Culture dish marking method for cell photoelectric correlation experiment and application of culture dish marking method

Abstract

The invention discloses a culture dish marking method for a cell photoelectric correlation experiment and application of the culture dish marking method, and the method comprises the following steps: placing a stainless steel carrying net with a Marker pattern in a target area at the bottom of a culture dish, and taking the stainless steel carrying net as a mask plate; putting the culture dish with the mask plate into a metal ion sputtering instrument, taking platinum as a sputtering target material, and starting the metal ion sputtering instrument to carry out ion sputtering so as to form a Marker formed by a platinum plating layer at the bottom of the culture dish; and taking out the culture dish and removing the mask plate to obtain the culture dish with the Marker for the cell photoelectric correlation experiment. The method is easy and convenient to operate, low in cost, high in mark transfer rate and safe to use, no glass slag is left after resin separation, and the separated resin embedding block does not damage a slicing knife during slicing.

Description

Technical Field

[0001] This invention relates to the field of special consumables preparation technology for cell experiments, specifically to a method for labeling culture dishes for cell photoelectric correlation experiments and its application. Background Technology

[0002] Cell photoelectrochemical correlation assays are a special type of experiment in cell biology that correlates and registers fluorescence signals acquired by optical equipment such as confocal microscopes and super-resolution fluorescence microscopes with cellular ultrastructures acquired by electron microscopes, thereby obtaining the true ultrastructural localization information of fluorescently labeled components. This experiment requires the use of special culture dishes with markers at the bottom, and the markers must be able to achieve imprint transfer in subsequent procedures to assist in cell division and localization.

[0003] Commercially available culture dishes with markers primarily use photolithography to create a textured structure (very shallow grooves or protrusions) on the bottom surface of the glass-bottomed culture dish as a marker. Taking ibidi brand products as an example, the preparation process involves multiple steps, including the fabrication of a marker mask (containing a 50 / 500μm grid or positioning pattern), photoresist coating, UV exposure to transfer the mask pattern to the photoresist layer, development, etching to form groove markers, sputtering of a metal film (such as chromium or titanium) to enhance contrast, and stripping off excess photoresist. The process is cumbersome, relies on chemical processes and a large amount of specialized equipment, has a long preparation cycle, and the marker area is fixed, resulting in poor flexibility.

[0004] In photoelectric correlation experiments, liquid resin is added to the culture dish and polymerized and hardened. Then, the bottom of the glass dish is separated from the resin embedding block containing cells, allowing the marker-induced bumps to imprint onto the resin for subsequent auxiliary positioning. There are two main methods for separating the glass from the resin embedding block, both of which have the following problems: One method, the liquid nitrogen freezing method, involves immersing the culture dish in liquid nitrogen and then manually separating the glass from the resin. This easily leads to irregular breakage of the glass, with some areas tightly adhering to the resin and unable to be separated. Forced separation can damage the resin and the internal contents. Some cells are difficult to completely separate due to the tiny indentations or protrusions on the glass. The fine glass fragments remaining on the resin surface can enter the subsequent sectioning process, causing permanent damage to the expensive diamond ultrathin microtome. Secondly, the hydrofluoric acid dissolution method requires immersing the culture dish in hydrofluoric acid for several hours to fully dissolve the glass material and then separate it. However, hydrofluoric acid poses a high risk of toxicity. In addition, if the treatment is insufficient, jelly-like glass fragments will remain on the uneven parts of the resin surface. The remaining glass fragments can still damage the microtome, and prolonged immersion can cause the resin block to become damp, affecting the quality of ultrathin sections.

[0005] Therefore, the petri dishes in the relevant technologies have the following drawbacks: complex manufacturing process and high degree of equipment and technical expertise; glass is not easy to completely separate during use, affecting the success rate of experiments and damaging expensive diamond microtome blades; and highly toxic reagents are required for use in experiments, posing risks to operation and management. Summary of the Invention

[0006] To address the shortcomings of existing culture dish preparation processes, such as complex processes, difficult separation, low safety, and poor flexibility, this invention provides a culture dish labeling method and its application that is simple to operate, low in cost, has a high label transfer rate, and is safe to use for cell photoelectric correlation experiments.

[0007] To achieve the above objectives, the present invention provides a method for labeling culture dishes for cell photoelectric correlation experiments, which includes the following steps: S1. Place a stainless steel mesh with a marker pattern in the target area at the bottom of the petri dish, and use the stainless steel mesh as a mask template. S2. Place the culture dish containing the mask template into a metal ion sputtering instrument, use platinum as the sputtering target, and start the metal ion sputtering instrument to perform ion sputtering to form a marker composed of platinum coating at the bottom of the culture dish. S3. Remove the culture dish and the mask template to obtain a marker-bearing culture dish for cell photoelectric correlation experiments.

[0008] According to an embodiment of the present invention, a method for labeling culture dishes for cell photoelectrocorrelation experiments is disclosed. This method directly uses a stainless steel mesh as a mask template and combines it with platinum ion sputtering to form a marker, eliminating the need for photoresist and complex photolithography equipment. The number of steps is greatly reduced, the preparation cycle is shortened, and the process can be completed within 20 minutes. The preparation can be carried out at any time in an electron microscopy room equipped with a metal ion sputtering instrument. The success rate of culture dish preparation is 100%, and the process cost is low, reducing the production cost. The marker imprint obtained by platinum ion sputtering has a high transfer success rate. Because this method can maintain the integrity and smoothness of the glass substrate surface without unevenness, it can achieve complete separation of resin and glass without glass debris residue. The separated resin-embedded blocks do not damage the microtome during sectioning. At the same time, since the marker material is Pt with a Mohs hardness of 4.0-4.5, it will not damage conventional glass cutters (Mohs hardness 5.5-6.0) and diamond cutters during ultrathin sectioning. This method allows for flexible selection of polystyrene or glass-bottomed cell culture dishes according to experimental needs, with readily available and inexpensive basic consumables. The pattern (such as grid size and shape) of the stainless steel mesh can be flexibly chosen, and markers can be added at any desired location. Multiple marker areas can also be freely set within the same culture dish, increasing experimental flexibility and sample size. This method avoids the use of highly toxic chemicals such as hydrofluoric acid, reducing operational and management risks.

[0009] Optionally, the metal ion sputtering instrument is set to a working distance of 65 mm and a sputtering thickness of 4 nm. This ensures sputtering uniformity with a 65 mm working distance, while the 4 nm thickness provides good light transmittance, supporting normal optical signal acquisition requirements, and avoiding problems such as marker transfer failure due to excessive thickness or weak imprint signals due to excessive thinness.

[0010] Optionally, the culture dish can be a polystyrene culture dish or a confocal microscopy-specific glass-bottom culture dish. This supports both polystyrene culture dishes and confocal microscopy-specific glass-bottom culture dishes, allowing users to flexibly choose according to their experimental needs and improving experimental compatibility.

[0011] Another embodiment of the present invention provides a marker-bearing culture dish prepared using the above-described culture dish marking method. The marker-bearing culture dish includes a culture dish body and a platinum marker layer formed on the bottom of the culture dish body; the thickness of the platinum marker layer is 4 nm ± 0.5 nm. The platinum marker layer has a thin film structure, which can be completely transferred to the resin embedding block during subsequent separation, without glass fragments remaining due to uneven structure, thus avoiding damage to expensive diamond ultrathin scalpels.

[0012] Another embodiment of the present invention provides a method for using the above-mentioned marker-equipped culture dish, which includes the following steps: (1) Sterilize the culture dish with marker by ultraviolet irradiation, then inoculate the target cells into the sterilized culture dish with marker and perform the pre-set experimental treatment; after the experimental treatment is completed, use an optical microscope to take bright field images and fluorescence information images of the cells in the culture dish with marker, and record the positional correspondence between the marker and the cells. (2) Add 2.5% glutaraldehyde solution to the culture dish with marker to chemically fix the cells in the culture dish, and then embed the cells in the culture dish in situ; (3) After embedding, perform separation operation according to the type of the culture dish body. If the culture dish body is a polystyrene culture dish, break the bottom of the culture dish to separate the resin embedding block carrying the platinum marker from the polystyrene material; if the culture dish body is a confocal glass bottom culture dish, put the culture dish into liquid nitrogen. After the temperature of the culture dish and the temperature of the liquid nitrogen are balanced, break off the PCR tube that is upside down on the bottom of the dish marker area to completely separate the resin embedding block carrying the platinum marker from the glass material in the PCR tube. (4) Based on the position of the platinum marker on the resin embedding block, combined with the correspondence between the marker and the cell position recorded in step (1), the target cell is located and ultrathin sectioning and electron microscopy imaging analysis are performed.

[0013] Therefore, this method eliminates the need for hydrofluoric acid throughout the entire process, thus removing operational and management risks associated with highly toxic reagents. It also features customized separation methods for different materials (breaking the bottom of polystyrene culture dishes and using liquid nitrogen to separate PCR tubes in glass-bottomed culture dishes), ensuring thorough separation without any debris residue and protecting the microtome. By recording the positional relationship between the marker and the cells, and combining this with the marker on the resin block, the target cells can be precisely located, solving the problem of difficult cell localization in existing technologies and improving the success rate of photoelectric correlation experiments.

[0014] Optionally, in step (2), the in situ embedding of cells in the culture dish includes performing the following operations in sequence: a) Buffer washing: The fixed cell samples were soaked and washed with phosphate buffer for 5 minutes each time, and the washing was repeated 3 times. b) First fixation with osmium tetroxide: Add 1% osmium tetroxide solution to the petri dish and place the petri dish in a refrigerator at 4°C for 1.5 h. c) First double-distilled water wash: Soak and wash the osmium tetroxide-fixed cell samples with double-distilled water for 3 minutes each time, and repeat the wash 5 times; d) Dithiomethylhydrazine reaction: Add 1% dithiomethylhydrazine solution to a petri dish and react for 20 min at room temperature and in the dark. e) Second double-distilled water rinse: Repeat the double-distilled water rinse operation in step c); f) Second fixation with osmium tetroxide: Add 1% osmium tetroxide solution to the petri dish and react in a refrigerator at 4°C for 40 min; g) Third double-distilled water rinse: Repeat the double-distilled water rinse operation in step c); h) Uranium acetate staining: Add 2% uranium acetate aqueous solution to the petri dish and stain overnight at 4°C. i) Fourth double-distilled water rinse: Repeat the double-distilled water rinse operation in step c); j) Dehydration and Resin Infiltration: After the fourth double-distilled water washing, the cell samples were subjected to the following operations in sequence: Cell samples were soaked sequentially in ethanol concentration gradients of 30%-50%-70%-90%, with a soaking time of 7 min for each concentration gradient; Cell samples were then dehydrated by soaking in 100% ethanol for 7 min each time, repeated 4 times; Cell samples were then soaked sequentially in a mixture of 100% ethanol and 812 resin at a volume ratio of 3:1, 1:1, and 1:3, with infiltration times of 1 h, 2 h, and 2 h, respectively; The mixture was removed, and pure 812 resin was added to soak the cell samples, incubating overnight; Then, fresh pure 812 resin was added, and incubation continued for 4 h; k) Resin embedding and polymerization: The embedding operation is performed according to the type of culture dish: If the culture dish is a polystyrene culture dish, remove the residual 812 resin in the culture dish and add 400 μL of pure 812 resin to the culture dish; if the culture dish is a confocal glass-bottom culture dish, take an open PCR tube, fill the tube with pure 812 resin, and invert it on the marker area at the bottom of the culture dish; after the operation is completed, put the culture dish in an oven at 70℃ to carry out the resin polymerization reaction for 24 h.

[0015] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0016] Figure 1 The images show the actual stainless steel marker carrier mesh according to Embodiment 1 of the present invention, the actual images of the culture dish with marker before and after metal ion sputtering, and the enlarged view of the marker area of ​​the culture dish with marker. Figure 2 The images shown are physical photos and enlarged views of the resin-embedded block separated from a glass-bottomed culture dish with a marker, according to Embodiment 1 of the present invention. Figure 3 The images show the Marker region captured by the ultra-high resolution fluorescence microscope according to Embodiment 2 of the present invention, the Marker region captured during the embedding process, and the embedding block to be ultrathinly sliced. Figure 4 The image shows the cell growth in a glass-bottomed culture dish with a 4 nM Pt Marker according to Example 2 of the present invention, where A represents cell seeding for 4 hours, B represents cell seeding for 24 hours, and C represents cell seeding for 60 hours. Figure 5 This is a comparison of the nuclear fluorescence intensity of cells with and without Pt coverage areas according to Embodiment 2 of the present invention; Figure 6 The fluorescence attenuation in a 4 nm Marker culture dish according to Example 2 of the present invention; Figure 7 The fluorescence decay in a glass-bottomed culture dish with an 8 nM Pt Marker according to Example 2 of the present invention is shown. Figure 8 The image shows the marker separation in a glass-bottomed culture dish with an 8 nM Pt Marker according to Embodiment 2 of the present invention, where A represents the separation with a 4 nM Pt Marker, B represents the separation with an 8 nM Pt Marker, and C is a magnified view of B. Figure 9This describes the separation of resin in commercially available ibidi glass-bottomed culture dishes with markers. Figure 10 The stability test of the marker in the glass-bottomed culture dish with 4 nM Pt marker is shown in the following figures: A is untreated, B is soaked in phosphate buffer for 24 hours, C is soaked in ultrapure water for 24 hours, and D is soaked in 100% ethanol for 24 hours. Detailed Implementation

[0017] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0018] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0019] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0020] The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely descriptive and do not limit the present invention in any way.

[0021] Example 1 A platinum marker layer was formed at the bottom of the culture dish using metal ion sputtering combined with masking techniques. The specific steps are as follows: Step S1: Placement of the mask template Select the appropriate petri dish type (polystyrene petri dish or confocal glass-bottomed petri dish) according to experimental requirements, ensuring the bottom is clean and free of impurities. Place a stainless steel marker mesh as a mask flat on the target area at the bottom of the petri dish (single or multiple areas can be freely selected), ensuring the mesh completely adheres to the bottom. In this embodiment, the stainless steel marker mesh is the ZB-200F4 model from Beijing Zhongxing Bairui Technology Co., Ltd. (including a preset grid / positioning pattern). Step S2: Metal ion sputtering treatment Place the culture dish containing the mask from step S1 into a Leica ACE600 metal ion sputtering instrument and set the parameters as follows: working distance 65 mm; sputtering target platinum (Pt); sputtering thickness 4 nm; vacuum value 8 × 10⁻⁶. -3 mbar, current 35mA; start the instrument for ion sputtering (about 20 minutes) to ensure uniform coating.

[0022] Step S3: Mask Removal and Petri Dish Acquisition After sputtering is complete, remove the culture dish and carefully remove the mask to obtain a culture dish with a platinum marker on the bottom.

[0023] The results are as follows Figure 1 As shown, Figure 1 A shows the stainless steel marker mesh used in this embodiment, approximately 3 mm in diameter with square holes of approximately 100 μm side length. The mesh has seven English letters for cell localization. B shows the marker-bearing culture dish prepared using ion sputtering in this embodiment. The upper part of B shows the commercially available culture dish with the mask placed on it, while the lower part shows the marker-bearing culture dish after ion sputtering. The culture dishes used in this embodiment are a commercially available polystyrene culture dish (left) and a confocal glass-bottomed culture dish (right). C shows a magnified view of the marker area on the culture dish after marking in this embodiment, where the metal marker is clearly visible.

[0024] Therefore, it can be seen that the culture dish obtained by the embodiment has a uniform thin film structure in the target area at the bottom of the culture dish body, without obvious unevenness, and a thickness of 4 nm; the material is Pt, with a Mohs hardness of 4.0-4.5, stable chemical properties, no cytotoxicity, and can be completely transferred to the resin embedding block.

[0025] In addition, resin embedding blocks were prepared using commercially available ibidi glass-bottomed culture dishes with markers as a comparison, such as... Figure 9 As shown, Figure 9 In Figure A, the left marker area contains residual glass fragments and damage to the resin surface. Figure 9 Scratches were generated when the glass base separated from the resin in Figure B.

[0026] Buffer solution, ultrapure water, and 100% ethanol were added to the prepared glass-bottomed culture dish containing a 4 nM Pt Marker. After soaking for 24 hours, the Marker area was photographed. Figure 10 As shown, the marker did not exhibit any deformation, detachment, or curling.

[0027] Example 2: How to use a petri dish with a marker Step 1: Sterilization and Cell Inoculation The marker-bearing culture dishes obtained in Example 1 were sterilized by ultraviolet irradiation (≥30 minutes). The target cells are inoculated and cultured and treated according to a preset protocol (such as drug stimulation or gene transfection). In this embodiment, the preset protocol is transient transfection of the cells. Successfully transfected cells express a red fluorescent fusion protein. The fusion protein is diffusely distributed in the cytoplasm when untreated. After drug treatment, the fusion protein will be localized to secondary lysosomes, forming bright red patches.

[0028] Step 2: Optical Imaging and Position Recording Bright-field and fluorescence images were captured using an optical microscope to clearly record the positional correspondence (coordinates / relative distance) between the marker and the target cells, which was used for subsequent localization of positive cells.

[0029] Step 3: Cell fixation Add 2.5% glutaraldehyde solution to the above-mentioned marker culture dish to chemically fix the cells in the dish, and fix at room temperature for 1 hour.

[0030] Step 4: In situ embedding of cells in the culture dish Follow these steps in order: a) Buffer washing: Immerse the fixed cell samples in 0.1M phosphate buffer (PB) for 5 minutes each time, repeat 3 times; b) First fixation with osmium tetroxide: Add 1% osmium tetroxide solution to the petri dish and place the petri dish in a refrigerator at 4°C for 1.5 h. c) Double distilled water washing: Soak and wash the osmium tetroxide-fixed cell samples in double distilled water for 3 minutes each time, repeating 5 times; d) Dithiomethylhydrazine reaction: Add 1% dithiomethylhydrazine solution to a petri dish and react for 20 min at room temperature and in the dark. e) Second double-distilled water rinse: Repeat the double-distilled water rinse operation in step c); f) Second fixation with osmium tetroxide: Add 1% osmium tetroxide solution to the petri dish and react in a refrigerator at 4°C for 40 min; g) Third double-distilled water rinse: Repeat the double-distilled water rinse operation in step c); h) Uranium acetate staining: Add 2% uranium acetate aqueous solution to the petri dish and stain overnight at 4°C. i) Fourth double-distilled water rinse: Repeat the double-distilled water rinse operation in step c); j) Dehydration and osmosis: The cell samples after the fourth double-distilled water washing were subjected to the following operations in sequence; Gradient ethanol dehydration: 30%→50%→70%→90% ethanol, 7 minutes / gradient; Dehydration with anhydrous ethanol: 100% ethanol, 7 minutes / time × 4 times; Resin infiltration: Soak in 100% ethanol and 812 resin at a volume ratio of 3:1 (1 h) → 1:1 (2 h) → 1:3 (2 h); incubate overnight in pure 812 resin, then replace with new 812 resin and continue incubation for 4 h; k) Embedding and aggregation: Polystyrene petri dish: Add 400 μL of pure 812 resin; Glass-bottomed culture dish: Fill an open PCR tube with 812 resin and invert it onto the marker area; Polymerize in a 70℃ oven for 24 hours.

[0031] Step 5: Separation operation after polymerization Polystyrene petri dish: The bottom of the petri dish can be broken with a hammer to completely shatter it, allowing the resin block to completely separate from the polystyrene material, carrying the metal marker. Glass-bottomed culture dish: Immerse the culture dish in liquid nitrogen, and after the temperature is balanced, break off the PCR tube. The resin embedding block carrying the metal marker will then be completely separated from the glass.

[0032] The results are as follows Figure 2 As shown, Figure 2 Image A shows a resin-embedded block separated from a culture dish with a marker-coated glass bottom, with no glass debris remaining on the surface; Image B shows a resin-embedded block separated from a culture dish with a marker-coated glass bottom, showing that the marker imprint has been completely transferred to the surface of the resin-embedded block; Image C is a magnified view of the surface of the resin-embedded block, showing that the transferred metal marker is clear and complete.

[0033] Step 6: Localization and Electron Microscopy Imaging Based on the correspondence between the markers imprinted on the resin embedding blocks in step 5 and the optical microscope images, cells of interest can be accurately located for ultrathin sectioning and subsequent electron microscopy imaging.

[0034] The results are as follows Figure 3 As shown, Figure 3 Image A shows a marker region image taken with a high-resolution fluorescence microscope, with the target cells showing red fluorescence; Image B shows a marker region image taken during the embedding process, where the target cells can be identified by the marker; Image C shows the embedding block about to be ultrathinly sectioned, where the metal marker imprint in the culture dish is clearly and completely transferred to the resin surface, allowing for accurate positioning of the target cells based on the marker.

[0035] like Figure 4 As shown, Figure 4The images show cell growth. Image A shows cells 4 hours after seeding, image B shows cells 24 hours after seeding, and image C shows cells 60 hours after seeding. Cells were seeded in glass-bottomed culture dishes with 4 nM Pt Markers and cultured for 60 hours. Images were taken of the same area, with lighter areas representing areas without Pt coverage and darker areas representing areas with Pt coverage. Cells in the Pt-covered areas showed normal growth and proliferation, with little difference in morphology and growth rate compared to cells in the uncovered areas. Furthermore, the Pt Markers remained stable and unchanged after 60 hours of immersion in the culture medium, without detachment or curling.

[0036] Furthermore, in step S2 of Example 1, a working distance of 65 mm and a sputtering thickness of 4 nm ensure uniform coating. Using a working distance of 55 mm or smaller, however, can easily lead to uneven coating due to obstruction from the sidewalls of the culture dish. Choosing a coating thickness greater than 6 nm will cause the marker to adhere poorly to the glass substrate, resulting in decreased light transmittance and severely weakened fluorescence signal. Specifically, as shown... Figures 5-8 As shown, Figure 5 The fluorescence decay in the culture dish with a 4 nm marker is shown. Figure 5 In the left image, the cells in the marker region have blue fluorescent signals representing cell nuclei and green fluorescent signals representing lysosomes. The right image shows a slight decrease in fluorescence intensity in the Pt-covered area (nuclei indicated by the red arrow) compared to the uncovered area (nuclei indicated by the yellow arrow), but this intensity is still sufficient for localization. Then, by quantifying and statistically analyzing the nuclear fluorescence intensity of 60 cells from each of the Pt-covered and uncovered areas, the following results were obtained: Figure 6 The chart shown indicates that when the marker thickness is 4 nm, the transmittance is higher than 75%.

[0037] The fluorescence decay in the culture dish with the 8 nM Pt Marker is as follows: Figure 7 As shown, Figure 7 In the bright-field image, the dark areas represent Pt-covered areas, and the light areas represent areas without Pt coverage. The blue fluorescence on the right represents the results of DAPI staining of cell nuclei. Figure 7 As can be seen, when the Pt Marker thickness is 8 nM, the intensity of the nuclear fluorescence signal is significantly attenuated compared to the area without Pt coverage, while when the Pt Marker thickness is 4 nM, the fluorescence signal is only slightly attenuated.

[0038] Marker separation in a culture dish with an 8 nM Pt Marker is as follows: Figure 8 As shown, Figure 8In Figure A, the 4nM Pt Marker was successfully separated, leaving a clear imprint on the resin surface; in Figure B, the 8nM Pt Marker was not successfully separated, resulting in an incomplete and unclear Marker imprint on the resin surface and unstable transfer performance; Figure C is a magnified view of a portion of Figure B, showing incomplete Marker imprints, which may be blurred or missing.

[0039] In summary, the method according to embodiments of the present invention forms a marker on a petri dish without the need for photoresist and complex photolithography equipment, significantly reducing the number of steps and shortening the preparation cycle. It can be completed within 20 minutes and can be performed at any time in an electron microscope room equipped with a metal ion sputtering system. The success rate of petri dish preparation is 100%, and the process cost is low, reducing production costs. The marker imprint obtained by platinum ion sputtering has a high transfer success rate because this method maintains the integrity and smoothness of the glass substrate surface without any unevenness, thus achieving complete separation of resin and glass without glass residue. The separated resin-embedded block does not damage the microtome during sectioning. Furthermore, since the marker material is Pt with a Mohs hardness of 4.0-4.5, it will not damage conventional glass cutters (Mohs hardness 5.5-6.0) or diamond cutters during ultrathin sectioning. This method allows for flexible selection of polystyrene or glass-bottomed cell culture dishes according to experimental needs, with readily available and inexpensive basic consumables. The pattern (such as grid size and shape) of the stainless steel mesh can be flexibly chosen, and markers can be added at any desired location. Multiple marker areas can also be freely set within the same culture dish, increasing experimental flexibility and sample size. This method avoids the use of highly toxic chemicals such as hydrofluoric acid, reducing operational and management risks.

[0040] 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.

[0041] 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 method for labeling culture dishes for cell photoelectric correlation experiments, characterized in that, Includes the following steps: S1. Place a stainless steel mesh with a marker pattern in the target area at the bottom of the petri dish, and use the stainless steel mesh as a mask template. S2. Place the culture dish containing the mask template into a metal ion sputtering instrument, use platinum as the sputtering target, start the metal ion sputtering instrument to perform ion sputtering, so as to form a marker composed of platinum coating at the bottom of the culture dish. S3. Remove the culture dish and the mask template to obtain a marker-bearing culture dish for cell photoelectric correlation experiments.

2. The petri dish labeling method as described in claim 1, characterized in that, The metal ion sputtering instrument is set to a working distance of 65 mm and a sputtering thickness of 4 nm.

3. The petri dish labeling method as described in claim 1, characterized in that, The culture dish is a polystyrene culture dish or a confocal glass-bottomed culture dish.

4. A marker-labeled culture dish prepared using any one of the culture dish labeling methods described in claims 1-3, characterized in that, The marker-bearing culture dish includes a culture dish body and a platinum marker layer formed on the bottom of the culture dish body; the thickness of the platinum marker layer is 4 nm ± 0.5 nm.

5. A method of using the marker-equipped petri dish as described in claim 4, characterized in that, Includes the following steps: (1) Sterilize the culture dish with marker by ultraviolet irradiation, then inoculate the target cells into the sterilized culture dish with marker and perform the pre-set experimental treatment; after the experimental treatment is completed, use an optical microscope to take bright field images and fluorescence information images of the cells in the culture dish with marker, and record the positional correspondence between the marker and the cells. (2) Add 2.5% glutaraldehyde solution to the culture dish with marker to chemically fix the cells in the culture dish, and then embed the cells in the culture dish in situ; (3) After embedding, perform separation operation according to the type of the culture dish body. If the culture dish body is a polystyrene culture dish, break the bottom of the culture dish to separate the resin embedding block carrying the platinum marker from the polystyrene material; if the culture dish body is a confocal glass bottom culture dish, put the culture dish into liquid nitrogen. After the temperature of the culture dish and the temperature of the liquid nitrogen are balanced, break off the PCR tube that is upside down in the marker area at the bottom of the dish to completely separate the resin embedding block carrying the platinum marker from the glass material in the PCR tube. (4) Based on the position of the platinum marker on the resin embedding block, combined with the correspondence between the marker and the cell position recorded in step (1), the target cell is located and ultrathin sectioning and electron microscopy imaging analysis are performed.

6. The method of use as described in claim 5, characterized in that, In step (2), the in situ embedding of cells in the culture dish includes the following operations performed sequentially: a) Buffer washing: The fixed cell samples were soaked and washed with phosphate buffer for 5 minutes each time, and the washing was repeated 3 times. b) First fixation with osmium tetroxide: Add 1% osmium tetroxide solution to the petri dish and place the petri dish in a refrigerator at 4°C for 1.5 h. c) First double-distilled water wash: Soak and wash the osmium tetroxide-fixed cell samples with double-distilled water for 3 minutes each time, and repeat the wash 5 times; d) Dithiomethylhydrazine reaction: Add 1% dithiomethylhydrazine solution to a petri dish and react for 20 min at room temperature and in the dark. e) Second double-distilled water rinse: Repeat the double-distilled water rinse operation in step c); f) Second fixation with osmium tetroxide: Add 1% osmium tetroxide solution to the petri dish and react in a refrigerator at 4°C for 40 min; g) Third double-distilled water rinse: Repeat the double-distilled water rinse operation in step c); h) Uranium acetate staining: Add 2% uranium acetate aqueous solution to the petri dish and stain overnight at 4°C. i) Fourth double-distilled water rinse: Repeat the double-distilled water rinse operation in step c); j) Dehydration and Resin Infiltration: After the fourth double-distilled water washing, the cell samples were subjected to the following operations in sequence: Cell samples were soaked sequentially in ethanol concentration gradients of 30%-50%-70%-90%, with a soaking time of 7 min for each concentration gradient; Cell samples were then dehydrated by soaking in 100% ethanol for 7 min each time, repeated 4 times; Cell samples were then soaked sequentially in a mixture of 100% ethanol and 812 resin at a volume ratio of 3:1, 1:1, and 1:3, with infiltration times of 1 h, 2 h, and 2 h, respectively; The mixture was removed, and pure 812 resin was added to soak the cell samples, incubating overnight; Then, fresh pure 812 resin was added, and incubation continued for 4 h; k) Resin embedding and polymerization: The embedding operation is performed according to the type of culture dish: If the culture dish is a polystyrene culture dish, remove the residual 812 resin in the culture dish and add 400 μL of pure 812 resin to the culture dish; if the culture dish is a confocal glass-bottom culture dish, take an open PCR tube, fill the tube with pure 812 resin, and invert it on the marker area at the bottom of the culture dish; after the operation is completed, put the culture dish in an oven at 70℃ to carry out the resin polymerization reaction for 24 h.

Images