Conical optical illumination-based reflective scanning super-resolution optical microscopy system and method

Abstract

Disclosed in the present invention are a conical optical illumination-based reflective scanning super-resolution optical microscopy system and method. The system comprises a sample displacement module, an optical microscopic imaging module, an annular focused beam generation module, a beam scanning module, a super-resolution focused illumination and collection module, a super-resolution optical imaging module and a computer. By means of using the advantages of annular focused beam illumination, high focusing efficiency of traditional lenses, and high beam scanning speed, high-efficiency super-resolution focused illumination with an extended depth of focus is achieved, and further traditional optical lenses can be used to achieve fast super-resolution optical microscopic imaging; fast label-free far-field super-resolution two-dimensional microscopic imaging is implemented by means of two-dimensional beam scanning; by means of performing label-free far-field super-resolution two-dimensional microscopic imaging on samples at different axial positions, super-resolution two-dimensional microscopic images of different cross sections of samples are obtained, thereby achieving three-dimensional tomography. The present invention can be applied to fast label-free super-resolution microscopic imaging of biological samples, and can also be applied to other fields such as industrial super-resolution microscopic inspection.

Description

Reflective Scanning Super-Resolution Optical Microscopy System and Method Based on Cone Illumination Technical Field

[0001] This invention belongs to the field of visible light microscopy, specifically relating to a reflective scanning super-resolution optical microscopy system and method based on light cone illumination. Background Technology

[0002] Existing microscopy techniques can be broadly categorized into near-field microscopy and far-field microscopy. Near-field microscopy includes methods such as microsphere-assisted imaging and near-field scanning microscopy. While near-field microscopy can achieve super-resolution imaging, its working distance is smaller than the working wavelength, significantly limiting its practical applications. Far-field super-resolution techniques, with their large working distances, overcome the limitations of near-field microscopy. Existing far-field super-resolution microscopy techniques include stimulated emission depletion microscopy, local activation microscopy, and random light reconstruction optical microscopy. However, these techniques all require fluorescent molecular labeling of the sample. Label-free far-field super-resolution microscopy, with its large working distance, lack of sample labeling requirement, and ease of use, has become an important development trend in super-resolution optical microscopy. In recent years, a few experiments have been reported on label-free far-field super-resolution microscopy based on super-diffraction devices. These systems all rely on novel super-resolution devices as illumination; however, the focusing efficiency of super-resolution devices is typically low, and these systems largely depend on moving sample scanning, resulting in slow imaging speeds. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing a reflective scanning super-resolution optical microscopy system and method based on light cone illumination, so as to achieve better resolution while improving reliability and imaging speed.

[0004] In a first aspect, the reflective scanning super-resolution optical microscopy system based on light cone illumination according to the present invention includes:

[0005] The sample displacement module is used to move the sample.

[0006] The optical microscopy imaging module is used to perform wide-field imaging of the sample and determine the super-resolution scanning imaging area;

[0007] A ring-shaped focused beam generating module is used to generate a ring-shaped focused beam. This module, along the light propagation direction, sequentially includes a light source module, a first linear polarizer, a conical lens, a first positive lens, a second positive lens, and a third positive lens. The first positive lens and the conical lens are placed coaxially and spaced apart. The front focal plane of the second positive lens coincides with the rear focal plane of the first positive lens, and the rear focal plane of the second positive lens coincides with the front focal plane of the third positive lens. The second and third positive lenses constitute a first 4f system. The light source module generates a collimated laser beam. This collimated laser beam, after passing through the first linear polarizer, the conical lens, and the first positive lens, forms a linearly polarized ring-shaped focused beam, and a circular focusing line is formed on the rear focal plane of the first positive lens.

[0008] The beam scanning module includes a beam scanning galvanometer, a beam scanning lens, a first telescope, and a reflecting mirror. The center of the beam scanning galvanometer coincides with the back focal plane of the third positive lens. The front focal plane of the beam scanning lens is located at the center of the beam scanning galvanometer. The back focal plane of the beam scanning lens coincides with the front focal plane of the first telescope, forming a second 4f system. The back focal plane of the first telescope, after being reflected by the reflecting mirror, coincides with the back focal plane of the objective lens in the super-resolution focusing illumination and collection module. The beam scanning galvanometer contains a controller for connecting to a computer, which controls the beam scanning galvanometer to perform two-dimensional vibration scanning.

[0009] The super-resolution focusing illumination and collection module is used to convert the ring-shaped focused beam into a super-resolution conical focused beam, forming a long focal depth super-resolution focal spot to illuminate the sample, collect the super-resolution signal light generated by the reflection and scattering of the sample, converge and collimate the super-resolution light signals reflected and scattered, and complete the polarization conversion; the super-resolution focusing illumination and collection module includes an objective lens, and the sample is located on the front focal plane of the objective lens.

[0010] A super-resolution optical imaging module is used to acquire the returned super-resolution signal light and convert it into an electrical signal. This module also includes a polarizing beam splitter, a spatial filter, a second linear polarizer, a second telescope lens, an optical fiber head, a photomultiplier tube, and a high-speed acquisition card. The polarizing beam splitter is located between a first positive lens and a second positive lens. The spatial filter is located on the focal plane formed after the light is focused by the second positive lens and reflected by the polarizing beam splitter. The second linear polarizer is located between the spatial filter and the second telescope lens. The front end of the optical fiber head coincides with the rear focal plane of the second telescope lens. The optical fiber head is connected to the photomultiplier tube, which is connected to the high-speed acquisition card, which is used to connect to a computer.

[0011] The computer is used to control the sample displacement module to move the sample, and synchronously control the beam scanning galvanometer to scan and receive the electrical signals output by the super-resolution optical imaging module, and process them to obtain a super-resolution microscopic image of the sample in the super-resolution scanning imaging area.

[0012] Optionally, when the annular focused beam generated by the annular focused beam generation module is reflected by the beam scanning galvanometer, passes through the scanning lens and the first sleeve lens, and is then reflected by the reflecting mirror to the rear focal plane of the objective lens; when the beam scanning galvanometer vibrates, the center of the annular focused beam coincides with the center of the rear focal plane of the objective lens, and the incident angle of the annular focused beam changes with the vibration of the beam scanning galvanometer; when the beam scanning galvanometer performs two-dimensional scanning, the annular focused beam, after being converted by the objective lens, forms a conical focused beam, which in turn forms a long focal depth focused light field, forming a super-resolution focused spot on the front focal plane of the objective lens. This focused spot will achieve continuous two-dimensional scanning on the front focal plane of the objective lens with the vibration of the beam scanning galvanometer; and by controlling the position of the rear focal plane of the objective lens in the Z-axis direction, the spatial range of continuous two-dimensional scanning on the front focal plane of the objective lens can be controlled.

[0013] Preferably, the super-resolution focusing illumination and collection module further includes a quarter-wave plate; the super-resolution optical imaging module further includes a polarization beam splitter; the optical microscopic imaging module includes a second beam splitter; the circularly focused linearly polarized beam emitted from the beam scanning module passes through the quarter-wave plate and forms a circularly polarized circularly focused line with an outer diameter smaller than the diameter of the rear focal plane of the objective lens on the rear focal plane. The circularly polarized circularly focused line is converted into a super-resolution conical focused beam after passing through the objective lens, forming a super-resolution focused spot on the front focal plane of the objective lens. This super-resolution focused spot illuminates the sample. The super-resolution signal light generated by the reflection and scattering of the sample is collected by the objective lens and then converged and collimated. It is then converted into a linearly polarized ring-shaped focused beam and a linearly polarized collimated beam orthogonal to the polarization of the incident beam by the quarter-wave plate, i.e., a linearly polarized super-resolution signal beam. This linearly polarized super-resolution signal beam is transmitted through the second beam splitter in the opposite direction of the original optical path, passes through the beam scanning module, enters the ring-shaped focused beam generation module, and is reflected by the polarization beam splitter placed in the optical path into the super-resolution optical imaging module.

[0014] When the beam scanning galvanometer in the beam scanning module performs a two-dimensional scan, the ring-shaped focused beam is transformed into a conical focused beam by the objective lens. The super-resolution focused spot formed on the front focal plane of the objective lens will continuously scan two dimensions on the front focal plane of the objective lens as the beam scanning galvanometer vibrates. The horizontal coordinate X(t) and vertical coordinate Y(t) of the position of the super-resolution focused spot illuminating the sample are functions of time t. The reflected light E collected by the objective lens... 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)).

[0015] Preferably, the super-resolution focused light spot is an annular focused light field. The inner and outer surfaces of the annular focused light field are two conical surfaces that are coaxial and have the same cone angle. The numerical aperture NA of the annular focused light field c , and satisfies: NA c < NA, where NA represents the numerical aperture of the objective lens.

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

[0017] First, the sample displacement module is a two-dimensional displacement stage. The two-dimensional displacement stage is connected to a computer. The sample is horizontally fixed on the two-dimensional displacement stage. The computer controls the two-dimensional displacement stage to drive the sample to move two-dimensionally in the XY plane to achieve preliminary positioning of the sample. The super-resolution focusing illumination and collection module further includes an axial nano-positioner. The axial nano-positioner is connected to the computer. The objective lens is installed on the axial nano-positioner. The computer controls the axial nano-positioner to move the objective lens in the Z direction. The beam scanning module achieves two-dimensional scanning of the sample, and combines with the axial nano-positioner to achieve three-dimensional tomography scanning.

[0018] Second, the sample displacement module is a three-dimensional displacement stage. The three-dimensional displacement stage is connected to a computer. The sample is horizontally fixed on the three-dimensional displacement stage. The computer controls the three-dimensional displacement stage to drive the sample to move in three directions of X, Y, and Z. In the X and Y directions, preliminary positioning of the sample is achieved. The beam scanning module achieves two-dimensional scanning of the sample, and combines with the displacement of the three-dimensional displacement stage in the Z direction to achieve three-dimensional tomography scanning, avoiding the imaging impact caused by the微小 movement of the objective lens, and having a better imaging effect.

[0019] Preferably, the super-resolution optical imaging module further includes a five-dimensional adjustment bracket. The fiber optic head is installed on the five-dimensional adjustment bracket. Adjusting the five-dimensional adjustment bracket can make the super-resolution signal light entering the fiber optic head reach the maximum;

[0020] The inner diameter of the optical fiber at the front end face of the fiber optic head is smaller than the first zero radius of the focal spot of the super-resolution signal light on the rear focal plane of the second sleeve lens, so as to better ensure that the super-resolution imaging resolution reaches the optimal.

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

[0022] The annular focused beam generating module further includes a field lens. The object-side principal plane of the field lens coincides with the rear focal plane of the first positive lens, and the image-side principal plane of the field lens coincides with the front focal plane of the second positive lens respectively, so as to change the propagation direction of the marginal rays of the annular focused beam; to adapt to different objective lenses and make the annular focused beam transmit through the objective lens without obstruction.

[0023] Preferably, the optical microscopy imaging module includes an incoherent light source, a collimating lens, a first beam splitter, a second beam splitter, a third telescope, and a digital camera. The digital camera is connected to a computer. The incoherent light source is located at the front focal point of the collimating lens. The light emitted from the incoherent light source is collimated by the collimating lens, reflected by the first beam splitter, and then passes sequentially through the second beam splitter and a quarter-wave plate to reach the objective lens. It converges into incoherent light at the front focal plane of the objective lens to illuminate the sample. The incoherent light generated by the reflection and scattering from the sample is collected by the objective lens and then passes sequentially through the quarter-wave plate, the second beam splitter, the first beam splitter, and the third telescope before entering the digital camera. This images the sample onto the digital camera and transmits the image to the computer via a camera signal cable. The computer then displays a wide-field microscopic image of the sample. This facilitates wide-field observation of the sample to determine the required super-resolution scanning imaging area.

[0024] Preferably, the spatial filter has three structures.

[0025] The first type of spatial filter is a ring-type spatial filter, which includes a glass substrate and a concentric circular aluminum film and a first annular aluminum film covering the glass substrate. The diameter of the circular aluminum film is equal to 2R. in The inner diameter of the first annular aluminum film is equal to 2R. out The outer diameter is equal to the diameter of the glass substrate, and the glass substrate has a transmittance of more than 90% for light waves with wavelength λ. The circular aluminum film and the first annular aluminum film both have a transmittance of 0 for light waves with wavelength λ. Only the super-resolution signal light generated by the sample reflection is allowed to pass through, i.e., the bright-field imaging light signal is allowed to pass through, thus achieving bright-field imaging. Wherein, R... in R represents the inner radius of the circular focal line at the back focal plane of the objective lens. out It represents the outer radius of the circular focusing line at the back focal plane of the objective lens.

[0026] The second type of spatial filter is a ring-shaped shielding spatial filter, which includes a glass substrate and a second ring-shaped aluminum film covering the glass substrate. The inner diameter of the second ring-shaped aluminum film is equal to 2R. in The outer diameter is equal to 2R. out The glass substrate has a transmittance of more than 90% for light waves with wavelength λ, while the second annular aluminum film has a transmittance of 0 for light waves with wavelength λ. Only the super-resolution signal light generated by the scattering of the sample is allowed to pass through, that is, the dark-field imaging light signal is allowed to pass through, thus achieving dark-field imaging.

[0027] The third type of spatial filter is a phase plate, which is made by fabricating a phase plate with a thickness of t and inner and outer radii of R on the surface of a glass substrate. in and R out A spatial filter, in R in and R outThe transmittance T of light with wavelength λ within the circular ring is 10%-20%, and its phase change relative to air at the same altitude is π / 2 or -π / 2. This is used to achieve phase-contrast imaging.

[0028] Secondly, the reflective scanning super-resolution optical microscopy method based on light cone illumination described in this invention employs the aforementioned reflective scanning super-resolution optical microscopy system, and the method includes:

[0029] Step 1: Use an optical microscopy imaging module to perform wide-field imaging of the sample and determine the super-resolution scanning imaging area;

[0030] Step 2: After determining the super-resolution scanning imaging area, the ring focusing beam generation module generates a ring focusing beam, and the super-resolution focusing illumination and collection module converts the ring focusing beam into a super-resolution conical focusing 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 converges and collimates the super-resolution light signals reflected and scattered, and completes polarization conversion.

[0031] Step 3: The computer-controlled sample displacement module moves the sample to the position to be scanned and turns on the beam scanning galvanometer to scan. At the same time, the super-resolution optical imaging module acquires the collimated and converted super-resolution signal light and converts it into an electrical signal for input into the computer.

[0032] Step 4: The computer processes the input electrical signal and combines it with the coordinates X(t) and Y(t) of the super-resolution illumination focal spot on 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 microscopic image I(X(t),Y(t)) of the sample in the super-resolution scanning imaging area.

[0033] The present invention has the following advantages:

[0034] (1) High-efficiency super-resolution focusing illumination: This invention utilizes a meticulously designed annular focusing beam generation module to efficiently generate annular focusing beams. Subsequently, using a super-resolution focusing illumination and collection module, this annular focusing beam is converted into a super-resolution conical focusing beam, forming a high-precision super-resolution focal spot to illuminate the sample. This process fully leverages the high focusing efficiency of traditional lenses, achieving high-efficiency super-resolution focusing illumination and significantly improving the signal-to-noise ratio of the image. Simultaneously, the generated super-resolution conical focusing beam forms a long depth-of-focus super-resolution focusing light field behind the objective lens, facilitating the reduction of alignment requirements for the illumination beam, focal plane, and sample.

[0035] (2) Rapid 2D Scanning: The beam scanning module employs beam scanning galvanometer technology to achieve rapid 2D scanning of the beam. This scanning method is not only fast but also highly accurate, enabling super-resolution imaging of a large area in a short time. This feature greatly improves imaging efficiency, allowing users to quickly acquire large amounts of high-quality image data.

[0036] (3) Label-free far-field super-resolution microscopy: Compared with traditional microscopy techniques that require fluorescent molecular labeling, a major highlight of this invention is its ability to achieve rapid far-field super-resolution two-dimensional microscopy imaging of unlabeled samples. This function not only expands the applicability of the technology but also simplifies experimental procedures and reduces the requirements for sample processing. At the same time, due to the large working distance, it makes applications in fields such as biological and industrial detection more convenient and efficient.

[0037] (4) Three-dimensional tomographic imaging capability: By performing label-free far-field super-resolution two-dimensional microscopic imaging on samples at different axial positions, this invention can acquire 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 samples and revealing the spatial distribution of complex samples, and is particularly suitable for fields such as biomedical research and materials science.

[0038] (5) High reliability and imaging quality: This invention ensures the reliability and stability of imaging by precisely controlling the optical path coordination and signal processing between modules. From the generation of the ring-shaped focusing beam, beam scanning, super-resolution focusing illumination to signal collection and processing, each step has been carefully designed and optimized to guarantee a high level of final imaging quality. In addition, the optical components and signal acquisition equipment used have high performance indicators, further improving the overall performance and imaging quality of the system.

[0039] In summary, the reflective scanning super-resolution optical microscopy system and method based on light cone illumination of this invention exhibits significant advantages in terms of efficient super-resolution focusing illumination, rapid two-dimensional scanning, label-free far-field super-resolution microscopy imaging, three-dimensional tomography capabilities, super-resolution three-dimensional imaging capabilities, as well as high reliability and imaging quality, providing strong technical support for fields such as biomedicine and industrial inspection. Attached Figure Description

[0040] Figure 1 is a schematic diagram of the reflective scanning super-resolution optical microscopy system based on light cone illumination in Example 1.

[0041] Figure 2 is a schematic diagram of the control of the ring-focused beam size by the conical lens, the third positive lens, and the first 4f system in Embodiment 1.

[0042] Figure 3 is a schematic diagram of the formation of a long focal depth super-resolution focused light field in Example 1.

[0043] Figure 4 is a parameter relationship diagram of the long focal depth super-resolution focused light field in Example 1.

[0044] Figure 5 is a schematic diagram of the super-resolution focused spot scanning on the focal plane of the objective lens in Example 1.

[0045] Figure 6 is a front view and cross-sectional schematic diagram of the spatial filter in Embodiment 1.

[0046] Figure 7 shows the relationship between the full width at half maximum (FWHM) of the objective lens and the focal spot and the system resolution under the ring-focused beam illumination in Example 1.

[0047] Figure 8 is a schematic diagram of the reflective scanning super-resolution optical microscopy system based on light cone illumination in Example 2.

[0048] Figure 9 is a front view and cross-sectional schematic diagram of the spatial filter in Embodiment 3.

[0049] Figure 10 is a front view and cross-sectional schematic diagram of the spatial filter in Embodiment 4.

[0050] Figure 11 is a front view and cross-sectional schematic diagram of the spatial filter in Embodiment 5.

[0051] In the diagram: 1. Laser; 2. First fiber optic jumper; 3. Fiber optic collimator; 4. First linear polarizer; 5. Conical lens; 6. First positive lens; 7. Second positive lens; 8. Third positive lens; 9. Beam scanning galvanometer; 10. Beam scanning lens; 11. First telescopic lens; 12. Mirror; 13. Quarter-wave plate; 14. Axial nanopositioner; 15. Objective lens; 16. Sample; 17. Two-dimensional displacement stage; 18. Scanning signal line; 19. Polarizing beam splitter; 20. Spatial filter; 201. Glass substrate; 202. 203. Circular aluminum film; 204. First annular aluminum film; 205. Second annular aluminum film; 21. Second linear polarizer; 22. Second telescope lens; 23. Fiber optic head; 24. Five-dimensional adjustment frame; 25. Second fiber optic patch cord; 26. Photomultiplier tube; 27. Acquisition signal line; 28. High-speed acquisition card; 29. ​​Data line; 30. Incoherent light source; 31. Collimating lens; 32. First beam splitter; 33. Second beam splitter; 34. Third telescope lens; 35. Digital camera; 36. Camera signal line; 37. Three-dimensional displacement stage; 38. Field lens. Detailed Implementation

[0052] To gain a more detailed understanding of the features and technical content of the embodiments of the present invention, the implementation of the embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The accompanying drawings are for reference and illustration only and are not intended to limit the embodiments of the present invention.

[0053] Example 1

[0054] As shown in Figure 1, the reflective scanning super-resolution optical microscopy system based on light cone illumination in this embodiment includes:

[0055] The sample displacement module is used to move sample 16.

[0056] The optical microscopy imaging module is used to perform wide-field imaging of sample 16 and determine the super-resolution scanning imaging area.

[0057] Ring-shaped focused beam generation module, used to generate a ring-shaped focused beam.

[0058] The beam scanning module is used to achieve rapid two-dimensional scanning of the beam.

[0059] The super-resolution focusing illumination and collection module is used to convert the ring-shaped focused beam into a super-resolution conical focused beam to form a super-resolution focal spot to illuminate the sample 16, collect the super-resolution signal light generated by the reflection and scattering of the sample 16, and collimate and convert it.

[0060] The super-resolution optical imaging module is used to acquire the returned super-resolution signal light and convert it into an electrical signal.

[0061] A computer (not shown in the figure) is used to control the sample displacement module to move the sample 16. The beam scanning galvanometer 9 is synchronously controlled to scan and receive the electrical signals output by the super-resolution optical imaging module, and the super-resolution microscopic image of the sample 16 in the super-resolution scanning imaging area is obtained through processing.

[0062] This system leverages the advantages of ring-shaped focused beam illumination, high focusing efficiency of traditional lenses, and fast beam scanning speed to achieve highly efficient super-resolution focused illumination. This enables rapid super-resolution optical microscopy imaging using traditional optical lenses. It achieves label-free far-field super-resolution two-dimensional microscopic imaging through two-dimensional beam scanning. By performing label-free far-field super-resolution two-dimensional microscopic imaging on samples at different axial positions, it obtains super-resolution two-dimensional microscopic images of different cross-sections of the samples, thereby achieving three-dimensional tomography. This system can be applied to label-free super-resolution rapid microscopic imaging of biological samples, as well as to industrial super-resolution microscopic detection and other fields.

[0063] The following is a detailed explanation of each module:

[0064] As shown in Figures 1 and 2, the optical microscopy imaging module includes an incoherent light source 30, a collimating lens 31, a first beam splitter 32, a second beam splitter 33, a third telescope 34, and a digital camera 35. The digital camera 35 is connected to a computer via a camera signal line 36. The incoherent light source 30 is located at the front focal point of the collimating lens 31. The light emitted from the incoherent light source 30 is collimated by the collimating lens 31, reflected by the first beam splitter 32, and then passes sequentially through the second beam splitter 33 and the quarter-wave plate 13 to reach the objective lens 15. It is then converged into incoherent light on the front focal plane of the objective lens 15 to illuminate the sample 16. The incoherent light generated by the reflection and scattering of the sample 16 is collected by the objective lens 15 and then passes sequentially through the quarter-wave plate 13, the second beam splitter 33, the first beam splitter 32, and the third telescope 34 before entering the digital camera 35. This images the sample 16 onto the digital camera 35 and are then sent to the computer, where the computer displays a wide-field microscopic image of the sample 16.

[0065] As shown in Figures 1 and 2, the annular focusing beam generation module includes, along the light propagation direction, a laser 1, an fiber collimator 3, a first linear polarizer 4, a conical lens 5, a first positive lens 6, a scene 38, a second positive lens 7, and a third positive lens 8. The laser 1 is connected to the fiber collimator 3 via a first fiber jumper 2. The coherent light emitted by the laser 1 is transmitted to the fiber collimator 3 via the first fiber jumper 2, and the fiber collimator 3 collimates the coherent light before outputting a collimated laser beam. The first positive lens 6 and the conical lens 5 are placed coaxially and spaced apart. The front focal plane F2 of the second positive lens 7 coincides with the rear focal plane F1' of the first positive lens 6, and the rear focal plane (F'2) of the second positive lens 7 coincides with the front focal plane (F3) of the third positive lens 8. The second positive lens 7 and the third positive lens 8 constitute a first 4f system. The light source module (laser 1 and fiber collimator 3) generates a collimated laser beam. This collimated laser beam passes through the first linear polarizer 4, the conical lens 5, and the first positive lens 6 to form a linearly polarized annular focused beam, and a circular focal line is formed at the rear focal plane of the first positive lens 6. The object-side principal plane of the field lens 38 coincides with the rear focal plane of the first positive lens 6, and the image-side principal plane of the field lens 38 coincides with the front focal plane of the second positive lens 7, thereby changing the propagation direction of the edge light rays of the annular focused beam; to adapt to different objectives 15, so that the annular focused beam can be transmitted through the objectives 15 without obstruction.

[0066] As shown in Figures 1 and 2, the beam scanning module includes a beam scanning galvanometer 9, a scanning lens 10, a first telescope 11, and a reflecting mirror 12. The center of the beam scanning galvanometer 9 coincides with the back focal plane of the third positive lens 8; the front focal plane of the scanning lens 10 is located at the center of the beam scanning galvanometer 9; the back focal plane of the scanning lens 10 coincides with the front focal plane of the first telescope 11, forming a second 4f system; the back focal plane of the first telescope 11, after being reflected by the reflecting mirror 12, coincides with the back focal plane of the objective lens 15 in the super-resolution focusing illumination and collection module. The beam scanning galvanometer 9 contains a controller for connecting to a computer (not shown in the figures). The computer controls the beam scanning galvanometer 9 to perform two-dimensional vibration scanning through the controller.

[0067] When the annular focused beam generated by the annular focused beam generation module is reflected by the beam scanning galvanometer 9, passes through the scanning lens 10 and the first sleeve lens 11, and is reflected by the reflecting mirror 12 to the rear focal plane of the objective lens 15; when the beam scanning galvanometer 9 vibrates, the center of the annular focused beam coincides with the center of the rear focal plane of the objective lens 15, and the incident angle of the annular focused beam changes with the vibration of the beam scanning galvanometer 9; when the beam scanning galvanometer 9 performs two-dimensional scanning, the annular focused beam is transformed by the objective lens 15, forming a conical focused light field behind the objective lens 15, thereby generating a long focal depth focused light field, and forming a super-resolution focused light spot on the front focal plane of the objective lens 15. This super-resolution focused light spot will achieve continuous two-dimensional scanning on the front focal plane of the objective lens 15 with the vibration of the beam scanning galvanometer 9; and by controlling the position of the rear focal plane of the objective lens 15 in the Z-axis direction, the spatial range of continuous two-dimensional scanning on the front focal plane of the objective lens 15 can be controlled.

[0068] As shown in Figures 1 and 2, the super-resolution focusing illumination and collection module includes a quarter-wave plate 13 and an objective lens 15. The back focal plane of the objective lens 15 coincides with the back focal plane of the first telescope lens 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 lens, which can be a dry microscope, water microscope, oil microscope, or solid microscope.

[0069] As shown in Figures 1 and 2, the sample displacement module is a two-dimensional displacement stage 17. The two-dimensional displacement stage 17 is connected to a computer (not shown in the figures) via 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 move the sample 16 in two dimensions within the XY plane. When the sample displacement module is the two-dimensional displacement stage 17, the super-resolution focusing illumination and collection module also includes an axial nanopositioner 14. The axial nanopositioner 14 is connected to the computer via a positioning signal line, and the objective lens 15 is mounted on the axial nanopositioner 14. The computer controls the axial nanopositioner 14 to move the objective lens 15 in the Z direction.

[0070] As shown in Figures 1 and 2, the super-resolution optical imaging module includes a polarizing beam splitter 19, a spatial filter 20, a second linear polarizer 21, a second telescope 22, an optical fiber head 23, a photomultiplier tube 26, and a high-speed acquisition card 28. The polarizing beam splitter 19 is located between the field lens 38 and the second positive lens 7. The spatial filter 20 is located on the focal plane formed after the light is focused by the second positive lens 7 and reflected by the polarizing beam splitter 19. The second linear polarizer 21 is located between the spatial filter 20 and the second telescope 22. The front end of the optical fiber head 23 coincides with the rear focal plane of the second telescope 22. The optical fiber head 23 is connected to the photomultiplier tube 26 via a second optical fiber jumper 25. The photomultiplier tube 26 is connected to the high-speed acquisition card 28 via a signal acquisition cable 27. The high-speed acquisition card 28 is connected to a computer via a data cable 29.

[0071] As shown in Figure 6, in one possible embodiment, the spatial filter 20 is a ring-type spatial filter, which includes a glass substrate 201 and a concentric circular aluminum film 202 and a first annular aluminum film 203 covering the glass substrate 201. The thickness t of both the circular aluminum film 202 and the first annular aluminum film 203 is greater than 50 nm, and the diameter of the circular aluminum film 202 is equal to 2R. in The inner diameter of the first annular 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 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.

[0072] In this embodiment of the application, the reflection-scanning super-resolution optical microscopy method based on light cone illumination employs the aforementioned reflection-scanning super-resolution optical microscopy system, and the method includes:

[0073] 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:

[0074] 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.

[0075] Step 2: After determining the super-resolution scanning imaging area, the ring-shaped focusing beam generation module generates a ring-shaped focusing beam. The super-resolution focusing illumination and collection module converts the ring-shaped focusing beam into a super-resolution conical focusing beam, forming a super-resolution focal spot to illuminate sample 16. It collects the super-resolution signal light reflected and scattered by sample 16, converges and collimates the reflected and scattered super-resolution light signals, and completes polarization conversion. Specifically:

[0076] The incoherent light source 30 does not emit light, and the laser 1 is turned on, emitting coherent light with wavelength λ. This light is transmitted to the fiber collimator 3 via the first fiber jumper 2. The fiber collimator 3 collimates the coherent light and outputs a collimated laser beam. This collimated laser beam passes through the first linear polarizer 4, the conical lens 5, and the first positive lens 6 to form a ring-shaped focused beam (a P-polarized ring beam). After passing through the first 4f system, the P-polarized ring beam passes through the beam scanning module and then through the quarter-wave plate 13, forming a circular focusing line on the back focal plane of the objective lens 15 with an outer diameter equal to the diameter of the back focal plane of the objective lens 15. This circularly polarized circular focusing line, after passing through the objective lens 15, is converted into a super-resolution conical focused beam, forming a super-resolution focused spot on the front focal plane of the objective lens 15. The full width at half maximum (FWHM) of the light spot is less than 0.5λ / NA, and the first zero-point radius of the super-resolution focal spot (i.e., the distance from the peak intensity position of the focal spot to the first zero-point position) is less than 0.61λ / NA. This super-resolution focused light spot illuminates the sample 16. The super-resolution signal light generated by the reflection and scattering of the sample 16 is collected by the objective lens 15 and then converged and collimated. It is then converted by the quarter-wave plate 13 into an S-polarized ring-focused beam and an S-polarized collimated beam orthogonal to the incident linearly polarized ring-focused beam, respectively. The S-polarized ring-focused beam and the S-polarized collimated beam pass through the second beam splitter 33 in the opposite direction of the original optical path, pass through the beam scanning module, enter the ring-focused beam generation module, and are reflected by the polarization beam splitter 19 placed in the optical path into the super-resolution optical imaging module.

[0077] Figure 2 illustrates the method for generating a P-polarized annular focused beam. Specifically: the fiber collimator 3 collimates the coherent light and outputs a collimated laser beam with radius R (different numerical aperture fiber collimators can be used to obtain collimated laser beams with different radii, thus obtaining super-resolution conical focused beams with different focal depths). The collimated laser beam with radius R passes through the first linear polarizer 4 and becomes a P-polarized collimated beam with radius R. This P-polarized collimated beam passes through a conical lens 5 and a first positive lens 6 placed coaxially with a spacing of d, forming a P-polarized annular focused beam, which forms a central radius R' on the back focal plane of the first positive lens 6. c The circular focal line can be adjusted by changing the focal length of the first positive lens 6, thereby changing the central radius R' of the circular focal line. c The P-polarized ring-focused beam is further projected onto the rear focal plane of the third positive lens 8 through the first 4f system, forming a central radius of R''. c The circular focal line. Where, R'' c =R' c×f3 / f2, where f2 represents the focal length of the second positive lens 7 and f3 represents the focal length of the third positive lens 8. By selecting second positive lenses 7 and third positive lenses 8 with different focal lengths, circular focal lines of different sizes can be obtained. Further, through the second 4f system composed of the beam scanning lens 10 and the first sleeve lens 11, the circular focal line is projected onto the rear focal plane F' of the first sleeve lens 11 t (which is also the rear focal plane of the objective lens 15). The magnification of the second 4f system is K. Therefore, the central radius R of the finally formed circular focal line can be controlled by controlling f1, f2, and f3 c = KR' c ×f3 / f2.

[0078] Fig. 3 shows the illumination working schematic diagram of forming a long depth-of-focus super-resolution focused light field. The left-handed circularly polarized annular focused beam illuminates the rear focal plane of the objective lens 15, and a circular focal line with a central diameter of 2R is formed on the rear focal plane of the objective lens 15 c , the inner radius of the circular focal line is R in , the outer radius is R out , and the central radius R c = (R out + R in ) / 2. After passing through the objective lens 15, the left-handed circularly polarized annular focused beam is converted into a super-resolution conical focused beam with a cone angle of θ c , and a long depth-of-focus super-resolution focused light field is formed along the optical axis before and after the front focal plane of the objective lens 15. The numerical aperture NA of the super-resolution conical focused beam c = n × sin(θ c ), NA c < NA; where, sin() is the sine function and n is the refractive index of the medium between the objective lens 15 and the sample 16. The full width at half maximum of the long depth-of-focus super-resolution focused light field is less than 0.5λ / NA, and the first zero radius of the super-resolution focal spot of the long depth-of-focus super-resolution focused light field (the distance from the peak position of the focal spot intensity to the first zero position) is less than 0.61λ / NA. When 0 < NA c < 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, it is necessary to satisfy: 0.947NA ≤ NA c < NA. The resolution of the super-resolution microscopy is determined by NA c , the larger NA c and the closer it is to NA, the closer the resolution of the super-resolution microscopy can be to the limit resolution 0.25λ / NA of the reflection confocal system. The larger NA c corresponds to the larger R c (R c is less than the radius of the rear focal plane of the objective lens 15), and the corresponding Rout , R in The closer it is (i.e., the smaller the width of the circular focus line), the narrower the spatial frequency of the super-resolution focusing light field with a long depth of focus. In practice, we control the radius R of the collimated laser beam and adjust the distance d between the cone lens 5 and the first positive lens 6 to control R' c , and then control the radius of the circular focus line. Then, since the S-polarized annular focusing beam corresponding to the circular focus line will undergo obvious diffraction during propagation, resulting in an increase in width, it is impossible to ensure that the circular focus line transmitted to the rear focal plane of the objective lens 15 can maintain a very small width; therefore, it is necessary to add a first 4f system composed of the second positive lens 7 and the third positive lens 8 to adjust the size of the S-polarized annular focusing beam (corresponding to the circular focus line), so that the numerical aperture NA c of the finally formed super-resolution conical focusing beam satisfies: 0.947NA ≤ NA c < NA. The first 4f system composed of the second positive lens 7 and the third positive lens 8 plays a role in adjusting the size of the circular focus line of the S-polarized annular focusing beam on the one hand, and acts as a transmission relay for the S-polarized annular focusing beam on the other hand, so as to ensure that after the circular focus line on the front focal plane of the first 4f system is adjusted, it is accurately projected onto the rear focal plane of the first 4f system (corresponding to the rear focal plane of the objective lens 15), thereby avoiding the size distortion and the fluctuation of the light field intensity distribution caused by the diffraction effect during the direct transmission of the S-polarized annular focusing beam, ensuring the high-quality illumination of the objective lens by the S-polarized annular focusing beam, and further ensuring that the generated super-resolution focal spot reaches the minimum to ensure that the system reaches the best resolution.

[0079] Given the numerical aperture NA of the objective lens 15, to ensure that the cone angle of the super-resolution conical focusing beam after passing through the objective lens 15 is θ c , it can be achieved in the following way: First, determine NA r and NA according to the required resolution d c , specifically d r = 0.5λ / (NA c + NA), where NA c ≤ NA, and NA c represents the numerical aperture corresponding to the circular focus line on the rear focal plane of the objective lens 15. Determine R c according to NA c (the determination method belongs to the prior art and can be achieved through actual measurement); then, according to R c = Kf1tan(β)f3 / f2 (where β is the cone angle of the conical beam emitted by the cone lens 5, and tan() is the tangent function), determine f1, f2 and f3 (f3 needs to have sufficient length so that the third positive lens 8 will not have spatial interference with subsequent optical elements.

[0080] Figure 4 shows the parameters FWHM (full width at half maximum), r0 (radius of the first null point), and SR (sidelobe peak ratio, i.e., the ratio of the maximum sidelobe intensity to the center intensity) of the super-resolution focused light field at a depth of field of objective lens 15 when NA = 0.95, and their corresponding NA. c The functional relationship. It can be seen that when NA c When R ≥ 0.9, the resulting long focal depth super-resolution focused light field has FWHM ≤ 0.399λ (less than 0.5λ / NA), r0 ≤ 0.426λ (less than 0.61λ / NA), and SR = 0.162. Therefore, by controlling R... c Make it satisfy: 0.9≤NA c With a value <0.95, a long focal depth super-resolution illumination field with FWHM≤0.399λ, r0≤0.426λ, and SR=0.162 can be achieved.

[0081] Figure 5 shows a schematic diagram of the super-resolution focused beam scanning on the focal plane of objective lens 15. The beam scanning module realizes two-dimensional scanning of the ring beam. At different times, the ring focused beam is incident on objective lens 15 at different angles. When the ring focused beam reaches the rear focal plane of objective lens 15, the ring focused beam always forms a ring centered on the center of the rear focal plane. At a given time t, the tilt angle of the incident ring focused beam is θ. s (t), at this time, a super-long depth-of-focus super-resolution focusing spot with a full width at half maximum (FWHM) length of 2ΔZ is formed before and after the focal plane (Z direction) behind objective lens 15. The offset of this focusing spot in the XY direction (relative to the optical axis Z) is d(t). By changing the incident angle θ s (t), which can change the offset d(t). At different times, the intensity distribution of the illumination ring-shaped focused beam on the back focal plane of the objective lens is always a ring centered on the center of the back focal plane, and the inner and outer radii of this ring are R and R, respectively. in and R out .

[0082] Figure 7 shows the relationship between the full width at half maximum (FWHM) of the objective lens (numerical aperture NA > 1) and the system resolution under ring-focused beam illumination. The vertical axis represents the FWHM of the objective lens's point spread function (NPF), in wavelength λ; the horizontal axis represents the lateral FWHM of the super-resolution focused spot, in wavelength λ; and the contour lines represent the system resolution (i.e., the minimum linewidth of the amplitude-type grating that the system can resolve, with an imaging fringe contrast better than 11%), in wavelength λ. When the FWHM of the objective lens (which can be a single-point focusing super-diffraction lens) is 0.6λ and the lateral FWHM of the super-resolution focused spot (the portion illuminating the sample) is both 0.38λ, the system resolution can reach 0.25λ~0.26λ; when both the FWHM of the objective lens's NPF and the lateral FWHM of the super-resolution focused spot are 0.35λ, the system resolution is better than 0.2λ.

[0083] Step 3: The computer-controlled beam scanning module performs a two-dimensional scan of the beam, thereby enabling the super-resolution focused spot to perform a two-dimensional scan on the front focal plane of objective lens 15. At the same time, the super-resolution optical imaging module simultaneously acquires the collimated and converted super-resolution signal light and converts it into an electrical signal for input into the computer.

[0084] Step 4: The computer processes the input electrical signal and combines it with the coordinates X(t) and Y(t) of the super-resolution focused spot moving on 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.

[0085] In this embodiment, the specific processing steps three and four are as follows: The S-polarized beam (i.e., the super-resolution signal light reflected by the polarization beam splitter 19 after convergence, collimation, and conversion) 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 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 fTwo-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.

[0086] Example 2

[0087] As shown in Figure 8, most of the structure of the reflective scanning super-resolution optical microscopy system based on light cone illumination in this embodiment is the same as that in Embodiment 1. 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 the scanning signal line 18. The sample 16 is horizontally fixed on the three-dimensional displacement stage 37. The computer controls the three-dimensional displacement stage 37 to move the sample 16 in the Z-axis direction.

[0088] As shown in Figure 8, the reflection-based super-resolution optical microscopy method based on light cone illumination in this embodiment is basically the same as that in Embodiment 1. The only difference is that in the specific processing of steps three and four, the computer controls the three-dimensional displacement stage 37 to move the sample 16 slightly in the Z direction, changing the Z-position of the front focal plane of the objective lens 15 in the sample 16. f Every time it moves a little, the high-speed acquisition card 28 acquires a signal once, obtaining different Z values. 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.

[0089] Example 3

[0090] As shown in Figure 9, in this embodiment, most of the structure of the reflective scanning super-resolution optical microscopy system based on light cone illumination is the same as in Embodiment 1. The difference is that the spatial filter 20 is a ring-shaped shielding spatial filter, which includes a glass substrate 201 and a second annular aluminum film 204 covering the glass substrate 201. The thickness t of the second annular aluminum film 204 is greater than 50 nm, and the inner diameter of the second annular aluminum film 204 is equal to 2R. in The outer diameter is equal to 2R. out The glass substrate 201 has a transmittance of more than 90% for light waves with wavelength λ, while the second annular aluminum film 204 has a transmittance of 0 for light waves with wavelength λ. Only the super-resolution signal light generated by the scattering of sample 16 is allowed to pass through, that is, the dark-field imaging light signal is allowed to pass through, thus achieving dark-field imaging. The reflection-scanning super-resolution optical microscopy method based on light cone illumination in this embodiment is basically the same as that in Embodiment 1, except that in step 3, after the P-polarized collimated beam is filtered by the spatial filter 20, only the super-resolution signal light generated by the scattering of sample 16 is allowed to converge on the back focal plane of the second sleeve lens 22 and be coupled into the fiber optic head 23.

[0091] Example 4

[0092] As shown in Figure 10, most of the structure of the reflective scanning super-resolution optical microscopy system based on light cone illumination in this embodiment is the same as that in Embodiment 1. The difference is that the spatial filter 20 is a phase plate, which is a plate with a thickness of t = t1 + t2 and inner and outer radii R respectively, prepared on the upper surface of the glass substrate. in and R out A spatial filter. In R in and R out Within the circular ring, the transmittance T of light with wavelength λ is 10%-20%, and its phase change relative to air at the same height is π / 2 (or -π / 2). The transparent medium with thickness t1 (transmittance greater than 90% at the working wavelength λ) has a thickness of 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 requirement of a transmittance of 10%-20% for light with wavelength λ.

[0093] Example 5

[0094] As shown in Figure 11, most of the structure of the reflective scanning super-resolution optical microscopy system based on light cone illumination in this embodiment is the same as that in Embodiment 1. The difference is that the spatial filter 20 is a phase plate, which is fabricated on the upper surface of a glass substrate with inner and outer radii of R. in and R out A spatial filter. In R in and R out The inner ring contains a metal film of thickness t2, with a transmittance T of 10%-20% for light waves at wavelength λ. In other regions outside the ring, there is a transparent medium of thickness t1 (with a transmittance greater than 90% at the working wavelength λ), with a thickness of t1=λ / 4(n-1), where n is the refractive index of the transparent medium, and its phase change relative to air at the same height is π / 2 (or -π / 2).

[0095] Example 6

[0096] In this embodiment, the structure of the reflective scanning super-resolution optical microscopy system based on light cone illumination is mostly the same as that in Embodiment 1, except that field lens 38 is not required.

[0097] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A reflective scanning super-resolution optical microscopy system based on light cone illumination, characterized in that, include: The sample displacement module is used to move the sample (16); An optical microscopy imaging module is used to perform wide-field imaging of the sample (16) and determine the super-resolution scanning imaging area; A ring-shaped focused beam generating module is used to generate a ring-shaped focused beam. The ring-shaped focused beam generating module includes, in sequence along the light propagation direction, a light source module, a first linear polarizer (4), a conical lens (5), a first positive lens (6), a second positive lens (7), and a third positive lens (8). The first positive lens (6) and the conical lens (5) are placed coaxially and spaced apart. The front focal plane of the second positive lens (7) coincides with the rear focal plane of the first positive lens (6), and the rear focal plane of the second positive lens (7) coincides with the front focal plane of the third positive lens (8). The second positive lens (7) and the third positive lens (8) constitute a first 4f system. The light source module generates a collimated laser beam. After passing through the first linear polarizer (4), the conical lens (5), and the first positive lens (6), the collimated laser beam forms a linearly polarized ring-shaped focused beam and forms a circular focusing line on the rear focal plane of the first positive lens (6). The beam scanning module includes a beam scanning galvanometer (9), a beam scanning lens (10), a first telescope (11), and a reflector (12). The center of the beam scanning galvanometer (9) coincides with the back focal plane of the third positive lens (8). The front focal plane of the beam scanning lens (10) is located at the center of the beam scanning galvanometer (9). The back focal plane of the beam scanning lens (10) coincides with the front focal plane of the first telescope (11), and the two constitute a second 4f system. The back focal plane of the first telescope (11) coincides with the back focal plane of the objective lens (15) in the super-resolution focusing illumination and collection module after being reflected by the reflector (12). The beam scanning galvanometer (9) contains a controller for connecting to a computer. The computer controls the beam scanning galvanometer (9) to perform two-dimensional vibration scanning through the controller of the beam scanning galvanometer (9). The super-resolution focusing illumination and collection module is used to convert the ring-shaped focusing beam into a super-resolution conical focusing beam to form a long focal depth super-resolution focal spot to illuminate the sample (16), collect the super-resolution signal light generated by the reflection and scattering of the sample (16), converge and collimate the super-resolution light signals reflected and scattered, and complete the polarization conversion; the super-resolution focusing illumination and collection module includes an 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 is used to acquire the returned super-resolution signal light and convert it into an electrical signal. The super-resolution optical imaging module also includes a polarization beam splitter (19), a spatial filter (20), a second linear polarizer (21), a second telescope (22), an optical fiber head (23), a photomultiplier tube (26), and a high-speed acquisition card (28). The polarization beam splitter (19) is located between the first positive lens (6) and the second positive lens (7). The spatial filter (20) is located on the focal plane formed after the light is focused by the second positive lens (7) and reflected by the polarization beam splitter (19). The second linear polarizer (21) is located between the spatial filter (20) and the second telescope (22). The front end face of the optical fiber head (23) coincides with the back focal plane of the second telescope (22). The optical fiber head (23) is connected to the photomultiplier tube (26). The photomultiplier tube (26) is connected to the high-speed acquisition card (28). The high-speed acquisition card (28) is used to connect to a computer. A computer is used to control the sample displacement module to move the sample (16), and to scan and receive the electrical signals output by the super-resolution optical imaging module by synchronously controlling the beam scanning galvanometer (9), and to process and obtain the super-resolution microscopic image of the sample in the super-resolution scanning imaging area.

2. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 1, characterized in that: When the ring-focused beam generated by the ring-focused beam generation module is reflected by the beam scanning galvanometer (9), passes through the scanning lens (10) and the first sleeve lens (11), and is reflected by the reflecting mirror (12) to the back focal plane of the objective lens (15); when the beam scanning galvanometer (9) vibrates, the center of the ring-focused beam coincides with the center of the back focal plane of the objective lens (15), and the incident angle of the ring-focused beam changes with the vibration of the beam scanning galvanometer (9); when the beam scanning galvanometer (9) performs two-dimensional scanning, the ring-focused beam is transformed by the objective lens (15) and forms a conical focused light field behind the objective lens (15), thereby generating a long focal depth focused light field, and forming a super-resolution focused light spot on the front focal plane of the objective lens (15). This focused light spot will achieve 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 reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 2, characterized in that: The super-resolution focusing illumination and collection module further includes a quarter-wave plate (13); the super-resolution optical imaging module further includes a polarization beam splitter (19); the optical microscopy imaging module includes a second beam splitter (33); after the linearly polarized annular focused beam emitted from the beam scanning module passes through the quarter-wave plate (13), a circularly polarized circular focused line with an outer diameter smaller than the diameter of the rear focal plane of the objective lens (15) is formed on the rear focal plane of the objective lens (15). This circularly polarized circular focused line is converted into a super-resolution conical focused beam through the objective lens (15) and forms a super-resolution focused spot on the front focal plane of the objective lens (15); this super-resolution focused spot illuminates the sample (16), and the super-resolution signal light generated by reflection and scattering from the sample (16) is collected by the objective lens (15), converged and collimated respectively, and then converted into a linearly polarized annular focused beam and a linearly polarized collimated beam orthogonal to the incident linearly polarization through the quarter-wave plate (13), that is, a linearly polarized super-resolution signal beam. This linearly polarized super-resolution signal beam transmits through the second beam splitter (33) along the original optical path in the reverse direction, passes through the beam scanning module, enters the annular focused beam generation module, and is reflected by the polarization beam splitter (19) placed in the optical path and enters the super-resolution optical imaging module; When the beam scanning galvanometer (9) in the beam scanning module performs two-dimensional scanning, the annular focused beam is converted into a conical focused beam by the objective lens, and the super-resolution focused spot formed on the front focal plane of the objective lens (15) will vibrate along with the beam scanning galvanometer (9), and thus continuous two-dimensional scanning is achieved on the front focal plane of the objective lens (15); the abscissa X(t) and ordinate Y(t) of the position where the super-resolution focused spot illuminates the sample (16) are functions of time t, and the reflected light Er(t) and scattered light Es(t) collected by the objective lens (15) are also functions of time t. The reflected light Er(t) and scattered light Es(t) reflect the sample structure and refractive index information at the corresponding positions.

4. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 2, characterized in that: The super-resolution focused spot is an annular focused light field. The inner and outer surfaces of this annular focused light field are two coaxial and equal conical angle conical surfaces. The numerical aperture NAc corresponding to this annular focused light field satisfies: NAc < NA; where NA represents the numerical aperture of the objective lens (15).

5. The reflection scanning super-resolution optical microscopy system based on light cone illumination according to claim 1, characterized in that: The sample displacement module is a two-dimensional displacement stage (17). The two-dimensional displacement stage (17) is connected to a computer. The sample (16) is horizontally fixed on the two-dimensional displacement stage (17). The computer controls the two-dimensional displacement stage (17) to drive the sample (16) to move two-dimensionally in the XY plane to achieve the initial positioning of the sample; the super-resolution focusing illumination and collection module further includes an axial nano-positioner (14). The axial nano-positioner (14) is connected to a computer. The objective lens (15) is installed on the axial nano-positioner (14). The computer controls the axial nano-positioner (14) to drive the objective lens (15) to move in the Z direction; Alternatively, the sample displacement module is a three-dimensional displacement stage (37), which is connected to a computer. The sample (16) is horizontally fixed on the three-dimensional displacement stage (37), and the computer controls the three-dimensional displacement stage (37) to move the sample (16) in the X, Y, and Z directions.

6. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 5, characterized in that: The super-resolution optical imaging module also includes a five-dimensional adjustment frame (24), and the fiber head (23) is mounted on the five-dimensional adjustment frame (24). Adjusting the five-dimensional adjustment frame (24) can maximize the super-resolution signal light entering the fiber head (23). The inner diameter of the fiber at the front end of the fiber head (23) is smaller than the first zero-point radius of the focal spot of the super-resolution signal light on the back focal plane of the second telescope (22).

7. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 1, characterized in that: The light source module includes a laser (1) and an optical fiber collimator (3). The laser (1) is connected to the optical fiber collimator (3) through a first optical fiber jumper (2). The coherent light emitted by the laser (1) 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 annular focusing beam generating module also includes a field lens (38), the object-side principal plane of which coincides with the back focal plane of the first positive lens (6), and the image-side principal plane of the field lens (38) coincides with the front focal plane of the second positive lens (7) to change the propagation direction of the edge light rays of the annular focusing beam.

8. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 3, characterized in that: The optical microscopic imaging module also includes an incoherent light source (30), a collimating lens (31), a first beam splitter (32), a third telescope (34), and a digital camera (35). The digital camera (35) is connected to a computer. The incoherent light source (30) is located at the front focal point of the collimating lens (31). The light emitted from the incoherent light source (30) is collimated by the collimating lens (31), reflected by the first beam splitter (32), and then passes through the second beam splitter (33) and the quarter-wave plate (13) in sequence to reach the objective lens (1). 5), and the incoherent light is focused on the front focal plane of the objective lens (15) to illuminate the sample (16). The incoherent light generated by the reflection and scattering of the sample (16) is collected by the objective lens (15) and passes through the quarter-wave plate (13), the second beam splitter (33), the first beam splitter (32), and the third telescope (34) in sequence before entering the digital camera (35), 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 reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 5, characterized in that: The spatial filter (20) is a ring-type spatial filter, which includes a glass substrate (201) and a concentric circular aluminum film (202) and a first annular aluminum film (203) covering the glass substrate. The diameter of the circular aluminum film (202) is equal to 2Rin, the inner diameter of the first annular aluminum film (203) is equal to 2Rout, and the outer diameter is equal to the diameter of the glass substrate (201). The transmittance of the glass substrate to light waves with wavelength λ is greater than 90%, and the transmittance of the circular aluminum film (202) and the first annular aluminum film (203) to light waves with wavelength λ is 0. Alternatively, the spatial filter (20) is a ring-shaped shielding spatial filter, which includes a glass substrate (201) and a second ring-shaped aluminum film (204) covering the glass substrate. The inner diameter of the second ring-shaped aluminum film is equal to 2Rin and the outer diameter is equal to 2Rout. 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. Alternatively, the spatial filter (20) is a phase plate, which is a spatial filter with a thickness of t and inner and outer radii of Rin and Rout respectively, prepared on the upper surface of a glass substrate. The transmittance T of light with wavelength λ in the ring between Rin and Rout is 10%-20%, and its phase change relative to air at the same height is π / 2 or -π / 2. Wherein, Rin represents the inner radius of the circular focal line at the back focal plane of the objective lens, and Rout represents the outer radius of the circular focal line at the back focal plane of the objective lens.

10. A reflective scanning super-resolution optical microscopy method based on light cone illumination, employing the reflective scanning super-resolution optical microscopy system as described in any one of claims 1 to 9, the method comprising: Step 1: Use an optical microscopy imaging module to perform wide-field imaging on the sample (16) to determine the super-resolution scanning imaging area; Step 2: After determining the super-resolution scanning imaging area, the ring focusing beam generation module generates a ring focusing beam, and the super-resolution focusing illumination and collection module converts the ring focusing beam into a super-resolution conical focusing beam to form a super-resolution focal spot to illuminate the sample (16), collects the super-resolution signal light generated by the reflection and scattering of the sample (16), and converges and collimates the super-resolution light signals reflected and scattered, and completes polarization conversion. Step 3: The computer-controlled sample displacement module moves the sample (16) to the position to be scanned and turns on the beam scanning galvanometer (9) to scan. At the same time, the super-resolution optical imaging module acquires the super-resolution signal light after convergence, collimation and conversion, and converts it into an electrical signal for input into the computer. Step 4: The computer processes the input electrical signal and combines it with the coordinates X(t) and Y(t) of the super-resolution illumination focal spot moving on 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.

Images