High-fidelity expansion microscopic imaging method for nuclear pore compound

Abstract

The invention relates to a high-fidelity expansion microscopic imaging method of a nuclear pore complex, which is characterized in that the signal intensity and the dyeing coverage are obviously enhanced through the immunostaining conditions of 37 DEG C temperature and prolonged incubation time, and the permeability of an antibody in iterative expansion gel is improved; by adopting a secondary anchoring cross-linking condition that 2.8% paraformaldehyde and 10% acrylamide are proportioned, under the condition that the expansion rate is not reduced, the structural heterogeneity index is further reduced, and the marking integrity is improved; through treatment of 92.5 mM SDS under the denaturation condition of 85 DEG C for 1.5 h, full denaturation can be achieved, and deformation and signal loss caused by protein breakage can be reduced. During imaging, the signal-to-noise ratio of fluorescence after single expansion is improved by using a water immersion objective lens, so that a clear annular NPC structure can be obtained by using a confocal microscope; therefore, the image fidelity and the signal-to-noise ratio are remarkably improved, and a technical basis is provided for subsequently realizing near-molecule-scale NPC imaging under a confocal microscope.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, and particularly relates to a high-fidelity expansion microscopy imaging method of nuclear pore complex. BACKGROUND

[0002] Nuclear pore complex (NPC) is a key protein complex on the nuclear membrane responsible for nucleocytoplasmic transport, and its structural heterogeneity is closely related to physiological and pathological processes such as cell stress and viral infection. Recent pioneering research on HIV transport through the NPC (Zila et al., Cell , 2021; Dickson et al., Nature , 2024; Fu et al., Nature , 2024; Kreysing et al., Cell , 2025) highlights the need to develop imaging tools that can resolve the structure of the NPC with high resolution and high fidelity.

[0003] Although electron microscopy (EM) can provide near-atomic resolution, it relies on signal averaging, limiting insights into individual NPC variability. Single-molecule localization microscopy (SMLM) can provide detailed visualization of the NPC with benchmark resolution and labeling fidelity (Thevathasan et al. Nat. Methods , 2019), but the technology is demanding and often difficult to obtain. Expansion microscopy (ExM) provides a promising alternative; ExM technology achieves super-resolution imaging by physically expanding the sample. However, existing solutions (such as iU-ExM, Louvel et al. Nat. Commun. , 2023) have at least two major limitations that need to be considered: one is structural distortion; although efforts are made to achieve isotropic expansion, all ExM variants produce NPC images with varying degrees of structural distortion. This makes data interpretation complex when studying NPC heterogeneity at the nanoscale. Distinguishing biological variations from artifacts during expansion remains a major challenge. So far, there have been some attempts to minimize distortion caused by ExM. One strategy uses a more uniform Tetra-gel network, significantly improving local isotropy, but requires custom synthesis of specialized chemicals (Gao et al. Nat. Nanotechnol. , 2021). In another strategy, researchers introduced a fluorescent grid GelMap into the pre-expansion gel to determine and correct local deformation (Damstra et al. Nat. MethodsHowever, neither of these methods has been validated to reduce nanoscale distortions of protein complexes like NPCs. Second, the efficiency of fluorescent labeling is poor, mainly affected by the following factors: loss of antigens during sample preparation, difficulty of the label to effectively penetrate the hydrogel matrix, and rapid photobleaching of commonly used fluorophores. These challenges hinder the achievement of maximum labeling density, thus limiting the reliability of nanoscale imaging. Label retention ExM (LR-ExM) innovatively employs trifunctional anchors to improve labeling coverage (Shi et al, J Cell Biol, 2021). However, these key chemical reagents are limited to use in specialized laboratories with advanced chemical capabilities.

[0004] Therefore, it is an urgent technical problem to develop a high-fidelity, high-labeling efficiency, and simple operation method for NPC imaging. SUMMARY

[0005] The present application aims to at least partially solve one of the technical problems in the related art. To this end, one object of the present application is to provide a high-fidelity expansion microscopy imaging method for nuclear pore complexes. In view of the problems of existing ExM technology in NPC imaging, such as large structural distortion, low fluorescence signal-to-noise ratio, and insufficient labeling efficiency, a high-fidelity expansion microscopy imaging method for nuclear pore complexes is provided by multi-parameter collaborative optimization.

[0006] To this end, the present application provides a high-fidelity expansion microscopy imaging method for nuclear pore complexes, comprising the following steps: (1) Build a gel chamber fixed with human osteosarcoma cells U2OS; (2) First anchoring: use paraformaldehyde and acrylamide to anchor the nuclear pore complex in human osteosarcoma cells U2OS for the first time; (3) First gelation: place the gel chamber after anchoring in step (2) in a wet box on ice, add gel monomer solution and initiator / accelerator, and after placing on ice for 15 minutes, incubate at 37°C for 45 minutes to complete gelation; (4) Denaturation treatment: add the gel obtained in step (3) to denaturation buffer and incubate in a 85°C water bath for 1.5 hours, the denaturation buffer includes 92.5 mM sodium dodecyl sulfate, 200 mM NaCl and 50 mM Tris-BASE, pH=6.8; (5) Immunostaining: place the gel in a primary antibody solution containing 2% BSA and incubate at 37°C for 24 hours; add fluorescent secondary antibody and incubate at 37°C for 12 hours; (6) First expansion: place the gel after immunostaining in step (5) in ultrapure water for expansion, ensuring sufficient expansion; (7) First re-embedding: the expanded gel is placed on ice, neutral gel solution is added, and oscillation treatment is performed 3 times, each for 10 minutes, and then placed in a humidity chamber for incubation at 37°C for 1 hour to complete gelation; (8) Second anchoring: the nuclear pore complex is subjected to second anchoring using a PBS solution containing 2.8% paraformaldehyde and 10% acrylamide; (9) Second re-embedding: the gel after the second anchoring is placed on ice, neutral gel solution is added, and oscillation treatment is performed 3 times, each for 10 minutes, and then placed in a humidity chamber for incubation at 37°C for 1 hour to complete gelation; (10) DHEBA decrosslinking: the gel after the second re-embedding is placed in NaOH, oscillated at room temperature for 1 hour, and then washed with PBS until neutral; (11) Second expansion: the gel after the decrosslinking in step (10) is placed in PBS and co-incubated with Hoechst 33342 dye for 2 hours, then washed with 0.1% Tween-20 PBS, and then expanded with ultrapure water until complete expansion; (12) Imaging: the expanded gel sample is imaged, and the image is collected using a water immersion objective to obtain an expanded microscopic image of the nuclear pore complex.

[0007] According to the high-fidelity expanded microscopic imaging method of the nuclear pore complex, the signal intensity and staining coverage are significantly enhanced by the immunostaining conditions of 37°C temperature and prolonged incubation time, and the penetration of the antibody in the iterative expanded gel is improved; the structural heterogeneity index (HI) is further reduced and the labeling integrity is improved without reducing the expansion ratio by using the second anchoring crosslinking condition of a ratio of 2.8% paraformaldehyde and 10% acrylamide; the denaturation condition of 92.5 mM SDS, 85°C, and 1.5 hours is used for treatment, which can sufficiently denature the protein and reduce the deformation and signal loss caused by protein breakage; in imaging, the use of a water immersion objective improves the fluorescence signal-to-noise ratio after single expansion, so that a clear ring-shaped NPC structure can be obtained using a confocal microscope; thereby the image fidelity and signal-to-noise ratio are significantly improved, and a technical foundation is provided for subsequent near-molecular-scale NPC imaging under a confocal microscope. Furthermore, the method exceeds the common ExM scheme in imaging quality, and realizes the visualization of NPC substructures with a resolution of less than 10 nm without the need for transfection tags.

[0008] In addition, according to the high-fidelity expanded microscopic imaging method of the nuclear pore complex according to the above embodiments of the present application, the following additional technical features can also be provided: Optionally, in step (1), the gel chamber fixed with human osteosarcoma cells U2OS includes the following steps: U2OS cells were seeded on coverslips to form cell coverslips; Two coverslips were stacked to form a spacer, and two spacers were fixed on both sides of the cell coverslips, a gel solution was added, and a coverslip was placed above the chamber to form a gel chamber; The U2OS cells in the gel chamber were prefixed with paraformaldehyde, permeabilized with Triton X-100, and then refixed, followed by a second permeabilization.

[0009] Alternatively, in step (2), the first anchoring is: placing the cells in a PBS anchoring solution containing 1.4% FA and 2% acrylamide, and incubating at 37°C for 3 hours.

[0010] Alternatively, in step (3), the gel monomer solution and initiator / accelerator include: 10% acrylamide, 19% sodium acrylate, 0.1% DHEBA, 0.25% APS / TEMED.

[0011] Alternatively, in step (4), the deformation treatment is: transferring the gel to a 12-well plate, adding denaturation buffer, and placing at room temperature for 15 minutes, then transferring to a centrifuge tube, adding fresh denaturation buffer, and incubating in a 85°C water bath for 1.5 hours.

[0012] Alternatively, in step (5), the fluorescent secondary antibody is CF583R dye.

[0013] Alternatively, in step (5), the immunostaining is: swelling the gel in ultrapure water, replacing the ultrapure water 3 times for 20 minutes each time, and then shrinking twice in PBS for 15 minutes each time; then placing the gel in a primary antibody solution containing 2% BSA, incubating at 37°C for 24 hours, and then adding a fluorescent secondary antibody and incubating at 37°C for 12 hours.

[0014] Alternatively, in step (6), the first swelling is: swelling the gel in ultrapure water, replacing the ultrapure water 3 times for 30 minutes each time, and then incubating in ultrapure water overnight to ensure full expansion.

[0015] Alternatively, in steps (7) and (9), the neutral gel solution includes 10% acrylamide, 0.05% DHEBA, and 0.1% APS / TEMED.

[0016] Alternatively, it further includes a step of analyzing the swelling micrograph of the nuclear pore complex: NPC segmentation: two rounds of DBSCAN density clustering are used to identify and refine the NPC and its internal Nup signals; Radius measurement: the distribution radius of the Nup signal centroid around the center point is estimated by a nonlinear least squares fitting method; Heterogeneity index: the coefficient of variation (CV) of the distance of each Nup centroid to the NPC center was calculated to assess the structural consistency; Coverage distribution: based on the eight-fold symmetry of the NPC, the distribution activity of the signal in each angular sector was counted to estimate the integrity of the ring structure; Other image processing: supplementary analysis was performed with ImageJ.

[0017] Additional aspects and advantages of the present application will be partially given in the following description, partially will become apparent from the following description, or will be learned by practice of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 The flow chart of the Python-based automated NPC analysis according to the embodiment 1 of the present application is shown; Figure 2 The optimization of the SDS concentration, FA / AA anchoring concentration, antibody incubation condition and lens type of the NPC-ExM according to the embodiment 2 of the present application, wherein, Figure 2 a shows the comparison chart of the effect after changing the objective lens; Figure 2 b is the comparison chart of the effect of prolonging the antibody incubation time; Figure 2 c-2f are the test result charts of the FA / AA anchoring concentration of the second anchoring and the related statistical charts; Figure 2 g-2j are the test result charts of the SDS concentration in the denaturant and the related statistical charts; Figure 3 The secondary antibody dye screening of the NPC-ExM according to the embodiment 3 of the present application, wherein Figure 3 a and 3b are the result charts and statistical results of the screening of a plurality of secondary antibody fluorescent dyes; Figure 3 c-3e is the half-height width analysis of the images using two relatively good fluorescent dyes; Figure 3 f-3g is the comparison chart of the Airyscan and SIM imaging using CF583R dye; Figure 4 The experimental flow chart of the NPC-ExM according to the embodiment 4 of the present application is shown; Figure 5 The comparison of the NPC-ExM, TREx, iU-ExM, Magnify and Pan-ExM according to the embodiment 4 of the present application, wherein, Figure 5 a and 5b are the result charts of the non-inflated cells and the nuclei of the cells inflated by a plurality of inflation microtechnologies; Figure 5 c and 5e are the result charts of the nuclear pore complex imaging of the non-inflated cells and the cells inflated by a plurality of inflation microtechnologies and the single molecule localization microtechnology and the related statistical result charts; Figure 6Schematic view of a gel chamber according to an embodiment of the present application. DETAILED DESCRIPTION

[0019] The technical solutions of the present application are described below through specific examples. It should be understood that the one or more method steps mentioned in the present application do not exclude the existence of other method steps before and after the described combination steps or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool to identify each method step, and is not intended to limit the arrangement order of each method step or to define the scope of the implementation of the present application, and the change or adjustment of the relative relationship without substantial change of the technical content is also regarded as the scope of the implementation of the present application.

[0020] In order to better understand the above technical solutions, the exemplary embodiments of the present application are described in more detail below. Although exemplary embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0021] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market; the experiments involved are all conventional experimental methods unless otherwise specified.

[0022] The cell line used in the embodiments of the present application is human osteosarcoma cell U2OS (ATCC HTB-96) for constructing the expansion microscopy imaging system. The cells are inoculated on #1.5 specification coverslips (NEST, item number 801008).

[0023] For antibodies, primary antibodies (primary) targeting different nucleoporins (Nup) were purchased from Proteintech, including anti-Nup96 (Cat# 12329-1-AP, dilution 1:150), anti-Nup62 (Cat# 13916-1-AP, dilution 1:150), and anti-Nup153 (Cat# 14189-1-AP, dilution 1:100). Fluorescently labeled secondary antibodies used include: Alexa Fluor 488 (Thermo Fisher, Cat# A32731, dilution 1:500), Alexa Fluor 568 and Alexa Fluor 647 (Thermo Fisher, Cat# A11036 or A21245, dilution 1:500), CF583R (Biotium, Cat# 96084, dilution 1:500), and ATTO647N (ROCKLAND, Cat# 611-156-122, dilution 1:200).

[0024] Hoechst 33342 (Thermo Fisher, Cat# H139, dilution 1:1000) was used for nuclear staining.

[0025] Monomers and cross-linking agents used for the preparation of swollen gels: acrylamide (Cat# A9099), sodium acrylate (Cat# 408220), N,N'-dihydroxyethyl acrylamide (DHEBA, Cat# 410987), N,N'-methylenebisacrylamide (BIS, Cat# 146072), and ammonium persulfate (APS, Cat# A3678) and tetramethyl ethylenediamine (TEMED, Cat# T7024) as initiator and catalyst for the polymerization reaction. Formaldehyde (Cat# F8775) was used for cell fixation and anchoring.

[0026] The application will now be described with reference to the following specific examples, which are intended to be purely exemplary and not limiting in any way.

[0027] Example 1. Construction of Python-based automated NPC analysis Python (https: / / www.python.org / ) and related image processing libraries [NumPy (https: / / numpy.org / ), scikit-learn (https: / / scikit-learn.org / stable / index.html), SciPy (https: / / scipy.org / )] were used to construct an automated analysis pipeline for the quantitative analysis of NPC structural features.

[0028] like Figure 1 As shown, the analysis process includes the following steps: (1) NPC segmentation: The original image (a) is used to identify and refine the NPC (b) and its internal Nup signal (c) by two rounds of DBSCAN density clustering, and the filtered image (e) is obtained and converted into a point cloud (f). NUP is identified (g), the centroid of NUP is found and a circle is drawn (h1), and the distance between the centroids is measured (h2); (2) Radius measurement: The distribution radius of the Nup signal centroid around the center point is estimated by nonlinear least squares fitting method (i); (3) Heterogeneity index: The coefficient of variation (CV) of the distance from each Nup centroid to the NPC center is calculated to evaluate the structural consistency; (4) Coverage distribution: Based on the octave symmetry of the NPC (j), the distribution activity of the signal in each angular sector is statistically analyzed (k) to estimate the integrity of the ring structure; (5) Other image processing: ImageJ is used to perform supplementary analysis of indicators such as half-width and full height.

[0029] This process systematically quantifies the morphological fidelity, symmetry integrity, and signal coverage of NPCs, supporting the evaluation of the advantages of the NPC-ExM method in nanoscale structural analysis.

[0030] Example 2: Optimization of SDS concentration, FA / AA anchoring concentration, antibody incubation conditions, and lens type for NPC-ExM The experimental flow of NPC-ExM includes the following steps: (1) Cell plating U2SO cells at a density of approximately 40% were seeded onto #1.5 coverslips to form cell coverslips. To ensure good cell adhesion, the cells were incubated in McCoy's 5A medium (Basal Media, L630KJ) for at least 18 hours before fixation.

[0031] (2) Gel cell construction Two coverslips, each measuring 22 × 22 mm (#1.5 size), were stacked together and secured to either side of the cell coverslip using strong adhesive. After adding the gel solution, another coverslip (#1.5 size, 22 × 22 mm) was placed above the chamber to form a gel chamber. Figure 6 As shown in the figure. The device forms an initial gel approximately 170 μm thick.

[0032] (3) Cell fixation Cells were pre-fixed in freshly prepared 2.4% paraformaldehyde (FA) PBS at room temperature for 30 seconds, followed by infiltration with 0.4% Triton X-100 PBS for 3 minutes. Cells were then washed twice with PBS for 5 minutes each time. Cells were then fixed again with freshly prepared 2.4% FA PBS for 20 minutes. Cells were then washed twice with PBS for 5 minutes each time. Cells were then infiltrated with 0.2% Triton X-100 PBS for 10 minutes, and washed twice more with PBS for 5 minutes each time.

[0033] (4) First anchoring The fixed cells were placed in a PBS anchoring solution containing 1.4% FA and 2% acrylamide (AA) and incubated at 37°C for 3 hours.

[0034] (5) First gelation After the initial anchoring, the cells were washed twice with PBS for 5 minutes each time. The gel chambers were placed in a humidified chamber on ice. Then, monomeric solutions (MS) were added: 10% AA, 19% sodium acrylate (SA), 0.1% DHEBA, and 0.25% APS / TEMED (1×PBS solution). The chambers were incubated on ice for 15 minutes, and then at 37°C for 45 minutes to complete gelation.

[0035] (6) Degradation treatment Carefully transfer the gel to a 12-well plate, add denaturing buffer (37 mM, 92.5 mM or 200 mM sodium dodecyl sulfate, 200 mM NaCl, 50 mM Tris-BASE, pH=6.8), and incubate at room temperature for 15 minutes. Then transfer to a 1.5 mL centrifuge tube, add fresh denaturing buffer (37 mM, 92.5 mM or 200 mM sodium dodecyl sulfate, 200 mM NaCl, 50 mM Tris-BASE, pH=6.8), and incubate in an 85°C water bath for 1.5 hours.

[0036] (7) Immunostaining After denaturation, the gel was swelled in ultrapure water (replaced 3 times, 20 minutes each time, at room temperature), and then shrank twice with PBS (15 minutes each time, at room temperature). The gel was then placed in a primary antibody solution containing 2% BSA and incubated overnight at 4°C or for 24 hours at 37°C. Then, fluorescent secondary antibody was added, and the gel was incubated at 37°C for 24 hours or for 12 hours.

[0037] After each antibody staining, wash three times with 0.1% Tween-20 PBS for 15 minutes each time.

[0038] (8) First expansion After staining, place the gel in ultrapure water to expand (replace 3 times, 30 minutes each time, at room temperature), and then incubate overnight in ultrapure water to ensure full expansion.

[0039] (9) Neutral gel re-embedding The expanded gel was placed on ice, and fresh neutral gelation solution (10% AA, 0.05% DHEBA, 0.1% APS / TEMED, ddH2O) was added and shaken three times for 10 minutes each time. Then, a coverslip was placed and the gel was incubated in a humidity chamber at 37°C for 1 hour to complete gelation.

[0040] (10) Second anchoring reaction The gel was placed in PBS solutions containing 1.4% FA and 2% AA, 2.8% FA and 4% AA, 5.6% FA and 8% AA, 2.8% FA and 10% AA, or 2.8% FA and 20% AA, and incubated with shaking at 37°C for 5 hours, followed by washing with PBS for 30 minutes at room temperature.

[0041] (11) Second gel re-embedding The neutral gel was placed on ice, and fresh second gelation solution (10% AA, 19% SA, 0.1% BIS, 0.1% APS / TEMED in PBS) was added and shaken three times for 10 minutes each time. Then, a coverslip was placed and the mixture was incubated in a humidity chamber at 37°C for 1 hour to complete gelation.

[0042] (12) DHEBA decrosslinking treatment Place the gel in 200 mM NaOH and shake at room temperature for 1 hour. Then wash with PBS 3 times for 30 minutes each time until neutral.

[0043] (13) Second expansion The gel was incubated with Hoechst 33342 dye in PBS for 2 hours, then washed three times with 0.1% Tween-20 PBS (15 minutes each time). Finally, it was swelled with ultrapure water (replaced three times, 30 minutes each time) until complete swelling.

[0044] (14) Imaging Confocal microscopy imaging was performed using a 40× water objective and a 63× oil objective, respectively. The images obtained after imaging were analyzed using the NPC analysis method described in Example 1.

[0045] The results are as follows Figure 2 As shown, using a water immersion objective instead of a traditional oil immersion objective significantly improves the fluorescence signal-to-noise ratio after a single expansion, enabling the acquisition of clear annular NPC structures using confocal microscopy. Figure 2 a andFigure 2 b). To address the issue of poor antibody permeability in iterative swelling gels, the applicant set the staining temperature to 37°C and extended the antibody incubation time to 12-24 hours, significantly enhancing signal intensity and staining coverage. Figure 2 b). By testing different ratios of formaldehyde (FA) and acrylamide (AA) concentrations, 2.8% FA and 10% AA were determined to be the optimal ratio. This ratio further reduced the structural heterogeneity index (HI) and improved label integrity without decreasing the expansion ratio. Figure 2 c. Figure 2 d、 Figure 2 e Figure 2 f). Furthermore, by lowering the commonly used SDS concentration from 200 mM to 92.5 mM and operating at 85°C for 1.5 hours, it was determined that this concentration effectively denatured the protein while minimizing deformation and signal loss due to protein breakage, resulting in significantly better imaging performance than the high SDS concentration condition. Figure 2 g、 Figure 2 h、 Figure 2 i、 Figure 2 j).

[0046] Example 3: Screening of secondary antibody dyes for NPC-ExM The experimental flow of NPC-ExM includes the following steps: (1) Cell plating U2SO cells at a density of approximately 40% were seeded onto #1.5 coverslips to form cell coverslips. To ensure good cell adhesion, the cells were incubated in McCoy's 5A medium (Basal Media, L630KJ) for at least 18 hours before fixation.

[0047] (2) Gel cell construction Two coverslips, each a size 1.5 (22 × 22 mm), were stacked and secured to either side of the cell coverslip using strong adhesive. After adding the gel solution, another coverslip (also a size 1.5, 22 × 22 mm) was placed above the chamber to form a gel chamber. This apparatus produced an initial gel approximately 170 μm thick.

[0048] (3) Cell fixation Cells were pre-fixed in freshly prepared 2.4% paraformaldehyde (FA) PBS at room temperature for 30 seconds, followed by infiltration with 0.4% Triton X-100 PBS for 3 minutes. Cells were then washed twice with PBS for 5 minutes each time. Cells were then fixed again with freshly prepared 2.4% FA PBS for 20 minutes. Cells were then washed twice with PBS for 5 minutes each time. Cells were then infiltrated with 0.2% Triton X-100 PBS for 10 minutes, and washed twice more with PBS for 5 minutes each time.

[0049] (4) First anchoring The fixed cells were placed in a PBS anchoring solution containing 1.4% FA and 2% acrylamide (AA) and incubated at 37°C for 3 hours.

[0050] (5) First gelation After the initial anchoring, the cells were washed twice with PBS for 5 minutes each time. The gel chambers were then placed in a humidified chamber on ice. Monomer solution (MS) was then added: 10% AA, 19% sodium acrylate (SA), 0.1% DHEBA, and 0.25% APS / TEMED (1xPBS solution). The chambers were incubated on ice for 15 minutes, then at 37°C for 45 minutes to complete gelation.

[0051] (6) Degradation treatment Carefully transfer the gel to a 12-well plate, add denaturing buffer (92.5 mM sodium dodecyl sulfate, 200 mM NaCl, 50 mM Tris-BASE, pH=6.8), and incubate at room temperature for 15 minutes. Then transfer to a 1.5 mL centrifuge tube, add fresh denaturing buffer (92.5 mM sodium dodecyl sulfate, 200 mM NaCl, 50 mM Tris-BASE, pH=6.8), and incubate in an 85°C water bath for 1.5 hours.

[0052] (7) Immunostaining After denaturation, the gel was swelled in ultrapure water (replaced 3 times, 20 minutes each time, at room temperature), and then shrank twice with PBS (15 minutes each time, at room temperature). The gel was then placed in a primary antibody solution containing 2% BSA and incubated at 37°C for 24 hours. Then add the fluorescent secondary antibody [the fluorescent secondary antibodies are PlussAlexaFluor488 (Thermo Fisher, catalog number A32731, dilution 1:500), ATTO488 (Sigma, catalog number 36098-1ML-F, dilution 1:500), AlexaFluor568 (Thermo Fisher, catalog number A11036, dilution 1:500), CF583R (Biotium, catalog number #96084, dilution 1:500), AlexaFluor647 (Thermo Fisher, catalog number A21245, dilution 1:500) or ATTO647N (ROCKLAND, catalog number 611-156-122, dilution 1:200)] and incubate at 37°C for 12 hours.

[0053] After each antibody staining, wash three times with 0.1% Tween-20 PBS for 15 minutes each time.

[0054] (8) First expansion After staining, place the gel in ultrapure water to expand (replace 3 times, 30 minutes each time, at room temperature), and then incubate overnight in ultrapure water to ensure full expansion.

[0055] (9) Neutral gel re-embedding The expanded gel was placed on ice, and fresh neutral gelation solution (10% AA, 0.05% DHEBA, 0.1% APS / TEMED, ddH2O) was added and shaken three times for 10 minutes each time. Then, a coverslip was placed and the gel was incubated in a humidity chamber at 37°C for 1 hour to complete gelation.

[0056] (10) Second anchoring reaction The gel was placed in a PBS solution containing 2.8% FA and 10% AA and incubated with shaking at 37°C for 5 hours, and then washed with PBS for 30 minutes at room temperature.

[0057] (11) Second gel re-embedding The neutral gel was placed on ice, and fresh second gelation solution (10% AA, 19% SA, 0.1% BIS, 0.1% APS / TEMED in PBS) was added and shaken three times for 10 minutes each time. Then, a coverslip was placed and the mixture was incubated in a humidity chamber at 37°C for 1 hour to complete gelation.

[0058] (12) DHEBA decrosslinking treatment Place the gel in 200 mM NaOH and shake at room temperature for 1 hour. Then wash with PBS 3 times for 30 minutes each time until neutral.

[0059] (13) Second expansion The gel was incubated with Hoechst 33342 dye in PBS for 2 hours, then washed three times with 0.1% Tween-20 PBS (15 minutes each time). Finally, it was swelled with ultrapure water (replaced three times, 30 minutes each time) until complete swelling.

[0060] (14) Imaging Super-resolution microscopy was performed using structured light illumination microscopy (SIM), and the images obtained after imaging were simultaneously analyzed using the NPC analysis method described in Example 1.

[0061] The results are as follows Figure 3 As shown, the fluorescence retention of several commercial dyes before and after neutral gelation was compared. AF647 showed an intensity decrease of over 80%. In contrast, CF583R and Atto647N were the most bleach-resistant dyes tested. Inspired by this result, the performance of CF583R and AF568 in NPC-ExM was compared using Ariyscan microscopy. The enhanced fluorescence signal-to-noise ratio of CF583R indeed resulted in a significant improvement in image quality, with clearer outlines and smaller average half-width at half-maximum (FWHM) of Nup96 aggregates compared to AF568. More interestingly, when used in conjunction with SIM, CF583R enabled NPC-ExM to resolve features of subnuclear pore complexes, and occasionally even distinguish individual Nup96 molecules in the corners of single nucleopore complexes. In conclusion, the choice of dye CF583R, which enhances the fluorescence signal-to-noise ratio, combined with structured illumination microscopy (SIM), enables sub-10 nm resolution imaging without the need for transfection with labeled proteins, allowing observation of fine features of NPC substructures.

[0062] Example 5: Comparison of NPC-ExM, TREx, iU-ExM, Magnify, and Pan-ExM The experimental procedure for NPC-ExM is as follows: Figure 4 As shown, the specific steps include: (1) Cell plating U2SO cells at a density of approximately 40% were seeded onto #1.5 coverslips to form cell coverslips. To ensure good cell adhesion, the cells were incubated in McCoy's 5A medium (Basal Media, L630KJ) for at least 18 hours before fixation.

[0063] (2) Gel cell construction Two coverslips, each measuring 22 × 22 mm (#1.5 size), were stacked together and secured to either side of the cell coverslip using strong adhesive. After adding the gel solution, another coverslip (#1.5 size, 22 × 22 mm) was placed above the chamber to form a gel chamber. Figure 6 As shown in the figure. The device forms an initial gel approximately 170 μm thick.

[0064] (3) Cell fixation Cells were pre-fixed in freshly prepared 2.4% paraformaldehyde (FA) PBS at room temperature for 30 seconds, followed by infiltration with 0.4% Triton X-100 PBS for 3 minutes. Cells were then washed twice with PBS for 5 minutes each time. Cells were then fixed again with freshly prepared 2.4% FA PBS for 20 minutes. Cells were then washed twice with PBS for 5 minutes each time. Cells were then infiltrated with 0.2% Triton X-100 PBS for 10 minutes, and washed twice more with PBS for 5 minutes each time.

[0065] (4) First anchoring The fixed cells were placed in a PBS anchoring solution containing 1.4% FA and 2% acrylamide (AA) and incubated at 37°C for 3 hours.

[0066] (5) First gelation After the initial anchoring, the cells were washed twice with PBS for 5 minutes each time. The gel chambers were then placed in a humidified chamber on ice. Monomer solution (MS) was then added: 10% AA, 19% sodium acrylate (SA), 0.1% DHEBA, and 0.25% APS / TEMED (1xPBS solution). The chambers were incubated on ice for 15 minutes, then at 37°C for 45 minutes to complete gelation.

[0067] (6) Degradation treatment Carefully transfer the gel to a 12-well plate, add denaturing buffer (92.5 mM sodium dodecyl sulfate, 200 mM NaCl, 50 mM Tris-BASE, pH=6.8), and incubate at room temperature for 15 minutes. Then transfer to a 1.5 mL centrifuge tube, add fresh denaturing buffer (2.5% sodium dodecyl sulfate, 200 mM NaCl, 50 mM Tris-BASE, pH=6.8), and incubate in an 85°C water bath for 1.5 hours.

[0068] (7) Immunostaining After denaturation, the gel was swelled in ultrapure water (replaced 3 times, 20 minutes each time, at room temperature), and then shrank twice with PBS (15 minutes each time, at room temperature). The gel was then placed in a primary antibody solution containing 2% BSA and incubated at 37°C for 24 hours. Then, fluorescent secondary antibody (CF583R dye) was added, and the gel was incubated at 37°C for 12 hours.

[0069] After each antibody staining, wash three times with 0.1% Tween-20 PBS for 15 minutes each time.

[0070] (8) First expansion After staining, place the gel in ultrapure water to expand (replace 3 times, 30 minutes each time, at room temperature), and then incubate overnight in ultrapure water to ensure full expansion.

[0071] (9) Neutral gel re-embedding The expanded gel was placed on ice, and fresh neutral gelation solution (10% AA, 0.05% DHEBA, 0.1% APS / TEMED, ddH2O) was added and shaken three times for 10 minutes each time. Then, a coverslip was placed and the gel was incubated in a humidity chamber at 37°C for 1 hour to complete gelation.

[0072] (10) Second anchoring reaction The gel was placed in a PBS solution of 2.8% FA and 10% AA and incubated with shaking at 37°C for 5 hours, and then washed with PBS for 30 minutes at room temperature.

[0073] (11) Second gel re-embedding The neutral gel was placed on ice, and fresh second gelation solution (10% AA, 19% SA, 0.1% BIS, 0.1% APS / TEMED in PBS) was added and shaken three times for 10 minutes each time. Then, a coverslip was placed and the mixture was incubated in a humidity chamber at 37°C for 1 hour to complete gelation.

[0074] (12) DHEBA decrosslinking treatment Place the gel in 200 mM NaOH and shake at room temperature for 1 hour. Then wash with PBS 3 times for 30 minutes each time until neutral.

[0075] (13) Second expansion The gel was incubated with Hoechst 33342 dye in PBS for 2 hours, then washed three times with 0.1% Tween-20 PBS (15 minutes each time). Finally, it was swelled with ultrapure water (replaced three times, 30 minutes each time) until complete swelling.

[0076] (14) Imaging Confocal microscopy imaging was performed, and the images obtained after imaging were simultaneously analyzed using the NPC analysis method described in Example 1.

[0077] Single-molecule localization microscopy (SMLM), one of the best imaging techniques for NPC imaging, was used as the ground truth. Several common ExM schemes with suitable theoretical expansion coefficients were selected and compared with the NPC-ExM scheme proposed in this application.

[0078] The results are as follows Figure 5 As shown, a cross-sectional comparison was conducted on four mature ExM methods—TREx, iU-ExM, Magnify, and Pan-ExM. Only TREx and iU-ExM achieved satisfactory NPC imaging results, but they still suffer from significant structural distortion and incomplete labeling issues. Figure 5 c). Among them, iU-ExM performed best, with higher amplification rate and better structural fidelity. Therefore, the applicant focused on comparing the SMLM, iU-ExM, and NPC-ExM schemes. The analysis results show that, through approximately 15-fold isotropic expansion, NPC-ExM achieved an imaging resolution of approximately 20 nanometers under confocal microscopy, reducing the structural deviation from single-molecule localization microscopy (SMLM) to 13.6%, significantly better than iU-ExM's 50%. Simultaneously, NPC-ExM significantly improved the proportion of high-quality NPC images (Coverage distribution ≥ 6), with a difference of only approximately 17.6% compared to SMLM, while the difference for iU-ExM was approximately 49.2%. Figure 5 a, Figure 5 b、 Figure 5 d、 Figure 5 e).

[0079] In summary, the NPC-ExM method according to embodiments of the present invention significantly enhances signal intensity and staining coverage through immunostaining conditions of 37°C and extended incubation time, improving antibody permeability in iteratively expanded gels. The secondary anchoring and crosslinking conditions using a ratio of 2.8% paraformaldehyde and 10% acrylamide further reduce the structural heterogeneity index (HI) and improve label integrity without reducing the expansion magnification. Denaturation treatment with 92.5 mM SDS at 85°C for 1.5 hours ensures sufficient denaturation while minimizing deformation and signal loss due to protein breakage. The use of a water immersion objective in imaging improves the fluorescence signal-to-noise ratio after a single expansion, enabling clear visualization of the ring-shaped NPC structure using confocal microscopy. This significantly improves image fidelity and signal-to-noise ratio, providing a technical foundation for achieving near-molecular-scale NPC imaging under confocal microscopy. Furthermore, this method surpasses current common ExM schemes in imaging quality and achieves sub-10 nm resolution visualization of NPC substructures without the need for transfection tags.

[0080] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. The illustrative expressions of the above terms in this specification should not be construed as necessarily referring to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate the different embodiments or examples described in this specification.

[0081] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A high-fidelity dilatational microscopy imaging method for nuclear pore complexes, characterized in that, Includes the following steps: (1) Constructing gel chambers immobilized with human osteosarcoma cells U2OS; (2) First anchoring: The nuclear pore complex in human osteosarcoma cells U2OS was first anchored using paraformaldehyde and acrylamide; (3) First gelation: Place the anchored gel chamber in step (2) in a humid box on ice, add gel monomer solution and initiator / accelerator, place on ice for 15 minutes, and incubate at 37°C for 45 minutes to complete gelation; (4) Denaturation treatment: Add the gel obtained in step (3) to denaturation buffer and incubate in an 85°C water bath for 1.5 hours. The denaturation buffer includes 92.5 mM sodium dodecyl sulfate, 200 mM NaCl and 50 mM Tris-BASE, pH=6.

8. (5) Immunostaining: Place the gel in a primary antibody solution containing 2% BSA and incubate at 37°C for 24 hours; add fluorescent secondary antibody and incubate at 37°C for 12 hours; (6) First expansion: Place the gel after immunostaining in step (5) in ultrapure water to expand, ensuring full expansion; (7) First re-embedding: Place the expanded gel on ice, add neutral gel solution, shake three times for 10 minutes each time, then place it in a humidity chamber and incubate at 37°C for 1 hour to complete gelation; (8) Second anchoring: The nuclear pore complex was second anchored using a PBS solution containing 2.8% paraformaldehyde and 10% acrylamide; (9) Second embedding: Place the gel after the second anchoring on ice, add a neutral gel solution, shake three times for 10 minutes each time, and then place it in a humidity chamber and incubate at 37°C for 1 hour to complete the gelation. (10) DHEBA decrosslinking: The gel after the second re-embedding was placed in NaOH and shaken at room temperature for 1 hour, and then washed with PBS until neutral. (11) Second expansion: Place the decrosslinked gel from step (10) in PBS and incubate with Hoechst 33342 dye for 2 hours, then wash with 0.1% Tween-20 PBS, and then expand with ultrapure water until the expansion is complete; (12) Imaging: The expanded gel sample was imaged using a water immersion objective to obtain an expanded microscopic image of the nuclear pore complex.

2. The high-fidelity dilatational microscopy imaging method for nuclear pore complexes as described in claim 1, characterized in that, In step (1), constructing gel chambers immobilized with human osteosarcoma cells U2OS includes the following steps: U2OS cells were seeded onto coverslips to form cell coverslips; Two coverslips are stacked to form a spacer. The two spacers are fixed on both sides of the cell coverslip. A gel solution is added, and a coverslip is placed above the chamber to form a gel chamber. U2OS cells in the gel chambers were pre-fixed with paraformaldehyde, permeated with Triton X-100, and after repeated fixation, a second permeation was performed.

3. The high-fidelity dilatational microscopy imaging method for nuclear pore complexes as described in claim 1, characterized in that, In step (2), the first anchoring is performed by placing the cells in a PBS anchoring solution containing 1.4% FA and 2% acrylamide and incubating at 37°C for 3 hours.

4. The high-fidelity dilatational microscopy imaging method for nuclear pore complexes as described in claim 1, characterized in that, In step (3), the gel monomer solution and initiator / accelerator include: 10% acrylamide, 19% sodium acrylate, 0.1% DHEBA, and 0.25% APS / TEMED.

5. The high-fidelity dilatational microscopy imaging method for nuclear pore complexes as described in claim 1, characterized in that, In step (4), the denaturation process is as follows: transfer the gel to a 12-well plate, add denaturation buffer, place at room temperature for 15 minutes, then transfer to a centrifuge tube, add fresh denaturation buffer, and incubate in an 85°C water bath for 1.5 hours.

6. The high-fidelity dilatational microscopy imaging method for nuclear pore complexes as described in claim 1, characterized in that, In step (5), the fluorescent secondary antibody is CF583R dye.

7. The high-fidelity dilatational microscopy imaging method for nuclear pore complexes as described in claim 1, characterized in that, In step (5), the immunostaining is performed as follows: the gel is swelled in ultrapure water, the ultrapure water is changed 3 times, each time for 20 minutes, and then shrunken twice with PBS for 15 minutes each time; then the gel is placed in a primary antibody solution containing 2% BSA and incubated at 37°C for 24 hours, and then fluorescent secondary antibody is added and incubated at 37°C for 12 hours.

8. The high-fidelity dilatational microscopy imaging method for nuclear pore complexes as described in claim 1, characterized in that, In step (6), the first expansion is as follows: the gel is placed in ultrapure water to expand, the ultrapure water is changed 3 times, each time for 30 minutes, and then incubated in ultrapure water overnight to ensure sufficient expansion.

9. The high-fidelity dilatational microscopy imaging method for nuclear pore complexes as described in claim 1, characterized in that, In steps (7) and (9), the neutral gel solution comprises 10% acrylamide, 0.05% DHEBA, and 0.1% APS / TEMED.

10. The high-fidelity dilatational microscopy imaging method for nuclear pore complexes as described in claim 1, characterized in that, The method also includes the step of analyzing the expansion microscopic images of the nuclear pore complex: NPC segmentation: Two rounds of DBSCAN density clustering are used to identify and refine NPCs and their internal Nup signals; Radius measurement: The distribution radius of the Nup signal centroid around the center point is estimated by nonlinear least squares fitting method; Heterogeneity index: Calculate the coefficient of variation (CV) of the distance from each Nup centroid to the NPC center to assess structural consistency; Coverage distribution: Based on the octave symmetry of NPC, the distribution activity of statistical signals in sectors at various angles is used to estimate the integrity of the ring structure; Other image processing: supplementary analysis using ImageJ.

Images