Metasurface structure and total internal reflection fluorescence microscope

Abstract

The invention relates to the technical field of microscopic imaging, in particular to a metasurface structure and a total internal reflection fluorescence microscope. The metasurface structure is applied to the total internal reflection fluorescence microscope, the total internal reflection fluorescence microscope comprises a microscope objective, the metasurface structure comprises a silicon dioxide substrate, silicon columns and a coating material layer, and the silicon columns are arranged on the silicon dioxide substrate in an array distribution mode; the coating material layer covers the surfaces of the silicon column and the silicon dioxide substrate, and the metasurface structure can reflect fluorescence signals with wavelengths in a preset range to a microscope objective and transmit laser or fluorescence signals with other wavelengths. The metasurface structure provided by the embodiment of the invention can reflect the fluorescence signal with the wavelength in the preset range to the microscope objective and transmit the laser or fluorescence signal with other wavelengths, so that the fluorescence signal with the wavelength in the preset range can enter the microscope objective, and the detection sensitivity of the total internal reflection fluorescence microscope is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of microscopy imaging technology, and particularly to a metasurface structure and a total internal reflection fluorescence microscope. Background Art

[0002] In the process of life science moving from the macroscopic to the microscopic, the total internal reflection fluorescence microscope plays an important role. With the development of optoelectronic technology, functional materials, and information technology, it has become possible to image biomolecules such as proteins and nucleic acids at the subcellular level, which is of great significance for revealing life activities, disease diagnosis, and developing new treatment strategies.

[0003] As a commonly used imaging characterization instrument, the wide-field fluorescence imaging microscope is interfered by background fluorescence from outside the focal plane during imaging, thus limiting its application at the subcellular level. The total internal reflection fluorescence microscope (TIRFM) uses an evanescent wave for excitation, with a shallow depth and a small excitation volume, which can significantly reduce the background signal and produce high-contrast images. Applied in the field of live cell fluorescence imaging, it can localize single molecules in live cells at the nanoscale.

[0004] TIRFM is the most widely used microscopy imaging instrument in the field of single molecule imaging. In the TIRFM optical system, an evanescent wave with a very small depth generated by total reflection when light travels from an optically denser medium to an optically less dense medium is used to excite fluorescence, so that the excitation region is limited near the cell membrane, reducing the excitation of intracellular fluorescent molecules. Compared with the traditional wide-field fluorescence imaging microscope, TIRFM has the characteristics of high signal-to-noise ratio and contrast.

[0005] Since its invention, the total internal reflection fluorescence microscope has been widely used in biomedical research. However, in single molecule detection applications, due to the limitation of the objective numerical aperture on the fluorescence signal entering the detection system, only the fluorescence signal within a smaller aperture angle can be detected by the detector, which limits the detection sensitivity. When the concentration of the fluorescent molecule to be measured is extremely low or even a single fluorescent molecule, the signal-to-noise ratio will decrease, making it difficult to extract the target signal from the high-noise background. The detection limit of existing single molecule methods based on total internal reflection fluorescence microscopes is limited within the picomolar range, which is several orders of magnitude higher than the concentration of biomarkers present in many diseases. The sensitivity of single molecule detection using TIRFM needs to be further improved. Summary of the Invention

[0006] The purpose of the present invention is to at least solve the problem of relatively low detection sensitivity of the total internal reflection fluorescence microscope in the prior art, and this purpose is achieved through the following technical solutions.

[0007] A first aspect of an embodiment of the present application provides a metasurface structure, which is applied to a total internal reflection fluorescence microscope. The total internal reflection fluorescence microscope includes a microscope objective. The metasurface structure includes:

[0008] A silica substrate;

[0009] Silicon pillars, which are arranged on the silica substrate in an array distribution; and

[0010] A coating material layer, which covers the surfaces of the silicon pillars and the silica substrate. The metasurface structure can reflect fluorescence signals within a preset wavelength range to the microscope objective and transmit laser or fluorescence signals of other wavelengths.

[0011] For the metasurface structure of the embodiment of the present application, by providing a silica substrate, arranging silicon pillars in an array distribution on the silica substrate, and providing a coating material layer that covers the surfaces of the silicon pillars and the silica substrate, the metasurface structure can reflect fluorescence signals within a preset wavelength range to the microscope objective and transmit laser or fluorescence signals of other wavelengths. Then, the fluorescence signals within the preset wavelength range can enter the microscope objective, enhancing the detection sensitivity of the total internal reflection fluorescence microscope.

[0012] According to the metasurface structure of the present application, it also has the following additional technical features.

[0013] Optionally, the silicon pillars are obtained by growing from the surface of the silica substrate.

[0014] Optionally, the size of the silicon pillars and / or the distance between adjacent silicon pillars can be designed through a simulation algorithm so that the metasurface structure can reflect fluorescence signals of different wavelengths.

[0015] Optionally, the raw material of the coating material layer includes a transparent polymer material.

[0016] A second aspect of an embodiment of the present application provides a total internal reflection fluorescence microscope, which includes the metasurface structure mentioned in the above embodiment.

[0017] According to the total internal reflection fluorescence microscope of the present application, it also has the following additional technical features.

[0018] Optionally, the total internal reflection fluorescence microscope further includes a microscope objective; a sample to be measured can be placed between the microscope objective and the metasurface structure. Among them, the metasurface structure is used to reflect fluorescence signals. When the sample to be measured is placed between the microscope objective and the metasurface structure, the fluorescence signals generated after the laser provided by the microscope objective irradiates the sample to be measured are reflected by the metasurface structure; the fluorescence signals reflected by the metasurface structure can be transmitted to the microscope objective.

[0019] Optionally, the total internal reflection fluorescence microscope further includes an illumination system for providing a laser to the microscope objective; wherein, the illumination system includes:

[0020] An illumination light source for generating an incident light beam:

[0021] A first dichroic sheet for splitting incident light beams of two different wavelengths into a first light beam and a second light beam according to the wavelength;

[0022] A first shutter and a second shutter, wherein the first shutter and the second shutter are respectively disposed on the propagation paths of the first light beam and the second light beam, the first shutter is used to selectively pass the first light beam, and the second shutter is used to selectively pass the second light beam;

[0023] A first beam expander group including a first convex lens and a second convex lens, the first beam expander group being used to expand the first light beam;

[0024] A second beam expander group including a third convex lens and a fourth convex lens, the second beam expander group being used to expand the second light beam;

[0025] A second dichroic sheet for combining the first light beam and the second light beam and providing them to the microscope objective of the total internal reflection fluorescence microscope.

[0026] Optionally, the illumination system further includes:

[0027] A sixth mirror for reflecting the second light beam passing through the first dichroic sheet so that the first light beam and the second light beam are parallel.

[0028] Optionally, the illumination system further includes a first reflection assembly and a second reflection assembly. The first reflection assembly includes a third mirror, a fourth mirror and a fifth mirror, and the first light beam is reflected by the first reflection assembly to the second dichroic sheet. The second reflection assembly includes a seventh mirror and an eighth mirror, and the second light beam is reflected by the second reflection assembly to the second dichroic sheet. Among them, the first light beam reflected by the first reflection assembly and the second light beam reflected by the second reflection assembly are perpendicular to each other, and the first light beam and the second light beam are combined by the second dichroic sheet and enter the microscope objective.

[0029] Optionally, the illumination system further includes:

[0030] A first aperture for adjusting the size of the light spot of the first light beam; and

[0031] A second aperture, which is used to adjust the size of the spot of the second light beam. Description of the Drawings

[0032] By reading the following detailed description of the preferred embodiments, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of illustrating the preferred embodiments and are not considered to be a limitation of the present invention. Moreover, throughout the drawings, the same reference numerals are used to denote the same components.

[0033] Figure 1 Schematic diagram of the microstructure unit of the metasurface structure provided by the embodiment of the present invention;

[0034] Figure 2 Overall schematic diagram of the metasurface structure provided by the embodiment of the present invention;

[0035] Figure 3 is Figure 2 Partial enlarged structural schematic diagram of the metasurface structure shown in

[0036] Figure 4 is Figure 3 SEM image of the partial structure of the metasurface shown;

[0037] Figure 5 Schematic diagram of the optical path structure of the total internal reflection fluorescence microscope provided by the embodiment of the present invention;

[0038] Figure 6 is Figure 1 Simulated reflection spectrum diagram of the metasurface structure shown in

[0039] Figure 7 is Figure 1 Actually measured reflected light spectrum diagram of the metasurface structure shown in

[0040] Figure 8 Imaging diagram of the Streptavidin-Cy5 dye sample by the total internal reflection fluorescence microscope in the prior art;

[0041] Figure 9 Imaging diagram of the Streptavidin-Cy5 dye sample by the total internal reflection fluorescence microscope provided by the embodiment of the present invention in Figure 8 the same area;

[0042] Figure 10 Statistical distribution diagram of the fluorescence intensity of the signal points on the image by the total internal reflection fluorescence microscope provided by the embodiment of the present invention and the total internal reflection fluorescence microscope in the prior art;

[0043] Figure 11Simulated reflection spectrum of a dual-wavelength reflection (excitation at 561 nm and 647 nm) metasurface structure.

[0044] The reference numerals in the figure are as follows:

[0045] 1000, total internal reflection fluorescence microscope;

[0046] 100, metasurface structure;

[0047] 110, silica substrate;

[0048] 120, silicon pillar;

[0049] 130, coating material layer;

[0050] 300, illumination system;

[0051] 301, illumination light source;

[0052] 302, achromatic lens;

[0053] 303, first mirror;

[0054] 304, second mirror;

[0055] 305, first dichroic filter;

[0056] 306, first neutral density filter;

[0057] 307, first shutter;

[0058] 308, first convex lens;

[0059] 309, second convex lens;

[0060] 310, first aperture;

[0061] 311, third mirror;

[0062] 312, fourth mirror;

[0063] 313, fifth mirror;

[0064] 314, first auxiliary convex lens;

[0065] 315, sixth mirror;

[0066] 316, second neutral density filter;

[0067] 317, second shutter;

[0068] 318, third convex lens;

[0069] 319, fourth convex lens;

[0070] 320. Second aperture stop

[0071] 321. Seventh mirror

[0072] 322. Eighth mirror

[0073] 323. Second auxiliary convex lens

[0074] 324. Second dichroic sheet

[0075] 325. Microscope objective

[0076] 2000. Sample to be measured Detailed implementation manners

[0077] The exemplary embodiments of the present application will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present application can be more thoroughly understood and the scope of the present application can be completely conveyed to those skilled in the art.

[0078] It should be noted that in the description of the present invention, the terms "first" and "second" are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0079] The detection sensitivity of total internal reflection fluorescence microscopy in existing single molecule detection technologies is limited by the signal-to-noise ratio. As the concentration of the molecule to be measured decreases, the signal-to-noise ratio will decrease, making it difficult to extract the target signal from the high-noise background. The detection limit of existing single molecule methods based on total internal reflection fluorescence microscopy is limited within the picomolar range, which is several orders of magnitude higher than the concentration of biomarkers present in many diseases.

[0080] To solve this technical problem and improve the detection sensitivity of total internal reflection fluorescence microscopy, an embodiment of the present application provides a metasurface structure 100 applied to a total internal reflection fluorescence microscope 1000. Among them, Figure 1 is a schematic diagram of the microstructure unit of the metasurface structure 100 provided by the embodiment of the present invention, Figure 2 is an overall schematic diagram of the metasurface structure 100 provided by the embodiment of the present invention, Figure 3 is Figure 2 a partially enlarged structural schematic diagram of the metasurface structure 100 shown in Figure 4 at point A, Figure 3 is Figure 1 、 Figure 2 、 Figure 3 andFigure 4 As shown, the first aspect of the embodiments of the present application provides a metasurface structure 100, and the metasurface structure 100 is applied to a total internal reflection fluorescence microscope 1000, and the total internal reflection fluorescence microscope 1000 includes a microscope objective 325.

[0081] Here, the total internal reflection fluorescence microscope 1000 is an optical imaging technology that utilizes the characteristic of the evanescent wave generated on the other side of the medium after total internal reflection of light to excite fluorescent molecules in an extremely thin region and observe the fluorescent molecules. In the visible light range, the penetration depth of the evanescent wave is 50 nm to 100 nm. Due to the exponentially decaying characteristic of the excitation light, only the sample region extremely close to the total reflection surface is excited to generate fluorescence, greatly reducing the interference of background light noise, thereby obtaining high-quality imaging quality and reliable observation data.

[0082] The metasurface structure 100 of the present application includes components such as a silica substrate 110, silicon pillars 120, and a coating material layer 130. Among them, the number of silicon pillars 120 is multiple and is distributed on the silica substrate 110 in an array form. The coating material layer 130 covers the surfaces of the silicon pillars 120 and the silica substrate 110, and the metasurface structure 100 can reflect the fluorescence signal within a preset range of wavelengths to the microscope objective 325 and transmit the laser or fluorescence signals of the remaining wavelengths.

[0083] It should be noted that the preset range of wavelengths here can be a single wavelength or multiple wavelengths, so that the metasurface structure 100 can be applicable to fluorescent dyes with various emission wavelengths for single-molecule fluorescence enhancement imaging. The remaining wavelengths refer to other wavelengths except the preset range of wavelengths.

[0084] The silica substrate 110 refers to a substrate made of silica. Among them, silica is an inorganic compound with stable chemical properties, good insulation properties, and mechanical properties.

[0085] It should be emphasized that the silicon pillars 120 here are in a cylindrical structure and can be embedded by the coating material layer 130, which can avoid the damage of the silicon pillars 120 by external objects during use and form protection for the silicon pillars 120.

[0086] Optionally, as Figure 1 shown, the silicon pillars 120 are obtained by growing from the surface of the silica substrate 110. As Figure 5 shown, in actual application, the coating material layer 130 faces the sample to be measured 2000.

[0087] In a specific embodiment, as Figure 3As shown, the silicon pillars 120 can be arranged at equal intervals on one surface of the silica substrate 110. That is to say, the distance between each silicon pillar 120 and its adjacent silicon pillars 120 is the same.

[0088] The number of silicon pillars 120 is multiple, and the multiple silicon pillars 120 are arranged in a multi-row and multi-column structure and are evenly distributed on the surface of the silica substrate 110. During use, the surface of the coating material layer 130 faces the sample to be measured 2000.

[0089] The array distribution of the silicon pillars 120 enables the metasurface structure 100 to reflect fluorescence signals within a preset wavelength range and transmit laser or fluorescence signals of other wavelengths.

[0090] In a specific embodiment, the raw material of the coating material layer 130 includes a transparent polymer material such as SU8. That is to say, the coating material layer 130 can be made of SU8 material. Among them, SU8 is a negative photoresist mainly composed of epoxy resin, photosensitizer and diluent. Among them, epoxy resin is the main component of SU8, which is responsible for providing the structure and mechanical strength of the colloid. The role of the photosensitizer is to cause a chemical reaction after SU8 is exposed to light to form a pattern or structure. The diluent is used to adjust the viscosity of the photoresist to make SU8 more suitable for specific application requirements.

[0091] In a preferred embodiment, the metasurface structure 100 of the present application can have a substantially flat upper surface, which is the upper surface of the coating material layer 130. Thus, the metasurface structure 100 of the present application can reflect fluorescence signals of specific wavelengths and transmit signals of other wavelengths.

[0092] In some embodiments, the size of the silicon pillars 120 (including the diameter and height of the silicon pillars 120) and / or the distance between adjacent silicon pillars 120 can be designed through a simulation algorithm so that the metasurface structure 100 can reflect fluorescence signals of different wavelengths. Thus, different metasurface structures 100 can be designed for the fluorescence signal wavelengths of different samples to be measured 2000. Correspondingly, each sample to be measured 2000 can be detected with high sensitivity.

[0093] Taking the sample to be measured 2000 containing Cy5 fluorescent dye as an example, the metasurface structure 100 needs to reflect the 670nm fluorescence signal. Figure 2 The area where the metasurface structure 100 is arranged is within the middle rectangular frame. In this 1mm 2 area, 6.25 million silicon pillars 120 are arranged, and the spacing between adjacent two silicon pillars 120 is 0.4μm.

[0094] Accordingly, the metasurface structure 100 can reflect the fluorescence signal at 670 nm emitted by the Cy5 fluorescent dye. Among them, Cy5 is a reactive dye used to label the amino groups of peptides, proteins, and oligonucleotides.

[0095] The metasurface structure 100 of the present application has a millimeter-scale size, which can meet the detection requirements of biological samples. The silicon pillars 120 are embedded in the coating material layer 130 made of SU8 material, and the structure is stable. It can be directly contacted with aqueous solution samples, so that the single metasurface structure 100 can be reused.

[0096] For fluorescent dyes with different emission wavelengths, the parameters such as the radius, height, and spacing of the silicon pillars 120 of the metasurface structure 100 are designed through a simulation algorithm to adjust the reflection wavelength of the metasurface structure 100, so that the maximum reflectance wavelength of the metasurface structure 100 matches the fluorescence wavelength, realizing the reflection of the fluorescence signals emitted by different fluorescent dyes. In addition, for multi-wavelength applications, a metasurface structure 100 matching multiple wavelengths needs to be designed.

[0097] In addition, the metasurface structure 100 of the present application can be used in cooperation with a microfluidic chip for the detection of low-abundance disease markers. Moreover, the metasurface structure 100 of the present application can be used in various scenarios that require reflecting fluorescent signals of specific wavelengths, such as PALM (Photoactivated Localization Microscopy) / STORM (Stochastic Optical Reconstruction Microscopy).

[0098] The second aspect of the embodiments of the present application also provides a total internal reflection fluorescence microscope 1000. Figure 5 It is a schematic structural diagram of the optical path of the total internal reflection fluorescence microscope 1000 provided by the embodiments of the present invention. As Figure 5As shown, when the metasurface structure 100 of the present application is in use, the sample to be measured 2000 can be placed between the microscopic objective lens 325 of the total internal reflection fluorescence microscope 1000 and the metasurface structure 100. When in use, the coating material layer 130 faces the sample to be measured 2000. At this time, the laser can irradiate the sample to be measured 2000 through the microscopic objective lens 325 and excite the sample to be measured 2000 to generate a fluorescence signal. On the one hand, this fluorescence signal can be transmitted to the microscopic objective lens 325. On the other hand, this fluorescence signal can also be transmitted to the microscopic objective lens 325 through the reflection of the metasurface structure 100. Specifically, the fluorescence signal reflected by the metasurface structure 100 can pass through the sample to be measured 2000 and, after passing through the sample to be measured 2000, be transmitted to the microscopic objective lens 325. Thus, the total internal reflection fluorescence microscope 1000 of the present application can increase the fluorescence signal entering the microscopic objective lens 325, effectively improve the sensitivity and signal-to-noise ratio of fluorescence signal detection, and thus achieve high-sensitivity detection of the sample to be measured 2000.

[0099] As Figure 5 shown in the structure, the total internal reflection fluorescence microscope 1000 may include structures such as a microscopic objective lens 325 and an illumination system 300.

[0100] The microscopic objective lens 325 of the present application can focus the laser on the sample surface and collect the fluorescence signal. Thus, the laser generated by the illumination system 300 can be provided to the microscopic objective lens 325 and irradiate the sample to be measured 2000 through the microscopic objective lens 325. Moreover, after the fluorescence signal reflected by the metasurface structure 100 passes through the sample to be measured 2000, it is transmitted to the EMCCD (Electron-Multiplying Charge Coupled Device) detector through the microscopic objective lens 325.

[0101] Referring again to Figure 5 , the illumination system 300 of the present application includes an illumination light source 301, a first dichroic sheet 305, a first shutter 307, a second shutter 317, a first beam expander group, a second beam expander group, and a second dichroic sheet 324. Among them, the illumination light source 301 is used to generate an incident light beam. The first shutter 307 and the second shutter 317 are respectively provided on the propagation paths of the first light beam and the second light beam. The first shutter 307 is used to select and pass the first light beam, the second shutter 317 is used to select and pass the second light beam, the first beam expander group is used to expand the first light beam, the second beam expander group is used to expand the second light beam, and the second dichroic sheet 324 is used to combine the first light beam and the second light beam and provide them to the microscopic objective lens 325 of the total internal reflection fluorescence microscope 1000.

[0102] Preferably, the incident light beam of the present application can be a laser beam. The illumination light source 301 of the present application can generate laser beams of different wavelengths. In a specific embodiment, the illumination light source 301 can generate laser beams of multiple wavelengths.

[0103] Optionally, the laser beam generated by the illumination light source 301 is provided to the first dichroic plate 305 via an achromatic lens 302 and two reflective mirrors. The laser beams of different wavelengths generated by the illumination light source 301 can be collimated by the achromatic lens 302. The two reflective mirrors are a first reflective mirror 303 and a second reflective mirror 304, which can change the propagation direction of the laser beam.

[0104] The achromatic lens 302 is a lens specially designed to reduce chromatic aberration. It can focus light of different wavelengths on the same plane, thereby making the light purer.

[0105] After the laser beam's direction is adjusted by the first and second reflectors 303 and 304, the first dichroic plate 305 splits the incident beam into two perpendicular beams, transmitted and reflected, based on their wavelengths. The reflected light is the first beam, and the transmitted light is the second beam. The transmitted light is then reflected by the sixth reflector 315, aligning the first and second beams.

[0106] Next, the first light beam will propagate through the first neutral density filter 306, and similarly, the second light beam will propagate through the second neutral density filter 316. At this time, the intensity of the first light beam will be adjusted by the first neutral density filter 306, and accordingly, the intensity of the second light beam will be adjusted by the second neutral density filter 316.

[0107] The first shutter 307 is disposed on the propagation path of the first light beam and is used to select the first light beam. The second shutter 317 is disposed on the propagation path of the second light beam and is used to select the second light beam. Specifically, the first shutter 307 and the second shutter 317 of the present application can be controlled by a computer, respectively, and can not only select different laser beams, but also modulate continuous wave laser light into pulsed light.

[0108] The first beam expander group of the present application is used to expand the first light beam; the second beam expander group is used to expand the second light beam; in a specific embodiment, the first beam expander group and the second beam expander group have the same structure, both of which include two convex lenses.

[0109] Specifically, the first beam expander lens group includes a first convex lens 308 and a second convex lens 309 .

[0110] Specifically, the second beam expander lens group includes a third convex lens 318 and a fourth convex lens 319 .

[0111] Optionally, the lighting system 300 of the present application may further include a first aperture 310 and a second aperture 320; the first aperture 310 is used to adjust the lateral dimension of the spot of the first light beam; the second aperture 320 is used to adjust the lateral dimension of the spot of the second light beam. Specifically, along the propagation direction of the first light beam, the first aperture 310 is located on the downstream side of the second convex lens 309, that is, along the propagation direction of the second light beam, the second aperture 320 is located on the downstream side of the fourth convex lens 319.

[0112] Optionally, the lighting system 300 of the present application further includes a first reflection component and a second reflection component. The first light beam is reflected by the first reflection component to the second dichroic sheet 324, and the second light beam is reflected by the second reflection component to the second dichroic sheet 324, wherein the first light beam reflected by the first reflection component and the second light beam reflected by the second reflection component are perpendicular to each other. Along the propagation direction of the first light beam, the first reflection component is located on the downstream side of the first aperture 310, and along the propagation direction of the second light beam, the second reflection component is located on the downstream side of the second aperture 320.

[0113] Optionally, the first reflection component includes a third mirror 311, a fourth mirror 312, and a fifth mirror 313. The third mirror 311, the fourth mirror 312, and the fifth mirror 313 are used to change the propagation direction of the first light beam. A first auxiliary convex lens 314 and a second dichroic sheet 324 are sequentially provided on the downstream side of the fifth mirror 313.

[0114] Optionally, the second reflection component includes a seventh mirror 321 and an eighth mirror 322. The seventh mirror 321 and the eighth mirror 322 are used to change the propagation direction of the second light beam. A second auxiliary convex lens 323 and a second dichroic sheet 324 are sequentially provided on the downstream side of the eighth mirror 322.

[0115] Along the propagation direction of the first light beam, the first auxiliary convex lens 314 is located on the downstream side of the first reflection component, and the second auxiliary convex lens 323 is located on the downstream side of the second reflection component. The first auxiliary convex lens 314 is used to focus the first light beam onto the rear focal plane of the microscope objective 325; the second auxiliary convex lens 323 is used to focus the second light beam onto the rear focal plane of the microscope objective 325. Accordingly, by adjusting the height of the first auxiliary convex lens 314, the position of the spot of the first light beam on the rear focal plane of the microscope objective 325 can be adjusted, so as to adjust the angle at which the excitation light enters the sample to be measured from the microscope objective 325, and realize TIRF (Total Internal Reflection Fluorescence) illumination. Similarly, by adjusting the height of the second auxiliary convex lens 323, the position of the spot of the second light beam on the rear focal plane of the microscope objective 325 can also be adjusted.

[0116] Specifically, the first light beam passing through the first auxiliary convex lens 314 and the second light beam passing through the second auxiliary convex lens 323 both propagate to the second dichroic sheet 324. The second dichroic sheet 324 is used to combine the first light beam and the second light beam and provide them to the microscopic objective lens 325 of the total internal reflection fluorescence microscope 1000. Thus, the first light beam and the second light beam are incident on the sample to be measured 2000 by the microscopic objective lens 325 at the TIR angle (Total Internal Reflection Angle), exciting the generation of fluorescence signals. The fluorescence signals propagating upward are reflected by the metasurface structure 100 placed above the sample to be measured 2000, and then collected by the microscopic objective lens 325 and enter the EMCCD detector, thereby increasing the solid angle of signal detection, amplifying the single-molecule fluorescence signal, improving the signal-to-noise ratio and detection sensitivity, reducing the detection limit, and can be used to detect low-abundance disease markers.

[0117] In the total internal reflection fluorescence microscope 1000 of the present application, by setting the metasurface structure 100 that reflects specific fluorescence wavelengths, the signal-to-noise ratio and sensitivity of the total internal reflection fluorescence microscope 1000 can be improved, and the detection limit can be reduced; the size of the microstructure of the metasurface structure 100 is designed by a simulation algorithm. By designing parameters such as the size of the silicon pillars 120 and the interval between two adjacent silicon pillars 120, the wavelength of the reflected light can be adjusted, and single or multi-wavelength reflection can be achieved, which is applicable to fluorescent dyes with various emission wavelengths for single-molecule fluorescence enhancement imaging.

[0118] Next, in combination with Figure 5 a more detailed introduction to the propagation path of the incident light beam will be given.

[0119] The illumination light source 301 of the illumination system 300 generates laser beams of different wavelengths. After the laser beams are collimated by the achromatic lens 302, the directions are adjusted by the first mirror 303 and the second mirror 304. The first dichroic sheet 305 divides the incident light beam into mutually perpendicular transmitted light and reflected light according to different wavelengths. The reflected light is the first light beam, and the transmitted light is the second light beam. Among them, the first light beam successively passes through the first neutral density filter 306, the first shutter 307, the first convex lens 308, the second convex lens 309, the first aperture 310, the third mirror 311, the fourth mirror 312, the fifth mirror 313 and the first auxiliary convex lens 314 and then is incident on the second dichroic sheet 324. Then, the first light beam is reflected by the second dichroic sheet 324 to the microscopic objective lens 325.

[0120] Continue to refer to Figure 5As shown, the second light beam sequentially passes through the sixth mirror 315, the second neutral density filter 316, the second shutter 317, the third convex lens 318, the fourth convex lens 319, the second aperture 320, the seventh mirror 321, the eighth mirror 322, the second auxiliary convex lens 323, and the second dichroic sheet 324, and is transmitted by the second dichroic sheet 324 to the microscope objective 325.

[0121] The excitation light formed by the first light beam and the second light beam is incident on the sample to be measured 2000 at the TIR angle by the microscope objective 325, exciting a fluorescence signal, and the fluorescence signal propagating upward is reflected by the metasurface structure 100 placed above the sample to be measured 2000 and collected by the microscope objective 325 and then enters the EMCCD detector.

[0122] The following matters need to be noted in the specific implementation process of the total internal reflection fluorescence microscope 1000.

[0123] It is necessary to design the metasurface structure 100 with specific parameters for dyes of specific fluorescence wavelengths so that the fluorescence wavelength matches the maximum reflectivity wavelength of the metasurface structure 100.

[0124] For multi-wavelength applications, it is necessary to design the metasurface structure 100 that matches multiple wavelengths. The wavelength of the reflected light can be adjusted by designing parameters such as the size of the silicon pillars 120, such as the height and diameter of the silicon pillars 120, and the interval between two adjacent silicon pillars 120.

[0125] The metasurface structure 100 in this application is an optical metasurface, which has multiple microstructure units. The size of the microstructure units is designed by a simulation algorithm. By designing parameters such as the size of the silicon pillars 120 and the interval between two adjacent silicon pillars 120, the wavelength of the reflected light can be adjusted, and single or multi-wavelength reflection can be achieved. It is applicable to fluorescent dyes of various emission wavelengths for single-molecule fluorescence enhancement imaging. In addition, the manufacturing cost of the metasurface structure 100 in this application is relatively low, and it has a high commercial application prospect.

[0126] Continue to refer to Figure 6 , Figure 6 For Figure 1 is the simulated reflection spectrum diagram of the metasurface structure 100 shown in Figure 7 For Figure 1 is the actually measured reflected light spectrum diagram of the metasurface structure 100 shown in Figure 7 It can be seen from Figure 6 that the actually measured reflected light spectrum diagram of the processed metasurface structure 100 is in good agreement with the simulated reflection spectrum diagram shown in

[0127] Continue to refer toFigure 8 and Figure 9 as shown, where Figure 8 is an imaging diagram of the Streptavidin-Cy5 (streptavidin labeled with pentamethine cyanine dye) dye sample by the total internal reflection fluorescence microscope 1000 in the prior art, Figure 9 is an imaging diagram of the Streptavidin-Cy5 dye sample by the metasurface-assisted total internal reflection fluorescence microscope 1000 provided in the embodiment of the present invention in Figure 8 the same region. Streptavidin labeled with pentamethine cyanine dye is a complex combining Cy5 fluorescent dye and streptavidin.

[0128] From Figure 8 and Figure 9 it can be seen that for the total internal reflection fluorescence microscope 1000 assisted by the metasurface structure 100, compared with the traditional total internal reflection fluorescence microscope, the observed fluorescence signal contrast and signal-to-noise ratio are significantly improved, and a larger number of single molecule fluorescence points can be observed (as shown within the dashed boxes in Figure 8 and Figure 9 ).

[0129] Figure 10 is a statistical distribution diagram of the fluorescence intensity of the signal points on the images of 60 Streptavidin-Cy5 dye samples taken by the total internal reflection fluorescence microscope 1000 assisted by the metasurface structure 100 provided in the embodiment of the present invention and the total internal reflection fluorescence microscope in the prior art. Among them, meta represents the metasurface structure group, contol represents the control group in the prior art, the abscissa is the signal intensity, and the ordinate is the number of fluorescence points. From the comparison of the two results, it can be seen that with the assistance of the metasurface structure 100, the total internal reflection fluorescence microscope 1000 can observe fluorescence signal points with greater intensity and a larger number of signal points.

[0130] Figure 11 is the theoretical reflection spectrum diagram of the dual-wavelength (excitation at 561 nm and 647 nm) enhanced metasurface structure 100, Figure 11 the size of the silicon pillars 120 in Figure 6 is different from the size of the silicon pillars 120 of the metasurface structure 100 in Figure 11 . In

[0131] As described above, it is only the preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claimed rights.

Claims

1. A metasurface structure is applied to a total internal reflection fluorescence microscope, and the total internal reflection fluorescence microscope includes a microscope objective lens, characterized in that, The metasurface structure includes: A silica substrate; Silicon pillars, which are arranged on the silica substrate in an array distribution; and A coating material layer that covers the surfaces of the silicon pillars and the silica substrate. The metasurface structure can reflect fluorescence signals within a preset wavelength range to the microscope objective lens and transmit laser or fluorescence signals of other wavelengths.

2. The metasurface structure according to claim 1, wherein The silicon pillars are obtained by growing from the surface of the silica substrate.

3. The metasurface structure according to claim 1, characterized in that, The size of the silicon pillars and / or the distance between adjacent silicon pillars can be designed through a simulation algorithm so that the metasurface structure can reflect fluorescence signals of different wavelengths.

4. The metasurface structure according to claim 1, wherein The raw material of the coating material layer includes a transparent polymer material.

5. A total internal reflection fluorescence microscope, characterized in that, Including the metasurface structure according to any one of claims 1-4.

6. The total internal reflection fluorescence microscope according to claim 5, characterized in that, It further includes a microscope objective lens; a sample to be measured can be placed between the microscope objective lens and the metasurface structure. Among them, the metasurface structure is used to reflect fluorescence signals. When the sample to be measured is placed between the microscope objective lens and the metasurface structure, the fluorescence signals generated after the laser provided outward by the microscope objective lens irradiates the sample to be measured are reflected by the metasurface structure; the fluorescence signals reflected by the metasurface structure can be transmitted to the microscope objective lens.

7. The total internal reflection fluorescence microscope according to claim 6, wherein It further includes an illumination system, and the illumination system is used to provide laser to the microscope objective lens; wherein, the illumination system includes: An illumination light source for generating an incident light beam: A first dichroic sheet, which is used to divide incident light beams of two different wavelengths into a first light beam and a second light beam according to the wavelength; A first shutter and a second shutter. Among them, the first shutter and the second shutter are respectively arranged on the propagation paths of the first light beam and the second light beam. The first shutter is used to select and pass the first light beam, and the second shutter is used to select and pass the second light beam; A first beam expander group, which includes a first convex lens and a second convex lens and is used to expand the first light beam; A second beam expander group, which includes a third convex lens and a fourth convex lens and is used to expand the second light beam; and A second dichroic sheet, which is used to combine the first light beam and the second light beam and provide them to the microscope objective lens of the total internal reflection fluorescence microscope.

8. The total internal reflection fluorescence microscope according to claim 7, wherein The illumination system further includes: A sixth mirror, which is used to reflect the second light beam passing through the first dichroic sheet so that the first light beam and the second light beam are parallel.

9. The total internal reflection fluorescence microscope according to claim 8, characterized in that, The illumination system further includes a first reflection component and a second reflection component. The first reflection component includes a third mirror, a fourth mirror and a fifth mirror, and the first light beam is reflected by the first reflection component to the second dichroic sheet; the second reflection component includes a seventh mirror and an eighth mirror, and the second light beam is reflected by the second reflection component to the second dichroic sheet. Among them, the first light beam reflected by the first reflection component and the second light beam reflected by the second reflection component are perpendicular to each other, and the first light beam and the second light beam are combined by the second dichroic sheet and enter the microscope objective lens.

10. The total internal reflection fluorescence microscope according to any one of claims 7 to 9, characterized in that The illumination system further includes: A first aperture, the first aperture being used for adjusting the size of the spot of the first light beam; and A second aperture, the second aperture being used for adjusting the size of the spot of the second light beam.

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