Rapid non-destructive testing method of nanopore membrane based on dark-field scattering optical method

Abstract

The application provides a kind of nanometer hole nuclear pore membrane rapid nondestructive testing method based on dark field scattering optical method, comprising: using dark field optical microscope to focus on the nuclear pore membrane to be detected, after focusing, adjust to dark field mode, obtain the scattering light image of the nuclear pore membrane to be detected under dark field mode;Determine the image area and the number of scattering light spots in the image based on the scattering light image, obtain the density of the nuclear pore membrane;Move the dark field optical microscope light spot center to the single hole position to collect dark field scattering spectrum, obtain the single hole dark field scattering spectrum intensity I ;Based on the relationship between scattering spectrum intensity I And aperture D Formula I = aD 2 +b , the aperture of the nuclear pore module is calculated by the single hole dark field scattering spectrum intensity I Therefore, the application has the advantages of simple operation, low cost, fast detection speed, etc., and can be widely applied in the field of nanotechnology and material science.

Description

Technical Field

[0001] This invention relates to a rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics, and relates to the fields of membrane materials and nuclear technology. Background Technology

[0002] In the fields of nanotechnology and materials science, the density, distribution, and size of nanopores are crucial parameters affecting their performance and applications. Nuclear pore membranes are a special type of membrane material with a nanoporous structure, widely used in the biomedical and biotechnology fields. The nanopores in nuclear pore membranes can be used for material sieving across scales, with size thresholds ranging from tens of micrometers to angstroms, and are widely used in numerous membrane material fields such as wastewater treatment, food preservation, and ion separation.

[0003] Rapid detection of nuclear pore membranes is of great significance for basic research and commercial application. Rapid detection involves two stages of nuclear pore membrane production: irradiation detection and etching detection. Rapid, non-destructive testing of the distribution, density, and size of nanopores in nuclear pore membranes is essential for their commercial functionality. Generally, nuclear pore membranes with micron-sized pores far exceeding the optical diffraction limit can be rapidly detected using bright-field optical microscopy, which typically requires the nuclear pore density to not exceed 10-1. 7 pores / cm 2 Otherwise, etching crosslinking will lead to inaccurate detection. With the increasing demand for high-density and small-pore nuclear pore membranes, more precise detection equipment is needed. The characterization methods commonly used for these nuclear pore membranes include scanning electron microscopy (SEM) and atomic force microscopy (AFM).

[0004] While the methods described above can provide high-resolution images, they also have some limitations: 1) Complex sample preparation: SEM typically requires metal sputtering of samples, and AFM also requires specialized sample preparation to maintain low surface roughness. 2) High equipment operating costs: These microscopes are expensive and require specialized operators and maintenance, resulting in high overall operating costs. 3) Limited detection range: Although SEM and AFM can operate under normal conditions, their scanning range is relatively small, making it difficult to rapidly characterize large-area samples. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. Therefore, in response to the above-mentioned problems, the object of the present invention is to provide a rapid non-destructive testing method for nanopore nuclear pore membranes based on dark-field scattering optics, capable of achieving efficient and non-destructive testing of nanopores.

[0006] To achieve the aforementioned objectives, the present invention employs the following technical solution: a rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics, comprising:

[0007] The nuclear pore membrane to be tested was focused using a dark-field optical microscope. After focusing, the microscope was switched to dark-field mode to acquire the scattered light image of the nuclear pore membrane to be tested in dark-field mode.

[0008] The density of the nuclear pore membrane is obtained by determining the image area and the number of scattered light spots in the image based on the scattered light image;

[0009] By moving the center of the dark-field optical microscope spot to the position of a single aperture, dark-field scattering spectra are acquired to obtain the intensity of the single-aperture dark-field scattering spectrum. I ;

[0010] Based on scattering spectral intensity I With aperture D relational formula I=aD 2 +b Spectral intensity through single-aperture dark field scattering I The pore size of the nuclear pore motif was calculated, where, a and b Fitting parameters.

[0011] In some possible implementations, a dark-field optical microscope is used to focus the nuclear pore membrane to be tested. After focusing, the microscope is switched to dark-field mode, and a scattered light image of the nuclear pore membrane to be tested is acquired in dark-field mode, including:

[0012] The nuclear pore membrane under test was located by low-magnification focusing using a low-magnification objective lens of a dark-field optical microscope, and then the nuclear pore membrane under test was refocused using a high-magnification objective lens after low-magnification focusing.

[0013] After high-magnification focusing, the dark-field optical microscope is switched to dark-field mode to increase the brightness of the light source. At the same time, the focus is finely adjusted to make the dark-field spot the brightest, and the scattered light image of the nuclear pore membrane is acquired in dark-field mode.

[0014] In some possible implementations, the low-power objective lens is a 10x or 20x objective lens; the high-power objective lens is a 50x or 100x objective lens.

[0015] In some possible implementations, the density of the nuclear pore membrane is obtained by determining the image area and the number of scattered light spots in the image based on the scattered light image, including:

[0016] The length and width of the scattered light image are measured using the actual scale of the dark-field optical microscope to obtain the image area.

[0017] The number of scattered light spots in the scattered light image can be counted either by setting a program or manually.

[0018] The density of the nuclear pore membrane is calculated by dividing the total number of scattered light spots by the image area.

[0019] Some possible implementations also include a step of processing the scattered light image, specifically: adjusting the contrast of the scattered light image to make the scattered light spot of the nuclear pore membrane clear.

[0020] In some possible implementations, the intensity of the scattering spectrum I With aperture D relational formula I=aD 2 +b The acquisition process includes:

[0021] Preparation of nuclear pore membrane samples;

[0022] The prepared nuclear pore membrane sample is flattened and attached to a glass slide;

[0023] Determining the pore size of nuclear pore membrane samples using scanning electron microscopy D ;

[0024] Dark-field optical microscopy was used to acquire dark-field scattering spectra, background signal spectra, and light source spectra at a single aperture location;

[0025] Calculate the intensity of dark field scattering spectrum in a single aperture I = (Dark field scattering spectrum - Background signal spectrum) / (Light source spectrum - Background signal spectrum);

[0026] Based on the intensity of dark field scattering spectrum of a single hole I And the pore size of the nuclear pore membrane sample obtained by scanning electron microscopy D Perform data fitting and obtain fitting parameters. a and b Thus, the intensity of the dark field scattering spectrum of a single aperture is obtained. I With aperture D relational formula I=aD 2 +b .

[0027] In some possible implementations, the preparation of nuclear pore membrane samples includes:

[0028] The nuclear pore membrane irradiated with fast heavy ions was expanded by chemical etching to achieve a pore size exceeding 30 nm and a flux not exceeding 5 × 10⁻⁶. 8 ions / cm 2 After etching, the nuclear pore membrane is ultrasonically cleaned in an aqueous solution, and the remaining water droplets are evaporated to obtain the nuclear pore membrane sample.

[0029] In some possible implementations, dark-field optical microscopy acquires dark-field scattering spectra, background signal spectra, and light source spectra at a single aperture location, including:

[0030] Move the center of the dark-field optical microscope spot to a single aperture position to collect dark-field scattering spectra;

[0031] After collecting the dark-field scattering spectrum of the non-pore locations on the nuclear pore membrane, the light source was turned off and the background signal spectrum was collected.

[0032] The nuclear pore membrane sample to be tested was replaced with a reflector, and the light source spectrum was collected in bright field mode.

[0033] Because the present invention adopts the above technical solution, it has the following characteristics:

[0034] 1. This invention utilizes dark-field optical microscopy to perform rapid imaging and spectral testing of samples, enabling rapid detection of nanopore uniformity and pore density. Furthermore, by analyzing the scattered light imaging distribution and spectral intensity, it achieves quantitative detection of nanopore pore size.

[0035] 2. This invention fully leverages the advantages of dark-field optics in high signal-to-noise ratio imaging and convenient spectral testing. Through dark-field imaging, spectral testing, and data analysis, it enables rapid detection of nanoscale pore sizes in nuclear pore membranes.

[0036] 3. This invention provides the advantages of high signal-to-noise ratio imaging and convenient spectral testing by leveraging dark-field optics. It improves the non-destructive and rapid detection of nanoscale pores in nuclear pore membranes through dark-field spectral imaging and quantitative spectral testing. Since the dark-field scattering optics method is not dependent on the type and thickness of nuclear pore membranes, it can be used for the rapid detection of different types of nuclear pore membranes.

[0037] 4. This invention utilizes dark-field scattering optical imaging and spectral detection to perform rapid dark-field detection of nuclear pore membranes with pore sizes ranging from hundreds to tens of nanometers through dark-field scattering light analysis. It obtains key parameters such as uniformity, density, and pore size of the nuclear pore membrane, solving the problems of low resolution in traditional bright-field optical imaging and low efficiency in characterization by precision instruments such as electron microscopes and atomic force microscopes. It can achieve non-destructive and rapid pore size detection of nanopores in nuclear pore membranes larger than 30 nanometers, and has extremely high practical value.

[0038] 5. This invention characterizes nanopore pore size based on dark-field scattering optics, enabling efficient and non-destructive testing of nanopores. By imaging the sample with a dark-field optical microscope and analyzing the intensity and distribution of scattered light, quantitative detection of nanopore pore size can be achieved.

[0039] In summary, this invention has the advantages of simple operation, low cost, and fast detection speed, and can be widely applied in the fields of nanotechnology and materials science. Attached Figure Description

[0040] Various other advantages and benefits will become apparent to those skilled in the art upon reading the following detailed description of preferred embodiments. The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts. In the drawings:

[0041] Figure 1 The distribution of nuclear pore membranes observed in dark-field mode under a 100x objective lens in an embodiment of the present invention;

[0042] Figure 2 The image shows the dark-field detection of the hierarchical pore size nuclear pore membrane implemented in this invention. The upper left is an SEM image, the upper middle is a bright-field microscopy image, and the upper right is a dark-field scattering image. The lower left is the dark-field scattering spectrum of individual nanopores with different pore sizes, the lower middle is the relationship between the dark-field scattering spectrum and the pore size, and the lower right is a schematic diagram of the dark-field objective lens.

[0043] Figure 3 For the pore size limit detection of this embodiment of the invention, the first row of images are SEM images of the nuclear pore membrane as the pore size decreases sequentially, the second row of images are dark field scattering optical representations, and the third row of images are dark field scattering spectral results of different nanopores at the corresponding pore size.

[0044] Figure 4 This is a schematic diagram illustrating the mathematical relationship between the aperture diameter and the integral of the scattered light intensity in an embodiment of the present invention. Detailed Implementation

[0045] It should be understood that the terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used herein may also include the plural forms. The terms “comprising,” “including,” “containing,” and “having” are inclusive and therefore indicate the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The method steps, processes, and operations described herein are not construed as requiring them to be performed in a particular order described or illustrated unless the order of performance is explicitly indicated. It should also be understood that additional or alternative steps may be used.

[0046] Although terms such as first, second, third, etc., may be used in this document to describe multiple elements, components, regions, layers, and / or segments, these elements, components, regions, layers, and / or segments should not be limited by these terms. These terms may be used only to distinguish one element, component, region, layer, or segment from another. Unless the context clearly indicates otherwise, terms such as "first," "second," and other numerical terms used herein do not imply order or sequence. Therefore, the first element, component, region, layer, or segment discussed below may be referred to as the second element, component, region, layer, or segment without departing from the teachings of the exemplary embodiments.

[0047] For ease of description, spatial relative terms may be used in the text to describe the relationship of one element or feature relative to another element or feature as shown in the figure. These relative terms include, for example, "inside," "outside," "middle," "outer," "below," "above," etc. Such spatial relative terms are intended to include different orientations of the device in use or operation, other than those depicted in the figure.

[0048] In recent years, dark-field optical microscopy has attracted widespread attention due to its ability to provide high-contrast images without damaging samples. Dark-field optical microscopy avoids direct incident light entering the objective lens; instead, it images the sample through scattered light, thus enhancing the contrast of sample details. This method is particularly suitable for observing minute structures and defects in samples. Dark-field scattering optics utilizes the dark-field imaging technology of dark-field optical microscopy to characterize nanostructures by analyzing the intensity and distribution of scattered light from the sample. Compared to traditional methods, dark-field optical microscopy has the following advantages: 1) Non-destructive testing: Dark-field imaging does not require complex sample pretreatment, enabling testing without damaging the sample. 2) Simple operation: Dark-field optical microscopy is relatively simple to operate, does not require a high-vacuum environment, and is suitable for various laboratory conditions. 3) Low cost: Dark-field optical microscopy equipment is inexpensive and easy to maintain, making it suitable for large-scale application. 4) Rapid imaging: Dark-field optical microscopy can rapidly image large-area samples, making it suitable for high-throughput testing.

[0049] To address the challenge of rapid detection of nuclear pore membranes with pore sizes ranging from hundreds to tens of nanometers, this invention provides a rapid, non-destructive detection method for nanopore nuclear pore membranes based on dark-field scattering optics. The method includes: focusing the nuclear pore membrane under test using a dark-field optical microscope; adjusting the microscope to dark-field mode after focusing; acquiring a scattered light image of the membrane in dark-field mode; determining the image area and the number of scattered light spots in the image based on the scattered light image to obtain the density of the nuclear pore membrane; and moving the center of the dark-field optical microscope spot to a single pore location to acquire a dark-field scattering spectrum, thereby obtaining the intensity of the single-pore dark-field scattering spectrum. I Based on scattering spectral intensityI With aperture D relational formula I=aD 2 +b Spectral intensity through single-aperture dark field scattering I The pore size of the nuclear pore membrane is calculated. Therefore, this invention fully leverages the advantages of dark-field optics in high signal-to-noise ratio imaging and convenient spectral testing, achieving rapid detection of nanoscale pore sizes in nuclear pore membranes through dark-field imaging, spectral testing, and data analysis.

[0050] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0051] This embodiment provides a rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics. By subtracting reflected optical signals in dark-field optics to enhance the signal-to-noise ratio, it achieves rapid detection of nuclear pore membranes within a specific pore size range. The specific implementation process includes:

[0052] S1. Sample preparation.

[0053] In this embodiment, the specific process of sample preparation is as follows:

[0054] S11. Expand the nuclear pore membrane after fast heavy ion irradiation using chemical etching to achieve a pore size exceeding 30 nm, and ensure that the flux (representing the sub-beam within a unit area irradiated by the heavy ion beam within a certain time) does not exceed 5 × 10⁻⁶. 8 ions / cm 2 Specifically, this involves multiple rounds of irradiation etching, specifically using fast heavy ion irradiation at 5×10⁻⁶ ppm. 6 ions / cm 2 Sensitized etching to 50-100 nm, the above irradiation etching was repeated 3 to 4 times. After etching, the nuclear pore membrane was ultrasonically cleaned in an aqueous solution, the residual water droplets were evaporated, and the prepared nuclear pore membrane sample was flatly attached to a glass slide.

[0055] S12. Determine the pore size of the above nuclear pore membrane samples using scanning electron microscopy. D .

[0056] S2, Optical Imaging.

[0057] In this embodiment, the specific process of optical imaging is as follows:

[0058] The nuclear pore membrane sample is positioned by low-magnification focusing using a 10x objective lens of a dark-field optical microscope. After focusing, a high-magnification objective lens is used to refocus the sample. A 50x or 100x objective lens is typically chosen as an example, but this is not the only option; the magnification should be selected based on the specific situation. For the detection of nanopores smaller than 100 nm, a 100x objective lens must be selected. After focusing, the dark-field optical microscope is switched to dark-field mode (dark-field mode is a special mode for microscopes that subtracts the reflected signal from the light source, effectively reducing the surrounding background signal and improving detection sensitivity). The brightness of the light source is increased, and the focus is fine-tuned to maximize the brightness of the dark-field spot. The scattered light image of the nanopore is then acquired in dark-field mode.

[0059] S3, Data Processing.

[0060] In this embodiment, the specific data processing procedure is as follows:

[0061] S31. Process the acquired scattered light image, specifically by adjusting the image contrast to make the scattered light spot of the nanopore clear and to determine the irradiation uniformity.

[0062] S32. Determine the image area and count the number of scattered light spots in the image based on the scale of the dark-field optical microscope image. Specifically, measure the length and width of the image according to the actual scale of the dark-field optical microscope (instrument parameters, which can be calibrated) to obtain the image area. The number of scattered light spots can be measured by setting a program or manually counting.

[0063] S33. Calculate the density of the nuclear pore membrane.

[0064] In this embodiment, the density of the nuclear pore membrane is the total number of scattered light spots divided by the area of ​​the image.

[0065] S4, Spectral Acquisition.

[0066] In this embodiment, the specific process of spectral acquisition is as follows:

[0067] S41. After acquiring the scattered light image using dark-field mode, if rapid aperture assessment is required, spectral testing needs to be performed. The center of the dark-field optical microscope spot is moved to the location of a single aperture for dark-field scattering spectral acquisition. The grating is set to 150 g / mm, and the spectral center is at 750 nm. The acquisition integration time and number of integrations are set according to the signal intensity. For nanopores smaller than 100 nm, the integration time is set to 30 s, and the number of integrations is 5. This is an example, but not limited to this. A 20-megapixel fast camera is used to capture dark-field light scattering imaging of the nanopores, and a visible-light grating spectrometer is used to perform spectral testing on the detection area.

[0068] S42. After collecting the dark-field scattering spectrum at the non-pore location on the nuclear pore membrane, turn off the light source and collect the background signal spectrum.

[0069] S43. Replace the nuclear pore membrane sample with a reflector and collect the light source spectrum in bright field mode.

[0070] S5, Spectral Data Processing.

[0071] In this embodiment, the specific process of spectral data processing is as follows:

[0072] Calculate the spectral intensity: Single-aperture dark-field scattering spectral intensity = (Dark-field scattering spectrum - Background signal spectrum) / (Light source spectrum - Background signal spectrum). Considering the possibility of random errors at a single wavelength, the calculation range is selected as 550 nm-750 nm.

[0073] By fitting the data of the dark-field scattering spectral intensity of a single aperture with the aperture size obtained from scanning electron microscopy, a mathematical relationship between the dark-field scattering spectral intensity of a single aperture and the aperture size is obtained: I=aD 2 +b In the formula, a and b Fitting parameters.

[0074] S6. Aperture characterization.

[0075] In this embodiment, the specific implementation process of aperture characterization is as follows: based on the obtained single-aperture dark-field scattering spectral intensity, the fitting parameters are determined. a and b In the case of, through I=aD 2 +b The pore size of the nuclear pore membrane to be tested can then be obtained.

[0076] In summary, this invention provides a method for imaging and spectral analysis using dark-field scattered light. In this method, the pore size of the nuclear pore membrane exceeds 30 nm, which is the detection limit obtained through experimental testing, and the flux does not exceed 5 × 10⁻⁶. 8 ions / cm 2 The optical microscope selected is an optical microscope with bright and dark field capabilities, equipped with a reflective optical path, and 10x, 50x, and 100x objectives.

[0077] The following detailed embodiments illustrate the specific applications of the rapid non-destructive testing method for nanopore nuclear membranes based on dark field scattering optics of the present invention.

[0078] Example 1: This example provides a rapid, non-destructive testing method for nanopore nuclear pore membranes based on dark-field scattering optics, used for measuring the density of nuclear pore membranes, including:

[0079] S1. Sample Preparation: A 19-micron-thick polyterephthalate (PET) sample was irradiated with a fast heavy ion (Kr) beam at a set flux of 1 × 10⁻⁶. 7ions / cm 2 Etch it in an 8 M sodium hydroxide solution at 80 degrees Celsius for 2 minutes. After etching, rinse it with water and dry the surface with a hair dryer.

[0080] S2. Sample Placement: Place the sample flat on the glass slide, put it on the microscope sample stage, and focus using the 20x, 50x, and 100x bright field modes in sequence.

[0081] S3. Dark-field imaging: With the objective lens at 100x magnification, switch the optical microscope to dark-field mode, adjust the light source brightness and fine-tune the focus to achieve the clearest outline of the light spot in the field of view, and then take a picture to acquire the image. Figure 1 As shown.

[0082] S4. Calculation of nuclear pore membrane density (irradiation flux): The number of scattered light spots is approximately 1134. The field of view size of the 100x objective image is 129.36 μm × 86.63 μm. The nuclear pore membrane density is calculated by dividing the two values, resulting in a value of 1.015 × 10⁷ ions / cm². 2 .

[0083] Example 2: This example provides a rapid non-destructive testing method for nanopore nuclear membranes based on dark-field scattering optics, used for pore size measurement, including:

[0084] S1. Standard preparation: A 15-micron polycarbonate (PC) membrane was irradiated with a fast heavy ion Xe beam at a flux of 5 × 10⁶ ions / cm. 2 Then, sensitize both sides with ultraviolet light for two hours each, followed by chemical etching in a 5M sodium hydroxide solution under ultrasonication and a 50°C water bath for about 3-5 minutes. After etching, clean with deionized water using ultrasound and dry the surface with a hairdryer. Repeat the above irradiation-sensitization-etching-cleaning-drying process twice to obtain a hierarchical porous sample.

[0085] S2. Sample Placement: The sample will be attached to the glass slide using electrostatic adsorption. The microscope will be adjusted to bright field under low magnification objective lens, and the sample will be focused at 100x magnification to locate the sampling and testing position. Figure 1 For etching apertures below 100 nm, a 100x objective lens must be used.

[0086] S3. Dark-field imaging: Adjust the microscope to dark-field mode, adjust the light source brightness and fine focus to achieve the clearest outline of the light spot in the field of view, and then take a picture to obtain the image size, such as... Figure 2 As shown in the upper right corner.

[0087] S4. Dark-field spectroscopy test: The dark-field spectrum of a single nanopore was acquired, with the grating set to 150 g / mm and the spectral center at 750 nm. The acquisition integration time and number of integrations were set according to the signal intensity. For nanopores smaller than 100 nm, the integration time was set to 30 s and the number of integrations was 1. The results are as follows. Figure 2 As shown in the lower left corner.

[0088] S5. Reference Signal Acquisition: After acquiring the dark-field scattering spectrum of the non-pore locations on the nuclear pore membrane, turn off the light source and acquire the background signal spectrum. Replace the sample with a reflector and acquire the light source spectrum in bright-field mode.

[0089] S6. Spectral Data Processing: Calculation of spectral intensity: Single-aperture dark-field scattering spectral intensity = (Dark-field scattering spectrum - Background signal spectrum) / (Light source spectrum - Background signal spectrum). Considering the possibility of random errors at a single wavelength, the calculation range is selected as 550 nm-750 nm. Calibration is performed with scanning electron microscope images. Figure 2 Top left), the mathematical relationship between the aperture size and spectral intensity of the statistical aperture ( Figure 2 (Lower middle section) is used as the basis for aperture measurement. The mathematical relationship obtained by fitting the figure is: I =5.4×10 -4 × D 2 +3.

[0090] S7. The intensity of the single-pore dark-field scattering spectrum of the nuclear pore membrane to be tested. I By substituting the given mathematical relationships, the pore size of the nuclear pore membrane under test can be obtained under these conditions.

[0091] Example 3: This example provides a rapid non-destructive testing method for nanopore nuclear membranes based on dark-field scattering optics, including:

[0092] S1. Sample Preparation: PET ion track membranes irradiated with fast heavy ions were selected, with an irradiation dose of 3 × 10⁻⁶. 7 ions / cm2. The sample was sensitized in both directions for 2 hours, and then ultrasonically etched in a 50°C sodium hydroxide solution for 40, 60, 80, 100, and 120 seconds. The sample was then electrostatically adhered to a glass slide, and the microscope was adjusted to focus at 100x bright field magnification.

[0093] S2. Dark-field imaging: Set the microscope to dark-field mode and perform dark-field imaging of each sample under a 100x objective lens to collect the dark-field scattering spectra of different samples. Set the grating to 150 g / mm and the spectral center to 750 nm. Adjust the acquisition integration time and integration count according to the signal intensity. For nanopores smaller than 100 nm, set the integration time to 30 s and the integration count to 5.

[0094] S3. Dark-field imaging and density analysis: Adjust the microscope to dark-field mode, adjust the light source brightness and fine focusing to achieve the clearest outline of the light spot in the field of view, and take a picture to acquire the image, such as... Figure 3 The second row of the image is shown.

[0095] S4. Dark-field spectroscopy test: Collect the dark-field spectrum of a single nanopore, set the grating to 150 g / mm, and the spectral center to 750 nm. Set the acquisition integration time and integration times according to the signal intensity. For nanopores below 100 nm, the integration time is set to 30 s and the integration times are 5.

[0096] S5. Reference Signal Acquisition: After acquiring the dark-field scattering spectrum of the non-pore locations on the nuclear pore membrane, turn off the light source and acquire the background signal spectrum. Replace the sample with a reflector and acquire the light source spectrum in bright-field mode.

[0097] S6. Spectral Data Processing: Calculate the spectral intensity. Single-aperture dark-field scattering spectral intensity = (dark-field scattering spectrum - background signal spectrum) / (source spectrum - background signal spectrum), such as... Figure 3 The third row of figures is shown. Considering the possibility of random errors at a single wavelength, the calculation range is selected as 550 nm-750 nm. This is then compared with scanning electron microscope images. Figure 2 (Top left) The mathematical relationship between the aperture size and spectral intensity is statistically analyzed and used as the basis for aperture measurement. The mathematical relationship obtained by fitting the data in the figure is as follows: I =4.27×10 -4 × D 2 +0.097, as Figure 4 As shown, the detection limit of this method is 27.5 nm.

[0098] S7. The intensity of the single-pore dark-field scattering spectrum of the nuclear pore membrane to be tested. I By substituting the given mathematical relationships, the pore size of the nuclear pore membrane under test can be obtained under these conditions.

[0099] The various embodiments in this specification are described in a progressive manner. Similar or identical parts between embodiments can be referred to interchangeably. Each embodiment focuses on describing the differences from other embodiments. In the description of this specification, the terms "a preferred embodiment," "furthermore," "specifically," "in this embodiment," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the embodiments in this specification. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described can be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0100] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics, characterized in that, include: The nuclear pore membrane to be tested was focused using a dark-field optical microscope. After focusing, the microscope was switched to dark-field mode to acquire the scattered light image of the nuclear pore membrane to be tested in dark-field mode. The density of the nuclear pore membrane is obtained by determining the image area and the number of scattered light spots in the image based on the scattered light image; By moving the center of the dark-field optical microscope spot to the position of a single aperture, dark-field scattering spectra are acquired to obtain the intensity of the single-aperture dark-field scattering spectrum. I ; Based on scattering spectral intensity I With aperture D relational formula I=aD 2 +b Spectral intensity through single-aperture dark field scattering I The pore size of the nuclear pore motif was calculated, where, a and b Fitting parameters.

2. The rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics according to claim 1, characterized in that, The nuclear pore membrane to be tested was focused using a dark-field optical microscope. After focusing, the microscope was switched to dark-field mode, and the scattered light image of the nuclear pore membrane to be tested was acquired in dark-field mode, including: The nuclear pore membrane under test was located by low-magnification focusing using a low-magnification objective lens of a dark-field optical microscope, and then the nuclear pore membrane under test was refocused using a high-magnification objective lens after low-magnification focusing. After high-magnification focusing, the dark-field optical microscope is switched to dark-field mode to increase the brightness of the light source. At the same time, the focus is finely adjusted to make the dark-field spot the brightest, and the scattered light image of the nuclear pore membrane is acquired in dark-field mode.

3. The rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics according to claim 2, characterized in that, Low-power objectives use 10x or 20x objectives; high-power objectives use 50x or 100x objectives.

4. The rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics according to claim 1, characterized in that, Based on the scattered light image, the image area and the number of scattered light spots in the image are determined to obtain the density of the nuclear pore membrane, including: The length and width of the scattered light image are measured using the actual scale of the dark-field optical microscope to obtain the image area. The number of scattered light spots in the scattered light image can be counted either by setting a program or manually. The density of the nuclear pore membrane is calculated by dividing the total number of scattered light spots by the image area.

5. The rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics according to claim 1, characterized in that, It also includes a step of processing the scattered light image, specifically: adjusting the contrast of the scattered light image to make the scattered light spot of the nuclear pore membrane clear.

6. The rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics according to claim 1, characterized in that, Scattering spectral intensity I With aperture D relational formula I=aD 2 +b The acquisition process includes: Preparation of nuclear pore membrane samples; The prepared nuclear pore membrane sample is flattened and attached to a glass slide; Determining the pore size of nuclear pore membrane samples using scanning electron microscopy D ; Dark-field optical microscopy was used to acquire dark-field scattering spectra, background signal spectra, and light source spectra at a single aperture location; Calculate the intensity of dark field scattering spectrum in a single aperture I = (Dark field scattering spectrum - Background signal spectrum) / (Light source spectrum - Background signal spectrum); Based on the intensity of dark field scattering spectrum of a single hole I And the pore size of the nuclear pore membrane sample obtained by scanning electron microscopy D Perform data fitting and obtain fitting parameters. a and b Thus, the intensity of the dark field scattering spectrum of a single aperture is obtained. I With aperture D relational formula I=aD 2 +b .

7. The rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics according to claim 6, characterized in that, Preparation of nuclear pore membrane samples includes: The nuclear pore membrane irradiated with fast heavy ions was expanded by chemical etching to achieve a pore size exceeding 30 nm and a flux density not exceeding 5 × 10⁻⁶. 8 ions / cm 2 After etching, the nuclear pore membrane is ultrasonically cleaned in an aqueous solution, and the remaining water droplets are evaporated to obtain the nuclear pore membrane sample.

8. The rapid non-destructive testing method for nanoporous and nuclear pore membranes based on dark-field scattering optics according to claim 6, characterized in that, Dark-field optical microscopy acquires dark-field scattering spectra, background signal spectra, and light source spectra at a single aperture location, including: Move the center of the dark-field optical microscope spot to a single aperture position to collect dark-field scattering spectra; After collecting the dark-field scattering spectrum of the non-pore locations on the nuclear pore membrane, the light source was turned off and the background signal spectrum was collected. The nuclear pore membrane sample to be tested was replaced with a reflector, and the light source spectrum was collected in bright field mode.

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