Annular focused scanning illumination for wide-field super-resolution optical microscopy systems and methods

Abstract

The application discloses a kind of annular focusing scanning illumination's reflection wide field super-resolution optical microscopic system and method, system includes sample displacement module, annular light beam generation module, light beam scanning module, super-resolution focusing illumination and collection module, optical microscopic imaging module, phase contrast module and computer.By utilizing annular light beam illumination, the advantage of high focusing efficiency of conventional lens, fast light beam scanning speed, realize fast, efficient super-resolution focusing illumination, and then can be realized fast super-resolution optical microscopic imaging using conventional optical lens;By two-dimensional light beam scanning illumination, realize non-labeled far-field super-resolution two-dimensional microscopic fast wide field imaging;By the non-labeled far-field super-resolution two-dimensional microscopic wide field imaging of sample at different axial positions, obtain the super-resolution two-dimensional microscopic wide field imaging of different cross sections of sample, and then realize three-dimensional tomography.The application can be applied to non-labeled super-resolution microscopic fast imaging of biological sample, and can also be applied to industrial related super-resolution microscopic detection and other fields.

Description

Technical Field

[0001] This invention belongs to the field of visible light microscopy, specifically relating to a wide-field super-resolution optical microscopy system and method with ring-shaped focused scanning illumination. Background Technology

[0002] Existing microscopy techniques can be broadly categorized into near-field microscopy and far-field microscopy. Near-field microscopy includes methods such as microsphere-assisted imaging and near-field scanning microscopy. While near-field microscopy can achieve super-resolution imaging, its working distance is smaller than the working wavelength, significantly limiting its practical applications. Far-field super-resolution techniques, with their large working distances, overcome the limitations of near-field microscopy. Existing far-field super-resolution microscopy techniques include stimulated emission depletion microscopy, local activation microscopy, and random light reconstruction optical microscopy. However, these techniques all require fluorescent molecular labeling of the sample. Label-free far-field super-resolution microscopy offers advantages such as large working distance, no need for sample labeling, and ease of use, thus becoming an important development trend in super-resolution optical microscopy. In recent years, a few experiments have been reported on label-free far-field super-resolution microscopy based on super-diffraction devices. These systems all rely on novel super-resolution devices as illumination. However, the focusing efficiency of super-resolution devices is typically low, and most of these systems depend on moving sample scanning, resulting in slow imaging speeds.

[0003] Therefore, it is necessary to develop a reflective wide-field super-resolution optical microscopy system and method with ring-shaped focused scanning illumination. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by providing a wide-field super-resolution optical microscopy system and method with ring-shaped focused scanning illumination, so as to achieve better resolution while improving reliability, imaging speed, signal-to-noise ratio, and environmental adaptability.

[0005] In a first aspect, the present invention provides a ring-shaped focused scanning illumination reflective wide-field super-resolution optical microscopy system, comprising: The sample displacement module is used to move the sample. Ring beam generation module, used to generate ring beams; The beam scanning module is used to achieve rapid two-dimensional scanning of the beam, thereby enabling rapid two-dimensional super-resolution scanning illumination. The super-resolution focusing illumination and collection module includes a first objective lens, with the sample located on the front focal plane of the first objective lens; it is used to convert the ring beam into a super-resolution ring focused beam, form a super-resolution focal spot to illuminate the sample, collect the super-resolution signal light generated by the reflection and scattering of the sample, and collimate and convert it. An optical microscopy imaging module includes a non-super-resolution illumination source, a collimating lens, a second beam splitter, a first beam splitter, a second telescope, and a digital camera. It is used for non-super-resolution wide-field imaging of samples, determining the super-resolution imaging region, and realizing real-time acquisition of both non-super-resolution wide-field and super-resolution wide-field imaging. The non-super-resolution illumination source is located at the front focal point of the collimating lens. A phase contrast module is used to generate the reference light required for phase contrast microscopy and to achieve super-resolution phase contrast wide-field imaging in conjunction with an optical microscopy imaging module. The phase contrast module includes a second quarter-wave plate, a third positive lens, and a second reflecting mirror arranged in sequence, and the front focal plane of the third positive lens coincides with the back focal plane of the first telescope after reflection by the first reflecting mirror and the first beam splitter; or the phase contrast module includes a second quarter-wave plate, a second objective lens, and a second reflecting mirror arranged in sequence, and the exit pupil plane of the second objective lens coincides with the back focal plane of the first telescope after reflection by the first reflecting mirror and the first beam splitter. The computer is used to control the sample displacement module to move the sample, and synchronously control the beam scanning galvanometer to scan and acquire non-super-resolution wide-field or super-resolution wide-field imaging from the optical microscopy imaging module, and process the obtained microscopic images.

[0006] Optionally, the optical microscopic imaging module further includes a second linear polarizer disposed between the second sleeve lens and the second beam splitter; The non-super-resolution illumination source of the optical microscopy imaging module 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, reflected by the second beam splitter, reflected by the first beam splitter, and reaches the first objective lens. It is then focused on the front focal plane of the first objective lens to provide non-super-resolution illumination to the sample. The non-super-resolution illumination light reflected and scattered by the sample is collected by the first objective lens and then passes through the first beam splitter, the second beam splitter, the second linear polarizer, and the second telescopic lens in sequence before entering the digital camera, so that the sample is imaged on the digital camera to obtain a non-super-resolution wide-field imaging result.

[0007] Optionally, the back focal plane of the third positive lens of the phase contrast module coincides with the reflecting surface of the second mirror. When the phase contrast module is needed, a portion of the ring beam from the beam scanning module is reflected into the phase contrast module by the first beam splitter. This linearly polarized ring beam passes through the second quarter-wave plate, is focused on the back focal plane of the third positive lens, and is reflected by the second mirror. After passing through the third positive lens, the second quarter-wave plate, and the first beam splitter in sequence, it enters the optical microscopic imaging module as reference light to participate in super-resolution phase contrast wide-field imaging on the target surface of the digital camera. 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 super-resolution signal light collected by the super-resolution focusing illumination and collection module is reflected by the first beam splitter into the optical microscopic imaging module. After passing through the second beam splitter and the second telescope in sequence, a super-resolution wide-field image of the sample is formed on the target surface of the digital camera. After being acquired by the digital camera, it is sent to the computer for processing and display. When performing super-resolution phase-contrast wide-field imaging, the non-super-resolution illumination source is turned off, and the light source unit and beam scanning module are turned on for beam scanning. The super-resolution signal light collected by the super-resolution focusing illumination and collection module is reflected by the first beam splitter and enters the optical microscopy imaging module. Simultaneously, the reference light reflected back from the phase-contrast module enters the optical microscopy imaging module through the first beam splitter. The two beams are parallel or coaxial and pass sequentially through the second beam splitter and the second telescope, interfering on the target surface of the digital camera to form a super-resolution phase-contrast wide-field image of the sample. This image is then acquired by the digital camera and sent to the computer for processing and display. When performing super-resolution imaging and non-super-resolution imaging fusion, both the non-super-resolution illumination source and the light source unit need to be turned on simultaneously.

[0008] Optionally, the super-resolution focusing illumination and collection module further includes a first quarter-wave plate; after the linearly polarized ring beam emitted from the beam scanning module passes through the first quarter-wave plate, a circularly polarized ring beam is formed on the exit pupil surface of the first objective lens, which is concentric with the exit pupil of the first objective lens and has an outer diameter less than or equal to the exit pupil diameter of the first objective lens. After passing through the first objective lens, the circularly polarized ring beam is converted into a super-resolution ring focused beam and forms a super-resolution focused spot on the front focal plane of the first objective lens. The super-resolution focused spot illuminates the sample. The super-resolution signal light generated by the reflection and scattering of the sample is collected and collimated by the first objective lens and then converted into an S-linearly polarized collimated beam orthogonal to the incident linear polarization by the first quarter-wave plate. After being reflected by the first beam splitter, the S-linearly polarized collimated beam enters the optical microscopic imaging module and is transmitted through the second beam splitter and the second telescope to form a super-resolution wide-field image on the target surface of the digital camera. When the beam scanning galvanometer in the beam scanning module performs a two-dimensional scan, the super-resolution focused spot formed by the circularly polarized ring beam on the front focal plane of the first objective lens will achieve rapid and continuous two-dimensional scanning illumination on the front focal plane of the first objective lens as the beam scanning galvanometer vibrates. The full width at half maximum (FWHM) of the resulting super-resolution focused spot is less than 0.5λ / NA, and the first null radius of the super-resolution focal spot (i.e., the distance from the peak intensity position of the focal spot to the first null position) is less than 0.61λ / NA, where λ represents the wavelength of the collimated beam, and NA represents the numerical aperture of the first objective lens.

[0009] Optionally, when the system is in the unmarked super-resolution intensity wide-field imaging mode, the first beam splitter is a polarization beam splitter, thereby improving the efficiency of reflected and transmitted light.

[0010] Optionally, the ring beam generating module includes a light source unit, a first linear polarizer, a beam expander, and a ring spatial filter arranged sequentially along the light propagation direction; the light source unit includes a super-resolution illumination source and an optical fiber collimator, the super-resolution illumination source being connected to the optical fiber collimator; the light emitted by the super-resolution illumination source is transmitted to the optical fiber collimator, which collimates the light and outputs a collimated beam; the ring spatial filter is placed coaxially and spaced apart from the beam expander; the light source unit generates a collimated beam, which, after passing through the first linear polarizer, the beam expander, and the ring spatial filter, forms a linearly polarized ring beam; Alternatively, the ring beam generating module includes a light source unit, a first linear polarizer, a beam expander, a ring spatial filter, a first positive lens, and a second positive lens arranged sequentially along the light propagation direction; the light source unit includes a super-resolution illumination source and an optical fiber collimator, the super-resolution illumination source being connected to the optical fiber collimator; the light emitted by the super-resolution illumination source is transmitted to the optical fiber collimator, which collimates the light and outputs a collimated beam; the ring spatial filter and the beam expander are placed coaxially and spaced apart, the front focal plane of the first positive lens is located behind the exit surface of the ring spatial filter, and the rear focal plane of the first positive lens coincides with the front focal plane of the second positive lens; the light source unit generates a collimated beam, which, after passing through the first linear polarizer, the beam expander, and the ring spatial filter, forms a linearly polarized ring beam; Alternatively, the ring beam generating module may further include a light source unit, a first linear polarizer, a first conical lens, a second conical lens, and a ring spatial filter arranged sequentially along the light propagation direction; the light source unit includes a super-resolution illumination source and an optical fiber collimator, the super-resolution illumination source being connected to the optical fiber collimator; the light emitted by the super-resolution illumination source is transmitted to the optical fiber collimator, which collimates the light and outputs a collimated beam; the ring spatial filter is placed coaxially and spaced apart from the beam expander, the first conical lens, and the second conical lens; the light source unit generates a collimated beam, which, after passing through the first linear polarizer, the beam expander, and the ring spatial filter, forms a linearly polarized ring beam; the use of the conical lens group can improve the light energy utilization rate, and the spatial filter after the conical lens group also filters out the light energy within the inner diameter of the ring beam generated by the conical lens, reducing the participation of non-ring light components in imaging, thereby improving the imaging quality. Alternatively, the ring beam generating module may further include a light source unit, a first linear polarizer, a first conical lens, a second conical lens, a ring spatial filter, a first positive lens, and a second positive lens arranged sequentially along the light propagation direction. The light source unit includes a super-resolution illumination source and an fiber optic collimator, with the super-resolution illumination source connected to the fiber optic collimator. Light emitted from the super-resolution illumination source is transmitted to the fiber optic collimator, which collimates the light and outputs a collimated beam. The ring spatial filter is coaxially spaced with the beam expander, the first conical lens, and the second conical lens. The front focal plane of the first positive lens is located behind the exit surface of the ring spatial filter, and the rear focal plane of the first positive lens coincides with the front focal plane of the second positive lens. The light source unit generates a collimated beam, which, after passing through the first linear polarizer, the beam expander, and the ring spatial filter, forms a linearly polarized ring beam. The use of the conical lens group can improve light energy utilization. The spatial filter following the conical lens group also filters out light energy within the inner diameter of the ring beam generated by the conical lens, reducing the participation of non-ring light components in imaging, thereby improving image quality. 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).

[0011] Optionally, the beam scanning module includes a beam scanning galvanometer, a beam scanning lens, a first sleeve lens, and a first reflecting mirror; the center of the beam scanning galvanometer coincides with the back focal plane of the second positive lens; the front focal plane of the beam scanning lens is located at the center of the beam scanning galvanometer; the back focal plane of the beam scanning lens coincides with the front focal plane of the first sleeve lens; the back focal plane of the first sleeve lens, after being reflected by the first reflecting mirror, coincides with the exit pupil plane of the first objective lens; the beam scanning galvanometer includes a controller for connecting 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; When the ring beam generated by the ring beam generation module is reflected by the beam scanning galvanometer, and then through the beam scanning lens and the first sleeve lens, it is reflected by the first reflecting mirror to the exit pupil of the first objective lens; when the beam scanning galvanometer vibrates, the center of the ring beam always coincides with the center of the exit pupil of the first objective lens, and the incident angle of the ring beam changes with the vibration of the beam scanning galvanometer; when the beam scanning galvanometer performs two-dimensional scanning, the ring beam will form a focused spot on the front focal plane of the first objective lens, and this focused spot will achieve rapid and continuous two-dimensional scanning of the sample on the front focal plane of the first objective lens with the vibration of the beam scanning galvanometer.

[0012] Optionally, the super-resolution focused spot is an annular focused light field, the inner and outer surfaces of which are two coaxial conical surfaces, and the inner numerical aperture NA corresponding to the annular focused light field is... in External numerical aperture (NA) out For a given resolution d r d needs to be satisfied. r =0.5λ / (NA in +NA); when 0.947NA≤NA in <NA,NA out The optimal resolution is achieved when 0 ≤ NA; where NA represents the numerical aperture of the first objective lens 15. In practice, as long as 0 ≤ NA is satisfied... in <NA out ≤NA can achieve super-resolution wide-field imaging, but cannot achieve the optimal resolution.

[0013] Optionally, to achieve three-dimensional tomographic scanning of the sample, the sample displacement module has two structures.

[0014] 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 two-dimensional displacement stage to move the sample in two dimensions 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 first objective lens is mounted on the axial nanopositioner, and the computer controls the axial nanopositioner to move the first objective lens in the Z direction. The beam scanning module realizes two-dimensional scanning of the sample, and combined with the axial nanopositioner, realizes three-dimensional tomographic scanning.

[0015] The second method involves a three-dimensional displacement module, which is connected to a computer. The sample is horizontally fixed on the three-dimensional displacement stage, and the computer controls the stage to move the sample in the X, Y, and Z directions. The X and Y directions are used for initial sample positioning; the beam scanning module performs two-dimensional scanning of the sample, and the Z-direction displacement of the three-dimensional displacement stage enables three-dimensional tomographic scanning. This avoids the imaging effects caused by minute movements of the first objective lens, resulting in better imaging performance.

[0016] Secondly, the present invention provides a reflective wide-field super-resolution optical microscopy method with ring-focused scanning illumination, employing a reflective wide-field super-resolution optical microscopy system with ring-focused scanning illumination as described in the present invention. The method includes at least one of the following: label-free super-resolution intensity wide-field imaging mode, label-free super-resolution phase-contrast wide-field imaging mode, simultaneous acquisition mode of label-free super-resolution intensity wide-field imaging and non-super-resolution wide-field imaging, and simultaneous acquisition mode of label-free super-resolution phase-contrast wide-field imaging and non-super-resolution wide-field imaging. In label-free super-resolution intensity wide-field imaging mode: Step 11: Use an optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample to determine the super-resolution imaging region; Step 12: Turn off the phase contrast module so that it does not participate in imaging; Step 13: After determining the super-resolution imaging area, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam to form a super-resolution focal spot to illuminate the sample, collect the super-resolution signal light generated by the sample reflection and scattering, and collimate and convert it. Step 14: The computer-controlled sample displacement module moves the sample to the position to be scanned and turns on the beam scanning module to scan. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light to form a super-resolution wide-field image on the target surface of the digital camera. Step 15: The computer reads the super-resolution wide-field image captured by the digital camera, processes it, and displays it; In label-free super-resolution phase-contrast wide-field imaging mode: Step 21: Use an optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample to determine the super-resolution imaging region; Step 22: Turn on the phase contrast module to participate in imaging; Step 23: After determining the super-resolution imaging area, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam to form a super-resolution focal spot to illuminate the sample, collect the super-resolution signal light generated by the sample reflection and scattering, and collimate and convert it. Step 24: The computer-controlled sample displacement module moves the sample to the position to be scanned and turns on the beam scanning module to scan. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light. The super-resolution signal light and the reference light generated by the phase contrast module interfere on the target surface of the digital camera to form super-resolution phase contrast wide-field imaging. Step 25: The computer reads the super-resolution phase-contrast wide-field image captured by the digital camera, processes it, and displays it; In the simultaneous acquisition mode of label-free super-resolution intensity wide-field imaging and non-super-resolution wide-field imaging: Step 31: Turn on the non-super-resolution illumination source, use the optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample, determine the super-resolution imaging area, and keep the non-super-resolution wide-field imaging on at all times. Step 32: Turn off the phase contrast module so that it does not participate in imaging; Step 33: After determining the super-resolution imaging area, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam to form a super-resolution focal spot to illuminate the sample, collect the super-resolution signal light generated by the sample reflection and scattering, and collimate and convert it. Step 34: The computer-controlled sample displacement module moves the sample to the position to be scanned and turns on the beam scanning module to scan. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light to form a super-resolution wide-field image on the target surface of the digital camera. Meanwhile, after illuminating the sample with non-super-resolution illumination light, a non-super-resolution wide-field image is formed on the target surface of the digital camera. Step 35: The computer reads the image formed by fusing the unmarked super-resolution intensity wide-field image and the non-super-resolution wide-field image captured by the digital camera, and processes and displays it; In the simultaneous acquisition mode of label-free super-resolution phase-contrast wide-field imaging and non-super-resolution wide-field imaging: Step 41: Turn on the non-super-resolution illumination source and use the optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample, determine the super-resolution imaging area, and keep the non-super-resolution wide-field imaging on at all times. Step 42: Turn on the phase contrast module to participate in imaging; Step 43: After determining the super-resolution imaging area, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam to form a super-resolution focal spot to illuminate the sample, collect the super-resolution signal light generated by the sample reflection and scattering, and collimate and convert it. Step 44: The computer-controlled sample displacement module moves the sample to the position to be scanned and turns on the beam scanning module to perform scanning. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light. The super-resolution signal light and the reference light generated by the phase contrast module interfere on the target surface of the digital camera to form super-resolution phase contrast wide-field imaging. Meanwhile, after the sample is illuminated by non-super-resolution illumination light, non-super-resolution wide-field imaging is formed on the target surface of the digital camera. Step 45: The computer reads the image formed by fusing the unmarked super-resolution phase-contrast wide-field image and the non-super-resolution wide-field image captured by the digital camera, and processes and displays it. In steps 21 and 41, when the sample is a transparent sample, the sample is fixed on the reflective surface of the reflective slide, and the reflective slide is fixed on the sample displacement module.

[0017] The present invention has the following advantages: (1) High-efficiency super-resolution focusing illumination: This invention can efficiently generate a ring beam through a carefully designed ring beam generation module. Subsequently, using a super-resolution focusing illumination and collection module, the ring beam is converted into a super-resolution ring 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 high-efficiency super-resolution focusing illumination and significantly improving the signal-to-noise ratio of imaging.

[0018] (2) 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 illumination method is not only fast, enabling illumination of a large area in a short time, but also allows for direct acquisition of super-resolution wide-field imaging of the illuminated area sample via a digital camera. This feature greatly improves imaging efficiency, signal-to-noise ratio, system stability, and reliability, enabling users to quickly acquire large amounts of high-quality image data.

[0019] (3) Fast super-resolution wide field imaging: Compared with the confocal mode using photomultiplier tubes, the present invention does not require a high-speed acquisition card and photomultiplier tubes, which reduces the acquisition time of a single frame image, or greatly improves the field of view of a single frame image under the premise of the same single frame image acquisition speed.

[0020] (4) 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 wide-field, 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 and large field of view (equal to the field of view of the first objective lens), its application in fields such as biological and industrial detection becomes more convenient and efficient.

[0021] (5) Three-dimensional tomographic imaging capability: By performing label-free far-field super-resolution two-dimensional wide-field microscopic imaging on samples at different axial positions, this invention can acquire super-resolution two-dimensional microscopic images of different cross-sections of the sample, and then realize three-dimensional tomographic imaging through data processing. This function is of great significance for analyzing the internal structure of samples and revealing the spatial distribution of complex samples, and is particularly suitable for fields such as biomedical research and materials science.

[0022] (6) High reliability and imaging quality: This invention ensures the reliability and stability of imaging by precisely controlling the optical path coordination and signal processing between modules. From the generation of the ring 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.

[0023] (7) The present invention can achieve the acquisition of label-free super-resolution intensity wide field imaging, the acquisition of label-free super-resolution phase contrast wide field imaging, the simultaneous acquisition of label-free super-resolution intensity wide field imaging and non-super-resolution wide field imaging, and the simultaneous acquisition of label-free super-resolution phase contrast wide field imaging and non-super-resolution wide field imaging.

[0024] In summary, the ring-focused scanning illumination reflective wide-field super-resolution optical microscopy system and method of the present invention exhibit significant advantages in terms of efficient and rapid two-dimensional scanning super-resolution illumination, label-free far-field super-resolution microscopy imaging, three-dimensional tomography imaging capability, super-resolution three-dimensional imaging capability, as well as high reliability, imaging field of view, imaging speed, and imaging quality, providing strong technical support for fields such as biomedicine and industrial inspection. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the reflective wide-field super-resolution optical microscopy system with ring-shaped focusing scanning illumination in Example 1.

[0026] Figure 2 This is a schematic diagram of the beam expander, annular space filter, and first 4f system controlling the size of the annular beam in Embodiment 1.

[0027] Figure 3 This is a schematic diagram of the ring space filter structure in Embodiment 1 and Embodiment 2.

[0028] Figure 4 This is a schematic diagram of the cone lens group, the ring space filter, and the first 4f system controlling the size of the ring beam in Embodiment 1.

[0029] Figure 5 This is a schematic diagram of the formation of a super-resolution focused light spot in Example 1.

[0030] Figure 6 This is a parameter relationship diagram of the super-resolution focused spot in Example 1.

[0031] Figure 7 This is a schematic diagram of the super-resolution focused spot scanning on the focal plane of the first objective lens in Example 1.

[0032] Figure 8This is a graph showing the relationship between the full width at half maximum (FWHM) of the first objective lens and the focal spot and the system resolution under ring beam illumination in Example 1.

[0033] Figure 9 This is a schematic diagram of the reflective wide-field super-resolution optical microscopy system with ring-shaped focusing scanning illumination in Example 2.

[0034] Figure 10 This is a schematic diagram of the reflective wide-field super-resolution optical microscopy system with ring-shaped focusing scanning illumination in Example 4.

[0035] In the figure: 1. Super-resolution illumination source, 2. First fiber optic patch cord, 3. Fiber optic collimator, 4. First linear polarizer, 5. Beam expander, 6. Circular spatial filter, 7. First positive lens, 8. Second positive lens, 9. Beam scanning galvanometer, 10. Beam scanning lens, 11. First telescope, 12. First mirror, 13. First quarter-wave plate, 14. Axial nanopositioner, 15. First objective lens, 16. Sample, 17. Two-dimensional displacement stage, 18. Scanning signal line, 19. First beam splitter, 20. Second telescope, 21. Digital camera, 22. Camera signal line, 23. Non-super-resolution illumination source, 24. Collimating lens, 25. Second beam splitter, 26. Second quarter-wave plate, 27. Third positive lens, 28. Second mirror, 29. Three-dimensional displacement stage, 30. First conical lens, 31. Second conical lens, 32. Second objective lens, 33. Optical axis. Detailed Implementation

[0036] 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.

[0037] Example 1 like Figure 1 As shown in the embodiments of this application, a ring-shaped focused scanning illumination reflective wide-field super-resolution optical microscopy system includes: Ring beam generation module, used to generate ring beams.

[0038] The beam scanning module is used to achieve rapid two-dimensional scanning of the beam, thereby enabling rapid two-dimensional super-resolution scanning illumination.

[0039] The sample displacement module is used to move sample 16.

[0040] The optical microscopy imaging module is used to perform non-super-resolution wide-field imaging on sample 16, determine the super-resolution imaging region, and realize real-time acquisition of non-super-resolution wide-field imaging and real-time acquisition of super-resolution wide-field imaging.

[0041] The super-resolution focusing illumination and collection module is used to convert the ring beam into a super-resolution ring focused beam, form a super-resolution focal spot to illuminate the sample 16, collect the super-resolution signal light generated by the reflection and scattering of the sample 16, and collimate and convert it.

[0042] The phase contrast module is used to generate the reference light required for phase contrast microscopy. Combined with the optical microscopy imaging module, it enables super-resolution phase contrast wide-field imaging.

[0043] A computer (not shown in the figure) is used to control the sample displacement module to move the sample 16, and to scan and acquire non-super-resolution wide-field imaging or super-resolution wide-field imaging of the optical microscopy imaging module by synchronously controlling the beam scanning galvanometer 9, and to process the obtained microscopic images.

[0044] This system leverages the advantages of ring beam illumination, high focusing efficiency of traditional lenses, and fast beam scanning speed to achieve efficient and rapid super-resolution scanning illumination. This enables rapid super-resolution optical microscopy wide-field imaging using traditional optical microscopy systems. By performing two-dimensional super-resolution beam scanning illumination on sample 16, combined with a traditional microscopy system, it achieves label-free far-field super-resolution two-dimensional microscopic rapid wide-field imaging. Furthermore, by performing label-free far-field super-resolution two-dimensional microscopic wide-field imaging on samples at different axial positions, it obtains super-resolution two-dimensional microscopic images of different cross-sections of the samples, thereby achieving three-dimensional tomography. This system can be applied to label-free super-resolution rapid microscopic imaging of biological samples, as well as to industrial super-resolution microscopic detection and other related fields.

[0045] The following is a detailed explanation of each module: like Figure 1 and Figure 2 As shown, the ring beam generating module, along the direction of light propagation, sequentially includes a light source unit, a first linear polarizer 4, a beam expander 5, a ring spatial filter 6, a first positive lens 7, and a second positive lens 8. 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 via a first optical fiber jumper 2. 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 beam expander 5 and the ring spatial filter 6 are placed coaxially and spaced apart. The front focal plane F1 of the first positive lens 7 is behind the exit surface of the ring spatial filter 6, and the rear focal plane (F'1) of the first positive lens 7 coincides with the front focal plane (F2) of the second positive lens 8. The first positive lens 7 and the second positive lens 8 constitute a first 4f system. The light source unit generates a collimated beam, which, after passing through the first linear polarizer 4, the beam expander 5, and the ring spatial filter 6, forms a linearly polarized ring beam.

[0046] In one possible embodiment, the super-resolution illumination source 1 can be a coherent light source (such as a laser) or a partially coherent light source (such as a photodiode).

[0047] like Figure 1 and Figure 2 As shown, the beam scanning module includes a beam scanning galvanometer 9, a beam scanning lens 10, a first sleeve mirror 11, and a first reflecting mirror 12. The center of the beam scanning galvanometer 9 coincides with the rear focal plane of the second positive lens 8; the front focal plane of the beam scanning lens 10 is located at the center of the beam scanning galvanometer 9; and the rear focal plane of the beam scanning lens 10 coincides with the front focal plane of the first sleeve mirror 11. 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.

[0048] like Figure 1 and Figure 2 As shown, the super-resolution focusing illumination and collection module includes a first quarter-wave plate 13 and a first objective lens 15. The rear focal plane of the first telescope 11 in the beam scanning module, after being reflected by the first reflecting mirror 12, coincides with the exit pupil plane of the first objective lens 15. The sample 16 is located on the front focal plane of the first objective lens 15. The first objective lens 15 can be a dry lens, water lens, oil lens, or fixed lens. The linearly polarized ring beam output from the beam scanning module is output as a circularly polarized ring beam after passing through the first quarter-wave plate 13. This circularly polarized ring beam is then converted into a super-resolution ring-focused beam after passing through the first objective lens 15.

[0049] 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 first objective lens 15 is mounted on the axial nanopositioner 14, and the computer controls the axial nanopositioner 14 to move the first objective lens 15 in the Z direction.

[0050] like Figure 1 and Figure 2As shown, the optical microscopy imaging module includes a first beam splitter 19, a second telescope 20, a digital camera 21, a non-super-resolution illumination source 23, a collimating lens 24, and a second beam splitter 25. The digital camera 21 is connected to a computer. The non-super-resolution illumination source 23 is located at the front focal point of the collimating lens 24. The light emitted from the non-super-resolution illumination source 23 is collimated by the collimating lens 24, reflected by the second beam splitter 25, reflected by the first beam splitter 19, and then by the first quarter-wave plate 13 to reach the first objective lens 15. The light is then focused into non-super-resolution illumination light on the front focal plane of the first objective lens 15 to illuminate the sample 16. The non-super-resolution illumination light reflected and scattered by the sample 16 is collected by the first objective lens 15 and then reflected by the first quarter-wave plate 13, the first beam splitter 19, the second beam splitter 25, and the second telescope 20 before entering the digital camera 21. This images the sample 16 onto the digital camera 21 and are then sent to the computer, where the computer displays the non-super-resolution wide-field microscopic image of the sample 16.

[0051] The phase contrast module generates the reference light required for phase contrast microscopy. Combined with the optical microscopy imaging module, it enables super-resolution phase contrast wide-field imaging. It comprises a second quarter-wave plate 26, a third positive lens 27, and a second reflecting mirror 28 arranged sequentially. The front focal plane of the third positive lens 27 coincides with the rear focal plane of the first telescope lens 11 after reflection by the first reflecting mirror 12 and the first beam splitter 19. The rear focal plane of the third positive lens 27 coincides with the reflecting surface of the second reflecting mirror 28. The third positive lens 27 and the second reflecting mirror 28 constitute a reflective 4f system. When using this phase contrast module, the first beam splitter 19 reflects a portion of the linearly polarized annular beam from the beam scanning module into the phase contrast module. This linearly polarized annular beam, after passing through the second quarter-wave plate 26, is focused onto the rear focal plane of the third positive lens 27. Further reflected by the second reflecting mirror 28, it is collimated again by the third positive lens 27, forming another annular beam. This beam then passes through the second quarter-wave plate 26 and serves as reference light entering the optical microscopy imaging module. When performing super-resolution intensity wide-field imaging, the non-super-resolution illumination source 23 and phase contrast module are turned off, and the light source unit and beam scanning module are turned on to perform beam scanning. The super-resolution signal light collected by the super-resolution focusing illumination and collection module is reflected by the first beam splitter 19 and enters the optical microscopic imaging module. After passing through the second beam splitter 25 and the second telescope 20 in sequence, a super-resolution wide-field image of sample 16 is formed on the target surface of digital camera 21. After being acquired by digital camera 21, it is sent to computer for processing and display through camera signal line 22. When performing super-resolution phase-contrast wide-field imaging, the non-super-resolution illumination source 23 is turned off, and the light source unit, phase-contrast module, and beam scanning module are turned on for beam scanning. The super-resolution signal light collected by the super-resolution focusing illumination and collection module is reflected by the first beam splitter 19 and enters the optical microscopic imaging module. Simultaneously, the reference light reflected back from the phase-contrast module enters the optical microscopic imaging module through the first beam splitter 19. The two beams are parallel or coaxial, and after passing through the second beam splitter 25 and the second telescope 20 in sequence, they interfere on the target surface of the digital camera 21, thereby forming a super-resolution phase-contrast wide-field image of the sample 16. After being acquired by the digital camera 21, the image is sent to the computer for processing and display via the camera signal line 22. When performing super-resolution imaging and non-super-resolution imaging fusion, the non-super-resolution illumination source 23 and the light source unit need to be turned on simultaneously.

[0052] In this embodiment of the application, a reflective wide-field super-resolution optical microscopy method with ring-focused scanning illumination is provided. The method employs the aforementioned reflective wide-field super-resolution optical microscopy system with ring-focused scanning illumination. The method includes at least one of the following: label-free super-resolution intensity wide-field imaging mode, label-free super-resolution phase-contrast wide-field imaging mode, simultaneous acquisition mode of label-free super-resolution intensity wide-field imaging and non-super-resolution wide-field imaging, and simultaneous acquisition mode of label-free super-resolution phase-contrast wide-field imaging and non-super-resolution wide-field imaging.

[0053] In use, the on / off state of the non-super-resolution illumination source 23 is determined, and the super-resolution illumination source 1 is turned on. The super-resolution illumination source 1 emits light with a wavelength of λ, which is transmitted to the fiber collimator 3 through the first fiber optic jumper 2. The fiber collimator 3 collimates the light and outputs a collimated beam. This collimated beam passes through the first linear polarizer 4, the beam expander 5, and the annular spatial filter 6 to form a linearly polarized annular beam (taking a P-polarized annular beam as an example). After passing through the first 4f system, this P-polarized annular beam passes through the beam scanning module and then through the first quarter-wave plate 13. On the exit pupil surface of the first objective lens 15, it forms a circularly polarized annular beam that is concentric with the exit pupil of the first objective lens 15 and has an outer diameter less than or equal to the exit pupil diameter of the first objective lens 15. After passing through the first objective lens 15, this circularly polarized annular beam is converted into a super-resolution beam. A ring-shaped focused beam forms a super-resolution focused spot on the front focal plane of the first objective lens 15. The full width at half maximum (FWHM) of the super-resolution focused spot is less than 0.5λ / NA, and the first zero-point radius of the super-resolution focal spot (i.e., the distance from the peak intensity position of the focal spot to the first zero-point position) is less than 0.61λ / NA. This super-resolution focused spot illuminates the sample 16. The super-resolution signal light generated by the reflection and scattering of the sample 16 is collected and collimated by the first objective lens 15, and then converted into an S-polarized collimated beam orthogonal to the incident linearly polarized ring beam by the first quarter-wave plate 13. The S-polarized collimated beam is reflected by the first beam splitter 19 and enters the optical microscopy imaging module. The second telescope 20 in the optical microscopy imaging module realizes super-resolution wide-field imaging of the sample 16. In phase-contrast mode, the phase-contrast module needs to be activated to allow the super-resolution signal light and reference light to interfere on the target surface of the digital camera 21, forming a super-resolution phase-contrast wide-field image of sample 16. The super-resolution wide-field image or super-resolution interferometric image of sample 16 is then acquired through the digital camera 21. The resolution of this super-resolution wide-field image or super-resolution phase-contrast wide-field image in the XY plane is close to the limiting resolution of 0.25λ / NA for reflective confocal systems. The computer-controlled axial nanopositioner 14 moves the first objective lens 15 in the Z direction, changing the Z-position of the first objective lens 15's front focal plane within the sample. f To obtain different Z f A two-dimensional super-resolution microscopic image in the XY plane at a given location can be used to obtain a three-dimensional tomographic microscopic image, with a resolution close to the limit resolution of 0.25λ / NA of a reflective confocal system. When performing super-resolution imaging and non-super-resolution imaging fusion, both the non-super-resolution illumination source 23 and the light source unit need to be turned on simultaneously.

[0054] The following is a detailed explanation of each imaging mode: Imaging Mode 1: Label-free super-resolution intensity wide-field imaging mode Step 11: Perform non-super-resolution wide-field imaging on sample 16 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 23 is collimated by the collimating lens 24, reflected by the second beam splitter 25, and then reflected by the first beam splitter 19 and the first quarter-wave plate 13 before reaching the first objective lens 15. There, it converges at the front focal plane of the first objective lens 15 to form non-super-resolution illumination light, illuminating the sample 16. The non-super-resolution illumination light reflected and scattered by the sample 16 is collected by the first objective lens 15 and then reflected by the first quarter-wave plate 13, the first beam splitter 19, the second beam splitter 25, and the second telescopic lens 20 before entering the digital camera 21. This images the sample 16 onto the digital camera 21 and are transmitted to the computer via the camera signal line 22. The computer displays a non-super-resolution 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 into the super-resolution imaging region.

[0055] Step 12: Turn off the phase contrast module so that it does not participate in imaging. An opaque baffle can be added to its optical path.

[0056] Step 13: After determining the super-resolution imaging region, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam, forming a super-resolution focal spot. The beam scanning module is then activated to perform two-dimensional super-resolution scanning illumination on sample 16, collecting the super-resolution signal light reflected and scattered by sample 16, and collimating and converting it. Specifically: Non-super-resolution illumination source 23 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 (taking a P-polarized collimated beam as an example). This collimated beam passes through the first linear polarizer 4, beam expander 5, and annular spatial filter 6 to form a linearly polarized annular beam (i.e., a P-polarized annular beam). This P-polarized annular beam passes through the first 4f system, the beam scanning module, and the first quarter-wave plate 13, forming a circularly polarized annular beam concentric with the exit pupil of the first objective lens 15 and with an outer diameter less than or equal to the exit pupil diameter of the first objective lens 15 on the exit pupil surface. This circularly polarized annular beam is then converted into a circularly polarized annular beam after passing through the first objective lens 15. A super-resolution annular focused beam forms a super-resolution focused spot on the front focal plane of the first objective lens 15. The full width at half maximum (FWHM) of the super-resolution focused spot is less than 0.5λ / NA, and the first zero-point radius of the super-resolution focal spot (i.e., the distance from the peak intensity position of the focal spot to the first zero-point position) is less than 0.61λ / NA. This super-resolution focused spot illuminates the sample 16. The super-resolution signal light generated by the reflection and scattering of the sample 16 is collected and collimated by the first objective lens 15, and then converted into an S-polarized collimated beam orthogonal to the incident linearly polarized annular beam by the first quarter-wave plate 13. The S-polarized collimated beam is reflected by the first beam splitter 19 and enters the optical microscopic imaging module, and the beam scanning module is activated to achieve super-resolution wide-field imaging of the sample 16.

[0057] In one possible embodiment, the first beam splitter 19 is a polarization beam splitter, which can improve the light energy utilization of the system's super-resolution imaging.

[0058] Step 14: The computer-controlled sample displacement module moves the sample 16 to the position to be scanned and performs scanning. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light to form a super-resolution wide-field image on the target surface of the digital camera 21.

[0059] Step 15: The computer reads the super-resolution wide-field image captured by the digital camera 21, and processes and displays it.

[0060] Imaging Mode 2: Label-free super-resolution phase-contrast wide-field imaging mode Step 21: Perform non-super-resolution wide-field imaging on sample 16 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 23 is collimated by the collimating lens 24, reflected by the second beam splitter 25, and then sequentially passes through the first beam splitter 19 and the first quarter-wave plate 13 to reach the first objective lens 15. There, it converges at the front focal plane of the first objective lens 15 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 first objective lens 15 and sequentially passes through the first quarter-wave plate 13, the first beam splitter 19, the second beam splitter 25, and the second telescopic lens 20 before entering the digital camera 21. This images the sample 16 onto the digital camera 21 and are transmitted to the computer via the camera signal line 22. 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 and place it within the super-resolution imaging region.

[0061] Step 22: Turn on the phase contrast module to participate in imaging, and connect its optical path to the system.

[0062] Step 23: After determining the super-resolution imaging region, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam, forming a super-resolution focal spot. The beam scanning module is then activated to perform two-dimensional super-resolution scanning illumination on sample 16, collecting the super-resolution signal light reflected and scattered by sample 16, and collimating and converting it. Specifically: Non-super-resolution illumination source 23 does not emit light. Super-resolution illumination source 1 is turned on, emitting light with wavelength λ. This light is transmitted to fiber collimator 3 via the first fiber optic jumper 2. Fiber collimator 3 collimates the light and outputs a collimated beam (taking a P-polarized collimated beam as an example). This collimated beam passes through the first linear polarizer 4, beam expander 5, and annular spatial filter 6 to form a linearly polarized annular beam (i.e., a P-polarized annular beam). This P-polarized annular beam passes through the first 4f system, the beam scanning module, and the first quarter-wave plate 13. It then forms a circularly polarized annular beam concentric with the exit pupil of the first objective lens 15, with an outer diameter less than or equal to the exit pupil diameter of the first objective lens 15. After passing through the first objective lens 15, this circularly polarized annular beam is converted into a super-resolution annular focused beam, forming a super-resolution focused spot on the front focal plane of the first objective lens 15. The full width at half maximum (FWHM) of the super-resolution focused spot is less than 0.5λ / NA. The first zero-point radius of the spot (i.e., the distance from the peak intensity position of the focal spot to the first zero-point position) is less than 0.61λ / NA. This super-resolution focused spot illuminates the sample 16. The super-resolution signal light generated by the reflection and scattering of the sample 16 is collected and collimated by the first objective lens 15. It is then converted into an S-polarized collimated beam orthogonal to the incident linearly polarized ring beam by the first quarter-wave plate 13. The S-polarized collimated beam is reflected by the first beam splitter 19 and enters the optical microscopic imaging module. At the same time, the linearly polarized ring beam reflected by the first beam splitter 19 enters the phase contrast module and passes through the second quarter-wave plate 26, the third positive lens 27, and the second reflecting mirror 28 in sequence. After being reflected by the second reflecting mirror 28, it passes through the third positive lens 27 and the second quarter-wave plate 26 in sequence and enters the optical microscopic imaging module as a reference light. The super-resolution signal light and the reference light interfere on the target surface of the digital camera 21 and the beam scanning module is activated to form a super-resolution phase contrast wide-field imaging of the sample 16.

[0063] Step 24: The computer-controlled sample displacement module moves the sample 16 to the position to be scanned and performs scanning. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light. The super-resolution signal light and the reference light generated by the phase contrast module interfere on the target surface of the digital camera 21 to form a super-resolution phase contrast wide-field imaging.

[0064] Step 25: The computer reads the super-resolution phase-contrast wide-field image captured by the digital camera 21, and processes and displays it.

[0065] Imaging Mode 3: Simultaneous Acquisition Mode of Label-Free Super-Resolution Intensity Wide-Field Imaging and Non-Super-Resolution Wide-Field Imaging Step 31: Turn on the non-super-resolution illumination source 23, and use the optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample 16 to determine the super-resolution imaging region, while keeping the non-super-resolution wide-field imaging on at all times. Specifically: The super-resolution illumination source 1 is initially turned off, emitting no light. The light emitted by the non-super-resolution illumination source 23 is collimated by the collimating lens 24, reflected by the second beam splitter 25, and then sequentially passes through the first beam splitter 19 and the first quarter-wave plate 13 to reach the first objective lens 15. There, it converges at the front focal plane of the first objective lens 15 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 first objective lens 15 and sequentially passes through the first quarter-wave plate 13, the first beam splitter 19, the second beam splitter 25, and the second telescopic lens 20 before entering the digital camera 21. This images the sample 16 onto the digital camera 21 and are transmitted to the computer via the camera signal line 22. 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 and place it within the super-resolution imaging region.

[0066] Step 32: Turn off the phase contrast module so that it does not participate in imaging. An opaque baffle can be added to its optical path.

[0067] Step 33: After determining the super-resolution imaging region, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam, forming a super-resolution focal spot. The beam scanning module is then activated to perform two-dimensional super-resolution scanning illumination on sample 16, collecting the super-resolution signal light reflected and scattered by sample 16, and collimating and converting it. Specifically: Non-super-resolution illumination source 23 emits light, and super-resolution illumination source 1 is turned on. Super-resolution illumination source 1 emits light with wavelength λ, which is transmitted to fiber collimator 3 via the first fiber optic jumper 2. Fiber collimator 3 collimates the light and outputs a collimated beam (taking a P-polarized collimated beam as an example). This collimated beam passes through the first linear polarizer 4, beam expander 5, and annular spatial filter 6 to form a linearly polarized annular beam (i.e., a P-polarized annular beam). This P-polarized annular beam passes through the first 4f system, the beam scanning module, and the first quarter-wave plate 13, forming a circularly polarized annular beam concentric with the exit pupil of the first objective lens 15 and with an outer diameter less than or equal to the exit pupil diameter of the first objective lens 15 on the exit pupil surface. After passing through the first objective lens 15, this circularly polarized annular beam is converted into a super-resolution annular beam. A super-resolution focused beam is formed on the front focal plane of the first objective lens 15. The full width at half maximum (FWHM) of the super-resolution focused beam 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-resolution focused beam illuminates the sample 16. The super-resolution signal light generated by the reflection and scattering of the sample 16 is collected and collimated by the first objective lens 15, and then converted into an S-polarized collimated beam orthogonal to the incident linearly polarized ring beam by the first quarter-wave plate 13. The S-polarized collimated beam is reflected by the first beam splitter 19 and enters the optical microscopic imaging module. The digital camera 21 simultaneously acquires the super-resolution wide-field imaging light signal and the non-super-resolution wide-field imaging light signal of the sample 16.

[0068] Step 34: The computer-controlled sample displacement module moves the sample 16 to the position to be scanned and performs scanning. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light to form a super-resolution wide-field image on the target surface of the digital camera 21. Meanwhile, the sample 16 is illuminated by non-super-resolution illumination light to form a non-super-resolution wide-field image on the target surface of the digital camera 21.

[0069] Step 35: The computer reads the image formed by fusing the unlabeled super-resolution intensity wide-field image and the non-super-resolution wide-field image captured by the digital camera 21, and processes and displays it.

[0070] Imaging Mode 4: Simultaneous Acquisition Mode of Label-Free Super-Resolution Phase-Contrast Wide-Field Imaging and Non-Super-Resolution Wide-Field Imaging Step 41: Turn on the non-super-resolution illumination source 23, and use the optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample 16 to determine the super-resolution imaging area, while keeping the non-super-resolution wide-field imaging on at all times. Specifically: The super-resolution illumination source 1 is initially turned off, emitting no light. The light emitted by the non-super-resolution illumination source 23 is collimated by the collimating lens 24, reflected by the second beam splitter 25, and then sequentially passes through the first beam splitter 19 and the first quarter-wave plate 13 to reach the first objective lens 15. There, it converges at the front focal plane of the first objective lens 15 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 first objective lens 15 and sequentially passes through the first quarter-wave plate 13, the first beam splitter 19, the second beam splitter 25, and the second telescopic lens 20 before entering the digital camera 21. This images the sample 16 onto the digital camera 21 and are transmitted to the computer via the camera signal line 22. 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 and place it within the super-resolution imaging region.

[0071] Step 42: Turn on the phase contrast module to participate in imaging, and connect its optical path to the system.

[0072] Step 43: After determining the super-resolution imaging region, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam, forming a super-resolution focal spot to perform two-dimensional scanning illumination on sample 16. The module collects the super-resolution signal light reflected and scattered by sample 16, and collimates and converts it. Specifically: Non-super-resolution illumination source 23 emits light, and super-resolution illumination source 1 is turned on. Super-resolution illumination source 1 emits light with wavelength λ, which is transmitted to fiber collimator 3 via the first fiber optic jumper 2. Fiber collimator 3 collimates the light and outputs a collimated beam (taking a P-polarized collimated beam as an example). This collimated beam passes through the first linear polarizer 4, beam expander 5, and annular spatial filter 6 to form a linearly polarized annular beam (i.e., a P-polarized annular beam). This P-polarized annular beam passes through the first 4f system, the beam scanning module, and the first quarter-wave plate 13, forming a circularly polarized annular beam concentric with the exit pupil of the first objective lens 15 and with an outer diameter less than or equal to the exit pupil diameter of the first objective lens 15. After passing through the first objective lens 15, this circularly polarized annular beam is converted into a super-resolution annular focused beam, forming a super-resolution focused spot on the front focal plane of the first objective lens 15. The full width at half maximum (FWHM) of the super-resolution focused spot is less than 0.5λ / NA. The first zero-point radius of the super-resolution focal spot (i.e., the distance from the peak intensity position of the focal spot to the first zero-point position) is less than 0.61λ / NA. This super-resolution focused spot illuminates the sample 16. The super-resolution signal light generated by the reflection and scattering of the sample 16 is collected and collimated by the first objective lens 15, and then converted into an S-polarized collimated beam orthogonal to the incident linearly polarized ring beam by the first quarter-wave plate 13. This S-polarized collimated beam is reflected by the first beam splitter 19 and enters the optical microscopic imaging module. Simultaneously, the linearly polarized ring beam reflected by the first beam splitter 19 enters the phase contrast module, and then passes sequentially through the second quarter-wave plate 26, the third positive lens 27, and the second reflecting mirror 28. After being reflected by the second reflecting mirror 28, it passes sequentially through the third positive lens 27 and the second quarter-wave plate 26 and enters the optical microscopic imaging module as a reference light. The super-resolution signal light and the reference light interfere on the target surface of the digital camera 21 to form a super-resolution interference image of the sample 16. The digital camera 21 simultaneously acquires super-resolution wide-field imaging and non-super-resolution wide-field imaging of the sample 16.

[0073] Step 44: The computer-controlled sample displacement module moves the sample 16 to the position to be scanned and performs scanning. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light. The super-resolution signal light and the reference light generated by the phase contrast module interfere on the target surface of the digital camera 21 to form a super-resolution phase contrast wide-field imaging. Meanwhile, after the sample 16 is illuminated by non-super-resolution illumination light, a non-super-resolution wide-field imaging is formed on the target surface of the digital camera 21. Step 45: The computer reads the image formed by fusing the unlabeled super-resolution phase-contrast wide-field image and the non-super-resolution wide-field image captured by the digital camera 21, and processes and displays it.

[0074] In this embodiment of the application, in steps 21 and 41, when sample 16 is a transparent sample, sample 16 is fixed on the reflective surface of a reflective glass slide (not shown in the figure), and the reflective glass slide is fixed on the sample displacement module.

[0075] like Figure 2 As shown, a method for generating a ring beam with a width of W' (i.e., the difference between the outer and inner radii of a P-polarized ring beam) and an inner radius of R' is presented. in The outer radius is R' out The method for P-polarized ring beams. Specifically: The fiber collimator 3 collimates the light and outputs a collimated beam with radius R (different numerical aperture fiber collimators can be used to obtain collimated beams with different radii). The collimated beam with radius R becomes a linearly polarized collimated beam with radius R after passing through the first linear polarizer 4 (taking a P-polarized collimated beam as an example). This P-polarized collimated beam then passes through the coaxially placed beam expander 5 and the ring spatial filter 6 to form an inner radius of R''. in The outer radius is R'' out The P-polarized annular beam is further passed through a first 4f system composed of a first positive lens 7 and a second positive lens 8, forming an inner radius of R'. in The outer radius is R' out A P-polarized ring beam; where R' in =R'' in × f 2 / f 1, R' out =R'' out × f 2 / f 1, and the ring width W'=R' out -R' in ; f 1 represents the focal length of the first positive lens 7. f 2 represents the focal length of the second positive lens 8. The inner and outer radii R'' of the first positive lens 7, the second positive lens 8, and the annular space filter 6 are selected with different focal lengths. in and R'' out P-polarized ring beams of different sizes can be obtained. Therefore, the inner and outer radii R'' of the ring spatial filter 6 can be controlled. in and R'' out The focal length of the first positive lens 7 f 1. The focal length of the second positive lens 8 f 2. To control the ring width W' and inner radius R' of the formed P-polarized ring beam. in and outer radius R' out The P-polarized annular beam is magnified (or reduced) again after passing through the beam scanning lens 10 and the first sleeve lens 11, with a magnification of K=f t / f s (K<1 for shrinkage, K>1 for magnification), therefore the inner and outer radii of the final P-polarized ring beam are R and R, respectively. in and R out And R in =KR'' in × f 2 / f 1, R out =KR'' out × f 2 / f 1. To meet the needs of different objectives in producing super-resolution focused beams, because objectives from different manufacturers and of different types have varying inner radius R of the annular beam. in and outer radius R out The requirements are different.

[0076] like Figure 5 The diagram illustrates the illumination process for forming a super-resolution focused beam. The resulting circularly polarized ring beam is considered as having an inner radius of R. in The outer radius is R out Taking a left-handed circularly polarized ring beam as an example, when the left-handed circularly polarized ring beam illuminates the first objective lens 15, it is transformed into a super-resolution ring-focused beam after passing through the first objective lens 15. A super-resolution focused spot is formed on the front focal plane of the first objective lens 15. This super-resolution focused spot is a ring-focused light field, with a full width at half maximum (FWHM) of less than 0.5λ / NA and a first null radius of less than 0.61λ / NA. The inner and outer surfaces of this ring-focused light field are two coaxial conical surfaces, with an inner cone angle of θ. in The external cone angle is θ out NA in NA represents the internal numerical aperture corresponding to the annular focused light field. out NA represents the external numerical aperture corresponding to the annular focused light field. in =n×sin(θ in ), NA out =n×sin(θ out ), where n represents the refractive index of the medium between the first objective lens 15 and the sample 16, and sin() represents the sine function. When 0 ≤ NA in <NA,NA out When NA = 0.25λ / 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, NA... out It needs to be equal to NA (i.e., NA) out =NA, corresponding to the outer diameter R of a left-handed circularly polarized ring beam. outis equal to the exit pupil diameter of the first objective lens 15), and 0.947NA ≤ NA in <NA. Since R out is equal to the exit pupil diameter of the first objective lens 15, after the first objective lens 15 is determined, NA out is determined (corresponding to R out being determined), θ out is also determined. At this time, the resolution of super-resolution microscopy is determined by NA in (corresponding to R in ). The larger R in is, the smaller the ring width W is, and the higher the resolution of super-resolution microscopy is. In practice, as long as 0 ≤ NA in <NA out ≤ NA, super-resolution wide-field imaging can be achieved, but the best resolution cannot be reached.

[0077] To achieve the best resolution, the closer the value of NA in is to NA, the smaller the ring width W is, the smaller the transverse size of the super-resolution focal spot (i.e., the super-resolution focused spot) of the annular focused light field is, and the closer the resolution of super-resolution microscopy 0.5λ / (NA + NA in ) can be to the limit resolution 0.25λ / NA of the reflection confocal system. In practice, by controlling the radius R of the collimated beam and adjusting the annular spatial filter 6R'' in 、R'' out ; However, since the P-polarized annular beam with a small ring width will undergo obvious diffraction during propagation, resulting in an increase in the ring width, it is impossible to ensure that the circularly polarized annular beam propagating to the exit pupil plane of the first objective lens 15 can maintain a very small ring width. Therefore, the radius R of the collimated beam cannot be too small, but to make the circularly polarized annular beam propagating to the exit pupil plane of the first objective lens 15 maintain a very small ring width to achieve the best resolution, it is necessary to add a first 4f system composed of the first positive lens 7 and the second positive lens 8 to control the size of the P-polarized annular beam, combined with a second 4f system composed of the beam scanning lens 10 and the first sleeve lens 11, so that the annular focused light field satisfies: NA out = NA, and 0.947NA ≤ NA in<NA. The first 4f system composed of the first positive lens 7 and the second positive lens 8 plays a role in controlling the size of the P-polarized annular beam on the one hand (that is, it can well compress the width of the P-polarized annular beam at the exit end of the annular spatial filter 6 to be close to the diffraction limit width of the beam and finally project it onto the exit pupil plane of the first objective lens), and on the other hand plays a role in relaying the transmission of the P-polarized annular beam, so as to ensure that the size of the P-polarized annular beam on the front focal plane of the first 4f system is accurately projected onto the rear focal plane of the first 4f system, thereby avoiding the annular size distortion and the fluctuation of the light field intensity distribution caused by the diffraction effect during the direct transmission of the P-polarized annular beam, ensuring the high-quality illumination of the first objective lens by the P-polarized annular beam, and further ensuring that the generated super-resolution focal spot reaches the minimum to ensure that the system reaches the best resolution.

[0078] As Figure 2 shown, to ensure that the inner cone angle of the inner and outer surfaces of the annular focused light field formed after passing through the first objective lens 15 is θ in and the outer cone angle is θ out , it can be achieved through the following methods: First, according to the required NA out and NA in , determine the corresponding R out and R in (the determination method belongs to the prior art), and select a suitable first objective lens 15 according to NA out = NA; then, according to R in = R'' in × f 2 / f 1 × f t / f s and R out = R'' out × f 2 / f 1 × f t / f s , determine the corresponding R'' in and R'' out .

[0079] As Figure 5 shown, to ensure that the inner cone angle of the inner and outer surfaces of the annular focused light field formed after passing through the first objective lens 15 is θ in and the outer cone angle is θ out , it can be achieved through the following methods: First, according to the required NA out and NA in , determine the corresponding R out and R in(The method of determination belongs to the prior art), and according to NA out =NA, select a suitable first objective lens 15; then, according to R in =R'' in × f 2 / f 1× f t / f s R out =R'' out × f 2 / f 1× f t / f s Determine R'' corresponding to the ring space filter 6. in and R'' out .

[0080] like Figure 6 As shown, when the numerical aperture NA of the first objective lens 15 is 0.95, and NA out When the beam width at half maximum (FWHM), r0 (radius of the first null point), and SR (sidelobe peak ratio, the ratio of the maximum sidelobe intensity to the center intensity) of the super-resolution focused spot are equal to those of NA. in The functional relationship. It can be seen that when NA in When the radius is ≥0.9, the resulting super-resolution focused spot has a full width at half maximum (FWHM) ≤0.388λ (less than 0.5λ / NA), r0 ≤0.415λ (less than 0.61λ / NA), and SR ≤0.162. The FWHM and first null radius of the super-resolution focused spot are both smaller than the corresponding full width at half maximum (FWHM) of 0.5λ / NA and first null radius of 0.61λ / NA for the focused light field of a conventional lens. Therefore, by controlling the inner and outer radii of the left-hand circularly polarized ring beam to satisfy: NA out =0.95 and 0.9≤NA in With a value <0.95, a super-resolution illumination field with FWHM≤0.388λ, r0≤0.415λ, and SR≤0.162 can be achieved.

[0081] like Figure 7As shown, this is a schematic diagram of the super-resolution focused spot scanning on the focal plane of the first objective lens 15. The beam scanning module realizes two-dimensional scanning of the ring beam. Within the same scanning cycle, at different times, the ring beam is incident on the first objective lens 15 at different angles. When the ring beam reaches the exit pupil of the first objective lens 15, the ring beam always forms a ring centered on the center of the exit pupil. As shown in Figure a, the illumination focusing situation with two different incident angles is given. At two different times t1 and t2, the incident angles of the ring beam are θ1 and θ2, respectively, forming a super-resolution focused spot at different positions on the front focal plane of the first objective lens 15. As shown in Figure b, at different times, the light intensity distribution of the ring beam on the exit pupil of the first objective lens 15 is always a ring centered on the center of the exit pupil, and the inner and outer radii of this ring are R and R, respectively. in and R out . Figure 7 The downward arrow indicates optical axis 33.

[0082] like Figure 8 The figure shows the relationship between the full width at half maximum (FWHM) of the first objective lens 15 (numerical aperture NA > 1) and the system resolution under ring beam illumination. The vertical axis represents the FWHM of the objective lens point spread function, in wavelength λ; the horizontal axis represents the lateral FWHM of the super-resolution focused spot, in wavelength λ; and the contour lines represent the system resolution (i.e., the minimum linewidth of the amplitude-type grating that the system can resolve, with an imaging fringe contrast better than 11%), in wavelength λ. When the FWHM of the objective lens point spread function is 0.6λ and the lateral FWHM of the super-resolution focused spot (the portion illuminating the sample) is both 0.38λ, the system resolution can reach 0.25λ~0.26λ; when both the FWHM of the objective lens point spread function and the lateral FWHM of the super-resolution focused spot are 0.35λ, the system resolution is better than 0.2λ.

[0083] Example 2 like Figure 9 As shown in the embodiment of this application, most of the structure of a ring-focused scanning illumination reflective wide-field super-resolution optical microscopy system is the same as that of Embodiment 1. The difference is that the sample displacement module is a three-dimensional displacement stage 29, which is connected to a computer. The sample 16 is horizontally fixed on the three-dimensional displacement stage 29, and the computer controls the three-dimensional displacement stage 29 to move the sample 16 in the X, Y, and Z directions.

[0084] The rest is the same as in Example 1.

[0085] Example 3 In this embodiment, the structure of a ring-focused scanning illumination reflective wide-field super-resolution optical microscopy system is largely the same as that in Embodiment 1, except that: the ring beam generation module includes a light source unit, a first linear polarizer 4, a beam expander 5, and a ring spatial filter 6 arranged sequentially along the light propagation direction; the light source unit includes a super-resolution illumination source 1 and an optical fiber collimator 3, with the super-resolution illumination source 1 connected to the optical fiber collimator 3; the light emitted from the super-resolution illumination source 1 is transmitted to the optical fiber collimator 3, which collimates the light and outputs a collimated beam. The ring spatial filter 6 is placed coaxially and spaced apart from the beam expander 5; the light source unit generates a collimated beam, which, after passing through the first linear polarizer 4, the beam expander 5, and the ring spatial filter 6, forms a linearly polarized ring beam.

[0086] The rest is the same as in Example 1 or Example 2.

[0087] Example 4 like Figure 10 As shown in the embodiment of this application, most of the structure of a ring-focused scanning illumination reflective wide-field super-resolution optical microscopy system is the same as that of Embodiment 1. The difference is that the third positive lens 27 is replaced by the second objective lens 32, which is the same as the first objective lens 15, and the exit pupil surface of the second objective lens 32 coincides with the back focal plane of the first telescope lens 11 after reflection by the first reflecting mirror 12 and the first beam splitter 19.

[0088] The rest is the same as in Example 1 or Example 2.

[0089] Example 5 In this embodiment, most of the structure of a ring-focused scanning illumination reflective wide-field super-resolution optical microscopy system is the same as that in Embodiment 1. The difference is that in the unmarked super-resolution intensity wide-field imaging mode, the first beam splitter 19 may not use a polarization beam splitter, and the first linear polarizer 4 may be removed from the light source module. The first quarter-wave plate 13 may be removed from the super-resolution focused illumination and collection module.

[0090] The rest is the same as in Example 1 or Example 2.

[0091] Example 6 In this embodiment, the structure of a ring-focused scanning illumination reflection wide-field super-resolution optical microscopy system is mostly the same as that in Embodiment 1. The difference is that in the unmarked super-resolution intensity wide-field imaging mode, a second linear polarizer (not shown in the figure) can be added between the second telescope 20 and the second beam splitter 25 in the optical microscopy imaging module, so that only the super-resolution signal light reflected back from the sample participates in the super-resolution intensity wide-field imaging.

[0092] The rest is the same as in Example 1 or Example 2.

[0093] Example 7 like Figure 4 As shown in the embodiment of this application, the structure of a ring-focused scanning illumination reflective wide-field super-resolution optical microscopy system is mostly the same as that of Embodiment 1, except that: the ring beam generation module includes a light source unit, a first linear polarizer 4, a first conical lens 30, a second conical lens 31, a ring spatial filter 6, a first positive lens 7, and a second positive lens 8 arranged sequentially along the light propagation direction; the light source unit includes a super-resolution illumination source 1 and an optical fiber collimator 3, the super-resolution illumination source 1 being connected to the optical fiber collimator 3; the light emitted from the super-resolution illumination source 1 is transmitted to the optical fiber collimator 3, and the optical fiber collimator 3 collimates the light and outputs a collimated beam. The ring spatial filter 6 is placed coaxially and spaced apart from the beam expander 5, the first conical lens 30, and the second conical lens 31, the front focal plane of the first positive lens 7 being located behind the exit surface of the ring spatial filter 6, and the rear focal plane of the first positive lens 7 coinciding with the front focal plane of the second positive lens 8; the light source unit generates a collimated beam, which, after passing through the first linear polarizer 4, the beam expander 5, and the ring spatial filter 6, forms a linearly polarized ring beam.

[0094] like Figure 3 As shown, a schematic diagram of the ring spatial filter 6 is presented, with a radius of R'' in and R'' out The circular area between the two parts is translucent, while the rest is opaque.

[0095] like Figure 4 As shown, a method for generating a ring beam with a width of W' (i.e., the difference between the outer and inner radii of a P-polarized ring beam) and an inner radius of R' is presented. in The outer radius is R' out The method for P-polarized ring beams. Specifically: The fiber collimator 3 collimates the light and outputs a collimated beam with radius R (different numerical aperture fiber collimators can be used to obtain collimated beams with different radii). The collimated beam with radius R becomes a linearly polarized collimated beam with radius R after passing through the first linear polarizer 4 (taking a P-polarized collimated beam as an example). This P-polarized collimated beam then passes through the coaxially placed first conical lens 30, second conical lens 31, and annular spatial filter 6 to form an inner radius of R''. in The outer radius is R'' out The P-polarized annular beam is further passed through a first 4f system composed of a first positive lens 7 and a second positive lens 8, forming an inner radius of R'. in The outer radius is R' out A P-polarized ring beam; where R' in =R'' in × f 2 / f1, R' out =R'' out × f 2 / f 1, and the ring width W'=R' out -R' in ; f 1 represents the focal length of the first positive lens 7. f 2 represents the focal length of the second positive lens 8. By selecting different focal lengths for the first positive lens 7 and the second positive lens 8, P-polarized ring beams of different sizes can be obtained. Therefore, by controlling the distance d between the first conical lens 30 and the second conical lens 31, and the focal length of the first positive lens 7, P-polarized ring beams of different sizes can be obtained. f 1. The focal length of the second positive lens 8 f 2. To control the ring width W' and inner radius R' of the formed P-polarized ring beam. in and outer radius R' out The P-polarized annular beam is magnified (or reduced) again after passing through the beam scanning lens 10 and the first sleeve lens 11, with a magnification of K= f t / f s (K<1 for shrinkage, K>1 for magnification), therefore the inner and outer radii of the final P-polarized ring beam are R and R, respectively. in and R out And R in =KR'' in × f 2 / f 1, R out =KR'' out × f 2 / f 1. To meet the needs of different objectives in producing super-resolution focused beams, because objectives from different manufacturers and of different types have varying inner radius R of the annular beam. in and outer radius R out The requirements are different.

[0096] The rest is the same as in Example 1 or Example 2.

[0097] Example 8 In this embodiment, the structure of a ring-focused scanning illumination reflective wide-field super-resolution optical microscopy system is largely the same as that in Embodiment 1, except that: the ring beam generation module includes a light source unit, a first linear polarizer 4, a first conical lens 30, a second conical lens 31, and a ring spatial filter 6 arranged sequentially along the light propagation direction; the light source unit includes a super-resolution illumination source 1 and an optical fiber collimator 3, with the super-resolution illumination source 1 connected to the optical fiber collimator 3; the light emitted from the super-resolution illumination source 1 is transmitted to the optical fiber collimator 3, which collimates the light and outputs a collimated beam. The ring spatial filter 6 is placed coaxially and spaced apart from the beam expander 5, the first conical lens 30, and the second conical lens 31; the light source unit generates a collimated beam, which, after passing through the first linear polarizer 4, the beam expander 5, and the ring spatial filter 6, forms a linearly polarized ring beam.

[0098] The rest is the same as in Example 1 or Example 2.

[0099] Example 9 In this embodiment, the structure of a ring-focused scanning illumination reflective wide-field super-resolution optical microscopy system is largely the same as in Embodiment 1, except that the beam scanning lens 10 and the first telescope lens 11 in the beam scanning module can be replaced by two conventional positive lenses. That is, the beam scanning lens 10 is replaced by a fourth positive lens (not shown in the figure), and the first telescope lens 11 is replaced by a fifth positive lens (not shown in the figure).

[0100] The rest is the same as in Example 1.

[0101] The above description is merely a specific embodiment of the present invention. In particular, since the focal plane of an actual lens has a certain degree of redundancy, certain adjustments can be made near the described position to optimize the overall system. However, 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 wide-field super-resolution optical microscopy system with ring-shaped focusing scanning illumination, characterized in that, include: The sample displacement module is used to move the sample (16); Ring beam generation module, used to generate ring beams; The beam scanning module is used to achieve rapid two-dimensional scanning of the beam; The super-resolution focusing illumination and collection module includes a first objective lens (15), wherein the sample (16) is located on the front focal plane of the first objective lens (15); it is used to convert the ring beam into a super-resolution ring focused beam, form a 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 collimate and convert it. An optical microscopy imaging module includes a non-super-resolution illumination source (23), a collimating lens (24), a second beam splitter (25), a first beam splitter (19), a second telescope (20), and a digital camera (21); it is used to perform non-super-resolution wide-field imaging on a sample (16), determine the super-resolution imaging region, and realize real-time acquisition of non-super-resolution wide-field imaging and real-time acquisition of super-resolution wide-field imaging; the non-super-resolution illumination source (23) is located at the front focal point of the collimating lens (24); A phase contrast module is used to generate the reference light required for phase contrast microscopy and to achieve super-resolution phase contrast wide-field imaging in conjunction with an optical microscopy imaging module; the phase contrast module includes a second quarter-wave plate (26), a third positive lens (27), and a second reflecting mirror (28) arranged in sequence, and the front focal plane of the third positive lens (27) coincides with the back focal plane of the first telescope (11) after reflection by the first reflecting mirror (12) and the first beam splitter (19); or the phase contrast module includes a second quarter-wave plate (26), a second objective lens (32), and a second reflecting mirror (28) arranged in sequence, and the exit pupil surface of the second objective lens (32) coincides with the back focal plane of the first telescope (11) after reflection by the first reflecting mirror (12) and the first beam splitter (19); A computer is used to control the sample displacement module to move the sample (16), and to scan and acquire non-super-resolution wide-field imaging or super-resolution wide-field imaging of the optical microscopy imaging module by synchronously controlling the beam scanning galvanometer (9), and to process the obtained microscopic images.

2. The annular focused scanning illumination reflective wide-field super-resolution optical microscopy system according to claim 1, characterized in that: The optical microscopy imaging module also includes a second linear polarizer (30) disposed between the second telescope (20) and the second beam splitter (25). The non-super-resolution illumination source (23) of the optical microscopy imaging module is located at the front focal point of the collimating lens (24). The light emitted by the non-super-resolution illumination source (23) is collimated by the collimating lens (24), reflected by the second beam splitter (25), reflected by the first beam splitter (19), and reaches the first objective lens (15). The light is then focused on the front focal plane of the first objective lens (15) to provide non-super-resolution illumination to the sample (16). The non-super-resolution illumination light reflected and scattered by the sample (16) is collected by the first objective lens (15) and then reflected by the first beam splitter (19), the second beam splitter (25), the second linear polarizer (30), and the second telescope (20) before entering the digital camera (21), so that the sample (16) is imaged on the digital camera (21) and a non-super-resolution wide-field imaging result is obtained.

3. The annular focusing scanning illumination reflective wide-field super-resolution optical microscopy system according to claim 1, characterized in that: The back focal plane of the third positive lens (27) of the phase contrast module coincides with the reflecting surface of the second mirror (28). When the phase contrast module is needed, a portion of the linearly polarized ring beam from the beam scanning module is reflected into the phase contrast module by the first beam splitter (19). After passing through the second quarter-wave plate (26), the linearly polarized ring beam is focused on the back focal plane of the third positive lens (27), and after being reflected by the second mirror (28), it passes through the third positive lens (27), the second quarter-wave plate (26), and the first beam splitter (19) in sequence, and then enters the optical microscopic imaging module as a reference light to participate in the super-resolution phase contrast wide-field imaging on the target surface of the digital camera (21).

4. The annular focusing scanning illumination reflective wide-field super-resolution optical microscopy system according to claim 1, characterized in that: The super-resolution focusing illumination and collection module also includes a first quarter-wave plate (13). After the linearly polarized annular beam emitted from the beam scanning module passes through the first quarter-wave plate (13), it forms a circularly polarized annular beam on the exit pupil surface of the first objective lens (15) that is concentric with the exit pupil of the first objective lens (15) and has an outer diameter less than or equal to the exit pupil diameter of the first objective lens (15). This circularly polarized annular beam is converted into a super-resolution annular focused beam after passing through the first objective lens (15) and forms a super-resolution focused beam on the front focal plane of the first objective lens (15). The super-resolution focused light spot illuminates the sample (16). The super-resolution signal light generated by the reflection and scattering of the sample (16) is collected and collimated by the first objective lens (15). It is then converted into an S-polarized collimated beam orthogonal to the polarization of the incident light by the first quarter-wave plate (13). After being reflected by the first beam splitter (19), the S-polarized collimated beam enters the optical microscopic imaging module and is transmitted through the second beam splitter (25) and the second telescope (20) to form a super-resolution wide-field image on the target surface of the digital camera (21). When the beam scanning galvanometer (9) in the beam scanning module performs two-dimensional scanning, the super-resolution focused spot formed by the circularly polarized ring beam on the front focal plane of the first objective lens (15) will achieve rapid continuous two-dimensional scanning illumination on the front focal plane of the first objective lens (15) as the beam scanning galvanometer (9) vibrates.

5. The annular focused scanning illumination reflective wide-field super-resolution optical microscopy system according to claim 4, characterized in that: When the system is in the unmarked super-resolution intensity wide-field imaging mode, the first beam splitter (19) is a polarization beam splitter.

6. The annular focusing scanning illumination reflective wide-field super-resolution optical microscopy system according to claim 1, characterized in that: The ring beam generating module includes a light source unit, a first linear polarizer (4), a beam expander (5), and a ring spatial filter (6) arranged sequentially along the light propagation direction; 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); the light emitted by the super-resolution illumination source (1) is transmitted to the optical fiber collimator (3), the optical fiber collimator (3) collimates the light and outputs a collimated beam; the ring spatial filter (6) is placed coaxially and spaced apart from the beam expander (5); the light source unit generates a collimated beam, which forms a linearly polarized ring beam after passing through the first linear polarizer (4), the beam expander (5), and the ring spatial filter (6); Alternatively, the ring beam generating module may include a light source unit, a first linear polarizer (4), a beam expander (5), a ring spatial filter (6), a first positive lens (7), and a second positive lens (8) arranged sequentially along the direction of light propagation; the light source unit includes a super-resolution illumination source (1) and an optical fiber collimator (3), the super-resolution illumination source (1) being connected to the optical fiber collimator (3); the light emitted by the super-resolution illumination source (1) is transmitted to the optical fiber collimator (3), the optical fiber collimator (3) collimates the light and outputs a collimated beam; the ring spatial filter (6) and the beam expander (5) are placed coaxially and spaced apart, the front focal plane of the first positive lens (7) is located behind the exit surface of the ring spatial filter (6), and the rear focal plane of the first positive lens (7) coincides with the front focal plane of the second positive lens (8); the light source unit generates a collimated beam, which forms a linearly polarized ring beam after passing through the first linear polarizer (4), the beam expander (5), and the ring spatial filter (6); Alternatively, the ring beam generating module may also include a light source unit, a first linear polarizer (4), a first conical lens (33), a second conical lens (34), and a ring spatial filter (6) arranged sequentially along the light propagation direction; the light source unit includes a super-resolution illumination source (1) and an optical fiber collimator (3), the super-resolution illumination source (1) being connected to the optical fiber collimator (3); the light emitted by the super-resolution illumination source (1) is transmitted to the optical fiber collimator (3), the optical fiber collimator (3) collimates the light and outputs a collimated beam; the ring spatial filter (6) is placed coaxially and spaced apart from the beam expander (5), the first conical lens (33), and the second conical lens (34); the light source unit generates a collimated beam, which forms a linearly polarized ring beam after passing through the first linear polarizer (4), the beam expander (5), and the ring spatial filter (6); Alternatively, the ring beam generating module may further include a light source unit, a first linear polarizer (4), a first conical lens (33), a second conical lens (34), a ring spatial filter (6), a first positive lens (7), and a second positive lens (8) arranged sequentially along the light propagation direction; the light source unit includes a super-resolution illumination source (1) and an optical fiber collimator (3), the super-resolution illumination source (1) being connected to the optical fiber collimator (3); the light emitted by the super-resolution illumination source (1) is transmitted to the optical fiber collimator (3), and the optical fiber collimator (3)... The light is collimated and then output as a collimated beam. The annular spatial filter (6) is placed coaxially and spaced apart from the beam expander (5), the first conical lens (33), and the second conical lens (34). The front focal plane of the first positive lens (7) is located behind the exit surface of the annular spatial filter (6), and the rear focal plane of the first positive lens (7) coincides with the front focal plane of the second positive lens (8). The light source unit generates a collimated beam, which forms a linearly polarized annular beam after passing through the first linear polarizer (4), the beam expander (5), and the annular spatial filter (6). The super-resolution illumination source (1) is a coherent light source or a partially coherent light source.

7. The annular focused scanning illumination reflective wide-field super-resolution optical microscopy system according to claim 6, 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 reflecting mirror (12). The center of the beam scanning galvanometer (9) coincides with the back focal plane of the second positive lens (8). The front focal plane of the beam scanning lens (10) is located at the center of the beam scanning galvanometer (9). The back focal plane of the beam scanning lens (10) coincides with the front focal plane of the first telescope (11). The back focal plane of the first telescope (11) coincides with the exit pupil of the first objective lens (15) after being reflected by the first reflecting mirror (12). The beam scanning galvanometer (9) contains a controller for connecting to a computer. The computer controls the beam scanning galvanometer (9) to enter the beam scanning mode through the controller. Two-dimensional vibration scanning is performed. When the linearly polarized ring beam generated by the ring beam generation module is reflected by the beam scanning galvanometer (9), passes through the beam scanning lens (10) and the first sleeve lens (11), and is reflected by the first reflecting mirror (12) to the exit pupil surface of the first objective lens (15); when the beam scanning galvanometer (9) vibrates, the center of the linearly polarized ring beam always coincides with the center of the exit pupil surface of the first objective lens (15), and the incident angle of the linearly polarized ring beam changes with the vibration of the beam scanning galvanometer (9); when the beam scanning galvanometer (9) performs two-dimensional scanning, the ring beam will form a focused spot on the front focal plane of the first objective lens (15), and the focused spot will achieve continuous two-dimensional scanning on the front focal plane of the first objective lens (15) with the vibration of the beam scanning galvanometer (9); Alternatively, the beam scanning module may include a beam scanning galvanometer (9), a fourth positive lens, a fifth positive lens, and a first reflecting mirror (12); the center of the beam scanning galvanometer (9) coincides with the back focal plane of the second positive lens (8); the front focal plane of the fourth positive lens is located at the center of the beam scanning galvanometer (9); the back focal plane of the fourth positive lens coincides with the front focal plane of the fifth positive lens; the back focal plane of the fifth positive lens coincides with the exit pupil of the first objective lens (15) after being reflected by the first reflecting mirror (12); the beam scanning galvanometer (9) contains a controller, which 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); When the linearly polarized ring beam generated by the ring beam generation module is reflected by the beam scanning galvanometer (9), and after passing through the fourth positive lens and the fifth positive lens, it is reflected by the first reflecting mirror (12) to the exit pupil surface of the first objective lens (15); when the beam scanning galvanometer (9) vibrates, the center of the linearly polarized ring beam always coincides with the center of the exit pupil surface of the first objective lens (15), and the incident angle of the linearly polarized ring beam changes with the vibration of the beam scanning galvanometer (9); when the beam scanning galvanometer (9) performs two-dimensional scanning, the ring beam will form a focused spot on the front focal plane of the first objective lens (15), and the focused spot will achieve continuous two-dimensional scanning on the front focal plane of the first objective lens (15) with the vibration of the beam scanning galvanometer (9).

8. The annular focusing scanning illumination reflective wide-field super-resolution optical microscopy system according to claim 4, characterized in that: The super-resolution focused spot is a ring-shaped focused light field, the inner and outer surfaces of which are two coaxial conical surfaces. The inner numerical aperture NA corresponding to this ring-shaped focused light field is... in External numerical aperture (NA) out For a given resolution d r d needs to be satisfied. r =0.5λ / (NA in +NA); when 0.947NA≤NA in <NA,NA out When =NA, the optimal resolution can be achieved; where NA represents the numerical aperture of the first objective lens (15).

9. The annular focusing scanning illumination reflective wide-field super-resolution 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 drive the sample (16) to move 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 first objective lens (15) is mounted on the axial nanopositioner (14). The computer controls the axial nanopositioner (14) to drive the first objective lens (15) to move in the Z direction. Alternatively, the sample displacement module is a three-dimensional displacement stage (29). The three-dimensional displacement stage (29) is connected to a computer. The sample (16) is horizontally fixed on the three-dimensional displacement stage (29). The computer controls the three-dimensional displacement stage (29) to move the sample (16) in the X, Y and Z directions.

10. A reflective wide-field super-resolution optical microscopy method with ring-shaped focused scanning illumination, characterized in that: The reflective wide-field super-resolution optical microscopy system using ring-focused scanning illumination as described in any one of claims 1 to 9 includes at least one of the following: label-free super-resolution intensity wide-field imaging mode, label-free super-resolution phase-contrast wide-field imaging mode, simultaneous acquisition mode of label-free super-resolution intensity wide-field imaging and non-super-resolution wide-field imaging, and simultaneous acquisition mode of label-free super-resolution phase-contrast wide-field imaging and non-super-resolution wide-field imaging. In label-free super-resolution intensity wide-field imaging mode: Step 11: Use an optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample (16) to determine the super-resolution imaging region; Step 12: Turn off the phase contrast module so that it does not participate in imaging; Step 13: After determining the super-resolution imaging area, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam to form a super-resolution focal spot to illuminate the sample (16), collect the super-resolution signal light generated by the reflection and scattering of the sample (16), and collimate and convert it. Step 14: The computer-controlled sample displacement module moves the sample (16) to the position to be scanned, and turns on the beam scanning module to perform beam scanning. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light and forms a super-resolution wide field image on the target surface of the digital camera (21). Step 15: The computer reads the super-resolution wide-field image captured by the digital camera (21), processes it, and displays it; In label-free super-resolution phase-contrast wide-field imaging mode: Step 21: Use an optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample (16) to determine the super-resolution imaging region; Step 22: Turn on the phase contrast module to participate in imaging; Step 23: After determining the super-resolution imaging area, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam to form a super-resolution focal spot to illuminate the sample (16), collect the super-resolution signal light generated by the reflection and scattering of the sample (16), and collimate and convert it. Step 24: The computer-controlled sample displacement module moves the sample (16) to the position to be scanned and turns on the beam scanning module to perform beam scanning. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light. The super-resolution signal light and the reference light generated by the phase contrast module interfere on the target surface of the digital camera (21) to form super-resolution phase contrast wide-field imaging. Step 25: The computer reads the super-resolution phase-contrast wide-field image captured by the digital camera (21), processes and displays it; In the simultaneous acquisition mode of label-free super-resolution intensity wide-field imaging and non-super-resolution wide-field imaging: Step 31: Turn on the non-super-resolution illumination source (23), use the optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample (16), determine the super-resolution imaging area, and always keep the non-super-resolution wide-field imaging on. Step 32: Turn off the phase contrast module so that it does not participate in imaging; Step 33: After determining the super-resolution imaging area, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam to form a super-resolution focal spot to illuminate the sample (16), collect the super-resolution signal light generated by the reflection and scattering of the sample (16), and collimate and convert it. Step 34: The computer-controlled sample displacement module moves the sample (16) to the position to be scanned, and turns on the beam scanning module to perform beam scanning. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light and forms a super-resolution wide-field image on the target surface of the digital camera (21). Meanwhile, after the sample (16) is illuminated by non-super-resolution illumination light, a non-super-resolution wide-field image is formed on the target surface of the digital camera (21). Step 35: The computer reads the image formed by fusing the unmarked super-resolution intensity wide-field image and the non-super-resolution wide-field image captured by the digital camera (21), and processes and displays it; In the simultaneous acquisition mode of label-free super-resolution phase-contrast wide-field imaging and non-super-resolution wide-field imaging: Step 41: Turn on the non-super-resolution illumination source (23), use the optical microscopy imaging module to perform non-super-resolution wide-field imaging on the sample (16), determine the super-resolution imaging area, and always keep the non-super-resolution wide-field imaging on. Step 42: Turn on the phase contrast module to participate in imaging; Step 43: After determining the super-resolution imaging area, the ring beam generation module generates a ring beam, and the super-resolution focusing illumination and collection module converts the ring beam into a super-resolution ring focused beam to form a super-resolution focal spot to illuminate the sample (16), collect the super-resolution signal light generated by the reflection and scattering of the sample (16), and collimate and convert it. Step 44: The computer-controlled sample displacement module moves the sample (16) to the position to be scanned, and the beam scanning module is turned on to perform beam scanning. At the same time, the optical microscopic imaging module acquires the collimated and converted super-resolution signal light. The super-resolution signal light and the reference light generated by the phase contrast module interfere on the target surface of the digital camera (21) to form super-resolution phase contrast wide-field imaging. At the same time, after the sample (16) is illuminated by non-super-resolution illumination light, non-super-resolution wide-field imaging is formed on the target surface of the digital camera (21). Step 45: The computer reads the image formed by fusing the unmarked super-resolution phase-contrast wide-field image and the non-super-resolution wide-field image captured by the digital camera (21), and processes and displays it; In steps 21 and 41, when the sample (16) is a transparent sample, the sample (16) is fixed on the reflective surface of the reflective slide and the reflective slide is fixed on the sample displacement module.

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