A two-dimensional ten-nanometer positioning method and device based on polarization switching hollow spot illumination

Abstract

The present invention discloses a two-dimensional ten-nanometer-level positioning method and device based on polarization-switched hollow light spot illumination, and relates to the field of super-resolution technology. It includes: the laser generated by the laser periodically switches the polarization state through an electro-optical modulator, and is modulated by a 0‑2π vortex phase plate to focus on the sample to form a transverse hollow light spot. The hollow light spots with different polarization states have different light intensity distributions. The polarization state of the hollow light spot is switched, and time-sharing illumination is performed to obtain the distribution of the number of photons excited by the fluorescent molecules under different light intensity distributions, and the two-dimensional spatial information of the sample is reconstructed using an algorithm. The present invention uses a polarization-switched hollow light spot for illumination, changes the light intensity distribution of the fluorescent molecule excitation light in a time-sharing manner, and detects the two-dimensional position of the fluorescent molecule. Compared with the traditional method, the method of changing the light intensity distribution by polarization switching of the hollow light spot simplifies the optical path and reduces costs, while achieving high-precision two-dimensional positioning of the fluorescent molecule.

Description

Technical Field

[0001] The present invention relates to the field of super-resolution technology, and in particular to a two-dimensional ten-nanometer level positioning method and device based on polarization switching hollow spot illumination. Background Art

[0002] In 2014, the Nobel Prize in Chemistry was awarded to German scientist Stefan W. Hell and American scientists Eric Betzig and William E. Moerner for their outstanding contributions in the field of optical super-resolution. The "stimulated emission depletion" (STED) microscopy system based on point scanning proposed by Stefan W. Hell achieved a resolution beyond the optical diffraction limit by minimizing the system PSF. In 2017, Hell published a paper in Science magazine, innovatively combining the advantages of random coordinate positioning and fixed coordinate resolution, and proposed a new optical super-resolution technology called minimum light flux microscopy (MINFLUX). The random coordinate positioning technology locates by Gaussian center fitting fluorescence, and the fixed coordinate resolution technology uses a hollow beam to deplete fluorescence, thereby reducing the system's PSF. MINFLUX only uses a hollow beam to excite fluorescence and locate the center of a dark spot with a fixed coordinate, achieving the effect of single-molecule positioning. Compared with traditional methods, the positioning accuracy of MINFLUX is greatly improved, and it helps to solve the problem of photobleaching.

[0003] MINFLUX is different from the previous single-molecule Gaussian fitting positioning algorithm. It moves the laser spot and makes a probability estimate based on the different photon number distributions obtained to fit the position of a single molecule. The main purpose of moving the laser spot is to change the light intensity distribution received by the fluorescent molecule. The position of the fluorescent molecule can be solved by combining the prior light intensity distribution information and the photon number distribution obtained under different light intensity distributions. In this process, the laser spot needs to be moved at high speed through an electro-optical deflector, which greatly increases the difficulty of optical path construction and equipment costs.

[0004] Therefore, it is an urgent problem for those skilled in the art to propose a two-dimensional ten-nanometer positioning method and device based on polarization-switching hollow spot illumination to solve the difficulties existing in the prior art. Summary of the invention

[0005] In view of this, the present invention provides a two-dimensional ten-nanometer positioning method and device based on polarization switching hollow light spot illumination. The method of changing the light intensity distribution by polarization switching of the hollow light spot simplifies the optical path and reduces costs, while achieving high-precision two-dimensional positioning of fluorescent molecules.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A two-dimensional ten-nanometer-level positioning method based on polarization-switched hollow spot illumination comprises the following steps:

[0008] S1. Use a Glan prism to adjust the polarization of the collimated laser beam so that the laser beam is a linearly polarized laser beam;

[0009] S2. Periodically modulating the polarization state of the linearly polarized laser beam to achieve polarization switching of the linearly polarized laser beam;

[0010] S3. performing transverse phase modulation on the linearly polarized laser beam after polarization switching obtained in S2 to obtain a transverse hollow spot laser beam;

[0011] S4. Projecting a transverse hollow spot laser beam onto the sample to scan the sample;

[0012] S5. using a single photon counter to receive the signal light generated by the sample excited by the transverse hollow spot laser beam at different positions, and obtaining the signal photon number information;

[0013] S6. Based on the signal photon number information, use the algorithm to reconstruct the two-dimensional spatial information of the sample.

[0014] In the above method, optionally, in S2, an electro-optic modulator is used to periodically modulate the polarization state of the linearly polarized laser beam to achieve polarization switching of the linearly polarized laser beam.

[0015] In the above method, optionally, in S3, a 0-2π vortex phase plate is used to perform transverse phase modulation on the linearly polarized laser beam after polarization switching obtained in S2 to obtain a transverse hollow spot laser beam;

[0016] The principle of transverse phase modulation of the 0-2π vortex phase plate is: modulation function f(ρ,φ)=φ, where ρ is the distance between a point on the light beam and the optical axis, and φ is the angle between the polar coordinate vector of the position in the section perpendicular to the optical axis of the light beam and the x-axis.

[0017] In the above method, optionally, the sample in S4 is a fluorescent molecule.

[0018] A two-dimensional ten-nanometer-level positioning device based on polarization switching hollow spot illumination, a two-dimensional ten-nanometer-level positioning method based on polarization switching hollow spot illumination using any of the above items, comprising: an excitation light source, an electric sample stage carrying a sample to be measured, and a microscope objective lens projecting light onto the electric sample stage, wherein the excitation light source and the microscope objective lens are provided in sequence:

[0019] a laser for exciting the light source beam;

[0020] A collimating lens for collimating and expanding the laser beam output through the single-mode optical fiber;

[0021] Glan prism for converting laser beam into linearly polarized light;

[0022] An electro-optic modulator for adjusting the polarization state of a laser beam;

[0023] A telescopic system for magnifying the light spot;

[0024] A 0-2π vortex phase plate for transverse phase modulation of the laser beam;

[0025] 4f system for realizing conjugation between 0-2π vortex phase plate and piezoelectric deflection mirror;

[0026] A 1 / 2 wave plate used to adjust the polarization characteristics of the outgoing light beam of the 4f system;

[0027] A mirror for reflecting the laser beam;

[0028] a dichroic mirror for transmitting the fluorescence signal;

[0029] A piezoelectric deflection mirror for deflecting the optical path of the laser beam after polarization phase modulation to scan the selected area of ​​the sample;

[0030] 4f system for achieving conjugation between the piezoelectric deflection mirror and the microscope objective entrance pupil;

[0031] Achromatic quarter-wave plate to compensate for the change in polarization state caused by the dichroic mirror;

[0032] The device is also provided with a controller for controlling the electro-optic modulator and the piezoelectric deflection mirror and a detection system for collecting signal light emitted by the sample to be tested.

[0033] The above device, optionally, the 4f system includes a scanning lens and a field lens;

[0034] The numerical aperture of the microscope objective is NA=1.4.

[0035] The above device, optionally, the detection system comprises:

[0036] A narrow-band filter for filtering out stray light and part of the laser light in the fluorescence emitted by the dichroic mirror;

[0037] a focusing lens for focusing the filtered fluorescence beam onto a detector;

[0038] A spatial filter for spatially filtering the signal light, the spatial filter being located at a focal plane of the focusing lens;

[0039] A single photon counter used to detect the intensity signal of the signal beam.

[0040] In the above device, optionally, the spatial filter adopts a pinhole or a multimode optical fiber. If a pinhole is adopted, the diameter of the pinhole used is smaller than the diameter of an Airy disk;

[0041] The detector uses avalanche photodiode APD.

[0042] It can be seen from the above technical solutions that, compared with the prior art, the present invention provides a two-dimensional ten-nanometer-level positioning method and device based on polarization-switched hollow spot illumination, which has the following beneficial effects:

[0043] (1) A two-dimensional ten-nanometer positioning method based on polarization-switching hollow spot illumination was proposed for the first time;

[0044] (2) The hollow spot of the excitation light is polarized and modulated. The light intensity distribution of the hollow spot is different under different polarization states, and there is no need to use an electro-optical deflector to move the hollow spot at high speed to achieve changes in the light intensity distribution;

[0045] (3) The present invention uses an electro-optic modulator to periodically switch the polarization state of the light beam, and then focuses it on the sample to form a transverse hollow light spot after modulation by a 0-2π vortex phase plate. By switching the polarization state of the hollow light spot, time-sharing illumination can be performed. The detector collects the fluorescence signals excited under different polarization states, and combines the maximum likelihood estimation algorithm and the prior hollow light spot light intensity distribution information to reconstruct the two-dimensional precise position information of the fluorescent molecules. Compared with the traditional method of using an electro-optical deflector to change the position of the hollow light spot to achieve a change in light intensity distribution, the method of changing the light intensity distribution by switching the polarization of the hollow light spot simplifies the optical path and reduces costs, while achieving high-precision two-dimensional positioning of fluorescent molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0047] Figure 1 A flow chart of a two-dimensional ten-nanometer-level positioning method based on polarization-switched hollow spot illumination provided by the present invention;

[0048] Figure 2 A schematic diagram of the structure of a two-dimensional ten-nanometer-level positioning device based on polarization-switching hollow spot illumination provided by the present invention;

[0049] Figure 3Schematic diagram of light intensity distribution of the horizontal hollow light spot provided by the present invention under different polarization states, wherein 3a is a schematic diagram of circular polarization, 3b is a schematic diagram of linear polarization in the X-axis direction, 3c is a schematic diagram of linear polarization in the Y-axis direction, 3d is a schematic diagram of linear polarization at an angle of 45° between the X-axis and the Y-axis, and 3e is a schematic diagram of linear polarization at an angle of 135° between the X-axis and the Y-axis;

[0050] Figure 4 A schematic diagram of a selected area scanning mode provided by the present invention;

[0051] Figure 5 A schematic diagram of the Cramer-Rao bound (CRB) for positioning accuracy provided by the present invention;

[0052] Among them, 1-laser, 2-single-mode optical fiber, 3-collimating lens, 4-Glan prism, 5-electro-optic modulator, 6-first telescope system, 7-0~2π vortex phase plate, 8-first reflector, 9-second telescope system, 10-1 / 2 wave plate, 11-dichroic mirror, 12-narrow-band filter, 13-focusing lens, 14-spatial filter, 15-single-photon counter, 16-piezoelectric deflection mirror, 17-second reflector, 18-scanning lens, 19-field lens, 20-achromatic 1 / 4 wave plate, 21-microscope objective, 22-electric sample stage. DETAILED DESCRIPTION

[0053] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0054] Reference Figure 1 As shown, the present invention discloses a two-dimensional ten-nanometer-level positioning method based on polarization switching hollow spot illumination, comprising the following steps:

[0055] S1. Use the Glan prism 4 to adjust the polarization of the collimated laser beam so that the laser beam is a linearly polarized laser beam;

[0056] S2. Periodically modulating the polarization state of the linearly polarized laser beam to achieve polarization switching of the linearly polarized laser beam;

[0057] S3. performing transverse phase modulation on the linearly polarized laser beam after polarization switching obtained in S2 to obtain a transverse hollow spot laser beam;

[0058] S4. Projecting a transverse hollow spot laser beam onto the sample to scan the sample;

[0059] S5. Using a single photon counter 15 to receive the signal light excited by the transverse hollow spot laser beam at different positions of the sample, and obtaining the signal photon number information;

[0060] S6. Based on the signal photon number information, use the algorithm to reconstruct the two-dimensional spatial information of the sample.

[0061] Furthermore, in S2, the electro-optic modulator 5 is used to periodically modulate the polarization state of the linearly polarized laser beam to achieve polarization switching of the linearly polarized laser beam.

[0062] Furthermore, in S3, the linearly polarized laser beam after polarization switching obtained in S2 is subjected to transverse phase modulation by using a 0-2π vortex phase plate 7 to obtain a transverse hollow spot laser beam;

[0063] The principle of transverse phase modulation of the 0-2π vortex phase plate 7 is: modulation function f(ρ,φ)=φ, where ρ is the distance between a point on the light beam and the optical axis, and φ is the angle between the polar coordinate vector of the position in the section perpendicular to the optical axis of the light beam and the x-axis.

[0064] Furthermore, the sample in S4 is a fluorescent molecule.

[0065] In a specific embodiment, a two-dimensional ten-nanometer positioning device based on polarization-switching hollow spot illumination includes a laser 1, a single-mode optical fiber 2, a collimating lens 3, a Glan prism 4, an electro-optic modulator 5, a first telescope system 6, a 0-2π vortex phase plate 7, a first reflector 8, a second telescope system 9, a 1 / 2 wave plate 10, a dichroic mirror 11, a narrow-band filter 12, a focusing lens 13, a spatial filter 14, a single-photon counter 15, a piezoelectric deflection mirror 16, a second reflector 17, a scanning lens 18, a field lens 19, an achromatic 1 / 4 wave plate 20, a microscope objective 21, and an electric sample stage 22.

[0066] Among them, the single-mode optical fiber 2, the collimating lens 3, the Glan prism 4, the electro-optic modulator 5, the first telescope system 6, the 0-2π vortex phase plate 7, and the first reflector 8 are sequentially located on the optical axis of the output light beam of the laser 1.

[0067] Among them, the second telescopic system 9, the 1 / 2 wave plate 10, the dichroic mirror 11, and the piezoelectric deflection mirror 16 are sequentially located on the optical axis of the outgoing light of the 0-2π vortex phase plate 7 after being deflected by the first reflector 8.

[0068] Among them, the narrowband filter 12, the focusing lens 13, the spatial filter 14, and the single photon counter 15 are sequentially located on the optical axis of the light beam after being reflected by the dichroic mirror 11.

[0069] The second reflector 17 is located on the optical axis of the outgoing light beam of the piezoelectric deflection mirror 16 .

[0070] Among them, the scanning lens 18 , the field lens 19 , the achromatic quarter wave plate 20 , the microscope objective lens 21 , and the electric sample stage 22 are sequentially located on the optical axis of the light emitted by the piezoelectric deflection mirror 16 after being deflected by the second reflector 17 .

[0071] Specifically, the numerical aperture NA of the microscope objective lens 21 is 1.4; the diameter of the spatial filter 14 used is 0.8 Airy disk diameter, and the single photon counter 15 uses an avalanche photodiode (APD) as a detector.

[0072] The specific two-dimensional ten-nanometer-level positioning method based on polarization switching hollow spot illumination is as follows: the laser beam emitted from the laser 1 is first introduced into the single-mode optical fiber 2 and collimated by the collimating lens 3. The collimated beam is converted into linearly polarized light through the Glan prism 4 and incident on the electro-optic modulator 5. The electro-optic modulator 5 will periodically change the polarization state of the light spot during the positioning process. After that, the beam passes through the first telescope system 6 to amplify the light spot. The amplified beam passes through the 0-2π vortex phase plate 7 and is modulated into a transverse hollow light spot. After the beam is reflected by the first reflector 8, the 0-2π vortex phase plate 7 is conjugated with the piezoelectric deflection mirror 16 through the second telescope system 9. The 1 / 2 wave plate 10 is used to adjust the polarization characteristics of the 4f system output beam. After the beam passes through the dichroic mirror 11, it is incident on the piezoelectric deflection mirror 16.

[0073] The light beam emitted by the piezoelectric deflection mirror 16 is deflected by the second reflector 17 and then focused by the scanning lens 18 and collimated by the field lens 19. It then passes through the achromatic quarter wave plate 20 to compensate for the change in polarization state caused by the dichroic mirror 11, and is finally projected onto the sample to be measured on the electric sample stage 22 through the microscope objective 21.

[0074] The light field distribution of the incident light near the focus of the microscope objective 21 can be determined by the Debye integral, as follows:

[0075]

[0076] In the formula, is the coordinate in the cylindrical coordinate system with the focal position of the microscope objective lens 21 as the origin, represents the electric vector intensity at (r2, φ2, z2), i is an imaginary unit, C is a normalization constant, θ is the beam aperture angle, φ is the angle between the polar coordinate vector of the beam vertical to the Z-axis section and the x-axis, A1(θ, φ) is the amplitude distribution of the incident light, and A2(θ, φ) characterizes the structure of the microscope objective 21. It represents the polarization information of the incident light, k = 2π / λ, and n is the refractive index of the medium.

[0077] It can be found from the above calculation that when the incident light beam is circularly polarized, the light spot formed on the sample to be measured after being focused by the microscope objective 21 is a donut-shaped hollow light spot, and its transverse light field (X-axis and Y-axis cross-sectional light field) is distributed as follows: Figure 3 shown.

[0078] The signal light emitted by the sample to be tested is collected by the microscope objective 21, and then passes through the achromatic quarter wave plate 20, the field lens 19, the scanning lens 18, the second reflector 17, the piezoelectric deflection mirror 16 in sequence, and finally reflected by the dichroic mirror 11. The signal light beam reflected by the dichroic mirror 11 is filtered out of stray light by the narrow-band filter 12, and then is focused by the focusing lens 13, and then passes through the spatial filter 14, and is finally received by the single photon counter 15.

[0079] By adjusting the voltage by the controller, the electro-optic modulator 5 can be used to periodically make the hollow light spot obtain different polarization states, so that the single molecule can be excited under different light intensity distributions to obtain the two-dimensional light emission position of the molecule. The light intensity distribution of the hollow light spot under different polarization states is shown in FIG. Figure 3 As shown, 3a is a schematic diagram of circular polarization, 3b is a schematic diagram of linear polarization in the X-axis direction, 3c is a schematic diagram of linear polarization in the Y-axis direction, 3d is a schematic diagram of linear polarization at a 45° angle between the X-axis and the Y-axis, and 3e is a schematic diagram of linear polarization at a 135° angle between the X-axis and the Y-axis.

[0080] The controller adjusts the piezoelectric deflection mirror 16 to scan the selected area of ​​the sample to be tested. The scanning mode is as follows: Figure 4 shown.

[0081] By using the maximum likelihood probability estimation function on the collected data and performing inverse analysis, a more accurate location of the fluorescent molecules can be obtained. Figure 5 The single-molecule localization accuracy (CRB) of the simulation calculation is demonstrated when the number of collected photons N is 500.

[0082] The principle of using maximum likelihood probability estimation to obtain the position of fluorescent molecules is: in one positioning cycle, since the excitation dark spot is modulated into multiple polarization states, the intensity distribution of the dark spot under different polarization states is different, so the fluorescent molecule is exposed to different light excitation intensities multiple times, so the detector will receive different numbers of photons, thus forming a specific distribution of photons in a positioning cycle. For each spatial point in the area to be positioned, the probability of generating the same photon number distribution as the experimental result when the fluorescent molecule is at that point can be calculated. According to the principle of maximum likelihood probability estimation, the spatial point corresponding to the maximum probability is selected, which is the most likely location of the fluorescent molecule. The probability can be calculated according to the following formula, because the distribution of the number of photons is essentially a polynomial distribution:

[0083]

[0084] Where N is the total number of photons collected, k is the total number of polarization states of the excitation spot in a positioning cycle, and n is i represents the number of photons collected in the ith polarization state in one positioning cycle, p i represents the probability that the received photon is generated by the fluorescent molecule aligned with the spot in the i-th polarization state, I i Represents the intensity of the excitation light that the fluorescent molecules are exposed to when the polarization state of the i-th light spot is reached.

[0085] and Figure 1 Corresponding to the method described above, the embodiment of the present invention further provides a two-dimensional ten-nanometer-level positioning device based on polarization switching hollow spot illumination, which is used to Figure 1 The specific implementation of the method is shown in the following diagram: Figure 2 As shown, specifically including:

[0086] An excitation light source, an electric sample stage 22 carrying a sample to be tested, and a microscope objective lens 21 projecting light onto the electric sample stage 22. Between the excitation light source and the microscope objective lens 21, there are arranged:

[0087] A laser 1 for exciting a light source beam;

[0088] A collimating lens 3 for collimating and expanding the laser beam outputted through the single-mode optical fiber 2;

[0089] Glan prism 4 for converting laser beam into linearly polarized light;

[0090] An electro-optic modulator 5 for adjusting the polarization state of the laser beam;

[0091] A telescopic system for magnifying the light spot;

[0092] A 0-2π vortex phase plate 7 for transverse phase modulation of the laser beam;

[0093] A 4f system for realizing conjugation between the 0-2π vortex phase plate 7 and the piezoelectric deflection mirror 16;

[0094] A 1 / 2 wave plate 10 provided for adjusting the polarization characteristics of the outgoing light beam of the 4f system;

[0095] A mirror for reflecting the laser beam;

[0096] A dichroic mirror 11 for transmitting the fluorescent signal;

[0097] A piezoelectric deflection mirror 16 for deflecting the optical path of the laser beam after polarization phase modulation to scan the selected area of ​​the sample;

[0098] A 4f system for achieving conjugation of the entrance pupil of the piezoelectric deflection mirror 16 and the microscope objective lens 21;

[0099] An achromatic quarter wave plate 20 for compensating for the change in polarization state caused by the dichroic mirror 11;

[0100] A controller for controlling the electro-optic modulator 5 and the piezoelectric deflection mirror 16 and a detection system for collecting signal light emitted by the sample to be tested are also provided.

[0101] Furthermore, the 4f system includes a scanning lens 18 and a field lens 19; the numerical aperture NA of the microscope objective lens 21 is 1.4.

[0102] Furthermore, the detection system comprises:

[0103] A narrow-band filter 12 for filtering out stray light and part of the laser light in the fluorescence emitted by the dichroic mirror 11;

[0104] A focusing lens 13 for focusing the filtered fluorescent light beam onto the detector;

[0105] A spatial filter 14 for performing spatial filtering on the signal light, the spatial filter 14 being located at the focal plane of the focusing lens 13;

[0106] A single photon counter 15 is used to detect the light intensity signal of the signal light beam.

[0107] Furthermore, the spatial filter 14 uses a pinhole or a multimode optical fiber. If a pinhole is used, the diameter of the pinhole used is smaller than the diameter of an Airy disk;

[0108] The detector uses avalanche photodiode APD.

[0109] In this specification, each embodiment is described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the embodiments can be referred to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant parts can be referred to the method part.

[0110] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A two-dimensional ten-nanometer positioning method based on polarization switching hollow spot illumination, characterized in that: The following steps are involved: S1. Use a Glan prism to adjust the polarization of the collimated laser beam so that the laser beam is a linearly polarized laser beam; S2. Periodically modulating the polarization state of the linearly polarized laser beam to achieve polarization switching of the linearly polarized laser beam; S3. performing transverse phase modulation on the linearly polarized laser beam after polarization switching obtained in S2 to obtain a transverse hollow spot laser beam; S4. Projecting a transverse hollow spot laser beam onto the sample to scan the sample; S5. using a single photon counter to receive the signal light generated by the sample excited by the transverse hollow spot laser beam at different positions, and obtaining the signal photon number information; S6. Based on the signal photon number information, use the algorithm to reconstruct the two-dimensional spatial information of the sample; The voltage is adjusted by the controller, and the electro-optic modulator is used to periodically make the hollow light spot obtain different polarization states, with a period of 45°-0°-90°, so that the single molecule is excited under different light intensity distributions to obtain the two-dimensional light emission position of the molecule; The collected data were subjected to a maximum likelihood probability estimation function and inversely solved to obtain the localization of the fluorescent molecules.

2. According to claim 1, a two-dimensional ten-nanometer positioning method based on polarization switching hollow spot illumination is characterized in that: In S2, an electro-optic modulator is used to periodically modulate the polarization state of the linearly polarized laser beam to achieve polarization switching of the linearly polarized laser beam.

3. The two-dimensional ten-nanometer-level positioning method based on polarization-switched hollow spot illumination according to claim 1 is characterized in that: In S3, a 0-2π vortex phase plate is used to perform transverse phase modulation on the linearly polarized laser beam after polarization switching obtained in S2 to obtain a transverse hollow spot laser beam; The principle of transverse phase modulation of the 0-2π vortex phase plate is: modulation function f(ρ,φ)=φ, where ρ is the distance between a point on the light beam and the optical axis, and φ is the angle between the polar coordinate vector of the position in the section perpendicular to the optical axis of the light beam and the x-axis.

4. The two-dimensional ten-nanometer-level positioning method based on polarization-switching hollow spot illumination according to claim 1 is characterized in that: The samples in S4 are fluorescent molecules.

5. A two-dimensional ten-nanometer-level positioning device based on polarization-switching hollow spot illumination, characterized in that: A two-dimensional ten-nanometer-level positioning method based on polarization-switching hollow spot illumination as described in any one of claims 1 to 4 is applied, comprising: An excitation light source, an electric sample stage for carrying the sample to be tested, and a microscope objective lens for projecting light onto the electric sample stage. Between the excitation light source and the microscope objective lens, there are: a laser for exciting the light source beam; A collimating lens for collimating and expanding the laser beam output through the single-mode optical fiber; Glan prism for converting laser beam into linearly polarized light; An electro-optic modulator for adjusting the polarization state of a laser beam; A telescopic system for magnifying the light spot; A 0-2π vortex phase plate for transverse phase modulation of the laser beam; 4f system for realizing conjugation between 0-2π vortex phase plate and piezoelectric deflection mirror; A 1 / 2 wave plate used to adjust the polarization characteristics of the outgoing light beam of the 4f system; A mirror for reflecting the laser beam; a dichroic mirror for transmitting fluorescence signals; A piezoelectric deflection mirror for deflecting the optical path of the laser beam after polarization phase modulation to scan the selected area of ​​the sample; 4f system for achieving conjugation between the piezoelectric deflection mirror and the microscope objective entrance pupil; Achromatic quarter-wave plate to compensate for the change in polarization state caused by the dichroic mirror; The device is also provided with a controller for controlling the electro-optic modulator and the piezoelectric deflection mirror and a detection system for collecting signal light emitted by the sample to be tested.

6. A two-dimensional ten-nanometer-level positioning device based on polarization-switching hollow spot illumination according to claim 5, characterized in that: The 4f system includes a scan lens and a field lens; The numerical aperture of the microscope objective is NA=1.

4.

7. The two-dimensional ten-nanometer-level positioning device based on polarization-switching hollow spot illumination according to claim 5, characterized in that: The detection system includes: A narrow-band filter for filtering out stray light and part of the laser light in the fluorescence emitted by the dichroic mirror; a focusing lens for focusing the filtered fluorescence beam onto a detector; A spatial filter for spatially filtering the signal light, the spatial filter being located at a focal plane of the focusing lens; A single photon counter used to detect the intensity signal of the signal beam.

8. The two-dimensional ten-nanometer-level positioning device based on polarization-switching hollow spot illumination according to claim 7, characterized in that: The spatial filter uses a pinhole or a multimode optical fiber. If a pinhole is used, the diameter of the pinhole used is smaller than the diameter of an Airy disk. The detector uses avalanche photodiode APD.

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