Reflection-type super-resolution optical microscopy system and method based on light-pyramid scanning illumination

Abstract

The application discloses a reflection type super-resolution optical microscopic system and method based on light cone scanning illumination, and the system comprises a sample displacement module, a ring-shaped focused light beam generation module, a light beam scanning module, a super-resolution focused illumination and collection module, an optical microscopic imaging module and a computer. By utilizing the advantages of the ring-shaped focused light beam illumination, the high focusing efficiency of the traditional lens and the high scanning speed of the light beam, efficient and fast long-focus-depth super-resolution focused scanning illumination is realized, and then the traditional optical microscopic system can be used to realize fast super-resolution optical microscopic imaging. Two-dimensional light beam scanning is used to realize non-labeled far-field super-resolution two-dimensional microscopic fast imaging. By performing non-labeled far-field super-resolution two-dimensional microscopic imaging on samples at different axial positions, super-resolution two-dimensional microscopic imaging of different cross sections of the sample is obtained, and then three-dimensional tomography is realized. The application can be applied to non-labeled super-resolution microscopic fast imaging of biological samples and can also be applied to the field of industrial related super-resolution microscopic detection.

Description

Technical Field

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

[0002] Microscopy, a key tool in scientific research and industrial testing, is currently mainly divided into two categories: near-field microscopy and far-field microscopy. Near-field microscopy techniques, such as microsphere-assisted imaging and near-field scanning microscopy, can achieve a certain degree of super-resolution imaging, but their inherent limitation—working distance shorter than the working wavelength—significantly restricts their versatility and flexibility in practical applications. Far-field microscopy, while offering a longer working distance, often requires fluorescent molecular labeling, which not only increases experimental complexity and cost but may also cause irreversible damage to the sample. Furthermore, existing far-field super-resolution microscopy techniques, such as stimulated emission depletion microscopy, localized activation microscopy, and random light reconstruction optical microscopy, while achieving some progress, still suffer from insufficient resolution and low focusing efficiency.

[0003] Specifically, insufficient resolution is a key problem in microscopy. Traditional microscopy techniques struggle to overcome the diffraction limit, resulting in limited imaging resolution. While near-field microscopy can achieve super-resolution imaging, its limited working distance restricts its applications. Far-field microscopy, although with a large working distance, often requires fluorescent molecular labeling, and its resolution still needs improvement.

[0004] Meanwhile, low focusing efficiency is another problem that needs to be addressed. In existing label-free far-field super-resolution microscopy techniques based on super-diffraction devices, the focusing efficiency of the super-resolution devices is generally low, resulting in limited image quality.

[0005] Furthermore, labeling complexity and cost are also major challenges facing far-field microscopy. Fluorescent molecular labeling not only increases experimental complexity and cost but can also cause irreversible damage to samples. Therefore, developing label-free far-field super-resolution microscopy techniques has become a current research hotspot. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a reflective super-resolution optical microscopy system and method based on light conic scanning illumination, so as to achieve better resolution while improving reliability and imaging speed.

[0007] In a first aspect, the present invention provides a reflective super-resolution optical microscopy system based on light conic scanning illumination, comprising: The sample displacement module is used to move the sample. Ring-shaped focused beam generation module, used to generate a ring-shaped focused beam; The beam scanning module is used to achieve rapid two-dimensional scanning of the beam; The super-resolution focusing illumination and collection module is used to convert the ring-shaped focused beam into a super-resolution conical focused beam to 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, converge and collimate the super-resolution light signals reflected and scattered, and complete the polarization conversion; the super-resolution focusing illumination and collection module includes an objective lens, and the sample is located on the front focal plane of the objective lens; The computer is used to control the sample displacement module to move the sample, and synchronously control the beam scanning galvanometer to scan and receive the electrical signals output by the super-resolution optical imaging module, and process them to obtain a super-resolution microscopic image of the sample in the super-resolution imaging area. An optical microscopy imaging module is used for wide-field imaging of samples, determining the super-resolution imaging region, and achieving real-time acquisition of super-resolution images. This module includes a first beam splitter, a second telescope, a digital camera, a non-super-resolution illumination source, a collimating lens, and a second beam splitter. The non-super-resolution illumination source is located at the front focal point of the collimating lens. Light emitted from the non-super-resolution illumination source is collimated by the collimating lens, reflected by the second beam splitter, and then reflected by the first beam splitter and a quarter-wave plate before reaching the objective lens. It converges at the front focal plane of the objective lens to form non-super-resolution illumination light to illuminate the sample. The non-super-resolution illumination light generated by the sample's reflection and scattering is collected by the objective lens and then sequentially... After passing through a quarter-wave plate, the first beam splitter, the second beam splitter, and the second telescope, the light enters the digital camera, where it is imaged and sent to a computer for displaying a wide-field microscopic image. During super-resolution imaging, the non-super-resolution illumination source is turned off, and the ring-shaped focusing beam generation module and beam scanning module are activated 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 microscopic imaging module. It then passes through the second beam splitter and the second telescope, forming a super-resolution image of the sample on the target surface of the digital camera. This image is then acquired by the digital camera and sent to the computer for processing and display via a data cable.

[0008] Optionally, the annular focused beam generating module includes, in sequence along the light propagation direction, a light source unit, a first linear polarizer, a conical lens, a first positive lens, a second positive lens, and a third positive lens, wherein the first positive lens and the conical lens are placed coaxially and spaced apart, the front focal plane of the second positive lens coincides with the rear focal plane of the first positive lens, and the rear focal plane of the second positive lens coincides with the front focal plane of the third positive lens. The second positive lens and the third positive lens constitute a first 4f system. The light source unit generates a collimated beam, which, after passing through the first linear polarizer, the conical lens, and the first positive lens, forms a linearly polarized annular focused beam and forms a circular focusing line on the rear focal plane of the first positive lens.

[0009] Optionally, the beam scanning module includes a beam scanning galvanometer, a beam scanning lens, a first sleeve lens, and a reflecting mirror. The center of the beam scanning galvanometer coincides with the back focal plane of the third positive lens. The front focal plane of the beam scanning lens is located at the center of the beam scanning galvanometer. The back focal plane of the beam scanning lens coincides with the front focal plane of the first sleeve lens, forming a second 4f system. The back focal plane of the first sleeve lens, after being reflected by the reflecting mirror, coincides with the back focal plane of the objective lens in the super-resolution focusing illumination and collection module. The beam scanning galvanometer contains a controller for connecting to a computer, which controls the beam scanning galvanometer to perform two-dimensional vibration scanning. The annular focused beam generated by the annular focusing beam generation module is reflected by the beam scanning galvanometer, passes through the scanning lens and the first sleeve lens, and is then reflected by the reflecting mirror to the rear focal plane of the objective lens. When the beam scanning galvanometer vibrates, the center of the annular focused beam coincides with the center of the rear focal plane of the objective lens, and the incident angle of the annular focused beam changes with the vibration of the beam scanning galvanometer. The annular focused beam is transformed by the objective lens, forming a conical focused light field behind the objective lens, thereby generating a long focal depth super-resolution focused light field, and forming a super-resolution focused light spot on the front focal plane of the objective lens. When the beam scanning galvanometer performs two-dimensional scanning, this focused light spot will continuously scan two dimensions on the front focal plane of the objective lens with the vibration of the beam scanning galvanometer. By controlling the position of the rear focal plane of the objective lens in the Z-axis direction, the spatial range of continuous two-dimensional scanning on the front focal plane of the objective lens can be controlled.

[0010] Optionally, the super-resolution focusing illumination and collection module further includes a quarter-wave plate; the linearly polarized annular focused beam emitted from the beam scanning module passes through the quarter-wave plate and forms a circularly polarized circular focused line with an outer diameter smaller than the diameter of the rear focal plane of the objective lens on the rear focal plane. This circularly polarized circular focused line is transformed into a super-resolution conical focused beam after passing through the objective lens and forms a super-resolution focused spot on the front focal plane of the objective lens. This super-resolution focused spot illuminates the sample. The super-resolution signal light generated by the reflection and scattering of the sample is collected by the objective lens and then converged and collimated. It is then transformed by the quarter-wave plate into a linearly polarized annular focused beam and a linearly polarized collimated beam orthogonal to the polarization of the incident beam, i.e., a linearly polarized super-resolution signal beam. This linearly polarized super-resolution signal beam is transmitted and reflected by the first beam splitter and enters the optical microscopic imaging module. The ring-shaped focused beam is transformed into a super-resolution conical focused beam by the objective lens. When the beam scanning galvanometer in the beam scanning module performs a two-dimensional scan, the super-resolution focused spot formed on the front focal plane of the objective lens will vibrate with the beam scanning galvanometer, thereby achieving continuous two-dimensional scanning illumination on the front focal plane of the objective lens.

[0011] Optionally, in intensity super-resolution microscopy mode: the first beam splitter employs a polarization beam splitter to improve the efficiency of reflected and transmitted light.

[0012] Optionally, the super-resolution focused spot is an annular focused light field, wherein the inner and outer surfaces of the annular focused light field are two coaxial conical surfaces with equal cone angles, and the numerical aperture NA corresponding to the annular focused light field is... c For a given resolution d r d needs to be satisfied. r =0.5λ / (NA c +NA); when 0.947NA≤NA c The optimal resolution is achieved when the value is ≤NA; where NA represents the numerical aperture of the objective lens.

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

[0014] The first type is a two-dimensional displacement stage, which is 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 in the XY plane to achieve the initial positioning of the sample. The super-resolution focusing illumination and collection module also includes an axial nanopositioner, which is connected to a computer. The objective lens is mounted on the axial nanopositioner, and the computer controls the axial nanopositioner to move the objective lens in the Z direction. The 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 XY plane is parallel to the focal plane of the objective lens. The X and Y directions are used for initial positioning of the sample. The beam scanning module performs two-dimensional scanning of the sample, and the three-dimensional tomographic scanning is achieved by combining the Z-direction displacement of the three-dimensional displacement stage. This avoids the imaging effects caused by minute movements of the objective lens, resulting in better imaging quality.

[0015] Optionally, the light source unit includes a super-resolution illumination source and an optical fiber collimator. The super-resolution illumination source is connected to the optical fiber collimator via a first optical fiber jumper. The light emitted by the super-resolution illumination source is transmitted to the optical fiber collimator via the first optical fiber jumper, and the optical fiber collimator collimates the light and outputs a collimated beam.

[0016] Optionally, the annular focusing beam generating module further includes a field lens, the object-side principal plane of which coincides with the rear focal plane of the first positive lens, and the image-side principal plane of which coincides with the front focal plane of the second positive lens, to change the propagation direction of the edge rays of the annular focusing beam. This adapts to different objectives, allowing the annular focusing beam to pass through the objectives without obstruction. Alternatively, the annular focusing beam generating module may further include a spatial filter and a field lens arranged sequentially between the first positive lens and the second positive lens along the optical path. The spatial filter is coaxially arranged with the first positive lens and is used to control the convergence angle of the beam while filtering out stray light generated by the cone tip of the cone lens. The object-side principal plane of the field lens coincides with the back focal plane of the first positive lens, and the image-side principal plane of the field lens coincides with the front focal plane of the second positive lens, so as to change the propagation direction of the edge light rays of the annular focusing beam. Super-resolution illumination sources can be coherent light sources (such as lasers) or partially coherent light sources (such as photodiodes).

[0017] Secondly, the reflective scanning super-resolution optical microscopy method based on light cone illumination described in this invention employs the reflective scanning super-resolution optical microscopy system as described in this invention, and the method includes two imaging modes: Imaging Mode 1: Label-free super-resolution intensity imaging Step 1: Use an optical microscopy imaging module to perform wide-field imaging of the sample and determine the super-resolution imaging region; Step 2: After determining the super-resolution imaging area, the ring focusing beam generation module generates a ring focusing beam, and the super-resolution focusing illumination and collection module converts the ring focusing beam into a super-resolution conical focusing beam to form a super-resolution focal spot to illuminate the sample. It collects the super-resolution signal light generated by the reflection and scattering of the sample, and converges and collimates the super-resolution light signals reflected and scattered, and completes polarization conversion. Step 3: 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 super-resolution optical imaging module acquires the super-resolution signal light after convergence, collimation and conversion, and converts it into an electrical signal for input into the computer. Step 4: The computer reads the super-resolution image captured by the digital camera, processes it, and displays it; Imaging Mode 2: Simultaneous acquisition with non-super-resolution wide-field imaging Step 1: Use an optical microscopy imaging module to perform wide-field imaging of the sample and determine the super-resolution imaging region; Step 2: After determining the super-resolution imaging area, simultaneously activate the light source units of the non-super-resolution illumination source and the ring focusing beam generation module. The ring focusing beam generation module generates a ring focusing beam, and the super-resolution focusing illumination and collection module converts the ring focusing beam into a super-resolution conical focusing beam, forming a super-resolution focal spot to illuminate the sample. It collects the super-resolution signal light generated by the sample's reflection and scattering, converges and collimates the reflected and scattered super-resolution light signals, and completes polarization conversion. Simultaneously, the non-super-resolution illumination source illuminates the sample and collects the non-super-resolution signal light generated by the sample's reflection and scattering. Step 3: 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 super-resolution optical imaging module acquires the super-resolution signal light after convergence, collimation and conversion, and converts it into an electrical signal for input into the computer. Step 4: The computer reads the image formed by fusing the super-resolution image and the non-super-resolution image captured by the digital camera, and processes and displays it.

[0018] The present invention has the following advantages: (1) The present invention can acquire super-resolution images at the optical microscopy imaging module. Specifically, when super-resolution imaging is performed, the non-super-resolution illumination source is turned off, the ring focusing beam generation module and the beam scanning module are turned on, and the beam is scanned. 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 microscopy imaging module, and passes through the second beam splitter and the second telescope in sequence to form a super-resolution image of the sample on the target surface of the digital camera. After being acquired by the digital camera, the image is sent to the computer for processing and display via a data cable.

[0019] (2) The present invention can simultaneously perform non-super-resolution wide-field imaging and non-label super-resolution microscopy, superimpose two images on a digital camera, and acquire them simultaneously through the digital camera.

[0020] (3) This invention enables highly efficient super-resolution focused illumination: Through a carefully designed annular focused beam generation module, this invention can efficiently generate an annular focused beam. Subsequently, using the super-resolution focused illumination and collection module, this annular focused beam is converted into a super-resolution conical focused beam, forming a high-precision super-resolution focal spot to illuminate the sample. This process fully utilizes the high focusing efficiency of traditional lenses, achieving highly efficient super-resolution focused illumination and significantly improving the signal-to-noise ratio of imaging. At the same time, the generated super-resolution conical focused beam forms a long depth-of-focus super-resolution focused light field behind the objective lens, which facilitates reducing the alignment requirements of the illumination beam, focal plane, and sample.

[0021] (4) This invention enables rapid two-dimensional scanning super-resolution illumination: The beam scanning module employs beam scanning galvanometer technology to achieve rapid two-dimensional scanning super-resolution illumination of the beam. This scanning method is not only fast but also highly accurate, enabling super-resolution imaging of a large area in a short time. This feature greatly improves imaging efficiency, allowing users to quickly acquire a large amount of high-quality image data.

[0022] (5) This invention enables label-free far-field super-resolution microscopy: Compared with traditional microscopy techniques that require fluorescent molecular labeling, a major highlight of this invention is its ability to achieve rapid far-field super-resolution two-dimensional microscopy imaging of unlabeled samples. This function not only expands the applicability of the technology but also simplifies experimental procedures and reduces the requirements for sample processing. At the same time, due to the large working distance, it makes applications in fields such as biological and industrial detection more convenient and efficient.

[0023] (6) This invention enables three-dimensional tomographic imaging: By performing label-free far-field super-resolution two-dimensional microscopic imaging on samples at different axial positions, this invention can acquire super-resolution two-dimensional microscopic images of different cross-sections of the sample, and then realize three-dimensional tomographic imaging through data processing. This function is of great significance for analyzing the internal structure of samples and revealing the spatial distribution of complex samples, and is particularly suitable for fields such as biomedical research and materials science.

[0024] (7) This invention features high reliability and imaging quality: By precisely controlling the optical path coordination and signal processing between modules, this invention ensures the reliability and stability of imaging. From the generation of the ring-focused beam and rapid scanning super-resolution illumination to signal collection and processing, each step has been carefully designed and optimized to guarantee a high level of final imaging quality. In addition, the optical components and signal acquisition equipment used have high performance indicators, further improving the overall performance and imaging quality of the system.

[0025] (8) This invention can be applied to label-free super-resolution microscopic rapid imaging of biological samples, and can also be applied to industrial super-resolution microscopic detection and other fields.

[0026] In summary, the reflective super-resolution optical microscopy system and method based on light conic scanning illumination of this invention exhibits 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 capabilities, super-resolution three-dimensional imaging capabilities, as well as high reliability and imaging quality, providing strong technical support for fields such as biomedicine and industrial inspection. Attached Figure Description

[0027] Figure 1 This is one of the structural schematic diagrams of the reflective super-resolution optical microscopy system based on light cone scanning illumination in Embodiment 1.

[0028] Figure 2 This is the second schematic diagram of the reflective super-resolution optical microscopy system based on light cone scanning illumination in Example 1.

[0029] Figure 3 This is one of the schematic diagrams for controlling the size of the annular focused beam in Example 1.

[0030] Figure 4 This is the second schematic diagram of controlling the size of the annular focused beam in Example 1.

[0031] Figure 5 This is the third schematic diagram of controlling the size of the annular focused beam in Example 1.

[0032] Figure 6 This is a schematic diagram of the spatial filter structure in Example 1; Figure 7 This is a schematic diagram of the formation of a long focal depth super-resolution focused light field in Example 1.

[0033] Figure 8 This is a parameter relationship diagram of the long focal depth super-resolution focused light field in Example 1.

[0034] Figure 9 This is a schematic diagram of the super-resolution focused spot scanning on the focal plane of the objective lens in Example 1.

[0035] Figure 10 This is a graph showing the relationship between the full width at half maximum (FWHM) of the objective lens and the focal spot and the system resolution under ring-focused beam illumination in Example 1.

[0036] Figure 11 This is a schematic diagram of the reflective super-resolution optical microscopy system based on light cone scanning illumination in Example 2.

[0037] In the figure: 1. Super-resolution illumination source; 2. First fiber optic jumper; 3. Fiber optic collimator; 4. First linear polarizer; 5. Conical lens; 6. First positive lens; 7. Second positive lens; 8. Third positive lens; 9. Beam scanning galvanometer; 10. Beam scanning lens; 11. First telescopic lens; 12. Mirror; 13. Quarter-wave plate; 14. Axial nanopositioner; 15. Objective lens; 16. Sample; 17. Two-dimensional displacement stage; 18. Scanning signal line; 19. The first... 20. Beam splitter, 21. Second telescope lens, 22. Digital camera, 23. Camera signal line, 24. Non-super-resolution illumination source, 25. Collimating lens, 26. Second beam splitter, 27. Three-dimensional displacement stage, 28. Field lens, 29. Spatial filter, 30. Super-resolution conical focusing beam, 31. Optical axis, 32. Super-resolution focal spot, 33. Back focal plane of objective lens, 34. Circular focusing beam, 35. Front focal plane of objective lens, 36. Center of back focal plane of objective lens, 37. Z-axis. Detailed Implementation

[0038] 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.

[0039] Example 1 like Figure 1and Figure 2 As shown in this embodiment, a reflective super-resolution optical microscopy system based on light cone scanning illumination includes: The sample displacement module is used to move sample 16.

[0040] The optical microscopy imaging module is used to perform wide-field imaging of sample 16, determine the super-resolution imaging area, and acquire super-resolution images of sample 16.

[0041] Ring-shaped focused beam generation module, used to generate a ring-shaped focused beam.

[0042] The beam scanning module is used to achieve rapid two-dimensional scanning of the beam.

[0043] The super-resolution focusing illumination and collection module is used to convert the ring-shaped focusing beam 33 into a super-resolution conical focusing beam 29, forming a super-resolution focal spot 31 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.

[0044] A computer (not shown in the figure) is used to control the sample displacement module to move the sample 16. The beam scanning galvanometer 9 is synchronously controlled to scan and receive the electrical signals output by the super-resolution optical imaging module, and the super-resolution microscopic image of the sample 16 in the super-resolution imaging area is obtained through processing.

[0045] This system leverages the advantages of ring-shaped focused 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 imaging using traditional optical microscopy systems. Furthermore, by performing two-dimensional super-resolution beam scanning illumination on samples and combining it with traditional microscopy systems, it achieves label-free far-field super-resolution two-dimensional microscopic imaging. By performing label-free far-field super-resolution two-dimensional microscopic imaging on samples at different axial positions, it obtains super-resolution two-dimensional microscopic images of different cross-sections of the samples, thereby achieving 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.

[0046] The following is a detailed explanation of each module: like Figure 3As shown, the annular focusing beam generation module includes, in sequence along the light propagation direction, a super-resolution illumination source 1, an optical fiber collimator 3, a first linear polarizer 4, a conical lens 5, a first positive lens 6, a second positive lens 7, and a third positive lens 8. The super-resolution illumination source 1 is connected to the optical fiber collimator 3 via a first optical fiber jumper 2; the light emitted from the super-resolution illumination source 1 is transmitted to the optical fiber collimator 3 via the first optical fiber jumper 2, and the optical fiber collimator 3 collimates the light and outputs a collimated beam. The first positive lens 6 and the conical lens 5 are placed coaxially and spaced apart. The front focal plane (F2) of the second positive lens 7 coincides with the rear focal plane (F'1) of the first positive lens 6, and the rear focal plane (F'2) of the second positive lens 7 coincides with the front focal plane (F3) of the third positive lens 8. The second positive lens 7 and the third positive lens 8 constitute the first 4f system. The light source unit (super-resolution illumination source 1 and fiber collimator 3) generates a collimated beam. After passing through the first linear polarizer 4, the conical lens 5 and the first positive lens 6, the collimated beam forms a linearly polarized annular focused beam and forms a first circular focusing line on the focal plane behind the first positive lens 6.

[0047] like Figure 1 As shown, in one embodiment, the annular focusing beam generating module further includes a field lens 27. The object-side principal plane of the field lens 27 coincides with the rear focal plane of the first positive lens 6, and the image-side principal plane of the field lens 27 coincides with the front focal plane of the second positive lens 7, thereby changing the propagation direction of the edge rays of the annular focusing beam. This adapts to different objectives 15, allowing the annular focusing beam to pass through the objectives 15 without obstruction.

[0048] like Figure 2 and Figure 5 As shown, in another example, the ring-shaped focusing beam generating module, in addition to the super-resolution illumination source 1, fiber collimator 3, first linear polarizer 4, conical lens 5, first positive lens 6, second positive lens 7, third positive lens 8 and field lens 27, also includes a spatial filter 28 coaxially disposed between the first positive lens 6 and the field lens 27, which is used to control the beam convergence angle and filter out stray light generated by the tip of the conical lens.

[0049] like Figure 4 As shown, in another example, the annular focusing beam generating module includes a spatial filter 28 in addition to the super-resolution illumination source 1, fiber collimator 3, first linear polarizer 4, conical lens 5, first positive lens 6, second positive lens 7, and third positive lens 8, but does not include the field lens 27. The spatial filter 28 is coaxially disposed between the first positive lens 6 and the second positive lens 7.

[0050] 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).

[0051] like Figure 1 and Figure 2 As shown, the beam scanning module includes a beam scanning galvanometer 9, a scanning lens 10, a first telescope 11, and a reflecting mirror 12. The center of the beam scanning galvanometer 9 coincides with the back focal plane of the third positive lens 8; the front focal plane of the scanning lens 10 is located at the center of the beam scanning galvanometer 9; the back focal plane of the beam scanning lens 10 coincides with the front focal plane of the first telescope 11, forming a second 4f system; the back focal plane of the first telescope 11, after being reflected by the reflecting mirror 12, coincides with the back focal plane of the objective lens 15 in the super-resolution focusing illumination and collection module. The beam scanning galvanometer 9 contains a controller for connecting to a computer (not shown in the figure). The computer controls the beam scanning galvanometer 9 to perform two-dimensional vibration scanning through the controller.

[0052] The annular focused beam generated by the annular focused beam generation module is reflected by the beam scanning galvanometer 9, passes through the scanning lens 10 and the first sleeve lens 11, and is then reflected by the reflecting mirror 12 to the rear focal plane of the objective lens 15. When the beam scanning galvanometer 9 vibrates, the center of the annular focused beam coincides with the center 35 of the rear focal plane of the objective lens, and the incident angle of the annular focused beam changes with the vibration of the beam scanning galvanometer 9. The annular focused beam is transformed by the objective lens 15, forming a conical focused light field behind the objective lens 15, thereby generating a long focal depth super-resolution focused light field, and forming a super-resolution focused light spot on the front focal plane of the objective lens 15. When the beam scanning galvanometer 9 performs two-dimensional scanning, this super-resolution focused light spot will achieve continuous two-dimensional scanning on the front focal plane of the objective lens 15 with the vibration of the beam scanning galvanometer 9. By controlling the position of the rear focal plane 32 of the objective lens in the Z-axis 36 direction, the spatial range of continuous two-dimensional scanning on the front focal plane of the objective lens 15 can be controlled.

[0053] like Figure 1 As shown, the super-resolution focusing illumination and collection module includes a quarter-wave plate 13 and an objective lens 15. The back focal plane of the objective lens 15 coincides with the back focal plane of the first telescope lens 11, and the sample 16 is located on the front focal plane of the objective lens 15. The objective lens 15 can be a dry lens, a water lens, an oil lens, or a fixed lens.

[0054] like Figure 1As 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 via a camera signal line 22. 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 a quarter-wave plate 13 before reaching the objective lens 15. The light is then focused into non-super-resolution illumination light on the front focal plane of the objective lens 15 to illuminate the sample 16. The non-super-resolution illumination light generated by the reflection and scattering of the sample 16 is collected by the objective lens 15 and then reflected by the 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 a wide-field microscopic image of the sample 16.

[0055] 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 19 into the optical microscopic imaging module, and then passes through the second beam splitter 25 and the second telescope 20 in sequence to form a super-resolution image of sample 16 on the target surface of digital camera 21. After being acquired by digital camera 21, the image is sent to computer for processing and display via data cable 22.

[0056] like Figure 1 As shown, the sample displacement module is a two-dimensional displacement stage 17, which is connected to a computer (not shown in the figure) via a scanning signal line 18. The sample 16 is horizontally fixed on the two-dimensional displacement stage 17, and the computer controls the two-dimensional displacement stage 17 to move the sample 16 in two dimensions within the XY plane. When the sample displacement module is a two-dimensional displacement stage 17, the super-resolution focusing illumination and collection module also includes an axial nanopositioner 14, which is connected to the computer via a positioning signal line. The objective lens 15 is mounted on the axial nanopositioner 14, and the computer controls the axial nanopositioner 14 to move the objective lens 15 in the Z direction.

[0057] In this embodiment, the reflective super-resolution optical microscopy method based on light conic scanning illumination employs the aforementioned reflective scanning super-resolution optical microscopy system. This method includes two imaging modes: Imaging Mode 1: Label-free super-resolution intensity imaging Step 1: Wide-field imaging of sample 16 is performed using an optical microscopy imaging module to determine the super-resolution imaging region. Specifically: The super-resolution illumination source 1 is initially turned off, emitting no light. The light emitted by the non-super-resolution illumination source 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 quarter-wave plate 13 before reaching the objective lens 15. This light converges at the front focal plane of the objective lens 15 to form non-super-resolution illumination light, illuminating the sample 16. The non-super-resolution illumination light generated by the reflection and scattering from the sample 16 is collected by the objective lens 15 and then reflected by the 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 into the super-resolution imaging region.

[0058] Step 2: After determining the super-resolution imaging region, the annular focusing beam generation module generates an annular focusing beam. The super-resolution focusing illumination and collection module converts the annular focusing beam into a super-resolution conical focusing beam 29, forming a super-resolution focusing spot to illuminate the sample 16. It collects the super-resolution light signals reflected and scattered by the sample 16, converges and collimates the reflected and scattered super-resolution light signals, and completes polarization conversion. 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. This collimated beam passes through the first linear polarizer 4, the conical lens 5, and the first positive lens 6 to form a ring-shaped focused beam (assumed to be a P-polarized ring-shaped focused beam). This P-polarized ring-shaped focused beam passes through the first 4f system, then through the beam scanning module, and finally through the quarter-wave plate 13. It then forms a circularly polarized circularly focused line on the back focal plane of the objective lens 15 with an outer diameter smaller than the diameter of the back focal plane of the objective lens 15. This circularly polarized circularly focused line, after passing through the objective lens 15, is converted into a super-resolution beam. A conical focusing beam 29 forms a super-resolution focused spot on the front focal plane of the 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 light signal generated by the reflection and scattering of the sample 16 is collected and collimated by the objective lens 15, and then converted into an S-polarized collimated beam orthogonal to the incident linearly polarized ring beam by a quarter-wave plate 13. This S-polarized collimated beam is reflected by the first beam splitter 19 and enters the optical microscopic imaging module to achieve super-resolution imaging of the sample 16.

[0059] Imaging Mode 2: Simultaneous acquisition of label-free super-resolution intensity imaging and non-super-resolution wide-field imaging Step 1: Wide-field imaging of sample 16 is performed using an optical microscopy imaging module to determine the super-resolution imaging region. Specifically: The super-resolution illumination source 1 is initially turned off, emitting no light. The light emitted by the non-super-resolution illumination source 23 is collimated by the collimating lens 24, reflected by the second beam splitter 25, and then passes sequentially through the first beam splitter 19 and the quarter-wave plate 13 to reach the objective lens 15. It converges at the front focal plane of the objective lens 15 to form non-super-resolution illumination light, illuminating the sample 16. The non-super-resolution illumination light generated by the reflection and scattering from the sample 16 is collected by the objective lens 15 and then passes sequentially through the 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.

[0060] Step 2: After determining the super-resolution imaging region, the ring-shaped focusing beam generation module generates a ring-shaped focusing beam. The super-resolution focusing illumination and collection module converts the ring-shaped focusing beam into a super-resolution conical focusing beam, forming a super-resolution focusing spot to illuminate sample 16. It collects the super-resolution light signals reflected and scattered by sample 16, converges and collimates the reflected and scattered super-resolution light signals, and completes polarization conversion. Specifically: The non-super-resolution illumination light source 23 emits light, while the super-resolution illumination light source 1 is turned on. The super-resolution illumination light 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. After passing through the first linear polarizer 4, the axicon lens 5, and the first positive lens 6, the collimated beam forms an annular focused beam (assumed to be a P-polarized annular beam). The P-polarized annular focused beam passes through the first 4f system, the beam scanning module, and then passes through the quarter-wave plate 13, and a circularly polarized circular focused line with an outer diameter smaller than or equal to the diameter of the rear focal plane 32 of the objective lens 15 is formed on the rear focal plane of the objective lens 15. After passing through the objective lens 15, the circularly polarized circular focused line is converted into a super-resolution conical focused beam, and a super-resolution focused spot is formed on the front focal plane of the objective lens 15. The full width at half maximum of the super-resolution focused spot is less than 0.5λ / NA, and the first zero radius of the super-resolution focal spot of the super-resolution focused spot (i.e., the distance from the peak position of the focal spot intensity to the first zero position) is less than 0.61λ / NA. The 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 objective lens 15, and then converted into an S-polarized collimated beam orthogonal to the incident linearly polarized annular beam through the quarter-wave plate 13. The S-polarized collimated beam is reflected by the first beam splitter 19 and enters the optical microscopy imaging module to achieve super-resolution imaging of the sample 16. At the same time, the reflected and scattered light obtained by non-super-resolution illumination also enters the optical microscopy imaging module to achieve non-super-resolution wide-field imaging. At this time, the super-resolution image and the non-super-resolution image are automatically fused on the digital camera 21.

[0061] As Figure 6 shown, in one embodiment, in the annular focused beam generation module, if a spatial filter 28 is placed after the first positive lens 6. The spatial filter 28 is transmissive with a radius r within the range of R1 < r < R2 centered on the optical axis, and the rest is non-transmissive. It is used to control the angles θ1 and θ2 of the outgoing beam and filter out the stray light generated by the tip of the axicon lens 5 at the same time.

[0062] In another embodiment, the annular focused beam generation module may not include the spatial filter 28.

[0063] As Figure 2 shown, a method for generating a P-polarized annular focused beam is given. Specifically: the fiber collimator 3 collimates the light and outputs a collimated beam with a radius of R (by selecting fiber collimators with different numerical apertures, collimated beams with different radii can be obtained, and then super-resolution conical focused beams with different focal depths can be obtained). The collimated beam with a radius of R becomes a P-polarized collimated beam with a radius of R after passing through the first linear polarizer 4; the P-polarized collimated beam passes through the coaxial placement with a spacing of dAfter the conical lens 5 and the first positive lens 6, a P-polarized ring-shaped focused beam is formed, forming a central radius R' on the rear focal plane of the first positive lens 6. c The first circular focal line can have its center radius R' changed by altering the focal length of the first positive lens 6. c The P-polarized ring-focused beam is further projected onto the rear focal plane of the third positive lens 8 through the first 4f system, forming a central radius of R''. c The second circular focal line. Where, R'' c =R' c × f 3 / f 2, f 2 represents the focal length of the second positive lens 7. f 3 represents the focal length of the third positive lens 8. By selecting second positive lenses 7 and third positive lenses 8 with different focal lengths, second circular focal lines of different sizes can be obtained. Furthermore, through the second 4f system composed of beam scanning lens 10 and first telescope 11, the second circular focal line is projected onto the back focal plane F' of the first telescope 11. t (Also the back focal plane of objective lens 15), the magnification of the second 4f system is K. Therefore, the magnification can be controlled by adjusting d, f 1. f 2. f 3 controls the center radius R of the final formed third circular focal line. c =KR' c × f 3 / f 2.

[0064] like Figure 7 The diagram illustrates the illumination process for forming a super-resolution focused light field with a long focal depth. A circularly polarized ring-shaped focused beam (assumed to be a left-handed circularly polarized ring-shaped focused beam) illuminates the back focal plane of objective lens 15, forming a light field with a central diameter of 2R on the back focal plane of objective lens 15. c A circularly polarized circularly focused line, the inner radius of which is R. in The outer radius is R out Center radius R c =(R out +R in The left-hand circularly polarized ring-shaped focused beam, after passing through objective lens 15, is transformed into a super-resolution conical focused beam with a cone angle of θ. c A long depth-of-focus super-resolution focused light field is formed along the optical axis 30 before and after the front focal plane of objective lens 15. The numerical aperture NA of the super-resolution conical focused beam is... c =n×sin(θ c ), NA c≤NA; where sin() is the sine function, and n is the refractive index of the medium between objective lens 15 and sample 16. The full width at half maximum (FWHM) of the super-resolution focused light field with a long focal depth is less than 0.5λ / NA, and the radius of the first null point of the super-resolution focused focal spot (the distance from the peak intensity position of the focal spot to the first null point position) is less than 0.61λ / NA. When 0 ≤ NA c When λ / NA is less than or equal to 0.947λ / NA, the actual resolution of super-resolution microscopy is greater than 0.25λ / NA and less than 0.5λ / NA. To achieve super-resolution microscopy with practical application and even better resolution, the following condition must be met: 0.947λ ≤ NA. c ≤NA. The resolution of super-resolution microscopy is ≤NA. c Decision, NA c The larger and closer the value is to NA, the closer the resolution of super-resolution microscopy can get to the limit resolution of 0.25λ / NA for reflective confocal systems. c The larger the value, the greater the corresponding value of R. c The larger (R) c (smaller than the back focal plane radius of objective lens 15), and the corresponding R out R in The closer the focal length is to the point of polarization (i.e., the smaller the width of the circularly polarized focal line), the narrower the spatial frequency of the super-resolution focused light field at long focal depths. In practice, we control the radius R of the collimated beam and adjust the distance between the conical lens 5 and the first positive lens 6. d To control R' c This allows for the control of the radius of the circularly polarized circular focusing line. However, because the P-polarized annular focused beam corresponding to the circularly polarized circular focusing line undergoes significant diffraction during propagation, resulting in a larger width, it becomes impossible to guarantee that the circularly polarized circular focusing line reaching the back focal plane of objective lens 15 will maintain a minimal width. Therefore, it is necessary to add a first 4f system composed of the second positive lens 7 and the third positive lens 8 to adjust the size of the P-polarized annular focused beam (corresponding to the circularly polarized circular focusing line), so that the numerical aperture NA of the final super-resolution conical focused beam is... c Satisfies: 0.947NA≤NA c ≤NA. The first 4f system, composed of the second positive lens 7 and the third positive lens 8, serves two purposes: firstly, it adjusts the size of the circular focal line of the P-polarized ring-focused beam; secondly, it acts as a relay for the transmission of the P-polarized ring-focused beam. This ensures that the circular focal line of the first 4f system, after adjustment, is accurately projected onto the rear focal plane of the first 4f system (corresponding to the rear focal plane of the objective lens 15). This avoids size distortion and light field intensity distribution fluctuations caused by diffraction during direct transmission of the P-polarized ring-focused beam, ensuring high-quality illumination of the objective lens by the P-polarized ring-focused beam. Consequently, it ensures that the generated super-resolution focal spot 31 is minimized, thus guaranteeing the system achieves optimal resolution.

[0065] Given the numerical aperture NA of objective lens 15, how can we ensure that the cone angle of the super-resolution conical focused beam after passing through objective lens 15 is θ? c This can be achieved as follows: First, based on the desired resolution d... r Determine NA c And NA, specifically d r =0.5λ / (NA c +NA), where NA c ≤NA, NA c This represents the numerical aperture corresponding to the circularly polarized circular focusing line on the back focal plane of objective lens 15. (Based on NA) c Determine R c (The determination method belongs to the prior art and can be achieved through actual measurement); then, according to R c =K f 1tan( β ) f 3 / f 2 (of which) β Let be the cone angle of the conical beam emitted from conical lens 5, and tan() be the tangent function. f 1. f 2 and f 3 ( f 3 needs to be of sufficient length so that the third positive lens 8 will not spatially interfere with subsequent optical elements.

[0066] like Figure 8 As shown, the parameters FWHM (full width at half maximum), r0 (radius of the first null point), and SR (sidelobe peak ratio, i.e., the ratio of the maximum sidelobe intensity to the center intensity) of the super-resolution focused light field at a depth of field of objective lens 15 are given as follows: when the numerical aperture NA = 0.95. c The functional relationship. It can be seen that when NA c When R ≥ 0.9, the resulting long focal depth super-resolution focused light field has FWHM ≤ 0.399λ (less than 0.5λ / NA), r0 ≤ 0.426λ (less than 0.61λ / NA), and SR = 0.162. Therefore, by controlling R... c Make it satisfy: 0.9≤NA c With a value ≤0.95, a long focal depth super-resolution illumination field with FWHM≤0.399λ, r0≤0.426λ, and SR=0.162 can be achieved.

[0067] like Figure 9The diagram shows a schematic of the super-resolution focused beam scanning on the focal plane of objective lens 15. The beam scanning module performs two-dimensional scanning of the circularly polarized circularly focused beam. At different times, the circularly polarized circularly focused beam is incident on objective lens 15 at different angles. When the circularly polarized circularly focused beam reaches the rear focal plane 32 of the objective lens, it always forms a ring centered on the center of the rear focal plane. At a given time t, the tilt angle of the incident circularly polarized circularly focused beam is θ. s (t), at this time, a super-long depth-of-focus super-resolution focusing spot with a full width at half maximum (FWHM) length of 2ΔZ is formed before and after the objective lens back focal plane 32 (Z direction). The offset of this super-long depth-of-focus super-resolution focusing spot in the XY direction (relative to the optical axis Z) is d(t). By changing the incident angle θ s (t), which can change the offset d(t). At different times, the intensity distribution of the circularly polarized ring-shaped focused beam on the back focal plane of the objective lens is always a ring centered on the center of the back focal plane, and the inner and outer radii of this ring are R and R, respectively. in and R out .

[0068] like Figure 10 The figure shows the relationship between the full width at half maximum (FWHM) of the objective lens (numerical aperture NA > 1) and the system resolution under circularly polarized ring-focused beam illumination. The vertical axis represents the FWHM of the objective lens's point spread function (MPF), in wavelength λ; the horizontal axis represents the lateral FWHM of the super-resolution 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 MPF of the objective lens (which can be a single-point focusing super-diffraction lens) is 0.6λ and the lateral FWHM of the super-resolution focused spot (the portion illuminating the sample) is both 0.38λ, the system resolution can reach 0.25λ~0.26λ; when both the MPF of the objective lens and the lateral FWHM of the super-resolution focused spot are 0.35λ, the system resolution is better than 0.2λ.

[0069] Step 3: The computer-controlled beam scanning module performs a two-dimensional scan of the beam, thereby enabling the super-resolution focused spot to perform two-dimensional scanning illumination on the front focal plane 34 of the objective lens. At the same time, the optical imaging module simultaneously acquires the super-resolution image of the sample 16 and inputs it into the computer.

[0070] Step 4: The computer processes the input super-resolution image. Using a sample displacement module, the sample is moved along the Z-axis (36°), thus acquiring a three-dimensional super-resolution microscopic image of the sample.

[0071] In this embodiment, the specific processing steps of steps three and four are as follows: When performing super-resolution imaging, the non-super-resolution illumination source is turned off, and the light source unit and beam scanning module are turned on to perform beam scanning. The super-resolution light signal collected by the super-resolution focusing illumination and collection module is reflected by the first beam splitter 19 into the optical microscopic imaging module, and then passes through the second beam splitter 25 and the second telescopic lens 20 in sequence to form a super-resolution image of sample 16 on the target surface of digital camera 21. After being acquired by digital camera 21, it is sent to computer for processing and display via data cable 22. The computer controls the beam scanning galvanometer 9 in the beam scanning module to perform two-dimensional scanning. The super-resolution focused light spot formed by the circularly polarized ring focusing beam on the front focal plane of objective lens 15 will achieve continuous two-dimensional scanning on the front focal plane of objective lens 15 as the beam scanning galvanometer 9 vibrates. The horizontal coordinate X(t) and vertical coordinate Y(t) of the position of the super-resolution focused light spot on sample 16 are functions of time t. The reflected light E collected by objective lens 15 r (t) and scattered light E s (t) is also a function of time, and the reflected light E r (t) and scattered light E s The image (t) reflects information such as the sample structure and refractive index at the corresponding positions (x-axis X(t) and y-axis Y(t)). The resolution of this image in the XY plane is close to the limiting resolution of a reflective confocal system (0.25λ / NA). A computer-controlled axial nanopositioner 14, carrying the objective lens 15, moves in the Z-direction, changing the Z-position of the objective lens's front focal plane 34 within the sample. f To obtain different Z f Two-dimensional super-resolution microscopic images in the XY plane at a given location can be used to obtain three-dimensional tomographic microscopic images, with a resolution close to the limit resolution of 0.25λ / NA of a reflective confocal system.

[0072] Example 2 like Figure 11 As shown, in this second embodiment, most of the structure of a reflective super-resolution optical microscopy system based on light cone scanning illumination is the same as that in the first embodiment. The difference is that the sample displacement module is a three-dimensional displacement stage 26. The three-dimensional displacement stage 26 is connected to the computer through the scanning signal line 18. The sample 16 is horizontally fixed on the three-dimensional displacement stage 26. The computer controls the three-dimensional displacement stage 26 to move the sample 16 in the Z-axis 36 direction.

[0073] like Figure 11 As shown, in this second embodiment, the reflective super-resolution optical microscopy method based on light conic scanning illumination is basically the same as in the first embodiment. The only difference is that in the specific processing of steps three and four, the computer controls the three-dimensional displacement stage 37 to move the sample 16 slightly in the Z direction, changing the Z-position of the objective lens front focal plane 34 in the sample 16. fEach time the camera moves a point, digital camera 21 captures different Z values. f Two-dimensional super-resolution microscopic images in the XY plane at a given location can be used to obtain three-dimensional tomographic microscopic images, with a resolution close to the limit resolution of 0.25λ / NA of a reflective confocal system.

[0074] Example 3 In Embodiment 3 of this application, most of the structure of a reflective super-resolution optical microscopy system based on light cone scanning illumination is the same as that in Embodiment 1. The difference is that in the intensity super-resolution microscopy imaging mode, the first beam splitter 19 adopts a polarization beam splitter to improve the efficiency of reflected and transmitted light.

[0075] In other embodiments, the first beam splitter 19 may also be a non-polarizing beam splitter.

[0076] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A reflective super-resolution optical microscopy system based on light cone scanning illumination, characterized in that, include: The sample displacement module is used to move the sample (16); Ring-shaped focused beam generation module, used to generate a ring-shaped focused beam; The beam scanning module is used to achieve rapid two-dimensional scanning of the beam; The super-resolution focusing illumination and collection module is used to convert the ring-shaped focusing beam into a super-resolution conical focusing beam (29) to form a super-resolution focal spot (31) to illuminate the sample (16), collect the super-resolution signal light generated by the reflection and scattering of the sample (16), converge and collimate the super-resolution light signals reflected and scattered, and complete the polarization conversion; the super-resolution focusing illumination and collection module includes an objective lens (15), and the sample (16) is located on the front focal plane of the objective lens (15); A computer is used to control the sample displacement module to move the sample (16), and to scan and receive the electrical signals output by the super-resolution optical imaging module by synchronously controlling the beam scanning galvanometer (9), and to process and obtain the super-resolution microscopic image of the sample in the super-resolution imaging area. An optical microscopy imaging module is used to perform wide-field imaging of the sample (16), determine the super-resolution imaging area, and realize the real-time acquisition of super-resolution images. The optical microscopy imaging module includes a 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 non-super-resolution illumination source (23) 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 then by a quarter-wave plate (13) to reach the objective lens (15). The light is then converged into non-super-resolution illumination light on the front focal plane of the objective lens (15) to illuminate the sample (16). The non-super-resolution illumination light generated by the reflection and scattering of the sample (16) is collected by the objective lens (15). After collection, the sample (16) passes through a quarter-wave plate (13), the first beam splitter (19) for reflection, the second beam splitter (25), and the second telescope (20) in sequence before entering the digital camera (21). The sample (16) is then imaged on the digital camera (21) and sent to the computer, where the computer displays a wide-field microscopic image of the sample (16). When performing super-resolution imaging, the non-super-resolution illumination source (23) is turned off, and the ring focusing beam generation module 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. It then passes through the second beam splitter (25) and the second telescope (20) in sequence, forming a super-resolution image of the sample (16) on the target surface of the digital camera (21). After being acquired by the digital camera (21), the image is sent to the computer for processing and display via the data cable (22).

2. The reflective super-resolution optical microscopy system based on light cone scanning illumination according to claim 1, characterized in that: The ring-shaped focused beam generating module includes, in sequence along the light propagation direction, a light source unit, a first linear polarizer (4), a conical lens (5), a first positive lens (6), a second positive lens (7), and a third positive lens (8). The first positive lens (6) and the conical lens (5) are placed coaxially and spaced apart. The front focal plane of the second positive lens (7) coincides with the rear focal plane of the first positive lens (6), and the rear focal plane of the second positive lens (7) coincides with the front focal plane of the third positive lens (8). The second positive lens (7) and the third positive lens (8) constitute a first 4f system. The light source unit generates a collimated beam. After passing through the first linear polarizer (4), the conical lens (5), and the first positive lens (6), the collimated beam forms a linearly polarized ring-shaped focused beam and forms a circular focusing line on the rear focal plane of the first positive lens (6).

3. The reflective super-resolution optical microscopy system based on light cone scanning illumination according to claim 1, characterized in that: The beam scanning module includes a beam scanning galvanometer (9), a beam scanning lens (10), a first telescope (11), and a reflector (12). The center of the beam scanning galvanometer (9) coincides with the back focal plane of the third positive lens (8). The front focal plane of the beam scanning lens (10) is located at the center of the beam scanning galvanometer (9). The back focal plane of the beam scanning lens (10) coincides with the front focal plane of the first telescope (11), and the two constitute a second 4f system. The back focal plane of the first telescope (11) coincides with the back focal plane of the objective lens (15) in the super-resolution focusing illumination and collection module after being reflected by the reflector (12). The beam scanning galvanometer (9) contains a controller for connecting to a computer. The computer controls the beam scanning galvanometer (9) to perform two-dimensional vibration scanning through the controller of the beam scanning galvanometer (9). When the ring-focused beam generated by the ring-focused beam generation module is reflected by the beam scanning galvanometer (9), passes through the scanning lens (10) and the first sleeve lens (11), and is reflected by the reflecting mirror (12) to the back focal plane of the objective lens (15); when the beam scanning galvanometer (9) vibrates, the center of the ring-focused beam coincides with the center of the back focal plane (32) of the objective lens, and the incident angle of the ring-focused beam changes with the vibration of the beam scanning galvanometer (9); when the beam scanning galvanometer (9) performs two-dimensional scanning, the ring-focused beam is transformed by the objective lens (15) and forms a conical focused light field behind the objective lens (15), thereby generating a long focal depth super-resolution focused light field, and forming a super-resolution focused light spot on the front focal plane of the objective lens (15). This focused light spot will achieve continuous two-dimensional scanning on the front focal plane of the objective lens (15) with the vibration of the beam scanning galvanometer (9).

4. The reflective super-resolution optical microscopy system based on light cone scanning illumination according to claim 2, characterized in that: The super-resolution focusing illumination and collection module also includes a quarter-wave plate (13); after the linearly polarized ring-shaped focused beam emitted from the beam scanning module passes through the quarter-wave plate (13), it forms a circularly polarized circular focusing line with an outer diameter smaller than the diameter of the rear focal plane of the objective lens (15) on the rear focal plane (32). After passing through the objective lens (15), the circularly polarized circular focusing line is transformed into a super-resolution conical focused beam (29) and forms a super-resolution focused spot on the front focal plane of the objective lens (15). The super-resolution focused spot illuminates the sample (16). The super-resolution signal light generated by the reflection and scattering of the sample (16) is collected by the objective lens (15) and then converged and collimated. After passing through the quarter-wave plate (13), it is transformed into a linearly polarized ring-shaped focused beam and a linearly polarized collimated beam orthogonal to the polarization of the incident beam, i.e., a linearly polarized super-resolution signal beam. The linearly polarized super-resolution signal beam is transmitted and reflected by the first beam splitter (19) and enters the optical microscopic imaging module. The ring-shaped focused beam is converted into a conical focused beam by the objective lens (15). When the beam scanning galvanometer (9) in the beam scanning module performs a two-dimensional scan, the super-resolution focused spot formed on the front focal plane (34) of the objective lens will vibrate with the beam scanning galvanometer (9), thereby achieving continuous two-dimensional scanning illumination on the front focal plane of the objective lens (15).

5. The reflective super-resolution optical microscopy system based on light cone scanning illumination according to claim 4, characterized in that: The first beam splitter (19) is a polarization beam splitter.

6. The reflective super-resolution optical microscopy system based on light cone scanning illumination according to claim 3, 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 with equal cone angles. The numerical aperture NA corresponding to this ring-shaped focused light field is... c For a given resolution d r d needs to be satisfied. r =0.5λ / (NA c +NA); when 0.947NA≤NA c The optimal resolution is achieved when the value is ≤NA; where NA represents the numerical aperture of the objective lens (15).

7. The reflective super-resolution optical microscopy system based on light cone scanning illumination according to claim 1, characterized in that: The sample displacement module is a two-dimensional displacement stage (17). The two-dimensional displacement stage (17) is connected to the computer. The sample (16) is horizontally fixed on the two-dimensional displacement stage (17). The computer controls the two-dimensional displacement stage (17) to move the sample (16) in two dimensions in the XY plane to achieve the initial positioning of the sample. The super-resolution focusing illumination and collection module also includes an axial nanopositioner (14). The axial nanopositioner (14) is connected to the computer. The objective lens (15) is mounted on the axial nanopositioner (14). The computer controls the axial nanopositioner (14) to move the objective lens (15) in the Z direction. Alternatively, the sample displacement module is a three-dimensional displacement stage (26), which is connected to a computer. The sample (16) is horizontally fixed on the three-dimensional displacement stage (26), and the computer controls the three-dimensional displacement stage (26) to move the sample (16) in the X, Y, and Z directions. The XY plane is parallel to the focal plane of the objective lens (15).

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

9. The reflective super-resolution optical microscopy system based on light cone scanning illumination according to claim 1, characterized in that: The annular focusing beam generating module also includes a field lens (27), the object-side principal plane of the field lens (27) coincides with the back focal plane of the first positive lens (6), and the image-side principal plane of the field lens (27) coincides with the front focal plane of the second positive lens (7) respectively, so as to change the propagation direction of the edge light of the annular focusing beam. Alternatively, the annular focusing beam generating module may also include a spatial filter (28) and a field lens (27) arranged sequentially between the first positive lens (6) and the second positive lens (7) along the optical path. The spatial filter (28) is coaxially arranged with the first positive lens (6) and is used to control the convergence angle of the beam and filter out stray light generated by the cone tip of the cone lens. The object principal plane of the field lens (27) coincides with the back focal plane of the first positive lens (6), and the image principal plane of the field lens (27) coincides with the front focal plane of the second positive lens (7) to change the propagation direction of the edge light of the annular focusing beam.

10. A reflective scanning super-resolution optical microscopy method based on light cone illumination, employing the reflective super-resolution optical microscopy system based on light cone scanning illumination as described in any one of claims 1 to 9, the method comprising two imaging modes: Imaging Mode 1: Label-free super-resolution intensity imaging Step 1: Use an optical microscopy imaging module to perform wide-field imaging on the sample (16) to determine the super-resolution imaging area; Step 2: After determining the super-resolution imaging area, the ring focusing beam generation module generates a ring focusing beam, and the super-resolution focusing illumination and collection module converts the ring focusing beam into a super-resolution conical focusing beam (29) to form a super-resolution focal spot (31) to illuminate the sample (16), collects the super-resolution signal light generated by the reflection and scattering of the sample (16), and converges and collimates the super-resolution light signals reflected and scattered, and completes polarization conversion. Step 3: The computer-controlled sample displacement module moves the sample (16) to the position to be scanned and turns on the beam scanning module to scan. At the same time, the super-resolution optical imaging module acquires the super-resolution signal light after convergence, collimation and conversion, and converts it into an electrical signal for input into the computer. Step 4: The computer reads the super-resolution image captured by the digital camera (21), processes it, and displays it; Imaging Mode 2: Simultaneous acquisition of label-free super-resolution intensity imaging and non-super-resolution wide-field imaging Step 1: Use an optical microscopy imaging module to perform wide-field imaging on the sample (16) to determine the super-resolution imaging area; Step 2: After determining the super-resolution imaging area, simultaneously turn on the light source units of the non-super-resolution illumination source (23) and the ring focusing beam generation module, so that the ring focusing beam generation module generates a ring focusing beam. The super-resolution focusing illumination and collection module converts the ring focusing beam into a super-resolution conical focusing beam (29), forming a super-resolution focal spot (31) to illuminate the sample (16), collects the super-resolution signal light generated by the reflection and scattering of the sample (16), and converges and collimates the super-resolution light signals reflected and scattered, and completes polarization conversion; at the same time, the non-super-resolution illumination source (23) illuminates the sample (16) and collects the non-super-resolution signal light generated by the reflection and scattering of the sample (16); Step 3: The computer-controlled sample displacement module moves the sample (16) to the position to be scanned and turns on the beam scanning module to scan. At the same time, the super-resolution optical imaging module acquires the super-resolution signal light after convergence, collimation and conversion, and converts it into an electrical signal for input into the computer. Step 4: The computer reads the super-resolution image and the non-super-resolution image captured by the digital camera (21) and processes and displays the resulting image.

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