; A microscope objective used to focus the light emitted from the tube lens to the stage; The detection optical path sub-module includes: Bandpass filter used to filter out stray light in the output light of the dichroic mirror; A doublet lens for converging the light emitted from the bandpass filter; The second 4F lens group used to amplify the two-way outgoing light of the doublet lens; A parallel filter fiber for spatially filtering the two paths of light emitted by the second 4F lens group; a second detector for detecting the beam exiting the parallel filtered fiber; The distance between the parallel filter fibers is 375um. In order to ensure that 60% of the energy of the Airy disk is collected, the magnification of the entire system is set to 800 times, so the distance between the two light spots on the object surface should be 470nm±50nm. 3. The real-time fluorescence radiation differential super-resolution microscopy device based on parallel spot scanning according to claim 1 or 2, wherein the microscope objective lens has a numerical aperture NA=1.49, a magnification of 100 times, and a tube mirror. The focal length of the scan lens is 200mm, and the focal length of the scan lens is 50mm. 4. A real-time fluorescence radiation differential super-resolution microscopy method using the real-time fluorescence radiation differential super-resolution microscopy device based on parallel spot scanning according to any one of claims 1-3, wherein the method comprises the following steps : (1) The laser beam emitted by the laser is collimated and divided into S-polarized light and P-polarized light using a polarization beam splitter; (2) Using a quarter-wave plate to modulate the S-polarized light into a circularly polarized solid spot; (3) Phase modulation is performed on the P polarized light to modulate into vortex polarized light; (4) The modulated P-polarized light is further modulated into a circularly polarized hollow spot by using a quarter-wave plate; (5) According to the diffraction limit formula of the circular hole, in order to ensure that the solid spot and the hollow spot will not interfere with each other, the first plane mirror is used to make the excitation light of the solid spot and the excitation light of the hollow spot staggered on the object surface by at least 200 nm or more; (6) utilize solid spot excitation light and hollow spot excitation light to carry out two-dimensional scanning to fluorescent sample simultaneously, filter out stray light and excitation light, collect fluorescence signal, obtain the positive confocal fluorescence intensity figure I ₁ ( x, y) and the negative confocal fluorescence intensity map I ₂ (x, y) obtained by the modulation of the hollow spot; (7) The two fluorescence intensity maps are shifted and matched, and the super-resolution image I(x,y) is obtained according to the formula I(x,y)=I ₁ (x,y)-γI ₂ (x,y), where ,

is the maximum value of the first signal light intensity I ₁ (x, y),

is the maximum value of the second signal light intensity I ₂ (x, y); when I(x, y) is a negative number, set I(x, y)=0. 5. real-time fluorescence radiation differential super-resolution microscopy method according to claim 4, is characterized in that, in described step (1), utilize half wave plate to adjust the light splitting ratio of two beams of polarized light, make S polarized light The light intensity of light is weaker than that of P-polarized light. 6. real-time fluorescence radiation differential super-resolution microscopy method according to claim 4, is characterized in that, in described step (3), utilizes vortex phase plate to carry out phase modulation to P polarized light, and modulation function is

where ρ is the distance between a point on the beam and the optical axis,

is the angle between the position polar coordinate vector and the X axis in the vertical optical axis section of the beam. 7 . The real-time fluorescence radiation differential super-resolution microscopy method according to claim 4 , wherein, in the step (6), an avalanche photodiode or a photomultiplier tube is used to collect the fluorescence signal. 8 .

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