Reflective scanning super-resolution optical microscopy system and method based on annular illumination

Abstract

Disclosed in the present invention are a reflective scanning super-resolution optical microscopy system and method based on annular illumination. The system comprises a sample displacement module, an optical microscopy imaging module, an annular beam generation module, a beam scanning module, a super-resolution focusing illumination and collection module, a super-resolution optical imaging module and a computer. The advantages of annular beam illumination, high focusing efficiency of conventional lenses and fast beam scanning speed are utilized to achieve high-efficiency super-resolution focusing illumination, so as to further achieve fast super-resolution optical microscopic imaging by using conventional optical lenses; two-dimensional beam scanning is used to achieve non-labeled far-field super-resolution two-dimensional microscopic rapid imaging; and non-labeled far-field super-resolution two-dimensional microscopic imaging is performed on samples at different axial positions to acquire super-resolution two-dimensional microscopic imaging of different cross sections of the samples, so as to achieve three-dimensional tomography. The present invention can be applied to non-labeled super-resolution microscopic rapid imaging of biological samples, and can also be applied to industrial-related super-resolution microscopic detection and other fields.

Description

Annular illumination based reflective scanning super-resolution optical microscopy system and method TECHNICAL FIELD

[0001] The present application belongs to the field of visible light microscopy, and particularly relates to an annular illumination based reflective scanning super-resolution optical microscopy system and method. BACKGROUND

[0002] Existing microscopy techniques can be roughly divided into two categories: near-field microscopy and far-field microscopy. Near-field microscopy includes microsphere-assisted imaging and near-field scanning microscopy. Near-field microscopy methods can achieve super-resolution microscopic imaging, but their working distance is less than the working wavelength, which greatly limits their practical application. Far-field super-resolution technology has a large working distance and can overcome the shortcomings of near-field microscopy. Existing far-field super-resolution microscopy techniques include stimulated emission depletion microscopy, local activation microscopy, and stochastic optical reconstruction microscopy. However, these techniques all require fluorescent molecule labeling of the sample under test. Non-labeled far-field super-resolution microscopy technology has a large working distance, does not require sample labeling, and is easy to use, so it has become an important development trend in super-resolution optical microscopy technology. In recent years, there have been a few experimental reports on non-labeled far-field super-resolution microscopy based on super-diffraction devices. These systems all rely on new super-resolution devices as illumination devices; however, the focusing efficiency of super-resolution devices is usually low, and most of these systems rely on moving sample scanning, which is slow in imaging speed. SUMMARY

[0003] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide an annular illumination based reflective scanning super-resolution optical microscopy system and method, so as to achieve better resolution while improving reliability and imaging speed.

[0004] In a first aspect, the annular illumination based reflective scanning super-resolution optical microscopy system provided by the present application comprises:

[0005] A sample displacement module for moving the sample;

[0006] An optical microscopic imaging module for wide-field imaging of the sample to determine the super-resolution scanning imaging area;

[0007] The annular light beam generating module is used for generating an annular light beam, and sequentially comprises a light source module, a first linear polarizer, a first conical lens, a second conical lens, a first positive lens and a second positive lens along a light propagation direction, wherein the second conical lens is coaxially and spaced apart from the first conical lens, a front focal plane of the first positive lens coincides with an exit end surface of the second conical lens, a back focal plane of the first positive lens coincides with a front focal plane of the second positive lens, and the first positive lens and the second positive lens constitute a 4f system; the light source module generates a collimated laser beam, which forms a linearly polarized annular light beam after passing through the first linear polarizer, the first conical lens and the second conical lens.

[0008] The light beam scanning module comprises a light beam scanning galvanometer, a light beam scanning lens, a first sleeve mirror and a reflecting mirror, wherein a central position of the light beam scanning galvanometer coincides with the back focal plane of the second positive lens; a front focal plane of the light beam scanning lens is located at the central position of the light beam scanning galvanometer; a back focal plane of the light beam scanning lens coincides with a front focal plane of the first sleeve mirror; a back focal plane of the first sleeve mirror coincides with an exit pupil surface of an objective lens in the super-resolution focusing illumination and collection module after being reflected by the reflecting mirror; the light beam scanning galvanometer comprises a controller, and the controller is used for being connected with a computer; the computer controls the light beam scanning galvanometer to perform two-dimensional vibration scanning through the controller of the light beam scanning galvanometer.

[0009] The super-resolution focusing illumination and collection module is used for converting the annular light beam into a super-resolution annular focused light beam, forming a super-resolution focal spot to illuminate a sample, collecting super-resolution signal light generated by reflection and scattering of the sample, and collimating and converting; the super-resolution focusing illumination and collection module comprises an objective lens, and the sample is located on a front focal plane of the objective lens.

[0010] The super-resolution optical imaging module is used for acquiring the collimated and converted super-resolution signal light and converting the super-resolution signal light into an electric signal; the super-resolution optical imaging module further comprises a spatial filter, a second linear polarizer, a second sleeve mirror, a fiber head, a photomultiplier tube and a high-speed acquisition card, a front end surface of the fiber head coincides with a back focal plane of the second sleeve mirror, the fiber head is connected with the photomultiplier tube, the photomultiplier tube is connected with the high-speed acquisition card, and the high-speed acquisition card is used for being connected with the computer.

[0011] The computer is used for controlling the sample displacement module to move the sample, synchronously controlling the light beam scanning galvanometer to scan and receiving an electric signal output by the super-resolution optical imaging module, and processing to obtain a super-resolution microscopic image of the sample in a super-resolution scanning imaging region.

[0012] Optionally, when the annular light beam generated by the annular light beam generating module is reflected by the beam scanning galvanometer, passes through the scanning lens and the first sleeve mirror, and is reflected by the mirror to the exit pupil plane of the objective lens; when the beam scanning galvanometer vibrates, the center of the annular light beam always coincides with the center of the exit pupil plane of the objective lens, and the incident angle of the annular light beam changes with the vibration of the beam scanning galvanometer; when the beam scanning galvanometer performs two-dimensional scanning, the annular light beam will form a focused spot on the front focal plane of the objective lens, and the focused spot will realize continuous two-dimensional scanning on the front focal plane of the objective lens with the vibration of the beam scanning galvanometer.

[0013] Preferably, the super-resolution focusing illumination and collection module further comprises a quarter-wave plate; the super-resolution optical imaging module further comprises a polarization beam splitter; the optical microscopic imaging module comprises a second beam splitter; the annular linearly polarized light beam emitted from the beam scanning module forms a circularly polarized annular light beam on the exit pupil plane of the objective lens after passing through the quarter-wave plate, the circularly polarized annular light beam has the same diameter as the exit pupil of the objective lens and is concentric with the exit pupil of the objective lens, the circularly polarized annular light beam is converted into a super-resolution annular focused light beam after passing through the objective lens, and a super-resolution focused spot is formed on the front focal plane of the objective lens, the full width at half maximum of the super-resolution focused spot is less than 0.5λ / NA, the first zero point radius of the super-resolution focused spot (i.e. the distance from the position of the peak value of the focal spot intensity to the first zero point) is less than 0.61λ / NA, the super-resolution focused spot illuminates the sample, the super-resolution signal light generated by reflection and scattering of the sample is collected and collimated by the objective lens, and then converted into a linearly polarized collimated light beam orthogonal to the incident linear polarization by the quarter-wave plate, the collimated light beam passes through the second beam splitter in the opposite direction of the original light path, enters the annular light beam generating module through the beam scanning module, and is reflected by the polarization beam splitter placed in the light path into the super-resolution optical imaging module;

[0014] When the beam scanning galvanometer in the beam scanning module performs two-dimensional scanning, the super-resolution focused spot formed by the annular light beam on the front focal plane of the objective lens will realize continuous two-dimensional scanning on the front focal plane of the objective lens with the vibration of the beam scanning galvanometer; the horizontal coordinate X(t) and the vertical coordinate Y(t) of the position of the super-resolution focused spot on the sample are functions of time t, and the reflected light E r (t) and the scattered light E s (t) collected by the objective lens are also functions of time, and the reflected light E r (t) and the scattered light E s (t) reflect the information of the sample structure and refractive index at the corresponding position (horizontal coordinate X(t) and vertical coordinate Y(t)). Wherein, λ represents the wavelength of the collimated laser beam, and NA represents the numerical aperture of the objective lens.

[0015] Preferably, the super-resolution focused spot is a ring-shaped focused light field, the inner and outer surfaces of the ring-shaped focused light field are coaxial two conical surfaces, and the inner numerical aperture NAin , outer numerical aperture NA out satisfies: 0.947NA≤NA in <NA, NA out =NA.

[0016] Preferably, to achieve three-dimensional tomographic scanning of the sample, the sample displacement module has two structures.

[0017] First, the sample displacement module is a two-dimensional displacement table, the two-dimensional displacement table is connected with the computer, the sample is horizontally fixed on the two-dimensional displacement table, the computer controls the two-dimensional displacement table to drive the sample to move in the XY plane, thereby realizing the initial positioning of the sample; the super-resolution focusing illumination and collection module further comprises an axial nanometer positioner, the axial nanometer positioner is connected with the computer, the objective lens is installed on the axial nanometer positioner, and the computer controls the axial nanometer positioner to drive the objective lens to move in the Z direction. The light beam scanning module realizes two-dimensional scanning of the sample, and the axial nanometer positioner realizes three-dimensional tomographic scanning.

[0018] Second, the sample displacement module is a three-dimensional displacement table, the three-dimensional displacement table is connected with the computer, the sample is horizontally fixed on the three-dimensional displacement table, the computer controls the three-dimensional displacement table to drive the sample to move in the X, Y and Z directions, wherein the X and Y directions realize the initial positioning of the sample; the light beam scanning module realizes two-dimensional scanning of the sample, and the Z direction displacement of the three-dimensional displacement table realizes three-dimensional tomographic scanning, thereby avoiding the influence of the slight movement of the objective lens on imaging, and the imaging effect is better.

[0019] Preferably, the super-resolution optical imaging module further comprises a five-dimensional adjustment frame, the optical fiber head is installed on the five-dimensional adjustment frame, and the five-dimensional adjustment frame is adjusted to maximize the super-resolution signal light entering the optical fiber head;

[0020] The inner diameter of the optical fiber at the front end surface of the optical fiber head is smaller than the first zero radius of the focal spot of the super-resolution signal light on the back focal plane of the second sleeve mirror, thereby better ensuring that the super-resolution imaging resolution reaches the optimum.

[0021] Preferably, the light source module comprises a laser and a fiber collimator, and the laser is connected with the fiber collimator through a first fiber jumper; the coherent light emitted by the laser is transmitted to the fiber collimator through the first fiber jumper, and the fiber collimator collimates and outputs the collimated laser beam after collimating the coherent light.

[0022] Preferably, the optical microscopic imaging module comprises a non-coherent light source, a collimating lens, a first beam splitter, a second beam splitter, a third nosepiece and a digital camera, the digital camera is connected with a computer, the non-coherent light source is located at the front focal point of the collimating lens; the light emitted by the non-coherent light source is collimated by the collimating lens, reflected by the first beam splitter, and then passes through the second beam splitter, a quarter-wave plate and an objective lens in sequence, and converges on the front focal plane of the objective lens to illuminate the sample with non-coherent light; the non-coherent light reflected and scattered by the sample is collected by the objective lens, and then passes through the quarter-wave plate, the second beam splitter, the first beam splitter and the third nosepiece in sequence to enter the digital camera, so that the sample is imaged on the digital camera and sent to the computer through a camera signal line, and the wide-field microscopic image of the sample is displayed by the computer. Thus, the sample can be conveniently observed in wide field to determine the required super-resolution scanning imaging area.

[0023] Preferably, the spatial filter has three structures.

[0024] The first spatial filter is a ring-shaped passing type spatial filter, which comprises a glass substrate and a circular aluminum film and a first ring-shaped aluminum film which are concentrically covered on the glass substrate, the diameter of the circular aluminum film is equal to 2R in , the inner diameter of the first ring-shaped aluminum film is equal to 2R out , and the outer diameter is equal to the diameter of the glass substrate, the transmittance of the glass substrate to light waves with a wavelength of λ is greater than 90%, and the transmittance of the circular aluminum film and the first ring-shaped aluminum film to light waves with a wavelength of λ is 0. Only the super-resolution signal light reflected by the sample is allowed to pass, that is, the bright-field imaging light signal is allowed to pass, and bright-field imaging is realized. Wherein, R in represents the inner radius of the annular light beam at the exit pupil of the objective lens, and R out represents the outer radius of the annular light beam at the exit pupil of the objective lens.

[0025] The second spatial filter is a ring-shaped shielding type spatial filter, which comprises a glass substrate and a second ring-shaped aluminum film covered on the glass substrate, the inner diameter of the second ring-shaped aluminum film is equal to 2R in , and the outer diameter is equal to 2R out , the transmittance of the glass substrate to light waves with a wavelength of λ is greater than 90%, and the transmittance of the second ring-shaped aluminum film to light waves with a wavelength of λ is 0. Only the super-resolution signal light scattered by the sample is allowed to pass, that is, the dark-field imaging light signal is allowed to pass, and dark-field imaging is realized.

[0026] The third spatial filter is a phase plate, which is a spatial filter with a thickness of t, an inner radius of R in and an outer radius of R out prepared on the upper surface of a glass substrate, R in and R outThe transmittance T of the light wave with a wavelength of λ in the annular ring is 10%-20%, and the phase change relative to air at the same height is π / 2.

[0027] In a second aspect, the annular illumination-based reflective scanning super-resolution optical microscopy method comprises the following steps:

[0028] Step one, using the optical microscopy imaging module to perform wide-field imaging on the sample to determine the super-resolution scanning imaging area;

[0029] Step two, after determining the super-resolution scanning imaging area, the annular light beam generating module generates an annular light beam, the super-resolution focusing illumination and collection module converts the annular light beam into a super-resolution annular focusing light beam to form a super-resolution focal spot to illuminate the sample, collects the super-resolution signal light generated by the reflection and scattering of the sample, and collimates and converts;

[0030] Step three, the computer controls the sample displacement module to move the sample to the position to be scanned, and turns on the light beam scanning galvanometer to scan, while the super-resolution optical imaging module acquires the collimated and converted super-resolution signal light and converts it into an electrical signal input into the computer;

[0031] Step four, the computer processes the input electrical signal and combines the super-resolution illumination focal spot on the coordinates X(t) and Y(t) of the sample movement and the signal light intensity I(t) obtained by the photomultiplier tube at the corresponding time t to form a super-resolution microscopy image I(X(t), Y(t)) of the sample in the super-resolution scanning imaging area.

[0032] The present application has the following advantages:

[0033] (1) Efficient super-resolution focusing illumination: the annular light beam generating module of the present application can efficiently generate an annular light beam. Subsequently, the annular light beam is converted into a super-resolution annular focusing light beam by the super-resolution focusing illumination and collection module to form a high-precision super-resolution focal spot to illuminate the sample. This process fully utilizes the high focusing efficiency of traditional lenses to achieve efficient super-resolution focusing illumination, significantly improving the signal-to-noise ratio of the image.

[0034] (2) Fast two-dimensional scanning: the light beam scanning module uses light beam scanning galvanometer technology to achieve fast two-dimensional scanning of the light beam. This scanning method not only has high speed but also high precision, and can perform super-resolution imaging on a large area in a short time. This feature greatly improves the imaging efficiency, enabling users to quickly obtain a large amount of high-quality image data.

[0035] (3) Non-labeled far-field super-resolution microscopic imaging: Compared with traditional microscopic techniques that require fluorescent molecular labeling, a major highlight of the present application is its ability to achieve fast imaging of non-labeled sample far-field super-resolution two-dimensional microscopy. This function not only expands the scope of application of the technology, but also simplifies the experimental steps and reduces the requirements for sample processing. At the same time, due to the large working distance, it makes the application in the fields of biological and industrial detection more convenient and efficient.

[0036] (4) Three-dimensional tomographic imaging capability: By performing non-labeled far-field super-resolution two-dimensional microscopic imaging on samples at different axial positions, the present application can obtain super-resolution two-dimensional microscopic images of different cross-sections of the sample, and then realize three-dimensional tomographic imaging through data processing. This function is of great significance for analyzing the internal structure of the sample and revealing the spatial distribution of complex samples, and is particularly suitable for biomedical research, materials science and other fields.

[0037] (5) High reliability and imaging quality: The present application precisely controls the light path cooperation between each module and the signal processing process to ensure the reliability and stability of the imaging. From the generation of the annular light beam, the scanning of the light beam, the super-resolution focused illumination to the collection and processing of the signal, each link is carefully designed and optimized to ensure a high level of final imaging quality. In addition, the optical elements and signal acquisition equipment used have high performance indicators, further improving the overall performance and imaging quality of the system.

[0038] In summary, the annular illumination-based reflective scanning super-resolution optical microscopic system and method of the present application has significant advantages in efficient super-resolution focused illumination, fast two-dimensional scanning, non-labeled far-field super-resolution microscopic imaging, three-dimensional tomographic imaging capability, and high reliability and imaging quality, providing strong technical support for the fields of biomedical research, industrial detection, etc. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 is a structural schematic diagram of the annular illumination-based reflective scanning super-resolution optical microscopic system in Example 1.

[0040] Figure 2 is a schematic diagram of the first and second conical lenses and the 4f system controlling the size of the annular light beam in Example 1.

[0041] Figure 3 is a schematic diagram of the formation of a super-resolution focused light spot in Example 1.

[0042] Figure 4 is a parameter relationship diagram of the super-resolution focused light spot in Example 1.

[0043] Figure 5 is a schematic diagram of the scanning of the super-resolution focused light spot on the focal plane of the objective lens in Example 1.

[0044] Figure 6 is a front view and sectional view schematic diagram of the spatial filter in Example 1.

[0045] Figure 7 is a diagram showing the relationship between the full width at half maximum of the objective lens and the focal spot and the system resolution in the case of annular beam illumination in Example 1.

[0046] Figure 8 is a diagram showing the structure of the annular illumination-based reflective scanning super-resolution optical microscopy system in Example 2.

[0047] Figure 9 is a diagram showing the front view and cross-sectional view of the spatial filter in Example 3.

[0048] Figure 10 is a diagram showing the front view and cross-sectional view of the spatial filter in Example 4.

[0049] Figure 11 is a diagram showing the front view and cross-sectional view of the spatial filter in Example 5. DETAILED DESCRIPTION

[0050] In order to enable a more detailed understanding of the features and technical content of the embodiments of the present application, the implementation of the embodiments of the present application will be described in detail below with reference to the accompanying drawings, which are for reference only and do not limit the embodiments of the present application.

[0051] Example 1

[0052] As shown in Figure 1, the annular illumination-based reflective scanning super-resolution optical microscopy system in the present embodiment includes a sample displacement module, an optical microscopy imaging module, an annular beam generation module, a beam scanning module, a super-resolution focused illumination and collection module, a super-resolution optical imaging module, and a computer. The sample displacement module is used to move the sample 16. The optical microscopy imaging module is used to perform wide-field imaging on the sample 16 to determine the super-resolution scanning imaging region. The annular beam generation module is used to generate an annular beam. The beam scanning module is used to achieve rapid two-dimensional scanning of the beam. The super-resolution focused illumination and collection module is used to convert the annular beam into a super-resolution annular focused beam, form a super-resolution focal spot, and illuminate the sample 16, collect the super-resolution signal light generated by reflection and scattering of the sample 16, and collimate and convert. The super-resolution optical imaging module is used to acquire the collimated and converted super-resolution signal light and convert it into an electrical signal. The computer (not labeled in the figure) is used to control the sample displacement module to move the sample 16. By synchronously controlling the beam scanning galvanometer 9 and receiving the electrical signal output by the super-resolution optical imaging module, the super-resolution microscopy image of the sample 16 in the super-resolution scanning imaging region is obtained after processing.

[0053] The system realizes efficient super-resolution focusing illumination by utilizing the advantages of annular beam illumination, high focusing efficiency of traditional lenses and fast scanning speed of light beams, and then can realize fast super-resolution optical microscopic imaging by using traditional optical lenses; two-dimensional light beam scanning is used to realize non-labeled far-field super-resolution two-dimensional microscopic fast imaging; non-labeled far-field super-resolution two-dimensional microscopic imaging of samples at different axial positions is performed to obtain super-resolution two-dimensional microscopic imaging of different cross sections of the sample, and then three-dimensional tomography is realized. The system can be applied to non-labeled super-resolution microscopic fast imaging of biological samples and can also be applied to the field of industrial related super-resolution microscopic detection.

[0054] The various modules are described in detail as follows:

[0055] As shown in FIGS. 1 and 2, the optical microscopic imaging module includes an incoherent light source 30, a collimating lens 31, a first beam splitter 32, a second beam splitter 33, a third sleeve mirror 34 and a digital camera 35, the digital camera 35 is connected to a computer through a camera signal line 36, and the incoherent light source 30 is located at the front focal point of the collimating lens 31. The light emitted by the incoherent light source 30 is collimated by the collimating lens 31 and reflected by the first beam splitter 32, and then passes through the second beam splitter 33, the quarter-wave plate 13 and the objective lens 15 in turn, and converges on the front focal plane of the objective lens 15 to form incoherent light for illuminating the sample 16. The incoherent light reflected and scattered by the sample 16 is collected by the objective lens 15 and then enters the digital camera 35 after passing through the quarter-wave plate 13, the second beam splitter 33, the first beam splitter 32 and the third sleeve mirror 34 in turn, so that the sample 16 is imaged on the digital camera 35 and sent to the computer. The computer displays the wide-field microscopic image of the sample 16.

[0056] As shown in FIGS. 1 and 2, the annular beam generation module includes a laser 1, a fiber collimator 3, a first linear polarizer 4, a first conical lens 5, a second conical lens 6, a first positive lens 7 and a second positive lens 8 in sequence along the light propagation direction. The laser 1 is connected to the fiber collimator 3 through a first fiber jumper 2; the coherent light emitted by the laser 1 is transmitted to the fiber collimator 3 through the first fiber jumper 2, and the fiber collimator 3 collimates the coherent light and outputs a collimated laser beam. The second conical lens 6 is coaxially and spaced apart from the first conical lens 5, the front focal plane F1 of the first positive lens 7 coincides with the exit end surface of the second conical lens 6, the rear focal plane (i.e. F'1) of the first positive lens 7 coincides with the front focal plane (i.e. F2) of the second positive lens 8, and the first positive lens 7 and the second positive lens 8 constitute a 4f system. The light source module generates a collimated laser beam, which forms a linearly polarized annular beam after passing through the first linear polarizer 4, the first conical lens 5 and the second conical lens 6.

[0057] As shown in FIG. 1 and FIG. 2, the light beam scanning module comprises a light beam scanning galvanometer 9, a scanning lens 10, a first sleeve mirror 11 and a reflecting mirror 12. The center position of the light beam scanning galvanometer 9 coincides with the back focal plane of the second positive lens 8; the front focal plane of the scanning lens 10 is located at the center position of the light beam scanning galvanometer 9; the back focal plane of the scanning lens 10 coincides with the front focal plane of the first sleeve mirror 11; the back focal plane of the first sleeve mirror 11 coincides with the exit pupil plane of the objective lens 15 in the super-resolution focusing illumination and collection module after being reflected by the reflecting mirror 12. The light beam scanning galvanometer 9 comprises a controller for connecting with a computer (not shown in the figure), and the computer controls the light beam scanning galvanometer 9 to perform two-dimensional vibration scanning through the controller of the light beam scanning galvanometer 9.

[0058] As shown in FIG. 1 and FIG. 2, the super-resolution focusing illumination and collection module comprises a quarter-wave plate 13 and an objective lens 15, the exit pupil plane of the objective lens 15 coincides with the back focal plane of the first sleeve mirror 11, and the sample 16 is located on the front focal plane of the objective lens 15. The objective lens 15 uses a conventional microscope objective, which can be a dry lens, a water lens, an oil lens or a solid lens.

[0059] As shown in FIG. 1 and FIG. 2, the sample displacement module is a two-dimensional displacement stage 17, the two-dimensional displacement stage 17 is connected with a computer (not shown in the figure) through a scanning signal line 18, the sample 16 is horizontally fixed on the two-dimensional displacement stage 17, and the computer controls the two-dimensional displacement stage 17 to drive the sample 16 to move in two dimensions in the XY plane. When the sample displacement module is the two-dimensional displacement stage 17, the super-resolution focusing illumination and collection module further comprises an axial nanometer positioner 14, the axial nanometer positioner 14 is connected with the computer through a positioning signal line, the objective lens 15 is installed on the axial nanometer positioner 14, and the computer controls the axial nanometer positioner 14 to drive the objective lens 15 to move in the Z direction.

[0060] As shown in FIG. 1 and FIG. 2, the super-resolution optical imaging module comprises a polarization beam splitter 19, a spatial filter 20, a second linear polarizer 21, a second sleeve mirror 22, a fiber head 23, a photomultiplier tube 26 and a high-speed acquisition card 28. The front end surface of the fiber head 23 coincides with the back focal plane of the second sleeve mirror 22, the fiber head 23 is connected with the photomultiplier tube 26 through a second optical fiber jumper 25, the photomultiplier tube 26 is connected with the high-speed acquisition card 28 through an acquisition signal line 27, and the high-speed acquisition card 28 is connected with a computer through a data line 29.

[0061] As shown in FIG. 6, in a possible embodiment, the spatial filter 20 is a ring-passing type spatial filter, which comprises a glass substrate 201 and a circular aluminum film 202 and a first ring-shaped aluminum film 203 which are concentrically covered on the glass substrate 201. The thickness t of the circular aluminum film 202 and the first ring-shaped aluminum film 203 is greater than 50 nm, the diameter of the circular aluminum film 202 is equal to 2R in , and the inner diameter of the first ring-shaped aluminum film 203 is equal to 2Rout The outer diameter is equal to the diameter of the glass substrate 201. The transmittance of the glass substrate 201 to light waves with wavelength λ is greater than 90%. The transmittance of the circular aluminum film 202 and the first annular aluminum film 203 to light waves with wavelength λ is 0.

[0062] In this embodiment of the application, the ring illumination-based reflective scanning super-resolution optical microscopy method employs the aforementioned reflective scanning super-resolution optical microscopy system, and the method includes:

[0063] Step 1: Wide-field imaging of sample 16 is performed using an optical microscopy imaging module to determine the super-resolution scanning imaging area. Specifically:

[0064] Laser 1 is initially turned off, emitting no coherent light. The light emitted from the incoherent light source 30 is collimated by collimating lens 31, reflected by first beam splitter 32, and then sequentially passes through second beam splitter 33 and quarter-wave plate 13 to reach objective lens 15. There, it converges into incoherent light at the front focal plane of objective lens 15 to illuminate sample 16. The incoherent light generated by reflection and scattering from sample 16 is collected by objective lens 15 and sequentially passes through quarter-wave plate 13, second beam splitter 33, first beam splitter 32, and third telescope 34 before entering digital camera 35. This images sample 16 onto digital camera 35 and are transmitted to computer via camera signal line 36. The computer displays a wide-field microscopic image of sample 16, facilitating wide-field observation of sample 16 and determining the required super-resolution scanning imaging region. If sample 16 is not within the super-resolution scanning imaging region, the computer can control a two-dimensional displacement stage 17 to move sample 16 and place it within the super-resolution scanning imaging region.

[0065] Step 2: After determining the super-resolution scanning imaging area, the ring beam generation module generates a ring beam. The super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam, forming a super-resolution focal spot to illuminate sample 16. The module collects the super-resolution signal light reflected and scattered by sample 16, and then collimates and converts it. Specifically:

[0066] The incoherent light source 30 does not emit light, the laser 1 is turned on, the laser 1 emits coherent light with a wavelength of λ, which is transmitted to the optical fiber collimator 3 through the first optical fiber jumper 2, the optical fiber collimator 3 collimates the coherent light and outputs a collimated laser beam, the collimated laser beam forms a ring-shaped light beam (a P-polarized ring-shaped light beam) after passing through the first linear polarizer 4, the first conical lens 5 and the second conical lens 6, the P-polarized ring-shaped light beam passes through the 4f system, then passes through the beam scanning module, and then passes through the quarter-wave plate 13, and finally forms a circularly polarized ring-shaped light beam on the exit pupil plane of the objective lens 15, which is concentric with the exit pupil of the objective lens 15 and has the same diameter as the exit pupil of the objective lens 15, the circularly polarized ring-shaped light beam passes through the objective lens 15 and is converted into a super-resolution ring-shaped focused light beam, which forms a super-resolution focused light spot on the front focal plane of the objective lens 15, the full width at half maximum of the super-resolution focused light spot is less than 0.5λ / NA, the first zero point radius of the super-resolution focused light spot (i.e. the distance from the peak position of the focal spot intensity to the first zero point position) is less than 0.61λ / NA, and the super-resolution focused light spot illuminates the sample 16, the super-resolution signal light reflected and scattered by the sample 16 is collected and collimated by the objective lens 15, converted into an S-polarized collimated light beam orthogonal to the incident linearly polarized ring-shaped light beam by the quarter-wave plate 13, and then enters the super-resolution optical imaging module through the polarization beam splitter 19 placed in the light path.

[0067] As shown in FIG. 2, a method for generating a P-polarized ring-shaped light beam with a ring width of W' (i.e. the difference between the outer radius and the inner radius of the ring-shaped light beam) and an inner radius of R' in , and an outer radius of R' out is given. Specifically:

[0068] The optical fiber collimator 3 collimates the coherent light and outputs a collimated laser beam with a radius of R (different numerical aperture optical fiber collimators can be selected to obtain collimated laser beams with different radii), the collimated laser beam with a radius of R becomes a P-polarized collimated light beam with a radius of R after passing through the first linear polarizer 4, the P-polarized collimated light beam passes through the coaxially placed first conical lens 5 and second conical lens 6 with a distance of d, and forms a P-polarized ring-shaped light beam with an inner radius of R'' in and an outer radius of R'' out , the inner radius R'' in and the outer radius R'' out of the P-polarized ring-shaped light beam can be changed by changing the distance d, while keeping the ring width W'' of the P-polarized ring-shaped light beam unchanged (i.e. W''=R'' out -R'' in =R). The P-polarized ring-shaped light beam further passes through the 4f system composed of the first positive lens 7 and the second positive lens 8, and forms a P-polarized ring-shaped light beam with an inner radius of R' in and an outer radius of R'out P-polarized annular beam; wherein R' in =R'' in ×f2 / f1, R' out =R'' out ×f2 / f1, and the ring width W' = R' out -R' in =R'×f2 / f1; fi represents the focal length of the first positive lens, and f2 represents the focal length of the second positive lens. Different sizes of P-polarized annular beams can be obtained by selecting the first positive lens 7 and the second positive lens 8 with different focal lengths. Therefore, the ring width W' of the formed P-polarized annular beam can be controlled by controlling the radius R' of the collimated laser beam, the focal length fi of the first positive lens 7, and the focal length f2 of the second positive lens 8, and the inner radius R' and the outer radius R' of the formed P-polarized annular beam can be controlled by the distance d, the focal length fi of the first positive lens 7, and the focal length f2 of the second positive lens 8. in out The beam is amplified (or reduced, the amplification factor K = f t / f s , K < 1 for reduction, K > 1 for amplification) again after passing through the beam scanning lens 10 and the first sleeve mirror 11, so that the inner and outer radii of the finally formed annular beam are R in and R out , respectively, and R in = KR'' in ×f2 / f1, R out = KR'' out ×f2 / f1, to meet the needs of different objectives to produce super-resolution focusing spots, because different manufacturers of objectives, different types of objectives have different requirements for the inner radius R in and the outer radius R out of the annular beam.

[0069] As shown in FIG. 3, the illumination work of forming a super-resolution focusing spot is given. The left-handed circularly polarized annular beam with an inner radius R in and an outer radius R out illuminates the objective 15, and is converted into a super-resolution annular focusing beam after passing through the objective 15, forming a super-resolution focusing spot on the front focal plane of the objective 15. The super-resolution focusing spot is an annular focusing light field, and the full width at half maximum of the annular focusing light field is less than 0.5λ / NA, and the first zero point radius of the super-resolution focusing spot of the annular focusing light field is less than 0.61λ / NA. The inner and outer surfaces of the annular focusing light field are two coaxial conical surfaces, the inner cone angle is θ in , and the outer cone angle is θ out , NA in represents the inner numerical aperture corresponding to the annular focusing light field, and NA out represents the outer numerical aperture corresponding to the annular focusing light field, and NA​in = n x sin(θ in ), NA out = n x sin(θ out ), n represents the refractive index of medium between the objective 15 and the sample 16, and sin() represents the sine function. When 0 < NA in < NA, NA out = NA, the actual resolution of the super-resolution microscopy is greater than 0.25λ / NA and less than 0.5λ / NA. To achieve super-resolution microscopy imaging with practical value and better resolution, NA out needs to be equal to NA (i.e. NA out = NA, corresponding to the outer diameter R out of the left-handed circularly polarized annular beam being equal to the exit pupil diameter of the objective 15), and 0.947NA ≤ NA in < NA. Since R out is equal to the exit pupil diameter of the objective 15, after the objective 15 is determined, NA out is determined (corresponding to R out being determined), θ out is also determined, and at this time the resolution of the super-resolution microscopy is determined by NA in (corresponding to R in ), the greater R in is, the smaller the ring width W is, and the higher the resolution of the super-resolution microscopy is.

[0070] To achieve the best resolution, the value of NA in is closer to NA, the ring width W is smaller, the transverse size of the super-resolution focal spot (i.e. the super-resolution focused light spot) of the annular focused light field is smaller, and the resolution 0.5λ / (NA+NA in ) of the super-resolution microscopy is closer to the limit resolution 0.25λ / NA of the reflective confocal system. In practice, the radius R of the collimated laser beam is controlled, and R' in , R' out are adjusted by controlling the distance d between the first cone lens 5 and the second cone lens 6; however, since the P-polarized annular beam with a small ring width will obviously diffract during propagation, resulting in a larger ring width, it is impossible to ensure that the P-polarized annular beam propagating to the exit pupil surface of the objective 15 can maintain an extremely small ring width. Therefore, the radius R of the collimated laser beam cannot be too small, but to make the P-polarized annular beam propagating to the exit pupil surface of the objective 15 maintain an extremely small ring width to achieve the best resolution, it is necessary to increase the 4f system composed of the first positive lens 7 and the second positive lens 8 to control the size of the P-polarized annular beam, and combine the 4f system composed of the scanning lens 10 and the first sleeve mirror 11 to make the annular focused light field satisfy: NA out = NA, and 0.947NA ≤ NA inThe 4f system composed of the first positive lens 7 and the second positive lens 8 plays a role of controlling the size of the P-polarized annular light beam on one hand (i.e. the width of the annular light beam at the exit end of the second conical lens 6 can be well compressed to close to the diffraction limit width of the light beam, and finally projected to the exit pupil plane of the objective lens), and plays a role of transmission relay of the P-polarized annular light beam on the other hand, so as to ensure that the size of the P-polarized annular light beam at the front focal plane of the 4f system is accurately projected to the rear focal plane of the 4f system, thereby avoiding the annular size distortion and the light field intensity distribution fluctuation of the P-polarized annular light beam due to the diffraction effect in the direct transmission, ensuring the high quality illumination of the P-polarized annular light beam to the objective lens, and further ensuring that the generated super-resolution focal spot reaches the minimum, so as to ensure that the system reaches the best resolution.

[0071] To ensure that the inner and outer cone angles of the annular focusing light field formed after the objective lens 15 are θ in and θ out respectively, the following can be implemented: first, according to the required NA out and NA in , the corresponding R out and R in are determined (the determination method belongs to the prior art), and a suitable objective lens 15 is selected according to NA out =NA ; then, the radius R of the collimated laser beam is selected, and f1 and f2 are determined according to R out -R in =R×f2 / f1×f t / f s , and the appropriate first positive lens 7 and second positive lens 8 are determined; finally, the interval d between the first conical lens 5 and the second conical lens 6 is determined according to R' in =R in ×f1 / f2×f s / f t , R' out =R out ×f1 / f2×f s / f t .

[0072] As shown in FIG. 4, when the numerical aperture NA of the objective lens 15 is 0.95, and NA out =NA , the functional relationship between the parameters FWHM (i.e. full width at half maximum), r0 (i.e. first zero radius) and SR (i.e. side lobe peak ratio, which is the ratio of the maximum side lobe intensity to the center intensity) of the super-resolution focusing spot and NA in is given. It can be seen that when NA inWhen ≥0.9, the FWHM of the generated super-resolution focused spot is ≤0.388λ (less than 0.5λ / NA), r0≤0.415λ (less than 0.61λ / NA), and SR≤0.162, and both the FWHM and the first zero point radius of the super-resolution focused spot are less than the FWHM 0.5λ / NA and the first zero point radius 0.61λ / NA of the corresponding conventional lens focused light field. Therefore, by controlling the inner and outer radii of the left-handed circularly polarized annular light beam to satisfy: NA out =0.95 and 0.9≤NA in <0.95, the super-resolution illumination light field with FWHM≤0.388λ, r0≤0.415λ, and SR≤0.162 can be achieved.

[0073] As shown in FIG. 5, a schematic diagram of the super-resolution focused spot scanning on the focal plane of the objective lens 15 is shown. The light beam scanning module realizes two-dimensional scanning of the annular light beam. At different times, the annular light beam is incident on the objective lens 15 at different angles. When the annular light beam reaches the exit pupil plane of the objective lens 15, the annular light beam is always in the form of a circular ring with the center of the exit pupil plane as the center. As shown in FIG. a, the illumination focusing conditions of two different incident angles are given. At two different times t1 and t2, the incident angles of the annular light beam are θ1 and θ2, respectively, and the super-resolution focused spots are formed at different positions on the front focal plane of the objective lens. As shown in FIG. b, at different times, the light intensity distribution of the illumination annular light beam on the exit pupil plane of the objective lens is always a circular ring with the center of the exit pupil as the center. The inner and outer radii of the circular ring are R in and R out , respectively.

[0074] As shown in FIG. 7, a diagram showing the relationship between the half-height full width of the focal spot and the system resolution under the illumination of the annular light beam and the objective lens (the numerical aperture NA of this objective lens is greater than 1) is shown. The vertical coordinate in the diagram is the half-height full width of the point spread function of the objective lens, and the unit is wavelength λ. The horizontal coordinate is the transverse half-height full width of the super-resolution focal spot, and the unit is wavelength λ. The contour line is the system resolution (i.e., the minimum line width of the amplitude grating that can be resolved by the system, and the imaging fringe contrast is better than 11%), and the unit is wavelength λ. When the half-height full width of the point spread function of the objective lens (which can be a single-point focusing super-diffraction lens) is 0.6λ, and the transverse (to the part of the sample illumination) half-height full width of the super-resolution focal spot is 0.38λ, the system resolution can reach 0.25λ-0.26λ. When the half-height full width of the point spread function of the objective lens and the transverse half-height full width of the super-resolution focal spot are both 0.35λ, the system resolution is better than 0.2λ.

[0075] Step three, the computer controls the light beam scanning module to perform two-dimensional scanning on the light beam, thereby realizing two-dimensional scanning of the super-resolution focused spot on the front focal plane of the objective lens 15, and simultaneously, the super-resolution optical imaging module synchronously acquires the collimated and converted super-resolution signal light, and converts it into an electrical signal input to the computer.

[0076] Step 4: The computer processes the input electrical signal and combines it with the coordinates of the super-resolution focusing spot movement to form a two-dimensional super-resolution microscopic image of the sample within the super-resolution scanning imaging area. By using a sample displacement module to move the sample along the Z-axis, a three-dimensional super-resolution microscopic image of the sample can be obtained.

[0077] In this embodiment, the specific processing steps three and four are as follows: The S-polarized collimated beam (i.e., the collimated and converted super-resolution signal light reflected by the polarization beam splitter 19) is filtered by the spatial filter 20 and then further passes through the second linear polarizer 21. Only the super-resolution signal light generated by the reflection of the sample 16 is focused on the back focal plane of the second telescope 22 and coupled into the fiber optic head 23. Then, it enters the photomultiplier tube 26 through the second fiber optic jumper 25, where it is converted into an analog electrical signal. This analog electrical signal is acquired by the high-speed acquisition card 28 through the acquisition signal line 27 and converted into a digital electrical signal, which is then input to the computer through the data line 29. The computer controls the beam scanning galvanometer 9 in the beam scanning module to perform two-dimensional scanning. The super-resolution focused spot formed by the ring beam on the front focal plane of the objective lens 15 will continuously scan two dimensions on the front focal plane of the objective lens 15 as the beam scanning galvanometer 9 vibrates. The x-coordinate X(t) and y-coordinate Y(t) of the position of the super-resolution focused spot illuminating the sample 16 are functions of time t, and the reflected light E collected by objective lens 15 is... r (t) and scattered light E s (t) is also a function of time, and the reflected light E r (t) and scattered light E s (t) reflects information such as the sample structure and refractive index at the corresponding positions (horizontal coordinate X(t) and vertical coordinate Y(t)). By performing a two-dimensional scan in the XY plane (with a scan step size smaller than the minimum resolution size of the system), the signal intensity of the corresponding coordinates is directly output as a coordinate function I(X(t),Y(t)) (where X and Y are the coordinates of the super-resolution focusing spot on the focal plane of objective lens 15), thus obtaining a two-dimensional super-resolution microscopic image of sample 16. The resolution of this image in the XY plane is close to the limit resolution of the reflective confocal system, 0.25λ / NA. The computer controls the axial nanopositioner 14, which moves the objective lens 15 in the Z direction, changing the Z-position Z of the sample where the front focal plane of objective lens 15 is located. f To obtain different Z f Two-dimensional super-resolution microscopic images in the XY plane at a given location can be used to obtain three-dimensional tomographic microscopic images, with a resolution close to the limit resolution of 0.25λ / NA of a reflective confocal system.

[0078] Example 2

[0079] As shown in Fig. 8, the most part of the structure of the annular illumination based reflective scanning super-resolution optical microscopy system in the embodiment is the same as that in Embodiment 1, and the difference is that the sample displacement module is a three-dimensional displacement stage 37, the three-dimensional displacement stage 37 is connected to the computer through a scanning signal line 18, the sample 16 is horizontally fixed on the three-dimensional displacement stage 37, and the computer controls the three-dimensional displacement stage 37 to drive the sample 16 to move in the Z-axis direction.

[0080] As shown in Fig. 8, the annular illumination based reflective scanning super-resolution optical microscopy method in the embodiment is basically the same as that in Embodiment 1, and the difference is only that in the specific processing process of steps three and four, the computer controls the three-dimensional displacement stage 37 to drive the sample 16 to move slightly in the Z direction, so as to change the Z direction position Z of the front focal plane of the objective lens 15 in the sample 16 f , and the high-speed acquisition card 28 acquires the signal once every time the sample moves a little, so as to obtain the two-dimensional super-resolution microscopic images in the XY plane at different Z f positions. Then, the three-dimensional tomographic microscopic images can be obtained, and the resolution is close to the limit resolution 0.25λ / NA of the reflective confocal system.

[0081] Embodiment three

[0082] As shown in Fig. 9, in the embodiment, the most part of the structure of the annular illumination based reflective scanning super-resolution optical microscopy system is the same as that in Embodiment 1, and the difference is that the spatial filter 20 is a ring-shaped blocking type spatial filter, which includes a glass substrate 201 and a second ring-shaped aluminum film 204 covering the glass substrate 201, the thickness t of the second ring-shaped aluminum film 204 is greater than 50 nm, the inner diameter of the second ring-shaped aluminum film 204 is equal to 2R in , and the outer diameter is equal to 2R out . The transmittance of the glass substrate 201 to light waves with a wavelength of λ is greater than 90%, and the transmittance of the second ring-shaped aluminum film 204 to light waves with a wavelength of λ is 0. Only the super-resolution signal light generated by the sample 16 is allowed to pass, that is, the dark field imaging light signal is allowed to pass, so as to realize dark field imaging. The annular illumination based reflective scanning super-resolution optical microscopy method in the embodiment is basically the same as that in Embodiment 1, and the difference is only that in step three, after the P-polarized collimated light beam is filtered by the spatial filter 20, only the super-resolution signal light generated by the sample 16 is converged on the back focal plane of the second sleeve mirror 22 and coupled into the optical fiber head 23.

[0083] Embodiment four

[0084] As shown in Fig. 10, in the embodiment, the most part of the structure of the annular illumination based reflective scanning super-resolution optical microscopy system is the same as that in Embodiment 1, and the difference is that the spatial filter 20 is a phase plate, which is a glass substrate on which a thickness t=t1+t2, inner and outer radii R in and Rout one spatial filter. In R in and R out , the transmittance T of light wave with wavelength λ in the annular region is 10%-20%, and the phase change relative to air of the same height is π / 2. The transparent medium with thickness t1 (transmittance greater than 90% at working wavelength λ) has thickness t1 = λ / 4(n-1), where n is the refractive index of the transparent medium; t2 is the thickness of the metal film, which should satisfy the transmittance of 10%-20% for light wave with wavelength λ.

[0085] Embodiment five

[0086] As shown in Fig. 11, the most structure of the reflection scanning super-resolution optical microscope system based on annular illumination in the embodiment is the same as that of embodiment one, and the difference is that the spatial filter 20 is a phase plate, which is a spatial filter with inner radius R in and outer radius R out on the surface of a glass substrate. In the annular region between R in and R out , there is a metal film with thickness t2, and the transmittance T of light wave with wavelength λ in the annular region is 10%-20%; in other regions outside the annular region, there is a transparent medium with thickness t1 (transmittance greater than 90% at working wavelength λ), and the thickness t1 = λ / 4(n-1), where n is the refractive index of the transparent medium, and the phase change relative to air of the same height is π / 2.

[0087] The above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited to this. Any change or replacement within the technical range disclosed by the present application can be easily thought by those skilled in the art, which should be covered in the protection scope of the present application.

Claims

1. A ring illumination based reflective scanning super-resolution optical microscopy system, characterized in that, The application relates to a super-resolution microscope, which comprises the following modules: a sample displacement module for moving a sample (16); an optical microscopic imaging module for wide-field imaging of the sample (16) and determining a super-resolution scanning imaging area; a ring-shaped light beam generating module, which comprises, in sequence along the light propagation direction, a light source module, a first linear polarizer (4), a first conical lens (5), a second conical lens (6), a first positive lens (7), and a second positive lens (8), wherein the second conical lens (6) is coaxially and separately arranged with the first conical lens (5), the front focal plane of the first positive lens (7) is coincident with the exit end surface of the second conical lens (6), the rear focal plane of the first positive lens (7) is coincident with the front focal plane of the second positive lens (8), and the first positive lens (7) and the second positive lens (8) form a 4f system; the light source module generates a collimated laser beam, which forms a linearly polarized ring-shaped light beam after passing through the first linear polarizer (4), the first conical lens (5), and the second conical lens (6); a light beam scanning module, which comprises a light beam scanning galvanometer (9), a light beam scanning lens (10), a first sleeve mirror (11), and a reflecting mirror (12), wherein the center position of the light beam scanning galvanometer (9) is coincident with the rear focal plane of the second positive lens (8); the front focal plane of the light beam scanning lens (10) is located at the center position of the light beam scanning galvanometer (9); the rear focal plane of the light beam scanning lens (10) is coincident with the front focal plane of the first sleeve mirror (11); the rear focal plane of the first sleeve mirror (11) is coincident with the exit pupil surface of an objective lens (15) in a super-resolution focusing illumination and collection module after being reflected by the reflecting mirror (12); the light beam scanning galvanometer (9) comprises a controller, which is connected with a computer; the computer controls the light beam scanning galvanometer (9) to perform two-dimensional vibration scanning through the controller of the light beam scanning galvanometer (9); a super-resolution focusing illumination and collection module, which is used for converting the ring-shaped light beam into a super-resolution ring-shaped focused light beam, forming a super-resolution focal spot to illuminate the sample (16), collecting super-resolution signal light generated by reflection and scattering of the sample (16), and collimating and converting; the super-resolution focusing illumination and collection module comprises the objective lens (15), and the sample (16) is located on the front focal plane of the objective lens (15); a super-resolution optical imaging module, which is used for acquiring the collimated and converted super-resolution signal light and converting the super-resolution signal light into an electric signal; the super-resolution optical imaging module further comprises a spatial filter (20), a second linear polarizer (21), a second sleeve mirror (22), a fiber head (23), a photomultiplier tube (26), and a high-speed acquisition card (28); the front end surface of the fiber head (23) is coincident with the rear focal plane of the second sleeve mirror (22); the fiber head (23) is connected with the photomultiplier tube (26); the photomultiplier tube (26) is connected with the high-speed acquisition card (28); and the high-speed acquisition card (28) is connected with a computer; the computer is used for controlling the sample displacement module to move the sample (16), controlling the light beam scanning galvanometer (9) to scan, receiving the electric signal output by the super-resolution optical imaging module, and processing to obtain a super-resolution microscopic image of the sample in the super-resolution scanning imaging area.

2. The annular illumination based reflective scanning super-resolution optical microscopy system according to claim 1, wherein: When the annular light beam generated by the annular light beam generating module is reflected by the beam scanning galvanometer (9), passes through the scanning lens (10) and the first sleeve mirror (11), and is reflected by the mirror (12) to the exit pupil plane of the objective lens (15), the center of the annular light beam always coincides with the center of the exit pupil plane of the objective lens (15) when the beam scanning galvanometer (9) vibrates, and the incident angle of the annular light beam changes with the vibration of the beam scanning galvanometer (9); when the beam scanning galvanometer (9) performs two-dimensional scanning, the annular light beam will form a focused spot on the front focal plane of the objective lens (15), and the focused spot will realize continuous two-dimensional scanning on the front focal plane of the objective lens (15) with the vibration of the beam scanning galvanometer (9).

3. The annular illumination based reflective scanning super-resolution optical microscopy system according to claim 2, wherein: The super-resolution focused illumination and collection module further comprises a quarter-wave plate (13); the super-resolution optical imaging module further comprises a polarization beam splitter (19); the optical microscopy imaging module comprises a second beam splitter (33); the linearly polarized annular light beam emitted from the beam scanning module forms a circularly polarized annular light beam on the exit pupil plane of the objective lens (15) after passing through the quarter-wave plate (13), which is concentric with the exit pupil of the objective lens (15) and has an outer diameter equal to the diameter of the exit pupil of the objective lens (15); the circularly polarized annular light beam is converted into a super-resolution annular focused light beam after passing through the objective lens (15) and forms a super-resolution focused spot on the front focal plane of the objective lens (15); the super-resolution focused spot illuminates the sample (16), and the super-resolution signal light reflected and scattered by the sample (16) is collected and collimated by the objective lens (15), converted into a linearly polarized collimated light beam orthogonal to the incident linear polarization by the quarter-wave plate (13), transmitted through the second beam splitter (33) in the opposite direction of the original light path, enters the annular light beam generating module after passing through the beam scanning module, and is reflected by the polarization beam splitter (19) placed in the light path into the super-resolution optical imaging module; When the light beam scanning mirror (9) in the light beam scanning module makes two-dimensional scanning, the super-resolution focused spot formed by the annular light beam on the front focal plane of the objective lens (15) will realize continuous two-dimensional scanning on the front focal plane of the objective lens (15) along with the vibration of the light beam scanning mirror (9); the horizontal coordinate X(t) and the vertical coordinate Y(t) of the position of the super-resolution focused spot on the sample (16) are functions of time t, and the reflected light E r (t) and the scattered light E s (t) collected by the objective lens (15) are also functions of time, and the reflected light E r (t) and the scattered light E s (t) reflect the sample structure and the refractive index information at the corresponding position.

4. The ring illumination based reflective scanning super resolution optical microscopy system of claim 2, wherein: The super-resolution focusing light spot is a ring-shaped focusing light field, inner and outer surfaces of the ring-shaped focusing light field are coaxial two conical surfaces, the ring-shaped focusing light field corresponds to an inner numerical aperture NA in , and an outer numerical aperture NA out , and satisfy: 0.947NA≤NA in <NA, NA out =NA; wherein, NA represents a numerical aperture of the objective lens (15).

5. The annular illumination based reflective scanning super-resolution optical microscopy system according to claim 1, wherein: The sample displacement module is a two-dimensional displacement stage (17), which is connected to a computer; the sample (16) is horizontally fixed on the two-dimensional displacement stage (17), and the computer controls the two-dimensional displacement stage (17) to drive the sample (16) to move in the XY plane for initial positioning of the sample; the super-resolution focused illumination and collection module further comprises an axial nanometer positioner (14), which is connected to the computer; the objective lens (15) is installed on the axial nanometer positioner (14), and the computer controls the axial nanometer positioner (14) to drive the objective lens (15) to move in the Z direction. Or the sample displacement module is a three-dimensional displacement table (37), the three-dimensional displacement table (37) is connected with the computer, the sample (16) is horizontally fixed on the three-dimensional displacement table (37), and the computer controls the three-dimensional displacement table (37) to drive the sample (16) to move in X, Y and Z directions.

6. The ring illumination based reflective scanning super resolution optical microscopy system of claim 5, wherein: The super-resolution optical imaging module further comprises a five-dimensional adjusting frame (24), and the optical fiber head (23) is installed on the five-dimensional adjusting frame (24), and adjusting the five-dimensional adjusting frame (24) can maximize the super-resolution signal light entering the optical fiber head (23). The optical fiber head (23) front end face optical fiber inner diameter is less than the first zero point radius of the focus spot of the super-resolution signal light on the back focal plane of the second sleeve mirror (22).

7. The ring illumination based reflective scanning super resolution optical microscopy system of claim 5, wherein: The light source module comprises a laser (1) and a fiber collimator (3), and the laser (1) is connected with the fiber collimator (3) through a first fiber jumper (2); the coherent light emitted by the laser (1) is transmitted to the fiber collimator (3) through the first fiber jumper (2), and the fiber collimator (3) collimates and outputs the coherent light as a collimated laser beam.

8. The ring illumination based reflective scanning super resolution optical microscopy system of claim 3, wherein: The optical microscopic imaging module further comprises an incoherent light source (30), a collimating lens (31), a first beam splitter (32), a third sleeve mirror (34) and a digital camera (35), the digital camera (35) is connected with the computer, and the incoherent light source (30) is located at the front focal point of the collimating lens (31); the light emitted by the incoherent light source (30) is collimated by the collimating lens (31) and reflected by the first beam splitter (32), and then passes through the second beam splitter (33) and the quarter-wave plate (13) to reach the objective lens (15) in sequence, and converges on the front focal plane of the objective lens (15) to form incoherent light to illuminate the sample (16), the incoherent light reflected and scattered by the sample (16) is collected by the objective lens (15), and then enters the digital camera (35) through the quarter-wave plate (13), the second beam splitter (33), the first beam splitter (32) and the third sleeve mirror (34) in sequence, so that the sample (16) is imaged on the digital camera (35) and sent to the computer, and the computer displays the wide-field microscopic image of the sample (16).

9. The ring illumination based reflective scanning super resolution optical microscopy system of claim 5, wherein: The spatial filter (20) is a ring-passing type spatial filter, which comprises a glass substrate (201) and a circular aluminum film (202) and a first ring-shaped aluminum film (203) overlaid on the glass substrate concentrically, the diameter of the circular aluminum film (202) is equal to 2R in , the inner diameter of the first ring-shaped aluminum film (203) is equal to 2R out , the outer diameter is equal to the diameter of the glass substrate (201), the transmittance of the glass substrate to light waves with a wavelength of λ is greater than 90%, and the transmittance of the circular aluminum film (202) and the first ring-shaped aluminum film (203) to light waves with a wavelength of λ is 0; wherein, R in represents the inner radius of the ring-shaped light beam at the exit pupil of the objective lens, and R out represents the outer radius of the ring-shaped light beam at the exit pupil of the objective lens. Or the spatial filter (20) is a ring-shaped blocking type spatial filter, which comprises a glass substrate (201) and a second ring-shaped aluminum film (204) covering the glass substrate, an inner diameter of the second ring-shaped aluminum film is equal to 2R in , and an outer diameter is equal to 2R out , the glass substrate (201) has a transmittance of more than 90% for light waves with a wavelength of λ, and the second ring-shaped aluminum film (204) has a transmittance of 0 for light waves with a wavelength of λ; wherein R in represents an inner radius of a ring-shaped light beam at an exit pupil of an objective lens, and R out represents an outer radius of the ring-shaped light beam at the exit pupil of the objective lens. Or the spatial filter (20) is a phase plate, which is a glass substrate on the surface of a thickness of t, the inner and outer radius of R in and R out A spatial filter, the transmittance T of the light wave with a wavelength of λ in the annulus between R in and R out is 10%-20%, and the phase change relative to the same height of air is π / 2.

10. A reflective scanning super-resolution optical microscopic method based on annular illumination, using the reflective scanning super-resolution optical microscopic system according to any one of claims 1 to 9, the method comprising: Step one, using the optical microscopic imaging module to perform wide-field imaging on the sample (16) to determine the super-resolution scanning imaging area; Step two, after determining the super-resolution scanning imaging area, the annular light beam generating module generates an annular light beam, the super-resolution focusing illumination and collection module converts the annular light beam into a super-resolution annular focused light beam to form a super-resolution focal spot to illuminate the sample (16), and collects the super-resolution signal light reflected and scattered by the sample (16) and converts it; Step three, the computer controls the sample displacement module to drive the sample (16) to move to the position to be scanned, and starts the light beam scanning galvanometer (9) to scan, while the super-resolution optical imaging module acquires the collimated and converted super-resolution signal light and converts it into an electrical signal input into the computer. Step four, the computer processes the inputted electrical signal, and combines the super-resolution illumination focal spot moving coordinate X(t) and Y(t) of the sample (16) and the signal light intensity I(t) obtained by the photomultiplier tube (26) at the corresponding time t, to form a super-resolution microscopic image I(X(t), Y(t)) of the sample in the super-resolution scanning imaging area.

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