Reflective scanning super-resolution optical microscopic system and method based on light cone illumination

Abstract

The invention discloses a reflective scanning super-resolution optical microscopy system and method based on light cone illumination. The system comprises a sample displacement module, an optical microscopy imaging module, an annular focusing light beam generation module, a light beam scanning module, a super-resolution focusing illumination and collection module, a super-resolution optical imaging module and a computer. By utilizing the advantages of annular focusing light beam illumination, high focusing efficiency of a traditional lens and high light beam scanning speed, high-efficiency long-focal-depth super-resolution focusing illumination is realized, and rapid super-resolution optical microscopic imaging can be realized by adopting a traditional optical lens; non-marking far-field super-resolution two-dimensional microscopic rapid imaging is realized through two-dimensional light beam scanning; non-label far-field super-resolution two-dimensional microscopic imaging is carried out on samples at different axial positions, super-resolution two-dimensional microscopic imaging of different cross sections of the samples is obtained, and then three-dimensional chromatography is achieved. The method can be applied to non-labeled super-resolution microscopic rapid imaging of biological samples, and can also be applied to the fields of industrial related super-resolution microscopic detection and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of visible light microscopy, and in particular relates to a reflective scanning super-resolution optical microscopy system and method based on light cone illumination. Background Art

[0002] Existing microscopy techniques can be roughly divided into two categories: near-field microscopy and far-field microscopy. Near-field microscopy includes methods such as microsphere-assisted imaging and near-field scanning microscopy. Near-field microscopy methods can achieve super-resolution microscopic imaging, but its working distance is less than the working wavelength, which greatly limits its practical application; far-field super-resolution technology has a large working distance and can overcome the shortcomings of near-field microscopy. Existing far-field super-resolution microscopy techniques include stimulated emission depletion microscopy, local activation microscopy, random light reconstruction optical microscopy, etc. However, these techniques require fluorescent molecular labeling of the sample to be detected. Non-labeled far-field super-resolution microscopy technology has a large working distance, does not require sample labeling, and is easy to use, so it has become an important development trend of super-resolution optical microscopy technology. In recent years, a few experiments have been reported on non-labeled far-field super-resolution microscopy based on super-diffraction devices. These systems all rely on new super-resolution devices as illumination devices; however, the focusing efficiency of super-resolution devices is usually low, and most of these systems rely on moving sample scanning, and the imaging speed is slow.

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

[0004] The purpose of the present invention is to provide a reflective scanning super-resolution optical microscopy system and method based on light cone illumination in view of the deficiencies in the prior art, so as to achieve better resolution while improving reliability and imaging speed.

[0005] In a first aspect, the reflective scanning super-resolution optical microscopy system based on light cone illumination of the present invention comprises: A sample displacement module, used to drive the sample to move; Optical microscopy module, used to perform wide-field imaging of the sample and determine the super-resolution scanning imaging area; An annular focused beam generating module is used to generate an annular focused beam. The annular focused beam generating module includes a light source module, a first linear polarizer, an aconic lens, a first positive lens, a second positive lens and a third positive lens in sequence along the light propagation direction, wherein the first positive lens and the aconic lens are coaxially arranged with an interval, the front focal plane of the second positive lens coincides with the back focal plane of the first positive lens, the back focal plane of the second positive lens coincides with the front focal plane of the third positive lens, and the second positive lens and the third positive lens form a first 4f system; the light source module generates a collimated laser beam, and the collimated laser beam forms a linearly polarized annular focused beam after passing through the first linear polarizer, the aconic lens and the first positive lens, and forms a circular focal line on the back focal plane of the first positive lens; A beam scanning module comprises a beam scanning galvanometer, a beam scanning lens, a first sleeve mirror and a reflector, wherein the center position of the beam scanning galvanometer coincides with the rear focal plane of the third positive lens; the front focal plane of the beam scanning lens is located at the center position of the beam scanning galvanometer; the rear focal plane of the beam scanning lens coincides with the front focal plane of the first sleeve mirror, and the two constitute a second 4f system; the rear focal plane of the first sleeve mirror coincides with the rear focal plane of the objective lens in the super-resolution focusing illumination and collection module after being reflected by the reflector; the beam scanning galvanometer comprises a controller, the controller is used to be connected to a computer, and the computer controls the beam scanning galvanometer to perform two-dimensional vibration scanning through the controller of the beam scanning galvanometer; A super-resolution focusing illumination and collection module is used to convert a ring-shaped focusing beam into a super-resolution conical focusing beam, form a long-focus-depth super-resolution focal spot to illuminate the sample, collect super-resolution signal light generated by reflection and scattering of the sample, respectively converge and collimate the reflected and scattered super-resolution light signals, and complete 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; A super-resolution optical imaging module is used to obtain the returned super-resolution signal light and convert it into an electrical signal; the super-resolution optical imaging module also includes a polarization beam splitter, a spatial filter, a second linear polarizer, a second sleeve mirror, an optical fiber head, a photomultiplier tube and a high-speed acquisition card, the polarization beam splitter is located between the first positive lens and the second positive lens, the spatial filter is located on the focal plane formed after the light is focused by the second positive lens and reflected by the polarization beam splitter; the second linear polarizer is located between the spatial filter and the second sleeve mirror, the front end face of the optical fiber head coincides with the rear focal plane of the second sleeve mirror, the optical fiber head is connected to the photomultiplier tube, the photomultiplier tube is connected to the high-speed acquisition card, and the high-speed acquisition card is used to connect to a computer; The computer is used to control the sample displacement module to drive the sample to move, and synchronously control the beam scanning galvanometer to scan and receive the electrical signal output by the super-resolution optical imaging module, and process it to obtain a super-resolution microscopic image of the sample in the super-resolution scanning imaging area.

[0006] Optionally, when the annular focused light beam generated by the annular focused light beam generating module is reflected by the light beam scanning galvanometer, passes through the scanning lens and the first sleeve mirror, and is reflected by the reflector to the rear focal plane of the objective lens; when the light beam scanning galvanometer vibrates, the center of the annular focused light beam coincides with the center of the rear focal plane of the objective lens, and the incident angle of the annular focused light beam changes with the vibration of the light beam scanning galvanometer; when the light beam scanning galvanometer performs two-dimensional scanning, the annular focused light beam is converted by the objective lens to form a conical focused light beam, and then forms a long-focus deep focused light field, which will form a super-resolution focused light spot on the front focal plane of the objective lens, and the focused light spot will realize continuous two-dimensional scanning on the front focal plane of the objective lens as the light beam scanning galvanometer vibrates; and by controlling the position of the rear focal plane of the objective lens in the Z-axis direction, the spatial range of continuous two-dimensional scanning on the front focal plane of the objective lens can be controlled.

[0007] Preferably, the super-resolution focusing illumination and collection module further includes a quarter-wave plate; the super-resolution optical imaging module further includes a polarization beam splitter; the optical microscopy imaging module includes a second beam splitter; after the circularly focused linearly polarized light beam emitted from the beam scanning module passes through the quarter-wave plate, a circularly polarized circularly focused line with an outer diameter smaller than the diameter of the rear focal plane of the objective lens is formed on the rear focal plane of the objective lens. The circularly polarized circularly focused line is converted into a super-resolution conical focused light beam through the objective lens, and a super-resolution focused light spot is formed on the front focal plane of the objective lens. The sample is illuminated by the super-resolution focused light spot. The super-resolution signal light generated by the reflection and scattering of the sample is collected by the objective lens, converged and collimated respectively, and then converted into a linearly polarized circularly focused light beam and a linearly polarized collimated light beam orthogonal to the incident linearly polarization through the quarter-wave plate, that is, a linearly polarized super-resolution signal light beam. The linearly polarized super-resolution signal light beam passes through the second beam splitter along the original optical path in the reverse direction, passes through the beam scanning module, enters the circularly focused light beam generation module, and is reflected by the polarization beam splitter placed in the optical path into the super-resolution optical imaging module; When the beam scanning galvanometer in the beam scanning module performs two-dimensional scanning, the circularly focused light beam is converted into a conical focused light beam by the objective lens, and the super-resolution focused light spot formed on the front focal plane of the objective lens will continuously perform two-dimensional scanning on the front focal plane of the objective lens as the beam scanning galvanometer vibrates; the abscissa X(t) and ordinate Y(t) of the position where the super-resolution focused light spot illuminates the sample are functions of time t, and the reflected light E r (t) and the scattered light E s (t) are also functions of time. The reflected light E r (t) and the scattered light E s (t) reflect information such as the sample structure and refractive index at the corresponding position (abscissa X(t) and ordinate Y(t)).

[0008] Preferably, the super-resolution focused light spot is a circularly focused light field. The inner and outer surfaces of the circularly focused light field are two coaxial conical surfaces with equal cone angles. The numerical aperture NA c of the circularly focused light field satisfies: NA c <NA, where NA represents the numerical aperture of the objective lens.

[0009] Preferably, to achieve three-dimensional tomographic scanning of the sample, the sample displacement module has two structures.

[0010] The first type is that the sample displacement module is a two-dimensional displacement stage, which is connected to a computer, and the sample is horizontally fixed on the two-dimensional displacement stage. The computer controls the two-dimensional displacement stage to drive the sample to move two-dimensionally in the XY plane to achieve the initial positioning of the sample; the super-resolution focusing illumination and collection module also includes an axial nanopositioner, which is connected to a computer, and the objective lens is installed on the axial nanopositioner. The computer controls the axial nanopositioner to move the objective lens in the Z direction. The beam scanning module realizes two-dimensional scanning of the sample, and realizes three-dimensional tomographic scanning in combination with the axial nanopositioner.

[0011] The second type is that the sample displacement module is a three-dimensional displacement stage. The three-dimensional displacement stage is connected to a computer. The sample is horizontally fixed on the three-dimensional displacement stage. The computer controls the three-dimensional displacement stage to drive the sample to move in the X, Y, and Z directions. The X and Y directions are used to achieve the initial positioning of the sample. The light beam scanning module achieves two-dimensional scanning of the sample, combined with the Z-direction displacement of the three-dimensional displacement stage to achieve three-dimensional tomographic scanning, avoiding the imaging influence caused by the slight movement of the objective lens and achieving better imaging effect.

[0012] Preferably, the super-resolution optical imaging module further comprises a five-dimensional adjustment frame, on which the optical fiber head is mounted, and the five-dimensional adjustment frame can be adjusted to maximize the super-resolution signal light entering the optical fiber head; The inner diameter of the optical fiber at the front end face of the optical fiber head is smaller than the first zero point radius of the focal spot of the super-resolution signal light on the rear focal plane of the second sleeve mirror, thereby better ensuring that the super-resolution imaging resolution reaches the optimal level.

[0013] Preferably, the light source module includes a laser and a fiber collimator, the laser is connected to the fiber collimator through a first fiber jumper; the coherent light emitted by the laser is transmitted to the fiber collimator through the first fiber jumper, and the fiber collimator collimates the coherent light and outputs a collimated laser beam; The annular focused light beam generating module also includes a field lens, the object side principal surface of the field lens coincides with the rear focal plane of the first positive lens, and the image side principal surfaces of the field lens coincide with the front focal planes of the second positive lens, respectively, so as to change the propagation direction of the edge light of the annular focused light beam; to adapt to different objective lenses, so that the annular focused light beam can be transmitted through the objective lens without obstruction.

[0014] Preferably, the optical microscopic imaging module includes an incoherent light source, a collimating lens, a first beam splitter, a second beam splitter, a third telescope and a digital camera, the digital camera is connected to a computer, and the incoherent light source is located at the front focus of the collimating lens; the light emitted by the incoherent light source is collimated by the collimating lens and reflected by the first beam splitter, and then passes through the second beam splitter and the quarter wave plate in sequence to reach the objective lens, and converges into incoherent light on the front focal plane of the objective lens to illuminate the sample, and the incoherent light generated by the reflection and scattering of the sample is collected by the objective lens, and then passes through the quarter wave plate, the second beam splitter, the first beam splitter, and the third telescope in sequence to enter the digital camera, so that the sample is imaged on the digital camera and sent to the computer through the camera signal line, and the computer displays the wide-field microscopic image of the sample. This facilitates wide-field observation of the sample to determine the required super-resolution scanning imaging area.

[0015] Preferably, the spatial filter has three structures.

[0016] The first spatial filter is a ring-type spatial filter, which includes a glass substrate and a concentric circular aluminum film and a first ring-shaped aluminum film covering the glass substrate, wherein the diameter of the circular aluminum film is equal to 2R in , the inner diameter of the first annular aluminum film is equal to 2R out , the outer diameter is equal to the diameter of the glass substrate, the transmittance of the glass substrate to the light wave with wavelength λ is greater than 90%, and the transmittance of the circular aluminum film and the first annular aluminum film to the light wave with wavelength λ is 0. Only the super-resolution signal light generated by the sample reflection is allowed to pass, that is, the bright field imaging light signal is allowed to pass, so as to realize bright field imaging. Among them, R in Represents the inner radius of the circular focal line at the rear focal plane of the objective lens, R out Represents the outer radius of the circular focus line at the rear focal plane of the objective.

[0017] The second spatial filter is an annular shielding type spatial filter, which includes a glass substrate and a second annular aluminum film covering the glass substrate, wherein the inner diameter of the second annular aluminum film is equal to 2R in , outer diameter equals 2R out The glass substrate has a transmittance greater than 90% for light waves with a wavelength of λ, and the second annular aluminum film has a transmittance of 0 for light waves with a wavelength of λ. Only the super-resolution signal light generated by sample scattering is allowed to pass, that is, the dark field imaging light signal is allowed to pass, thereby achieving dark field imaging.

[0018] The third type of spatial filter is a phase plate, which is a plate with a thickness of t and an inner and outer radius of R on the surface of a glass substrate. in and R out A spatial filter, in R in and R outThe transmittance T of the light wave with wavelength λ in the ring between them is 10%-20%, and its phase change relative to the air at the same height is π / 4 or -π / 4. It is used to realize phase contrast imaging.

[0019] In a second aspect, the reflective scanning super-resolution optical microscopy method based on light cone illumination of the present invention adopts the above-mentioned reflective scanning super-resolution optical microscopy system, and the method comprises: Step 1: Use an optical microscopy imaging module to perform wide-field imaging of the sample and determine the super-resolution scanning imaging area; Step 2: After determining the super-resolution scanning imaging area, the annular focused beam generating module generates an annular focused beam, and the super-resolution focused illumination and collection module converts the annular focused beam into a super-resolution conical focused beam to form a super-resolution focal spot to illuminate the sample, collect the super-resolution signal light generated by the sample reflection and scattering, respectively converge and collimate the reflected and scattered super-resolution light signals, and complete the polarization conversion; Step 3: The computer controls the sample displacement module to move the sample to the position to be scanned, and turns on the beam scanning galvanometer for scanning. At the same time, the super-resolution optical imaging module obtains the collimated and converted super-resolution signal light, and converts it into an electrical signal and inputs it into the computer; Step 4: The computer processes the input electrical signal and combines the coordinates X(t) and Y(t) of the super-resolution illumination focus moving on the sample and the signal light intensity I(t) obtained by the photomultiplier tube at the corresponding time t to form a super-resolution microscopic image I(X(t), Y(t)) of the sample in the super-resolution scanning imaging area.

[0020] The present invention has the following advantages: (1) High-efficiency super-resolution focused illumination: The present invention can efficiently generate a ring-shaped focused beam through a carefully designed ring-shaped focused beam generation module. Subsequently, the ring-shaped focused beam is converted into a super-resolution conical focused beam using a super-resolution focused illumination and collection module to form a high-precision super-resolution focal spot to illuminate the sample. This process fully utilizes the advantages of high focusing efficiency of traditional lenses, realizes high-efficiency super-resolution focused illumination, and significantly improves the signal-to-noise ratio of imaging. At the same time, the generated super-resolution conical focused beam forms a long-focus depth super-resolution focused light field behind the objective lens, which is convenient for reducing the alignment requirements of the illumination beam, focal plane, and sample.

[0021] (2) Fast 2D scanning: The beam scanning module uses beam scanning galvanometer technology to achieve fast 2D scanning of the beam. This scanning method is not only fast but also highly accurate, and can perform super-resolution imaging of a large area in a short time. This feature greatly improves the efficiency of imaging, allowing users to quickly obtain a large amount of high-quality image data.

[0022] (3) Label-free far-field super-resolution microscopy: Compared with traditional microscopy techniques that require fluorescent molecular labeling, a major highlight of this invention is that it can achieve far-field super-resolution two-dimensional microscopy rapid imaging of unlabeled samples. This function not only expands the scope of application of the technology, but also simplifies the experimental steps and reduces the requirements for sample processing. At the same time, due to the large working distance, the application in fields such as biological and industrial detection is more convenient and efficient.

[0023] (4) Three-dimensional tomography capability: By performing non-labeled far-field super-resolution two-dimensional microscopic imaging of samples at different axial positions, the present invention can obtain super-resolution two-dimensional microscopic images of different cross-sections of the sample, and then realize three-dimensional tomography 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 biomedical research, materials science and other fields.

[0024] (5) High reliability and imaging quality: The present invention ensures the reliability and stability of imaging by precisely controlling the optical path coordination and signal processing between modules. From the generation of annular focused beams, beam scanning, super-resolution focused illumination to signal collection and processing, each link has been carefully designed and optimized to ensure a high level of final imaging quality. In addition, the optical components and signal acquisition equipment used have high performance indicators, which further improves the overall performance and imaging quality of the system.

[0025] In summary, the reflective scanning super-resolution optical microscopy system and method based on light cone illumination of the present invention show significant advantages in terms of efficient super-resolution focused illumination, fast two-dimensional scanning, label-free far-field super-resolution microscopy imaging, three-dimensional tomography capability, super-resolution three-dimensional imaging capability, high reliability and imaging quality, etc., providing strong technical support for the fields of biomedicine, industrial detection, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the structure of the reflective scanning super-resolution optical microscopy system based on light cone illumination in Example 1.

[0027] Figure 2 Schematic diagram of controlling the size of the annular focused beam using the axicon, the third positive lens and the first 4f system in Example 1.

[0028] Figure 3 It is a schematic diagram of forming a long focal depth super-resolution focused light field in Example 1.

[0029] Figure 4 Parameter relationship diagram of the long focal depth super-resolution focused light field in Example 1.

[0030] Figure 5Schematic diagram of the super-resolution focused light spot scanning on the focal plane of the objective lens in Example 1.

[0031] Figure 6 Schematic diagram of the front view and cross section of the spatial filter in the first embodiment.

[0032] Figure 7 Graph showing the relationship between the objective lens, the full width at half maximum of the focal spot and the system resolution under the condition of annular focused beam illumination in Example 1.

[0033] Figure 8 Schematic diagram of the structure of the reflective scanning super-resolution optical microscopy system based on light cone illumination in Example 2.

[0034] Fig. 9 Schematic diagram of the front view and cross section of the spatial filter in the third embodiment.

[0035] Fig.10 Schematic diagram of the front view and cross section of the spatial filter in the fourth embodiment.

[0036] Fig.11 Schematic diagram of the front view and cross section of the spatial filter in the fifth embodiment.

[0037] In the figure: 1, laser, 2, first fiber jumper, 3, fiber collimator, 4, first linear polarizer, 5, conical lens, 6, first positive lens, 7, second positive lens, 8, third positive lens, 9, beam scanning galvanometer, 10, beam scanning lens, 11, first sleeve mirror, 12, reflector, 13, quarter wave plate, 14, axial nanopositioner, 15, objective lens, 16, sample, 17, two-dimensional translation stage, 18, scanning signal line, 19, polarization beam splitter, 20, spatial filter, 201, glass substrate, 202, Circular aluminum film, 203, first annular aluminum film, 204, second annular aluminum film, 21, second linear polarizer, 22, second sleeve mirror, 23, optical fiber head, 24, five-dimensional adjustment frame, 25, second optical fiber jumper, 26, photomultiplier tube, 27, acquisition signal line, 28, high-speed acquisition card, 29, data line, 30, incoherent light source, 31, collimating lens, 32, first beam splitter, 33, second beam splitter, 34, third sleeve mirror, 35, digital camera, 36, camera signal line, 37, three-dimensional translation stage, 38, field mirror. DETAILED DESCRIPTION

[0038] In order to enable a more detailed understanding of the features and technical contents of the embodiments of the present invention, the implementation of the embodiments of the present invention is described in detail below in conjunction with the accompanying drawings. The attached drawings are for reference only and are not intended to limit the embodiments of the present invention.

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

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

[0041] The annular focused beam generating module is used to generate an annular focused beam.

[0042] The beam scanning module is used to realize fast two-dimensional scanning of the light beam.

[0043] The super-resolution focusing illumination and collection module is used to convert the annular focusing beam into a super-resolution conical focusing 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.

[0044] The super-resolution optical imaging module is used to obtain the returned super-resolution signal light and convert it into an electrical signal.

[0045] The computer (not shown) is used to control the sample displacement module to move the sample 16. The computer controls the beam scanning galvanometer 9 to scan and receive the electrical signal output by the super-resolution optical imaging module, and processes the super-resolution microscopic image of the sample 16 in the super-resolution scanning imaging area.

[0046] This system achieves efficient super-resolution focused illumination by utilizing the advantages of annular focused beam illumination, high focusing efficiency of traditional lenses, and fast beam scanning speed, and can then use traditional optical lenses to achieve fast super-resolution optical microscopy imaging; non-labeled far-field super-resolution two-dimensional microscopy fast imaging is achieved through two-dimensional beam scanning; by performing non-labeled far-field super-resolution two-dimensional microscopy imaging on samples at different axial positions, super-resolution two-dimensional microscopy imaging of different cross sections of the sample is obtained, thereby achieving three-dimensional tomography. This system can be applied to non-labeled super-resolution microscopy fast imaging of biological samples, and can also be applied to industrial-related super-resolution microscopy detection and other fields.

[0047] The following is a detailed description of each module: like Figure 1 and Figure 2As shown, the optical microscopic imaging module includes an incoherent light source 30, a collimating lens 31, a first beam splitter 32, a second beam splitter 33, a third tube mirror 34 and a digital camera 35; the digital camera 35 is connected to the computer through a camera signal line 36, and the incoherent light source 30 is located at the front focus of the collimating lens 31. The light emitted by the incoherent light source 30 is collimated by the collimating lens 31 and reflected by the first beam splitter 32, and then passes through the second beam splitter 33 and the quarter wave plate 13 in sequence to reach the objective lens 15, and converges into incoherent light on the front focal plane of the objective lens 15 to illuminate the sample 16. The incoherent light generated by the reflection and scattering of the sample 16 is collected by the objective lens 15, and then passes through the quarter wave plate 13, the second beam splitter 33, the first beam splitter 32, and the third tube mirror 34 in sequence to enter the digital camera 35, so that the sample 16 is imaged on the digital camera 35 and sent to the computer, and the computer displays the wide-field microscopic image of the sample 16.

[0048] like Figure 1 and Figure 2 As shown, the annular focused beam generating module includes a laser 1, a fiber collimator 3, a first linear polarizer 4, a cone lens 5, a first positive lens 6, a scene 38, a second positive lens 7 and a third positive lens 8 in sequence along the light propagation direction. The laser 1 is connected to the fiber collimator 3 through the first fiber jumper 2; the coherent light emitted by the laser 1 is transmitted to the fiber collimator 3 through the first fiber jumper 2, and the fiber collimator 3 collimates the coherent light and outputs a collimated laser beam. The first positive lens 6 is coaxially spaced with the cone lens 5, the front focal plane F2 of the second positive lens 7 coincides with the back focal plane F1' of the first positive lens 6, the back focal plane (i.e. F'2) of the second positive lens 7 coincides with the front focal plane (i.e. F3) of the third positive lens 8, and the second positive lens 7 and the third positive lens 8 constitute a first 4f system. The light source module (laser 1 and fiber collimator 3) generates a collimated laser beam, which forms a linearly polarized annular focused beam after passing through the first linear polarizer 4, the aconical lens 5 and the first positive lens 6, and forms a circular focus line at the rear focal plane of the first positive lens 6. The object side principal surface of the field lens 38 coincides with the rear focal plane of the first positive lens 6, and the image side principal surface of the field lens 38 coincides with the front focal plane of the second positive lens 7, respectively, to change the propagation direction of the edge light of the annular focused beam; to adapt to different objective lenses 15, so that the annular focused beam can be transmitted through the objective lens 15 without obstruction.

[0049] like Figure 1 and Figure 2As shown, the beam scanning module includes a beam scanning galvanometer 9, a scanning lens 10, a first sleeve mirror 11 and a reflector 12. The center position of the beam scanning galvanometer 9 coincides with the rear focal plane of the third positive lens 8; the front focal plane of the scanning lens 10 is located at the center position of the beam scanning galvanometer 9; the rear focal plane of the scanning lens 10 coincides with the front focal plane of the first sleeve mirror 11, and the two constitute a second 4f system; the rear focal plane of the first sleeve mirror 11 coincides with the rear focal plane of the objective lens 15 in the super-resolution focusing illumination and collection module after being reflected by the reflector 12. The beam scanning galvanometer 9 contains a controller for connecting to a computer (not shown in the figure), and the computer controls the beam scanning galvanometer 9 to perform two-dimensional vibration scanning through the controller of the beam scanning galvanometer 9.

[0050] When the annular focused light beam generated by the annular focused light beam generating module is reflected by the light beam scanning galvanometer 9, passes through the scanning lens 10 and the first sleeve mirror 11, and is reflected by the reflector 12 to the rear focal plane of the objective lens 15; when the light beam scanning galvanometer 9 vibrates, the center of the annular focused light beam coincides with the center of the rear focal plane of the objective lens 15, and the incident angle of the annular focused light beam changes with the vibration of the light beam scanning galvanometer 9; when the light beam scanning galvanometer 9 performs two-dimensional scanning, the annular focused light beam is transformed by the objective lens 15 to form a conical focused light field behind the objective lens 15, thereby generating a long-focus deep focused light field and forming a super-resolution focused light spot on the front focal plane of the objective lens 15, the super-resolution focused light spot will realize continuous two-dimensional scanning on the front focal plane of the objective lens 15 with the vibration of the light beam scanning galvanometer 9; and by controlling the position of the rear focal plane of the objective lens 15 in the Z-axis direction, the spatial range of continuous two-dimensional scanning on the front focal plane of the objective lens 15 can be controlled.

[0051] like Figure 1 and Figure 2 As shown, the super-resolution focusing illumination and collection module includes a quarter wave plate 13 and an objective lens 15, the rear focal plane of the objective lens 15 coincides with the rear focal plane of the first sleeve lens 11, and the sample 16 is located on the front focal plane of the objective lens 15. The objective lens 15 uses a conventional microscope objective lens, which can be a dry lens, a water lens, an oil lens or a solid lens.

[0052] like Figure 1 and Figure 2 As shown, the sample displacement module is a two-dimensional displacement stage 17, which is connected to a computer via a scanning signal line 18 (not shown in the figure), and the sample 16 is horizontally fixed on the two-dimensional displacement stage 17. The computer controls the two-dimensional displacement stage 17 to drive the sample 16 to move two-dimensionally in the XY plane. When the sample displacement module is a two-dimensional displacement stage 17, the super-resolution focusing illumination and collection module also includes an axial nanopositioner 14, which is connected to a computer via a positioning signal line, and an objective lens 15 is mounted on the axial nanopositioner 14. The computer controls the axial nanopositioner 14 to drive the objective lens 15 to move in the Z direction.

[0053] like Figure 1 and Figure 2 As shown, the super-resolution optical imaging module includes a polarization beam splitter 19, a spatial filter 20, a second linear polarizer 21, a second sleeve mirror 22, an optical fiber head 23, a photomultiplier tube 26 and a high-speed acquisition card 28. The polarization beam splitter 19 is located between the field lens 38 and the second positive lens 7, and the spatial filter 20 is located on the focal plane formed after the light is focused by the second positive lens 7 and reflected by the polarization beam splitter 19; the second linear polarizer 21 is located between the spatial filter 20 and the second sleeve mirror 22, the front end surface of the optical fiber head 23 coincides with the rear focal plane of the second sleeve mirror 22, the optical fiber head 23 is connected to the photomultiplier tube 26 through the second optical fiber jumper 25, the photomultiplier tube 26 is connected to the high-speed acquisition card 28 through the acquisition signal 27, and the high-speed acquisition card 28 is connected to the computer through the data line 29.

[0054] like Figure 6 As shown, in a possible embodiment, the spatial filter 20 is a circular pass type spatial filter, which includes a glass substrate 201 and a circular aluminum film 202 and a first circular aluminum film 203 that are concentric and cover the glass substrate 201. The thickness t of the circular aluminum film 202 and the first circular aluminum film 203 is greater than 50nm, and the diameter of the circular aluminum film 202 is equal to 2R in , the inner diameter of the first annular aluminum film 203 is equal to 2R out , the outer diameter is equal to the diameter of the glass substrate 201, the transmittance of the glass substrate 201 to the light wave with a wavelength of λ is greater than 90%, and the transmittance of the circular aluminum film 202 and the first annular aluminum film 203 to the light wave with a wavelength of λ is 0.

[0055] In an embodiment of the present application, a reflective scanning super-resolution optical microscopy method based on light cone illumination adopts the above-mentioned reflective scanning super-resolution optical microscopy system, and the method includes: Step 1: Use an optical microscopy imaging module to perform wide-field imaging on the sample 16 to determine the super-resolution scanning imaging area. Specifically: The laser 1 is first turned off and does not emit coherent light. The light emitted by the incoherent light source 30 is collimated by the collimating lens 31 and reflected by the first beam splitter 32, and then passes through the second beam splitter 33 and the quarter wave plate 13 in sequence to reach the objective lens 15, and converges into incoherent light on the front focal plane of the objective lens 15 to illuminate the sample 16. The incoherent light generated by the reflection and scattering of the sample 16 is collected by the objective lens 15, and then passes through the quarter wave plate 13, the second beam splitter 33, the first beam splitter 32, and the third sleeve lens 34 in sequence to enter the digital camera 35, so that the sample 16 is imaged on the digital camera 35 and sent to the computer through the camera signal line 36. The computer displays the wide-field microscopic image of the sample 16, thereby facilitating wide-field observation of the sample 16, so as to facilitate determination of the required super-resolution scanning imaging area. If the sample 16 is not in the super-resolution scanning imaging area, the computer can control the two-dimensional displacement stage 17 to drive the sample 16 to move so that the sample 16 is located in the super-resolution scanning imaging area.

[0056] Step 2: After determining the super-resolution scanning imaging area, the annular focused beam generating module generates an annular focused beam, and the super-resolution focused illumination and collection module converts the annular focused beam into a super-resolution conical focused beam to form a super-resolution focal spot to illuminate the sample 16, collect the super-resolution signal light reflected and scattered by the sample 16, respectively converge and collimate the reflected and scattered super-resolution light signals, and complete the polarization conversion. Specifically: The incoherent light source 30 does not emit light, the laser 1 is turned on, and the laser 1 emits coherent light with a wavelength of λ, which is transmitted to the fiber collimator 3 through the first fiber jumper 2. The fiber collimator 3 collimates the coherent light and outputs a collimated laser beam. The collimated laser beam passes through the first linear polarizer 4, the conical lens 5, and the first positive lens 6 to form an annular focused beam (a P-polarized annular beam). After the P-polarized annular focused beam passes through the first 4f system, the beam scanning module, and the quarter-wave plate 13, a circular focusing line with an outer diameter smaller than the diameter of the rear focal plane of the objective lens 15 is formed on the rear focal plane of the objective lens 15. After passing through the objective lens 15, the circularly polarized circular focusing line is converted into a super-resolution conical focused beam, forming a super-resolution focused light spot on the front focal plane of the objective lens 15. The super-resolution focusing light spot The full width at half maximum of the light spot is less than 0.5λ / NA, and the first zero point radius of the super-resolution focal spot of the super-resolution focusing spot (i.e., the distance from the focal spot intensity peak position to the first zero point position) is less than 0.61λ / NA. The super-resolution focusing spot illuminates the sample 16, and the super-resolution signal light generated by reflection and scattering of the sample 16 is collected by the objective lens 15 and respectively converged and collimated, and then converted into an S-polarized annular focusing beam and an S-polarized collimated beam orthogonal to the incident linearly polarized annular focusing beam through the quarter-wave plate 13, and the S-polarized annular focusing and S-polarized collimated beams pass through the second beam splitter 33 along the opposite direction of the original optical path, pass through the beam scanning module, enter the annular focusing beam generating module, and are reflected by the polarization beam splitter 19 placed in the optical path to enter the super-resolution optical imaging module.

[0057] Figure 2 A method for generating a P-polarized annular focused beam is given. Specifically, the optical fiber collimator 3 collimates the coherent light and outputs a collimated laser beam with a radius of R (selecting optical fiber collimators with different numerical apertures can obtain collimated laser beams with different radii, and then obtain super-resolution conical focused beams with different focal depths). The collimated laser beam with a radius of R passes through the first linear polarizer 4 and becomes a P-polarized collimated beam with a radius of R. The P-polarized collimated beam passes through two coaxially placed optical fibers with a spacing of d After the conical lens 5 and the first positive lens 6, a P-polarized annular focused beam is formed, and a central radius R' is formed on the rear focal plane of the first positive lens 6. c The central radius R' of the circular focal line can be changed by changing the focal length of the first positive lens 6. c The P polarized annular focused beam is further projected onto the rear focal plane of the third positive lens 8 through the first 4f system, forming a central radius of R'' c The circular focal line of c =R' c × f 3 / f 2, f 2 represents the focal length of the second positive lens 7, f3 represents the focal length of the third positive lens 8. By selecting the second positive lens 7 and the third positive lens 8 with different focal lengths, circular focal lines of different sizes can be obtained. Further, through the second 4f system composed of the beam scanning lens 10 and the first sleeve lens 11, the circular focal line is projected onto the rear focal plane F' of the first sleeve lens 11 t (which is also the rear focal plane of the objective lens 15), and the magnification of the second 4f system is K. Therefore, by controlling f 1, f 2, f 3, the central radius R of the finally formed circular focal line can be controlled c =KR' c × f 3 / f 2.

[0058] Figure 3 The illumination working schematic of forming a super-resolution focusing light field with a long depth of focus is given. The left-handed circularly polarized annular focusing beam illuminates the rear focal plane of the objective lens 15, and a circular focal line with a central diameter of 2R is formed on the rear focal plane of the objective lens 15 c , the inner radius of the circular focal line is R in , the outer radius is R out , and the central radius R c =(R out +R in ) / 2. After passing through the objective lens 15, the left-handed circularly polarized annular focusing beam is converted into a super-resolution conical focusing beam with a cone angle of θ c , and a long-depth-of-focus super-resolution focusing light field is formed along the optical axis before and after the front focal plane of the objective lens 15. The numerical aperture NA of the super-resolution conical focusing beam c =n×sin(θ c ), NA c <NA; where, sin() is the sine function, and n is the refractive index of the medium between the objective lens 15 and the sample 16. The full width at half maximum of the long-depth-of-focus super-resolution focusing light field is less than 0.5λ / NA, and the first zero radius of the super-resolution focal spot of the long-depth-of-focus super-resolution focusing light field (the distance from the focal spot intensity peak position to the first zero position) is less than 0.61λ / NA. When 0 < NA c <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 imaging with practical value and better resolution, it is necessary to satisfy: 0.947NA ≤ NA c <NA. The resolution of super-resolution microscopy is determined by NA c , the larger NA c and the closer it is to NA, the closer the resolution of super-resolution microscopy can be to the limit resolution 0.25λ / NA of the reflection confocal system. The larger NA c is, the larger the corresponding Rc The larger (R c is less than the radius of the rear focal plane of the objective lens 15), and the corresponding R out 、R in is closer (i.e., the width of the circular focus line is smaller), the narrower the spatial frequency of the long-depth-of-field super-resolution focused light field. In practice, we control the radius R of the collimated laser beam and adjust the distance between the axicon lens 5 and the first positive lens 6 d to control R' c , and then control the radius of the circular focus line. Then, since the S-polarized annular focused beam corresponding to the circular focus line will undergo obvious diffraction during propagation, resulting in an increase in width, it is impossible to ensure that the circular focus line transmitted to the rear focal plane of the objective lens 15 can maintain an extremely small width; therefore, it is necessary to add a first 4f system composed of the second positive lens 7 and the third positive lens 8 to adjust the size of the S-polarized annular focused beam (corresponding to the circular focus line), so that the numerical aperture NA of the finally formed super-resolution conical focused beam c satisfies: 0.947NA ≤ NA c < NA. The first 4f system composed of the second positive lens 7 and the third positive lens 8 plays a role in adjusting the size of the circular focus line of the S-polarized annular focused beam on the one hand, and plays a role in relaying the transmission of the S-polarized annular focused beam on the other hand, so as to ensure that after the circular focus line on the front focal plane of the first 4f system is adjusted, it is accurately projected onto the rear focal plane of the first 4f system (corresponding to the rear focal plane of the objective lens 15), thereby avoiding the size distortion and light field intensity distribution fluctuation caused by the diffraction effect during the direct transmission of the S-polarized annular focused beam, ensuring the high-quality illumination of the objective lens by the S-polarized annular focused beam, and further ensuring that the generated super-resolution focal spot reaches the minimum to ensure that the system reaches the best resolution.

[0059] Given the numerical aperture NA of the objective lens 15, to ensure that the cone angle of the super-resolution conical focused beam after passing through the objective lens 15 is θ c , it can be achieved in the following way: First, determine NA r and NA according to the required resolution d c , specifically d r = 0.5λ / (NA c + NA), where NA c ≤ NA, NA c represents the numerical aperture corresponding to the circular focus line on the rear focal plane of the objective lens 15. Determine R c according to NA c (the determination method belongs to the prior art and can be achieved through actual measurement); then, according to R c = K f 1tan( β ) f 3 / f2 (of which β is the cone angle of the conical light beam emitted by the conical lens 5, tan() is the tangent function), determine f 1. f 2 and f 3 ( f 3 needs to be long enough so that the third positive lens 8 does not interfere spatially with subsequent optical elements.

[0060] Figure 4 The parameters FWHM (full width at half maximum), r0 (radius of the first zero point) and SR (sidelobe peak ratio, i.e. the ratio of the maximum sidelobe intensity to the center intensity) of the long focal depth super-resolution focused light field and NA are given when the numerical aperture NA of the objective lens 15 is 0.95. c It can be seen that when NA c ≥0.9, the generated long focal depth super-resolution focused light field has a FWHM≤0.399λ (less than 0.5λ / NA), r0≤0.426λ (less than 0.61λ / NA), and SR=0.162. c So that it satisfies: 0.9≤NA c <0.95, a long focal depth super-resolution illumination light field with FWHM≤0.399λ, r0≤0.426λ, and SR=0.162 can be achieved.

[0061] like Figure 5 As shown in FIG. 1 , it is a schematic diagram of scanning the super-resolution focused light spot on the focal plane of the objective lens 15. The beam scanning module realizes two-dimensional scanning of the annular beam. At different times, the annular focused beam enters the objective lens 15 at different angles. When the annular focused beam reaches the rear focal plane of the objective lens 15, the annular focused beam always presents a circular ring with the center of the rear focal plane as the center. At a given time t, the inclination angle of the incident annular focused beam is θ s (t), at this time, an ultra-long focal depth super-resolution focusing spot with a half-width length of 2△Z is formed before and after the rear focal plane of the objective lens 15 (in the Z direction). The offset of the focusing spot in the XY direction (relative to the optical axis Z) is d(t). By changing the incident angle θ s (t), that is, the offset d(t) can be changed. At different times, the light intensity distribution of the illuminating annular focused beam on the rear focal plane of the objective lens is always a circular ring centered at the center of the rear focal plane, and the inner and outer radii of the circular ring are R in and R out .

[0062] like Figure 7The figure shows the relationship between the objective lens (the numerical aperture NA of this objective lens is greater than 1), the half-width of the focal spot and the system resolution under the condition of annular focused beam illumination. The ordinate in the figure is the half-width of the point spread function of the objective lens, in units of wavelength λ, the abscissa is the lateral half-width of the super-resolution focal spot of the super-resolution focused spot, in units of wavelength λ, and the contour line is the system resolution (i.e., the minimum line width of the amplitude-type grating that the system can resolve, and the imaging fringe contrast is better than 11%), in units of wavelength λ. When the half-width of the point spread function of the objective lens (a single-point focusing super-diffraction lens can be used) is 0.6λ and the half-width of the lateral half-width of the super-resolution focal spot (the part that illuminates the sample) is 0.38λ, the system resolution can reach 0.25λ~0.26λ; when the half-width of the point spread function of the objective lens and the lateral half-width of the super-resolution focal spot are both 0.35λ, the system resolution is better than 0.2λ.

[0063] Step 3: The computer controls the beam scanning module to perform two-dimensional scanning on the beam, thereby realizing two-dimensional scanning of the super-resolution focused spot on the front focal plane of the objective lens 15. At the same time, the super-resolution optical imaging module synchronously obtains the collimated and transformed super-resolution signal light, and converts it into an electrical signal and inputs it into the computer.

[0064] Step 4: The computer processes the input electrical signal, combines the coordinates X(t) and Y(t) of the super-resolution focusing spot moving on the sample 16 and the signal light intensity I(t) obtained by the photomultiplier tube 26 at the corresponding time t, and forms a super-resolution microscopic image I(X(t), Y(t)) of the sample in the super-resolution scanning imaging area.

[0065] In the embodiment of the present application, the specific processing process of step three and step four is as follows: the S-polarized light beam (i.e., the super-resolution signal light reflected by the polarization beam splitter 19 after being converged, collimated, and converted) is filtered by the spatial filter 20 and further passes through the second linear polarizer 21, leaving only the super-resolution signal light generated by the reflection of the sample 16, which converges on the rear focal plane of the second sleeve mirror 22, and is coupled into the optical fiber head 23, and then enters the photomultiplier tube 26 through the second optical fiber jumper 25, and is converted into an analog electrical signal by the photomultiplier tube 26. The analog electrical signal is collected and converted into a digital electrical signal by the high-speed acquisition card 28 through the acquisition signal line 27, and is input into the computer through the data line 29. The computer controls the beam scanning galvanometer 9 in the beam scanning module to perform two-dimensional scanning, and the super-resolution focusing spot formed by the annular light beam on the front focal plane of the objective lens 15 will achieve continuous two-dimensional scanning on the front focal plane of the objective lens 15 as the beam scanning galvanometer 9 vibrates. The horizontal coordinate X(t) and the vertical coordinate Y(t) of the position where the super-resolution focused light spot illuminates the sample 16 are functions of time t. The reflected light E collected by the objective lens 15 r (t) and scattered light E s (t) is also a function of time, the reflected light E r (t) and scattered light Es (t) reflects the sample structure and refractive index information at the corresponding position (abscissa X(t) and ordinate Y(t)). By performing a two-dimensional scan in the XY plane (the scanning step is smaller than the minimum resolution size of the system), the signal intensity of the corresponding coordinates is directly output as the coordinate function I(X(t), Y(t)) (where X and Y are the coordinates of the super-resolution focusing spot on the focal plane of the objective lens 15), and a two-dimensional super-resolution microscopic image of the sample 16 can be obtained. The resolution of the image in the XY plane is close to the limiting resolution of the reflective confocal system, 0.25λ / NA. The computer controls the axial nanopositioner 14 to move the objective lens 15 in the Z direction, changing the Z position Z in the sample where the front focal plane of the objective lens 15 is located. f , get different Z f By using a two-dimensional super-resolution microscopic image in the XY plane at a certain position, a three-dimensional tomographic microscopic image can be obtained, whose resolution is close to the limiting resolution of the reflective confocal system, 0.25λ / NA.

[0066] Embodiment 2 like Figure 8 As shown, most of the structures of the reflective scanning super-resolution optical microscopy system based on light cone illumination in this embodiment are the same as those in the first embodiment, except that: the sample displacement module is a three-dimensional displacement stage 37, the three-dimensional displacement stage 37 is connected to the computer via a scanning signal line 18, the sample 16 is horizontally fixed on the three-dimensional displacement stage 37, and the computer controls the three-dimensional displacement stage 37 to drive the sample 16 to move in the Z-axis direction.

[0067] like Figure 8 As shown, the reflective scanning super-resolution optical microscopy method based on light cone illumination in this embodiment is basically the same as that in the first embodiment, the only difference is that in the specific processing of step 3 and step 4, the computer controls the three-dimensional displacement stage 37 to drive the sample 16 to move slightly in the Z direction, changing the Z direction position Z of the front focal plane of the objective lens 15 in the sample 16. f Every time it moves a little, the high-speed acquisition card 28 collects a signal to obtain different Z f By using a two-dimensional super-resolution microscopic image in the XY plane at a certain position, a three-dimensional tomographic microscopic image can be obtained, whose resolution is close to the limiting resolution of the reflective confocal system, 0.25λ / NA.

[0068] Embodiment 3 like Fig. 9 As shown, in this embodiment, most of the structures of the reflective scanning super-resolution optical microscopy system based on light cone illumination are the same as those in the first embodiment, except that: the spatial filter 20 is an annular blocking type spatial filter, which includes a glass substrate 201 and a second annular aluminum film 204 covering the glass substrate 201, the thickness t of the second annular aluminum film 204 is greater than 50nm, and the inner diameter of the second annular aluminum film 204 is equal to 2Rin , outer diameter equals 2R out , the transmittance of the glass substrate 201 to the light wave with a wavelength of λ is greater than 90%, and the transmittance of the second annular aluminum film 204 to the light wave with a wavelength of λ is 0. Only the super-resolution signal light generated by the scattering of the sample 16 is allowed to pass, that is, the dark field imaging light signal is allowed to pass, so as to realize dark field imaging. The reflective scanning super-resolution optical microscopy method based on light cone illumination in this embodiment is basically the same as that in the first embodiment, except that in step 3, after the P-polarized collimated light beam is filtered by the spatial filter 20, only the super-resolution signal light generated by the scattering of the sample 16 is concentrated on the rear focal plane of the second sleeve mirror 22 and coupled into the optical fiber head 23.

[0069] Embodiment 4 like Fig.10 As shown, most of the structures of the reflective scanning super-resolution optical microscopy system based on light cone illumination in this embodiment are the same as those in the first embodiment, except that: the spatial filter 20 is a phase plate, which is prepared on the upper surface of the glass substrate with a thickness of t=t1+t2 and an inner and outer radius of R in and R out A spatial filter. in and R out The transmittance T of the light wave with wavelength λ in the ring between is 10%-20%, and its phase change relative to the air at the same height is π / 4 (or -π / 4). The transparent medium with thickness t1 (transmittance greater than 90% at the working wavelength λ) has a thickness of t1=λ / 4(n-1), where n is the refractive index of the transparent medium; t2 is the thickness of the metal film, which should satisfy the transmittance of 10%-20% for the light wave with wavelength λ.

[0070] Embodiment 5 like Fig.11 As shown, most structures of the reflective scanning super-resolution optical microscopy system based on light cone illumination in this embodiment are the same as those in the first embodiment, except that: the spatial filter 20 is a phase plate, which is prepared on the upper surface of the glass substrate with inner and outer radii R in and R out A spatial filter. in and R out The metal film in the ring is of thickness t2, and its transmittance T at the wavelength λ is 10%-20%; the other areas outside the ring area are transparent media of thickness t1 (transmittance is greater than 90% at the working wavelength λ), and its thickness is t1=λ / 4(n-1), where n is the refractive index of the transparent medium, and its phase change relative to the air at the same height is π / 4 (or -π / 4).

[0071] Embodiment 6 Most structures of the reflective scanning super-resolution optical microscopy system based on light cone illumination in this embodiment are the same as those in the first embodiment, except that the field lens 38 is not required.

[0072] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims

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

2. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 1, characterized in that: When the annular focused light beam generated by the annular focused light beam generating module is reflected by the light beam scanning galvanometer (9), passes through the scanning lens (10) and the first sleeve mirror (11), and is reflected by the reflector (12) to the rear focal plane of the objective lens (15); when the light beam scanning galvanometer (9) vibrates, the center of the annular focused light beam coincides with the center of the rear focal plane of the objective lens (15), and the incident angle of the annular focused light beam changes with the vibration of the light beam scanning galvanometer (9); when the light beam scanning galvanometer (9) performs two-dimensional scanning, the annular focused light beam is transformed by the objective lens (15), and a conical focused light field is formed behind the objective lens (15), thereby generating a long-focus deep focused light field and forming a super-resolution focused light spot on the front focal plane of the objective lens (15), and the focused light spot will realize continuous two-dimensional scanning on the front focal plane of the objective lens (15) with the vibration of the light beam scanning galvanometer (9).

3. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 2, characterized in that: The super-resolution focusing illumination and collection module further comprises a quarter-wave plate (13); the super-resolution optical imaging module further comprises a polarization beam splitter (19); the optical microscopic imaging module comprises a second beam splitter (33); after the linearly polarized annular focusing beam emitted from the beam scanning module passes through the quarter-wave plate (13), a circularly polarized circular focusing line having an outer diameter smaller than the diameter of the rear focal plane of the objective lens (15) is formed on the rear focal plane of the objective lens (15); the circularly polarized circular focusing line is converted into a super-resolution conical focusing beam after passing through the objective lens (15), and a super-resolution focusing light spot is formed on the front focal plane of the objective lens (15). The super-resolution focused light spot illuminates the sample (16), and the super-resolution signal light generated by the reflection and scattering of the sample (16) is collected by the objective lens (15) and respectively converged and collimated, and then converted into a linearly polarized annular focused light beam and a linearly polarized collimated light beam orthogonal to the incident line polarization through a quarter-wave plate (13), that is, a linearly polarized super-resolution signal light beam. The linearly polarized super-resolution signal light beam is transmitted through the second beam splitter (33) in the opposite direction of the original light path, passes through the beam scanning module, enters the annular focused light beam generating module, and is reflected by the polarization beam splitter (19) placed in the light path to enter the super-resolution optical imaging module; When the beam scanning galvanometer (9) in the beam scanning module performs two-dimensional scanning, the annular focused beam is converted into a conical focused beam by the objective lens, and the super-resolution focused light spot formed on the front focal plane of the objective lens (15) will follow the vibration of the beam scanning galvanometer (9), thereby realizing continuous two-dimensional scanning on the front focal plane of the objective lens (15); the horizontal coordinate X(t) and the vertical coordinate Y(t) of the position where the super-resolution focused light spot illuminates the sample (16) are functions of time t, and the reflected light E collected by the objective lens (15) r (t) and scattered light E s (t) is also a function of time, the reflected light E r (t) and scattered light E s (t) reflects the sample structure and refractive index information at the corresponding position.

4. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 2, characterized in that: The super-resolution focused light spot is an annular 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 the annular focused light field is c , which satisfies: NA c <NA; where NA represents the numerical aperture of the objective lens (15).

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

6. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 5, characterized in that: The super-resolution optical imaging module further comprises a five-dimensional adjustment frame (24), the optical fiber head (23) being mounted on the five-dimensional adjustment frame (24), and the five-dimensional adjustment frame (24) being adjusted so that the super-resolution signal light entering the optical fiber head (23) reaches a maximum; The inner diameter of the optical fiber at the front end face of the optical fiber head (23) is smaller than the first zero point radius of the focal spot of the super-resolution signal light on the rear focal plane of the second sleeve mirror (22).

7. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 1, characterized in that: The light source module comprises a laser (1) and an optical fiber collimator (3); the laser (1) is connected to the optical fiber collimator (3) via a first optical fiber jumper (2); coherent light emitted by the laser (1) is transmitted to the optical fiber collimator (3) via the first optical fiber jumper (2); the optical fiber collimator (3) collimates the coherent light and outputs a collimated laser beam; The annular focused light beam generating module further comprises a field lens (38), wherein the object side principal surface of the field lens (38) coincides with the rear focal plane of the first positive lens (6), and the image side principal surfaces of the field lens (38) coincide with the front focal planes of the second positive lens (7), so as to change the propagation direction of the edge light of the annular focused light beam.

8. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 3, characterized in that: The optical microscopic imaging module further comprises an incoherent light source (30), a collimating lens (31), a first beam splitter (32), a third tube mirror (34) and a digital camera (35). The digital camera (35) is connected to a computer. The incoherent light source (30) is located at the front focus of the collimating lens (31). Light emitted by the incoherent light source (30) is collimated by the collimating lens (31) and reflected by the first beam splitter (32). The light then passes through the second beam splitter (33), the quarter wave plate (13) and reaches the objective lens (1 5), and converges into incoherent light on the front focal plane of the objective lens (15) to illuminate the sample (16). The incoherent light generated by the reflection and scattering of the sample (16) is collected by the objective lens (15), and then passes through the quarter-wave plate (13), the second beam splitter (33), the first beam splitter (32), and the third sleeve lens (34) in sequence before entering the digital camera (35), so that the sample (16) is imaged on the digital camera (35) and sent to a computer, which displays a wide-field microscopic image of the sample (16).

9. The reflective scanning super-resolution optical microscopy system based on light cone illumination according to claim 5, characterized in that: The spatial filter (20) is a circular pass-type spatial filter, comprising a glass substrate (201) and a circular aluminum film (202) and a first circular aluminum film (203) which are concentric and cover the glass substrate, wherein the diameter of the circular aluminum film (202) is equal to 2R in , the inner diameter of the first annular aluminum film (203) is equal to 2R out , an outer diameter equal to the diameter of the glass substrate (201), the glass substrate has a transmittance of greater than 90% for light waves with a wavelength of λ, and the circular aluminum film (202) and the first annular aluminum film (203) have a transmittance of 0 for light waves with a wavelength of λ; Alternatively, the spatial filter (20) is an annular shielding type spatial filter, comprising a glass substrate (201) and a second annular aluminum film (204) covering the glass substrate, wherein the inner diameter of the second annular aluminum film is equal to 2R in , outer diameter equals 2R out , the glass substrate (201) has a transmittance greater than 90% for light waves with a wavelength of λ, and the second annular aluminum film (204) has a transmittance of 0 for light waves with a wavelength of λ; Alternatively, the spatial filter (20) is a phase plate, which is a plate with a thickness of t and inner and outer radii of R and R respectively prepared on the upper surface of a glass substrate. in and R out A spatial filter, in R in and R out The transmittance T of the light wave with wavelength λ in the ring between is 10%-20%, and its phase change relative to the air at the same height is π / 4 or -π / 4; Among them, R in Represents the inner radius of the circular focal line at the rear focal plane of the objective lens, R out Represents the outer radius of the circular focus line at the rear focal plane of the objective.

10. A reflective scanning super-resolution optical microscopy method based on light cone illumination, using the reflective scanning super-resolution optical microscopy system according to any one of claims 1 to 9, the method comprising: Step 1: Use an optical microscopy imaging module to perform wide-field imaging on the sample (16) to determine a super-resolution scanning imaging area; Step 2: after determining the super-resolution scanning imaging area, the annular focused beam generating module generates an annular focused beam, and the super-resolution focused illumination and collection module converts the annular focused beam into a super-resolution conical focused beam 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), respectively converge and collimate the reflected and scattered super-resolution light signals, and complete the polarization conversion; Step 3: The computer controls the sample displacement module to move the sample (16) to the position to be scanned, and turns on the beam scanning galvanometer (9) to scan. At the same time, the super-resolution optical imaging module obtains the super-resolution signal light after being converged, collimated and converted, and converts it into an electrical signal and inputs it into the computer; Step 4: The computer processes the input electrical signal and combines the coordinates X(t) and Y(t) of the super-resolution illumination focal spot moving on the sample (16) and the signal light intensity I(t) obtained by the photomultiplier tube (26) at the corresponding time t to form a super-resolution microscopic image I(X(t), Y(t)) of the sample in the super-resolution scanning imaging area.

Images