Reflective super-deep field super-resolution wide-field optical microscopy system and method

Abstract

The application discloses a reflection type super-large depth of field super-resolution wide-field optical microscopic system and method, and the system comprises a sample displacement module, a ring-shaped focusing light beam generation module, a light beam scanning module, a super-large depth of field super-resolution wide-field optical microscopic imaging module, an optical microscopic imaging module and a computer. Through the advantages of the ring-shaped focusing light beam illumination, the high focusing efficiency of the traditional lens and the high scanning speed of the light beam, efficient and fast super-long focal depth super-resolution focusing scanning illumination is realized, and then the traditional optical microscopic system can be used to realize fast, super-large depth of field super-resolution wide-field optical microscopic imaging. The application can be applied to non-labeled super-resolution microscopic fast imaging of biological samples, and can also be applied to industrial related super-resolution microscopic detection, real-time imaging of micro-operation and other fields.

Description

Technical Field

[0001] This invention belongs to the field of visible light microscopy, specifically relating to a reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system and method. Background Technology

[0002] Microscopy, a key tool in scientific research and industrial testing, is currently mainly divided into two categories: near-field microscopy and far-field microscopy. Near-field microscopy techniques, such as microsphere-assisted imaging and near-field scanning microscopy, while capable of achieving a certain degree of super-resolution imaging, are inherently limited by their working distance being smaller than the working wavelength, significantly restricting their versatility and flexibility in practical applications. Far-field microscopy, although with a large working distance, often requires fluorescent molecular labeling, which not only increases experimental complexity and cost but may also cause irreversible damage to the sample. Furthermore, existing far-field super-resolution microscopy techniques, such as stimulated emission depletion microscopy, local activation microscopy, and random light reconstruction optical microscopy, while achieving some progress, still suffer from a small depth of field, preventing the realization of large depth-of-field super-resolution imaging. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of existing technologies by providing a reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system and method to achieve ultra-large depth-of-field super-resolution wide-field optical imaging.

[0004] In a first aspect, the present invention provides a reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system, comprising: The sample displacement module is used to move the sample. Ring-shaped focused beam generation module, used to generate a ring-shaped focused beam; The beam scanning module is used to achieve rapid two-dimensional scanning of the beam; 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 depth-of-field super-resolution focal spot to illuminate the sample, collect the ultra-long depth-of-field super-resolution signal light generated by the reflection and scattering of the sample, converge and collimate the ultra-long depth-of-field super-resolution light signals reflected and scattered, and complete polarization conversion; the super-resolution focusing illumination and collection module includes an objective lens, with the sample located on the front focal plane of the objective lens; The ultra-deep field super-resolution wide-field optical microscopy imaging module includes a second beam splitter, a fourth positive lens, a second conical lens, a second telescope, and a first digital camera. The second beam splitter reflects the ultra-deep field super-resolution signal light collected by the super-resolution focusing illumination and collection module into the ultra-deep field super-resolution wide-field optical microscopy imaging module. After passing through the fourth positive lens and the second conical lens, it is converted into a ring collimated beam. The ring collimated beam is then focused onto the target surface of the first digital camera by the second telescope, forming an ultra-deep field super-resolution wide-field optical imaging and realizing the real-time acquisition of super-resolution images. An optical microscopy imaging module is used to perform wide-field imaging of samples and determine the super-resolution imaging region. The optical microscopy imaging module includes a third beam splitter, a third telescope, a second digital camera, a non-super-resolution illumination source, and a collimating lens. The non-super-resolution illumination source is located at the front focal point of the collimating lens. The light emitted from the non-super-resolution illumination source is collimated by the collimating lens and reflected by the third beam splitter. It then passes sequentially through the first beam splitter, the second beam splitter, and the quarter-wave plate of the beam scanning module to reach the objective lens. The light is then converged into non-super-resolution illumination light on the front focal plane of the objective lens to illuminate the sample. The non-super-resolution illumination light generated by the reflection and scattering of 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, the third beam splitter, and the third telescope before entering the second digital camera. The sample is then imaged on the second digital camera and sent to a computer, which displays a wide-field microscopic image of the sample. 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 ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module, and process them to obtain super-resolution microscopic images of the sample in the super-resolution imaging area.

[0005] Optionally, the annular focused beam generating module includes, in sequence along the light propagation direction, a light source unit, a first linear polarizer, a conical lens, a first positive lens, a second positive lens, and a third positive lens, wherein 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 positive lens and the third positive lens constitute a first 4f system. The light source unit generates a collimated beam, which, after passing through the first linear polarizer, the conical lens, and the first positive lens, forms a linearly polarized annular focused beam and forms a first circular focusing line on the rear focal plane of the first positive lens. Alternatively, the ring-shaped focused beam generating module may include, in sequence along the direction of light propagation, a light source unit, a conical lens, a first positive lens, a second positive lens, and a third positive lens, wherein 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, the rear 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 constitute a first 4f system; the light source unit generates a collimated beam, which, after passing through the conical lens and the first positive lens, forms a ring-shaped focused beam and forms a first circular focusing line on the rear focal plane of the first positive lens.

[0006] Optionally, the beam scanning module includes a beam scanning galvanometer, a beam scanning lens, a first telescope, and a first beam splitter. The center of the beam scanning galvanometer coincides with the back focal plane of the third positive lens after reflection by the beam scanning galvanometer. 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 reflection by the first beam splitter, 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. When the ring-focused beam generated by the ring-focusing beam generation module is reflected by the beam scanning galvanometer, passes through the scanning lens and the first sleeve lens, and is reflected by the first beam splitter to the rear focal plane of the objective lens; when the beam scanning galvanometer vibrates, the center of the ring-focused beam coincides with the center of the rear focal plane of the objective lens, and the incident angle of the ring-focused beam changes with the vibration of the beam scanning galvanometer; the ring-focused beam is transformed by the objective lens to form a conical focused light field behind the objective lens, thereby generating an ultra-long depth of focus focused light field, and forming an ultra-long depth of focus super-resolution focused light spot in front of and behind the front focal plane of the objective lens. When the beam scanning galvanometer performs two-dimensional scanning, the ultra-long depth of focus focused light spot will continuously scan in two dimensions in the XY plane within the focal depth range of the ultra-long depth of focus super-resolution focused light spot in front of and behind the front focal plane of the objective lens in the optical axis direction, as the beam scanning galvanometer vibrates. The XY plane is parallel to the focal plane of the objective lens.

[0007] Optionally, the super-resolution focusing illumination and collection module further includes a quarter-wave plate; The linearly polarized ring-focused beam emitted from the beam scanning module, after passing through a quarter-wave plate, forms a circularly polarized circular focusing line on the rear focal plane of the objective lens with an outer diameter less than or equal to the diameter of the rear focal plane of the objective lens. This circularly polarized circular focusing line is transformed into a super-resolution conical focusing beam after passing through the objective lens, and forms an ultra-long depth-of-field super-resolution focusing spot in front of and behind the front focal plane of the objective lens. This ultra-long depth-of-field super-resolution focusing spot illuminates the sample. The super-resolution signal light generated by the reflection and scattering of the sample within the depth of focus range is collected by the objective lens and transformed by a quarter-wave plate into a linearly polarized ring-focused beam and a linearly polarized collimated beam orthogonal to the incident linearly polarized ring beam, i.e., a linearly polarized super-resolution signal beam, which is reflected by the second beam splitter and enters the ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module. The ring-shaped focusing beam is transformed into a super-resolution conical focusing beam by the objective lens. When the beam scanning galvanometer in the beam scanning module performs a two-dimensional scan, the ultra-long focal depth super-resolution focusing spot formed before and after the front focal plane of the objective lens will vibrate with the beam scanning galvanometer, thereby achieving continuous two-dimensional scanning illumination in the XY plane within the focal depth range of the ultra-long focal depth super-resolution focal spot before and after the front focal plane of the objective lens.

[0008] Optionally, the ultra-long depth-of-focus super-resolution focusing spot is a conical focusing light field, wherein the inner and outer surfaces of the conical focusing light field are two coaxial conical surfaces with equal cone angles, and the numerical aperture NA corresponding to the conical focusing light field is... c For a given resolution d r Need to satisfy: d r =0.5λ / (NA c +NA); when 0.947NA≤NA c The optimal resolution is achieved when the value is ≤NA; where NA represents the numerical aperture of the objective lens.

[0009] Preferably, to achieve deep-depth imaging scanning of the sample, the sample displacement module has two structures.

[0010] In the first embodiment, the sample displacement module is a two-dimensional displacement stage connected to a computer. The sample is horizontally fixed on the two-dimensional displacement stage, and the computer controls the stage to move the sample two-dimensionally within the XY plane, achieving initial positioning of the sample. The super-resolution focusing illumination and collection module also includes an axial nanopositioner connected to the computer. The objective lens is mounted on the axial nanopositioner, and the computer controls the axial nanopositioner to move the objective lens in the Z direction. The beam scanning module achieves two-dimensional scanning of the sample, and combined with the axial nanopositioner, achieves deep-depth imaging scanning.

[0011] The second method involves a sample displacement module that is a three-dimensional displacement stage connected to a computer. The sample is horizontally fixed on the three-dimensional displacement stage, and the computer controls the three-dimensional displacement stage to move the sample in the X, Y, and Z directions. The X and Y directions are used to achieve initial positioning of the sample. The beam scanning module performs two-dimensional scanning of the sample, and the Z-direction displacement of the three-dimensional displacement stage is combined with the large-depth imaging scan to avoid the imaging effect caused by the slight movement of the objective lens, resulting in better imaging effect.

[0012] Optionally, the light source unit includes a super-resolution illumination source and an optical fiber collimator. The super-resolution illumination source is connected to the optical fiber collimator via a first optical fiber jumper. The light emitted by the super-resolution illumination source is transmitted to the optical fiber collimator via the first optical fiber jumper, and the optical fiber collimator collimates the light and outputs a collimated beam. The super-resolution illumination source can be a coherent light source (such as a laser) or a partially coherent light source (such as a photodiode).

[0013] Optionally, the annular focusing beam generating module further includes a field lens, the object-side principal plane of which coincides with the back focal plane of the first positive lens, and the image-side principal plane of which coincides with the front focal plane of the second positive lens, so as to change the propagation direction of the edge light rays of the annular focusing beam and allow the annular focusing beam to pass through the objective lens without obstruction.

[0014] Optionally, the ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module further includes a second linear polarizer; When performing super-resolution imaging, the non-super-resolution illumination source is turned off, and the light source unit and beam scanning module are turned on to perform beam scanning. The ultra-long depth-of-field super-resolution signal light collected by the super-resolution focusing illumination and collection module is reflected by the second beam splitter into the ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module. After passing through the fourth positive lens, the second conical lens, the second linear polarizer, and the second telescope, it is converted into an annular collimated beam. The annular collimated beam is focused on the target surface of the first digital camera by the second telescope, forming an ultra-large depth-of-field super-resolution wide-field optical image. After being acquired by the first digital camera, it is sent to the computer for processing and display through the first data line.

[0015] Secondly, the present invention provides a reflective ultra-large depth-of-field super-resolution wide-field optical microscopy method, employing the reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system as described in the present invention. The method includes the following steps: Step 1: Use an optical microscopy imaging module to perform wide-field imaging of the sample and determine the super-resolution imaging region; Step 2: After determining the super-resolution 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 an ultra-long focal length super-resolution focusing spot to illuminate the sample, collect the ultra-long depth-of-field super-resolution signal light generated by the sample reflection and scattering, and complete the polarization conversion. Step 3: The computer-controlled sample displacement module moves the sample to the position to be scanned, and the beam scanning galvanometer is turned on to scan, acquire ultra-long depth-of-field super-resolution signal light, and send it into the ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module to form ultra-large depth-of-field super-resolution wide-field optical imaging on the target surface of the first digital camera. Step 4: The computer reads, processes, and displays the ultra-wide field optical image captured by the first digital camera with super-depth of field and super-resolution.

[0016] The present invention has the following advantages: (1) This system includes a sample displacement module, a ring-shaped focused beam generation module, a beam scanning module, an ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module, an optical microscopy imaging module, and a computer. By utilizing the advantages of ring-shaped focused beam illumination, high focusing efficiency of traditional lenses, and fast beam scanning speed, it achieves efficient and rapid ultra-long depth-of-field super-resolution focused scanning illumination, thereby enabling rapid, ultra-large depth-of-field super-resolution wide-field optical microscopy imaging using traditional optical microscopy systems. This invention can be applied to label-free super-resolution rapid microscopic imaging of biological samples, and can also be applied to industrial-related super-resolution microscopic detection, real-time micromanipulation imaging, and other fields.

[0017] (2) This invention possesses ultra-long depth-of-field imaging capability and large depth imaging capability. By utilizing the ultra-long depth of field formed by the conical focusing beam, ultra-long depth-of-field imaging of samples can be achieved. At the same time, this ultra-long depth-of-field light field has superior penetration and self-healing properties, which can greatly improve the imaging depth. 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.

[0018] (3) This invention enables highly efficient super-resolution focused illumination. Through a meticulously designed annular focused beam generation module, it efficiently generates an annular focused beam. Subsequently, using a super-resolution focused illumination and collection module, this annular focused beam is converted into a super-resolution conical focused beam, forming a high-precision super-resolution focal spot to illuminate the sample. This process fully utilizes the high focusing efficiency of traditional lenses, achieving highly efficient super-resolution focused illumination and significantly improving the signal-to-noise ratio of the image. Simultaneously, the generated super-resolution conical focused beam forms an ultra-long depth-of-focus super-resolution focused light field behind the objective lens, facilitating the reduction of alignment requirements for the illumination beam, focal plane, and sample.

[0019] (3) This invention enables rapid two-dimensional scanning super-resolution illumination. The beam scanning module employs beam scanning galvanometer technology to achieve rapid two-dimensional scanning super-resolution illumination 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 a large amount of high-quality image data.

[0020] (4) This invention enables 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.

[0021] (5) This invention features high reliability and imaging quality. By precisely controlling the optical path coordination and signal processing between modules, this invention ensures the reliability and stability of imaging. From the generation of the ring-focused beam and rapid scanning super-resolution 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.

[0022] In summary, the reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system and method described in this invention exhibit significant advantages in terms of efficient and rapid two-dimensional scanning super-resolution illumination, label-free far-field super-resolution microscopy imaging, ultra-long depth-of-field imaging capability, large depth imaging capability, super-resolution three-dimensional imaging capability, as well as high reliability and imaging quality, providing strong technical support for fields such as biomedicine and industrial inspection. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system in Example 1.

[0024] Figure 2 This 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.

[0025] Figure 3 This is a schematic diagram of the formation of an ultra-long depth-of-focus super-resolution focused light field in Example 1.

[0026] Figure 4 This is a parameter relationship diagram of the ultra-long focal depth super-resolution focusing light field in Example 1.

[0027] Figure 5This is a schematic diagram of the scanning of the ultra-long focal depth super-resolution focusing spot on the focal plane of the objective lens in Example 1.

[0028] Figure 6 This is a graph showing the relationship between the full width at half maximum (FWHM) of the objective lens and the focal spot and the system resolution under ring-focused beam illumination in Example 1.

[0029] Figure 7 This is a schematic diagram of the reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system in Example 2; In the diagram: 1. Super-resolution illumination source; 2. First fiber optic patch cord; 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. First beam splitter; 13. Quarter-wave plate; 14. Axial nanopositioner; 15. Objective lens; 16. Sample; 17. Two-dimensional displacement stage; 18. Scanning signal line; 19. Second beam splitter; 20. Fourth positive lens; 21. The first... 22. Second telescope lens, 23. First digital camera, 24. First data cable, 25. Non-super-resolution illumination source, 26. Collimating lens, 27. Third beam splitter, 28. Third telescope lens, 29. Second digital camera, 30. Second data cable, 31. Three-dimensional displacement stage, 32. Field lens, 33. Second linear polarizer, 34. Super-resolution conical focusing beam, 35. Optical axis, 36. Super-long depth of focal length super-resolution focal spot, 37. Center of the rear focal plane of the objective lens, 38. Rear focal plane of the objective lens, 39. Front focal plane of the objective lens. Detailed Implementation

[0030] 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.

[0031] Example 1 like Figure 1 As shown, the reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system in this embodiment includes: The sample displacement module is used to move sample 16.

[0032] The optical microscopy imaging module is used to perform wide-field imaging of sample 16, determine the super-resolution imaging area, and acquire super-resolution images of sample 16.

[0033] Ring-shaped focused beam generation module, used to generate a ring-shaped focused beam.

[0034] The beam scanning module is used to achieve rapid two-dimensional scanning of the beam.

[0035] The super-resolution focusing illumination and collection module is used to convert the ring-shaped focusing beam into a super-resolution conical focusing beam 34, forming a super-long depth-of-field super-resolution focal spot 36 to illuminate the sample 16, collect the super-long depth-of-field super-resolution signal light generated by the reflection and scattering of the sample 16, and convert it.

[0036] The ultra-deep-field super-resolution wide-field optical microscopy imaging module, combined with the super-resolution focusing illumination and collection module, enables ultra-deep-field super-resolution wide-field optical microscopy imaging. It includes a second beam splitter 19, a fourth positive lens 20, a second conical lens 21, a second linear polarizer 33, a second telescope 22, a first digital camera 23, and a first data line 24. The second beam splitter 19 reflects the ultra-deep-field super-resolution signal light collected by the super-resolution focusing illumination and collection module into the ultra-deep-field super-resolution wide-field optical microscopy imaging module. After passing through the fourth positive lens 20 and the second conical lens 21, it is converted into a ring-shaped collimated beam. This ring-shaped collimated beam is then converged onto the target surface of the first digital camera 23 by the second telescope 22, forming an ultra-deep-field super-resolution wide-field optical image.

[0037] A computer (not shown in the figure) is used to control the sample displacement module to move sample 16. The beam scanning galvanometer 9 is synchronously controlled to scan and receive the electrical signals output by the ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module, and the signals are processed to obtain ultra-long depth-of-field super-resolution microscopic images of sample 16 within the super-resolution imaging area and ultra-long focal depth range.

[0038] This system leverages the advantages of ring-focused beam illumination, high focusing efficiency of traditional lenses, fast beam scanning speed, and an ultra-long, super-resolution focused light field to achieve efficient and rapid ultra-long depth-of-field super-resolution scanning illumination. This enables rapid ultra-long depth-of-field super-resolution optical microscopy imaging using traditional optical microscopy systems. Furthermore, by performing two-dimensional super-resolution beam scanning illumination on samples and combining it with traditional microscopy systems, it achieves label-free far-field super-resolution two-dimensional microscopic rapid imaging. 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 deep-depth imaging. 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.

[0039] The following is a detailed explanation of each module: like Figure 1 and Figure 2As shown, the optical microscopy imaging module includes a non-super-resolution illumination source 25, a collimating lens 26, a third beam splitter 27, a third telescope 28, and a second digital camera 29; the second digital camera 29 is connected to a computer via a second data cable 30, and the non-super-resolution illumination source 25 is located at the front focal point of the collimating lens 26. The light emitted from the non-super-resolution illumination source 25 is collimated by the collimating lens 26 and reflected by the third beam splitter 27. It then passes sequentially through the first beam splitter 12, the second beam splitter 19, and the quarter-wave plate 13 to reach the objective lens 15. The light is then focused into non-super-resolution illumination light on the front focal plane 39 of the objective lens to illuminate the sample 16. The non-super-resolution illumination light generated by the reflection and scattering of the sample 16 is collected by the objective lens 15 and passes sequentially through the quarter-wave plate 13, the second beam splitter 19, the first beam splitter 12, the third beam splitter 27, and the third telescopic lens 28 before entering the second digital camera 29. This images the sample 16 onto the second digital camera 29 and are then sent to the computer, where the computer displays a wide-field microscopic image of the sample 16.

[0040] The ultra-deep-field super-resolution wide-field optical microscopy imaging module includes a second beam splitter 19, a fourth positive lens 20, a second conical lens 21, a second linear polarizer 33, a second telescope lens 22, a first digital camera 23, and a first data line 24. The second beam splitter 19 reflects the ultra-deep-field super-resolution signal light collected by the super-resolution focusing illumination and collection module into the ultra-deep-field super-resolution wide-field optical microscopy imaging module. After passing through the fourth positive lens 20, the second conical lens 21, the second linear polarizer 33, and the second telescope lens 22, it is converted into a ring-shaped collimated beam. This ring-shaped collimated beam is then converged by the second telescope lens 22 onto the target surface of the first digital camera 23, forming an ultra-deep-field super-resolution wide-field optical image.

[0041] When performing super-resolution imaging, the non-super-resolution illumination source 25 is turned off, and the light source unit and beam scanning module are turned on to perform beam scanning. The ultra-long depth-of-field super-resolution signal light collected by the super-resolution focusing illumination and collection module is reflected by the second beam splitter 19 into the ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module. After passing through the fourth positive lens 20 and the second conical lens 21, it is converted into an annular collimated beam. The annular collimated beam is converged on the target surface of the first digital camera 23 through the second telescope 22 to form an ultra-long depth-of-field super-resolution wide-field optical image. After being acquired by the first digital camera 23, it is sent to the computer for processing and display through the first data line 24.

[0042] like Figure 1 and Figure 2As shown, the annular focusing beam generation module includes, in sequence along the light propagation direction, a super-resolution illumination source 1, an optical fiber collimator 3, a first linear polarizer 4, a first conical lens 5, a first positive lens 6, a field lens 32, a second positive lens 7, and a third positive lens 8. The super-resolution illumination source 1 is connected to the optical fiber collimator 3 via a first optical fiber jumper 2; the light emitted from the super-resolution illumination source 1 is transmitted to the optical fiber collimator 3 via the first optical fiber jumper 2, and the optical fiber collimator 3 collimates the light and outputs a collimated beam. The first positive lens 6 and the first 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 (F'1) 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 the first 4f system. The light source unit (i.e., the super-resolution illumination source 1 and the fiber collimator 3) generates a collimated beam. This collimated 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 forms a first circular focused line at the rear focal plane of the first positive lens 6. When using a field lens (or without a field lens), the object-side principal plane of the field lens 32 coincides with the rear focal plane of the first positive lens 6, and the image-side principal plane of the field lens 32 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 to the maximum extent without obstruction.

[0043] like Figure 1 and Figure 2 As shown, the beam scanning module includes a beam scanning galvanometer 9, a scanning lens 10, a first telescope lens 11, and a first beam splitter 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 lens 11, forming a second 4f system; the back focal plane of the first telescope lens 11, after reflection by the first beam splitter 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 figure). The computer controls the beam scanning galvanometer 9 to perform two-dimensional vibration scanning through the controller.

[0044] The ring-focused beam generated by the ring-focusing 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 then reflected by the first beam splitter 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 37 of the back focal plane of the objective lens, and the incident angle of the ring-focused beam changes with the vibration of the beam scanning galvanometer 9. The ring-focused beam is transformed by the objective lens 15, forming a conical focused light field behind the objective lens 15, thereby generating an ultra-long depth-of-focus focused light field, and then... A super-long focal depth super-resolution focusing spot is formed before and after the front focal plane 39. When the beam scanning galvanometer 9 performs two-dimensional scanning, this super-long focal depth super-resolution focusing spot will continuously scan in the XY plane (parallel to the objective lens focal plane) within the focal depth range of the super-long focal depth super-resolution focusing spot before and after the front focal plane 39 of the objective lens as the beam scanning galvanometer 9 vibrates. By controlling the position of the rear focal plane 38 of the objective lens in the Z-axis direction (i.e., the optical axis 35 direction), the spatial range of continuous two-dimensional scanning at different optical axis positions before and after the front focal plane 39 of the objective lens can be controlled.

[0045] 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 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 in front of and behind the front focal plane 39 of the objective lens. The objective lens 15 can be a dry lens, a water lens, an oil lens, or a fixed lens.

[0046] 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 (not shown in the figure) 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 a two-dimensional displacement stage 17, the super-resolution focusing illumination and collection module also includes an axial nanopositioner 14, which is connected to the computer via a positioning signal line. The objective lens 15 is mounted on the axial nanopositioner 14, and the computer controls the axial nanopositioner 14 to move the objective lens 15 in the Z direction.

[0047] In one possible embodiment, the super-resolution illumination source can be a coherent light source (such as a laser) or a partially coherent light source (such as a photodiode).

[0048] In this embodiment of the application, the reflective ultra-large depth-of-field super-resolution wide-field optical microscopy method employs the aforementioned reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system, and the method includes: Step 1: Wide-field imaging of sample 16 is performed using an optical microscopy imaging module to determine the super-resolution imaging region. Specifically: The super-resolution illumination source 1 is initially turned off, emitting no light. The light emitted by the non-super-resolution illumination source 25 is collimated by the collimating lens 26, reflected by the third beam splitter 27, and then sequentially passes through the first beam splitter 12, the second beam splitter 19, and the quarter-wave plate 13 to reach the objective lens 15. It converges at the front focal plane 39 of the objective lens to form non-super-resolution illumination light, illuminating the sample 16. The non-super-resolution illumination light generated by reflection and scattering from the sample 16 is collected by the objective lens 15 and sequentially passes through the quarter-wave plate 13, the second beam splitter 19, the first beam splitter 12, the third beam splitter 27, and the third telescopic lens 28 before entering the second digital camera 29. This images the sample 16 onto the second digital camera 29 and are transmitted to the computer via the second data line 30. The computer displays a wide-field microscopic image of the sample 16, facilitating wide-field observation of the sample 16 and determining the desired super-resolution imaging region. If the sample 16 is not within the super-resolution imaging region, the computer can control the two-dimensional displacement stage 17 to move the sample 16 to the super-resolution imaging region.

[0049] Step 2: After determining the super-resolution imaging region, 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-long depth-of-field super-resolution focal spot to illuminate sample 16. It collects the super-long depth-of-field super-resolution signal light generated by reflection and scattering from sample 16 and completes polarization conversion. Specifically: Non-super-resolution illumination source 25 does not emit light, while super-resolution illumination source 1 is turned on, emitting light with wavelength λ. This light is transmitted to fiber optic collimator 3 via the first fiber optic jumper 2. The fiber optic collimator 3 collimates the light and outputs a collimated beam. This collimated 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 (assumed to be a P-polarized ring beam). After passing through the first 4f system, the beam scanning module, and the quarter-wave plate 13, this P-polarized ring beam forms a circularly polarized focused line on the back focal plane of objective lens 15 with an outer diameter less than or equal to the diameter of the back focal plane of objective lens 15. This circularly polarized focused line, after passing through objective lens 15, is converted into a super-resolution conical focused beam, which is focused on the front focal plane 3 of objective lens 15. A super-long depth-of-field super-resolution focusing spot is formed before and after 9. The full width at half maximum (FWHM) of this super-long depth-of-field super-resolution focusing 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 position of the focal spot intensity to the first zero-point position) is less than 0.61λ / NA. This super-long depth-of-field super-resolution focusing spot illuminates the sample 16. The super-long depth-of-field super-resolution signal light generated by the reflection and scattering of the sample 16 is collected by the objective lens 15 and converted into an S-polarized collimated beam orthogonal to the incident linearly polarized ring beam by the quarter-wave plate 13. The S-polarized collimated beam is reflected by the second beam splitter 19 and enters the super-large depth-of-field super-resolution wide-field optical microscopy imaging module to realize super-long depth-of-field super-resolution wide-field microscopy imaging of the sample 16.

[0050] like Figure 2 As shown, in another embodiment, the annular focusing beam generating module may also exclude the field lens 32.

[0051] Figure 2 A method for generating a P-polarized annular focused beam is presented. Specifically: An optical fiber collimator 3 collimates the light and outputs a collimated beam with radius R (different numerical aperture optical fiber collimators can be used to obtain collimated beams of different radii, thus resulting in super-resolution conical focused beams of different focal depths). This collimated beam with radius R passes through a first linear polarizer 4 to become a P-polarized collimated beam with radius R; this P-polarized collimated beam is then passed through a coaxially placed... d After the conical lens 5 and the first positive lens 6, a P-polarized ring-shaped focused beam is formed, forming a central radius R' on the rear focal plane of the first positive lens 6. c The first circular focal line can have its center radius R' changed by altering the focal length of the first positive lens 6. 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 second circular focal line. Where, R'' 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 second positive lenses 7 and third positive lenses 8 with different focal lengths, second circular focal lines of different sizes can be obtained. Furthermore, through the second 4f system composed of beam scanning lens 10 and first telescope 11, the second circular focal line is projected onto the back focal plane F' of the first telescope 11. t (Also the back 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 controls the center radius R of the final formed third circular focal line. c =KR' c × f 3 / f 2.

[0052] Figure 3 A schematic diagram of the illumination process for forming a super-resolution focused light field with extremely long focal depth is provided. A circularly polarized ring-shaped focused beam (assumed to be a left-handed circularly polarized ring-shaped focused beam) illuminates the back focal plane of objective lens 15, forming a light field with a central diameter of 2R on the back focal plane of objective lens 15. c A circularly polarized circular focusing line, the inner radius of which is R. in The outer radius is R out Center radius R c =(R out +R in ) / 2, the left-hand circularly polarized ring-shaped focused beam, after passing through objective lens 15, is transformed into a super-resolution conical focused beam with a cone angle of θ. c This creates an ultra-long depth-of-focus super-resolution focused light field along the optical axis before and after the objective lens's front focal plane 39. The numerical aperture NA of the super-resolution conical focused beam is... c =n×sin(θ c ), NA c ≤NA; where sin() is the sine function, and n is the refractive index of the medium between objective lens 15 and sample 16. The full width at half maximum (FWHM) of this ultra-long depth-of-focus super-resolution focusing light field is less than 0.5λ / NA, and the first null radius (distance from the peak intensity position of the focal spot to the first null position) of the ultra-long depth-of-focus super-resolution focusing light field is less than 0.61λ / NA. When 0 ≤ NA c When λ / NA is less than or equal to 0.947λ / NA, the actual resolution of super-resolution microscopy is greater than 0.25λ / NA and less than 0.5λ / NA. To achieve super-resolution microscopy with practical application and even better resolution, the following condition must be met: 0.947λ ≤ NA. c ≤NA. The resolution of super-resolution microscopy is ≤NA.c Decision, NA c The larger and closer the value is to NA, the closer the resolution of super-resolution microscopy can get to the limit resolution of 0.25λ / NA for reflective confocal systems. c The larger the value, the greater the corresponding value of R. c The larger (R) c (smaller than the back focal plane radius of objective lens 15), and the corresponding R out R in The closer the focal length is to the point of focus (i.e., the smaller the width of the circularly polarized focal line), the narrower the spatial frequency of the super-resolution focused light field. In practice, we control the radius R of the collimated beam and adjust the distance between the conical lens 5 and the first positive lens 6. d To control R' c This allows for the control of the radius of the circularly polarized circular focusing line. However, because the S-polarized annular focused beam corresponding to the circularly polarized circular focusing line undergoes significant diffraction during propagation, resulting in a larger width, it becomes impossible to guarantee that the circularly polarized circular focusing line reaching the back focal plane of objective lens 15 will maintain a minimal 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 focused beam (corresponding to the circularly polarized circular focusing line), so that the numerical aperture NA of the final super-resolution conical focused beam is... c Satisfies: 0.947NA≤NA c ≤NA. The first 4f system, composed of the second positive lens 7 and the third positive lens 8, serves two purposes: firstly, it adjusts the size of the circular focal line of the P-polarized ring-focused beam; secondly, it acts as a relay for the transmission of the P-polarized ring-focused beam. This ensures that the circular focal line of the first 4f system, after adjustment of its front focal plane, is accurately projected onto the rear focal plane of the first 4f system (corresponding to the rear focal plane of objective lens 15). This avoids size distortion and light field intensity distribution fluctuations caused by diffraction during direct transmission of the P-polarized ring-focused beam, ensuring high-quality illumination of the objective lens by the P-polarized ring-focused beam. Consequently, it minimizes the super-resolution focal spot, ensuring the system achieves optimal resolution.

[0053] Given the numerical aperture NA of objective lens 15, how can we ensure that the cone angle of the super-resolution conical focused beam after passing through objective lens 15 is θ? c This can be achieved as follows: First, based on the desired resolution d... r Determine NA c And NA, specifically d r =0.5λ / (NA c +NA), where NA c ≤NA, NA c This represents the numerical aperture corresponding to the circularly polarized circular focusing line on the back focal plane of objective lens 15. (Based on NA) c Determine Rc (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 (of which) β Let be the cone angle of the cone-shaped beam emitted from cone lens 5, and tan() be the tangent function. f 1. f 2 and f 3 ( f 3 needs to be of sufficient length so that the third positive lens 8 will not spatially interfere with subsequent optical elements.

[0054] Figure 4 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 with objective lens 15 and numerical aperture NA = 0.95 are given. c The functional relationship. It can be seen that when NA c When R ≥ 0.9, the resulting 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, an ultra-long depth-of-focus super-resolution illumination field can be achieved with FWHM≤0.399λ, r0≤0.426λ, and SR=0.162.

[0055] like Figure 5 The diagram shows the scanning of the ultra-long depth-of-focus super-resolution focused beam on the focal plane of objective lens 15. The beam scanning module performs two-dimensional scanning of the circularly polarized circularly focused beam. At different times, the circularly polarized circularly focused beam is incident on objective lens 15 at different angles. When the circularly polarized circularly focused beam reaches the rear focal plane 38 of the objective lens, it always forms a ring centered on the center of the rear focal plane. At a given time t, the tilt angle of the incident circularly polarized circularly 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) of 2ΔZ is formed before and after the objective lens's rear focal plane 38 (Z direction). The offset of this super-long depth-of-focus super-resolution focusing spot in the XY direction (relative to the optical axis Z) is Δ(t). By changing the incident angle θ s (t), which can change the offset Δ(t). At different times, the intensity distribution of the circularly polarized ring-shaped focused beam on the rear focal plane 38 of the objective lens is always a ring centered on the center of the rear focal plane, and the inner and outer radii of this ring are R and R, respectively. in and Rout .

[0056] like Figure 6 The figure shows the relationship between the full width at half maximum (FWHM) of the objective lens (numerical aperture NA > 1) and the system resolution under circularly polarized ring-focused beam illumination. The vertical axis represents the FWHM of the objective lens's point spread function (MPF), in wavelength λ; the horizontal axis represents the lateral FWHM of the super-resolution focal 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 focal 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 MPF and the lateral FWHM of the super-resolution focal spot are 0.35λ, the system resolution is better than 0.2λ.

[0057] 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, acquire ultra-long depth-of-field super-resolution signal light, and send it to the ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module to form ultra-large depth-of-field super-resolution wide-field optical imaging on the target surface of the first digital camera 23.

[0058] Step 4: The computer directly reads the ultra-large depth-of-field super-resolution wide-field optical image captured by the first digital camera 23 through the first data line 24, and processes and displays it.

[0059] In this embodiment, the specific processing steps of steps three and four are as follows: When performing super-resolution imaging, the non-super-resolution illumination source 25 is turned off, and the light source unit and beam scanning module are turned on to perform beam scanning. The ultra-long depth-of-field super-resolution signal light collected by the super-resolution focusing illumination and collection module is reflected by the second beam splitter 19 into the ultra-large depth-of-field super-resolution wide-field optical microscopic imaging module. It passes through the fourth positive lens 20, the second conical lens 21, the second linear polarizer 33, and the second telescopic lens 22 in sequence, forming an ultra-large depth-of-field super-resolution wide-field optical image of sample 16 on the target surface of the first digital camera 23. After being acquired by the first digital camera 23, it is sent to the computer for processing and display through the first data line 24. In the computer-controlled beam scanning module, beam scanning mirror 9 performs two-dimensional scanning. The ultra-long depth-of-focus super-resolution focused spot formed by the circularly polarized ring-focused beam at the front focal plane 39 of the objective lens will achieve continuous two-dimensional scanning super-resolution illumination in the XY plane (objective lens focal plane) within the focal depth range of the ultra-long depth-of-focus super-resolution focused spot before and after the front focal plane 39 of the objective lens, as the beam scanning mirror 9 vibrates. The abscissa X(t) and ordinate Y(t) of the position of the ultra-long depth-of-focus super-resolution focused spot on the sample 16 are functions of time t. The reflected light E collected by objective lens 15...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 The image (t) reflects information such as the sample structure and refractive index at the corresponding positions (x-axis X(t) and y-axis Y(t)). The resolution of this image in the XY plane is close to the limiting resolution of a reflective confocal system (0.25λ / NA). A computer-controlled axial nanopositioner 14 moves the objective lens 15 in the Z-direction, changing the Z-position of the objective lens 15's front focal plane within the sample. f To obtain different Z f By obtaining a two-dimensional super-resolution microscopic image in the XY plane within a focal depth range of 2△Z before and after the location, a large-depth imaging microscopic image can be obtained, with a resolution close to the limit resolution of 0.25λ / NA of the reflective confocal system.

[0060] Example 2 like Figure 7 As shown in the second embodiment, most of the structure of the reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system is the same as that in the first embodiment. The difference is that the sample displacement module is a three-dimensional displacement stage 31. The three-dimensional displacement stage 31 is connected to the computer through the scanning signal line 18. The sample 16 is horizontally fixed on the three-dimensional displacement stage 31. The computer controls the three-dimensional displacement stage 31 to move the sample 16 in the Z-axis direction.

[0061] like Figure 7 As shown, the reflection-based ultra-large depth-of-field super-resolution wide-field optical microscopy method 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 31 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 deep-depth imaging microscopic images, with a resolution close to the limit resolution of 0.25λ / NA of reflective confocal systems.

[0062] Example 3 In this third embodiment, most of the structure of a reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system is the same as that in the first embodiment, except that the first linear polarizer 4, the quarter-wave plate 13, and the second linear polarizer 33 are removed.

[0063] Example 4 In this fourth embodiment, most of the structure of a reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system is the same as that in the first embodiment, except that the second beam splitter 19 is replaced by a polarization beam splitter.

[0064] 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 ultra-large depth-of-field super-resolution wide-field optical microscopy system, characterized in that, include: The sample displacement module is used to move the sample (16); Ring-shaped focused beam generation module, used to generate a ring-shaped focused beam; The beam scanning module is used to achieve rapid two-dimensional scanning of the beam; The super-resolution focusing illumination and collection module is used to convert the ring focusing beam into a super-resolution conical focusing beam (34) to form a long depth-of-field super-resolution focal spot (35) to illuminate the sample (16), collect the ultra-long depth-of-field super-resolution signal light generated by the reflection and scattering of the sample (16), converge and collimate the ultra-long depth-of-field 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 (39) of the objective lens; The ultra-deep field super-resolution wide field optical microscopy imaging module includes a second beam splitter (19), a fourth positive lens (20), a second conical lens (21), a second telescope (22), and a first digital camera (23). The second beam splitter (19) reflects the ultra-deep field super-resolution signal light collected by the super-resolution focusing illumination and collection module into the ultra-deep field super-resolution wide field optical microscopy imaging module. After passing through the fourth positive lens (20) and the second conical lens (22), it is converted into an annular collimated beam. The annular collimated beam is then converged on the target surface of the first digital camera (23) through the second telescope (22) to form an ultra-deep field super-resolution wide field optical imaging. The optical microscopy imaging module is used to perform wide-field imaging of the sample (16), determine the super-resolution imaging area, and realize the real-time acquisition of super-resolution images. The optical microscopy imaging module includes a third beam splitter (27), a third telescope (28), a second digital camera (29), a non-super-resolution illumination source (25), and a collimating lens (26). The non-super-resolution illumination source (25) is located at the front focal point of the collimating lens (26). The light emitted from the non-super-resolution illumination source (25) is collimated by the collimating lens (26), reflected by the third beam splitter (27), and then passes sequentially through the first beam splitter (12) and the second beam splitter of the beam scanning module. (19) The quarter-wave plate (13) reaches the objective lens (15) and converges into non-super-resolution illumination light on the front focal plane (39) of the objective lens to illuminate the sample (16). The non-super-resolution illumination 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 (19), the first beam splitter (12), the third beam splitter (27), and the third telescope (28) in sequence before entering the second digital camera (29), so that the sample (16) is imaged on the second digital camera (29) and sent to the computer, and the computer displays the wide-field microscopic image of the sample (16). The 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 ultra-large depth-of-field super-resolution wide-field optical microscopy 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 imaging area.

2. The reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system according to claim 1, characterized in that: The ring-shaped focused beam generating module includes, in sequence along the light propagation direction, a light source unit, 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 unit generates a collimated beam. After passing through the first linear polarizer (4), the conical lens (5), and the first positive lens (6), the collimated beam forms a linearly polarized ring-shaped focused beam and forms a first circular focusing line on the rear focal plane of the first positive lens (6). Alternatively, the ring-shaped focused beam generating module may include, along the direction of light propagation, a light source unit, a conical lens (5), a first positive lens (6), a second positive lens (7), and a third positive lens (8), wherein 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), 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) constitute a first 4f system; the light source unit generates a collimated beam, which forms a linearly polarized ring-shaped focused beam after passing through the conical lens (5) and the first positive lens (6), and forms a first circular focusing line on the rear focal plane of the first positive lens (6).

3. The reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system according to claim 1, characterized in that: The beam scanning module includes a beam scanning galvanometer (9), a beam scanning lens (10), a first telescope (11), and a first beam splitter (12). The center of the beam scanning galvanometer (9) coincides with the back focal plane of the third positive lens (8) after reflection by the beam scanning galvanometer (9). 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 reflection by the first beam splitter (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). 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 first beam splitter (12) to the back 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 (37) of the back focal plane of the objective lens, and the incident angle of the annular focused beam changes with the vibration of the beam scanning galvanometer (9); the annular focused beam is converted by the objective lens (15) and... A conical focusing light field is formed behind the objective lens (15), which in turn generates an ultra-long focal depth focusing light field and forms an ultra-long focal depth super-resolution focusing spot in front of and behind the front focal plane (39) of the objective lens. When the beam scanning galvanometer (9) performs two-dimensional scanning, the ultra-long focal depth focusing spot will be continuously scanned in the XY plane in the focal depth range of the ultra-long focal depth super-resolution focusing spot in front of and behind the front focal plane of the objective lens (15) in the optical axis direction, as the beam scanning galvanometer (9) vibrates. The XY plane is parallel to the focal plane of the objective lens (15).

4. The reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system according to claim 1, characterized in that: The super-resolution focused illumination and collection module also includes a quarter-wave plate (13). The linearly polarized ring-focused beam emitted from the beam scanning module passes through a quarter-wave plate (13) and forms a circularly polarized circular focusing line on the back focal plane (38) of the objective lens with an outer diameter less than or equal to the diameter of the back focal plane of the objective lens (15). This circularly polarized circular focusing line is transformed into a super-resolution conical focusing beam after passing through the objective lens (15) and forms an ultra-long focal depth super-resolution focusing spot in front of and behind the front focal plane (39) of the objective lens. This ultra-long focal depth super-resolution focusing spot illuminates the sample (16). The super-resolution signal light generated by the reflection and scattering of the sample (16) within the focal depth range is collected by the objective lens (15) and transformed by the quarter-wave plate (13) into a linearly polarized ring-focused beam and a linearly polarized collimated beam orthogonal to the incident linearly polarized ring beam, i.e., a linearly polarized super-resolution signal beam, which is reflected by the second beam splitter (19) and enters the ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module. When the beam scanning galvanometer (9) in the beam scanning module performs two-dimensional scanning, the ring-shaped focused beam is converted into a super-resolution conical focused beam by the objective lens (15). The super-resolution focused spot with ultra-long focal depth formed before and after the front focal plane of the objective lens (15) will be continuously illuminated in the XY plane as the beam scanning galvanometer (9) vibrates.

5. The reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system according to claim 3, characterized in that: The ultra-long depth-of-focus super-resolution focusing spot is a conical focusing light field. The inner and outer surfaces of this conical focusing light field are two coaxial conical surfaces with equal cone angles. The numerical aperture NA corresponding to this conical focusing light field is... c For a given resolution d r Requires satisfying: d r =0.5λ / (NA c +NA); when 0.947NA≤NA c The optimal resolution is achieved when the value is ≤NA; where NA represents the numerical aperture of the objective lens (15).

6. The reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system 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 the computer. The sample (16) is horizontally fixed on the two-dimensional displacement stage (17). The computer controls the two-dimensional displacement stage (17) to move the sample (16) in two dimensions 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 (14). The axial nanopositioner (14) is connected to the computer. 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. Alternatively, the sample displacement module is a three-dimensional displacement stage (31), which is connected to a computer. The sample (16) is horizontally fixed on the three-dimensional displacement stage (31), and the computer controls the three-dimensional displacement stage (31) to move the sample (16) in the X, Y, and Z directions.

7. The reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system according to claim 2, characterized in that: The light source unit includes a super-resolution illumination source (1) and an optical fiber collimator (3). The super-resolution illumination source (1) is connected to the optical fiber collimator (3) through a first optical fiber jumper (2). The light emitted by the super-resolution illumination source (1) is transmitted to the optical fiber collimator (3) through the first optical fiber jumper (2). The optical fiber collimator (3) collimates the light and outputs a collimated beam. The super-resolution illumination source (1) is a coherent light source or a partially coherent light source.

8. The reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system according to claim 2, characterized in that: The annular focusing beam generating module also includes a field lens (32), 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 (32) 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.

9. The reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system according to claim 2, characterized in that: The ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module also includes a second linear polarizer (33). When performing super-resolution imaging, the non-super-resolution illumination source (25) is turned off, and the light source unit and beam scanning module are turned on to perform beam scanning. The ultra-long depth-of-field super-resolution signal light collected by the super-resolution focusing illumination and collection module is reflected by the second beam splitter (19) into the ultra-large depth-of-field super-resolution wide-field optical microscopic imaging module. After passing through the fourth positive lens (20), the second conical lens (21), the second linear polarizer (33), and the second telescope (22), it is converted into an annular collimated beam. The annular collimated beam is converged on the target surface of the first digital camera (23) through the second telescope (22) to form an ultra-large depth-of-field super-resolution wide-field optical imaging. After being acquired by the first digital camera (23), it is sent to the computer for processing and display through the first data line (24).

10. A reflective ultra-large depth-of-field super-resolution wide-field optical microscopy method, employing the reflective ultra-large depth-of-field super-resolution wide-field optical microscopy system as described in 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 the super-resolution imaging area; Step 2: After determining the super-resolution 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 an ultra-long focal length super-resolution focusing spot to illuminate the sample (16), collect the ultra-long depth-of-field super-resolution signal light generated by the reflection and scattering of the sample (16), and complete the 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, obtain the ultra-long depth-of-field super-resolution signal light, and send it into the ultra-large depth-of-field super-resolution wide-field optical microscopy imaging module to form an ultra-large depth-of-field super-resolution wide-field optical image on the target surface of the first digital camera (23). Step 4: The computer reads the ultra-deep field super-resolution wide field optical image captured by the first digital camera (23), and processes and displays it.

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