A Deep Frequency Shifting Super-Resolution Microscopic Imaging Chip and System with Omnidirectional Ultra-High Wave Vector Illumination

Abstract

This invention provides a deep frequency-shifting super-resolution microscopy imaging chip and system with omnidirectional ultra-high wave vector illumination, belonging to the field of optical super-resolution microscopy. The chip includes: a substrate coated with an adhesion layer and multiple thin films on the adhesion layer; subwavelength-scale circular gratings are etched on the substrate and adhesion layer. Excitation light is incident on the circular grating through the substrate, generating evanescent waves with ultra-high wave vectors in various directions. These evanescent waves are coupled to the upper surface through the multilayer thin films, exciting surface plasmons with ultra-high transverse wave vectors in corresponding directions for omnidirectional ultra-high wave vector illumination and deep frequency-shifting super-resolution microscopy imaging of samples placed on the upper surface of the multilayer thin films. The system includes: an excitation module, an omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy imaging chip, an imaging module, and an excitation control and image acquisition module. This invention enables omnidirectional ultra-high resolution imaging of samples with complex and fine structures, meeting the application needs of related fields.

Description

Technical Field

[0001] This invention belongs to the field of optical super-resolution microscopy technology, specifically relating to a deep frequency-shifting super-resolution microscopy imaging chip and system with omnidirectional ultra-high wave vector illumination. Background Technology

[0002] Sub-λ / 10 (λ represents the excitation wavelength) ultra-high resolution optical microscopy is of great significance in biomedical research and in revealing micro- and nano-scale effects. Frequency-shifting super-resolution microscopy overcomes the diffraction limit of existing objective imaging systems by altering the transverse wave vector of the illumination field interacting with the sample. Existing on-chip frequency-shifting super-resolution imaging methods rely on waveguide modulation of the transverse wave vector of the illumination field, eliminating the need for complex light field manipulation and special sample labeling, thus offering significant advantages in extremely high-resolution imaging.

[0003] However, the resolution of current on-chip frequency-shifting super-resolution imaging technology is determined by the range of microstructure units fabricated. For example, to achieve sub-ten-nanometer resolution, a large number of precisely repeating sub-ten-nanometer structural units need to be fabricated. This not only places extremely high demands on fabrication technology and increases manufacturing costs, but also presents significant challenges in practical operation. Furthermore, for samples with complex and intricate structures, such as microvascular networks in biological tissues or fine structures in nanomaterials, existing technologies often struggle to achieve high signal-to-noise ratio imaging while maintaining high resolution, thus limiting their widespread application in these fields.

[0004] To overcome these challenges, researchers are constantly exploring new imaging methods and technologies. Chinese patent application CN108828756A proposes a surface plasmon nonlinear structured light illumination super-resolution microscopy imaging method and apparatus. This method uses surface plasmon polariton (SPP) interference fringes excited by grooves to excite fluorescence. The illumination angle is adjusted to achieve phase modulation of the interference fringes. Utilizing the nonlinear emission characteristics of fluorescence, fluorescence images illuminated by different interference fringes are acquired to reconstruct a super-resolution image. Its advantage lies in achieving resolution comparable to STED (stimulated emission depletion microscopy) and STORM (stochastic optical reconstruction microscopy), while avoiding the complex laser manipulation and lengthy image acquisition processes of these technologies. However, despite the significant improvement in resolution, the reliance on grooved structures to excite SPP interference fringes also presents limitations. The generation of interference fringes is strictly limited by the illumination azimuth angle, meaning it is difficult to excite high-wavelength vectors in all spatial directions. Therefore, when the sample has a complex and fine structure in multiple directions, this technique may not be able to provide super-resolution imaging covering the entire spectrum, thus limiting its effectiveness in certain specific application scenarios.

[0005] In summary, although existing frequency-shifting super-resolution microscopy and groove-excited SPP interference fringe super-resolution imaging methods have made significant progress in improving the resolution of optical microscopy, there are still technical obstacles to overcome in order to excite omnidirectional high-wavelength vector light to meet the needs of fields such as biomedicine and materials science for ultra-high resolution and complex structure imaging technology. Summary of the Invention

[0006] In view of the above, the purpose of this invention is to provide a deep frequency-shifting super-resolution microscopy imaging chip and system with omnidirectional ultra-high wave vector illumination. By matching the wave vector with a circular subwavelength grating, an omnidirectional ultra-high wave vector SPP mode is excited on the surface of a metal-dielectric-metal (MIM) film, thereby illuminating the sample on the upper surface of the imaging chip. Specifically, through two-dimensional scanning of the optical system, omnidirectional, large-angle excitation light can be generated. Under the excited SPP illumination, the imaging objective lens can receive scattering information from all directions of the sample, achieving omnidirectional imaging of samples with complex and intricate structures.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a deep frequency-shifting super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination, comprising: a substrate coated with an adhesive layer and a multilayer thin film on the adhesive layer, wherein a subwavelength scale annular grating is etched on the substrate and the adhesive layer.

[0009] In this process, excitation light is incident on a circular grating through a substrate, generating evanescent waves in various directions through the grating. These evanescent waves are coupled to the upper surface through a multilayer thin film, exciting surface plasmons with ultra-high transverse wave vectors in the corresponding directions. This process is used to perform omnidirectional ultra-high wave vector illumination and deep frequency-shifting super-resolution microscopy imaging of samples placed on the upper surface of the multilayer thin film.

[0010] Preferably, the excitation light is a ring-shaped, large-angle excitation light with omnidirectional TM polarization.

[0011] Preferably, the diameter of the annular grating is less than or equal to the beam diameter of the large-angle excitation light of the annular grating.

[0012] Preferably, to more effectively excite SPP, the evanescent wave of the first-order diffraction order of the circular ring grating is used to match the transverse wave vector of the surface plasmons on the surface of the multilayer thin film. The formula for wave vector matching is: Where, k spp Let denoted as the transverse wave vector of the surface plasmon generated on the upper surface of the multilayer thin film, k0 as the wave vector of the excitation light in free space, n as the refractive index of the medium in the space where the excitation light is located, θ as the incident angle of the excitation light, λ as the wavelength of the excitation light, and d as the period of the annular grating.

[0013] Preferably, the substrate material is transparent in the selected excitation light band to reduce energy loss after the excitation light is transmitted from the substrate, improve the photon utilization rate of the excitation ultra-high wave vector SPP, and thus improve the intensity of frequency-shifted illumination to obtain imaging results with higher signal-to-noise ratio. For example, SiO2, Al2O3, etc. can be selected in the visible light band, and GaP, GaN, etc. can be selected in the near-infrared band.

[0014] Generally, the substrate thickness is 250–500 μm.

[0015] Preferably, the multilayer thin film is a multilayer structure with alternating metal and dielectric layers, and both the uppermost and lowermost layers being metal layers. The ultra-high transverse wave vector of the SPP generated by the multilayer structure can be calculated based on the dielectric constant and geometric thickness of the film layers at the excitation wavelength. The materials of the metal and dielectric layers are selected according to the excitation wavelength, thereby determining their dielectric constants; the geometric thickness of the film layers is calculated based on the boundary conditions and continuity equation of Maxwell's equations and the expected transverse wave vector.

[0016] Preferably, within the excitation light band, the metal film layer is selected from metal materials with complex refractive index and low loss, such as Ag or Au, with a thickness of 5–100 nm; the dielectric film layer is selected from dielectric materials with high refractive index, such as SiO2, Al2O3, TiO2, Si3N4, GaP, or GaN, with a thickness of 5–100 nm.

[0017] Optionally, appropriate micro / nano fabrication methods, including magnetron sputtering, atomic layer deposition, and chemical vapor deposition, can be selected based on the designed film geometry and material; suitable thickness control methods, including photoelectrode method, quartz crystal monitoring, single-wavelength monitoring, and broadband spectral scanning, can be selected based on the designed geometry to ensure the accuracy of the film thickness.

[0018] Optionally, in order to evaluate the optical performance of the prepared multilayer film structure, parameters such as film thickness uniformity and surface roughness should also be measured. Generally, appropriate preparation processes should be adopted during the preparation process to ensure the hardness, adhesion and stability of the film. For example, for metal Ag, which has poor hardness, poor adhesion and poor stability, a preparation process with high vacuum, low substrate temperature and fast evaporation rate should be selected to form the film.

[0019] Secondly, to achieve the above-mentioned objectives, embodiments of the present invention also provide a deep frequency-shifting super-resolution microscopy system with omnidirectional ultra-high wave vector illumination, employing the aforementioned omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy chip, comprising: an excitation module, an omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy chip, an imaging module, and an excitation control and image acquisition module.

[0020] The excitation module includes, in sequence, a laser source for emitting laser light, a collimating lens for collimating the laser into parallel light, a laser polarization adjustment unit for adjusting the polarization state of the parallel light, an omnidirectional galvanometer scanning unit for converting the polarization-adjusted parallel light into large-angle incident excitation light, a scanning lens for uniformly converging the large-angle incident excitation light within the back focal plane of the excitation objective, and a numerical aperture of NA for projecting the converged large-angle incident excitation light onto a deep frequency-shifting super-resolution microscopy chip with omnidirectional ultra-high wave vector illumination. illu The excitation objective lens is used for laser excitation. The laser polarization adjustment unit includes a linear polarizer and a quarter-wave plate to adjust the polarization state of the excitation light. The omnidirectional galvanometer scanning unit includes two one-dimensional scanning galvanometers and a 4F optical system positioned between them. Generally, the scanning axes of the two one-dimensional scanning galvanometers should be on opposite planes and spatially perpendicular, with their reflecting surfaces located on the conjugate plane of the 4F optical system. The scanning lens converges the beam deflected by the galvanometers onto the back focal plane of the excitation objective lens, thereby generating excitation light incident at a large angle.

[0021] The upper surface of the multilayer thin film in the omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy chip is used to place the sample.

[0022] The imaging module includes sequentially arranged numerical apertures of NA. img The imaging objective lens and its matching mirror, tube mirror, and camera are used to receive the omnidirectional scattering signals of the sample and transmit them to the camera to obtain a deep frequency-shifted illumination image.

[0023] The excitation control and image acquisition module includes a galvanometer driver and control card, and a computer. The galvanometer driver and control card drives and controls the one-dimensional scanning galvanometer to achieve two-dimensional scanning of the beam. The camera control software in the computer synchronously saves the deep frequency-shifting illumination image and processes it to obtain a deep frequency-shifting super-resolution microscopic image for subsequent analysis.

[0024] Preferably, the wavelength of the laser source is selected based on the dispersion curve of the deep frequency-shifting super-resolution microscopy chip with omnidirectional ultra-high wave vector illumination, so as to excite a higher wave vector.

[0025] Preferably, the collimating lens should ensure that the spot diameter d1 of the emitted parallel light is less than or equal to the working size of the one-dimensional scanning galvanometer.

[0026] Preferably, the laser polarization adjustment unit is used to adjust the excitation light incident on the omnidirectional ultra-high wave vector illumination deep frequency shift super-resolution microscopy chip to TM polarization.

[0027] Preferably, the beam diameter d of the excitation objective lens that produces the excitation light at a large angle of incidence illu The diameter d of the annular grating of the deep frequency-shifting super-resolution microscopy chip with omnidirectional ultra-high wave vector illuminationgrating Matching, satisfying Where f illu f represents the equivalent focal length of the excitation objective. scan d1 represents the focal length of the scanning lens, and d2 represents the diameter of the spot of the parallel light emitted from the collimating lens.

[0028] Generally, the maximum angle of the excitation light produced by the excitation objective is θ. max Satisfying NA illu =n·sinθ max NA illu The numerical aperture of the excitation objective is represented by , and n represents the refractive index of the excitation objective.

[0029] Preferably, the camera in the imaging detection module is a high-sensitivity camera, such as an EMCCD or sCMOS research-grade camera.

[0030] Thirdly, to achieve the above-mentioned objectives, embodiments of the present invention also provide a method for deep frequency-shifting super-resolution microscopy with omnidirectional ultra-high wave vector illumination, implemented using the aforementioned omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy system, comprising the following steps:

[0031] The laser spot emitted from the laser source is adjusted to a size that matches the working dimensions of the one-dimensional scanning galvanometer, and the polarization state at the deep frequency-shifting super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination is adjusted to TM polarization.

[0032] The sample with a complex and intricate structure to be imaged is placed on the upper surface of a multilayer thin film in a deep frequency-shifting super-resolution microscopy chip with omnidirectional ultra-high wave vector illumination. It is also important to ensure that the center of the circular grating at the bottom of the chip is aligned with the exit pupil center of the excitation objective to minimize illumination azimuth errors.

[0033] The omnidirectional galvanometer scanning unit is controlled to perform a two-dimensional scan on the back focal plane of the excitation objective, thereby exciting omnidirectional ultra-high surface plasmons with ultra-high transverse wave vectors to illuminate the sample, and then the imaging objective receives the omnidirectional scattering signal of the sample.

[0034] The camera synchronously acquires the deep frequency shift illumination image corresponding to each scanning position and saves it to the computer. Based on whether the sample is fluorescently labeled, the corresponding reconstruction algorithm is used to calculate the deep frequency shift super-resolution microscopic image.

[0035] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0036] The deep frequency-shifting super-resolution microscopy chip provided by this invention can perform omnidirectional ultra-high wave vector illumination and deep frequency-shifting super-resolution microscopy imaging of samples placed on the surface of multilayer thin films. In the microscopy imaging system designed using the chip, the two-dimensional scanning of the omnidirectional galvanometer scanning unit and the excitation objective of the high numerical aperture (NA) provide omnidirectional, large-angle annular incident light. This ultra-high wave vector (SPP) illumination is excited by the annular grating of the deep frequency-shifting super-resolution microscopy chip, thereby illuminating samples with complex and fine structures, achieving omnidirectional ultra-high wave vector illumination for deep frequency-shifting super-resolution microscopy imaging. Compared to super-resolution microscopy chips excited by grooves or one-dimensional gratings, this invention uses a thin film structure with alternating metal dielectrics to generate a comparable level of ultra-high wave vector (SPP) illumination. Furthermore, the large-angle light from the annular scanning incident on the annular grating at the bottom of the chip provides an omnidirectional illumination field for the sample. Therefore, this invention has significant advantages in balancing omnidirectional illumination and ultra-high wave vector illumination. Combined with corresponding image reconstruction algorithms, it is expected to be applied to super-resolution imaging of complex and fine-structured samples with sub-λ / 10 resolution. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a schematic diagram of the structure of the omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy chip provided in the embodiments of the present invention, and a schematic diagram of the structure of the circular grating in the chip.

[0039] Figure 2 This is a schematic diagram of a deep frequency-shifting super-resolution microscopy system with omnidirectional ultra-high wave vector illumination provided in an embodiment of the present invention.

[0040] Figure 3 This is a schematic diagram illustrating the generation method of omnidirectional TM polarization excitation light in the omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy system provided in this embodiment of the invention.

[0041] Figure 4 This is a schematic flowchart of the deep frequency-shifting super-resolution microscopy method with omnidirectional ultra-high wave vector illumination provided in an embodiment of the present invention.

[0042] Figure 5 This is a schematic diagram comparing the spectrum range that the imaging system and imaging chip provided in this embodiment of the invention can detect with the spectrum range that existing frequency-shifting illumination methods can detect. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not limit the scope of protection of this invention.

[0044] Figure 1 This is a schematic diagram of the structure of the omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy chip provided in an embodiment of the present invention, as well as a schematic diagram of the structure of the circular annular grating in the chip. Figure 1 As shown, the embodiment provides a deep frequency-shifting super-resolution microscopy imaging chip with omnidirectional ultra-high wave vector illumination, including: a substrate coated with an adhesive layer and a multilayer thin film on the adhesive layer, and a subwavelength scale annular grating etched on the substrate and the adhesive layer.

[0045] In this process, omnidirectional TM polarization excitation light is incident on a circular ring grating through a substrate. The first-order diffraction order generated by the ring grating is an evanescent wave. The substrate of the grating should be transparent in the selected excitation light band and needs to match the refractive index of the image space medium of the excitation objective. Considering the actual micro / nano fabrication steps, an adhesion layer needs to be deposited on the substrate first to enhance the stability of the upper metal film. In this embodiment, the adhesion layer selected is a 2nm thick chromium film. Considering the diffraction efficiency of the grating, its first-order evanescent wave is used to match the wave vector on the upper surface of the film. The formula for wave vector matching is expressed as: Where k spp Let denoted as , k0 be the transverse wave vector of the SPP generated on the surface of the multilayer thin film, n be the refractive index of the medium in the space containing the excitation light, θ be the incident angle of the excitation light, λ be the wavelength of the excitation light, and d be the period of the annular grating. The diameter d of the annular grating to be fabricated is also given. grating It should be related to the beam diameter d of the excitation light illu Matching, d grating ≤d illu .

[0046] A metal-dielectric-metal MIM structure is sequentially fabricated on a circular grating. Ultimately, an ultra-high transverse wave vector (SPP) mode is generated between the topmost metal film and the air medium. The resulting ultra-high transverse wave vector (k) is... x Using the effective refractive index n of the entire MIM structure eff Let n represent eff =k x / k0. The metallic material should be selected based on the complex refractive index of the excitation light band, and the dielectric material should be selected considering the excitation light wavelength, determined according to its SPP mode cutoff wavelength. In this embodiment, Ag is used as the metallic material, and TiO2 or GaN is used as the dielectric material. The thickness of the metal and dielectric materials are ultimately related to the ultra-high transverse wave vector excited. Based on Maxwell's equations and continuity conditions, k1, k2, k3 of each interface in the multilayer film and the final SPP transverse wave vector k of the upper surface excited are calculated sequentially. spp .

[0047] In this embodiment, the chip fabrication method is as follows: Based on the geometric thickness of the metal and dielectric, this embodiment uses atomic layer deposition (ALD) to deposit multiple thin films; based on k spp The period of the annular grating is calculated and the fabrication process is determined. In this embodiment, electron beam lithography (EBL) is used to fabricate the annular grating. Table 1 shows the effective refractive index, the ultra-high wave vector that can be generated, and the period of the annular grating for a MIM structure composed of dielectric and metal materials (the metal layer in contact with the adhesion layer is 50 nm, the dielectric layer is 10 nm, and the upper surface metal layer is 10 nm) at a laser wavelength of 561 nm.

[0048] Table 1. Film composition, effective refractive index, transverse wave vector, and grating period of the chip.

[0049]

[0050] Figure 2 This is a schematic diagram of the omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy system provided in an embodiment of the present invention, as shown below. Figure 2 As shown, the embodiment also provides a deep frequency-shifting super-resolution microscopy imaging system with omnidirectional ultra-high wave vector illumination, including: an excitation module, an omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy imaging chip, an imaging module, and an excitation control and image acquisition module. Specifically, it includes: a laser source 201, a collimating lens 202, a polarization adjustment unit 203, an omnidirectional galvanometer scanning unit 204, a scanning lens 205, an excitation objective lens 206, an omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy imaging chip 207, an imaging objective lens 208, a reflecting mirror 209, a tube mirror 210, a camera 211, a computer 212, and a galvanometer driver and control card 213. 201-206 belong to the excitation module, 208-211 belong to the imaging module, and 212-213 belong to the excitation control and image acquisition module.

[0051] In this embodiment, the laser source 201 is a 561nm single-mode fiber laser. The collimating lens 202 generates parallel light with a spot diameter of 10mm. The polarization adjustment unit 203, including a linear polarizer and a quarter-wave plate, modulates the polarization state of the excitation light so that it is TM polarized when incident on the omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy chip 207. The omnidirectional galvanometer scanning unit 204 includes two one-dimensional scanning galvanometers and a 4F optical system located between them. The scanning axes of the two one-dimensional scanning galvanometers should be perpendicular to each other, and the reflecting surfaces of the two one-dimensional scanning galvanometers are located on the conjugate surface of the 4F optical system. The scanning lens 205 generates a uniform convergence point within the back focal plane of the excitation objective 206. The excitation objective 206 is used to generate excitation light incident at a large angle, and its exit pupil center should be located at the center of the circular grating of the omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy chip 207. In this embodiment, a one-dimensional scanning galvanometer with a working size of 10mm and a scanning lens with a focal length of 200mm are selected. The diameter d of the beam incident on the bottom grating of the chip is... grating ≤90μm.

[0052] In this embodiment, under a laser light source with a wavelength of 561 nm, the selected excitation objective 206 has a numerical aperture of NA. illu =1.49, an Olympus oil immersion lens with a magnification of 100×, whose image space medium is immersion oil (its refractive index n) e =1.518), and correspondingly, the substrate material is SCHOTT N-BK7 glass (its refractive index n = 1.518). e =1.51872). For example Figure 3 As shown, the plane 215 on which the chip is placed coincides with the focal plane of the excitation objective 206. By controlling the two-dimensional scanning of the omnidirectional galvanometer scanning unit 204, the parallel beam is converged by the scanning lens 205 onto the back focal plane 214 of the excitation objective 206 and undergoes a circular scan. Finally, parallel light with a large angle is emitted from the excitation objective 206, thereby exciting the omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy chip 207 to generate ultra-high wave vector illumination light. As the omnidirectional galvanometer scanning unit 204 performs the two-dimensional scanning, the large-angle excitation light is incident on the annular grating region 216 at the bottom of the omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy chip 207, exciting an omnidirectional ultra-high wave vector illumination field to illuminate the sample with a complex and fine structure placed on the upper surface of the multilayer thin film in the omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy chip 207.

[0053] In this embodiment, the omnidirectional scattering signal of the sample is received by the imaging objective 208 and transmitted to the tube mirror 210 via the reflecting mirror 209 to form a real image of the sample. The tube mirror 210 then projects the real image onto the camera 211, which acquires the deep frequency-shifted illumination image. The camera 211 is an sCMOS research-grade camera. The numerical aperture of the imaging objective 208 is NA. img =0.8. When using the two ultra-high wave vector illumination chips described in Table 1 to illuminate unlabeled samples, the achievable resolutions are 43.59 nm and 50.72 nm, respectively. The formula for calculating the resolution is: This system is expected to enable super-resolution microscopy imaging with sub-λ / 10 (56.1 nm) resolution.

[0054] In this embodiment, the driving and control of the one-dimensional scanning galvanometer is achieved through the galvanometer driver and control card 213 to realize the two-dimensional scanning of the beam. The camera control software in the computer 212 is used to synchronously save the deep frequency shift illumination image and process it to obtain a deep frequency shift super-resolution microscopic image for subsequent analysis.

[0055] Based on the aforementioned omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy system, this embodiment of the invention also provides an omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy method, such as... Figure 4 As shown, it includes the following steps:

[0056] S1, adjust the laser spot emitted from the laser source to a spot size that matches the working size of the one-dimensional scanning galvanometer, and adjust the polarization state at the deep frequency-shifting super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination to TM polarization.

[0057] S2, the sample with a complex and fine structure to be imaged is placed on the upper surface of a multilayer thin film in a deep frequency-shifting super-resolution microscopy chip with omnidirectional ultra-high wave vector illumination.

[0058] S3 controls the omnidirectional galvanometer scanning unit to perform a two-dimensional scan on the back focal plane of the excitation objective, thereby exciting omnidirectional ultra-high surface plasmons with ultra-high transverse wave vectors to illuminate the sample, and then the imaging objective receives the omnidirectional scattering signal of the sample.

[0059] S4. The camera synchronously acquires the deep frequency shift illumination image corresponding to each scanning position and saves it to the computer. Based on whether the sample is fluorescently labeled, the corresponding reconstruction algorithm is used to calculate the deep frequency shift super-resolution microscopic image.

[0060] To verify the effectiveness of the chip and system provided by this invention, a sample with a complex and fine structure was placed on the imaging chip. The sample was illuminated by omnidirectional ultra-high-frequency vector lighting and controlled by a numerical aperture (NA) sensor. imgThe imaging objective lens with an aperture of 0.8 receives a deep frequency-shifted illumination image of the sample from all directions. The spectrum range detected using spatial free-light large-angle omnidirectional frequency-shifted illumination is 501 (without a chip); the spectrum range detected using a single-layer natural high-refractive-index waveguide GaP frequency-shifted illumination is 502 (without a ring grating and multilayer film structure); the spectrum range detected using a grooved-excited multilayer film structure for ultra-high wave vector frequency-shifted illumination is 503; and the spectrum range detected using the chip in this embodiment for omnidirectional ultra-high wave vector frequency-shifted illumination is 504. Figure 5 As shown. Figure 5 The spectrum shifted by ultra-high frequency vector illumination in a single azimuth will overlap with the spectrum shifted by illumination in adjacent azimuths. This overlap depends on the step size of the large-angle ring light excitation, i.e., the scanning step size of the scanning galvanometer. In this embodiment, the smaller the scanning step size of the scanning galvanometer, the more comprehensive the spectral information of the fine structure of the sample can be detected. Compared with existing frequency-shifting illumination methods, the chip in this embodiment can increase the spectral range detected by existing microscopes by several times (approximately 4 times compared to GaP waveguide illumination and approximately 12 times compared to spatial free light illumination). Combined with the hardware settings of the imaging system, it can perform omnidirectional, fine ultra-high frequency spectrum detection.

[0061] This embodiment fully demonstrates the advantages of the present invention in super-resolution imaging of sub-λ / 10 complex and fine-structured samples, thereby enabling the observation and analysis of samples with complex and fine structures at a resolution that surpasses that of traditional imaging techniques, in order to meet the application needs of higher-resolution and more widely applicable imaging technologies in fields such as biomedicine and materials science.

[0062] The specific embodiments described above illustrate the technical solution and beneficial effects of the present invention in detail. It should be understood that the above description is only the most preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A deep frequency-shifting super-resolution microscopy chip with omnidirectional ultra-high wave vector illumination, characterized in that, include: A substrate coated with an adhesive layer and a multilayer film on the adhesive layer, with subwavelength scale circular gratings etched on the substrate and the adhesive layer; In this process, the omnidirectional TM polarized ring-shaped large-angle excitation light is incident on the circular ring grating through the substrate, and the maximum angle of the excitation light is θ. max Satisfying NA illu =n·sinθ max NA illu denoted by , n represents the numerical aperture of the excitation objective lens generating the excitation light, and denoted by , n represents the refractive index of the excitation objective lens. Evanescent waves in various directions are generated through a circular ring grating. These evanescent waves are coupled to the upper surface of the multilayer thin film, exciting surface plasmons with ultra-high transverse wave vectors in corresponding directions. This is used for omnidirectional ultra-high wave vector illumination and deep frequency-shifting super-resolution microscopy imaging of samples placed on the upper surface of the multilayer thin film. Specifically, the transverse wave vectors of the surface plasmons on the upper surface of the multilayer thin film are matched using the first-order diffraction evanescent waves of the circular ring grating. The wave vector matching formula is: Where, k spp Let denoted as the transverse wave vector of the surface plasmon generated on the upper surface of the multilayer thin film, k0 as the wave vector of the excitation light in free space, n as the refractive index of the medium in the space where the excitation light is located, θ as the incident angle of the excitation light, λ as the wavelength of the excitation light, and d as the period of the annular grating.

2. The omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopic imaging chip according to claim 1, characterized in that, The diameter of the circular grating is less than or equal to the beam diameter of the large-angle excitation light of the ring.

3. The omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy imaging chip according to claim 1, characterized in that, The substrate material is transparent in the selected excitation wavelength range.

4. The omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy chip according to claim 1, characterized in that, Multilayer thin films are multilayer structures with alternating metal and dielectric layers, and the top and bottom layers are both metal layers.

5. The omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy chip according to claim 4, characterized in that, Metal films are made of metal materials with complex refractive index and low loss, while dielectric films are made of dielectric materials with high refractive index.

6. A deep frequency-shifting super-resolution microscopy system with omnidirectional ultra-high wave vector illumination, employing the deep frequency-shifting super-resolution microscopy chip with omnidirectional ultra-high wave vector illumination as described in any one of claims 1-5, characterized in that, include: The excitation module, the omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy imaging chip, the imaging module, and the excitation control and image acquisition module; The excitation module includes, in sequence, a laser source for emitting laser light, a collimating lens for collimating the laser light into parallel light, a laser polarization adjustment unit for adjusting the polarization state of the parallel light, an omnidirectional galvanometer scanning unit for converting the polarization-adjusted parallel light into large-angle incident excitation light, a scanning lens for uniformly converging the large-angle incident excitation light within the back focal plane of the excitation objective, and an excitation objective for projecting the converged large-angle incident excitation light onto an omnidirectional ultra-high wave vector illumination deep frequency shift super-resolution microscopic imaging chip; wherein, the laser polarization adjustment unit includes a linear polarizer and a quarter-wave plate, and the omnidirectional galvanometer scanning unit includes two one-dimensional scanning galvanometers and a 4F optical system located between the two one-dimensional scanning galvanometers; The upper surface of the multilayer thin film in the omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopy chip is used to place the sample. The imaging module includes an imaging objective lens and a matching mirror, tube mirror, and camera arranged in sequence, used to receive the omnidirectional scattering signal of the sample and transmit it to the camera to obtain a deep frequency-shifted illumination image. The excitation control and image acquisition module includes a galvanometer driver and control card, and a computer. The galvanometer driver and control card drives and controls the one-dimensional scanning galvanometer to achieve two-dimensional scanning of the beam. The camera control software in the computer synchronously saves the deep frequency-shifting illumination image and processes it to obtain a deep frequency-shifting super-resolution microscopic image for subsequent analysis.

7. The omnidirectional ultra-high wave vector illumination deep frequency shifting super-resolution microscopic imaging system according to claim 6, characterized in that, The beam diameter d of the excitation objective lens that produces excitation light at large angles of incidence illu The diameter d of the annular grating of the deep frequency-shifting super-resolution microscopy chip with omnidirectional ultra-high wave vector illumination grating Matching, satisfying Where f illu f represents the equivalent focal length of the excitation objective. scan d1 represents the focal length of the scanning lens, and d2 represents the diameter of the spot of parallel light emitted from the collimating lens.

8. A method for deep frequency-shifting super-resolution microscopy with omnidirectional ultra-high wave vector illumination, implemented using the omnidirectional ultra-high wave vector illumination deep frequency-shifting super-resolution microscopy system as described in claim 6 or 7, characterized in that, Includes the following steps: The laser spot emitted from the laser source is adjusted to a size that matches the working dimensions of the one-dimensional scanning galvanometer, and the polarization state at the deep frequency-shifting super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination is adjusted to TM polarization. The sample with a complex and fine structure to be imaged is placed on the upper surface of a multilayer thin film in a deep frequency-shifting super-resolution microscopy chip with omnidirectional ultra-high wave vector illumination. The omnidirectional galvanometer scanning unit is controlled to perform a two-dimensional scan on the back focal plane of the excitation objective, thereby exciting omnidirectional ultra-high surface plasmons with ultra-high transverse wave vectors to illuminate the sample, and then the imaging objective receives the omnidirectional scattering signal of the sample. The camera synchronously acquires the deep frequency shift illumination image corresponding to each scanning position and saves it to the computer. Based on whether the sample is fluorescently labeled, the corresponding reconstruction algorithm is used to calculate the deep frequency shift super-resolution microscopic image.

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