A frequency-shifted label-free far-field super-resolution optical wide-field microscopic imaging method

Abstract

The application discloses a frequency-shifted label-free far-field super-resolution optical wide-field microscopic imaging method, which is based on large-area uniform high-frequency body plasmon (BPPs) mode field excited by metal / dielectric multilayer film structure material to perform frequency-shifted illumination on a sample, a plurality of images containing low-frequency and high-frequency information of the sample are obtained by adjusting an illumination wave vector, a series of picture intensity distributions with some overlaps in a frequency domain space are iteratively spliced, a spatial spectrum of the object covering a maximum detectable wave vector range is obtained, and finally a super-resolution label-free sample microscopic image is reconstructed by inverse Fourier transform. The application has the advantages of novel principle, simple structure, easy operation, wide working bandwidth, high efficiency, low cost and the like, and opens up a new idea for realizing higher-quality label-free optical microscopic imaging.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microscopic imaging, and relates to a frequency-shifted label-free super-resolution microscopic imaging method, in particular to a frequency-shifted label-free far-field super-resolution optical wide-field microscopic imaging method. BACKGROUND

[0002] Traditional optical systems are limited by the Abbe diffraction limit. How to improve the spatial resolution to below 100 nm or even to the molecular scale and realize high-contrast super-resolution microscopic imaging has become one of the main research directions of optical microscopic imaging technology. Super-resolution microscopic imaging technology is divided into fluorescent labeling and non-fluorescent labeling methods. Super-resolution fluorescence microscopic technology can improve the spatial resolution to the nanometer level, but super-resolution fluorescence microscopic technology relies too much on fluorescent dyes, and at the same time, fluorescent dyes have many problems such as long time consumption, inactivation, uneven staining, and photobleaching when labeling samples, which limits the application and development of super-resolution fluorescence microscopic technology. More worrying is that when the sample is a non-biological sample such as a micro-nano structure material or an integrated circuit chip that cannot be dyed, super-resolution fluorescence microscopic technology is no longer applicable.

[0003] In label-free microscopic imaging technology, frequency-shifted technology can break through the bandwidth limitation of traditional imaging and detection devices, and has significant advantages in large field of view and high speed. The frequency-shifted label-free super-resolution microscopic method uses evanescent wave illumination to shift the high-frequency spatial information of the sample to the low-frequency passband range that can be received by the microscope, and restores the super-resolution optical microscopic image through a frequency spectrum splicing algorithm. The frequency-shifted label-free super-resolution microscopic method does not require fluorescent labeling and can realize super-resolution imaging of non-biological samples, and has a wide application prospect.

[0004] The existing frequency-shifted label-free super-resolution microscopic technology includes nanometer optical fiber illumination super-resolution technology, nanowire ring illumination super-resolution microscopic technology, and waveguide grating illumination super-resolution microscopic technology. The above existing methods all belong to local illumination methods, and the illumination field area is limited, the evanescent wave excitation efficiency is limited, the spatial spectrum is not pure enough, and the transverse resolution improvement capability is limited by the refractive index of the substrate medium material. In addition, these methods also have certain difficulties in practical application, for example, the nanowire ring illumination method is not conducive to large-scale integrated production. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application provides a frequency-shifted label-free far-field super-resolution optical wide-field microscopic imaging method, which is based on large-area uniform high-frequency body plasmon (BPPs) mode field excited by a metal / dielectric multilayer film structure material to perform frequency-shifted illumination on a sample, a plurality of images containing low-frequency and high-frequency information of the sample are obtained by adjusting the illumination wave vector, the image intensity distribution of a series of images with overlapping frequency spectrum is iteratively spliced in the frequency domain space to obtain the object space spectrum covering the maximum detectable wave vector range, and finally an undeformed sample microscopic image with super-resolution is reconstructed by inverse Fourier transform.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] An illuminating device for frequency-shifted label-free far-field super-resolution optical wide-field microscopic imaging comprises, from bottom to top, a transparent substrate layer, an excitation grating and a metal / dielectric multilayer film structure layer, wherein:

[0008] The transparent substrate layer is processed with the excitation grating, and the metal / dielectric multilayer film structure layer is arranged on the excitation grating, and the metal / dielectric multilayer film structure layer is formed by alternately stacking a metal film layer and a dielectric film layer;

[0009] The illumination light source is irradiated from the back of the transparent substrate to the surface of the excitation grating, and the high-frequency diffraction subwave excited by the grating structure only has one 1st or 2nd order falling into the band-pass range of the metal / dielectric multilayer film.

[0010] A method for frequency-shifted label-free far-field super-resolution optical wide-field microscopic imaging using the above-mentioned illuminating device, comprising the following two methods:

[0011] Method A,

[0012] Step one, design two spatial filter windows [-kx max ,-kx min ] and [kx min ,kx max ] of high-frequency BPPs in the metal / dielectric multilayer film layer structure, the wavelength bandwidth of the illumination light source, and filter out the -1st order in the excitation grating diffraction order:

[0013] The vacuum wave vector under the working wavelength λ of the illumination light source is k0, the incident angle of the central light is θ, the excitation grating wave vector is k g , the refractive index of the substrate is n, and the design rules of the two spatial filter windows [-kx max ,-kx min ] and [kx min ,kx max ] of high-frequency BPPs in the metal / dielectric multilayer film layer structure and the wavelength bandwidth of the illumination light source are as follows:

[0014] 1) Design the excitation grating period to make kg High frequency cutoff frequency kx of BPPs window at working wavelength λ max ;

[0015] 2) Selection of incident angle θ range: at working wavelength λ, select nsinθk0-k g of wave vector to be located in BPPs window [-kx max , -kx min ] at this wavelength, while satisfying nsinθk0-mk g located outside two BPPs windows for any positive or negative integer m not equal to 1;

[0016] 3) The design rule for any angle and wavelength combination within the wavelength bandwidth of the illumination light source satisfies rule 2);

[0017] In this step, the excitation grating structure can also be designed to cooperate with the spatial filtering window of BPPs in the metal / dielectric multilayer film structure, so that the high-frequency BPPs field is excited by filtering out the-2 order;

[0018] In this step, the above rules apply to the case of one-dimensional excitation grating. When a two-dimensional excitation grating is used, the above design method is still followed, and the two-dimensional excitation grating satisfies the excitation of high-frequency BPPs in each direction, but the grating structure such as grating ring group needs to be redesigned, and then the appropriate direction angle illumination is selected to cooperate with the grating, and only one order is still filtered out;

[0019] Step two, place the sample of microscopic imaging on the metal / dielectric multilayer film structure;

[0020] Step three, use a single-wavelength light source as an illumination light source, and tilt the excitation grating from the center of the transparent substrate layer at an incident angle θ designed in step one, with a maximum angle adjustment range of 0°-90°. After the high-frequency diffraction sub-wave excited by the grating structure is spatially filtered by the metal / dielectric multilayer film structure, a large-area and uniform high-frequency BPPs mode field is excited. Based on the large-area and uniform high-frequency BPPs mode field excited by the metal / dielectric multilayer film structure material, the sample is frequency-shifted, and a series of images containing low-frequency and high-frequency information of the sample are obtained by adjusting the illumination wave vector. The intensity distribution of the pictures with some overlap in the frequency domain is iteratively spliced to obtain the object spatial spectrum covering the maximum detectable wave vector range. Finally, the super-resolution and non-distortion sample microscopic image is reconstructed by inverse Fourier transform, wherein:

[0021] The illumination wave vector is continuously adjusted with the change of angle, and the purity of the illumination wave vector depends on the cooperation design of the excitation grating period and the spatial spectrum filtering passband of the metal / dielectric multilayer film structure;

[0022] The incident angle and wavelength of the illumination source will change the wave vector of the BPPs. By changing the incident angle and wavelength, multiple images under different BPPs independent illumination can be obtained. The adjustment range of the BPPs wave vector needs to include low and high frequencies. At this time, the spatial frequency range that can be detected will also shift with the BPPs wave vector. According to the detection frequency spectrum corresponding to different BPPs, the frequency spectrum is recovered.

[0023] Method B,

[0024] Step one, design two spatial filtering windows [-kx max ,-kx min ] and [kx min ,kx max ] of the high-frequency BPPs in the metal / dielectric multilayer film layer structure, the wavelength bandwidth of the illumination source, and filter out the +1 order in the excited grating diffraction order:

[0025] The vacuum wave vector under the working wavelength λ of the illumination source is k0, the incident angle of the central light is θ, the wave vector of the excited grating is k g , the refractive index of the substrate is n, and the design rules of the two spatial filtering windows [-kx max ,-kx min ] and [kx min ,kx max ] of the high-frequency BPPs in the metal / dielectric multilayer film layer structure and the wavelength bandwidth of the illumination source are as follows:

[0026] 1) Design the excited grating period so that k g is located in the [kx min ,kx max ] of the BPPs window under the working wavelength λ;

[0027] 2) Selection of the incident angle θ range: under the working wavelength λ, select nsinθk0+k g is located in the corresponding wavelength BPPs window [kx min ,kx max ], while satisfying nsinθk0+mk g is located outside the two BPPs windows for any positive or negative integer m not equal to 1;

[0028] 3) The design rules for any angle and wavelength combination within the wavelength bandwidth of the illumination source satisfy rule 2);

[0029] In this step, the excited grating structure can also be designed to cooperate with the spatial filtering window of the BPPs in the metal / dielectric multilayer film layer structure, so that the +2 order is filtered out to realize the excitation of the high-frequency BPPs field;

[0030] In this step, the above rules apply to the case of one-dimensional excitation grating, when using two-dimensional excitation grating, still follow the above design method, two-dimensional excitation grating in each direction meets the excitation of high frequency BPPs in this direction, but the grating structure needs to be redesigned, such as grating ring group, and then cooperate with the grating direction to select the appropriate direction angle illumination, still only filter out one order;

[0031] Step two, place the sample of microscopic imaging on the metal / dielectric multilayer film structure layer;

[0032] Step three, use a variety of wavelength lasers (different laser wavelength range <400nm) or a wide waveband small divergence angle visible light source (light source wavelength bandwidth <400nm) as an illumination light source, tilt the excitation grating from the center of the bottom of the transparent substrate layer along the incident angle θ designed in step one, the maximum angle adjustment range is 0°-90°, after the high frequency diffraction subwave excited by the grating structure is filtered by the metal / dielectric multilayer film structure, a large area and uniform high frequency BPPs mode field is excited, based on the large area and uniform high frequency BPPs mode field excited by the metal / dielectric multilayer film structure material, the sample is frequency shifted, a plurality of images containing low frequency and high frequency information of the sample are obtained by adjusting the illumination wave vector, the picture intensity distribution of a series of frequency spectrum is iteratively spliced in the frequency domain space, the object spatial spectrum covering the maximum detectable wave vector range is obtained, and finally the super-resolution and non-distortion sample microscopic image is reconstructed by inverse Fourier transform.

[0033] The illumination wave vector will be adjusted according to the working performance of the illuminator at different wavelengths;

[0034] The change of the incident angle and wavelength of the illumination light source will cause the change of the wave vector of BPPs, by changing different incident angles and wavelengths, a plurality of images under the independent illumination of different BPPs can be obtained; the adjustment range of the wave vector of BPPs needs to include low frequency and high frequency, at this time, the detectable spatial spectrum range will also be translated with the wave vector of BPPs, and the frequency spectrum is recovered according to the detection spectrum corresponding to different BPPs.

[0035] In the application, the illumination light source can be incoherent, natural polarized light, laser, and the wavelength range is from ultraviolet to visible light.

[0036] In the application, the monochromatic light source can be a mercury lamp, a halogen lamp, a monochromatic LED lamp or an argon lamp with a filter.

[0037] In the application, when the illumination light source is an infrared waveband light source, a hyperbolic dispersion material under the corresponding working waveband can be used to replace the metal / dielectric multilayer film structure material to realize the spatial filtering characteristic.

[0038] In the present application, by matching multiple wavelengths and multiple illumination angles, different wave vector size illumination BPPs are provided, and when illuminating a sample, the corresponding low frequency and high frequency spatial information is respectively collected in the far field, the picture intensity distribution with a series of overlapping frequency spectrum is iteratively spliced in the frequency domain space, the object spatial spectrum covering the maximum detectable wave vector range is obtained, and finally the super-resolution non-deformation sample microscopic image is reconstructed by inverse Fourier transform.

[0039] In the present application, the metal in the metal / dielectric multilayer film structure can be a good conductor material: Au, Ag or Al, and the thickness is more than 10nm; the dielectric can be a low-loss optical film layer material, including but not limited to SiO2, and can contain higher refractive index materials such as SiC, etc., and the thickness is more than 10nm.

[0040] In the present application, the material used for the excitation grating can be a high refractive index medium with n>2, or a metal, including but not limited to Ag, Si, Cr or Cu.

[0041] In the present application, the excitation grating is used for modulating the spatial spectrum of the illumination light, and its structure includes but is not limited to one-dimensional metal structure, two-dimensional metal structure, one-dimensional high refractive index medium grating structure, two-dimensional high refractive index medium grating structure, and can also be a uniform distribution of nanostructures composed of non-transparent materials (metal materials, etc.), which can be one-dimensional nanostructure or two-dimensional nanostructure, and the size and spacing are not greater than the wavelength of the illumination light, and the shape can be regular geometric bodies such as ring grating structure or irregular arbitrary surface type.

[0042] In the present application, changing the size and spacing of the nanostructure in the excitation grating can adjust the spatial frequency and transmission efficiency of the excitation diffraction sub-wave.

[0043] In the present application, changing the metal material and dielectric material in the metal / dielectric multilayer film structure or changing the thickness of the metal film layer and the dielectric film layer or changing the film number of the metal / dielectric multilayer film structure can adjust the window position and cutoff frequency of the spatial bandpass filter.

[0044] In the present application, a transparent dielectric layer can be separately applied as a protective layer outside the uppermost metal film layer of the metal / dielectric multilayer film structure, and the thickness is 10nm-40nm.

[0045] In the present application, the excitation grating is prepared by magnetron sputtering, EBL, FIB, photolithography or nanoimprint, the filling is performed by flip process or filling process, and the metal / dielectric multilayer film structure is prepared by magnetron sputtering, thermal evaporation and other film coating technologies.

[0046] The principle of the present application is as follows:

[0047] Monochromatic transverse magnetic plane wave is irradiated to the excitation grating surface from the back of the substrate, and the high-frequency diffraction sub-wave excited by the grating structure is filtered by the metal / dielectric multilayer film structure in the spatial spectrum, thereby exciting a large-area and uniform high-frequency BPPs mode field. The BPPs mode is generated by the mutual coupling of surface plasmon (SP) fields existing at the interfaces between adjacent metal / dielectric layers in the metal / dielectric multilayer film structure. Unlike the characteristic of the SP mode being localized on the surface of the metal film layer, the BPPs mode penetrates the entire three-dimensional artificial electromagnetic super material space, presents a super-diffraction transmission characteristic in space in the metal / dielectric multilayer film structure, and presents an exponential decay when leaving the metal / dielectric multilayer film structure, thereby showing an evanescent wave characteristic. Through the design of the metal / dielectric multilayer film structure, the wave vector of the BPPs is much higher than that of the evanescent wave generated by total reflection. Based on the equivalent medium theory (EMT), the metal / dielectric multilayer film structure is approximated as an anisotropic medium with hyperbolic dispersion characteristics, and the spatial frequency k x The high-pass filter characteristic is that the low-frequency BPPs mode and the SP mode localized on the surface of the metal layer cannot be transmitted in the artificial electromagnetic super material, and only the high-frequency BPPs mode higher than the cutoff frequency can transmit through the metal / dielectric multilayer film structure and be used as an illumination field for illuminating a sample. When a grating or other nanostructure is used, the high-frequency diffraction sub-wave generated after illumination can be coupled to the BPPs mode in the metal / dielectric multilayer film structure. Through the design of a large-area grating or other nano-excitation structure in cooperation with the metal / dielectric multilayer film structure, a large-area and uniform high-frequency BPPs mode field can be excited, which is used as a wide-field microscopic imaging illumination source for a label-free sample. By cooperating with multiple wavelengths and multiple illumination angles to provide illumination BPPs with different wave vectors, the low-frequency and high-frequency spatial information of the far field is collected respectively when illuminating the sample, the picture intensity distribution with a series of overlapping frequency spectrums is iteratively spliced in the frequency domain space, the object spatial spectrum covering the maximum detectable wave vector range is obtained, and finally the super-resolution and non-distortion sample microscopic image is reconstructed through inverse Fourier transform.

[0048] Compared with the prior art, the present application has the following advantages:

[0049] 1. The present application directly uses a large-area ultraviolet interference lithography technology or other methods to prepare an excitation grating structure on an entire transparent substrate material, and has the characteristics that a large-area and uniform high-frequency BPPs mode field can be excited through the design of a large-area grating or other nano-excitation structure in cooperation with the metal / dielectric multilayer film structure, which is used as a wide-field microscopic imaging illumination source for a label-free sample, the transverse wave vector of which is much higher than that of the evanescent wave supported by a waveguide or other dielectric material, the resolution is greatly improved, and the resolution is theoretically estimated to be improved to λ / 7.82, which is about 4 times that of an ordinary microscope.

[0050] 2. The present application can realize wide-field illumination through the grating structure located above the entire substrate material.

[0051] 3. The illumination structure can be effectively separated from the sample, and the imaging area can be cleaned without damaging the illumination structure, facilitating reuse of the illumination structure;

[0052] 4. The method is conducive to integrated mass production and cost reduction;

[0053] 5. The application has the advantages of novel principle, simple structure, easy operation, wide working bandwidth, high efficiency and low cost, and opens up a new way for realizing higher quality label-free optical microscopic imaging. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 It is a structural schematic diagram of the illuminating device of the application, in which: 1 is a transparent substrate layer, 2 is an excitation grating, 3 is a dielectric film layer, 4 is a metal film layer, and 5 is a sample.

[0055] Figure 2 It is a label-free microscopic imaging schematic diagram of the illuminating device of the application.

[0056] Figure 3 It is a BPPs excitation structural schematic diagram of the embodiment 1 of the application, (a) is a BPPs excitation structural schematic diagram, (b) is an OTF of an Ag / SiO2 metal / dielectric multilayer film structure under TM and TE polarization, (c) is an OTF of-1, 0 and +1 order, and (d) is an OTF ratio of different diffraction orders.

[0057] Figure 4 It is a far-field imaging simulation process and result of a 100nm center distance double-slit structure under BPPs illumination in the embodiment 1 of the application, (a) is an x-z cross-section light intensity distribution of evanescent waves scattered to the far field by the BPPs structure surface monitored by a cross-section monitor using the FDTD method, (b) is a far-field imaging light intensity distribution of the 100nm center distance double-slit structure, (c) is a frequency spectrum of the double-slit structure, and (d) is a double-slit structure scattering light frequency spectrum detected by the far field.

[0058] Figure 5 It is a far-field imaging contrast under double-slit center distance change in the embodiment 1 of the application, (a) is a far-field contrast change curve under 80-120nm center distance change, and (b) is a far-field imaging normalized intensity of the double-slit structure with a center distance of 90-110nm.

[0059] Figure 6 It is an OTF of the Ag / SiO2 metal / dielectric multilayer film structure in the embodiment 1 of the application in the wavelength range of 380-780nm.

[0060] Figure 7For the super-resolution label-free microscopic image under BPPs illumination of transverse wave vector 3.86k0 in Example 2 of the present application, (a) OTF of TM polarized incident hyperbolic dispersion material; (b) OTF of -1, 0 and +1 order; (c) far-field imaging light intensity distribution of double-slit structure with a center distance of 68 nm. DETAILED DESCRIPTION

[0061] The technical solutions of the present application are further described below in conjunction with examples, but are not limited thereto, and any modification or equivalent replacement to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application shall be encompassed in the protection scope of the present application.

[0062] Example 1

[0063] This example takes a device capable of generating a pure BPPs illumination field of 2.66k0 and used for double-slit micro-nano structure illumination with a center distance of 100 nm to realize far-field label-free microscopic imaging as an example.

[0064] The illumination light is a TM plane wave with a wavelength of 532 nm, and the device is sequentially composed of a SiO2 substrate, a periodic sub-wavelength Ag grating, and an Ag / SiO2 metal / dielectric multilayer structure from bottom to top. The BPPs excitation structure design is shown in Figure 3 (a), wherein the metal / dielectric multilayer structure is alternately stacked by 10 layers of 20 nm thick Ag films and 9 layers of 50 nm thick SiO2 films, the periodic sub-wavelength Ag grating has a period of 170 nm and a thickness of 40 nm, and the duty cycle is 1:1. At a wavelength of 532 nm, the dielectric constants of Ag and SiO2 are -10.18+0.83i and 2.13, respectively.

[0065] In the process of microscopic imaging, the illumination object excites scattered light, which carries the high-frequency spatial structure information of the sample. The relationship between the scattered light collected in the far field and the spatial structure information of the object participating in imaging can be approximately explained by the Edvald reflection sphere model, which is expressed as the product of the Fourier space spectrum of the object scattering potential at the illumination wave vector and a low-pass function, wherein the value range of the low-pass function is |k s |<NAk0, k s is the transverse wave vector of the scattered light, k0=2π / λ is the object space optical wave vector, and NA is the numerical aperture of the objective lens. The transverse wave vector k x of the conventional illumination light is at most NAk0, as shown by the dashed line in Figure 2 (a), and the gray shaded area range is the value range of the scattered light k s that can be detected in the far field, and the maximum range that can be detected in the far field can be up to 2NAk0, but because of the loss of high-order Fourier components, the far-field imaging resolution is limited to the diffraction limit. When evanescent wave illumination is used, because the transverse wave vector keva greater than k0, the center of the spectrum carried by the far-field scattered field is shifted to the thin solid line, as Figure 2 (b) the thick solid line, the maximum spatial frequency that can be detected in the far field is extended to the thick solid circle, k eva + NAk0. At this time, more high-frequency information outside the imaging system cutoff frequency is translated into the passband of the imaging system when the evanescent wave illuminates the object, so that the maximum detectable spatial frequency of the entire optical system is expanded, thereby overcoming the diffraction limit and significantly improving the resolution of microscopic far-field imaging. In general, the evanescent wave excitation method, k eva limited by the waveguide or the refractive index of the medium material, so the BPPs provides an illumination source with higher transverse spatial wave vector. Figure 2 (d) is a schematic diagram of imaging a sample using BPPs illumination. The BPPs illumination source is composed of a hyperbolic dispersion material combined with a subwavelength grating. TM polarized waves are incident at a specific angle to excite multiple diffraction order subwaves containing high frequency diffraction waves by illuminating the subwavelength grating. Spatial spectrum filtering is achieved through the hyperbolic dispersion material, which filters out stray orders, thereby exciting a large-area single wave vector pure BPPs field on the exit surface. Illuminating the object can translate high-frequency spatial information into the passband of the far-field detection imaging system, thereby improving imaging resolution.

[0066] The filtering characteristics of the high-frequency BPPs excitation structure are analyzed in detail using the rigorous coupled wave analysis method. When a TM polarized light illuminates a metal / dielectric multilayer film structure, such as Figure 3 (b) the thick solid line, at this time the OTF presents a spatial spectrum band-pass filter passband with a window of [-3.12k0, -1.46k0], [1.46k0, 3.12k0], which means that high-frequency wave vectors that meet the range can propagate in the hyperbolic dispersion material, while TE polarized light is incident, such as Figure 3 (b) the thin solid line, at this time the overall transmittance is approximately 0. When a subwavelength nano-excitation grating is loaded, multiple diffraction order subwaves will be excited under TM wave illumination, and their transverse wave vectors can be determined by the grating formula:

[0067] k x,m = k inc + mk g ;

[0068] where m is the diffraction order of the subwave, and

[0069] k inc = nsinθk0;

[0070]

[0071] At normal incidence, the -1, 0, +1 order transverse wave vectors are -3.12k0, 0k0, 3.12k0, which are outside the passband and cannot propagate to the exit interface; when the incidence angle is increased, a larger transverse wave vector k inc , so that the -1 order moves into the passband, while the 0 order and the +1 order are still outside the passband, thereby filtering out pure -1 order. Figure 3 (c) shows the OTF of the -1, 0, +1 orders at different angles of incidence, and it can be seen that in the range of 0° to 90°, the -1 order can be coupled into the hyperdispersion material, while other orders are relatively suppressed. At 49°, other orders are also coupled into the hyperdispersion material, as shown in Figure 3 (d), which is due to the 0 order wave vector being about 1.1k0, and there is a local SPs mode at this wave vector position in the OTF of the hyperdispersion material, so as to ensure that the -1 order wave vector is filtered out pure, the available incidence angle is selected to be 3° to 90°.

[0072] This embodiment takes a double-slit object as an example, and uses the finite difference time domain method, Figure 4 to show the modeling process and results of far-field imaging of a double-slit structure on a Cr film under BPPs illumination. The center distance of the double-slit structure is 100 nm, the single-slit width is 50 nm, the objective lens NA is 0.85, and the illumination wavelength is 532 nm. According to the diffraction limit, the object cannot be resolved under traditional illumination. When the BPPs illumination with a transverse wave vector of 2.66k0 as shown in Figure 3 (a) is used, Figure 4 (a) shows the cross-sectional field intensity distribution of the model established by using the FDTD method. The BPPs mode is modulated at the double-slit structure and scatters to the far field. The field distribution of the far-field scattered light in the FDTD method model is derived, and the far-field imaging is reversely calculated by using the angular spectrum diffraction theory as shown in Figure 4 (b), and it can be known that the double slit can be resolved under BPPs illumination. This embodiment also analyzes the frequency spectrum distribution of the far-field scattered light field. As shown in Figure 4 (c) and 4(d), for the frequency spectrum of the double-slit structure, the far-field detectable scattered light spectrum range under traditional illumination is in the region with the origin as the center and NA / λ as the radius, Figure 4 (c) as shown by the dashed line. The far-field scattered light field distribution spectrum under BPPs illumination is as shown in Figure 4 (d), which is consistent with the frequency spectrum distribution of the double-slit structure with the center being (k BPPs / 2π, 0) and the radius being NA / λ. The result proves that the frequency spectrum is shifted by a distance of k BPPs , which produces a frequency shift effect. In addition, for the same wave vector 2.66k0, the imaging effects of double slits with different center distances under the BPPs illumination are also simulated and tested. Figure 5It is shown that the resolution ability is not only for the double-slit center distance of 100 nm at the transverse wave vector of 2.66k0, but also for the double-slit structures with the center distance ranging from 95 nm to 110 nm according to the Rayleigh criterion. Meanwhile, this is also its limitation, each transverse wave vector corresponds to a smaller resolution interval (here, the double-slit center distance) that can be best resolved.

[0073] The working performance of the embodiment in a wide wavelength range also has good performance in the visible light range (380-780 nm). Figure 6 The optical transfer function of the metal / dielectric multilayer film structure in the visible light range (380-780 nm) is shown. It can be seen from the figure that the lower limit of the OTF window hardly changes with the incident wavelength, and the upper limit slowly decreases with the increase of the incident wavelength. At the same time, it is found that the high-frequency spatial wave vector BPPs mode can be excited in the visible light band. In addition, by optimizing the illumination structure material and parameters, the working wavelength can be extended to the ultraviolet light band, or in part of the wavelength band to low-frequency transmission. For the infrared band, a hyperbolic metamaterial can be used to replace the metal / dielectric multilayer film structure material to realize the corresponding filtering characteristics.

[0074] Embodiment 2:

[0075] In this embodiment, the high-frequency BPPs of higher wave vectors can be optimized and improved by further changing the metal / dielectric multilayer film structure and the excitation grating structure parameters, using the working mode B to optimize the thickness and the number of layers of the sub-wavelength grating and the metal / dielectric multilayer film structure, and the wavelength is 532 nm. The device from bottom to top is SiO2 substrate, periodic sub-wavelength Ag grating and Ag / SiO2 metal / dielectric multilayer film structure. 8 pairs of Ag / SiO2 metal / dielectric multilayer film structure with a thickness of 27 nm / 15 nm, plus a 27 nm Ag film. The OTF is shown in Figure 7 (a), and the filtering passband is [-4.65k0, -2.15k0] and [2.15k0, 4.65k0] under the TM wave of 532 nm. When the excitation grating with a period of 187 nm is loaded, the -1, 0, +1 order OTF is shown in Figure 7 (b). It can be seen that the +1 order wave vector is filtered out by changing the illumination angle under the grating period. When the incident angle is selected as 44°, the +1 order wave vector is 3.86k0. When the illumination center distance is 68 nm double-slit structure, the imaging field intensity is shown in Figure 7 (c), which proves that the illumination of BPPs with higher transverse wave vectors can significantly improve the resolution of far-field imaging.

[0076] The present application can provide different wave vector size illumination BPPs by matching multiple wavelengths and multiple illumination angles on the basis of examples 1 and 2, collect corresponding low-frequency and high-frequency spatial information in the far field when illuminating a sample, perform iterative splicing on a series of picture intensity distributions with spectral overlap in the frequency domain space, obtain the object spatial spectrum covering the maximum detectable wave vector range, and finally perform inverse Fourier transform to reconstruct the super-resolution non-deformation sample microscopic image. Through theoretical estimation, the resolution can be improved to about 4 times of that of an ordinary microscope.

Claims

1. An illumination device for shift-from-no-label far-field super-resolution optical wide-field microscopic imaging, characterized in that The lighting device comprises, from bottom to top, a transparent substrate, an excitation grating, and a metal / dielectric multilayer film structure. The lighting source illuminates the surface of the excitation grating from the back of the transparent substrate. The high-frequency diffraction wavelet excited by the grating structure is designed for single-order filtering: only one first or second order wavelet falls within the bandpass range of the metal / dielectric multilayer film. The incident angle of the lighting source is controlled... This makes for or integers, Located outside the bandpass range, wherein, Operating wavelength of the lighting source Vacuum wave vector below, To excite the grating wave vector, The refractive index of the substrate, The single-order filtering is of the diffraction wavelet order; and the single-order filtering makes the transverse wave vector of the illumination field reach a maximum of [missing information]. ; The size and pitch of the excitation grating are not greater than the wavelength of the illumination light, and the shape is a regular geometric body or an irregular arbitrary surface shape; The metal / dielectric multilayer film structure layer is formed by alternately stacking metal film layers and dielectric film layers; when the uppermost layer of the metal / dielectric multilayer film structure is a metal film layer, a transparent dielectric layer can be separately applied as a protective layer outside the metal film layer, and the thickness of the transparent dielectric layer is 10 nm to 40 nm.

2. The illumination device for frequency-shifting label-free far-field superresolution optical wide-field microscopic imaging according to claim 1, characterized in that In the metal / dielectric multilayer film structure, the metal is Au, Ag or Al, and the thickness is greater than or equal to 10 nm; and the dielectric is SiO2 or SiC, and the thickness is greater than or equal to 10 nm.

3. The illumination device for frequency-shifting label-free far-field superresolution optical wide-field microscopic imaging according to claim 1, characterized in that The material of the excitation grating is a high-refractive-index dielectric or metal with n>2.

4. The illumination device for frequency-shifting label-free far-field superresolution optical wide-field microscopic imaging according to claim 3, characterized in that The metal is Ag, Si, Cr or Cu.

5. The illumination device for marker-free far-field superresolution optical wide-field microscopic imaging by frequency shifting according to claim 1, characterized in that The excitation grating is prepared by magnetron sputtering, EBL, FIB, photolithography or nanoimprinting, the filling is performed by a flip process or a filling process, and the metal / dielectric multilayer film structure is prepared by magnetron sputtering or thermal evaporation.

6. A method of shift-frequency label-free far-field super-resolution optical wide-field microscopic imaging using the illumination device of any one of claims 1-5, characterized in that The method comprises the following steps: Step one, design two spatial filter windows of high frequency BPPs in metal / dielectric multilayer film layer structure With[ , the wavelength bandwidth of the illumination light source; design the range of incident angle , so that for Integer, Located outside the two BPPs window, filter out-1 order in the excitation grating diffraction order: The vacuum wave vector of the illumination light source at the working wavelength λ is , the incident angle of the central light is , the excitation grating wave vector is , the refractive index of the substrate is , two spatial filtering windows of high-frequency BPPs in the metal / dielectric multilayer film layer structure and , and the design rules of the illumination light source wavelength bandwidth are as follows: 1) the grating period is designed to excite at the operating wavelength the high frequency cutoff kx of the lower BPPs window max ; 2) angle of incidence Selection of the range: at the working wavelength , the wave vector of lies in the BPPs window at this wavelength , while satisfying for any positive or negative integer not equal to 1 , lies outside both BPPs windows; 3) The design rule for any angle and wavelength combination within the wavelength bandwidth of the illumination light source satisfies rule 2); Step two, placing the microimaging sample above the metal / dielectric multilayer film structure layer; Step three, using a single-wavelength light source as the illumination light source, the light ray from the center of the bottom of the transparent substrate layer along the incident angle designed in step one The inclined illumination excites the grating, and the high-frequency diffraction subwaves excited by the grating structure are filtered in the spatial frequency spectrum of the metal / dielectric multilayer film structure, thereby exciting a large-area and uniform high-frequency body plasmon mode field. Based on the large-area and uniform high-frequency BPPs mode field excited by the metal / dielectric multilayer film structure material, the sample is subjected to frequency shift illumination, the wave vector is adjusted to obtain a plurality of images containing low-frequency and high-frequency information of the sample, the intensity distribution of a series of pictures with some overlap in the frequency domain space is iteratively spliced, the object spatial spectrum covering the maximum detectable wave vector range is obtained, and finally the super-resolution and non-distorted sample microscopic image is reconstructed through inverse Fourier transform.

7. A method of shift-frequency label-free far-field super-resolution optical wide-field microscopic imaging using the illumination device of any one of claims 1-5, characterized in that The method comprises the following steps: Step one, design two spatial filter windows of high frequency BPPs in metal / dielectric multilayer film layer structure With , the wavelength bandwidth of the illumination light source, design the range of the incident angle , so that for integer, located outside the two BPPs windows, filter out the +1 order in the excitation grating diffraction order: The working wavelength of the illumination light source The vacuum wave vector under The incident angle of the central light line is The excitation grating wave vector is The refractive index of the substrate is Two spatial filtering windows of high-frequency BPPs in the metal / dielectric multilayer film layer structure And The design rules of the illumination light source wavelength bandwidth are as follows: 1) the grating period is designed to excite at the operating wavelength of the lower BPPs window mid; 2) angle of incidence Selection of the range: at the working wavelength Below, the selection Located in the corresponding wavelength BPPs window In the meantime, satisfy any positive and negative integer , Located outside the two BPPs window; 3) The design rule for any angle and wavelength combination within the wavelength bandwidth of the illumination light source satisfies rule 2); Step two, placing the microimaging sample above the metal / dielectric multilayer film structure layer; Step three, using a variety of wavelength laser or wide band small divergence angle of visible light source as illumination light source, from the bottom center of the transparent substrate layer along the incident angle designed in step one The inclined illumination excites the grating, and the high-frequency diffraction subwaves excited by the grating structure are filtered in the spatial frequency spectrum of the metal / dielectric multilayer film structure, thereby exciting a large-area and uniform high-frequency body plasmon mode field. Based on the large-area and uniform high-frequency BPPs mode field excited by the metal / dielectric multilayer film structure material, the sample is frequency-shifted and illuminated, the wave vector is adjusted to obtain a plurality of images containing low-frequency and high-frequency information of the sample, the intensity distribution of a series of pictures with some overlap in the frequency domain space is iteratively spliced, the object spatial spectrum covering the maximum detectable wave vector range is obtained, and finally the super-resolution and non-distorted sample microscopic image is reconstructed through inverse Fourier transform.

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