Mark-free axial super-resolution measurement system and method based on one-dimensional photonic crystal

Abstract

The invention discloses an unmarked axial super-resolution measurement system and method based on a one-dimensional photonic crystal, and belongs to the technical field of optical measurement. The system comprises a dual-wavelength light source, a one-dimensional photonic crystal composite structure, a signal acquisition module and a data processing module; the one-dimensional photonic crystal composite structure comprises a substrate and a 1DPC structure. The 1DPC structure is formed by alternately stacking high-refractive-index materials and low-refractive-index materials, and comprises at least two 1DPC periodic structures with different periodic parameters; when a dual-wavelength light source is used for vertical incidence from the substrate side, for a wavelength lambda 1, 1 DPC structure, the structure is emitted in a transverse wave vector kt1, and an evanescent field with the intensity attenuating along with the exponential distance away from the surface is formed on the upper surface of the one-dimensional photonic crystal composite structure; and for the DPC structure with the wavelength of lambda 2, 1, the DPC structure is emitted in a transverse vector kt2, and a transmission field is formed on the upper surface of the one-dimensional photonic crystal composite structure. The problem of inaccurate measurement caused by crosstalk of geometric dimension and axial position information of the sample is solved, and the defects that an existing system is complex, large in size and high in cost are overcome.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical measurement technology, in particular to a label-free axial super-resolution measurement system and method based on one-dimensional photonic crystal. BACKGROUND

[0002] The existing label-free axial super-resolution measurement technology has the following technical problems:

[0003] (1) Measurement error: the geometric size information of the sample may interfere with the axial position information. For example, a large particle and a small particle closer to the surface may produce similar scattering intensity or spatial spectrum distribution, resulting in measurement error.

[0004] (2) Complex and expensive system: most existing technologies rely on high numerical aperture (usually oil immersion) objective lens, complex light field modulation elements (such as spatial light modulator) and high-precision signal acquisition system, resulting in a large volume, high cost and complex operation of the entire measurement device, which is difficult to meet the development trend of optical measurement integration and lightweight.

[0005] (3) Limited application: for fluorescence technology, there are problems such as photobleaching, complicated fluorescent labeling process and possible impact on sample activity. Although the label-free technology has advantages, the signal-to-noise ratio and measurement accuracy are easily disturbed by various factors.

[0006] Therefore, the present application designs a label-free axial super-resolution measurement system and method based on one-dimensional photonic crystal to solve the above problems. SUMMARY

[0007] In view of the above shortcomings of the prior art, the present application provides a label-free axial super-resolution measurement system and method based on one-dimensional photonic crystal, which is lightweight, low-cost, easy to operate and accurate.

[0008] To achieve the above purpose, the present application realizes the following technical solutions:

[0009] The label-free axial super-resolution measurement system based on one-dimensional photonic crystal comprises a dual-wavelength light source, a one-dimensional photonic crystal composite structure, a signal acquisition module and a data processing module.

[0010] The dual-wavelength light source is an illumination light containing two wavelengths λ1 and λ2.

[0011] The one-dimensional photonic crystal composite structure comprises a substrate and a 1DPC structure from bottom to top.

[0012] The 1DPC structure is formed by alternately stacking high refractive index material and low refractive index material, and contains at least two 1DPC period structures with different period parameters.

[0013] When using a dual-wavelength light source to make a perpendicular incidence from the substrate side:

[0014] For wavelength λ1, the 1DPC structure makes it exit with a transverse wave vector kt1, which is greater than the light wave vector in the environment medium where the sample is located, forming an evanescent field on the surface of the one-dimensional photonic crystal composite structure, which decays exponentially with the distance from the surface;

[0015] For wavelength λ2, the 1DPC structure makes it exit with a transverse wave vector kt2, which is smaller than kt1, forming a transmission field on the surface of the one-dimensional photonic crystal composite structure;

[0016] The signal acquisition module is used to collect the scattered light generated by the interaction of the sample with the evanescent field and the transmission field, and to superimpose to form a color image;

[0017] The data processing module is used to extract the spatial spectrum features of the λ2 color channel in the color image to obtain the geometric size related information of the sample, to extract the total intensity of the scattered light of the λ1 color channel and exclude the size influence to obtain the axial position related information of the sample, and to analyze the chroma value of the sample point in the color image and obtain the axial position of the sample based on the chroma-position mapping relationship.

[0018] Further, the λ1 is 532nm, and the λ2 is 633nm.

[0019] Further, the 1DPC structure is formed by stacking 9 layers of high refractive index material and low refractive index material alternately.

[0020] Further, the high refractive index material uses Si3N4, and the low refractive index material uses SiO2.

[0021] Further, the signal acquisition module includes a standard microscopic objective and a color camera.

[0022] Further, the color camera is sCMOS or EMCCD.

[0023] In order to better achieve the purpose of the present application, the present application also provides a label-free axial super-resolution measurement method based on a one-dimensional photonic crystal, which comprises the following steps:

[0024] Step one, sample placement: placing the nano sample to be measured on the upper surface of the above-mentioned one-dimensional photonic crystal composite structure;

[0025] Step two, dual-wavelength illumination: using the light source module to make a perpendicular or nearly perpendicular incidence of illumination light containing λ1 and λ2 from below the one-dimensional photonic crystal composite structure;

[0026] Step three, light field generation and sample scattering: after the illumination light is modulated by the 1DPC structure, the evanescent field and the transmission field are generated in the sample area at the same time, the sample interacts with the two fields respectively, and two colors of scattered light corresponding to λ1 and λ2 are generated;

[0027] Step four, information collection: the two colors of scattered light are collected from above by the standard microscope objective and the color camera of the signal collection module, and are superimposed to form a color image;

[0028] Step five, axial position decoupling and reconstruction: the spatial spectrum features of the λ2 color channel in the color image are extracted by the data processing module to obtain the sample geometric size related information, the total intensity of the scattered light of the λ1 color channel is extracted and the size influence is excluded to obtain the axial position related information of the sample, and the chroma value of the sample point in the color image is analyzed, and the axial position of the sample is obtained based on the chroma-position mapping relationship;

[0029] Step six, result output.

[0030] Further, the nanosample is a polystyrene microsphere, a virus or an organelle.

[0031] Compared with the prior art, the present application has the following advantages: high accuracy: through dual-wavelength and dual-mode illumination, the sample geometric size and axial position information are effectively decoupled, the crosstalk error source is fundamentally eliminated, and the accuracy of the label-free axial measurement is significantly improved.

[0032] Lightweight and low cost: a thin film chip (one-dimensional photonic crystal composite structure) is used to replace the large and expensive high numerical aperture objective lens and the complex external light field modulation system in the traditional technology. The measurement process only relies on a standard microscope and a color camera, which greatly reduces the volume, complexity and cost of the system.

[0033] Simple operation and intuitive results: the complex axial position information is converted into intuitive color change, the operator can even make a semi-quantitative judgment by naked eye, the data processing process is simplified, the use threshold is reduced, and a new dimension of visual observation is provided for label-free axial super-resolution imaging. BRIEF DESCRIPTION OF DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creating any inventive labor.

[0035] Figure 1 The principle diagram for realizing "wavelength-lateral wave vector-chroma encoding" for the 1DPC structure.

[0036] Figure 2 The difference of intensity distribution in axial direction between the evanescent field generated by λ1 and the transmission field generated by λ2.

[0037] Figure 3 The schematic diagram of the mapping principle of "chromaticity-axial position" and the simulation effect in the present application. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings of the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the present application.

[0039] Embodiment one: please refer to the accompanying drawings of the specification Figure 1 The label-free axial super-resolution measurement system based on one-dimensional photonic crystal includes a dual-wavelength light source, a one-dimensional photonic crystal composite structure, a signal acquisition module and a data processing module.

[0040] The dual-wavelength light source is an illumination light containing two wavelengths λ1 and λ2, wherein λ1 is 532 nm and λ2 is 633 nm.

[0041] The one-dimensional photonic crystal composite structure includes a substrate and a 1DPC (one-dimensional photonic crystal) structure from bottom to top.

[0042] The 1DPC structure is formed by alternately stacking 9 layers of high refractive index material and low refractive index material, and contains at least two 1DPC period structures with different period parameters.

[0043] By accurately designing the material and thickness of each 1DPC period structure, the 1DPC structure has a specific photonic band gap and conduction band. The core function of the 1DPC structure is to realize the encoding of "wavelength-lateral wave vector", that is, when the dual-wavelength light source is vertically or nearly vertically incident from the substrate side:

[0044] For wavelength λ1 (for example, 532 nm), the 1DPC structure makes it exit with a larger lateral wave vector (kt1), which is larger than the light wave vector in the environment medium where the sample is located, thereby forming an evanescent field with an intensity exponentially decaying with the distance from the surface on the upper surface of the one-dimensional photonic crystal composite structure.

[0045] For wavelength λ2 (for example, 633 nm), the 1DPC structure makes it exit with a smaller lateral wave vector (kt2), thereby forming a transmission field on the upper surface of the one-dimensional photonic crystal composite structure.

[0046] wherein the high refractive index material can employ Si3N4, etc., and the low refractive index material can employ SiO2, etc.;

[0047] The signal acquisition module comprises a standard microscope objective and a color camera, which is sCMOS or EMCCD, for collecting the scattered light generated by the interaction of the sample and the evanescent field and the transmission field, and superimposing to form a color image.

[0048] The data processing module is used to extract the spatial spectrum features of the λ2 color channel in the color image to obtain the sample geometric size related information, to extract the total intensity of the scattered light of the λ1 color channel and exclude the size influence to obtain the sample axial position related information, and to analyze the chroma value of the sample points in the color image and obtain the sample axial position based on the chroma-position mapping relationship.

[0049] Embodiment two: a label-free axial super-resolution measurement method based on one-dimensional photonic crystal, comprising the following steps:

[0050] Step one, sample placement: placing the nanometer sample to be measured (such as polystyrene microspheres, viruses, organelles, etc.) on the upper surface of the above-mentioned one-dimensional photonic crystal composite structure;

[0051] Step two, dual-wavelength illumination: using the light source module to vertically or nearly vertically incident illumination light containing λ1 and λ2 from below the one-dimensional photonic crystal composite structure;

[0052] Step three, light field generation and sample scattering: after the illumination light is modulated by the 1DPC structure, the evanescent field (generated by λ1) and the transmission field (generated by λ2) are simultaneously generated in the sample area, and the sample interacts with the two fields respectively to generate two colors of scattered light corresponding to λ1 and λ2;

[0053] Step four, information acquisition: collecting the two colors of scattered light from above by the standard microscope objective and the color camera of the signal acquisition module, and superimposing to form a color image;

[0054] Step five, axial position decoupling and reconstruction: through the data processing module, the spatial spectrum features of the λ2 color channel in the color image are extracted to obtain the sample geometric size related information, the total intensity of the scattered light of the λ1 color channel is extracted and the size influence is excluded to obtain the sample axial position related information, and the chroma value of the sample points in the color image is analyzed, and the sample axial position is obtained based on the chroma-position mapping relationship; specifically including:

[0055] (1) Geometric size information extraction: the transmission field illumination generated by the λ2 wavelength will make the scattered light distribution have the spatial frequency shift (SFS) effect. By analyzing the λ2 color channel signal in the color image (especially its spatial spectrum distribution characteristics at the back focal plane of the objective), information strongly related to the geometric size of the sample can be obtained.

[0056] The core principle of geometric dimension determination is the spatial frequency shift (SFS) effect generated by the transmission field illumination of the wavelength λ2, combined with the Mie scattering theory and numerical simulation logic, to achieve size extraction through the quantitative correlation of "scattered light spectral characteristics-sample geometric size". When the sample is irradiated by the transmission field generated by the wavelength λ2 (the transverse wave vector kt2≤k b , k b is the environmental free wave vector), the geometric size of the sample directly modulates the spatial distribution of the scattered light, making the scattered light exhibit a strong SFS effect related to the size; by analyzing the spatial spectral characteristics corresponding to the effect and combining the pre-established calibration model, the geometric size of the sample can be inversely deduced.

[0057] (2) Axial position information extraction: the evanescent field generated by the wavelength λ1 (532 nm) is extremely sensitive to the axial position. The total intensity of the scattered light in the specific color channel (green channel, G channel) corresponding to λ1 in the color image directly reflects the axial position of the sample after excluding the size effect through the λ2 channel. The specific process is as follows: first, locate the pixel points or regions corresponding to the "unknown sample" in the color image; then, based on the dual-wavelength coding design (λ1=532 nm corresponds to the G channel), extract the G channel intensity value of the pixels in the region; finally, combine the size calibration result of the λ2 channel (633 nm corresponds to the R channel), and calculate the (x, y) chromaticity coordinates of the color of the corresponding region of the sample through the "intensity ratio transformation of target channel and reference channel" in standard colorimetry.

[0058] (3) Chromaticity-position mapping: due to the different attenuation curves of the light intensity of the two wavelengths with the axial height (exponential decay of evanescent field, transmission field basically unchanged), the color of the superimposed image (for example, from red to green) will change continuously with the axial height of the sample. Through pre-theoretical calculation or experimental calibration of standard samples, an accurate "image chromaticity coordinate-sample axial position" mapping function is established.

[0059] The mapping function of image chromaticity coordinate-sample axial position is constructed based on the exponential decay characteristics of the evanescent field intensity. Assuming that the chromaticity coordinate of the sample in the imaging system is (x, y), since the scattered light intensity I λ1 of the wavelength λ1 decays exponentially with the axial position z ( ), while the scattered light intensity I λ2 of the wavelength λ2 is not sensitive to the axial position change (I λ2 ≈C), then the axial position z of the sample and the chromaticity coordinate x (or y) satisfy the following semi-logarithmic function relationship:

[0060]

[0061] Wherein, A represents a characteristic attenuation coefficient, the value of which is determined by the angle of incident light, wavelength and the difference between the refractive index of the surface of one-dimensional photonic crystal and the ambient medium, and controls the rate of color change with height; B represents a system response weighting coefficient, which is used to represent the relative detection sensitivity of the imaging system to the scattered light of the two wavelengths; C represents a height correction coefficient; X λ1 represents the first limit chromaticity coordinate, i.e. the theoretical chromaticity coordinate when only the scattered light of wavelength λ1 (evanescent field) exists; X λ2 represents the second limit chromaticity coordinate, i.e. the theoretical chromaticity coordinate when only the scattered light of wavelength λ2 (transmission field) exists.

[0062] Or simplified as the form based on the red-green channel ratio:

[0063]

[0064] Wherein, is the penetration depth of the evanescent field, which depends on the incident angle and the interface refractive index; k is a constant coefficient related to the scattering cross section of the system and the sample; is the two-wavelength intensity ratio function extracted from the chromaticity coordinate (x, y) (for example or the G / R ratio in the RGB space). The function shows that the axial position is linearly related to the logarithm of the chromaticity characteristic value, and the undetermined coefficients A, B, C or k in the above formula can be determined by calibrating the standard nanoparticles.

[0065] (4) Obtain the (x, y) chromaticity coordinate of the color of the corresponding region of the sample based on step (2), and refer to the image chromaticity coordinate-sample axial position mapping function to obtain the axial position of the sample with sub-10-nanometer precision.

[0066] Step six, result output.

[0067] For unknown samples, only the color image of the sample needs to be collected, and the chromaticity value of the sample point in the image is analyzed, so that the axial position of the sample with sub-10-nanometer precision can be quickly and accurately obtained by referring to the image chromaticity coordinate-sample axial position mapping function.

[0068] The application encodes two different wavelengths of light by using one-dimensional photonic crystal (1DPC) "wavelength-lateral wave vector". One wavelength is encoded to generate an evanescent field, and the scattering intensity thereof is mainly related to the axial position of the sample; the other wavelength is encoded to generate a transmission field and realize spatial frequency shift (SFS), and the scattering spatial frequency spectrum feature thereof is mainly related to the geometric size of the sample. Through the combination of double wavelengths and double modes, the mutual coupling interference of size and position information is fundamentally eliminated.

[0069] The present application superimposes two wavelength scattering signals to form a color image. Because the scattering intensity of the two wavelengths varies at different axial heights, the sample will exhibit different colors at different axial heights. Thus, a direct mapping relationship between the image chromaticity coordinates and the axial position of the sample is established, so that the operator can directly determine the axial position of the sample according to the color by the human eye or a common color camera, realizing the visualization, lightness and low cost of the measurement.

[0070] Figure 1 Through the visualization method, the "structure design → wave vector regulation → signal coding" logic of the 1DPC structure is clearly presented: without complex external light field modulation devices, the lateral wave vector of the dual-wavelength can be accurately encoded by the difference in the layering period of Si3N4 / SiO2, and the abstract wave vector difference is converted into intuitive chromaticity difference.

[0071] Figure 2 Through intuitive curve comparison, it is clearly proved that the "near-surface high sensitivity attenuation" of λ1 evanescent field and the "wide range constant intensity" of λ2 transmission field are complementary and independent - this difference enables the dual-wavelength light field to "lock" the axial position and geometric size of the sample respectively, avoiding the "size and position crosstalk" problem in traditional technology from the physical level, and providing the underlying basis for the subsequent "chromaticity-position mapping" (such as near-surface sample green and far-surface sample red).

[0072] Figure 3 Through the triple presentation of "CIE color chart (quantitative mapping) + correlation curve (mathematical relationship) + simulation imaging (intuitive effect)", the feasibility of the "chromaticity-axial position" mapping is fully proved: at the physical level, based on the light field intensity difference of Figure 2 , the monotonic relationship of "z→I1 / I2" is ensured; at the chromaticity level, through the standard chromaticity transformation, the unique correspondence of "I1 / I2→(x, y)" is ensured; at the application level, through simulation imaging, the identifiability and resolution of "(x, y)→color" are verified; these three logical closed loops ultimately support the core advantages of the present application "no labeling, lightness and high accuracy".

[0073] The above embodiments are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement for part of the technical features; and these modifications or replacements will not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A label-free axial super-resolution measurement system based on a one-dimensional photonic crystal, characterized in that, It includes a dual-wavelength light source, a one-dimensional photonic crystal composite structure, a signal acquisition module, and a data processing module; The dual-wavelength light source is illumination light containing two wavelengths, λ1 and λ2; The one-dimensional photonic crystal composite structure includes a substrate and a 1DPC structure from bottom to top; The 1DPC structure is composed of alternating stacks of high-refractive-index materials and low-refractive-index materials, and includes at least two 1DPC periodic structures with different periodic parameters. When a dual-wavelength light source is incident perpendicularly from the substrate side: For wavelength λ1, the 1DPC structure causes it to emit a transverse wave vector kt1, which is greater than the light wave vector in the ambient medium where the sample is located, forming an evanescent field on the surface of the one-dimensional photonic crystal composite structure whose intensity decreases exponentially with the distance from the surface. For wavelength λ2, the 1DPC structure causes it to emit a transverse wave vector kt2, where kt2 is less than kt1, forming a transmission field on the surface of the one-dimensional photonic crystal composite structure. The signal acquisition module is used to collect scattered light generated by the interaction between the sample and the evanescent field and the transmission field, and superimpose them to form a color image; The data processing module is used to extract the spatial spectral features of the λ2 color channel in the color image to obtain sample geometric size correlation information, to extract the total intensity of scattered light from the λ1 color channel and eliminate the influence of size to obtain axial position correlation information of the sample, and to analyze the chromaticity values ​​of sample points in the color image and obtain the axial position of the sample based on the chromaticity-position mapping relationship.

2. The label-free axial super-resolution measurement system based on a one-dimensional photonic crystal according to claim 1, characterized in that, The λ1 is 532nm and the λ2 is 633nm.

3. The label-free axial super-resolution measurement system based on a one-dimensional photonic crystal according to claim 1, characterized in that, The 1DPC structure is composed of nine layers of alternating high-refractive-index and low-refractive-index materials.

4. The label-free axial super-resolution measurement system based on a one-dimensional photonic crystal according to claim 1, characterized in that, The high-refractive-index material is Si3N4, and the low-refractive-index material is SiO2.

5. The label-free axial super-resolution measurement system based on a one-dimensional photonic crystal according to claim 1, characterized in that, The signal acquisition module includes a standard microscope objective and a color camera.

6. The label-free axial super-resolution measurement system based on a one-dimensional photonic crystal according to claim 5, characterized in that, The color camera is either sCMOS or EMCCD.

7. A label-free axial super-resolution measurement method based on a one-dimensional photonic crystal, utilizing the label-free axial super-resolution measurement system based on a one-dimensional photonic crystal as described in any one of claims 1 to 6, characterized in that, Includes the following steps: Step 1, Sample Placement: Place the nanosample to be measured on the upper surface of the above-mentioned one-dimensional photonic crystal composite structure; Step 2, Dual-wavelength illumination: Illumination light containing λ1 and λ2 is incident vertically from below the one-dimensional photonic crystal composite structure using a light source module; Step 3, Light Field Generation and Sample Scattering: After the illumination light is modulated by the 1DPC structure, an evanescent field and a transmission field are generated simultaneously in the sample area. The sample interacts with the two fields respectively, producing scattered light of two colors corresponding to λ1 and λ2. Step 4: Information Acquisition: The signal acquisition module collects two colors of scattered light from above and superimposes them to form a color image; Step 5, Axial Position Decoupling and Reconstruction: The spatial spectral features of the λ2 color channel in the color image are extracted by the data processing module to obtain the geometric size correlation information of the sample. The total intensity of scattered light from the λ1 color channel is extracted and the size influence is eliminated to obtain the axial position correlation information of the sample. At the same time, the chromaticity values ​​of the sample points in the color image are analyzed, and the axial position of the sample is obtained based on the chromaticity-position mapping relationship. Step 6: Output the results.

8. The label-free axial super-resolution measurement method based on a one-dimensional photonic crystal according to claim 7, characterized in that, The nanosamples are polystyrene microspheres, viruses, or organelles.

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