Reflection-type label-free super-resolution optical microscopic system and method

Abstract

The invention relates to a reflective non-labeled super-resolution optical microscopic system and method. The reflective non-labeled super-resolution optical microscopic system comprises a large-numerical-aperture conical super-resolution focusing module, a super-resolution focusing light field illumination and collection module, a sample and mechanical scanning module, a super-resolution imaging module and a wide-field microscopic imaging module, the characteristics of super-resolution focal spot, super-long focal depth, high radial spatial frequency, narrow frequency passband and the like of a large-numerical-aperture conical super-resolution light beam are utilized, projection of a super-resolution light field on front focal planes of two objective lenses is realized by adopting a 4f system, and a marking-free reflective far-field super-resolution optical microscopic system and method are realized by combining a confocal working principle; non-marked far-field super-resolution two-dimensional microscopic imaging is realized through two-dimensional scanning; and performing non-label far-field super-resolution two-dimensional microscopic imaging on object planes at different axial positions to obtain super-resolution two-dimensional microscopic imaging of different cross sections of the sample, thereby realizing three-dimensional chromatography. According to the invention, non-label, super-resolution and tomography of the sample can be realized.

Description

Technical Field

[0001] The present invention belongs to the related fields of optical microscopy, optical processing technology, etc., and particularly relates to a reflective non-marking super-resolution optical microscopy system and method. 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. The near-field microscopy method 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 the advantage of 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 all require fluorescent molecular labeling of the samples being tested. Label-free far-field super-resolution microscopy technology has the advantages of a large working distance, no need to label samples, and is easy to use. Therefore, it has become an important development trend of super-resolution optical microscopy technology.

[0003] Technical background references:

[0004] [1] Edward TF Rogers, Jari Lindberg, Tapashree Roy, Salvatore Savo, John E. Chad, Mark R. Dennis and Nikolay I. Zheludev. "A super-oscillatory lens opticalmicroscope for subwavelength imaging," Nature Materials, Vol. 11, pp. 432-435 (2012).

[0005] [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).

[0006] [3]Y Yan, L Li, C Feng, W Guo, S Lee, M Hong. "Micronosphere-Coupled ScanningLaser Confocal Nanoscope for Sub-Diffraction-Limited Imaging at 25nm LateralResolution in the Visible Spectrum," ACS Nano, Vol.8, pp.1809-1816 (2014).

[0007] [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).

[0008] [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 EngineeR1g, Vol. 168, pp. 107690 (2023). Summary of the invention

[0009] The purpose of the present invention is to provide a reflective non-labeling super-resolution optical microscopy system and method to realize a reflective far-field super-resolution optical microscopy without labeling, and to realize three-dimensional tomographic imaging of samples with a lateral resolution less than the diffraction limit.

[0010] In a first aspect, a reflective non-labeling super-resolution optical microscopy system according to the present invention comprises a large numerical aperture conical super-resolution focusing module, a super-resolution focusing light field illumination and collection module, a sample and mechanical scanning module, a super-resolution imaging module and a wide-field microscopy imaging module arranged along an optical path;

[0011] The large numerical aperture conical super-resolution focusing module is used to generate a P-polarized conical super-resolution focusing light field; the large numerical aperture conical super-resolution focusing module comprises a laser, a fiber collimator, a linear polarizer, a first conical lens, a second conical lens and a super-resolution conical beam focusing lens which are sequentially arranged along the optical path, and the laser is connected to the fiber collimator via a first fiber jumper; coherent light with a wavelength of λ emitted by the laser sequentially passes through the fiber collimator, the linear polarizer, the first conical lens, the second conical lens and the super-resolution conical beam focusing lens to form a P-polarized conical super-resolution focusing light field; within the axial range of the P-polarized conical super-resolution focusing light field, the size of the super-resolution focusing light spot has a lateral full width at half maximum (FWHM) <0.5λ / NA a , where NA a is the numerical aperture of the super-resolution cone-shaped beam focusing lens, and NA a =n w sin(θ a ), where n w is the refractive index of the medium at the output end of the super-resolution cone beam focusing lens, sin(·) is a sine function, and the radius of the first zero point is less than 0.61λ / NA a , the first zero point radius is the distance from the focal spot intensity peak position to the first zero point position;

[0012] The super-resolution focused light field illumination and collection module is used to project the P-polarized conical super-resolution focused light field onto the surface of the sample to be detected, and to collect the scattered light and scattered light of the sample to be detected;

[0013] The sample and mechanical scanning module is used to place the sample to be tested and drive the movement of the sample to be tested;

[0014] The super-resolution imaging module is used to collect super-resolution signal light, perform photoelectric conversion and obtain super-resolution images of reflected light and scattered light;

[0015] The wide-field microscopic imaging module is used to realize wide-field microscopic imaging of the detected sample and to observe the detected sample.

[0016] Optionally, the super-resolution focused light field illumination and collection module comprises a first objective lens, a polarization beam splitter, a first quarter-wave plate, a first tube lens, a reflector, a second quarter-wave plate, an axial nanopositioner and a second objective lens; the front focal plane of the first objective lens is located within the axial range of the P-polarization conical super-resolution focused light field; after the P-polarization conical super-resolution focused light field passes through the first objective lens, an annular focused light spot is formed on the rear focal plane of the first objective lens; the first tube lens and the reflector constitute a set of reflective 4f systems, the front focal plane of the first tube lens coincides with and is coaxial with the rear focal plane of the first objective lens; the reflective surface of the reflector is located on the rear focal plane of the first tube lens, and the front focal plane of the first tube lens is reflected by the polarization beam splitter and coincides with the rear focal plane of the second objective lens. The planes coincide; the conical P-polarization super-resolution focusing light field generated by the large numerical aperture conical super-resolution focusing module is received by the first objective lens, transmitted through the polarization beam splitter, converted into circularly polarized light through the first quarter-wave plate, passed through the first tube lens and reflected by the reflector, the reflected circularly polarized light is orthogonal to the incident circularly polarized light (that is, if the incident is left-handed circularly polarized light, it is right-handed circularly polarized light after reflection, and vice versa), and then successively passed through the first tube lens and the first quarter-wave plate to be converted into S linear polarized light, the S linear polarized light is reflected by the polarization beam splitter, and then passed through the second quarter-wave plate to convert the light beam into circularly polarized light (with the same polarization direction as the circularly polarized light reflected by the reflector) and then entered the second objective lens. Due to the effect of the reflective 4f system, the annular focused light spot on the rear focal plane of the first objective lens is projected onto the rear focal plane of the second objective lens. The corresponding light field forms a circularly polarized (with the same polarization direction as the circularly polarized light reflected by the reflector) conical super-resolution focused light field after passing through the second objective lens, and generates a focused light spot with a lateral size smaller than the diffraction limit along the optical axis before and after the focal plane of the second objective lens. The focal depth is DOF ​​= Rctan(θ a )=(R out -R in )ctan(θ a ), where ctan(·) is the cotangent function, R is the radius of the optical fiber collimator output beam, and the lateral full width at half maximum (FWHM) of the focused spot is <0.5λ / NA a , the first zero point radius is less than 0.61λ / NA a , whose spatial frequency is only distributed in the radial spatial frequency F a Nearby, F a =NA a / λ, radial spatial frequency F a The full width at half maximum (FWHM) corresponding to the peak amplitude F <F a / 10.

[0017] Optionally, the optical distance from the rear focal plane of the first objective lens to the first tube lens is equal to the optical distance from the rear focal plane of the second objective lens to the first tube lens.

[0018] Optionally, the sample and mechanical scanning module comprises a two-dimensional nanometer scanning stage, on which the sample to be detected is horizontally fixed, and the two-dimensional nanometer scanning stage is controlled to move two-dimensionally in the XY plane, thereby driving the sample to be detected to move two-dimensionally;

[0019] When the sample and mechanical scanning module are two-dimensional nano-scanning platforms, the super-resolution focusing illumination and collection module also includes an axial nano-positioner, and the second objective lens is mounted on the axial nano-positioner. The axial movement of the second objective lens is achieved by controlling the axial nano-positioner. When in use, the two-dimensional nano-scanning platform is connected to a computer, and the computer controls the two-dimensional nano-scanning platform to drive the sample to be tested to move in the X and Y directions. At the same time, the axial nano-positioner is also connected to the computer through a signal line, and the computer controls the axial nano-positioner to drive the second objective lens to move in the Z direction.

[0020] Alternatively, the sample scanning module is a three-dimensional nanometer scanning platform, on which the sample to be tested is horizontally fixed, and the three-dimensional nanometer scanning platform is controlled to move in three directions, namely, X, Y, and Z, so as to drive the sample to be tested to move in three dimensions. When in use, the three-dimensional nanometer scanning platform is connected to a computer, and the computer controls the three-dimensional nanometer scanning platform to drive the sample to be tested to move in three directions, namely, X, Y, and Z.

[0021] Optionally, the super-resolution imaging module includes a first lens, a second lens, a spatial filter, a second tube lens, a beam splitter, an optical fiber head, a photomultiplier tube and a high-speed acquisition card; the first lens and the second lens constitute a 4f system, the rear focal plane of the first lens and the front focal plane of the second lens coincide and are coaxial; the front focal plane of the first lens coincides with the rear focal plane of the second objective lens; the rear focal plane of the second lens coincides with the spatial filter and is coaxial; the front end face of the optical fiber head is located on the rear focal plane of the second tube lens; the optical fiber head is connected to the photomultiplier tube, and the photomultiplier tube is connected to the high-speed acquisition card. The 4f system composed of the first lens and the second lens transmits the light field on the rear focal plane of the second objective lens to the spatial filter. The light field contains the reflected light and scattered light of the detected sample. After passing through the second tube lens, the reflected light is focused into a conical focused light beam and forms a focused light field on the rear focal plane of the second tube lens. After passing through the second tube lens, the scattered light also forms a focused light field on the rear focal plane of the second tube lens. The reflected light and the scattered light are reflected by the beam splitter to reach the end face of the optical fiber head and are coupled into the optical fiber head. The optical fiber jumper inputs the optical signal into the photomultiplier tube, and the photomultiplier tube converts the super-resolution signal light into an electrical signal. The electrical signal is collected and converted by the high-speed acquisition card and becomes a digital signal.

[0022] Optionally, the super-resolution imaging module further comprises a five-dimensional adjustment frame, and the optical fiber head is mounted on the five-dimensional adjustment frame. When there is no sample to be detected, the five-dimensional adjustment frame can be adjusted to maximize the super-resolution signal light entering the optical fiber head.

[0023] Optionally, the wide-field microscopic imaging module includes an incoherent light source, a collimating lens, a cubic beam splitter and a digital camera, the target surface of the digital camera is located at the rear focal plane of the second tube lens; the incoherent light emitted by the incoherent light source is collimated by the collimating lens and reflected by the cubic beam splitter, and then passes through the spatial filter, the second lens, the first lens, the polarization beam splitter, the second quarter-wave plate, and the second objective lens in sequence, and then forms a focused light field behind the second objective lens and performs incoherent illumination on the sample to be detected; the incoherent reflected light and scattered light generated by the sample to be detected are collected by the second objective lens, and then pass through the second quarter-wave plate, the polarization beam splitter, the first lens, the second lens, the spatial filter, the cubic beam splitter, the second tube lens and the beam splitter in sequence, and then forms a wide-field microscopic light field of the sample to be detected at the rear focal plane of the second tube lens, and the wide-field microscopic light field is acquired by the digital camera to form a wide-field microscopic image of the sample to be detected.

[0024] Optionally, in the bright field mode, the spatial filter is an aluminum film pattern with a thickness of t>50nm made on a transparent glass substrate; the spatial filter has a radius smaller than the outer radius R out and is larger than the inner radius R in No aluminum film is provided within the range, and the light wave transmittance of the portion without the aluminum film is greater than 90%; the light wave transmittance of the aluminum film pattern portion is 0, and only the ring-shaped light beam reflected from the sample to be tested is allowed to pass, that is, the bright field imaging light signal is allowed to pass;

[0025] In the dark field mode, the spatial filter is an aluminum film pattern with a thickness of t>50nm made on a transparent glass substrate; the outer radius of the aluminum film pattern is R out And the inner radius is R in The light wave transmittance of the aluminum film part is 0, and the light wave transmittance of the part without the aluminum film is greater than 90%, allowing only the light scattered back from the sample to pass, that is, allowing the dark field imaging light signal to pass;

[0026] In the phase contrast mode, the spatial filter is a phase contrast plate, which includes a glass substrate and an annular dielectric film covering the glass substrate. The annular dielectric film is a π phase ring. The phase change of the π phase ring relative to other regions is π, and the inner diameter of the π phase ring is equal to 2R in , outer diameter equals 2R out The glass substrate has a transmittance of greater than 90% for light waves with a wavelength of λ, and the π phase ring has a transmittance of 10% to 20% for light waves with a wavelength of λ.

[0027] In a second aspect, a reflective label-free super-resolution optical microscopy method according to the present invention uses a reflective label-free super-resolution optical microscopy system according to the present invention, and the method comprises the following steps:

[0028] The sample to be tested is arranged on the sample and mechanical scanning module;

[0029] Generate a P-polarized conical super-resolution focusing light field through a large numerical aperture conical super-resolution focusing module;

[0030] The P-polarized conical super-resolution focusing light field is projected onto the surface of the sample to be inspected through the super-resolution focusing light field illumination and collection module, and the reflected light passing through the sample to be inspected and the scattered light caused by the sample to be inspected are collected;

[0031] The collected reflected light and scattered light are subjected to super-resolution signal light collection, photoelectric conversion and super-resolution image acquisition through the super-resolution imaging module;

[0032] The wide-field microscopic imaging module is used to perform wide-field microscopic imaging of the sample to determine the super-resolution scanning imaging area.

[0033] Optionally, the method further includes: the coherent light emitted by the laser is transmitted to the optical fiber collimator through the first optical fiber jumper, and the optical fiber collimator collimates the light beam and outputs it; the collimated light beam passes through a linear polarizer to form a P linearly polarized collimated light beam; the P linearly polarized collimated light beam passes through a first cone lens and a second cone lens to form an annular light beam; the annular light beam passes through a super-resolution cone beam focusing lens to form a P polarized cone super-resolution focusing light field;

[0034] The difference between the inner and outer diameters of the annular beam is changed by changing the numerical aperture of the fiber collimator; the diameter of the annular beam is changed by changing the distance between the first conical lens and the second conical lens to ensure that the effective working area of ​​the super-resolution conical beam focusing lens is efficiently illuminated.

[0035] The present invention has the following advantages: the present invention makes full use of the super-resolution focal spot and ultra-long focal depth (Rctan (θ a )>2n w λ / NA a 2 ,n w The refractive index of the medium at the output end of the super-resolution cone beam focusing lens), high radial spatial frequency (greater than 0.9NA o ,NA o is the numerical aperture of the objective lens), the frequency passband is narrow (the full width at half maximum FWHM of the frequency band F <F a / 10) and other characteristics, combined with the confocal (illumination lens and collection lens confocal) working principle, a reflective far-field super-resolution optical microscopy system and method without marking is realized; and it can realize three-dimensional tomography of samples, with a lateral resolution less than the diffraction limit and a resolution better than 0.5λ / (NA o +NA a ), where NAo is the numerical aperture of the objective lens. The system can achieve non-labeling, super-resolution, and tomographic imaging of samples, and can be applied to the detection of biological samples and non-biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 A schematic diagram of the structure of a reflective non-labeling super-resolution optical microscopy system in one embodiment of the present application;

[0037] Figure 2 A schematic diagram of the structure of a reflective label-free super-resolution optical microscopy system in another embodiment of the present application;

[0038] Figure 3 This is a working principle diagram of a super-resolution cone-beam focusing lens in an embodiment of the present application;

[0039] Figure 4 Schematic diagram of a grating-type super-resolution cone-shaped beam focusing lens in an embodiment of the present application;

[0040] Figure 5 A schematic diagram of a metasurface-type super-resolution cone-shaped beam focusing lens in an embodiment of the present application;

[0041] Figure 6 A schematic diagram of the arrangement of super units in an embodiment of the present application;

[0042] Figure 7 for Figure 6 A partial schematic diagram of the middle supercell;

[0043] Figure 8 This is another schematic diagram of the arrangement of super units in the embodiment of the present application;

[0044] Fig. 9 Schematic diagram of the relationship between the focus spot parameters and the numerical aperture in the embodiment of the present application;

[0045] Fig.10 This is a schematic diagram of the principle of generating a P-polarized conical super-resolution focusing light field in an embodiment of the present application;

[0046] Fig.11 This is a schematic diagram of super-resolution illumination for super-resolution microscopy in an embodiment of the present application;

[0047] Fig.12 This is a schematic diagram of optical signal collection in an embodiment of the present application;

[0048] Fig.13 Schematic diagram of three modes of the spatial filter in the embodiment of the present application;

[0049] Fig.14is a graph showing the relationship between the full width at half maximum of the objective lens and the illumination lens and the system resolution under the condition of coherent light illumination in the embodiment of the present application;

[0050] In the figure: 1. laser, 2. first fiber jumper, 3. fiber collimator, 4. linear polarizer, 5. first conical lens, 6. second conical lens, 7. super-resolution conical beam focusing lens, 8. first objective lens, 9. polarization beam splitter, 10. first quarter wave plate, 11. first tube lens, 12. reflector, 13. second quarter wave plate, 14. axial nanopositioner, 15. second objective lens, 16. sample to be detected, 17. first lens, 18. second lens, 19. spatial filter, 20. second tube lens, 21. beam splitter, 22. fiber head, 23. five-dimensional adjustment frame, 24. second fiber jumper, 25. photomultiplier tube, 26. signal line, 27. high-speed acquisition card, 28. two-dimensional nano scanning stage, 29. incoherent light source, 30. collimating lens, 31. cubic beam splitter, 32. digital camera, 33. three-dimensional nano scanning stage. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0052] like Figure 1 As shown, in an embodiment of the present application, a reflective non-labeling super-resolution optical microscopy system includes a large numerical aperture conical super-resolution focusing module arranged along the optical path, a super-resolution focusing light field illumination and collection module, a sample and mechanical scanning module, a super-resolution imaging module and a wide-field microscopy imaging module.

[0053] The following is a detailed description of each module:

[0054] like Figure 1 As shown, in an embodiment of the present application, a large numerical aperture conical super-resolution focusing module is used to generate a P-polarized conical super-resolution focusing light field. The large numerical aperture conical super-resolution focusing module includes a laser 1, a fiber collimator 3, a linear polarizer 4, a first conical lens 5, a second conical lens 6 and a super-resolution conical beam focusing lens 7. The laser 1 is connected to the fiber collimator 3 through a first fiber jumper 2. The coherent light with a wavelength of λ emitted by the laser 1 is transmitted to the fiber collimator 3 through the first fiber jumper 2, and forms a collimated light beam after passing through the fiber collimator 3. The collimated light beam forms a P-linearly polarized collimated light beam through the linear polarizer 4. The P-linearly polarized collimated light beam forms an annular light beam after passing through the first conical lens 5 and the second conical lens 6. The annular light beam forms a P-polarized conical super-resolution focusing light field through the super-resolution conical beam focusing lens 7. Within the axial range of the P-polarized conical super-resolution focusing light field, the size of the super-resolution focusing spot has a lateral full width at half maximum FWHM<0.5λ / NA a , where NA ais the numerical aperture of the super-resolution cone-shaped beam focusing lens 7, and the corresponding super-resolution focusing light field cone angle is θ a , and there are NA a =n w sin(θ a ), n w is the medium refractive index at the output end of the super-resolution cone beam focusing lens 7, and the first zero point radius is less than 0.61λ / NA a , where the first zero point radius is the distance from the focal spot intensity peak position to the first zero point position.

[0055] like Figure 1 , Figure 2 and Figure 3 As shown, in the embodiment of the present application, the super-resolution focusing light field illumination and collection module is used to project the P-polarized conical super-resolution focusing light field formed by the super-resolution conical beam focusing lens 7 onto the surface of the sample 16 to be detected, and collect the reflected light (circularly polarized reflected light) and scattered light (circularly polarized scattered light) of the sample 16 to be detected. The super-resolution focusing light field illumination and collection module includes a first objective lens 8, a polarization beam splitter (P linear polarization transmission, S polarization reflection) 9, a first quarter-wave plate 10, a first tube lens 11, a reflector 12, a second quarter-wave plate 13 and a second objective lens 15. Among them, the front focal plane of the first objective lens 8 is located within the axial range of the P-polarized conical super-resolution focusing light field, and within this range, the size of the super-resolution focusing spot has a lateral full width at half maximum FWHM <0.5λ / NA a , the first zero point radius is less than 0.61λ / NA a. After the P-polarized conical super-resolution focusing light field is received by the first objective lens 8, a ring-shaped focusing spot is formed on the rear focal plane of the first objective lens 8. The first tube lens 11 and the reflector 12 constitute a set of reflective 4f systems, and the front focal plane of the first tube lens 11 coincides with and is coaxial with the rear focal plane of the first objective lens 8. The reflector 12 is perpendicular to its corresponding optical axis, and the reflection surface of the reflector 12 is located at the rear focal plane of the first tube lens 11. The front focal plane of the first tube lens 11 coincides with the rear focal plane of the second objective lens 15 after being reflected by the polarization beam splitter 9. The P-polarized conical super-resolution focusing light field generated by the large numerical aperture conical super-resolution focusing module is transmitted through the polarization beam splitter 9, converted into circularly polarized light through the first quarter-wave plate 10, and is reflected by the reflector 12 after passing through the first tube lens 11. The reflected circularly polarized light is orthogonal to the incident circularly polarized light (that is, if the incident light is left-handed circularly polarized light, it is right-handed circularly polarized light after reflection, and vice versa). Then, it is converted into S linearly polarized light through the first tube lens 11 and the first quarter-wave plate 10 in sequence. The S linearly polarized light is reflected by the polarization beam splitter 9 and then converted into circularly polarized light through the second quarter-wave plate 13 (with the same polarization direction as the circularly polarized light reflected by the reflector 12) before entering the second objective lens 15. Due to the effect of the reflective 4f system, the annular focused light spot on the rear focal plane of the first objective lens 8 is projected onto the rear focal plane of the second objective lens 15. The corresponding light field forms a circularly polarized (with the same polarization direction as the circularly polarized light reflected by the reflector 12) conical super-resolution focused light field after passing through the second objective lens 15, and generates a focused light spot with a lateral size smaller than the diffraction limit along the optical axis before and after the focal plane of the second objective lens 15, and its focal depth is Rctan(θ a )=(R out -R in )ctan(θ a )(where ctan(·) is the cotangent function, R is the radius of the light beam emitted by the fiber collimator 3), the lateral full width at half maximum FWHM of the focused spot is <0.5λ / NA a , the first zero point radius is less than 0.61λ / NA a , whose spatial frequency is only distributed in the radial spatial frequency F a =NA a / λ, the frequency F a The full width at half maximum (FWHM) corresponding to the peak amplitude F <F a / 10. The sample 16 to be tested is placed on the front focal plane of the second objective lens 15. All geometrical parameters and optical parameters of the first objective lens 8 and the second objective lens 15 are exactly the same.

[0056] In the embodiments of the present application, left-handed circularly polarized light and right-handed circularly polarized light are collectively referred to as circularly polarized light.

[0057] In the embodiment of the present application, the first objective lens 8 includes microscope objective lenses with different magnifications and numerical apertures, and a traditional microscope objective lens or a super-diffraction lens can be used, and a dry lens, a water lens, an oil lens or a solid lens can be used.

[0058] like Figure 1 As shown, in the embodiment of the present application, the sample and mechanical scanning module is used to load the sample 16 to be detected, and realize the two-dimensional movement of the sample 16 to be detected in the XY plane, and cooperate with the super-resolution imaging module to scan and image the sample 16 to be detected point by point. The sample and mechanical scanning module includes a two-dimensional nano scanning stage 28, and the sample 16 to be detected is horizontally fixed on the two-dimensional nano scanning stage 28. By controlling the two-dimensional nano scanning stage 28 to move two-dimensionally in the XY plane, the sample 16 to be detected is driven to move two-dimensionally. When the sample and mechanical scanning module is a two-dimensional nano scanning stage, the super-resolution focusing illumination and collection module also includes an axial nano positioner 14, and the second objective lens 15 is installed on the axial nano positioner 14. The axial movement of the second objective lens 15 is realized by controlling the axial nano positioner 14. When in use, the two-dimensional nano scanning stage 28 is connected to a computer, and the computer controls the two-dimensional nano scanning stage 28 to drive the sample 16 to be detected to move in the X and Y directions. At the same time, the axial nano positioner 14 is also connected to the computer through a signal line, and the computer controls the axial nano positioner 14 to drive the second objective lens 15 to move in the Z direction.

[0059] The sample 16 to be tested needs to be placed on the focal plane of the second objective lens 15 to form a conical super-resolution beam. By synchronously controlling the movement of the sample 16 to be tested on the XY plane and the signal acquisition of the super-resolution imaging module, a functional relationship I(x, y) is established between the spatial coordinates x and y on the XY plane and the synchronously acquired signal intensity I. This function is the super-resolution optical microscopic image of the sample 16 to be tested, and its resolution is less than the diffraction limit.

[0060] In the embodiment of the present application, the axial position Z of the focus of the second objective lens 15 in the sample is changed by the axial nanopositioner 14. f , get different Z f A three-dimensional tomographic image can be obtained by using a two-dimensional super-resolution image in the XY plane at a certain position, and its resolution in the XY plane is less than the diffraction limit.

[0061] like Figure 2 As shown, in a possible embodiment, the sample scanning module is a three-dimensional nanometer scanning platform 33, and the sample 16 to be tested is horizontally fixed on the three-dimensional nanometer scanning platform 33. By controlling the three-dimensional nanometer scanning platform 33 to move in the three directions of X, Y, and Z, the sample 16 to be tested is driven to move three-dimensionally. When in use, the three-dimensional nanometer scanning platform 33 is connected to a computer, and the computer controls the three-dimensional nanometer scanning platform 33 to drive the sample 16 to be tested to move in the three directions of X, Y, and Z.

[0062] like Figure 1 As shown, in the embodiment of the present application, the super-resolution imaging module is used to collect the reflected light of the detected sample 16 and the scattered light caused by the detected sample 16 into the super-resolution imaging module under the illumination of the sample by the conical super-resolution beam, and perform super-resolution signal light collection, photoelectric conversion and super-resolution image acquisition. The super-resolution imaging module includes a first lens 17, a second lens 18, a spatial filter 19, a second tube lens 20, a beam splitter 21, an optical fiber head 22, a five-dimensional adjustment frame 23, a second optical fiber jumper 24, a photomultiplier tube 25, a signal line 26 and a high-speed acquisition card 27. The first lens 17 and the second lens 18 constitute a 4f system. The rear focal plane of the first lens 17 and the front focal plane of the second lens 18 coincide and are coaxial. The front focal plane of the first lens 17 coincides with the rear focal plane of the second objective lens 15. The rear focal plane of the second lens 18 coincides with the spatial filter 19 and is coaxial. The front end face of the optical fiber head 22 is located on the rear focal plane of the second tube lens 20. The optical fiber head 22 is connected to the photomultiplier tube 25 through the second optical fiber jumper 24, and the photomultiplier tube 25 is connected to the high-speed acquisition card 27 through the signal line 26. The 4f system composed of the first lens 17 and the second lens 18 transmits the light field on the rear focal plane of the second objective lens 15 to the spatial filter 19, and the light field contains the reflected light and scattered light of the sample 16 to be detected. After passing through the second tube lens 20, the reflected light is focused into a conical focused light beam and forms a focused light field at the rear focal plane of the second tube lens 20; after passing through the second tube lens 20, the scattered light also forms a focused light field at the rear focal plane of the second tube lens 20. The reflected light and scattered light are reflected by the beam splitter 21 to reach the end face of the optical fiber head 22 and coupled into the optical fiber head 22 (the inner diameter of the optical fiber at the front end face of the optical fiber head 22 is smaller than the first zero point radius of the focal spot of the super-resolution signal light); when there is no sample 16 to be detected, a reflection plane is placed on the front focal plane of the second objective lens 15 and is perpendicular to the optical axis, and the laser 1 is operated (the non-correlated light source 29 is turned off), and the light entering the optical fiber head 22 is maximized by adjusting the five-dimensional adjustment frame 23. The optical fiber jumper 24 inputs the optical signal into the photomultiplier tube 25, and the photomultiplier tube 25 converts the super-resolution signal light into an electrical signal; the electrical signal is collected and converted by the high-speed acquisition card 27 through the signal line 26 and becomes a digital signal, and is sent to the computer (not shown in the figure) by the data line.

[0063] In the embodiment of the present application, the second objective lens 15 includes microscope lenses with different magnifications and numerical apertures. A traditional microscope lens or a super-diffraction lens can be used. A dry lens, a water lens, an oil lens or a solid lens can be used, and the second objective lens 15 has the same numerical aperture as the first objective lens 8.

[0064] like Figure 1As shown, in the embodiment of the present application, the wide-field microscopic imaging module is used to realize wide-field microscopic imaging of the sample 16 to be detected, and is used for observing the sample 16 to be detected. The wide-field microscopic imaging module includes an incoherent light source 29, a collimating lens 30, a cubic beam splitter 31 and a digital camera 32, and the target surface of the digital camera 32 is located at the rear focal plane of the second tube lens 20. The incoherent light emitted by the incoherent light source 29 is collimated by the collimating lens 30, reflected by the cubic beam splitter 31, and passes through the spatial filter 19, the second lens 18, the first lens 17, the polarization beam splitter 9, the second quarter-wave plate 13 and the second objective lens 15 in sequence, and forms a focused light field after the second objective lens 15 and performs incoherent illumination on the sample 16 to be detected. After the incoherent reflected light and scattered light generated by the detected sample 16 are collected by the second objective lens 15, they pass through the second quarter wave plate 13, the polarization beam splitter 9, the first lens 17, the second lens 18, the spatial filter 19, the cubic beam splitter 31, the second tube lens 20 and the beam splitter 21 in sequence, and form a wide-field microscopic light field of the detected sample 16 at the rear focal plane of the second tube lens 20. The wide-field microscopic light field is acquired by the digital camera 32 to form a wide-field microscopic image of the detected sample 16, and enters the computer through the camera signal line, and is displayed in real time on the computer. In addition, the detected sample 16 can be observed in real time in a wide field to facilitate the determination of the required super-resolution scanning area. The wide-field microscopic image field of the detected sample 16 is acquired by the digital camera 28 and enters the computer through the camera signal line.

[0065] In the embodiment of the present application, there are many different implementation methods of the super-resolution cone-beam focusing lens 7. The implementation methods are described below by taking a grating-type super-resolution cone-beam focusing lens and a metasurface-type super-resolution cone-beam focusing lens as examples.

[0066] like Figure 4 As shown, the grating type super-resolution cone beam focusing lens 7 is on a glass substrate with O as the center and an inner radius R in and outer radius R out The concentric annular grating is made in the annular region. The period of the grating is T 1 Should meet T 1 =λ / n w sin(θ a ). The duty cycle of the grating is 50% (ie the ratio between the transparent area and the grating period).

[0067] In a possible embodiment, the grating may be an amplitude grating or a phase grating.

[0068] If an amplitude type grating is used, the black part of the amplitude type grating is opaque (transmittance is 0), and the white part of the amplitude type grating is completely transparent (transmittance is (n s -n w )2 / (n s +n w ) 2 , where n s is the refractive index of the glass substrate, n w n w is the refractive index of the medium at the output end of the super-resolution cone beam focusing lens). The opaque part can be realized by a metal layer with a thickness of t. For example, when aluminum is used, when the thickness t = 50nm, the transmittance can be 0. If a phase-type grating is used, the grating can be realized by a medium. The black part of the phase-type grating is a medium with a thickness of t, and t should satisfy t = 0.5λ / (n g -n w ), where n g is the refractive index of the grating dielectric material.

[0069] like Figure 5 As shown, in a possible embodiment, the super-resolution cone-shaped beam focusing lens 7 is a meta-structured surface type, that is, a super-resolution cone-shaped beam focusing lens 7 is formed on a glass substrate with O as the center and an inner radius R in and outer radius R out The annular area is divided into concentric rings (the number of rings in the figure is only for reference and does not mean that there are only three rings), and the width of each ring is T 2 Among them, the center radius of the i-th ring is R i =R in +(i-0.5)T 2 , 1≤i≤M, M is the number of rings, the phase value in the i-th ring Where × is a multiplication sign, m is an integer, and the value of m is chosen so that m is an integer. The phase is realized by using a geometric phase all-medium metasurface. The rotation angle corresponding to the geometric phase all-medium metasurface in the i-th ring is (Angle with the x-axis in the plane on the substrate).

[0070] like Figure 7 As shown in a possible embodiment, a metastructure unit structure of a geometric phase all-dielectric metasurface is given, which is an all-dielectric cube prepared on a glass substrate with a thickness of t, and its length, width and height are L respectively. f , L s and H.

[0071] like Figure 6 As shown, the first arrangement of superstructure units is given, specifically:

[0072] The surface of the super-resolution cone beam focusing lens 7 is divided into K×L square areas of equal size (such as Figure 6The area divided by the dotted line in the middle), the size of each square area is T 2 ×T 2 .

[0073] For the square area N kl , where N kl represents the square area of ​​the kth row and lth column, where k = 1, 2, ..., K, l = 1, 2, ..., L, K represents the total number of rows, and L represents the total number of columns. Square area N kl The distance from the center of the lens to the center of the lens surface O is r kl , if R in +(i-0.5)T 2 ≤r kl <R in +(i+0.5)T 2 , that is, the square area N kl The center of is located in the i-th ring zone, then in this square area N kl A full dielectric cube is set at the center, and the angle between the length direction of the full dielectric cube and the x-axis is γ kl Should meet ( m is an integer whose value is such that ).

[0074] In a possible embodiment, a metastructure unit structure of a geometric phase all-dielectric metasurface is given, which is an all-dielectric cube prepared on a glass substrate with a thickness of t, and its length, width and height are L, respectively. f , L s and H, the unit size is T 2 ×T 2 , where T 2 <λ. The extinction coefficient of the glass substrate is less than 0.01 at the working wavelength λ; the material of the all-dielectric cube can be TiO 2 (titanium dioxide), a-Si (amorphous silicon), and Si (silicon) and other materials can be selected in the infrared band. When left (right) circularly polarized light is incident along the Z direction, the outgoing right (left) circularly polarized light will produce an additional phase Where γ is the angle between the length of the cube and the x-axis. f , L s , H and T 2 , the cross circular polarization transmittance can reach more than 90%.

[0075] like Figure 8 As shown in the figure, a second arrangement of superstructure units is given, specifically:

[0076] Each annular zone is divided into N parts along its circumference.i There are equally spaced regions A, N i =m i or N i =m i +1, m i = 2πR i / T 2 The integer part of . A full dielectric cube (whose length, width and height are L f , L s and H), and the angle between the length direction of the full dielectric cube and the x-axis is γ ij Should meet ( Where × is a multiplication sign and m is an integer whose value is such that ), where j = 1, 2, ..., N i .

[0077] like Fig. 9 As shown in the figure, the numerical aperture NA of the super-resolution cone-shaped beam focusing lens 7 is given. a =0.95, the corresponding focusing light field parameters FWHM (full width at half maximum), r 0 (first zero point radius) and SR (sidelobe peak ratio, i.e. the ratio of the maximum sidelobe intensity to the center intensity) and NA a =n w sin(θ a ), where sin(·) is the sine function. It can be seen that when NA a When it is greater than 0.9, the FWHM of the generated focused light field is less than 0.388λ (less than 0.5λ / NA a ), r 0 <0.415(less than 0.61λ / NA a ), SR < 0.162, the half-maximum width and the first zero point radius of the focused light field are both smaller than the half-maximum width of the focused light field of the traditional lens 0.5λ / NA a and the first zero point radius 0.61λ / NA a . This shows that the system has higher focusing accuracy and resolution.

[0078] like Fig.10 As shown in FIG. 1 , a method for generating a large numerical aperture conical coherent illumination beam is given. A P linearly polarized collimated beam with a radius R is formed by passing through a first conical lens 5 and a second conical lens 6 coaxially placed with a spacing d to form a conical beam with an inner radius and an outer radius of R respectively. in and R out The inner radius R of the annular beam can be changed by changing the distance d. in and outer radius R out, and keep the ring width of the annular beam unchanged, that is, the ring width is W = R out -R in = R; the annular beam vertically illuminates the coaxially placed super-resolution cone beam focusing lens 7, whose numerical aperture is NA a =n w sin(θ a ). After the annular beam passes through the super-resolution cone beam focusing lens 7, at its focal length f c A converging conical beam is formed at the point, and a P-polarized conical super-resolution focusing light field is formed on the optical axis.

[0079] like Fig.11 As shown, a schematic diagram of the projection of the super-resolution light field is given. The P-polarized conical super-resolution focusing light field is converged by the first objective lens 8 on its rear focal plane and forms an annular focusing light spot; the light field of the annular focusing light spot is converted into left-handed circularly polarized light (right-handed circularly polarized light) after passing through the polarization beam splitter 9 and the first quarter-wave plate 10, and the light beam is further converged by the first tube lens 11 to form an annular light beam, which is reflected by the reflector 12 located on the focal plane of the first tube lens 11 to form a right-handed circularly polarized light (left-handed circularly polarized light) annular light beam, which is coaxial with the optical axis. The light beam is further converged by the first tube lens 11 to form an annular focused light beam, and is converted into an S-polarized annular focused light beam through the first quarter-wave plate 10; after the S-polarized annular focused light beam is reflected by the polarization beam splitter 9, it is converted into right-handed circularly polarized light (left-handed circularly polarized light) through the second quarter-wave plate 13, and forms an annular focused light spot on the rear focal plane of the second objective lens 15, and the shape and size of the annular focused light spot are consistent with the annular focused light spot on the rear focal plane of the first objective lens 8; finally, a super-resolution conical focused light beam of right-handed circularly polarized light (left-handed circularly polarized light) is formed on the front focal plane of the second objective lens 15, and the lateral full width at half maximum FWHM of the focused light spot is <0.5λ / NA a , the first zero point radius is less than 0.61λ / NA a , whose spatial frequency is only distributed in the radial spatial frequency F a =NA a / λ, the frequency F a The full width at half maximum (FWHM) corresponding to the peak amplitude F <F a / 10. Fig.11 C1 in : represents the back focal plane of the first objective lens 8. C2 represents the back focal plane of the second objective lens 15. All geometrical parameters and optical parameters of the first objective lens 8 and the second objective lens 15 are exactly the same.

[0080] like Fig.12As shown, a schematic diagram of collecting super-resolution optical signals is given. The sample 16 to be detected is placed on the front focal plane of the second objective lens 15. The super-resolution conical focusing spot on the front focal plane of the second objective lens 15 is reflected and scattered by the sample 16 to form a left-handed circularly polarized light (right-handed circularly polarized light) super-resolution signal light containing reflected light and scattered light; after the super-resolution signal light is collected by the second objective lens 15, the reflected light is converged by the second objective lens 15 on its rear focal plane to form an annular focusing spot, and the scattered light is collimated by the second objective lens 15 and then emitted; after passing through the second quarter-wave plate 13, the super-resolution signal light is converted into a P linear polarized light beam and transmitted through the polarization beam splitter 9; the 4f system composed of the first lens 17 and the second lens 18 projects the annular focusing spot on the rear focal plane of the second objective lens 15 onto the spatial filter 19, and the annular focusing area of ​​the annular focusing spot corresponds to the reflected light of the sample 16 to be detected, while the other areas correspond to the scattered light of the sample 16 to be detected. The reflected light and the scattered light are selectively filtered by the spatial filter 19. The reflected light transmitted through the spatial filter 19 is converged by the second tube lens 20 on the rear focal plane to form a conical convergent beam, and a focused long focal depth spot is formed on the optical axis. The scattered light transmitted through the spatial filter 19 is converged by the second tube lens 20 on the rear focal plane to form a focused spot. When only reflected light passes through, the system works in the bright field microscopy mode; when only scattered light passes through, the system works in the dark field microscopy mode; when the signal light (reflected light and transmitted light) passes through the phase contrast plate, the system works in the phase contrast mode. Fig.12 In the figure, C2 represents the rear focal plane of the second objective lens 15, and C3 represents the front focal plane of the second objective lens 15.

[0081] like Fig.13 As shown, schematic diagrams of the spatial filter 19 in three different working modes of the system are given.

[0082] like Fig.13 As shown in a, in the bright field mode, the spatial filter 19 is an aluminum film pattern with a thickness of t>50nm made on a translucent glass substrate; no aluminum film is set on the spatial filter 19 within a range of radius less than the outer radius Rout and greater than the inner radius Rin, and the light wave transmittance of the part without the aluminum film is greater than 90%; the light wave transmittance of the aluminum film pattern part is 0, and only the reflected light of the annular light beam reflected from the detected sample 16 is allowed to pass, that is, the bright field imaging light signal is allowed to pass.

[0083] like Fig.13 As shown in b, in the dark field mode, the spatial filter 19 is an aluminum film pattern with a thickness of t>50nm made on a transparent glass substrate; the outer radius of the aluminum film pattern is R out And the inner radius is R inThe light wave transmittance of the aluminum film part is 0, and the light wave transmittance of the part without the aluminum film is greater than 90%, allowing only the light scattered back from the sample 16 to pass, that is, allowing the dark field imaging light signal to pass.

[0084] like Fig.13 c, in the phase contrast mode, the spatial filter 19 is a phase contrast plate, which includes a glass substrate and an annular dielectric film covering the glass substrate, the annular dielectric film is a π phase ring (whose phase change is π), the phase change of the π phase ring relative to other regions is π, and the inner diameter of the π phase ring 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 π phase ring has a transmittance of 10% to 20% for light waves with a wavelength of λ. in and R out The values ​​of are respectively the inner diameter and outer diameter of the annular focused light spot obtained on the plane where the spatial filter 19 is located (i.e., the rear focal plane of the second lens 18) when a plane mirror is placed at the front focal plane of the second objective lens 15 and the plane mirror is perpendicular to the optical axis.

[0085] like Fig.14 As shown in the figure, it is a relationship diagram between the half-height full width of the focused spot of the objective lens and the super-resolution cone beam focusing lens and the system resolution under coherent light illumination. In the figure, the ordinate is the half-height full width of the point spread function of the objective lens, the unit is wavelength λ, the abscissa is the transverse half-height full width of the super-resolution cone beam focus spot, the unit is wavelength λ, and the contour line is the system resolution (that is, the minimum line width of the amplitude type grating that the system can distinguish, and the imaging fringe contrast is better than 11%), the unit is wavelength λ. For example, when the half-height full width of the point focus spot of the objective lens is 0.6λ and the half-height full width of the transverse (the part illuminating the sample) of the super-resolution cone beam focus spot is 0.38λ, the system resolution can reach 0.25λ~0.26λ; when the half-height full width of the point spread function of the objective lens (which can use a single-point focusing super-diffraction lens) and the half-height full width of the transverse (the part illuminating the sample) of the super-resolution cone beam focus spot are both 0.35λ, the system resolution can be better than λ / 5.

[0086] Based on the traditional microscopy system, the present invention provides a reflective non-marking super-resolution optical microscopy system and method, which utilizes the super-resolution focal spot and ultra-long focal depth (the focal depth is Rctan(θ a )=(R out -R in )ctan(θ a ), where ctan(·) is the cotangent function, and R is the radius of the optical fiber collimator output beam), high radial spatial frequency (greater than 0.9n w / λ), narrow frequency passband (less than 0.2nw / λ), and uses a 4f system to achieve the transmission of super-resolution light fields between two objective lenses. Combined with the confocal (illumination lens and collection lens confocal) working principle, a reflective far-field super-resolution optical microscopy system and method without labeling is realized; and three-dimensional tomography of samples can be achieved, with a lateral resolution less than the diffraction limit. The system can achieve non-labeling, super-resolution, and tomography of samples, and can be applied to the detection of biological samples and non-biological samples.

[0087] In an embodiment of the present application, a reflective non-label super-resolution optical microscopy method is provided, which uses a reflective non-label super-resolution optical microscopy system as described in an embodiment of the present application, and the method comprises the following steps:

[0088] The sample 16 to be tested is set on the sample and mechanical scanning module. A P-polarized conical super-resolution focusing light field is generated by a large numerical aperture conical super-resolution focusing module. The P-polarized conical super-resolution focusing light field is projected onto the surface of the sample 16 to be tested by a super-resolution focusing light field illumination and collection module, and the reflected light passing through the sample 16 to be tested and the scattered light caused by the sample 16 to be tested are collected. The collected reflected light and scattered light are subjected to super-resolution signal light collection, photoelectric conversion and super-resolution image acquisition by a super-resolution imaging module. Wide-field microscopic imaging of the sample 16 to be tested is performed by a wide-field microscopic imaging module.

[0089] In the embodiment of the present application, the difference between the inner and outer diameters of the annular light beam can be changed by changing the numerical aperture of the fiber collimator 3; the diameter of the annular light beam can be changed by changing the distance between the first conical lens 5 and the second conical lens 6 to ensure that the effective working area of ​​the super-resolution conical beam focusing lens 7 is efficiently illuminated.

[0090] During use, the wide-field microscopy imaging module is first used to perform wide-field imaging of the sample to be inspected in order to determine the area of ​​super-resolution scanning imaging. After the area of ​​super-resolution scanning imaging is determined, the sample is scanned by the scanning control module and the super-resolution optical imaging module controlled by a computer, and the computer obtains the function I(x, y) within the scanning area, which is the super-resolution image of the sample to be inspected.

[0091] The present invention utilizes the characteristics of super-resolution focal spot, ultra-long focal depth, high radial spatial frequency, narrow frequency passband, etc. of large numerical aperture conical super-resolution beam, and adopts 4f system to realize the projection of super-resolution light field on the front focal plane of two objective lenses, and combines the confocal (illumination lens and collection lens confocal) working principle to realize a reflective far-field super-resolution optical microscopy system and method without marking; realizes non-marking far-field super-resolution two-dimensional microscopic imaging through two-dimensional scanning; and obtains super-resolution two-dimensional microscopic imaging of different cross sections of the sample by performing non-marking far-field super-resolution two-dimensional microscopic imaging on the object plane at different axial positions, thereby realizing three-dimensional tomography. The present invention can realize non-marking, super-resolution resolution, and tomographic imaging of samples, and can be applied to non-marking super-resolution microscopic imaging of biological samples, and can also be applied to fields such as industrial-related super-resolution microscopic detection.

[0092] The above-mentioned embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above-mentioned embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A reflective non-labeling super-resolution optical microscopy system, characterized in that: It includes a large numerical aperture cone super-resolution focusing module arranged along the optical path, a super-resolution focusing light field illumination and collection module, a sample and mechanical scanning module, a super-resolution imaging module and a wide-field microscopy imaging module; The large numerical aperture conical super-resolution focusing module is used to generate a P-polarized conical super-resolution focusing light field; the large numerical aperture conical super-resolution focusing module comprises a laser (1), a fiber collimator (3), a linear polarizer (4), a first conical lens (5), a second conical lens (6) and a super-resolution conical beam focusing lens (7) which are sequentially arranged along an optical path, and the laser (1) is connected to the fiber collimator (3); coherent light with a wavelength of λ emitted by the laser (1) passes through the fiber collimator (3), the linear polarizer (4), the first conical lens (5), the second conical lens (6) and the super-resolution conical beam focusing lens (7) in sequence to form a P-polarized conical super-resolution focusing light field; within the axial range of the P-polarized conical super-resolution focusing light field, the size of the super-resolution focusing light spot has a lateral full width at half maximum (FWHM) < 0.5λ / NA a , where NA a is the numerical aperture of the super-resolution cone-shaped beam focusing lens (7), and NA a =n w sin(θ a ), where n w is the refractive index of the medium at the output end of the super-resolution cone-shaped beam focusing lens (7), sin(•) is a sine function, and the radius of the first zero point is less than 0.61λ / NA a , the first zero point radius is the distance from the focal spot intensity peak position to the first zero point position; The super-resolution focused light field illumination and collection module is used to project the P-polarized conical super-resolution focused light field after the super-resolution conical beam focusing lens (7) onto the surface of the detected sample (16), and to collect reflected light and scattered light of the detected sample (16); The sample and mechanical scanning module is used to place the sample (16) to be tested and drive the movement of the sample (16) to be tested; The super-resolution imaging module is used to collect super-resolution signal light, perform photoelectric conversion and obtain super-resolution images of reflected light and scattered light; The wide-field microscopic imaging module is used to realize wide-field microscopic imaging of the sample (16) to be detected, and is used to observe the sample (16) to be detected.

2. The reflective label-free super-resolution optical microscopy system according to claim 1, characterized in that: The super-resolution focused light field illumination and collection module comprises a first objective lens (8), a polarization beam splitter (9), a first quarter-wave plate (10), a first tube lens (11), a reflector (12), a second quarter-wave plate (13), an axial nanopositioner (14) and a second objective lens (15); the front focal plane of the first objective lens (8) is located within the axial range of the P-polarized conical super-resolution focused light field; after the P-polarized conical super-resolution focused light field passes through the first objective lens (8), an annular focused light spot is formed on the rear focal plane of the first objective lens (8); the first tube lens (11) and the reflector (12) form a set of reflective 4f systems, the front focal plane of the first tube lens (11) coincides with and is coaxial with the rear focal plane of the first objective lens (8); the reflective surface of the reflector (12) is located at the first tube lens The front focal plane of the first tube lens (11) coincides with the rear focal plane of the second objective lens (15) after being reflected by the polarization beam splitter (9); the P-polarized conical super-resolution focusing light field generated by the large numerical aperture conical super-resolution focusing module is received by the first objective lens (8), transmitted through the polarization beam splitter (9), converted into circularly polarized light after passing through the first quarter-wave plate (10), and then reflected by the reflector (12) after passing through the first tube lens (11). The reflected circularly polarized light is orthogonal to the incident circularly polarized light, and then sequentially passes through the first tube lens (11) and the first quarter-wave plate (10) to be converted into S-linear polarized light. The S-linear polarized light is reflected by the polarization beam splitter (9), passes through the second quarter-wave plate (13), and then converts the light beam into circularly polarized light before entering the second objective lens (15).

3. The reflective label-free super-resolution optical microscopy system according to claim 2, characterized in that: The optical distance from the rear focal plane of the first objective lens (8) to the first tube lens (11) is equal to the optical distance from the rear focal plane of the second objective lens (15) to the first tube lens (11).

4. The reflective label-free super-resolution optical microscopy system according to claim 1, characterized in that: The sample and mechanical scanning module comprises a two-dimensional nanometer scanning platform (28), on which the sample (16) to be detected is horizontally fixed, and the two-dimensional nanometer scanning platform (28) is controlled to move two-dimensionally in the XY plane, thereby driving the sample (16) to be detected to move two-dimensionally; When the sample and mechanical scanning module is a two-dimensional nanometer scanning platform (28), the super-resolution focusing illumination and collection module further comprises an axial nanometer positioner (14), the second objective lens (15) is mounted on the axial nanometer positioner (14), and the axial movement of the second objective lens (15) is achieved by controlling the axial nanometer positioner (14); Alternatively, the sample scanning module is a three-dimensional nanometer scanning platform (33), the sample to be detected (16) is horizontally fixed on the three-dimensional nanometer scanning platform (33), and the three-dimensional nanometer scanning platform (33) is controlled to move in three directions of X, Y, and Z, thereby driving the sample to be detected (16) to move three-dimensionally.

5. The reflective label-free super-resolution optical microscopy system according to claim 1, characterized in that: The super-resolution imaging module comprises a first lens (17), a second lens (18), a spatial filter (19), a second tube lens (20), a beam splitter (21), an optical fiber head (22), an optical fiber jumper (24), a photomultiplier tube (25) and a high-speed acquisition card (27); the first lens (17) and the second lens (18) form a 4f system; the rear focal plane of the first lens (17) and the front focal plane of the second lens (18) are coincident and coaxial; the front focal plane of the first lens (17) is coincident with the rear focal plane of the second objective lens (15); the rear focal plane of the second lens (18) is coincident and coaxial with the spatial filter (19); the front end surface of the optical fiber head (22) is located on the rear focal plane of the second tube lens (20); the optical fiber head (22) is connected to the photomultiplier tube (25), and the photomultiplier tube (25) is connected to the high-speed acquisition card (27).

6. The reflective label-free super-resolution optical microscopy system according to claim 5, characterized in that: The super-resolution imaging module further comprises a five-dimensional adjustment frame (23), on which the optical fiber head (22) is mounted. When there is no sample (16) to be detected, the five-dimensional adjustment frame (23) can be adjusted so that the super-resolution signal light entering the optical fiber head (22) reaches a maximum.

7. The reflective label-free super-resolution optical microscopy system according to claim 1, characterized in that: The wide-field microscopic imaging module comprises an incoherent light source (29), a collimating lens (30), a cubic beam splitter (31) and a digital camera (32); the target surface of the digital camera (32) is located at the rear focal plane of the second tube lens (20); the incoherent light emitted by the incoherent light source (29) is collimated by the collimating lens (30) and reflected by the cubic beam splitter (31), and then passes through a spatial filter (19), a second lens (18), a first lens (17), a polarization beam splitter (9), a second quarter-wave plate (13), and a second objective lens (15) in sequence, and forms a focused light field behind the second objective lens (15) and focuses the light on the incoherent light. The sample to be tested (16) is subjected to incoherent illumination; the incoherent reflected light and scattered light generated by the sample to be tested (16) are collected by the second objective lens (15), and then sequentially pass through the second quarter-wave plate (13), the polarization beam splitter (9), the first lens (17), the second lens (18), the spatial filter (19), the cubic beam splitter (31), the second tube lens (20) and the beam splitter (21), and then form a wide-field microscopic light field of the sample to be tested (16) at the rear focal plane of the second tube lens (20), and the wide-field microscopic light field is captured by a digital camera (32) to form a wide-field microscopic image of the sample to be tested (16).

8. The reflective label-free super-resolution optical microscopy system according to claim 1, characterized in that: In the bright field mode, the spatial filter (19) is an aluminum film pattern with a thickness of t>50nm made on a light-transmitting glass substrate; the spatial filter (19) has a radius smaller than the outer radius R out and is larger than the inner radius R in No aluminum film is provided within the range, and the light wave transmittance of the portion without the aluminum film is greater than 90%; the light wave transmittance of the aluminum film pattern portion is 0, and only the ring light beam reflected from the sample (16) to be tested is allowed to pass, that is, the bright field imaging light signal is allowed to pass; In the dark field mode, the spatial filter (19) is an aluminum film pattern with a thickness of t>50nm made on a light-transmitting glass substrate; the outer radius of the aluminum film pattern is R out And the inner radius is R in The light wave transmittance of the aluminum film part is 0, and the light wave transmittance of the part without the aluminum film is greater than 90%, so that only the light scattered back from the sample to be tested (16) is allowed to pass, that is, the dark field imaging light signal is allowed to pass; In the phase contrast mode, the spatial filter (19) is a phase contrast plate, which comprises a glass substrate and an annular dielectric film covering the glass substrate, the annular dielectric film is a π phase ring, the phase change of the π phase ring relative to other regions is π, and the inner diameter of the π phase ring is equal to 2R in , outer diameter equals 2R out The glass substrate has a transmittance of more than 90% for light waves with a wavelength of λ, and the π phase ring has a transmittance of 10% to 20% for light waves with a wavelength of λ.

9. A reflective label-free super-resolution optical microscopy method, characterized in that: Using the reflective non-labeling super-resolution optical microscopy system as claimed in any one of claims 1 to 8, the method comprises the following steps: Placing the sample to be tested (16) on the sample and mechanical scanning module; Generate a P-polarized conical super-resolution focusing light field through a large numerical aperture conical super-resolution focusing module; Projecting a P-polarized conical super-resolution focused light field onto the surface of the sample to be detected (16) through a super-resolution focused light field illumination and collection module, and collecting reflected light passing through the sample to be detected (16) and scattered light caused by the sample to be detected (16); The collected reflected light and scattered light are subjected to super-resolution signal light collection, photoelectric conversion and super-resolution image acquisition through the super-resolution imaging module; The wide-field microscopic imaging module is used to perform wide-field microscopic imaging of the sample (16) to be detected, so as to determine the super-resolution scanning imaging area.

10. The reflective label-free super-resolution optical microscopy method according to claim 9, characterized in that: Also includes: The coherent light emitted by the laser (1) is transmitted to the optical fiber collimator (3) through the first optical fiber jumper (2), and the optical fiber collimator (3) collimates the light beam and outputs it; the collimated light beam passes through a linear polarizer (4) to form a P linear polarized collimated light beam; the P linear polarized collimated light beam passes through a first conical lens (5) and a second conical lens (6) to form a ring light beam; the ring light beam passes through a super-resolution conical beam focusing lens (7) to form a P polarized conical super-resolution focusing light field; The difference between the inner and outer diameters of the annular beam is changed by changing the numerical aperture of the optical fiber collimator (3); and the diameter of the annular beam is changed by changing the distance between the first cone lens (5) and the second cone lens (6), so as to ensure that the effective working area of ​​the super-resolution cone beam focusing lens (7) is efficiently illuminated.

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