Reflective scanning super-resolution optical microscopic system and method with ultra-long depth of field

Abstract

The invention discloses an ultra-long depth-of-field reflective scanning super-resolution optical microscopic system and method. The system comprises a sample displacement module, an optical microscopic imaging module, an annular focusing light beam generation module, a light beam scanning module, a super-resolution focusing illumination and collection module, a super-resolution optical imaging module and a computer. By utilizing the advantages of annular focusing light beam illumination, high focusing efficiency of a traditional objective lens and high light beam scanning speed, efficient super-long-focal-depth super-resolution focusing illumination is realized, and then quick super-long-depth-of-field super-resolution optical microscopic imaging can be realized by adopting a traditional optical lens. The method can be applied to non-labeled super-resolution microscopic rapid imaging of biological samples, and can also be applied to the fields of industrial related super-resolution microscopic detection and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of visible light microscopy, and in particular relates to a reflective scanning super-resolution optical microscopy system and method with an ultra-long depth of field. Background Art

[0002] Existing microscopy techniques can be roughly divided into two categories: near-field microscopy and far-field microscopy. Near-field microscopy includes methods such as microsphere-assisted imaging and near-field scanning microscopy. Near-field microscopy methods can achieve super-resolution microscopic imaging, but its working distance is less than the working wavelength, which greatly limits its 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, random light reconstruction optical microscopy, etc. However, these techniques require fluorescent molecular labeling of the sample to be detected. 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 of super-resolution optical microscopy technology. In recent years, a few experiments have been reported 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, and the imaging speed is slow. At the same time, the depth of field and resolution of existing super-resolution imaging are contradictory, making it difficult to take both into account.

[0003] Technical background references: [1] Edward TF Rogers, Jari Lindberg, TapashreeRoy, Salvatore Savo, John E. Chad, Mark R. Dennis and Nikolay I. Zheludev. “A super-oscillatorylens optical microscope for subwavelength imaging,” Nature Materials, Vol. 11, pp. 432-435 (2012). [2] Edward TF Rogers and Nikolay IZheludev. "Optical super-oscillations: sub-wavelength light focusing and super-resolution imaging," Journal of Optics, Vol. 15, pp. 094008(2013). [3] Y Yan, L Li, CFeng, W Guo, S Lee, M Hong. "Microsphere-CoupledScanning Laser Confocal Nanoscope for Sub-Diffraction-Limited Imaging at 25nm LateralResolution in the Visible Spectrum," ACS Nano, Vol. 8, pp. 1809-1816 (2014). [4] Fei Qin, Kun Huang, Jianfeng Wu, JinghuaTeng,Cheng-Wei Qiu, andMinghui Hong. “A Supercritical Lens Optical Label-Free Microscopy: Sub-Diffraction Resolution and Ultra-Long Working Distance,” Advanced Materials, Vol. 28, 1602721 (2017). [5] Yi Zhou, Kun Zhang, Jinlong Wang, Zhengguo Shang, Gaofeng Liang, Zhihai Zhang, Zhongquan Wen, Yufei Liu, Gang Chen, “Label-free super-resolution microscopy based on non-diffractionsuperoscillation beam (NDSB) illumination,” Optics and Lasers in Engineering, Vol. 168, pp. 107690(2023) For example, patent document CN118465998A discloses a reflective super-resolution optical microscopy system and method based on ultra-long focal depth illumination. However, this method uses a sample module to drive the sample to move and achieve scanning imaging. Therefore, the imaging speed is limited by the speed of sample movement, and it is difficult to achieve real-time imaging. Especially for samples in solution, the rapid movement of the sample module will cause the sample to be unable to follow the moving speed of the sample module, resulting in blurred imaging of the sample. At the same time, there is a contradiction between the depth of field and resolution of super-resolution imaging, and it is difficult to take both into account. Summary of the invention

[0004] The purpose of the present invention is to provide a reflective scanning super-resolution optical microscopy system and method with ultra-long depth of field in view of the deficiencies in the prior art, so as to simultaneously achieve ultra-long depth of field and super-resolution while improving reliability and imaging speed.

[0005] In a first aspect, the reflective scanning super-resolution optical microscopy system with ultra-long depth of field described in the present invention comprises: A sample displacement module, used to drive the sample to move; Optical microscopy module, used to perform wide-field imaging of the sample and determine the super-resolution scanning imaging area; An annular focused beam generating module is used to generate an annular focused beam. The annular focused beam generating module includes a light source module, a first linear polarizer, an aconic lens, a first positive lens, a second positive lens and a third positive lens in sequence along the light propagation direction, wherein the first positive lens and the aconic lens are coaxially arranged with an interval, the front focal plane of the second positive lens coincides with the back focal plane of the first positive lens, the back focal plane of the second positive lens coincides with the front focal plane of the third positive lens, and the second positive lens and the third positive lens form a first 4f system; the light source module generates a collimated laser beam, and the collimated laser beam forms a linearly polarized annular focused beam after passing through the first linear polarizer, the aconic lens and the first positive lens, and forms a circular focal line on the back focal plane of the first positive lens; A beam scanning module comprises a beam scanning galvanometer, a beam scanning lens, a first sleeve mirror and a reflector, wherein the center position of the beam scanning galvanometer coincides with the rear focal plane of the third positive lens; the front focal plane of the beam scanning lens is located at the center position of the beam scanning galvanometer; the rear focal plane of the beam scanning lens coincides with the front focal plane of the first sleeve mirror, and the two constitute a second 4f system; the rear focal plane of the first sleeve mirror coincides with the rear focal plane of the objective lens in the super-resolution focusing illumination and collection module after being reflected by the reflector; the beam scanning galvanometer comprises a controller, the controller is used to be connected to a computer, and the computer controls the beam scanning galvanometer to perform two-dimensional vibration scanning through the controller of the beam scanning galvanometer; The super-resolution focusing illumination and collection module is used to convert the annular focusing beam into a super-resolution conical focusing beam, form a super-resolution focal spot with an ultra-long focal depth to illuminate the sample, collect the super-resolution signal light generated by the reflection and scattering of the sample within the focal depth range, and focus and polarize it; the super-resolution focusing illumination and collection module includes an objective lens, and the sample is located in the ultra-long focal depth range before and after the front focal plane of the objective lens (the axial half-height full width is 2△Z), that is, the focal depth length is 2△Z (the half-height full width of the axial intensity distribution, △Z>4λ f / πD, where λ is the wavelength, f is the focal length, and D is the pupil diameter of the objective lens); A super-resolution optical imaging module is used to obtain the collimated and converted super-resolution signal light and convert it into an electrical signal; the super-resolution optical imaging module also includes a polarization beam splitter, a second linear polarizer, a second sleeve mirror, an optical fiber head, a photomultiplier tube and a high-speed acquisition card, the polarization beam splitter is located between the first linear polarizer and the axle lens, the polarization beam splitter, the second linear polarizer, the second sleeve mirror and the optical fiber head are arranged in sequence along the propagation direction of the light after being reflected by the polarization beam splitter, and the front end face of the optical fiber head coincides with the rear focal plane of the second sleeve mirror, the optical fiber head is connected to the photomultiplier tube, the photomultiplier tube is connected to the high-speed acquisition card, and the high-speed acquisition card is used to connect to a computer; The computer is used to control the sample displacement module to drive the sample to move, and synchronously control the beam scanning galvanometer to scan and receive the electrical signal output by the super-resolution optical imaging module, and process it to obtain a super-resolution microscopic image of the sample in the super-resolution scanning imaging area.

[0006] Optionally, the annular focused beam generating module also includes a field lens arranged between the first positive lens and the second positive lens, the object side principal surface of the field lens coincides with the rear focal plane of the first positive lens, and the image side principal surface of the field lens coincides with the front focal plane of the second positive lens, so as to change the propagation direction of the edge light of the annular focused beam.

[0007] Optionally, when the annular focused beam generated by the annular focused 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 reflector 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 is converted by the objective lens to form a conical focused beam behind the objective lens, thereby forming an ultra-long focal depth focused light field, and forming an ultra-long focal depth focused light spot with a half-height full width of 2△Z in the Z-axis direction before and after the front focal plane of the objective lens, where △Z>4λ f / πD, where λ is the wavelength, f is the focal length, and D is the pupil diameter of the objective lens. The ultra-long focal depth focused spot will achieve continuous XY two-dimensional scanning in the front focal plane of the objective lens as the beam scanning galvanometer vibrates, and the scanning range in the Z-axis direction covers the ultra-long focal depth coverage; the position of the rear focal plane of the objective lens in the Z-axis direction is controlled to control the scanning range of the objective lens in the Z-axis direction, that is, [Z f -△Z, Z f +△Z], where Z f is the position of the front focal plane of the objective lens (15) in the Z-axis direction.

[0008] 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 microscopy imaging module includes a second beam splitter; after the circular focusing line polarized light beam emitted from the light beam scanning module passes through the quarter wave plate, a circular polarized circular focusing line with an outer diameter smaller than the diameter of the rear focal plane of the objective lens is formed on the rear focal plane of the objective lens; the circular polarized circular focusing line is converted into a super-resolution conical focusing light beam after passing through the objective lens, and an ultra-long focal depth super-resolution focusing spot is formed before and after the front focal plane of the objective lens, and the focal depth length is 2△Z (half-maximum full width of the axial intensity distribution, △Z>4λ f / πD, where λ is the wavelength, f is the focal length, and D is the pupil diameter of the objective lens), the half-height full width of the ultra-long focal depth super-resolution focusing spot is less than 0.5λ / NA, and the first zero point radius of the super-resolution focal spot of the ultra-long focal depth super-resolution focusing spot (that is, the distance from the focal spot intensity peak position to the first zero point position) is less than 0.61λ / NA. The ultra-long focal depth super-resolution focusing spot illuminates the sample, and the super-resolution signal light generated by the reflection and scattering of the sample is collected by the objective lens and respectively converged and collimated, and then converted into a linearly polarized collimated light beam orthogonal to the incident line polarization through a quarter wave plate, that is, a linearly polarized super-resolution signal beam. The linearly polarized super-resolution signal beam passes through the second beam splitter along the opposite direction of the original optical path, passes through the beam scanning module, enters the annular focusing beam generating module, and is reflected by the polarization beam splitter placed in the optical path to enter the super-resolution optical imaging module; wherein, λ represents the wavelength of the collimated laser beam, and NA represents the numerical aperture of the objective lens.

[0009] When the beam scanning galvanometer in the beam scanning module performs two-dimensional scanning, the annular focused beam is converted into a conical focused beam by the objective lens. The ultra-long focal depth and super-resolution focused light spot formed before and after the front focal plane of the objective lens will follow the vibration of the beam scanning galvanometer, thereby realizing continuous two-dimensional XY scanning on the front focal plane of the objective lens. The scanning range on the Z axis is [Z f -△Z,Z f +△Z]; the horizontal coordinate X(t) and vertical coordinate Y(t) of the position of the ultra-long focal depth super-resolution focused spot on the sample are functions of time t, and the reflected light E collected by the objective lens r (t) and scattered light E s (t) is also a function of time, the reflected light E r (t) and scattered light E s (t) reflects the sample structure and refractive index information within the ultra-long focal depth at the corresponding position (X(t), Y(t)).

[0010] Preferably, the ultra-long focal depth super-resolution focusing spot is an annular focusing light field, the inner and outer surfaces of the annular focusing light field are two coaxial conical surfaces with the same cone angle, and the numerical aperture NA corresponding to the annular focusing light field is c , and satisfies: NA c<NA。

[0011] Preferably, to achieve three-dimensional scanning of the sample, the sample displacement module has two structures.

[0012] The first type is that the sample displacement module is a two-dimensional displacement stage, which is connected to a computer, and 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 the initial positioning of the sample; the super-resolution focusing illumination and collection module also includes an axial nanopositioner, which is connected to a computer, and the objective lens is installed on the axial nanopositioner. The computer controls the axial nanopositioner to move the objective lens in the Z direction. The beam scanning module realizes two-dimensional scanning of the sample, and realizes three-dimensional scanning in combination with the axial nanopositioner.

[0013] The second type is that 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 the X, Y, and Z directions. The X and Y directions are used to achieve the initial positioning of the sample. The light beam scanning module achieves two-dimensional scanning of the sample, combined with the Z-direction displacement of the three-dimensional displacement stage to achieve three-dimensional scanning, avoiding the imaging influence caused by the slight movement of the objective lens and achieving better imaging effect.

[0014] Preferably, the super-resolution optical imaging module further comprises a five-dimensional adjustment frame, on which the optical fiber head is mounted, and the five-dimensional adjustment frame can be adjusted to maximize the super-resolution signal light entering the optical fiber head; The inner diameter of the optical fiber at the front end face of the optical fiber head is smaller than the first zero point radius of the focal spot of the super-resolution signal light on the rear focal plane of the second sleeve mirror, thereby better ensuring that the super-resolution imaging resolution reaches the optimal level.

[0015] Preferably, the light source module includes a laser and a fiber collimator, the laser is connected to 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 the coherent light and outputs a collimated laser beam.

[0016] Preferably, the optical microscopic 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, and the incoherent light source is located at the front focus of the collimating lens; the light emitted by the incoherent light source is collimated by the collimating lens and reflected by the first beam splitter, and then passes through the second beam splitter and the quarter wave plate in sequence to reach the objective lens, and converges into incoherent light on the front focal plane of the objective lens to illuminate the sample, and the incoherent light generated by the reflection and scattering of 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 telescope in sequence to enter the digital camera, so that the sample is imaged on the digital camera and sent to the computer through the camera signal line, and the computer displays the wide-field microscopic image of the sample. This facilitates wide-field observation of the sample to determine the required super-resolution scanning imaging area.

[0017] In a second aspect, the reflective scanning super-resolution optical microscopy method with an ultra-long depth of field described in the present invention adopts the above-mentioned reflective scanning super-resolution optical microscopy system, and the method comprises the following steps: Step 1: Use an optical microscopy imaging module to perform wide-field imaging of the sample and determine the super-resolution scanning imaging area; Step 2: after determining the super-resolution scanning imaging area, the annular focused beam generating module generates an annular focused beam, and the super-resolution focused illumination and collection module converts the annular focused beam into a super-resolution conical focused beam, forming an ultra-long focal depth super-resolution focused light spot with a half-height full width of 2△Z in the Z-axis direction to illuminate the sample (16), collect the super-resolution signal light generated by the reflection and scattering of the sample within the ultra-long focal depth range, and respectively converge and collimate the emitted light and the scattered light, and convert the polarization state; Step 3: The computer controls the sample displacement module to move the sample to the position to be scanned, and turns on the beam scanning galvanometer for scanning. At the same time, the super-resolution optical imaging module obtains the collimated and converted super-resolution signal light, and converts it into an electrical signal and inputs it into the computer; Step 4: The computer processes the input electrical signal and combines the coordinates X(t) and Y(t) of the super-resolution illumination focus on the sample 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), Z±△Z) of the sample in the super-resolution scanning imaging area, where Z f is the Z-axis position of the objective lens focal plane.

[0018] The present invention has the following advantages: (1) High-efficiency super-resolution focused illumination: The present invention can efficiently generate a ring-shaped focused beam through a carefully designed ring-shaped focused beam generation module. Subsequently, the ring-shaped focused beam is converted into a super-resolution conical focused beam with an ultra-long focal depth by using a super-resolution focused illumination and collection module, forming a high-precision ultra-long focal depth super-resolution focal spot to illuminate the sample. This process fully utilizes the advantages of the high focusing efficiency of traditional objective lenses, realizes high-efficiency super-resolution focused illumination, and significantly improves the imaging depth of field and the imaging signal-to-noise ratio.

[0019] (2) Fast 2D scanning: The beam scanning module uses beam scanning galvanometer technology to achieve fast 2D scanning of the beam. This scanning method is not only fast but also highly accurate, and can perform super-resolution imaging of a large area in a short time. This feature greatly improves the efficiency of imaging, allowing users to quickly obtain a large amount of high-quality image data to obtain information about sample changes over time.

[0020] (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 that it can achieve ultra-long depth of field far-field super-resolution two-dimensional microscopy rapid imaging of unlabeled samples. 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 fields such as biological and industrial detection more convenient and efficient.

[0021] (4) Three-dimensional imaging capability: By performing non-labeled far-field super-resolution two-dimensional microscopic imaging on samples at different axial positions, the present invention can obtain super-resolution two-dimensional ultra-long depth-of-field microscopic images of different cross-sections of the sample, and then realize three-dimensional 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.

[0022] (5) When the structure distribution in the Z direction is not important, the ultra-long focal depth super-resolution focused beam can be used to directly scan the three-dimensional structure in one go to obtain ultra-long depth of field imaging of the sample without moving the sample or the objective lens in the Z axis. (6) High reliability and imaging quality: The present invention ensures the reliability and stability of imaging by precisely controlling the optical path coordination and signal processing between modules. From the generation of annular focused beams, beam scanning, super-resolution focused illumination to signal collection and processing, each link has been carefully designed and optimized to ensure a high level of final imaging quality. In addition, the optical components and signal acquisition equipment used have high performance indicators, which further improves the overall performance and imaging quality of the system.

[0023] In summary, the reflective scanning super-resolution optical microscopy system and method with ultra-long depth of field of the present invention show significant advantages in terms of efficient super-resolution focused illumination, ultra-long depth of field imaging, fast two-dimensional scanning, label-free far-field super-resolution microscopy imaging, three-dimensional imaging capability, super-resolution three-dimensional imaging capability, high reliability and imaging quality, etc., and provide strong technical support for the fields of biomedicine, industrial detection, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the structure of the reflective scanning super-resolution optical microscopy system with ultra-long depth of field in Example 1.

[0025] Figure 2 Schematic diagram of controlling the size of the annular focused beam using the axicon, the third positive lens and the first 4f system in Example 1.

[0026] Figure 3 It is a schematic diagram of forming an ultra-long focal depth and super-resolution focused light field in Example 1.

[0027] Figure 4 Parameter relationship diagram of the ultra-long focal depth super-resolution focused light field in Example 1.

[0028] Figure 5 Schematic diagram of scanning of the ultra-long focal depth and super-resolution focused light spot on the focal plane of the objective lens in Example 1.

[0029] Figure 6 Graph showing the relationship between the objective lens, the full width at half maximum of the focal spot and the system resolution under the condition of annular focused beam illumination in Example 1.

[0030] Figure 7 Schematic diagram of the structure of the reflective scanning super-resolution optical microscopy system with ultra-long depth of field in Example 2.

[0031] In the figure: 1, laser, 2, first fiber jumper, 3, fiber 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 sleeve mirror, 12, reflector, 13, quarter wave plate, 14, axial nanopositioner, 15, objective lens, 16, sample, 17, two-dimensional translation stage, 18, scanning signal line, 19, Polarization beam splitter, 20. Second linear polarizer, 21. Second sleeve mirror, 22. Fiber optic head, 23. Five-dimensional adjustment frame, 24. Second fiber optic jumper, 25. Photomultiplier tube, 26. Collection signal line, 27. High-speed acquisition card, 28. Incoherent light source, 29. Collimating lens, 30. First beam splitter, 31. Second beam splitter, 32. Third sleeve mirror, 33. Digital camera, 34. Camera signal line, 35. Three-dimensional translation stage, 36. Field mirror. DETAILED DESCRIPTION

[0032] In order to enable a more detailed understanding of the features and technical contents of the embodiments of the present invention, the implementation of the embodiments of the present invention is described in detail below in conjunction with the accompanying drawings. The attached drawings are for reference only and are not intended to limit the embodiments of the present invention.

[0033] Embodiment 1 like Figure 1 As shown, the reflective scanning super-resolution optical microscopy system with ultra-long depth of field in this embodiment includes: The sample displacement module is used to drive the sample 16 to move.

[0034] The optical microscopy imaging module is used to perform wide-field imaging on the sample 16 and determine the super-resolution scanning imaging area.

[0035] The annular focused beam generating module is used to generate an annular focused beam.

[0036] The beam scanning module is used to realize fast two-dimensional scanning of the light beam.

[0037] The super-resolution focusing illumination and collection module is used to convert the annular focusing beam into a super-resolution conical focusing beam, forming an ultra-long focal depth super-resolution focal spot to illuminate the sample 16, and collect the super-resolution signal light generated by the reflection and scattering of the sample 16, and focus and collimate it respectively, and then perform polarization conversion.

[0038] The super-resolution optical imaging module is used to obtain the super-resolution signal light after focusing, collimation and polarization conversion, and convert it into an electrical signal.

[0039] The computer (not shown) is used to control the sample displacement module to move the sample 16. The computer controls the beam scanning galvanometer 9 to scan and receive the electrical signal output by the super-resolution optical imaging module, and processes the super-resolution microscopic image of the sample 16 in the super-resolution scanning imaging area.

[0040] This system achieves efficient ultra-long focal depth super-resolution focused illumination by utilizing the advantages of annular focused beam illumination, high focusing efficiency of traditional objective lenses, and fast beam scanning speed, and can then use traditional optical lenses to achieve ultra-long depth of field and fast super-resolution optical microscopy imaging; achieve non-labeled far-field super-resolution two-dimensional microscopy fast imaging through two-dimensional beam scanning; obtain super-resolution two-dimensional microscopy images of different cross sections of samples by performing non-labeled far-field super-resolution two-dimensional microscopy imaging on samples at different axial positions, and then achieve three-dimensional; ultra-long focal depth super-resolution focused light fields can also be used to directly achieve one-time ultra-long depth of field imaging of three-dimensional structures. This system can be applied to non-labeled super-resolution microscopy fast imaging of biological samples, and can also be applied to industrial-related super-resolution microscopy detection and other fields.

[0041] The following is a detailed description of each module: like Figure 1 and Figure 2 As shown, the optical microscopic imaging module includes an incoherent light source 28, a collimating lens 29, a first beam splitter 30, a second beam splitter 31, a third tube mirror 32 and a digital camera 33. The digital camera 33 is connected to a computer. The incoherent light source 28 is located at the front focus of the collimating lens 29. The light emitted by the incoherent light source 28 is collimated by the collimating lens 29 and reflected by the first beam splitter 30. It then passes through the second beam splitter 31 and the quarter wave plate 13 in sequence to reach the objective lens 15, and converges 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 through the quarter wave plate 13, the second beam splitter 31, the first beam splitter 30, and the third tube mirror 32 in sequence to enter the digital camera 33, so that the sample 16 is imaged on the digital camera 33 and sent to the computer, and the computer displays the wide-field microscopic image of the sample 16.

[0042] like Figure 1 and Figure 2 As shown, the annular focused beam generating module includes a laser 1, a fiber collimator 3, a first linear polarizer 4, an aconic lens 5, a first positive lens 6, a field lens 36, a second positive lens 7 and a third positive lens 8 in sequence along the light propagation direction. The laser 1 is connected to the fiber collimator 3 through the 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 first positive lens 6 is coaxially spaced with the aconic lens 5, the front focal plane F2 of the second positive lens 7 coincides with the back focal plane F1' of the first positive lens 6, the back focal plane (i.e. F'2) of the second positive lens 7 coincides with the front focal plane (i.e. F3) of the third positive lens 8, and the second positive lens 7 and the third positive lens 8 constitute a first 4f system. The light source module (including the laser 1 and the fiber collimator 3) generates a collimated laser beam, which forms a linearly polarized annular focused beam after passing through the first linear polarizer 4, the aconical lens 5 and the first positive lens 6, and forms a circular focus line on the rear focal plane of the first positive lens 6. The first principal surface of the field lens 36 coincides with the rear focal plane of the first positive lens 6, and the second principal surface of the field lens 36 coincides with the front focal plane of the second positive lens 7 respectively; so as to change the propagation direction of the edge light of the annular focused beam to adapt to different objective lenses 15, so that the annular focused beam can be transmitted through the objective lens 15 without obstruction.

[0043] like Figure 1 and Figure 2As shown, the beam scanning module includes a beam scanning galvanometer 9, a beam scanning lens 10, a first sleeve mirror 11 and a reflector 12, wherein the center position 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 position 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 sleeve mirror 11, and the two constitute a second 4f system; the back focal plane of the first sleeve mirror 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, and the controller is used to connect to a computer, and the computer controls the beam scanning galvanometer 9 to perform two-dimensional vibration scanning through the controller of the beam scanning galvanometer 9.

[0044] like Figure 1 and Figure 2 As shown, the super-resolution focusing illumination and collection module includes a quarter wave plate 13 and an objective lens 15, the rear focal plane of the objective lens 15 coincides with the rear focal plane of the first sleeve lens 11, and the sample 16 is located in the ultra-long focal depth range before and after the front focal plane of the objective lens 15. The objective lens 15 uses a traditional microscope objective lens, which can be a dry lens, a water lens, an oil lens or a solid lens.

[0045] like Figure 1 and Figure 2 As shown, the sample displacement module is a two-dimensional displacement stage 17, which is connected to a computer via a scanning signal line 18 (not shown in the figure), and 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. When the sample displacement module is a two-dimensional displacement stage 17, the super-resolution focusing illumination and collection module also includes an axial nanopositioner 14, which is connected to a computer via a positioning signal line, and an objective lens 15 is mounted on the axial nanopositioner 14. The computer controls the axial nanopositioner 14 to drive the objective lens 15 to move in the Z direction.

[0046] like Figure 1 and Figure 2 As shown, the super-resolution optical imaging module includes a polarization beam splitter 19, a second linear polarizer 20, a second sleeve mirror 21, an optical fiber head 22, a photomultiplier tube 25 and a high-speed acquisition card 27. The polarization beam splitter 19 is located between the first linear polarizer 4 and the conical lens 5. The polarization beam splitter 19, the second linear polarizer 20, the second sleeve mirror 21 and the optical fiber head 22 are arranged in sequence along the propagation direction of the light after being reflected by the polarization beam splitter 19, and the front end face of the optical fiber head 22 coincides with the rear focal plane of the second sleeve mirror 21. The optical fiber head 22 is connected to the photomultiplier tube 25, the photomultiplier tube 25 is connected to the high-speed acquisition card 27, and the high-speed acquisition card 27 is used to connect to a computer.

[0047] In an embodiment of the present application, a reflective scanning super-resolution optical microscopy method with an ultra-long depth of field adopts the above-mentioned reflective scanning super-resolution optical microscopy system, and the method comprises: 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. Specifically: The laser 1 is first turned off and does not emit coherent light. The light emitted by the incoherent light source 28 is collimated by the collimating lens 29 and reflected by the first beam splitter 30, and then passes through the second beam splitter 31 and the quarter wave plate 13 in sequence to reach the objective lens 15, and converges 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 through the quarter wave plate 13, the second beam splitter 31, the first beam splitter 30, and the third sleeve mirror 32 in sequence to enter the digital camera 33, so that the sample 16 is imaged on the digital camera 33 and sent to the computer through the camera signal line 34. The computer displays the wide-field microscopic image of the sample 16, thereby facilitating wide-field observation of the sample 16, so as to facilitate determination of the required super-resolution scanning imaging area. If the sample 16 is not in the super-resolution scanning imaging area, the computer can control the two-dimensional displacement stage 17 to drive the sample 16 to move so that the sample 16 is located in the super-resolution scanning imaging area.

[0048] Step 2: After determining the super-resolution scanning imaging area, the annular focused beam generation module generates an annular focused beam, and the super-resolution focused illumination and collection module converts the annular focused beam into a super-resolution conical focused beam, forming an ultra-long focal depth super-resolution focused light spot with a half-height full width of 2△Z in the Z-axis direction to illuminate the sample 16, collect the super-resolution signal light reflected and scattered by the sample 16 within the ultra-long focal depth range, and respectively converge and collimate the emitted light and scattered light, and convert the polarization state. Specifically: The incoherent light source 28 does not emit light, the laser 1 is turned on, and the laser 1 emits coherent light with a wavelength of λ, which is transmitted to the fiber collimator 3 through the first fiber jumper 2. The fiber collimator 3 collimates the coherent light and outputs a collimated laser beam. The collimated laser beam passes through the first linear polarizer 4, the conical lens 5, and the first positive lens 6 to form an annular focused beam (a P-polarized annular beam). After the P-polarized annular focused beam passes through the first 4f system, the beam scanning module, and the quarter-wave plate 13, a circular focusing 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. After passing through the objective lens 15, the circularly polarized circular focusing line is converted into a super-resolution conical focused beam, and an ultra-long focal depth super-resolution focused light spot is formed before and after the front focal plane of the objective lens 15. The focal depth of the ultra-long focal depth super-resolution focused light spot is greater than 4λ. f / πD, the full width at half maximum is less than 0.5λ / NA, the first zero point radius of the super-resolution focal spot of the ultra-long focal depth super-resolution focusing spot (that is, the distance from the focal spot intensity peak position to the first zero point position) is less than 0.61λ / NA, the ultra-long focal depth super-resolution focusing spot illuminates the sample 16, the super-resolution signal light reflected and scattered by the sample 16 is collected by the objective lens 15, and after being converged and collimated respectively, it is converted into an S-polarized annular focused beam and a collimated beam orthogonal to the incident linearly polarized annular focused beam through the quarter wave plate 13, the S linearly polarized beam passes through the second beam splitter 31 along the opposite direction of the original optical path, passes through the beam scanning module, enters the annular focused beam generating module, and is reflected by the polarization beam splitter 19 placed in the optical path to enter the super-resolution optical imaging module.

[0049] Figure 2 A method for generating a P-polarized annular focused beam is given. Specifically, the fiber collimator 3 collimates the coherent light and outputs a collimated laser beam with a radius of R (by selecting fiber collimators with different numerical apertures, collimated laser beams with different radii can be obtained, and then super-resolution conical focused beams with different focal depths can be obtained). The collimated laser beam with a radius of R passes through the linear polarizer 4 and becomes a P-polarized collimated beam with a radius of R. The P-polarized collimated beam passes through two coaxially placed laser beams with a spacing of d After the conical lens 5 and the first positive lens 6, a P-polarized annular focused beam is formed, and a central radius R' is formed on the rear focal plane of the first positive lens 6. c The central radius R' of the circular focal line can be changed by changing the focal length of the first positive lens 6. c The P polarized annular 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 of c =R' c × f 3 / f 2, f 2 represents the focal length of the second positive lens 7, f 3 represents the focal length of the third positive lens 8. By selecting the second positive lens 7 and the third positive lens 8 with different focal lengths, circular focal lines of different sizes can be obtained. Further, the circular focal line is projected onto the rear focal plane F' of the first tube mirror 11 through the second 4f system composed of the beam scanning lens 10 and the first tube mirror 11. t (also the rear focal plane of objective lens 15), the magnification of the second 4f system is K. Therefore, it can be controlled by f 1. f 2. f 3 to control the center radius R of the final circular focal line c =KR' c × f3 / f 2。

[0050] Figure 3 The illumination working schematic for forming an ultra-long depth-of-focus super-resolution focusing light field is given. The left-handed circularly polarized annular focusing beam illuminates the rear focal plane of the objective lens 15, and a circular focusing line with a central diameter of 2R is formed on the rear focal plane of the objective lens 15. The inner radius of the circular focusing line is R c , the outer radius is R in , and the central radius R out = (R c + R out ) / 2. After passing through the objective lens 15, the left-handed circularly polarized annular focusing beam is transformed into a super-resolution conical focusing beam with a cone angle of θ in , and an ultra-long depth-of-focus super-resolution focusing light field is formed along the optical axis before and after the front focal plane of the objective lens 15. The numerical aperture NA c of the super-resolution conical focusing beam is NA c = n × sin(θ c ), and 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 ultra-long depth-of-focus super-resolution focusing light field is less than 0.5λ / NA, and the first zero radius of the super-resolution focal spot of the ultra-long depth-of-focus super-resolution focusing 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 , the larger the corresponding R c (R c is less than the radius of the rear focal plane of the objective lens 15), and the closer the corresponding R out and R in are (i.e., the smaller the width of the circular focusing line), the narrower the spatial frequency of the ultra-long depth-of-focus super-resolution focusing light field. In practice, we control the radius R of the collimated laser beam and adjust the focal length f 1 of the first positive lens 6 to control R' c, thereby controlling the radius of the circular focusing line. Then, since the annular focusing beam corresponding to the circular focusing line will undergo significant diffraction during propagation, resulting in an increase in width and unable to ensure that the circular focusing line transmitted to the rear focal plane of the objective lens 15 can maintain an extremely 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 efficiently and high-quality relay the annular focusing beam (corresponding to the circular focusing line), so that the numerical aperture NA of the finally formed super-resolution conical focusing beam c 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 focusing line of the annular focusing beam on the one hand, and plays a role in relaying the transmission of the annular focusing beam on the other hand, so as to ensure that the circular focusing line on the front focal plane of the first 4f system is accurately projected onto the rear focal plane of the first 4f system, and then further through the second 4f system composed of the scanning lens 10 and the first tube lens 11, the annular focusing beam is further projected onto 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 annular focusing beam, ensuring the high-quality illumination of the objective lens 15 by the annular focusing beam, and further ensuring that the generated super-resolution focal spot reaches the minimum and the depth of focus reaches the maximum, so as to ensure that the system reaches the best resolution and depth of field.

[0051] 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 focusing 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 = K f 1tan( β ) f 3 / f 2 (where β is the cone angle of the conical beam emitted by the cone lens 5, and tan() is the tangent function), determine f 1, f 2 and f 3 ( f 3 needs to have a sufficient length so that the third positive lens 8 will not have spatial interference with subsequent optical elements.

[0052] Figure 4 The parameters FWHM (full width at half maximum), r0 (radius of the first zero point) and SR (sidelobe peak ratio, i.e. the ratio of the maximum sidelobe intensity to the center intensity) of the ultra-long focal depth super-resolution focused light field and NA are given when the numerical aperture NA of the objective lens 15 is 0.95. c It can be seen that when NA c ≥0.9, the generated ultra-long focal depth super-resolution focused light field has a FWHM≤0.399λ (less than 0.5λ / NA), r0≤0.426λ (less than 0.61λ / NA), and SR=0.162. c So that it satisfies: 0.9≤NA c <0.95, you can achieve ultra-long focal depth super-resolution illumination light field with FWHM≤0.399λ, r0≤0.426λ, SR=0.162. Note: This part is exactly the same as the just-finalized patent. Please check again to see if some parameters are different.

[0053] like Figure 5 As shown in FIG. 1 , it is a schematic diagram of scanning the ultra-long focal depth super-resolution focused light spot on the focal plane of the objective lens 15. The beam scanning module realizes two-dimensional scanning of the annular beam. At different times, the annular focused beam enters the objective lens 15 at different angles. When the annular focused beam reaches the rear focal plane of the objective lens 15, the annular focused beam always presents a circular ring with the center of the rear focal plane of the objective lens 15 as the center. At a given time t, the inclination angle of the incident annular focused beam is θ s (t), at this time, an ultra-long focal depth super-resolution focusing spot with a half-width length of 2△Z is formed before and after the rear focal plane of the objective lens 15 (in the Z direction). The offset of the focusing spot in the XY direction (relative to the optical axis Z) is d(t). By changing the incident angle θ s (t), that is, the offset d(t) can be changed. At different times, the light intensity distribution of the illuminating annular focused beam on the rear focal plane of the objective lens is always a circular ring centered at the center of the rear focal plane, and the inner and outer radii of the circular ring are R in and R out .

[0054] like Figure 6The figure shows the relationship between the objective lens (the numerical aperture NA of this objective lens is greater than 1), the half-height full width of the focal spot and the system resolution under the condition of annular focused beam illumination. The ordinate in the figure is the half-height full width of the point spread function of the objective lens, in units of wavelength λ, the abscissa is the lateral half-height full width of the super-resolution focal spot of the ultra-long focal depth super-resolution focused spot, in units of wavelength λ, and the contour line is the system resolution (i.e., the minimum line width of the amplitude-type grating that the system can resolve, and the imaging fringe contrast is better than 11%), in units of wavelength λ. When the half-height full width of the point spread function of the objective lens (a single-point focusing super-diffraction lens can be used) is 0.6λ and the lateral half-height full width of the super-resolution focal spot (the part that illuminates the sample) 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 lateral half-height full width of the super-resolution focal spot are both 0.35λ, the system resolution is better than 0.2λ.

[0055] Step 3: The computer controls the sample displacement module to move the sample 16 to the position to be scanned, and turns on the beam scanning galvanometer 9 for scanning. At the same time, the super-resolution optical imaging module obtains the super-resolution signal light after convergence, collimation and conversion, and converts it into an electrical signal and inputs it into the computer.

[0056] Step 4: The computer processes the input electrical signal and combines 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), Z(t) of the sample in the super-resolution scanning imaging area. f ±△Z), where Z f ±△Z means the focal depth [Z f -△Z,Z f +△Z], where Z f is the Z-axis position of the focal plane of the objective lens 15.

[0057] In the embodiment of the present application, the specific processing process of step three and step four is as follows: the S-polarized light beam (i.e., the super-resolution signal light reflected by the polarization beam splitter 19, which is converged, collimated, and converted) passes through the second linear polarizer 20, converges on the rear focal plane of the second sleeve mirror 21, and is coupled into the optical fiber head 22, and then enters the photomultiplier tube 25 through the second optical fiber jumper 24, and is converted into an analog electrical signal by the photomultiplier tube 25. The analog electrical signal is collected and converted into a digital electrical signal by the high-speed acquisition card 27 through the acquisition signal line 26, and is input into the computer through the data line. The computer controls the beam scanning galvanometer 9 in the beam scanning module to perform two-dimensional scanning, and the super-resolution focusing spot with an ultra-long focal depth formed by the conical focusing light beam before and after the front focal plane of the objective lens 15 will realize continuous XY two-dimensional scanning in the front focal plane of the objective lens 15 with the vibration of the beam scanning galvanometer 9, and the scanning range on the Z axis [Z f -△Z,Z f+△Z]. The horizontal coordinate X(t) and the vertical coordinate Y(t) of the position where the ultra-long focal depth super-resolution focused light spot illuminates the sample 16 are functions of time t. The reflected light E collected by the objective lens 15 r (t) and scattered light E s (t) is also a function of time, the reflected light E r (t) and scattered light E s (t) reflects the sample structure and refractive index information within the ultra-long focal depth at the corresponding position (abscissa X(t) and ordinate Y(t)). By performing a two-dimensional scan in the XY plane (the scanning step is smaller than the minimum resolution size of the system), the signal intensity of the corresponding coordinates is used as the coordinate function I(X(t), Y(t), Z f ±△Z) (where X and Y are the coordinates of the center of the ultra-long focal depth super-resolution focusing spot on the focal plane of the objective lens 15) to obtain an ultra-long depth of field two-dimensional super-resolution microscopic image of the sample 16. The resolution of the image in the XY plane is close to the limiting resolution of the reflective confocal system 0.25λ / NA. f ±△Z means the focal depth [Z f -△Z,Z f +△Z]. The computer controls the axial nanopositioner 14 to move the objective lens 15 in the Z direction, changing the Z position Z of the front focal plane of the objective lens 15 in the sample. f , get different Z f By using a two-dimensional super-resolution microscopic image with an ultra-long depth of field in the XY plane at the position, a three-dimensional microscopic image can be obtained, and its resolution is close to the limiting resolution of the reflective confocal system 0.25λ / NA.

[0058] Embodiment 2 like Figure 7 As shown, most of the structures of the reflective scanning super-resolution optical microscopy system and method with ultra-long depth of field in this embodiment are the same as those in Embodiment 1, except that: the sample displacement module is a three-dimensional displacement stage 35, the three-dimensional displacement stage 35 is connected to the computer via a scanning signal line 18, the sample 16 is horizontally fixed on the three-dimensional displacement stage 35, and the computer controls the three-dimensional displacement stage 35 to drive the sample 16 to move in the Z-axis direction.

[0059] like Figure 7 As shown, the reflective scanning super-resolution optical microscopy method with ultra-long depth of field in this embodiment is basically the same as that in the first embodiment, the only difference is that in the specific processing of step 3 and step 4, the computer controls the three-dimensional displacement stage 35 to drive the sample 16 to move slightly in the Z direction, changing the Z direction position Z 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 27 collects a signal to obtain different Z fBy using a two-dimensional super-resolution microscopic image with an ultra-long depth of field in the XY plane at the position, a three-dimensional microscopic image can be obtained, and its resolution is close to the limiting resolution of the reflective confocal system 0.25λ / NA.

[0060] Embodiment 3 Most structures of the reflective scanning super-resolution optical microscopy system and method with ultra-long depth of field in this embodiment are the same as those in the first embodiment, except that the field lens 36 is not required.

[0061] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

1. A reflective scanning super-resolution optical microscopy system with ultra-long depth of field, characterized in that: include: A sample displacement module, used to drive the sample (16) to move; An optical microscopic imaging module, used for performing wide-field imaging of the sample (16) and determining a super-resolution scanning imaging area; An annular focused beam generating module is used to generate an annular focused beam. The annular focused beam generating module comprises 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) in sequence along the light propagation direction, wherein the first positive lens (6) and the conical lens (5) are coaxially arranged with an interval, the front focal plane of the second positive lens (7) coincides with the rear focal plane of the first positive lens (6), the rear focal plane of the second positive lens (7) coincides with the front focal plane of the third positive lens (8), and the second positive lens (7) and the third positive lens (8) form a first 4f system; the light source module generates a collimated laser beam, and the collimated laser beam forms a linearly polarized annular focused beam after passing through the first linear polarizer (4), the conical lens (5) and the first positive lens (6), and forms a circular focal line on the rear focal plane of the first positive lens (6); A beam scanning module comprises a beam scanning galvanometer (9), a beam scanning lens (10), a first sleeve mirror (11) and a reflector (12), wherein the center position of the beam scanning galvanometer (9) coincides with the rear focal plane of the third positive lens (8); the front focal plane of the beam scanning lens (10) is located at the center position of the beam scanning galvanometer (9); the rear focal plane of the beam scanning lens (10) coincides with the front focal plane of the first sleeve mirror (11), and the two constitute a second 4f system; the rear focal plane of the first sleeve mirror (11) coincides with the rear 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) comprises a controller, the controller is used to be connected to a computer, and the computer controls the beam scanning galvanometer (9) to perform two-dimensional vibration scanning through the controller of the beam scanning galvanometer (9); A super-resolution focusing illumination and collection module is used to convert a ring-shaped focusing beam into a super-resolution conical focusing beam to form a super-resolution focal spot with an ultra-long focal depth to illuminate a sample (16), collect super-resolution signal light generated by reflection and scattering of the sample (16) within the focal depth range, and focus and perform polarization conversion; the super-resolution focusing illumination and collection module comprises a quarter-wave plate (13), an axial nanopositioner (14) and an objective lens (15), and the sample (16) is located within the ultra-long focal depth range before and after the front focal plane of the objective lens (15); A super-resolution optical imaging module, used for acquiring super-resolution signal light after collimation and polarization conversion, and converting it into an electrical signal; the super-resolution optical imaging module also includes a polarization beam splitter (19), a second linear polarizer (20), a second sleeve mirror (21), an optical fiber head (22), a photomultiplier tube (25) and a high-speed acquisition card (27); The polarization beam splitter (19) is located between the first linear polarizer (4) and the conical lens (5); the polarization beam splitter (19), the second linear polarizer (20), the second sleeve mirror (21) and the optical fiber head (22) are arranged in sequence along the propagation direction of the light after being reflected by the polarization beam splitter (19); the front end surface of the optical fiber head (22) coincides with the rear focal plane of the second sleeve mirror (21); the optical fiber head (22) is connected to the photomultiplier tube (25); the photomultiplier tube (25) is connected to the high-speed acquisition card (27); and the high-speed acquisition card (27) is used to connect to a computer; The computer is used to control the sample displacement module to drive the sample (16) to move, to synchronously control the light beam scanning galvanometer (9) to scan and receive the electrical signal output by the super-resolution optical imaging module, and to process and obtain a super-resolution microscopic image of the sample in the super-resolution scanning imaging area.

2. The reflective scanning super-resolution optical microscopy system with ultra-long depth of field according to claim 1, characterized in that: The annular focused light beam generating module further comprises a field lens (36) arranged between the first positive lens (6) and the second positive lens (7), wherein the object-side principal surface of the field lens (36) coincides with the rear focal plane of the first positive lens (6), and the image-side principal surface of the field lens (36) coincides with the front focal plane of the second positive lens (7), so as to change the propagation direction of the edge light of the annular focused light beam.

3. The reflective scanning super-resolution optical microscopy system with ultra-long depth of field according to claim 1, characterized in that: When the annular focused light beam generated by the annular focused light beam generating module is reflected by the light beam scanning galvanometer (9), passes through the scanning lens (10) and the first sleeve mirror (11), and is reflected by the reflector (12) to the rear focal plane of the objective lens (15); when the light beam scanning galvanometer (9) vibrates, the center of the annular focused light beam coincides with the center of the rear focal plane of the objective lens (15), and the incident angle of the annular focused light beam changes with the vibration of the light beam scanning galvanometer (9); when the light beam scanning galvanometer (9) performs two-dimensional scanning, the annular focused light beam is transformed by the objective lens (15), and a conical focused light field is formed behind the objective lens (15), thereby generating an ultra-long focal depth focused light field, and forming an ultra-long focal depth super-resolution focused light spot with a half-height full width of 2△Z in the Z-axis direction before and after the front focal plane of the objective lens (15), wherein △Z>4λ f / πD, where λ is the wavelength, f is the focal length, and D is the pupil diameter of the objective lens. The focused light spot will achieve continuous XY two-dimensional scanning on the front focal plane of the objective lens (15) as the beam scanning galvanometer (9) vibrates, and the scanning range in the Z-axis direction is equal to the focal depth. The position of the rear focal plane of the objective lens (15) in the Z-axis direction is controlled to control the scanning range in the Z-axis direction of the objective lens (15), that is, [Z f -△Z, Z f +△Z], where Z f is the position of the front focal plane of the objective lens (15) in the Z-axis direction.

4. The reflective scanning super-resolution optical microscopy system with ultra-long depth of field according to claim 3, characterized in that: The super-resolution focusing 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 microscopic imaging module comprises a second beam splitter (33); after the linearly polarized annular focusing beam emitted from the beam scanning module passes through the quarter-wave plate (13), a circularly polarized circular focusing line having 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); the circularly polarized circular focusing line is converted into a super-resolution conical focusing beam after passing through the objective lens (15), and an ultra-long focal depth super-resolution focusing beam is formed before and after the front focal plane of the objective lens (15). Light spot; the ultra-long focal depth super-resolution focused light spot illuminates the sample (16); the super-resolution signal light generated by reflection and scattering of the sample (16) is collected by the objective lens (15) and converged and collimated respectively, and then converted into a linearly polarized annular focused light beam and a collimated light beam orthogonal to the incident line polarization through a quarter-wave plate (13), that is, a linearly polarized super-resolution signal light beam; the linearly polarized super-resolution signal light beam is transmitted through the second beam splitter (33) in the opposite direction of the original light path, passes through the beam scanning module, enters the annular focused light beam generating module, and is reflected by the polarization beam splitter (19) placed in the light path to enter the super-resolution optical imaging module; When the beam scanning galvanometer (9) in the beam scanning module performs two-dimensional scanning, the ultra-long focal depth super-resolution focused light spot formed in front and behind the front focal plane of the objective lens (15) will follow the vibration of the beam scanning galvanometer (9), thereby realizing continuous XY two-dimensional scanning in the front focal plane of the objective lens (15), and the scanning range on the Z axis is [Z f -△Z,Z f +△Z]; the horizontal coordinate X(t) and the vertical coordinate Y(t) of the position where the ultra-long focal depth super-resolution focused light spot illuminates the sample (16) are functions of time t, and the objective lens (15) collects the reflected light E within the ultra-long focal depth range. r (t) and scattered light E s (t) is also a function of time, the reflected light E r (t) and scattered light E s (t) reflects the sample structure and refractive index information within the ultra-long focal depth at the corresponding position (X(t), Y(t)).

5. The reflective scanning super-resolution optical microscopy system with ultra-long depth of field according to claim 3, characterized in that: The ultra-long focal depth and ultra-resolution focusing spot is an annular focusing light field, the inner and outer surfaces of which are two coaxial conical surfaces with equal cone angles, and the numerical aperture NA corresponding to the annular focusing light field is c , which satisfies: NA c <NA; where NA represents the numerical aperture of the objective lens (15).

6. The reflective scanning super-resolution optical microscopy system with ultra-long depth of field 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), and the computer controls the two-dimensional displacement stage (17) to drive the sample (16) to move two-dimensionally in the XY plane, thereby realizing the initial positioning of the sample in the XY direction; the super-resolution focusing illumination and collection module also includes an axial nanopositioner (14), the axial nanopositioner (14) is connected to the computer, the objective lens (15) is mounted on the axial nanopositioner (14), and the computer controls the axial nanopositioner (14) to drive the objective lens (15) to move in the Z direction, thereby realizing the initial positioning of the sample in the Z direction; Alternatively, the sample displacement module is a three-dimensional displacement platform (35), the three-dimensional displacement platform (35) is connected to a computer, the sample (16) is horizontally fixed on the three-dimensional displacement platform (35), and the computer controls the three-dimensional displacement platform (35) to drive the sample (16) to move in the three directions of X, Y, and Z to achieve initial positioning of the sample.

7. The reflective scanning super-resolution optical microscopy system with ultra-long depth of field according to claim 6, characterized in that: The super-resolution optical imaging module further comprises a five-dimensional adjustment frame (23), the optical fiber head (22) being mounted on the five-dimensional adjustment frame (23), and the five-dimensional adjustment frame (23) being adjusted so that the super-resolution signal light entering the optical fiber head (22) reaches a maximum; The inner diameter of the optical fiber at the front end face of the optical fiber head (22) is smaller than the first zero point radius of the focal spot of the super-resolution signal light on the rear focal plane of the second sleeve mirror (21).

8. The reflective scanning super-resolution optical microscopy system with ultra-long depth of field according to claim 6, characterized in that: The light source module comprises a laser (1) and an optical fiber collimator (3); the laser (1) is connected to the optical fiber collimator (3) via a first optical fiber jumper (2); coherent light emitted by the laser (1) is transmitted to the optical fiber collimator (3) via the first optical fiber jumper (2); the optical fiber collimator (3) collimates the coherent light and outputs a collimated laser beam.

9. The reflective scanning super-resolution optical microscopy system with ultra-long depth of field according to claim 4, characterized in that: The optical microscopy imaging module further comprises an incoherent light source (28), a collimating lens (29), a first beam splitter (30), a second beam splitter (31), a third tube mirror (32) and a digital camera (33). The digital camera (33) is connected to a computer. The incoherent light source (28) is located at the front focus of the collimating lens (29). Light emitted by the incoherent light source (28) is collimated by the collimating lens (29) and reflected by the first beam splitter (30), and then passes through the second beam splitter (31), the quarter wave plate (13) and the optical microscope (21). The incoherent light reaches the objective lens (15) and converges into incoherent light on the front focal plane of the objective lens (15) to illuminate the sample (16). The incoherent light generated by reflection and scattering of the sample (16) is collected by the objective lens (15), passes through the quarter-wave plate (13), the second beam splitter (31), the first beam splitter (30), and the third sleeve lens (32) in sequence, and then enters the digital camera (33), so that the sample (16) is imaged on the digital camera (33) and sent to a computer, and the computer displays a wide-field microscopic image of the sample (16).

10. A reflective scanning super-resolution optical microscopy method with an ultra-long depth of field, using the reflective scanning super-resolution optical microscopy system according to any one of claims 1 to 9, the method comprising the following steps: Step 1: Use an optical microscopy imaging module to perform wide-field imaging on the sample (16) to determine a super-resolution scanning imaging area; Step 2: after determining the super-resolution scanning imaging area, the annular focused beam generating module generates an annular focused beam, and the super-resolution focused illumination and collection module converts the annular focused beam into a super-resolution conical focused beam, forming an ultra-long focal depth super-resolution focused light spot with a half-height full width of 2△Z in the Z-axis direction to illuminate the sample (16), collect the super-resolution signal light generated by the reflection and scattering of the sample (16) within the ultra-long focal depth range, and respectively converge and collimate the emitted light and the scattered light, and convert the polarization state; Step 3: The computer controls the sample displacement module to move 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 obtains the super-resolution signal light after being converged, collimated and converted, and converts it into an electrical signal and inputs it into the computer; Step 4: The computer processes the input electrical signal and combines 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), Z) of the sample in the super-resolution scanning imaging area. f ±△Z), where Z f ±△Z means the focal depth [Z f -△Z,Z f +△Z], where Z f is the Z-axis position of the focal plane of the objective lens (15).

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