Method suitable for visible light imaging and synchrotron radiation single cell imaging and application thereof

Abstract

The invention provides a method suitable for visible light imaging and synchrotron radiation single cell imaging and application thereof, and the method comprises the following steps: (S1) labeling subcellular structures and / or target protein molecules in cells by using a primary antibody or biotin, and then labeling the cells by using a peroxidase probe material, the material of the peroxidase probe is selected from secondary antibody-peroxidase or streptavidin-peroxidase labeled cells; (S2) adding a working solution containing polymer monomer molecules and hydrogen peroxide into the cells marked in the step (S1), and reacting to generate a polymer; and (S3) after the reaction is stopped, carrying out visible light imaging and synchrotron radiation single cell imaging. According to the imaging method, visible light microscope observation and X-ray signal enhancement of marker sites can be realized at the same time, and three-dimensional high-resolution structure information of a subcellular level cell structure and three-dimensional high-resolution positioning information of biomacromolecules are provided.

Description

Technical Field

[0001] The present invention belongs to the field of biological imaging, and in particular relates to a method suitable for visible light imaging and synchrotron radiation single-cell imaging and its application. Background Art

[0002] As the most direct means of obtaining information about cell life activities, imaging is widely used in biological and medical research. With the continuous deepening of biological and medical research, researchers hope to obtain biological activity images at higher resolutions to achieve a more refined understanding of biological life processes.

[0003] Using optical or electron microscopes to image biological samples, we can obtain microscopic images of biological cells and tissues, thereby understanding the structure of biological tissues and clarifying various physiological functions of organisms. With the advancement of technology, more and more biological imaging methods have been developed, and biological imaging technology has become an indispensable method in cell biology research.

[0004] Bioimaging technology refers to the technical means of imaging the internal structure, function, metabolism, etc. of organisms in a non-invasive way. Current bioimaging technologies include: optical imaging, fluorescence imaging, magnetic resonance imaging, microscopic imaging, molecular imaging, photoacoustic imaging and ultrasonic imaging.

[0005] Traditional fluorescence microscopes have difficulty achieving resolutions below 200nm due to the optical diffraction limit of visible light. Synchrotron X-ray imaging technology relies on X-rays, which have shorter wavelengths and can achieve higher levels of resolution. However, due to the strong penetrating power of X-rays, low-contrast cell structures are difficult to identify, and different proteins with similar contrasts are difficult to distinguish. At the same time, due to the tight time of synchrotron radiation, there is an urgent need for research methods to pre-check imaging samples by conventional means. Therefore, it is very important to develop a probe labeling system that is easy to pre-check by conventional means and has good synchrotron X-ray signal intensity and labeling ability. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a method and application thereof suitable for visible light imaging and synchrotron radiation single-cell imaging. The imaging method of the present invention can enhance the X-ray signal of the labeling site, provide three-dimensional high-resolution structural information of the cell structure at the subcellular level and three-dimensional high-resolution positioning information of biomacromolecules, and also has visible light imaging signals, and can directly observe the labeling effect through a visible light imaging microscope, assist in synchrotron radiation pre-experiments, ensure the labeling effect of synchrotron radiation experiments, facilitate synchrotron radiation sample screening, make full use of scarce synchrotron radiation resources, and save experimental time costs and economic costs. In addition, this probe can be applied to a variety of protein markers and has a wide range of applicability.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method suitable for visible light imaging and synchrotron radiation single cell imaging, the labeling method comprising:

[0009] (S1) using a primary antibody or biotin to label intracellular subcellular structures and / or target protein molecules, and then using a peroxidase probe material to label cells, wherein the peroxidase probe material is selected from a secondary antibody-peroxidase or streptavidin-peroxidase to label cells;

[0010] (S2) adding a working solution containing polymer monomer molecules and hydrogen peroxide to the cells labeled in (S1) to generate polymers after reaction;

[0011] (S3) After terminating the reaction, visible light imaging and synchrotron radiation single-cell imaging were performed.

[0012] In the present invention, the primary antibody or biotin labeling plays a role in targeted recognition of target subcellular structures and targeted recognition of target biological molecules.

[0013] The present invention uses a polymer probe system to mark cells. The polymer probe has a high electron density and has stronger X-ray absorption than the cell's own structure. It can achieve X-ray signal enhancement at the marking site, and provide three-dimensional high-resolution structural information of subcellular level cell structure and three-dimensional high-resolution positioning information of biomacromolecules. At the same time, the polymer probe system will also produce visible light absorption signals, and the labeling effect can be directly observed by a visible light microscope. Therefore, it is possible to directly observe by a visible light microscope first, obtain pre-experimental results, screen samples that can be subsequently subjected to synchrotron radiation, and exclude samples that are not suitable for synchrotron radiation detection, so that scarce synchrotron radiation resources can be fully utilized, saving experimental time costs and economic costs.

[0014] Preferably, in (S1), the subcellular structure includes any one of mitochondria, endoplasmic reticulum, nucleus or nucleolus, or a combination of at least two thereof.

[0015] In the present invention, the target protein is labeled with a primary antibody of a characteristic protein of the target structure, such as TOMM20 for labeling mitochondria, Calnexin for labeling endoplasmic reticulum, and Ki67 for labeling nuclei and nucleoli. The labeling concentration varies according to the antibody manufacturer. For example, in the present invention, TOMM20 is diluted 1:200, Calnexin is diluted 1:200, and Ki67 is diluted 1:20. The diluent is a 30 mg / mL BSA solution dispersed in PBS, and the labeling condition is one hour at room temperature.

[0016] Preferably, the target protein molecule includes any one of the interferon-stimulated molecule STING, TOMM20 protein or calnexin protein Calnexin, or a combination of at least two of them.

[0017] In the present invention, when the primary antibody is used to label the target protein, the primary antibody of the interferon-stimulated molecule STING, TOMM20 protein or calcium-binding protein Calnexin is diluted 1:200, the diluent is a BSA solution with a concentration of 30 mg / mL dispersed in PBS, and the labeling condition is one hour at room temperature.

[0018] Preferably, the secondary antibody specifically binds to the primary antibody.

[0019] Preferably, in (S2), the polymer monomer molecule is diaminobenzidine or its hydrochloride (biphenyl-3,3',4,4'-tetramine tetrahydrochloride hydrate).

[0020] Preferably, the concentration of polymer monomer molecules in the working solution is 0.2-1 g / L, for example, it can be 0.2 g / L, 0.3 g / L, 0.4 g / L, 0.5 g / L, 0.6 g / L, 0.7 g / L, 0.8 g / L, 0.9 g / L or 1 g / L.

[0021] In the present invention, diaminobenzidine hydrochloride is used as the polymer monomer due to its poor solubility. Too low a concentration of the polymer monomer solution results in too low a labeling efficiency, while too high a concentration results in nonspecific labeling; within this range, the labeling rate is appropriate and the labeling specificity is good.

[0022] Preferably, in (S2), the concentration of hydrogen peroxide in the working solution is 5-15 mM, for example, 5 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM or 15 mM.

[0023] In the present invention, too low a concentration of hydrogen peroxide results in too low a labeling efficiency, while too high a concentration results in nonspecific labeling; within this range, the labeling rate is appropriate and the labeling specificity is good.

[0024] Preferably, in (S2), the working solution further comprises a buffer salt, and the buffer salt is selected from PBS (phosphate saline) and / or TBS (tris(hydroxymethyl)aminomethane-hydrochloride).

[0025] In the present invention, the working solution contains PBS buffer salt with a concentration of 136-138mM NaCl, 2.5-2.9mM KCl, 7-9mM Na2HPO4 and 1-2mM KH2PO4; preferably 137mM NaCl, 2.7mM KCl, 8mM Na2HPO4 and 1.5mM KH2PO4.

[0026] In the present invention, the working solution contains TBS buffer salt with a concentration of 136-138 mM NaCl, 2.5-2.9 mM KCl and 22-27 mM Tris; preferably 137 mM NaCl, 2.7 mM KCl and 25 mM Tris.

[0027] In the present invention, the polymer monomer, i.e., diaminobenzidine hydrochloride aqueous solution, is acidic, and the use of a buffered salt solution can stabilize the pH of the solution and prevent the acidic environment from damaging the cell structure. At the same time, a buffer concentration lower or higher than this concentration will cause the cells to swell or shrink.

[0028] Preferably, in (S2), the reaction temperature is 4-40°C, for example, 4°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C or 40°C, and the reaction time is 15-60 minutes, for example, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60, etc.

[0029] In the present invention, temperature affects the labeling rate and specificity. A temperature below 4°C results in too slow labeling efficiency, and a temperature above 40°C results in reduced labeling specificity. Time affects the labeling signal intensity. If the labeling time is less than 15 minutes, the synchrotron radiation X-ray imaging effect is poor and it is difficult to achieve the labeling function. If the labeling time is greater than 60 minutes, on the one hand, it leads to nonspecific labeling, and on the other hand, the signal is too strong, which makes it difficult for the synchrotron radiation X-ray to transmit imaging.

[0030] In the present invention, the working solution is removed to terminate the reaction, and the sample is washed three times with a phosphate buffer solution and then gradiently dehydrated and dried for synchrotron radiation X-ray imaging.

[0031] In the present invention, the step of gradient dehydration comprises: soaking the cells in aqueous solutions of ethanol with volume fractions of 30%, 50%, 70%, 80%, 90% and 100% in sequence for 5 minutes, and then drying naturally.

[0032] In the present invention, the gradient dehydration step is an optional step when performing visible light imaging; when performing synchrotron radiation imaging, gradient dehydration must be performed to avoid radiation damage during the imaging process. These gradients are selected to maintain cell morphology during the gradient dehydration process.

[0033] In a second aspect, the present invention provides application of the method suitable for visible light imaging and synchrotron radiation single-cell imaging described in the first aspect in cell imaging.

[0034] In the present invention, the method is suitable for labeling target structures or biological molecules in synchrotron radiation imaging, and the labeling effect can be verified by visible light imaging.

[0035] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The present invention provides good X-ray contrast as well as clear visible light imaging signals, can simplify synchrotron radiation pre-experiments, save precious synchrotron radiation machine time, and save experimental costs. At the same time, it has good imaging effects on subcellular structures and key biological molecules, expanding the application scope of synchrotron radiation imaging technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a synchrotron radiation X-ray imaging image of the probe prepared in Example 1 of the present invention.

[0039] Figure 2 This is a visible light microscope image of the mitochondrial outer membrane TOMM20 protein labeled in monkey kidney epithelial cells COS-7 according to Example 2 of the present invention.

[0040] Figure 3 Example 3: Visible light microscope image of Ki67 protein, a proliferation marker, labeling human osteosarcoma cells U2OS.

[0041] Figure 4 Example 4 Synchrotron radiation X-ray imaging image of the mitochondrial outer membrane TOMM20 protein labeled in human osteosarcoma cells U2OS.

[0042] Figure 5 Example 5 Synchrotron radiation X-ray imaging image of Calnexin protein labeled in monkey kidney epithelial cells COS-7.

[0043] Figure 6 Example 6 Synchrotron radiation X-ray imaging image of STING protein labeled in monkey kidney epithelial cells COS-7. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0045] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0046] The sources of the experimental materials in the following examples are as follows:

[0047] TOMM20 antibody: Novus, NBP2-67501.

[0048] Secondary antibody-horseradish peroxidase: Bioss, bs-0295G-HRP.

[0049] Biotinylated proliferative marker Ki67 antibody: Novus, NB500-170B.

[0050] Streptavidin-horseradish peroxidase: Aladdin, np156148.

[0051] Biotinylated STING protein antibody: Novus, NBP2-24683B.

[0052] Example 1

[0053] Extracellular peroxidase labeling

[0054] A) Dissolve 1 mg of streptavidin-horseradish peroxidase in 1 mL of deionized water.

[0055] B) Mix diaminobenzidine hydrochloride buffer and hydrogen peroxide buffer in equal proportions, which is called working solution. Diaminobenzidine hydrochloride buffer is prepared using solid and TBS (TBS buffer), and hydrogen peroxide buffer is prepared using 30% hydrogen peroxide and TBS. The final concentration of diaminobenzidine hydrochloride is 0.5 g / L, and the final concentration of hydrogen peroxide is 10 mM.

[0056] C) Mix the solution in step A and the working solution in equal proportions and react at room temperature for 15 minutes.

[0057] D) The precipitate was collected by centrifugation, washed with deionized water, and redispersed in deionized water for synchrotron radiation imaging.

[0058] See also Figure 1 , Figure 1 This is the synchrotron X-ray imaging image of the probe. Figure 1 It can be seen that the probe prepared in the example of the present invention has good X-ray contrast.

[0059] Example 2

[0060] Visible light imaging of mitochondrial outer membrane TOMM20 protein labeled in monkey kidney epithelial cells COS-7

[0061] A) Monkey kidney epithelial cells COS-7 were fixed with 4% paraformaldehyde for 15 minutes, labeled with mitochondrial outer membrane TOMM20 antibody, and then labeled with secondary antibody-horseradish peroxidase.

[0062] B) Mix diaminobenzidine hydrochloride buffer and hydrogen peroxide buffer in equal proportions, which is called working solution. Diaminobenzidine hydrochloride buffer is prepared using solid and PBS, and hydrogen peroxide buffer is prepared using 30% hydrogen peroxide and PBS. The final concentration of diaminobenzidine hydrochloride is 0.4 g / L, and the final concentration of hydrogen peroxide is 10 mM.

[0063] C) Add the working solution to the cells in step A and incubate at room temperature for 15 minutes.

[0064] D) Remove the working solution and wash three times with phosphate buffered saline for visible light imaging.

[0065] See also Figure 2 , Figure 2 This is the visible light microscope imaging image of this example, and the visible light imaging signal of the mitochondrial region is enhanced. Figure 2 It can be seen that the probe prepared in the example of the present invention has good visible light imaging contrast and achieves good labeling of the mitochondrial outer membrane TOMM20 protein.

[0066] Example 3

[0067] Visible light imaging of Ki67 protein, a proliferation marker, in human osteosarcoma U2OS cells

[0068] A) Human osteosarcoma U2OS cells were fixed with 4% paraformaldehyde for 15 minutes, labeled with biotinylated proliferation marker Ki67 antibody, and then labeled with streptavidin-horseradish peroxidase.

[0069] B) Mix diaminobenzidine hydrochloride buffer and hydrogen peroxide buffer in equal proportions, which is called working solution. Diaminobenzidine hydrochloride buffer is prepared using solid and PBS, and hydrogen peroxide buffer is prepared using 30% hydrogen peroxide and PBS. The final concentration of diaminobenzidine hydrochloride is 0.4 g / L, and the final concentration of hydrogen peroxide is 10 mM.

[0070] C) Add the working solution to the cells in step A and incubate at room temperature for 30 minutes.

[0071] D) Remove the working solution and wash three times with phosphate buffered saline for visible light imaging.

[0072] See also Figure 3 , Figure 3 This is the visible light microscope imaging image of this example, and the visible light imaging signal of the cell nucleus and nucleolus area is enhanced. Figure 3 It can be seen that the probe prepared in the example of the present invention has good visible light imaging contrast, realizes signal enhancement imaging of cell nuclei and nucleoli, and realizes good labeling of Ki67 protein, a proliferation marker accumulated in the cell nuclear region.

[0073] Example 4

[0074] Synchrotron Radiation Imaging of the Mitochondrial Outer Membrane TOMM20 Protein Labeled in Human Osteosarcoma Cells U2OS

[0075] A) Human osteosarcoma U2OS cells were fixed with 4% paraformaldehyde for 15 minutes, labeled with mitochondrial outer membrane TOMM20 antibody, and then labeled with secondary antibody-horseradish peroxidase.

[0076] B) Mix diaminobenzidine hydrochloride buffer and hydrogen peroxide buffer in equal proportions, which is called working solution. Diaminobenzidine hydrochloride buffer is prepared using solid and PBS, and hydrogen peroxide buffer is prepared using 30% hydrogen peroxide and PBS. The final concentration of diaminobenzidine hydrochloride is 0.5 g / L, and the final concentration of hydrogen peroxide is 10 mM.

[0077] C) Add the working solution to the cells in step A and incubate at room temperature for 30 minutes.

[0078] D) Remove the working solution and wash three times with phosphate buffered saline.

[0079] E) The cell sample in step D was immersed in 30%, 50%, 70%, 80%, 90%, and 100% ethanol aqueous solutions in turn for 5 minutes, and then dried naturally for synchrotron radiation X-ray imaging.

[0080] See also Figure 4 , Figure 4 This is the synchrotron radiation X-ray imaging of this example. The structure of the mitochondrial outer membrane is clear. Figure 4 It can be seen that the probe prepared in the example of the present invention has good synchrotron radiation X-ray contrast and specificity, and achieves specific labeling of mitochondrial outer membrane TOMM20 protein.

[0081] Example 5

[0082] Synchrotron Radiation Imaging of Endoplasmic Reticulum Labeled with COS-7 in Monkey Kidney Epithelial Cells

[0083] A) Monkey kidney epithelial cells COS-7 were fixed with 4% paraformaldehyde for 15 minutes, labeled with endoplasmic reticulum localized Calnexin antibody, and then labeled with secondary antibody-horseradish peroxidase.

[0084] B) Mix diaminobenzidine hydrochloride buffer and hydrogen peroxide buffer in equal proportions, which is called working solution. Diaminobenzidine hydrochloride buffer is prepared using solid and PBS, and hydrogen peroxide buffer is prepared using 30% hydrogen peroxide and PBS. The final concentration of diaminobenzidine hydrochloride is 0.5 g / L, and the final concentration of hydrogen peroxide is 10 mM.

[0085] C) Add the working solution to the cells in step A and incubate at room temperature for 30 minutes.

[0086] D) Remove the working solution and wash three times with phosphate buffered saline.

[0087] E) The cell sample in step D was immersed in 30%, 50%, 70%, 80%, 90%, and 100% ethanol aqueous solutions in turn for 5 minutes, and then dried naturally for synchrotron radiation X-ray imaging.

[0088] See also Figure 5 , Figure 5 This is the synchrotron radiation X-ray imaging of this example, the endoplasmic reticulum structure is clear. Figure 5 It can be seen that the probe prepared in the example of the present invention has good synchrotron radiation X-ray contrast and specificity, and achieves specific labeling of the endoplasmic reticulum.

[0089] Example 6

[0090] Synchrotron radiation imaging of STING protein labeled in monkey kidney epithelial cells COS-7

[0091] A) Monkey kidney epithelial cells COS-7 were fixed with 4% paraformaldehyde for 15 minutes, labeled with biotinylated STING protein antibody, and then labeled with streptavidin-horseradish peroxidase.

[0092] B) Mix diaminobenzidine hydrochloride buffer and hydrogen peroxide buffer in equal proportions, which is called working solution. Diaminobenzidine hydrochloride buffer is prepared using solid and PBS, and hydrogen peroxide buffer is prepared using 30% hydrogen peroxide and PBS. The final concentration of diaminobenzidine hydrochloride is 0.4 g / L, and the final concentration of hydrogen peroxide is 10 mM.

[0093] C) Add the working solution to the cells in step A and incubate at room temperature for 60 minutes.

[0094] D) Remove the working solution and wash three times with phosphate buffered saline.

[0095] E) The cell sample in step D was immersed in 30%, 50%, 70%, 80%, 90%, and 100% ethanol aqueous solutions in turn for 5 minutes, and then dried naturally for synchrotron radiation X-ray imaging.

[0096] See also Figure 6 , Figure 6 This is the synchrotron radiation X-ray imaging of this example, the endoplasmic reticulum structure is clear. Figure 6 It can be seen that the probe prepared in the example of the present invention has good synchrotron radiation X-ray contrast and specificity, and achieves specific labeling of the endoplasmic reticulum.

[0097] Example 7

[0098] This example performs extracellular peroxidase labeling, and the only difference from Example 1 is that in step B), the concentration of polymer monomer molecules in the working solution is 0.05 g / L.

[0099] Example 8

[0100] This example performs extracellular peroxidase labeling, and the only difference from Example 1 is that in step B), the concentration of polymer monomer molecules in the working solution is 2 g / L.

[0101] Example 9

[0102] This example performs extracellular peroxidase labeling, and the only difference from Example 1 is that in step B), the concentration of hydrogen peroxide in the working solution is 3 mM.

[0103] Example 10

[0104] This example performs extracellular peroxidase labeling, and the only difference from Example 1 is that in step B), the concentration of hydrogen peroxide in the working solution is 20 mM.

[0105] Embodiment 11

[0106] This example performs visible light imaging of the mitochondrial outer membrane TOMM20 protein labeled in monkey kidney epithelial cells COS-7. The only difference from Example 2 is that in step C), the reaction temperature is 4° C. and the reaction time is 80 minutes.

[0107] Example 12

[0108] This example performs visible light imaging of the mitochondrial outer membrane TOMM20 protein labeled in monkey kidney epithelial cells COS-7. The only difference from Example 2 is that in step C), the reaction temperature is 50° C. and the reaction time is 10 minutes.

[0109] The effects of different labeling methods were investigated in Examples 7-12, among which the imaging effects of the methods of Examples 7-12 were relatively poor. The labeling efficiency corresponding to the low concentration of the polymer monomer in Example 7 was too low, and the concentration in Example 8 was too high, resulting in nonspecific labeling. The low concentration of hydrogen peroxide in Example 9 resulted in too low labeling efficiency, and the high concentration in Example 10 resulted in nonspecific labeling. The labeling time was investigated in Examples 11 and 12. A short labeling time resulted in a low labeling density, and a long labeling time resulted in a strong nonspecific labeling. Therefore, only by controlling the amount of each component in the working solution and the labeling time within an appropriate range can a good imaging effect be achieved.

[0110] In summary, the present invention provides an imaging method that can enhance the X-ray signal of the labeling site, provide three-dimensional high-resolution structural information of the subcellular level cell structure and three-dimensional high-resolution positioning information of biological macromolecules. The above method can save experimental time and economic costs, is applicable to a variety of protein labels, and has wide applicability.

[0111] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method suitable for visible light imaging and synchrotron radiation single-cell imaging, characterized in that: The method comprises: (S1) using a primary antibody or biotin to label intracellular subcellular structures and / or target protein molecules, and then using a peroxidase probe material to label cells, wherein the peroxidase probe material is selected from a secondary antibody-peroxidase or streptavidin-peroxidase to label cells; (S2) adding a working solution containing polymer monomer molecules and hydrogen peroxide to the cells labeled in (S1) to generate polymers after reaction; (S3) After terminating the reaction, visible light imaging and synchrotron radiation single-cell imaging were performed.

2. The method for visible light imaging and synchrotron radiation single-cell imaging according to claim 1, characterized in that: In (S1), the subcellular structure includes any one of mitochondria, endoplasmic reticulum, nucleus or nucleolus, or a combination of at least two of them.

3. The method for visible light imaging and synchrotron radiation single-cell imaging according to claim 1 or 2, characterized in that: In (S1), the target protein molecule includes any one of the interferon-stimulated molecule STING, TOMM20 protein or calcium-binding protein Calnexin, or a combination of at least two of them.

4. The method for visible light imaging and synchrotron radiation single cell imaging according to any one of claims 1 to 3, characterized in that: In (S1), the secondary antibody specifically binds to the primary antibody.

5. The method for visible light imaging and synchrotron radiation single cell imaging according to any one of claims 1 to 4, characterized in that: In (S2), the polymer monomer molecule is diaminobenzidine or its hydrochloride.

6. The method for visible light imaging and synchrotron radiation single cell imaging according to any one of claims 1 to 5, characterized in that: In (S2), the concentration of polymer monomer molecules in the working solution is 0.2-1 g / L.

7. The method for visible light imaging and synchrotron radiation single cell imaging according to any one of claims 1 to 6, characterized in that: In (S2), the concentration of hydrogen peroxide in the working solution is 5-15 mM.

8. The method for visible light imaging and synchrotron radiation single cell imaging according to any one of claims 1 to 7, characterized in that: In (S2), the working solution further includes a buffer salt, and the buffer salt is selected from PBS and / or TBS.

9. The method for visible light imaging and synchrotron radiation single cell imaging according to any one of claims 1 to 8, characterized in that: In (S2), the reaction temperature is 4-40°C and the reaction time is 15-60 minutes.

10. Use of the method suitable for visible light imaging and synchrotron radiation single-cell imaging according to any one of claims 1 to 9 in cell imaging.

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