Deep frequency shift super-resolution microscopic imaging chip and system for omni-directional ultra-high wave vector illumination

Abstract

The invention provides a deep frequency shift super-resolution microscopic imaging chip and a deep frequency shift super-resolution microscopic imaging system for omni-directional ultra-high wave vector illumination, and belongs to the technical field of optical super-resolution microscopy. The chip comprises a substrate plated with an adhesion layer and multiple layers of films on the adhesion layer, and sub-wavelength-scale circular ring gratings are engraved on the substrate and the adhesion layer; exciting light enters the circular ring grating through the substrate, and evanescent waves of ultrahigh wave vectors in all directions are generated through the circular ring grating. The evanescent waves are coupled to the upper surface through the multi-layer thin film to excite surface plasmons with ultrahigh transverse wave vectors in the corresponding direction, and the surface plasmons are used for carrying out all-directional ultrahigh wave vector illumination and deep frequency shift super-resolution microscopic imaging on a sample placed on the upper surface of the multi-layer thin film. The system comprises an excitation module, a deep frequency shift super-resolution microscopic imaging chip for omni-directional ultra-high wave vector illumination, an imaging module and an excitation control and image acquisition module. According to the invention, omni-directional ultra-high resolution imaging of a complex fine structure sample can be realized, so that application requirements in related fields can be met.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical super-resolution microscopy, and in particular relates to a deep frequency-shifted super-resolution microscopy imaging chip and system with omnidirectional ultra-high wave vector illumination. Background Art

[0002] Sub-λ / 10 (λ represents the wavelength of the excitation light) ultra-high resolution optical microscopy is of great significance in biomedical research and in revealing micro- and nanoscale effects. Frequency-shifting super-resolution microscopy breaks through the diffraction limit of existing objective imaging systems by changing the transverse wave vector of the illumination light field acting on the sample. Existing on-chip frequency-shifting super-resolution imaging methods rely on the modulation of the transverse wave vector of the illumination field by waveguides, which does not require complex light field control and special sample labeling, and has significant advantages in ultra-high resolution imaging.

[0003] However, the resolution of the current on-chip frequency-shift super-resolution imaging technology is determined by the range of the processed microstructure units. For example, to achieve a sub-ten-nanometer resolution, a large number of sub-ten-nanometer precision repeating structure units need to be processed, which not only places extremely high demands on processing technology and increases manufacturing costs, but is also extremely difficult to operate in practice. In addition, for samples with complex fine structures, such as microvascular networks in biological tissues or fine structures in nanomaterials, existing technologies often find it difficult to achieve high signal-to-noise ratio imaging while ensuring high resolution, thus limiting its wide application in these fields.

[0004] In order to overcome the above difficulties, researchers are constantly exploring new imaging methods and technologies. The Chinese patent application with publication number CN108828756A proposes a surface plasmon nonlinear structured light illumination super-resolution microscopy imaging method and device, which uses the surface plasmon (SPP) interference fringes excited by the grooves to excite fluorescence, adjusts the illumination angle to achieve phase modulation of the interference fringes, and uses the characteristics of nonlinear fluorescence luminescence to collect fluorescence images illuminated by different interference fringes to reconstruct super-resolution images. Its advantage is that it can achieve resolution comparable to STED (stimulated emission depletion microscopy) and STORM (stochastic optical reconstruction microscopy), while avoiding the complex laser manipulation and long image acquisition process in these technologies. However, although this method has achieved remarkable results in improving resolution, its reliance on the characteristics of the groove structure to excite SPP interference fringes also brings limitations. The generation of interference fringes is strictly limited by the illumination azimuth, which means that it is difficult to excite high wave vector light in all directions in space. Therefore, when the sample has a complex fine structure in multiple directions, this technology may not be able to provide super-resolution imaging covering the full spectrum, thus limiting its effectiveness in certain specific application scenarios.

[0005] In summary, although the existing frequency-shifting super-resolution microscopy technology and the super-resolution imaging method based on SPP interference fringes excited by grooves have made important progress in improving the resolution of optical microscopy, it is still necessary to overcome technical barriers and stimulate all-round high-wavelength vector light to meet the needs of fields such as biomedicine and materials science for extremely high-resolution and complex structure imaging technology. Summary of the invention

[0006] In view of the above, the purpose of the present invention is to provide a deep frequency-shifted super-resolution microscopic imaging chip and system with omnidirectional ultra-high wave vector illumination, which can excite the omnidirectional ultra-high wave vector SPP mode on the surface of the metal-medium-metal (MIM) film layer through the circular sub-wavelength grating matching wave vector, and then illuminate the sample on the surface of the imaging chip. Among them, through the two-dimensional scanning of the optical system, omnidirectional and large-angle excitation light can be generated, and then under the excited SPP illumination, the imaging objective can receive the scattering information of the sample in all directions, and realize the omnidirectional imaging of samples with complex and fine structures.

[0007] In order to achieve the above-mentioned invention object, the technical solution provided by the present invention is as follows:

[0008] In a first aspect, an embodiment of the present invention provides a deep frequency-shifted 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 sub-wavelength-scale circular grating is engraved on the substrate and the adhesive layer;

[0009] Among them, the excitation light is incident on the circular grating through the substrate, and evanescent waves are generated in various directions through the circular grating. The evanescent waves are coupled to the upper surface through the multilayer film to excite surface plasmons with ultra-high lateral wave vectors in the corresponding direction, which are used to perform all-round ultra-high wave vector illumination and deep frequency-shift super-resolution microscopy imaging of the sample placed on the upper surface of the multilayer film.

[0010] Preferably, the excitation light is annular 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 annular large-angle excitation light.

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

[0013] Preferably, the material of the substrate is transparent in the selected excitation light band to reduce the energy loss of the excitation light after transmission from the substrate, improve the utilization rate of photons that excite the ultra-high wave vector SPP, and then increase 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 thickness of the substrate is 250 to 500 μm.

[0015] Preferably, the multilayer film is a multilayer structure in which metal film layers and dielectric film layers are alternately arranged and the topmost layer and the bottommost layer are both metal film layers. The ultra-high transverse wave vector of the SPP generated by the multilayer structure can be calculated based on the dielectric constant of the film layer at the wavelength of the excitation light and its geometric thickness. The materials of the metal film layer and the dielectric film layer are selected according to the wavelength of the excitation light, and then their dielectric constants are determined; the geometric thickness of the film layer is calculated based on the boundary conditions and continuity equations of the Maxwell equations and the expected transverse wave vector.

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

[0017] Optionally, select appropriate micro-nano processing methods according to the designed film layer geometric thickness and material, including magnetron sputtering, atomic layer deposition, chemical vapor deposition, etc.; select appropriate thickness control methods according to the designed geometric thickness, including photoelectrode value method, quartz crystal monitoring, single wavelength monitoring, wide spectrum scanning, etc., to ensure the accuracy of film layer thickness.

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

[0019] In the second aspect, to achieve the above-mentioned purpose of the invention, an embodiment of the present invention further provides a deep frequency-shifted super-resolution microscopy imaging system with omnidirectional ultra-high wavevector illumination, which adopts the above-mentioned deep frequency-shifted super-resolution microscopy imaging chip with omnidirectional ultra-high wavevector illumination, including: an excitation module, a deep frequency-shifted super-resolution microscopy imaging chip with omnidirectional ultra-high wavevector illumination, an imaging module, and an excitation control and image acquisition module.

[0020] The excitation module includes a laser light 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 in the rear focal plane of the excitation objective lens, and a laser beam having a numerical aperture of NA for emitting the converged large-angle incident excitation light onto a deep frequency-shifted super-resolution microscopic imaging chip for omnidirectional ultra-high wave vector illumination. illu The laser polarization adjustment unit includes a linear polarizer and a quarter wave plate, which are used 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 between the two one-dimensional scanning galvanometers. Generally, the scanning axes of the two one-dimensional scanning galvanometers should be on skew lines and vertical in space, and their reflection surfaces are on the conjugate plane of the 4F optical system. The scanning lens converges the light beam deflected by the galvanometer onto the back focal plane of the excitation objective lens, thereby generating excitation light with a large angle of incidence.

[0021] The upper surface of the multilayer film in the deep frequency-shift super-resolution microscopic imaging chip with all-round ultra-high wave vector illumination is used for placing samples.

[0022] The imaging module includes a numerical aperture NA set in sequence img The imaging objective lens and the matching reflector, tube lens, and camera are used to receive the omnidirectional scattering signal of the sample and transmit it to the camera to obtain a deep frequency-shifted illumination image.

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

[0024] Preferably, the wavelength of the laser light source is selected according to the dispersion curve of the deep frequency-shifted super-resolution microscopy imaging chip with omnidirectional ultra-high wave vector illumination to excite a higher wave vector.

[0025] Preferably, the collimating lens should ensure that the spot diameter d1 of the emitted parallel light is smaller 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 deep frequency-shifted super-resolution microscopy imaging chip with omni-directional ultra-high wave vector illumination to TM polarization.

[0027] Preferably, the beam diameter d of the excitation light incident at a large angle generated by the excitation objective lens is illu The diameter d of the annular grating of the deep frequency-shifted super-resolution microscopy imaging chip with omnidirectional ultra-high wave vector illuminationgrating Match, satisfy where f illu represents the equivalent focal length of the excitation objective, f scan represents the focal length of the scanning lens, and d1 represents the spot diameter of the parallel light emitted by the collimating lens.

[0028] Generally, the maximum angle of the excitation light generated by the excitation objective is θ max , satisfying NA illu =n×sinθ max , where represents , represents .

[0029] Preferably, the camera in the imaging detection module is a high-sensitivity camera, for example, an EMCCD, sCMOS or other scientific research-grade camera.

[0030] In a third aspect, to achieve the above-mentioned purpose of the invention, an embodiment of the present invention further provides a method for deep frequency shift super-resolution microscopy imaging with omnidirectional ultra-high wave vector illumination, which is implemented using the above-mentioned deep frequency shift super-resolution microscopy imaging system with omnidirectional ultra-high wave vector illumination, and includes the following steps:

[0031] The spot size of the laser emitted by the laser light source is adjusted to match the working size of the one-dimensional scanning galvanometer, and the polarization state at the deep frequency-shifted super-resolution microscopy imaging chip with all-round ultra-high wave vector illumination is adjusted to TM polarization;

[0032] The sample with complex fine structure to be imaged is placed on the upper surface of the multilayer film in the deep frequency shift super-resolution microscopy imaging chip with omnidirectional ultra-high wave vector illumination. It should also be noted that the center position of the circular grating at the bottom of the chip should be aligned with the center of the exit pupil of the excitation objective to reduce the error of the illumination azimuth.

[0033] Control the omnidirectional galvanometer scanning unit to perform two-dimensional scanning on the back focal plane of the excitation objective lens, and excite omnidirectional ultra-high surface plasmons with ultra-high transverse wave vectors to illuminate the sample, and then the imaging objective lens 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 synchronously. Depending 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 present invention has the following beneficial effects:

[0036] The deep frequency shift super-resolution microscopic imaging chip provided by the present invention can perform omnidirectional ultra-high wave vector illumination and deep frequency shift super-resolution microscopic imaging on the sample placed on the upper surface of the multilayer film, and in the microscopic imaging system obtained by using the chip design, the two-dimensional scanning of the omnidirectional galvanometer scanning unit and the excitation objective lens with a high numerical aperture NA can provide omnidirectional annular large-angle incident light, and the ultra-high wave vector SPP is excited by the annular grating of the deep frequency shift super-resolution microscopic imaging chip, thereby illuminating the sample with a complex fine structure, and realizing omnidirectional ultra-high wave vector illumination deep frequency shift super-resolution microscopic imaging. Compared with the super-resolution microscopic imaging chip of groove excitation and one-dimensional grating excitation, the present invention adopts a thin film structure of alternating metal media to produce a fairly high level of ultra-high wave vector SPP illumination mode, and in addition, the large-angle light of the annular scanning is incident on the annular grating at the bottom of the chip, which can provide an omnidirectional illumination field for the sample. Therefore, the present invention has significant advantages in taking into account both omnidirectional illumination and ultra-high wave vector illumination, and combined with the corresponding image reconstruction algorithm, it is expected to be applied to the super-resolution imaging of sub-λ / 10 complex fine structure samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0038] Figure 1 It is a schematic diagram of the structure of a deep frequency-shifted super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination and a schematic diagram of the structure of a circular ring grating in the chip provided by an embodiment of the present invention.

[0039] Figure 2 It is a schematic diagram of a deep frequency shift super-resolution microscopic imaging system with omnidirectional ultra-high wave vector illumination provided by an embodiment of the present invention.

[0040] Figure 3 It is a schematic diagram of a method for generating omnidirectional TM polarized excitation light in a deep frequency-shifted super-resolution microscopic imaging system with omnidirectional ultra-high wavevector illumination provided by an embodiment of the present invention.

[0041] Figure 4 It is a schematic flow chart of a method for deep frequency shift super-resolution microscopic imaging with omnidirectional ultra-high wave vector illumination provided by an embodiment of the present invention.

[0042] Figure 5 It is a schematic diagram comparing the spectrum range that can be detected by the imaging system and the imaging chip provided by the embodiment of the present invention with the spectrum range that can be detected by the existing frequency shift illumination method. DETAILED DESCRIPTION

[0043] To make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0044] Figure 1 Schematic diagram of the structure of the deep frequency shift super-resolution microscopic imaging chip with omni-directional ultra-high wave vector illumination and the structure of the circular ring grating in the chip provided by the embodiment of the present invention. Figure 1 As shown, the embodiment provides a deep frequency-shifted super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination, comprising: a substrate coated with an adhesion layer and a multilayer film on the adhesion layer, and a sub-wavelength-scale circular grating is engraved on the substrate and the adhesion layer.

[0045] Among them, the omnidirectional TM polarized excitation light is incident on the annular grating through the substrate, and the first diffraction order generated by the annular grating is the 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. Taking into account the actual micro-nano processing steps, it is first necessary to plate an adhesion layer on the substrate to enhance the stability of the metal film above. The adhesion layer selected in this embodiment is a chromium film with a thickness of 2nm. Taking into account the diffraction efficiency of the grating, the evanescent wave of its first diffraction order is used to match the wave vector on the surface of the film. The formula for wave vector matching is expressed as: where k spp is the transverse wave vector of the SPP generated on the surface of the multilayer film, k0 is the wave vector of the excitation light in free space, n is the refractive index of the medium in the space where the excitation light is located, θ is the incident angle of the excitation light, λ is the wavelength of the excitation light, and d is the period of the annular grating. The diameter d of the annular grating to be processed grating The beam diameter d of the excitation light should be illu Match, d grating ≤d illu .

[0046] The metal-medium-metal MIM structure is processed sequentially on the circular grating, and finally an ultra-high transverse wave vector SPP mode is generated between the top metal film and the air medium. The ultra-high transverse wave vector k x The effective refractive index n of the entire MIM structure is eff To indicate, n eff =k x / k0. Metal materials should be selected with a complex refractive index in the excitation light band. The selection of dielectric materials should take into account the wavelength of the excitation light and be determined according to the cutoff wavelength of its SPP mode. In this embodiment, Ag is used as the metal material and TiO2 or GaN is used as the dielectric material. The thickness of the metal and the dielectric is ultimately related to the ultra-high transverse wave vector excited. According to Maxwell's equations and continuity conditions, k1, k2, k3 of each interface in the multilayer film and the SPP transverse wave vector k excited on the surface are calculated in turn. spp .

[0047] The chip processing method in this embodiment is as follows: According to the geometric thickness of metal and dielectric, this embodiment uses atomic layer deposition (ALD) process to deposit multi-layer thin films; according to k spp The value is used to calculate the period of the annular grating and determine the processing technology of the grating. In this embodiment, the annular grating is processed by electron beam lithography (EBL). Table 1 shows the MIM structure composed of dielectric materials and metal materials (the metal layer in contact with the adhesion layer is 50nm, the dielectric layer is 10nm, and the upper surface metal layer is 10nm) at a laser wavelength of 561nm, as well as its effective refractive index, the ultra-high wave vector that can be generated, and the period of the annular grating.

[0048] Table 1 Chip film composition, effective refractive index, transverse wave vector and grating period

[0049]

[0050] Figure 2 Schematic diagram of a deep frequency shift super-resolution microscopic imaging system with omnidirectional ultra-high wave vector illumination provided by an embodiment of the present invention. Figure 2 As shown, the embodiment also provides a deep frequency shift super-resolution microscopic imaging system with omnidirectional ultra-high wave vector illumination, including: an excitation module, a deep frequency shift super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination, an imaging module, and an excitation control and image acquisition module. Specifically, it includes: a laser light 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, a deep frequency shift super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination 207, an imaging objective lens 208, a reflector 209, a tube lens 210, a camera 211, a computer 212, and a galvanometer drive 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 the embodiment, the laser light source 201 uses a 561nm single-mode fiber output 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 1 / 4 wave plate, modulates the polarization state of the excitation light to TM polarization when it is incident on the deep frequency shift super-resolution microscopy imaging chip 207 with all-round ultra-high wave vector illumination. The all-round galvanometer scanning unit 204 includes two one-dimensional scanning galvanometers and a 4F optical system between the two one-dimensional scanning galvanometers. The scanning axes of the two one-dimensional scanning galvanometers should be perpendicular to each other, and the reflection surfaces of the two one-dimensional scanning galvanometers are located on the conjugate plane of the 4F optical system. The scanning lens 205 generates a uniform convergence point in the rear focal plane of the excitation objective lens 206. The excitation objective lens 206 is used to generate excitation light with a large angle of incidence, and at the same time, its exit pupil center should be at the center of the circular grating of the deep frequency shift super-resolution microscopy imaging chip 207 with all-round ultra-high wave vector illumination. In the embodiment, a one-dimensional scanning galvanometer with a working size of 10 mm and a scanning lens with a focal length of 200 mm are selected, and the diameter of the beam incident on the grating at the bottom of the chip is d grating ≤90μm.

[0052] In the embodiment, under the laser light source with a wavelength of 561 nm, the excitation lens 206 selected in this embodiment has a numerical aperture NA illu = 1.49, the magnification is 100× for an Olympus oil immersion objective, and the image space medium is immersion oil (its refractive index n e =1.518), and accordingly, the substrate material is SCHOTT N-BK7 glass (its refractive index n e =1.51872). Figure 3 As shown, the plane 215 where the chip is placed coincides with the focal plane of the excitation objective lens 206. By controlling the two-dimensional scanning of the omnidirectional galvanometer scanning unit 204, the parallel light beams converge on the back focal plane 214 of the excitation objective lens 206 through the scanning lens 205 and perform annular scanning, and finally emit parallel light with a large angle from the excitation objective lens 206, thereby exciting the deep frequency shift super-resolution microscopic imaging chip 207 with omnidirectional ultra-high wave vector illumination to generate ultra-high wave vector illumination light. With the two-dimensional scanning of the omnidirectional galvanometer scanning unit 204, the large-angle excitation light is incident on the annular grating area 216 at the bottom of the deep frequency shift super-resolution microscopic imaging chip 207 with omnidirectional ultra-high wave vector illumination, exciting the omnidirectional ultra-high wave vector illumination field, so as to illuminate the sample with a complex fine structure placed on the upper surface of the multilayer film in the deep frequency shift super-resolution microscopic imaging chip 207 with omnidirectional ultra-high wave vector illumination.

[0053] In the embodiment, the omnidirectional scattered signal of the sample is received by the imaging objective lens 208 and transmitted to the tube lens 210 through the reflector 209 to form a real image of the sample. The tube lens 210 presents the real image of the sample on the camera 211, and the camera 211 collects the deep frequency shift illumination image. The camera 211 selects an sCMOS scientific research camera. The numerical aperture of the imaging objective lens 208 is NA img =0.8, when using the two ultra-high wave vector illumination chips described in Table 1 to illuminate samples without fluorescent labels, the achievable resolutions are 43.59nm and 50.72nm respectively, where the resolution calculation formula is: It is expected that this system can achieve super-resolution microscopic imaging with sub-λ / 10 (56.1nm) resolution.

[0054] In the embodiment, the one-dimensional scanning galvanometer is driven and controlled by the galvanometer drive and control card 213 to realize two-dimensional scanning of the light beam, and the camera control software in the computer 212 is used to synchronously save the deep frequency shift illumination image and process it to obtain the deep frequency shift super-resolution microscopic image for subsequent analysis.

[0055] According to the above-mentioned omnidirectional ultra-high wave vector illumination deep frequency shift super-resolution microscopic imaging system, the embodiment of the present invention also provides an omnidirectional ultra-high wave vector illumination deep frequency shift super-resolution microscopic imaging method, such as Figure 4 As shown, the following steps are included:

[0056] S1, adjusting the spot size of the laser emitted by the laser light source to a spot size that matches the working size of the one-dimensional scanning galvanometer, and adjusting the polarization state at the deep frequency-shifted super-resolution microscopy imaging chip with all-round ultra-high wave vector illumination to TM polarization;

[0057] S2, placing the sample with complex fine structure to be imaged on the upper surface of the multilayer film in the deep frequency shift super-resolution microscopy imaging chip with omnidirectional ultra-high wave vector illumination;

[0058] S3, controlling the omnidirectional galvanometer scanning unit to perform two-dimensional scanning on the back focal plane of the excitation objective lens, exciting omnidirectional ultra-high surface plasmons with ultra-high transverse wave vectors to illuminate the sample, and then the imaging objective lens 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 synchronously. According to the nature of whether the sample is fluorescently labeled, the corresponding reconstruction algorithm is used to solve the deep frequency shift super-resolution microscopic image.

[0060] In order to verify the effect of the chip and system provided by the present invention, a sample with a complex fine structure is placed on the imaging chip, and the sample is illuminated by omnidirectional ultra-high wave vector light, and the numerical aperture NA img=0.8 received by the imaging objective lens of the sample in all directions of deep frequency shift illumination. Comparison of the extreme spectrum range 501 detected by spatial free light large angle all directions of frequency shift illumination (without chip), the extreme spectrum range 502 detected by single layer of natural high refractive index waveguide GaP frequency shift illumination (without circular grating and multilayer film structure), the spectrum range 503 detected by ultra-high wave vector frequency shift illumination using groove-excited multilayer film structure, and the spectrum range 504 detected by all directions of ultra-high wave vector frequency shift illumination using the chip in this embodiment. Figure 5 shown. Figure 5 The spectrum shifted by ultra-high wave vector light illumination in a single azimuth will overlap with the spectrum shifted by illumination in an adjacent azimuth, and the amount of overlap depends on the step size of the large-angle annular light excitation, that is, 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 spectrum information of the fine structure of the sample can be detected. Compared with the existing frequency-shifting illumination method, the chip in this embodiment can increase the spectrum range detected by the existing microscope by multiples (about 4 times compared to GaP waveguide illumination, about 12 times compared to spatial free light illumination), and combined with the hardware settings of the imaging system, it can perform all-round fine ultra-high spectrum detection.

[0061] This embodiment fully demonstrates the advantages of the present invention in super-resolution imaging of sub-λ / 10 complex fine structure samples, so that samples with complex and fine structures can be observed and analyzed with a resolution that exceeds that of traditional imaging technologies, to meet the application needs of fields such as biomedicine and materials science for higher-definition and more widely applicable imaging technologies.

[0062] The specific implementation methods described above provide a detailed description of the technical solutions and beneficial effects of the present invention. It should be understood that the above is only the most preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, supplements and equivalent substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A deep frequency shift super-resolution microscopic imaging 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, wherein a sub-wavelength-scale circular grating is engraved on the substrate and the adhesive layer; Among them, the excitation light is incident on the circular grating through the substrate, and evanescent waves are generated in various directions through the circular grating. The evanescent waves are coupled to the upper surface through the multilayer film to excite surface plasmons with ultra-high lateral wave vectors in the corresponding direction, which are used for all-round ultra-high wave vector illumination and deep frequency-shift super-resolution microscopy imaging of the sample placed on the upper surface of the multilayer film.

2. The deep frequency shift super-resolution microscopy imaging chip with omni-directional ultra-high wave vector illumination according to claim 1, characterized in that: The excitation light is annular large-angle excitation light with omnidirectional TM polarization.

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

4. The deep frequency shift super-resolution microscopy imaging chip with omni-directional ultra-high wave vector illumination according to claim 1, characterized in that: The evanescent wave of the first-order diffraction of the ring grating is used to match the transverse wave vector of the surface plasmon on the surface of the multilayer film. The formula for wave vector matching is: Among them, k spp is the transverse wave vector of the surface plasmon generated on the surface of the multilayer film, k0 is the wave vector of the excitation light in free space, n is the refractive index of the medium in the space where the excitation light is located, θ is the incident angle of the excitation light, λ is the wavelength of the excitation light, and d is the period of the circular grating.

5. The deep frequency shift super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination according to claim 1, characterized in that: The material of the substrate is transparent in the selected excitation light wavelength band.

6. The deep frequency shift super-resolution microscopic imaging chip with omni-directional ultra-high wave vector illumination according to claim 1, characterized in that: The multilayer film is a multilayer structure in which metal film layers and dielectric film layers are alternately arranged, and both the uppermost layer and the lowermost layer are metal film layers.

7. The deep frequency shift super-resolution microscopic imaging chip with omni-directional ultra-high wave vector illumination according to claim 6, characterized in that: The metal film layer is made of a metal material with a complex refractive index and low loss, and the dielectric film layer is made of a dielectric material with a high refractive index.

8. An omnidirectional ultra-high wave vector illumination deep frequency shift super-resolution microscopic imaging system, using the omnidirectional ultra-high wave vector illumination deep frequency shift super-resolution microscopic imaging chip according to any one of claims 1 to 7, characterized in that: include: Excitation module, deep frequency shift super-resolution microscopy imaging chip with all-round ultra-high wave vector illumination, imaging module, and excitation control and image acquisition module; The excitation module includes a laser light 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 in the rear focal plane of the excitation objective lens, and an excitation objective lens for emitting the converged large-angle incident excitation light onto a deep frequency-shifted super-resolution microscopic imaging chip with omnidirectional ultra-high wave vector illumination; 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 between the two one-dimensional scanning galvanometers; The upper surface of the multilayer film in the deep frequency-shifted super-resolution microscopic imaging chip with all-round ultra-high wave vector illumination is used to place the sample; The imaging module includes an imaging objective lens and a matching reflector, a tube lens, and a camera, which are arranged in sequence, and are used to receive the omnidirectional scattering signal of the sample and transmit it to the camera to obtain a deep frequency shift illumination image; The excitation control and image acquisition module includes a galvanometer drive and control card and a computer. The galvanometer drive and control card drives and controls the one-dimensional scanning galvanometer to achieve two-dimensional scanning of the light beam, and the camera control software in the computer is used to synchronously save the deep frequency shift illumination image and process it to obtain the deep frequency shift super-resolution microscopic image for subsequent analysis.

9. The deep frequency shift super-resolution microscopic imaging system with omni-directional ultra-high wave vector illumination according to claim 8, characterized in that: The beam diameter d of the excitation light with large incident angle generated by the excitation objective illu The diameter d of the annular grating of the deep frequency-shifted super-resolution microscopy imaging chip with omnidirectional ultra-high wave vector illumination grating Match, satisfy where f illu represents the equivalent focal length of the excitation objective, f scan represents the focal length of the scanning lens, and d1 represents the spot diameter of the parallel light emitted by the collimating lens.

10. A method for deep frequency shift super-resolution microscopy with omnidirectional ultra-high wave vector illumination, implemented by using the deep frequency shift super-resolution microscopy with omnidirectional ultra-high wave vector illumination system according to claim 8 or 9, characterized in that: The following steps are involved: The spot size of the laser emitted by the laser light source is adjusted to match the working size of the one-dimensional scanning galvanometer, and the polarization state at the deep frequency-shifted super-resolution microscopy imaging chip with all-round ultra-high wave vector illumination is adjusted to TM polarization; The sample with complex fine structure to be imaged is placed on the upper surface of the multilayer film in the deep frequency shift super-resolution microscopy imaging chip with omnidirectional ultra-high wave vector illumination; Control the omnidirectional galvanometer scanning unit to perform two-dimensional scanning on the back focal plane of the excitation objective lens, and excite omnidirectional ultra-high surface plasmons with ultra-high transverse wave vectors to illuminate the sample, and then the imaging objective lens 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 synchronously. Depending 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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