3D Cell Culture Method for Ultra-High Resolution Sub-Organelle Imaging Research and Its Application

Abstract

The present invention discloses a 3D cell culture method for ultra-high resolution sub-organelle imaging research and its application, which comprises the following steps: Step (1): Drop the collagen I dilution solution onto the surface of the glass substrate for pre-incubation to form a pre-incubation layer; Step (2): Lay the collagen I and culture medium mixture on the surface of the pre-incubation layer for incubation to enable the collagen I to gel and form a network layer as the sedimentation layer; Step (3): Add the collagen I and cell mixture to the surface of the sedimentation layer for incubation to enable the collagen I to gel; Add cell culture medium and place it in an incubator for 5-7 days to allow the cells to grow and spread, complete the 3D cell culture, and form a 3D cell network layer. Through ultra-high resolution sub-organelle imaging, the sub-organelle structure can be presented, and the intracellular endocytosis kinetics of nanoparticles can be characterized and analyzed in real time for a long time.

Description

Technical Field

[0001] The present invention relates to the technical field of three-dimensional cell culture and microscopic imaging, and particularly relates to a 3D cell culture method for performing super-resolution subcellular organelle imaging research and its application. Background Art

[0002] Super-resolution subcellular organelle imaging technology can resolve the spatial distribution, dynamic behavior, and molecular interaction network of organelles at the nanoscale resolution, providing an important means for revealing key scientific issues such as intracellular protein colocalization, signal transduction mechanisms, and drug targeted delivery efficiency. Currently, in vitro models based on two-dimensional (2D) cell culture are still the mainstream experimental systems in the fields of cell biology, oncology, and nanomedicine research and development. However, there are significant differences between 2D cultured cells and in vivo cells in terms of morphology, function, and microenvironment responsiveness: In the 2D model, cells only contact the rigid substrate unilaterally, lacking the mechanical support and biochemical gradient of the three-dimensional (3D) extracellular matrix (ECM), and unable to simulate the dynamic cell-cell interaction and exocrine signal network in vivo. For example, characteristics such as hypoxia, acidic pH, and mechanical stress in the solid tumor microenvironment are difficult to reproduce in the 2D model, resulting in deviations between research conclusions on drug permeability, cell migration, etc. and the actual in vivo response.

[0003] To make up for this deviation, 3D cell culture technologies (such as scaffold models, cell spheroid models, and hydrogel models) have been widely introduced into in vitro research. Among them, the scaffold model constructs a biomimetic 3D network through natural ECM components such as collagen (Collagen I) and basement membrane extract (Matrigel), which can provide cell adhesion sites and simulate tissue mechanical properties; the cell spheroid model forms multicellular aggregates through suspension culture, reproducing the heterogeneity and metabolic gradient in the tumor microenvironment; the hydrogel model encapsulates cells using synthetic or natural polymer materials to achieve precise regulation of mechanical properties and degradation rates.

[0004] However, existing 3D culture systems face severe challenges when adapting to super-resolution imaging. Super-resolution imaging techniques all rely on high numerical aperture (NA) objective lenses. The higher the NA value, the higher the resolution. 60× / 1.4 NA oil immersion objective lenses and 100× / 1.45 NA oil immersion objective lenses are commonly used objective lenses. The working distance of these objective lenses (i.e., the distance between the objective lens and the sample) is usually only 0.16 - 0.25 mm. After deducting the thickness of the glass substrate of the culture vessel, which is 0.15 - 0.17 mm, the effective imaging area is limited to a narrow range of about 10 - 80 μm from the glass surface (subcellular scale). It should be noted that in studies based on 3D culture, the thicknesses of samples such as cells, cell spheres, and organoids are all greater than several hundred micrometers, far exceeding the imaging working distance of high NA value objective lenses. Therefore, super-resolution microscopes cannot be used to perform subcellular organelle imaging on large-volume 3D samples. Chinese Patent Publication No. CN112921000A discloses a 3D in vitro culture method for glioma based on a hanging drop culture model and studies the cell migration process based on a high-content imaging system. In this system, cells are the smallest research unit, and a low-resolution objective lens with a large depth of field and working distance is used, and subcellular organelle level research cannot be carried out.

[0005] Theoretically, the 3D cell culture technology based on the scaffold model can perform super-resolution by thinning the 3D culture system. However, mechanical compression after culture will destroy the integrity of the 3D structure. At the same time, the thin system will float in the solution away from the glass substrate of the culture dish due to the shaking of the culture medium liquid, thus exceeding the imaging working distance of the high NA value objective lens. In addition, during the 3D cell culture process of the thin system, cells are extremely likely to settle to the bottom of the dish and adhere to grow in 2D due to gravity.

[0006] In summary, developing an in vitro culture method that can both retain the authenticity of the 3D cell microenvironment and meet the requirements of super-resolution imaging technology has become an urgent need for interdisciplinary research. Summary of the Invention

[0007] The present invention provides a 3D cell culture method for super-resolution subcellular organelle imaging research. Through super-resolution subcellular organelle imaging, the subcellular organelle structure can be presented, and the intracellular endocytosis kinetics of nanoparticles can be characterized and analyzed in real time for a long time.

[0008] The technical solution of the present invention is as follows:

[0009] A 3D cell culture method for super-resolution subcellular organelle imaging research, comprising the following steps:

[0010] Step (1): Drop the collagen I (type I collagen) dilution solution onto the surface of the glass substrate for pre-incubation to form a pre-incubation layer;

[0011] Step (2): Spread the collagen I and culture medium mixture onto the surface of the pre-incubation layer and incubate it to form a gel of type I collagen to form a network layer, which is the sedimentation layer;

[0012] Step (3): Add the collagen I and cell mixture to the surface of the sedimentation layer and incubate it to form a gel of collagen I; add cell culture medium and place it in an incubator for 5 - 7 days to allow the cells to grow and spread, completing 3D cell culture and forming a 3D cell network layer.

[0013] The purpose of the pre-incubation step in Step (1) is to prevent the 3D cell network layer from detaching from the glass substrate, which is achieved by pre-bonding an extremely thin layer of collagen I gel fluff on the glass substrate.

[0014] Preferably, the collagen I diluent is collagen I diluted with an acetic acid solution; in the collagen I diluent, the concentration of collagen I is 10 - 100 μg / mL.

[0015] Preferably, the acetic acid solution is a 6 mM sterile acetic acid solution.

[0016] Preferably, in Step (1), the pre-incubation time is 2 - 10 h.

[0017] The concentration of collagen I in the pre-incubation layer and the incubation time need to be properly controlled. By adjusting the concentration of collagen I in the collagen I diluent and the pre-incubation time, the bonding density between the pre-incubation layer and the glass substrate can be adjusted. If the concentration is too low or the incubation time is too short, the gel network cannot form a stable bond with the glass substrate, and the 3D culture system will still be easily detached due to the shaking of the culture medium; if the concentration is too high or the incubation time is too long, the substrate bonding is thick, which will occupy the imaging working distance of the objective lens.

[0018] Preferably, the coating concentration of the pre-incubation layer on the surface of the glass substrate is 1 - 10 μg / cm 2 .

[0019] To obtain better imaging results using a super-resolution microscope (such as STORM), the thickness of the glass substrate is 0.15 - 0.17 mm; most preferably 0.17 mm.

[0020] The glass substrate is a confocal dish (matched with an inverted microscopic imaging system) or a cover slip (matched with an upright microscopic imaging system).

[0021] The purpose of the sedimentation layer is to prevent cells from settling to the bottom during the gelation process of the upper-layer collagen I and cell mixture, and to ensure that the 3D cultured cells for target imaging settle within the effective imaging range of the high-NA objective lens. Among them, the thickness and network density of the sedimentation layer are crucial. Note: The cells in the collagen I and cell mixture are cells digested by trypsin after traditional adherent culture. After digestion, the cells become round and smaller (10-20 μm), and due to gravity, they will settle in the network gaps of the sedimentation layer.

[0022] The collagen I and culture medium mixture is prepared from MEM (10×), NaHCO3, collagen I stock solution, and cell culture medium.

[0023] Preferably, in the collagen I and culture medium mixture, the concentration of type I collagen is 2-3 mg / mL, and the pH is 7.0. The network pores of the collagen I after gelation are relatively small at this concentration, which can not only effectively reduce the cell sedimentation rate but also ensure that the cells can spread after 5-7 days of cell culture.

[0024] Preferably, the incubation time of the sedimentation layer is 30-40 min. If the incubation time is too short, it is difficult for collagen I to form a gel and a network structure. If the incubation time is too long, a small amount of liquid in the thin system after gelation will be dried by the circulating air flow in the incubator, resulting in the collapse of the gel network structure.

[0025] Preferably, the thickness of the sedimentation layer is 50-70 μm.

[0026] The thickness of the sedimentation layer cannot be too thin, otherwise the digested round and smaller cells will settle to the bottom. The thickness of the sedimentation layer cannot be too thick (not exceeding 50-70 μm) to ensure that the settled cells can spread and grow within the imaging range of the objective lens. The thickness of the sedimentation layer for cell growth is < the working distance of the high numerical aperture objective lens - the thickness of the glass substrate.

[0027] Preferably, if the glass substrate is a confocal dish, the thickness of the sedimentation layer is controlled according to the inner diameter of the confocal dish and the volume of the collagen I and culture medium mixture added;

[0028] If the glass substrate is a cover slip, a double-sided adhesive ring with a specific thickness is pasted on the cover slip to control the thickness of the sedimentation layer.

[0029] The collagen I and cell mixture is prepared from MEM (10×), NaHCO3, type I collagen stock solution, and cell solution.

[0030] In step (3), in the collagen I and cell mixture, the concentration of collagen I is 2 - 3 mg / mL and the pH is 7.0.

[0031] If the concentration of collagen I is too low, it is likely to cause: after the cells are mixed with the gel-forming solution (neutralized collagen I solution), during the gel-forming process where collagen I transforms from a liquid state to a network structure in about 40 minutes, the cells may settle to the bottom of the glass slide and adhere to grow. Such cells will seriously affect the imaging of the target 3D cells. To avoid this situation, the density of collagen I can be increased to reduce the sedimentation rate. However, if the concentration of collagen I is too high, the high-density collagen I has a large scattering intensity, which affects the scattering imaging of gold nanoparticles. Secondly, the high-density collagen I will hinder the diffusion of gold nanoparticles or imaging dyes to the surrounding of the imaging cells during co-incubation, affecting the experiment and reducing the staining efficiency of the imaging cells. Finally, the cells have difficulty stretching in the high-density collagen I, affecting the imaging effect.

[0032] The concentration of collagen I in the collagen I and culture medium mixture is greater than the concentration of collagen I in the collagen I and cell mixture.

[0033] Preferably, the incubation time of the 3D cell network layer is 30 - 40 minutes. Too long or too short of this time will affect the formation of the 3D culture system.

[0034] To ensure the dark-field imaging effect, the total volume of the added gel-forming solution does not exceed 35 μL. The scattering background of the collagen I gel formed by this concentration and volume has a relatively small impact on the dark-field imaging of cells.

[0035] The described culture medium is a phenol red-free culture medium to avoid interference with imaging.

[0036] Preferably, the cell solution is a 3D cell sphere solution.

[0037] The present invention focuses on improving the traditional collagen I scaffold model. By precisely regulating the collagen concentration, matrix crosslinking degree, and cell seeding strategy, a 3D culture system with controllable thickness, no shedding, no trouble of sedimentation and adherent growth, and stable cell activity is constructed, which strictly matches the working distance of the high-power objective lens. At the same time, it ensures the diffusion efficiency of nanoparticles and the imaging signal-to-noise ratio, providing a reliable platform for the dynamic research of super-resolution subcellular organelles.

[0038] The 3D cells cultured by the present invention can be used for ultra-high resolution sub-organelle imaging, which can be extended to the culture and ultra-high resolution imaging of cell spheres and organoids, providing a reference for cell-based research in the fields of cell biology, oncology, clinical medicine, nanopharmaceuticals, etc.

[0039] The present invention also provides a method for ultra-high resolution sub-organelle imaging of 3D cells, including:

[0040] Observing the sub-organelles of the 3D cells cultured by the above culture method through high-resolution confocal imaging or STORM super-resolution imaging.

[0041] Further, the method for ultra-high resolution sub-organelle imaging of 3D cells includes:

[0042] Staining the organelles of the 3D cells cultured by the above culture method with a fluorescent probe, and imaging the stained 3D cells with a high-resolution confocal microscope or multi-channel excitation light;

[0043] Staining the organelles of the 3D cells cultured by the above culture method with a fluorescent probe, and imaging the stained 3D cells by separately exciting with the laser of a STORM super-resolution microscope.

[0044] The present invention also provides a method for real-time tracing and analyzing the whole process of intracellular endocytosis of gold nanoparticles, including:

[0045] Culturing 3D cells on a cover glass by the above culture method, co-incubating the gold nanoparticles with the 3D cells, and then using an upright dark-field microscope to real-time trace and observe the whole process of intracellular endocytosis of the particles.

[0046] The objective lens in the dark-field imaging system is an oil immersion lens with 60× NA adjustable, which can better match the oil immersion condenser while ensuring the signal-to-noise ratio; the detector is a color camera with a frame rate of 20 - 50 frames / s.

[0047] Extracting, analyzing, and visualizing the obtained dark-field imaging images using Image J, Matlab, and Origin software.

[0048] Compared with the prior art, the beneficial effects of the present invention are:

[0049] The 3D cell culture method of the present invention makes up for the gap between traditional 2D cultured cells and in vivo cells. The 3D cell culture method of the present invention uses conventional confocal dishes, cover glass - glass slides, and double-sided tape, with simple operation, no need for additional design and processing, and low consumable and time costs. The 3D cell culture method of the present invention has multiple advantages such as good reproducibility, low cell contamination rate, and no cell detachment.

[0050] The method for 3D cell culture of the present invention is directed to super-resolution imaging at the 3D sub-organelle level. This method breaks the limitation that it is difficult to perform super-resolution imaging on 3D cultured cells using an objective lens with a high NA value and a small working distance. Fluorescence imaging techniques such as high-resolution confocal (Zeiss LSM900 Airyscan 2) and super-resolution imaging techniques (AbbeLight STORM) can be used for super-resolution imaging of sub-organelles such as the cytoskeleton and mitochondria. It provides a methodological basis for the super-resolution imaging study of the sub-organelle structure and distribution of 3D cultured cells (cell spheres) and the co-localization study of intracellular interactions.

[0051] The method of the present invention also breaks the limitation that it is impossible to perform upright super-resolution and high signal-to-noise imaging on 3D cultured cells before. The whole process of endocytosis of gold nanoparticles in 3D cultured cells is traced and analyzed for a long time using the dual oil-immersion dark-field microscopy imaging technique. In summary, this method is applicable to various microscopy imaging systems and provides a methodological basis for the research based on 3D cultured cells. Brief Description of the Drawings

[0052] Figure 1 Flow schematic diagram of the 3D cell culture method for super-resolution sub-organelle imaging research;

[0053] Figure 2 Flow schematic diagram for pre-incubating a confocal dish or a cover glass;

[0054] Figure 3 Flow schematic diagram for the preparation of the sedimentation layer and the 3D cell culture layer;

[0055] Figure 4 Super-resolution cytoskeleton imaging map after cell immunofluorescence staining. (a) 2D cultured cells, (b) 3D cultured cells;

[0056] Figure 5 Statistical chart of the aspect ratio of 3D cultured cells and 2D cultured cells;

[0057] Figure 6 Single molecule point localization - cytoskeleton time series diagram of the STORM super-resolution imaging system for super-resolution imaging of the cytoskeleton of 3D cultured cells. The interval between adjacent two pictures is 500 frames; on the left side of each small picture is the single molecule dot map, and on the right side is the cytoskeleton map;

[0058] Figure 7 STORM super-resolution imaging map of microtubules and mitochondria of 3D cultured cells of the present invention. (a) Cell mitochondria map; (b) is the cell microtubule cytoskeleton map; (c) is the merge map of cell mitochondria and microtubule cytoskeleton;

[0059] Figure 8 Schematic diagram of the principle of the dark-field imaging system in Example 2;

[0060] Figure 9 It is a schematic diagram of the process for 3D cell culture based on the coverslip-slide system in Example 2;

[0061] Figure 10 It is an imaging diagram of the intracellular endocytosis process of nanoparticles in 3D-cultured cells by a dark-field microscopy imaging system;

[0062] Figure 11 It is a data analysis result diagram of the whole process of intracellular endocytosis of nanoparticles in 3D-cultured cells. (a) is a representative time mapping trajectory during the whole process of intracellular endocytosis of nanoparticles by 3D-cultured cells; (b) is a time series analysis diagram of displacement, velocity, and polarization angle during the whole process of intracellular endocytosis of nanoparticles by 3D-cultured cells formed by software such as Image J, Matlab, and Origin; (c) is a mean square displacement-time interval diagram during the whole process of intracellular endocytosis of nanoparticles by 3D-cultured cells. Detailed implementation manners

[0063] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the following embodiments are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0064] In the following embodiments, unless otherwise specified, they are all conventional methods. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0065] As Figure 1 shown, the present invention provides a 3D cell culture method for performing super-resolution subcellular organelle imaging research, including the following steps:

[0066] Step 1: Pre-incubate a confocal dish or a coverslip;

[0067] Step 2: Prepare a layer of cell sedimentation layer collagen I gel network;

[0068] Step 3: Inoculate a 3D cell collagen I network layer and perform 3D cell culture;

[0069] Step 4: Perform super-resolution imaging observation and analysis.

[0070] Example 1

[0071] A super-resolution imaging method for organelles of 3D-cultured cells includes the following steps:

[0072] 1) Pre-incubation of a confocal dish:

[0073] As Figure 2As shown, a confocal dish with a glass thickness of 0.17 mm was selected as the carrier, and collagen I diluted with 6 mM sterile acetic acid was dropped onto the glass and incubated in an incubator to pre-bond a thin layer of gelatin with an immeasurable thickness on the glass substrate.

[0074] Acetic acid with a purity of 99.99% was diluted with deionized water to a concentration of 6 mM, filtered through a 0.22 μm sterile filter, placed in an ultraviolet sterilization box for sterilization for 1 h, and then filtered and dispensed through a 0.22 μm sterile cell filter and stored at 4°C.

[0075] 6 mM sterile acetic acid at 4°C was added to a pre-cooled centrifuge tube, and the stock solution of collagen I (product number 354236) was taken into the centrifuge tube and mixed evenly. The final concentration of collagen I was 50 μg / mL. 200 μL of collagen I diluted with acetic acid was taken onto the round glass slide in a pre-cooled confocal dish, and the confocal dish was placed in a cell incubator and incubated for 6 h. Ensure that the entire operation process before placing it in the incubator is carried out in an environment below 4°C. The measures taken in this example include: ① Using a low-temperature liquid preparation module to pre-cool the aluminum centrifuge tube rack for PCR, which was placed in a -20°C refrigerator for a long time before the experiment and can maintain a temperature below 4°C for up to 8 h during the experiment; ② Ensuring that all pipette tips and utensils are pre-cooled before use.

[0076] After gently sucking out the excess collagen I diluted with acetic acid, it was washed 3 - 5 times with phosphate buffered saline (PBS) and air-dried naturally in a laminar flow hood for standby. The final coating concentration of the cover glass is 1 - 10 μg / cm 2 The coated cover glass can be stored at 4°C - 25°C for 3 months under sterile conditions.

[0077] 2) Preparation of the cell sedimentation layer (collagen I gel network layer):

[0078] The cell sedimentation layer is used to prevent or alleviate the sedimentation of cells to the bottom glass substrate for growth. As Figure 3 shown, a 2.5 mg / mL collagen I gel solution was prepared at 4°C and added to the pre-incubated confocal dish, and the culture dish was placed in an incubator and incubated for 40 min to form a network gel.

[0079] 10 μL of MEM (10×), 5 μL of NaHCO3 (7.5%), the stock solution of collagen I, and phenol red-free red blood cell medium were quickly added to a pre-cooled 600 μL centrifuge tube in sequence, and mixed evenly while adding. Similarly, all operations were ensured to be carried out at 4°C, and all consumables and reagents needed to be pre-cooled before the experiment.

[0080] After the pre-incubated confocal dish was placed on a pre-cooled aluminum centrifuge tube rack to cool down, the above-mentioned collagen I culture medium mixed solution was evenly spread on the round glass slide of the confocal dish. This operation needs to be strictly controlled at 4 °C. The volume needs to be strictly controlled to ensure that the thickness after gelation is less than the working distance after the objective glass slide is corrected for high-resolution objectives. In this example, the working distance after the objective glass slide is corrected is 0.12 mm, and the thickness of the sedimentation layer is selected to be 70 μm.

[0081] 3) Inoculate the 3D cell collagen I network layer and perform 3D cell culture: Prepare a collagen I gel and cell mixed solution at 4 °C and add it to the upper layer of the sedimentation layer of the confocal dish. Place the culture dish in a cell culture incubator and incubate for 40 min to allow the collagen I to gel. Subsequently, add 2 - 3 mL of phenol red-free culture medium to the culture dish and place the culture medium in the incubator for 5 - 7 days to allow the cells to grow and spread. Replace the cell culture medium every 2 - 3 days.

[0082] Quickly add 10 μL of MEM (10×), 5 μL of NaHCO3 (7.5%), collagen I stock solution, and cell solution to a pre-cooled 600 μL centrifuge tube in sequence, and mix evenly while adding. The final concentration of collagen I is 2 mg / mL. Similarly, all operations are ensured to be carried out at 4 °C, and all consumables and reagents need to be pre-cooled before the experiment.

[0083] 4) Observe the 3D cultured cells by high-resolution confocal system imaging: Perform organelle immunostaining on the 3D cultured cells and use a Zeiss LSM900 Airyscan 2 high-resolution confocal microscope for imaging.

[0084] Stain the microtubules, microfilaments, and cell nuclei of the 3D cultured cells with Actin-Tracker Red-555, Hoechst 33342 live cell staining solution, and Tubulin-Tracker Green immunofluorescent dye kit from Beyotime Biotechnology Co., Ltd. respectively. The specific operation strictly follows the operating procedure (protocol):

[0085] Microtubule staining:

[0086] a) Preparation of Tubulin-Tracker Green (500×) intermediate stock solution: Dissolve and take out an appropriate volume of Tubulin-Tracker Green (1000×) and add an equal volume of solubilizer;

[0087] b) Prepare the Tubulin-Tracker Green staining working solution in the following proportions: 2 μL of Tubulin-Tracker Green (500×), 10 μL of staining enhancer (100×), and 988 μL of Tubulin-Tracker Green diluent, and preheat it at 37°C;

[0088] c) Wash the cells 3 times with PBS;

[0089] d) Fluorescent labeling: Add the pre-warmed Tubulin-Tracker Green staining working solution at 37°C to the confocal dish for 3D-cultured cells, and co-incubate in an incubator at 37°C for 60 min;

[0090] e) Wash: Wash the cells 3 times with the Tubulin-Tracker Green diluent containing the staining enhancer (1×).

[0091] Microfilament staining:

[0092] a) Fixation: Add 500 μL of the universal cell fixative to the cells at 37°C for 10 min of fixation;

[0093] b) Wash: Wash the cells 3 times with PBS;

[0094] c) Staining: Dilute Actin-Tracker Green with the immunofluorescent staining secondary antibody diluent (P0108) from Beyotime at a ratio of 1:100, and add 200 μL dropwise to the confocal dish, and incubate in the dark at room temperature for 30 min;

[0095] d) Wash: Wash 3 times with the immunostaining washing solution (P0106) from Beyotime, 5 min each time.

[0096] Nucleus staining:

[0097] a) Staining: Dilute the Hoechst 33342 live cell staining solution (100×) with the immunostaining washing solution (P0106) from Beyotime to a final concentration of 1×, cover the cell sample, and incubate at room temperature for 5 min;

[0098] b) Wash: Wash 3 times with the immunostaining washing solution (P0106) from Beyotime, 5 min each time;

[0099] The Zeiss LSM900 Airyscan 2 high-resolution confocal microscope, based on a compound eye array detector, improves the ultimate resolution of traditional confocal microscopy from 200 nm to 120 nm. The small pinhole of 0.2 AU enhances the sectioning ability of the instrument, and the highly sensitive gallium arsenide phosphide array detector improves the signal-to-noise ratio of the instrument and is compatible with both strong and weak signals. In the 3D organelle fluorescence imaging experiment, an oil immersion objective lens with a 60× high numerical aperture is selected, the working distance of the slide is corrected to 0.12 mm, and the samples are excited with 405 nm, 488 nm, and 561 nm lasers. The laser intensities are set at 2%, 2.8%, and 4% respectively, and the gain values are all 800. Sequential scanning imaging is performed in Airyscan SR mode. Finally, the captured images are processed to obtain high-quality images, including Airyscan processing, deconvolution operation, adding scale bars, etc. As Figure 4 shown, the 3D cultured cells ( Figure 4 (b) in Figure 4 ) are more elongated than the 2D cells ( Figure 5 (a) in

[0100] ), and the cytoskeleton is in bundles. After statistics, the aspect ratio of 3D cultured cells is 6 times that of 2D cultured cells (as Figure 5 shown).

[0100] 5) Imaging and observing 3D cultured cells with a super-resolution microscopy system: Immunostain the organelles of 3D cultured cells and image them using an AbbeLight STORM super-resolution imaging system.

[0101] Use the Ningbo NanoWei fully automatic immunofluorescence sample maker to immunolabel the mitochondria and microtubules of 3D cultured cells according to the operating procedure (protocol). It should be noted that STORM imaging has strict requirements for the blinking characteristics of dyes. Preferably, in this embodiment, Alexa 647 is used to label mitochondria and NWCF561 is used to label microtubules. The specific steps are as follows:

[0102] a) Fixation: Add 500 μL of universal cell fixative to the cells at 37°C for 10 minutes of fixation;

[0103] b) Washing: Wash the cells 3 times with PBS;

[0104] c) Preparation of primary antibody dilution solution: The ratio of mitochondrial primary antibody to blocking buffer (BB) is 1:200, and the ratio of microtubule primary antibody to blocking buffer (BB) is 1:500;

[0105] d) Preparation of secondary antibody dilution solution: The ratio of Alexa Fluor 647 to blocking buffer (BB) is 1:200, and the ratio of NWSM 561 to blocking buffer (BB) is 1:50;

[0106] e) Place five solutions, namely the primary antibody diluent, secondary antibody diluent, BB, WB, and PBS, into the corresponding five wells of the sample preparation instrument respectively;

[0107] f) Install syringes on the gas path connectors corresponding to the five wells, insert the needles into the syringes, and then insert them into the corresponding wells respectively, ensuring that they are inserted to the bottom;

[0108] g) Click the start button in the automatic sampling module to start automatic sampling, and the green progress bar will appear in sequence;

[0109] h) After the automatic sampling is completed, click the stop button in the automatic sampling module, and the progress bar will change from green to gray;

[0110] i) Remove the five needles from the corresponding wells and insert them into the adapter respectively;

[0111] j) Click the start button in the sample preparation process to start sample preparation;

[0112] k) The sample preparation process is as follows:

[0113] i. Block with BB (500 μL) for 30 min;

[0114] ii. Incubate with the primary antibody (200 μL) for 60 min;

[0115] iii. Wash with WB (500 μL) three times, 10 min each time;

[0116] iv. Incubate with the secondary antibody (200 μL) for 40 min;

[0117] v. Wash with WB (500 μL) five times, 10 min each time;

[0118] vi. Wash with PBS (300 μL) for 5 min.

[0119] l) Post-fixation: Add 500 μL of universal cell fixative to the cells at room temperature, and the fixation duration is 10 min;

[0120] m) Washing: Wash the cells five times with sterile water;

[0121] n) Drying: Wash with 1 mL of sterile water five times, 5 min each time, and then leave the sample to air-dry at room temperature.

[0122] The AbbeLight STORM super-resolution imaging system was used to perform single-molecule localization imaging on mitochondria and microtubules respectively. This imaging system uses a 100× high NA value TIRF special objective lens, and the working distance after slide correction is 0.12 mm. After adding the STORM special imaging buffer, it was placed on the microscope for acquisition imaging. To avoid the influence of the short-wave high-energy 561 laser on the 647 dye, in this embodiment, the mitochondria labeled with Alexa 647 were first collected, the laser intensity was 50%, the exposure time was 50 ms, and the acquisition ended when more than 10,000 images were collected and the mitochondrial structure was intact. Then, the 561 laser was quickly turned on, the laser intensity was 80%, the exposure time was 50 ms, and the acquisition of NWCF561-labeled microtubules was started, and the acquisition ended when more than 10,000 images were collected and the microtubule structure was intact. Note that the laser intensity setting was set according to the channel scintillation situation before acquisition (as Figure 6 shown), that is, there are many single-molecule dots scintillating in the field of view, but the sample structure cannot be seen through the scintillating dots. The single-molecule dot map shows that the STORM dot scintillation effect is good, further verifying that our system can perform super-resolution subcellular organelle imaging.

[0123] The original images were subjected to super-resolution reconstruction and dual-channel drift correction. First, the original ROI.tif file of the Alexa 647 channel was imported, the Detection threshold was set, and Drift Correction was set to 0. Then, click START Batch to perform image reconstruction of the 647 channel. After completion, the original ROI.tif file of the NWCF561 channel was imported, the Detection threshold was set, and the drift data of the 647 channel was imported at Drift Correction. Click START Batch to perform image reconstruction of the 561 channel. Finally, in the "Batch" interface, load the "CoordTable*D.csv" files of the 647 and 561 channels in sequence, set the parameters for visual adjustment, and obtain the co-localization super-resolution image (as Figure 7 shown).

[0124] Example 2

[0125] A method for long-term real-time tracing and analyzing the whole process of gold nanoparticle endocytosis in 3D-cultured cells based on a dark-field imaging system:

[0126] 1) 3D cell culture based on a dual-oil-immersion high-resolution upright dark-field microscopy imaging system: Place a 0.17-mm coverslip in a culture dish. The steps are the same as in Example 1. Pre-incubate the coverslip to pre-bond a thin layer of flocking glue with an immeasurable thickness on the glass substrate. Subsequently, prepare a cell sedimentation layer of collagen I glue network layer in the double-sided adhesive ring to hinder or alleviate the cell sedimentation to grow on the bottom glass substrate. Finally, inoculate the 3D cell collagen I network layer and perform 3D cell culture.

[0127] To achieve the dark-field imaging effect with high resolution and high signal-to-noise ratio, this example selects a dual-oil-immersion upright dark-field imaging system with an oil objective lens and a dark-field condenser. The confocal dish is not suitable for this imaging system. Based on this, this example selects a coverslip + slide culture chamber.

[0128] To strictly control the thickness of the sedimentation layer to match the working distance of the objective lens, this example uses a non-toxic double-sided adhesive ring on the slide to achieve this. To match the thickness of the ring without leakage, this example selects a grooved slide, and a rectangular double-sided adhesive containing a hollow circle is pasted on the slide. The dimensions of the rectangle, hollow circle, and ring match the grooved slide ( Figure 9 )

[0129] The principle of the dark-field imaging system in Example 2 is as Figure 8 shown, Figure 8 where the yellow light is the incident light of the light source, and the red light is the scattered light of the sample set solution. During dark-field imaging, the incident light will not be collected and imaged by the objective lens, only the scattered light of the sample is collected and imaged by the objective lens. It is necessary to avoid the influence of stray light in the solution.

[0130] To avoid the collection of stray light in the solution by the objective lens, in addition to matching the working distance of the 60× oil objective lens, which is 0.22 mm, the thickness of the sample system should not be too thick. After weighing, the thickness of the ring in this example is 0.12 mm, and the thickness of the rectangle containing the hollow circle is 0.17 mm.

[0131] As Figure 9 shown, the preparation method of the coverslip + slide culture chamber is as follows: Purchase double-sided adhesives with thicknesses of 0.12 mm and 0.17 mm. Use a puncher to make a rectangular double-sided adhesive containing a hollow circle and a ring according to the set dimensions, and then place them in an ultraviolet sterilization box for sterilization. After sterilization, paste the double-sided adhesive ring and the rectangular double-sided adhesive containing the hollow circle on the coverslip and slide washed with piranha solution respectively, and then place them in an ultraviolet sterilization box for sterilization.

[0132] The steps of slide cleaning are as follows: Prepare piranha solution in a fume hood. After cooling, transfer it to a container equipped with a coverslip and a slide adapter, and immerse the slides. After overnight, rinse each slide with a large amount of water. Ultrasonically clean the slides with deionized water for 3 - 5 min, and then rinse each slide with deionized water. Repeat this step 3 - 5 times until water droplets do not adhere to the wall. Finally, immerse the slides in absolute ethanol, and after 2 h, dry each slide with a hair dryer and store it in an ultraviolet sterilization box.

[0133] In this example, to avoid the influence of stray light of the solution being collected by the objective lens on the dark - field imaging effect, the thickness of the sedimentation layer is selected as 50 μm (10 μL), and the thickness of the 3D cell collagen I network layer does not exceed 150 μm (30 μL).

[0134] 2) Tracking the process of intracellular endocytosis of gold nanoparticles by dark - field imaging: After the cells are fully spread, add 40 - 80 μL of the stock solution of gold nanoparticles (OD 10, Nanoseedz) to the culture dish and incubate in an incubator for 30 min. Subsequently, aspirate the cell culture medium solution containing nanoparticles from the culture dish and add it to the inner circle of the rectangle of the grooved slide. Use forceps to pick up the coverslip with 3D - cultured cells and invert it onto the slide to ensure fitting. After sealing with nail polish, place it under a dark - field microscope to track the whole process of intracellular endocytosis of nanoparticles (as Figure 10 shown), and the exposure time is 30 ms. Use Image J software to extract the trajectories of nanoparticles, and perform data processing with MATLAB and draw graphs with Origin for analysis.

[0135] The movement of gold nanoparticles during the whole process of intracellular endocytosis is mainly divided into 3 stages ( Figure 11 (b) in): Adsorption stage (0 - 3 s, cyan), interaction with the cell membrane stage (3 - 335 s, yellow), intracellular movement stage (335 - 738 s, green). At 0 - 2.5 s, gold nanoparticles diffuse in the culture medium in the collagen I gel network and approach the cells, and the radius of gyration (reflecting the movement range of the reaction particles) R g = 2.29 μm. At the 33rd frame, gold nanoparticles adsorb onto the cell membrane, and their translational motion begins to be restricted, R g = 0.10 μm. During the interaction with the cell membrane stage (3 - 340 s), gold nanoparticles move within a range of 1.5×3 μm 2 (R g = 0.83 μm). During the transmembrane stage (320 - 340 s), gold nanoparticles perform restricted motion (α < 1) within a small range (R g = 0.074 μm), and the diffusion coefficient (reflecting the speed of particle movement) D t = 0.00025 μm2 / s. After entering the cell, the gold nanoparticles first stay near the transmembrane site for ~30 s (R g = 0.11 μm), and then start to perform large-scale directional movement inside the cell (α = 1.58, R g = 3.60 μm). During the above several stages, the kinetic parameters of the gold nanoparticles first decrease and then increase, as shown in Table 1.

[0136]

[0137] The above-described embodiments have described in detail the technical solutions and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, supplements, equivalent replacements, etc. made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A 3D cell culture method for super-high resolution sub-organelle imaging research, characterized in that It includes the following steps: Step (1): Drop the collagen I dilution solution onto the surface of the glass substrate for pre-incubation to form a pre-incubation layer; in the collagen I dilution solution, the concentration of collagen I is 10-100 μg / mL; the pre-incubation time is 2-10 h; Step (2): Spread the collagen I and culture medium mixture onto the surface of the pre-incubation layer for incubation to form a gel of type I collagen to form a network layer as the sedimentation layer; in the collagen I and culture medium mixture, the concentration of collagen I is 2-3 mg / mL; the incubation time is 30-40 min; Step (3): Add the collagen I and cell mixture onto the surface of the sedimentation layer for incubation to form a gel of collagen I; in the collagen I and cell mixture, the concentration of collagen I is 2-3 mg / mL; the incubation time is 30-40 min; Add cell culture medium and place it in an incubator for 5-7 days to allow the cells to grow and spread, complete 3D cell culture, and form a 3D cell network layer.

2. The 3D cell culture method for super-high resolution sub-organelle imaging research according to claim 1, wherein The collagen I dilution solution is collagen I diluted with an acetic acid solution.

3. The 3D cell culture method for ultra-high resolution sub-organelle imaging research according to claim 1, characterized in that, The coating concentration of the pre-incubation layer on the glass substrate surface is 1-10 μg / cm 2 .

4. The 3D cell culture method for ultra-high resolution sub-organelle imaging research according to claim 1, characterized in that, The pH of the collagen I and culture medium mixture is 7.

0.

5. The 3D cell culture method for ultra-high resolution sub-organelle imaging research according to claim 1, characterized in that, The thickness of the sedimentation layer is 50-70 μm.

6. The 3D cell culture method for ultra-high resolution sub-organelle imaging research according to claim 1, wherein The pH of the collagen I and cell mixture is 7.

0.

7. A method for 3D cell super-resolution sub-organelle imaging, characterized in that, It includes: Observe the sub-organelles of the 3D cells cultured by the culture method according to any one of claims 1-6 through high-resolution confocal imaging or STORM super-resolution imaging.

8. The method for 3D cell super-resolution sub-organelle imaging according to claim 7, wherein, It includes: Stain the organelles of the 3D cells cultured by the above culture method with a fluorescent probe, and image the stained 3D cells with a high-resolution confocal microscope or multi-channel excitation light; Stain the organelles of the 3D cells cultured by the above culture method with a fluorescent probe, and image the stained 3D cells by laser excitation with a STORM super-resolution microscope respectively.

9. A method for real-time tracing and analyzing the whole process of intracellular endocytosis of gold nanoparticles, characterized in that It includes: Culture 3D cells on a coverslip by the culture method according to any one of claims 1-6. After co-incubating the gold nanoparticles with the 3D cells, use an upright dark-field microscope to perform real-time tracing observation on the whole process of intracellular uptake of the particles.

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