Bismuth citrate dyeing solution and preparation method thereof, dyeing composition and application thereof

Abstract

The application belongs to the technical field of electron microscope detection, and relates to a staining agent for electron microscope detection, and relates to a bismuth citrate staining solution, a preparation method thereof, a staining composition and application thereof. Bismuth citrate is added into an aqueous citric acid solution and uniformly mixed to form a white milky suspension; an alkaline solution is added into the white milky suspension until the white milky suspension becomes a clear and transparent solution. The application completely replaces a traditional lead citrate staining solution with a bismuth citrate system for the first time, and the most significant advantage thereof is that the staining solution is stable in property, and the problem of reaction with carbon dioxide in air to generate carbonate precipitate is completely avoided, so that pollution in an electron microscope staining process can be eradicated from the source. The staining effect of the staining solution is equivalent to that of the traditional lead citrate, and the operation is more simple, without any CO2 prevention device, so that the sample preparation efficiency and success rate are greatly improved, and the application has great popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of electron microscopy detection technology, and relates to staining agents for electron microscopy detection, specifically a bismuth citrate staining solution, its preparation method, staining composition, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In biological sample observation using transmission electron microscopy (TEM), staining ultrathin sections (typically 30–300 nm thick, but can be further categorized into 30–70 nm, 70–100 nm, 100–300 nm, etc., depending on the detection requirements) is a crucial step. The purpose is to increase the sample's ability to receive scattered electrons from different angles by specifically binding heavy metal ions to cellular components, thereby improving image contrast and resolution. Currently, the most widely used staining agent is lead citrate (such as Reynolds lead citrate), which has excellent staining effects on cell membranes, nucleic acids, and other structures.

[0004] However, lead citrate readily reacts with carbon dioxide (CO2) in the air to form insoluble lead carbonate precipitates. These precipitates deposit on the sample surface as fine, disordered particles, severely contaminating the sample, interfering with electron microscopy observation and image interpretation, and even leading to erroneous biological conclusions. This problem has plagued the field of electron microscopy for decades due to staining contamination. Currently, two main approaches are typically used to overcome this problem. The first approach mainly involves improvements to the staining apparatus, including the design and use of various complex isolation devices, such as using carbon dioxide absorbents (e.g., soda lime) during staining, creating a CO2-free environment in the petri dish (e.g., adding sodium hydroxide solution), or using more airtight staining boxes. However, these devices are complex in structure, cumbersome to operate, and expensive, and cannot completely eliminate contamination, severely reducing the efficiency of electron microscopy sample preparation. The second approach mainly involves optimizing the operating procedure, such as using paraffin mounting and strictly controlling the operation time, but these methods have limited effectiveness and are highly dependent on the operator's experience. Therefore, a new type of staining agent is needed that can fundamentally solve the carbon dioxide contamination problem while maintaining or even improving the staining effect. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a bismuth citrate staining solution, its preparation method, staining composition, and its application. The bismuth citrate staining solution provided by the present invention is stable, does not react with carbon dioxide in the air, and can prevent carbonate precipitation pollution at the source. Furthermore, its preparation method is simple, rapid, and easy to standardize.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: Firstly, a method for preparing a bismuth citrate staining solution includes the following steps: Bismuth citrate was added to an aqueous solution of citric acid and mixed thoroughly to form a white emulsion suspension. Add an alkaline solution to the white milky suspension until the white milky suspension becomes a clear and transparent solution.

[0007] The bismuth citrate staining solution provided by this invention is mainly used to replace lead citrate staining agent. Choosing bismuth citrate as the bismuth source avoids the introduction of other anions, which could affect the electron microscopy results. Adding bismuth citrate to the citric acid aqueous solution first, followed by the addition of an alkaline solution, helps control the alkalinity of the solution system and avoids excessive alkalinity affecting electron microscopy detection.

[0008] Secondly, a bismuth citrate staining solution is obtained by the preparation method described in the first aspect of the present invention.

[0009] This invention is the first to completely replace traditional lead citrate staining solution with a bismuth citrate system. Its most significant advantage lies in the stability of the staining solution, completely avoiding the problem of carbonate precipitation from reaction with carbon dioxide in the air, thus eliminating contamination at the source during electron microscopy staining. The staining effect of this solution is comparable to that of traditional lead citrate, and the operation is much simpler, requiring no CO2 protection devices, greatly improving sample preparation efficiency and success rate, and possessing significant value for widespread application.

[0010] Thirdly, a staining composition comprising the bismuth citrate staining solution and uranyl acetate as described in the second aspect of the present invention.

[0011] Fourthly, the use of a bismuth citrate staining solution as described in the second aspect of the present invention or a staining composition as described in the third aspect of the present invention in the preparation of staining agents for detecting biological samples under an electron microscope.

[0012] Fifthly, a staining method for biological samples used in electron microscopy includes the following steps: Provides the bismuth citrate staining solution as described in the second aspect of the present invention; The target biological sample was stained in the bismuth citrate staining solution.

[0013] The beneficial effects of this invention are as follows: 1. The bismuth citrate staining solution provided by this invention can eliminate contamination. This invention is the first to propose using bismuth citrate staining solution to replace lead citrate staining agent. The heavy metal element in the bismuth citrate staining solution is bismuth, which is stable, and its complexes do not react with carbon dioxide in the air, thus not forming insoluble carbonate precipitates. This fundamentally solves the staining contamination problem that has plagued the field of electron microscopy for decades.

[0014] 2. The bismuth citrate staining solution provided by this invention exhibits excellent staining effects. Experimental verification shows that the bismuth citrate staining solution provided by this invention has a staining effect on ultrastructures such as cell membranes, chromatin, and ribosomes that is comparable to traditional lead citrate staining agents in terms of contrast and resolution, and is even clearer on some structures.

[0015] 3. The bismuth citrate staining solution provided by this invention is simple to operate and highly efficient during the staining process. When using the bismuth citrate staining solution provided by this invention, no complex CO2 protection devices or cumbersome isolation procedures are required. Staining can be performed directly in a conventional experimental environment, greatly simplifying the process, reducing the technical requirements for operators, and significantly improving the efficiency and success rate of electron microscopy sample preparation.

[0016] 4. The bismuth citrate staining solution provided by this invention is safe and environmentally friendly. Bismuth is a low-toxicity metal, and its toxicity and environmental harm are far lower than that of lead. The bismuth citrate staining solution provided by this invention helps reduce the use of toxic lead reagents in laboratories, and is more in line with the green and safe concepts of modern laboratories. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0018] Figure 1 This is a schematic diagram of the appearance of the bismuth citrate staining solution prepared in Example 1 of the present invention; Figure 2 This is an electron micrograph of an ultrathin section of kidney tissue stained with lead citrate staining solution, as shown in Comparative Example 1 of this invention. Figure 3 This is an electron micrograph of an ultrathin section of kidney tissue stained with the bismuth citrate staining solution prepared in Example 1 of the present invention. Figure 4 This is an electron micrograph of an ultrathin section of kidney tissue stained with the bismuth citrate staining solution prepared in Example 2 of the present invention. Figure 5 This is an electron micrograph of an ultrathin section of HEK293 cells stained with bismuth citrate staining solution prepared in Example 1 in Experimental Example 3 of the present invention. Figure 6This is an electron micrograph of an ultrathin section of HEK293 cells stained with bismuth citrate staining solution prepared in Example 3 of this invention, as shown in Experimental Example 4 of this invention. Figure 7 This is an electron micrograph of an ultrathin section of heart tissue stained with the bismuth citrate staining solution prepared in Example 1 in Experimental Example 5 of the present invention.

[0019] Figure 8 The images shown are electron micrographs of ultrathin sections of rat kidney tissue stained with bismuth citrate staining solution prepared in Example 1 of this invention after being refrigerated for different times in Experiment Example 6 of this invention; A is a control image of freshly prepared staining solution, B is a staining image after being stored at 4℃ for 1 week, C is a staining image after being stored at 4℃ for 2 weeks, and D is a staining image after being stored at 4℃ for 4 weeks. Figure 9 The images shown are electron micrographs of HEK293 cell ultrathin sections stained with the bismuth citrate staining solution prepared in Example 1 of this invention after being left exposed for different times in Experiment 7 of this invention; A is a control image of freshly prepared staining solution, B is a staining image after being left exposed for 1 hour, C is a staining image after being left exposed for 4 hours, and D is a staining image after being left exposed for 24 hours. Detailed Implementation

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0021] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0022] In view of the problem that lead citrate staining agent can cause precipitation and interfere with the detection results during electron microscopy detection of stained organisms, this invention proposes a bismuth citrate staining solution, its preparation method, staining composition and its application.

[0023] A typical embodiment of the present invention provides a method for preparing a bismuth citrate staining solution, comprising the following steps: Bismuth citrate was added to an aqueous solution of citric acid and mixed thoroughly to form a white emulsion suspension. Add an alkaline solution to the white milky suspension until the white milky suspension becomes a clear and transparent solution.

[0024] In some embodiments, the citric acid concentration in the aqueous citric acid solution is 3-7% by mass. Firstly, under these conditions, bismuth citrate is ensured to dissolve sufficiently and maintain complexation stability. Citric acid acts as a ligand in this system, forming a stable, soluble complex with trivalent bismuth ions. If the concentration is too low, insufficient ligands are available, failing to completely complex bismuth ions and easily leading to the precipitation of bismuth hydroxide; if the concentration is too high, the solution viscosity becomes too high, inhibiting the complexation reaction kinetics and easily causing local pH abrupt changes during subsequent addition of alkaline solution, inducing irreversible precipitation. Secondly, these conditions match the ionic strength requirements for electron microscopy staining. This concentration range, in conjunction with the subsequently added alkaline solution (0.5-2M NaOH / KOH), stabilizes the pH of the final staining solution within the weakly alkaline range (~pH 12), ensuring that the heavy metal staining agent is within the effective range for contrast enhancement while avoiding damage to the ultrastructure of biological samples from excessively high pH levels.

[0025] In some embodiments, the ratio of bismuth citrate to citric acid aqueous solution is 0.05~0.15: 3~7, g / mL. Firstly, under these conditions, the concentration of heavy metal ions can be precisely controlled, balancing staining contrast and background noise. If the amount of bismuth citrate added is too small, the bismuth ion concentration is insufficient, resulting in inadequate electron staining density for structures such as cell membranes and chromatin, leading to poor image contrast. If the amount of bismuth citrate added is too large, there is an excess of free bismuth ions, which easily adsorb non-specifically onto the sample background, causing an "overall blackening" and obscuring fine structures. Furthermore, uncomplexed bismuth ions are easily hydrolyzed slowly upon contact with air, introducing new precipitation risks. Simultaneously, under these conditions, the molar ratio of citric acid to bismuth can be maintained at a complexation ratio of 2:1 to 3:1, ensuring the formation of the bismuth citrate complex anion (e.g., [Bi(C6H5O7)2)). 3- First, the structure is uniform and long-term stable. Second, when the volume of the citric acid aqueous solution is 3-7 mL, this volume corresponds to a 10 mL glass bottle reaction vessel. This ensures sufficient liquid phase space during shaking and mixing of the suspension, while avoiding liquid overflow when adjusting the pH with alkali. This conforms to routine laboratory operating practices and eliminates the need for custom-made special vessels. If the volume is too small (<3 mL), the solute concentration will be too high, the solubility will approach its limit, and the batch-to-batch reproducibility will be poor. If the volume is too large (>7 mL), it will dilute the effective concentration of the final staining solution and increase the reagent consumption and waste liquid disposal volume during subsequent staining. In addition, this volume of staining solution can prepare approximately 5-7 mL of staining solution per batch, which is just enough to meet the staining needs of dozens of ultrathin sections (each section only requires 10-20 μL of droplets), avoiding contamination or failure caused by repeated opening of storage bottles. Therefore, under these conditions, the effective staining concentration can be balanced, ensuring operational feasibility and batch stability.

[0026] In some embodiments, the alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution. Specifically, the concentration of the alkaline solution is 0.5~2 M. The unit M in this invention represents mol / L.

[0027] Another embodiment of the present invention provides a bismuth citrate staining solution, obtained by the above preparation method.

[0028] A third embodiment of the present invention provides a staining composition comprising the above-mentioned bismuth citrate staining solution and uranyl acetate.

[0029] A fourth embodiment of the present invention provides the application of the above-mentioned bismuth citrate staining solution or staining composition in the preparation of staining agents for electron microscopy detection of biological samples.

[0030] A fifth embodiment of the present invention provides a staining method for biological samples for electron microscopy, comprising the following steps: Provide the above-mentioned bismuth citrate staining solution; The target biological sample was stained in the bismuth citrate staining solution.

[0031] In some embodiments, staining with bismuth citrate staining solution is performed in an environment where carbon dioxide is not isolated.

[0032] In some embodiments, the staining method is a double staining method, which involves first staining with uranyl acetate and then staining with bismuth citrate.

[0033] In some embodiments, the target biological sample is an ultrathin section of biological tissue or an ultrathin section of cells. The thickness of the ultrathin section of the present invention is 30~300 nm, wherein, depending on the sample and observation (or detection) requirements, the thickness can be 30~70 nm, 70~100 nm, or 100~300 nm.

[0034] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0035] Example 1 A bismuth citrate staining solution is prepared as follows: (1) Measure 5 mL of 5% citric acid aqueous solution using a graduated cylinder and place it in a 10 mL clean glass bottle.

[0036] (2) Weigh 0.1g of bismuth citrate powder accurately using an analytical balance and add it to the citric acid solution from step (1). Tighten the cap and shake the solution using a vortex mixer for about 2 minutes. A uniform white milky suspension will be formed.

[0037] (3) Using a pipette, take 1M sodium hydroxide aqueous solution and add it dropwise to the emulsion suspension from step (2). After each drop, tighten the cap and shake well. As sodium hydroxide is added, the solution gradually changes from milky white to translucent, and finally becomes a clear and transparent solution. Record that approximately 5.5 mL of sodium hydroxide solution (1M) has been consumed. Stop adding alkali, and you will obtain the bismuth citrate staining solution.

[0038] The obtained bismuth citrate staining solution should be sealed and stored at room temperature or 4°C in a refrigerator, where it can be stored stably for several weeks.

[0039] Example 2 A bismuth citrate staining solution is prepared as follows: (1) Measure 3 mL of citric acid aqueous solution with a mass concentration of 3% using a graduated cylinder and place it in a 10 mL clean glass bottle.

[0040] (2) Weigh 0.05g of bismuth citrate powder accurately using an analytical balance and add it to the citric acid solution from step (1). Tighten the cap and shake the solution using a vortex mixer for about 2 minutes. A uniform white milky suspension will be formed.

[0041] (3) Using a pipette, draw up a 0.5M sodium hydroxide aqueous solution and add it dropwise to the emulsion suspension from step (2). After each drop, tighten the cap and shake well. As sodium hydroxide is added, the solution gradually changes from milky white to translucent, and finally becomes a clear and transparent solution, as shown in the image. Figure 1 As shown. Record that approximately 4.4 mL of sodium hydroxide solution (0.5 M) was consumed at this point. Stop adding alkali, and you will obtain the bismuth citrate staining solution.

[0042] The obtained bismuth citrate staining solution should be sealed and stored at room temperature or 4°C in a refrigerator, where it can be stored stably for several weeks.

[0043] Example 3 A bismuth citrate staining solution is prepared as follows: (1) Measure 7 mL of 7% citric acid aqueous solution using a graduated cylinder and place it in a 10 mL clean glass bottle.

[0044] (2) Weigh 0.15g of bismuth citrate powder accurately using an analytical balance and add it to the citric acid solution from step (1). Tighten the cap and shake the solution using a vortex mixer for about 2 minutes. A uniform white milky suspension will be formed.

[0045] (3) Using a pipette, take 2M potassium hydroxide aqueous solution and add it dropwise to the emulsion suspension from step (2). After each drop, tighten the cap and shake well. As potassium hydroxide is added, the solution gradually changes from milky white to translucent, and finally becomes a clear and transparent solution. Record that approximately 5.0 mL of potassium hydroxide solution (2M) has been consumed. Stop adding alkali, and you will have the bismuth citrate staining solution.

[0046] The obtained bismuth citrate staining solution should be sealed and stored at room temperature or 4°C in a refrigerator, where it can be stored stably for several weeks.

[0047] Experimental Example 1 Transmission electron microscopy observation of stained ultrathin sections of kidney tissue: Ultrathin sections (approximately 70 nm thick) of rat kidney tissue were mounted on a 200-mesh copper grid. First, uranium staining was performed: the copper grid, with the section side facing down, was floated on a droplet of saturated uranyl acetate aqueous solution and stained at room temperature in the dark for 10 minutes. The copper grid was then thoroughly rinsed three times with ultrapure water for 1 minute each time, and excess water was blotted away from the edges with filter paper. Next, bismuth staining was performed: the cleaned copper grid was similarly floated on a droplet of bismuth citrate staining solution prepared in Example 1 and stained at room temperature for 6 minutes. The copper grid was then rapidly and thoroughly rinsed three times with ultrapure water for 1 minute each time. The copper grid was placed in a clean petri dish and allowed to air dry naturally or dried using an infrared lamp. The dried sample was observed under a transmission electron microscope, and the results are as follows: Figure 3 As shown.

[0048] Experimental Example 2 Transmission electron microscopy observation of stained ultrathin sections of kidney tissue: Ultrathin sections (approximately 70 nm thick) of rat kidney tissue were mounted on a 200-mesh copper grid. First, uranium staining was performed: the copper grid, with the section side facing down, was floated on a droplet of saturated uranyl acetate aqueous solution and stained at room temperature in the dark for 10 minutes. The copper grid was then thoroughly rinsed three times with ultrapure water for 1 minute each time, and excess water was blotted away from the edges with filter paper. Next, bismuth staining was performed: the cleaned copper grid was similarly floated on a droplet of bismuth citrate staining solution prepared in Example 2 and stained at room temperature for 6 minutes. The copper grid was then rapidly and thoroughly rinsed three times with ultrapure water for 1 minute each time. The copper grid was placed in a clean petri dish and allowed to air dry naturally or dried using an infrared lamp. The dried sample was observed under a transmission electron microscope (FEI Talos F200C), and the results are as follows. Figure 4 As shown.

[0049] Comparative Example 1 Transmission electron microscopy observation of stained ultrathin sections of kidney tissue: Ultrathin sections (approximately 70 nm thick) of rat kidney tissue were mounted on a 200-mesh copper grid. First, uranium staining was performed: the copper grid, with the section side facing down, was floated on a droplet of saturated uranyl acetate aqueous solution and stained at room temperature in the dark for 10 minutes. The copper grid was then thoroughly rinsed three times with ultrapure water for 1 minute each time, and excess water was blotted away from the edges with filter paper. Next, lead staining was performed: the cleaned copper grid was similarly floated on a droplet of standard Reynolds lead citrate staining solution and stained at room temperature for 6 minutes. The copper grid was then rapidly and thoroughly rinsed three times with ultrapure water for 1 minute each time. The copper grid was placed in a clean petri dish and allowed to air dry naturally or dried using an infrared lamp. The dried sample was observed under a transmission electron microscope (FEI Talos F200C), and the results are as follows. Figure 2 As shown.

[0050] In the second step of staining in Experimental Examples 1-2 and Comparative Example 1, the staining was performed in an open petri dish. Figure 2 In the study, numerous randomly distributed, high-electron-density black particles (indicated by arrows) can be seen in cytoplasmic cavities and other areas. These particles are lead carbonate precipitates, which severely interfere with the normal observation of organelles. Figures 3-4 In the study, the staining effects of Examples 1 and 2 were comparable, with clean backgrounds and no contaminants. The ultrastructures such as the cell nucleus (N), mitochondria (Mt), and endoplasmic reticulum (ER) were clearly outlined, and the membrane structure contrast was good, demonstrating the excellent staining effect of bismuth citrate staining solution.

[0051] Experimental Example 3 Transmission electron microscopy observation of stained HEK293 cell ultrathin sections: Ultrathin sections (approximately 70 nm thick) of cultured HEK293 cells were mounted on a 200-mesh copper grid. First, uranium staining was performed: the copper grid, with the section side facing down, was floated on a droplet of saturated uranyl acetate aqueous solution and stained at room temperature in the dark for 10 minutes. The copper grid was then thoroughly rinsed three times with ultrapure water for 1 minute each time, and excess water was blotted away from the edges with filter paper. Next, bismuth staining was performed: the cleaned copper grid was similarly floated on a droplet of bismuth citrate staining solution prepared in Example 1 and stained at room temperature for 6 minutes. The copper grid was then rapidly and thoroughly rinsed three times with ultrapure water for 1 minute each time. The copper grid was placed in a clean culture dish and allowed to air dry naturally or dried using an infrared lamp. The dried sample was observed under a transmission electron microscope (FEI Talos F200C), and the results are as follows. Figure 5 As shown.

[0052] Figure 4 The results showed that after staining HEK293 cells with bismuth citrate staining solution, the cell membrane, mitochondria, endoplasmic reticulum, nuclear membrane (NE), ribosomes (Ribo) and other structures were clearly stained, with a clean background and no contaminating particles.

[0053] Test Example 4 Transmission electron microscopy observation of stained HEK293 cell ultrathin sections: Ultrathin sections (approximately 70 nm thick) of cultured HEK293 cells were mounted on a 200-mesh copper grid. First, uranium staining was performed: the copper grid, with the section side facing down, was floated on a droplet of saturated uranyl acetate aqueous solution and stained at room temperature in the dark for 10 minutes. The copper grid was then thoroughly rinsed three times with ultrapure water for 1 minute each time, and excess water was blotted away from the edges with filter paper. Next, bismuth staining was performed: the cleaned copper grid was similarly floated on a droplet of bismuth citrate staining solution prepared in Example 3 and stained at room temperature for 6 minutes. The copper grid was then rapidly and thoroughly rinsed three times with ultrapure water for 1 minute each time. The copper grid was placed in a clean culture dish and allowed to air dry naturally or dried using an infrared lamp. The dried sample was observed under a transmission electron microscope (FEI Talos F200C), and the results are as follows. Figure 6 As shown.

[0054] Figure 6 In the study, the nuclear membrane (NE), ribosomes (Ribo), mitochondria (MT) structures showed good contrast and were free of carbonate contamination, demonstrating their effectiveness. Figure 5 The effect is the same.

[0055] Experimental Example 5 Transmission electron microscopy observation of stained ultrathin sections of cardiac tissue: Ultrathin sections (approximately 70 nm thick) of rat heart tissue were mounted on a 200-mesh copper grid. First, uranium staining was performed: the copper grid, with the section side facing down, was floated on a droplet of saturated uranyl acetate aqueous solution and stained at room temperature in the dark for 10 minutes. The copper grid was then thoroughly rinsed three times with ultrapure water for 1 minute each time, and excess water was blotted away from the edges with filter paper. Next, bismuth staining was performed: the cleaned copper grid was similarly floated on a droplet of bismuth citrate staining solution prepared in Example 1 and stained at room temperature for 6 minutes. The copper grid was then rapidly and thoroughly rinsed three times with ultrapure water for 1 minute each time. The copper grid was placed in a clean petri dish and allowed to air dry naturally or dried using an infrared lamp. The dried sample was observed under a transmission electron microscope (FEI Talos F200C), and the results are as follows. Figure 7 As shown.

[0056] Figure 7 The results showed that after staining the heart tissue with bismuth citrate staining solution, the structures of mitochondria, myofibrils (MF), Z lines, etc. in the cardiac cells had good contrast and no carbonate precipitation contamination.

[0057] Experimental Example 6 Stability test: The bismuth citrate staining solution prepared in Example 1 was placed under the following conditions: Staining was performed after 1 week, 2 weeks, and 4 weeks of sealed storage at 4℃.

[0058] The staining process was the same as in Experiment 3, namely: Ultrathin sections (approximately 70 nm thick) of rat kidney tissue were mounted on a 200-mesh copper grid. First, uranium staining was performed: the copper grid, with the section side facing down, was floated on a droplet of saturated uranyl acetate aqueous solution and stained at room temperature in the dark for 10 minutes. The copper grid was then thoroughly rinsed three times with ultrapure water for 1 minute each time, and excess water was blotted away from the edges with filter paper. Next, bismuth staining was performed: the cleaned copper grid was similarly floated on a droplet of bismuth citrate staining solution prepared in Example 1 and stained at room temperature for 6 minutes. The copper grid was then rapidly and thoroughly rinsed three times with ultrapure water for 1 minute each time. The copper grid was placed in a clean petri dish and allowed to air dry naturally or dried using an infrared lamp. The dried sample was observed under a transmission electron microscope (FEI Talos F200C), and the results are as follows. Figure 8 As shown.

[0059] Figure 8 A, B, C, and D in the diagram indicate that the staining solution is chemically stable under refrigerated storage conditions and its staining ability is not affected by long-term storage, proving that the staining solution has excellent stability under refrigerated storage conditions.

[0060] Experimental Example 7 Stability test: The bismuth citrate staining solution prepared in Example 1 was placed under the following conditions: Staining was performed after the food was left exposed at room temperature for 1 hour, 4 hours, and 24 hours.

[0061] The staining process was the same as in Experiment 3, namely: Ultrathin sections (approximately 70 nm thick) of cultured HEK293 cells were mounted on a 200-mesh copper grid. First, uranium staining was performed: the copper grid, with the section side facing down, was floated on a droplet of saturated uranyl acetate aqueous solution and stained at room temperature in the dark for 10 minutes. The copper grid was then thoroughly rinsed three times with ultrapure water for 1 minute each time, and excess water was blotted away from the edges with filter paper. Next, bismuth staining was performed: the cleaned copper grid was similarly floated on a droplet of bismuth citrate staining solution prepared in Example 1 and stained at room temperature for 6 minutes. The copper grid was then rapidly and thoroughly rinsed three times with ultrapure water for 1 minute each time. The copper grid was placed in a clean culture dish and allowed to air dry naturally or dried using an infrared lamp. The dried sample was observed under a transmission electron microscope (FEI Talos F200C), and the results are as follows. Figure 9 As shown.

[0062] Figure 9A, B, C, and D in the table indicate that the staining solution has strong resistance to CO2 pollution. Even when exposed to air (containing CO2) for a long time, it will not produce carbonate precipitation and background pollution like lead citrate, proving that the staining solution has excellent open-air stability.

[0063] The above examples, comparative examples, and experimental cases demonstrate that the bismuth citrate staining solution provided by this invention can replace lead citrate staining agent for staining ultrathin sections of biological samples used for electron microscopy, and that this staining solution can solve the problem of precipitation caused by reaction with carbon dioxide, which interferes with detection. The staining mechanism of the bismuth citrate staining solution provided by this invention is presumably as follows: trivalent bismuth ions form stable complexes with protein carboxyl groups and nucleic acid phosphate groups in biological tissues, providing high electron density, thereby achieving specific staining; at the same time, bismuth carbonate is extremely unstable under staining conditions, therefore the staining solution does not react with CO2 in the air to form precipitate.

[0064] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a bismuth citrate staining solution, characterized in that, Includes the following steps: Bismuth citrate was added to an aqueous solution of citric acid and mixed thoroughly to form a white emulsion suspension. Add an alkaline solution to the white emulsion suspension until the white emulsion suspension becomes a clear and transparent solution; The mass concentration of citric acid in an aqueous solution is 3-7%. The ratio of bismuth citrate to citric acid aqueous solution is 0.05~0.15: 3~7, g / mL; the molar ratio of citric acid to bismuth is maintained at a complexation ratio of 2:1 to 3:1 to form a bismuth citrate complex anion; The alkaline solution is a sodium hydroxide solution or a potassium hydroxide solution.

2. A bismuth citrate staining solution, characterized in that, Obtained by the preparation method described in claim 1.

3. A dyeing composition, characterized in that, It includes the bismuth citrate staining solution as described in claim 2 and uranyl acetate.

4. The use of the bismuth citrate staining solution of claim 2 or the staining composition of claim 3 in the preparation of staining agents for electron microscopy of biological samples.

5. A staining method for biological samples for electron microscopy examination, characterized in that, Includes the following steps: Provide the bismuth citrate staining solution as described in claim 2; The target biological sample was stained in the bismuth citrate staining solution.

6. The staining method as described in claim 5, characterized in that, Staining was performed using bismuth citrate staining solution in an environment where carbon dioxide was not isolated.

7. The staining method as described in claim 5, characterized in that, The staining method is a double staining method, which involves first staining with uranyl acetate and then staining with bismuth citrate.

8. The staining method as described in claim 5, characterized in that, The target biological sample is an ultrathin section of biological tissue or an ultrathin section of cells.

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