Single molecule detection method for marking electron transfer of shewanella and application of single molecule detection method
By employing single-molecule super-resolution fluorescence microscopy and Nile blue fluorescent probes, the problem of high spatiotemporal resolution detection of electron transport processes in Shewanella has been solved, enabling nanoscale resolution observation and dynamic monitoring, thereby improving the efficiency of bioremediation and energy production.
Patent Information
- Application Number
- CN202511033995.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-25
- Publication Date
- 2025-11-11
AI Technical Summary
Existing technologies are insufficient to achieve high spatiotemporal resolution and high sensitivity in detecting electron transport processes in Shewanella cells, and cannot accurately observe nanoscale subcellular structures and dynamic changes, thus limiting their application in bioremediation and energy production.
Single-molecule super-resolution fluorescence microscopy and Nile blue fluorescent probes, combined with PAINT technology, were used to label Shewanella cells with single-molecule fluorescence, enabling observation and dynamic monitoring at nanometer-level resolution.
Breaking through the diffraction limit of traditional optical microscopes, this study accurately observed the binding sites and dynamic changes of Nile blue on Shewanella cells, improving detection accuracy, revealing the close correlation between electron transport activity and dynamic distribution, and optimizing pollutant degradation efficiency and microbial fuel cell performance.
Smart Images
Figure CN120927635A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biophotonics technology, specifically relating to a single-molecule detection method for labeling electron transfer in Shewanella bacteria and its application, which can be widely used in environmental monitoring, bioremediation, and bioenergy production. Background Technology
[0002] Electron transport within Shewanella oneidensis MR-1 cells is closely related to its cell membrane structures, including cytochromes, and plays a crucial role in pollutant degradation and microbial fuel cell power generation. However, existing technologies for real-time detection of Shewanella cell state and electron transport processes have limitations. On the one hand, traditional detection methods lack specific probes capable of precisely targeting Shewanella cells and achieving high spatiotemporal resolution, making it difficult to clearly observe the microscopic structures and dynamic changes related to electron transport within the cell. On the other hand, existing technologies lack sufficient sensitivity to effectively capture subtle molecular-level changes during electron transport, thus limiting research on the electron transport mechanism of Shewanella and significantly hindering its further development in practical applications such as bioremediation and energy production.
[0003] For centuries, the invention and application of optical microscopes have provided a crucial tool for studying cell morphology and function, greatly advancing the field of biology. However, in the study of electron transport mechanisms in Shewanella, traditional optical microscopes face significant challenges due to diffraction limitations: their longitudinal resolution is approximately 500-800 nm, and their lateral resolution is approximately 200-300 nm, making it impossible to observe nanoscale subcellular structures (such as cytochrome complexes and cell membrane nanodomains related to electron transport), thus hindering the accurate analysis of electron transport pathways and dynamic processes in Shewanella. Summary of the Invention
[0004] To address the shortcomings of existing technologies, this invention provides a single-molecule detection method for labeling electron transport in Shewanella bacteria. This method can detect the cell state and electron transport process of Shewanella bacteria in real time, providing key technical support for the study of Shewanella cell physiological activities and related application development. Based on super-resolution optical imaging technology, this method observes bacterial electron transport at the nanoscale, revealing a close correlation between the dynamic distribution of the fluorescent probe (Nile blue) on Shewanella cells and the cell's electron transport activity.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] This invention provides a single-molecule detection method for labeling electron transfer in Shewanella, comprising the following steps: using single-molecule super-resolution fluorescence microscopy, adding a fluorescent probe to a Shewanella sample, wherein the fluorescent probe specifically binds to Shewanella cells and is excited to emit fluorescence, thereby labeling Shewanella cells with single-molecule fluorescence.
[0007] Preferably, the single-molecule super-resolution fluorescence microscopy technique employs PAINT (points accumulation for imaging in nanoscale topography) super-resolution technology.
[0008] Preferably, the fluorescent probe is a Nile blue fluorescent probe. When the fluorescent probe is preferably a Nile blue fluorescent probe, the excitation wavelength is preferably 637 nm. The concentration of the Nile blue fluorescent probe is preferably 0.01-0.05 μM.
[0009] Specifically, the detection method includes the following steps: after cleaning the coverslip, spin-coating polylysine solution, then adding a suspension of Shewanella cells labeled with a fluorescent probe or adding a suspension of Shewanella cells and a fluorescent probe, exciting the fluorescent probe with excitation light using a single-molecule super-resolution fluorescence microscope, collecting the fluorescence signal, and generating a super-resolution image based on the single-molecule localization method.
[0010] Furthermore, the method for preparing the Shewanella cell suspension is as follows: Shewanella is cultured to the mid-exponential growth stage, the cells are collected by centrifugation, washed repeatedly by centrifugation with PBS buffer, and finally the cells are resuspended in PBS to obtain the Shewanella cell suspension.
[0011] Furthermore, the preparation method of the fluorescently labeled Shewanella cell suspension is as follows: Shewanella is cultured to the mid-exponential growth stage, the cells are collected by centrifugation, washed repeatedly by centrifugation with PBS buffer, and finally the cells are resuspended in PBS to obtain a Shewanella cell suspension. A fluorescent probe is then added to the cell suspension to obtain a fluorescently labeled Shewanella cell suspension.
[0012] For example, Shewanella is cultured on TSB medium.
[0013] In the above testing methods, glass is the preferred material for the coverslip.
[0014] In the above detection method, the concentration of the polylysine solution is preferably 0.05-0.15 mg / mL, and the spin-coating speed is preferably 1000-2000 rpm. Preferably, an electron transport promoter or an electron transport inhibitor may be added to the coverslip to increase or decrease the number of single-molecule fluorescent labels undergoing electron transport on Shewanella cells. When the fluorescent probe is preferably a Nile blue fluorescent probe, the electron transport promoter is preferably riboflavin (Rbf); the electron transport inhibitor is carbonyl cyanide 3-chlorophenylhydrazone (CCCP).
[0015] Preferably, after adding an electron transport promoter or an electron transport inhibitor, images are taken at regular intervals using a single-molecule super-resolution fluorescence microscope, and the images are analyzed using the ThunderSTORM plugin of the open-source image analysis platform ImageJ.
[0016] More specifically, the detection method includes:
[0017] Step (1), preparation of Shewanella cell samples: Shewanella was inoculated into TSB medium and cultured to the mid-exponential growth stage. Then, the bacterial suspension was taken, and the bacterial suspension was washed by centrifugation with PBS. The supernatant was discarded, and the bacterial suspension was washed by centrifugation with PBS three times. Then, PBS was added to the bacterial precipitate to obtain Shewanella cell suspension.
[0018] Step (2): Use ozone to clean the coverslip, spin-coat polylysine solution onto the coverslip, drop the Shewanella cell suspension obtained in step (1) onto the coverslip, incubate at room temperature for a period of time, then wash away unfixed bacteria with PBS, and then drop PBS again to maintain the normal physiological and metabolic state of the bacteria; then drop Nile blue fluorescent probe to obtain the coverslip sample.
[0019] Step (3) Place the coverslip sample obtained in step (2) into a single-molecule super-resolution fluorescence microscope, turn on the laser, and use excitation light with a wavelength of 637 nm to excite the Nile blue fluorescent probe to emit fluorescence, thereby marking the Shewanella sample.
[0020] In step (2), the incubation time at room temperature is generally 5 minutes.
[0021] In step (3), the power of the excitation light of the exciter is preferably 40 or 80 mW.
[0022] Preferably, images are taken at regular intervals using a single-molecule super-resolution fluorescence microscope, and the images are analyzed using the ThunderSTORM plugin of the open-source image analysis platform ImageJ.
[0023] This invention also provides the application of the aforementioned single-molecule detection method for labeling Shewanella electron transport in environmental monitoring, bioremediation, or bioenergy production. For example, when the above detection method is applied to environmental monitoring, by real-time monitoring of the dynamic changes in electron transport during the degradation of organic pollutants by Shewanella, the intensity, distribution, and time correlation of the single-molecule fluorescent labeling signal can be analyzed to quantitatively assess pollutant degradation efficiency and microbial metabolic activity.
[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0025] (1) The single-molecule detection method for labeling Shewanella electron transport provided by the present invention adopts single-molecule super-resolution fluorescence microscopy and Nile blue specific binding, realizing high spatiotemporal resolution monitoring at the single-molecule level, breaking through the diffraction limit of traditional optical microscopes, and can accurately observe the binding site and dynamic changes of Nile blue on Shewanella cells, providing nanometer-resolution visualization data for studying cell electron transport processes.
[0026] (2) Effectively eliminates non-specific signal interference, greatly improves detection accuracy, and can accurately capture subtle molecular changes during cell electron transport;
[0027] (3) The binding of Nile blue to Shewanella cells can effectively reflect the efficiency of bacterial electron transport. By detecting the changes in Nile blue fluorescence signal, the electron transport activity of Shewanella cells was analyzed, revealing the close correlation between the dynamic distribution of Nile blue on Shewanella cells and the cell electron transport activity.
[0028] (4) Based on the above characteristics, technologies for regulating the electron transfer process of Shewanella can be further developed to optimize the degradation efficiency of pollutants in bioremediation and improve the power generation performance of microbial fuel cells in energy production. At the same time, by utilizing the correlation between fluorescence signals and electron transfer activity, biosensors for the detection of heavy metal ions can be developed, expanding the application field of this detection technology. Attached Figure Description
[0029] Figure 1 The above are the bright field images of the Nile blue fluorescent probe used in Shewanella cells and the super-resolution localization images before and after the addition of the electron transport promoter riboflavin (Rbf) according to Example 2 of the present invention; wherein, (1) is the bright field image, (2) is before the addition of Rbf, and (3) and (4) are after 4 and 6 min of Rbf addition, respectively;
[0030] Figure 2The images shown are the bright field image of the Nile blue fluorescent probe used in Shewanella cells according to Example 2 of the present invention and the super-resolution localization images before and after the addition of the electron transfer inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP); wherein, (1) is the bright field image, (2) is before the addition of CCCP, and (3) and (4) are after 9 and 12 min of CCCP addition, respectively. Detailed Implementation
[0031] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0032] This invention provides a single-molecule detection method for labeling electron transport in Shewanella, comprising the following steps: using single-molecule super-resolution fluorescence microscopy, adding a fluorescent probe to a Shewanella sample; the fluorescent probe specifically binds to Shewanella cells and is excited to emit fluorescence, thereby labeling the Shewanella cells with single-molecule fluorescence. The fluorescent probe can bind to the cell membrane of Shewanella and be excited to emit fluorescence, dynamically monitoring the changes in the number of single-molecule fluorescent labels undergoing electron transport on Shewanella cells, thereby achieving dynamic labeling of the electron transport capability of Shewanella.
[0033] According to the present invention, the super-resolution imaging technology employed has been proven to be a core method for studying electron transport in Shewanella. Specifically, single-molecule localization microscopy (SMLM), based on the non-overlapping point spread functions (PSFs) of individual fluorescent molecules, can calculate molecular spatial coordinates with high precision. Its positioning accuracy is limited only by the signal-to-noise ratio (SNR), and is not constrained by light wavelength or pixel size, thus enabling nanometer-scale resolution observation.
[0034] As an important branch of SMLM technology, PAINT (points accumulation for imaging innanoscale topography) technology utilizes the dynamic binding-dissociation behavior of lipophilic fluorescent molecules on samples to achieve spatiotemporally sparse distribution of fluorophores at concentrations from pM to nM, thereby reconstructing high-resolution images. This technology, through the continuous exchange mechanism between fluorescent probes and bleached molecules in the imaging buffer, ensures both high-density labeling of target structures and precise localization through fluorescent molecule scintillation. After acquiring and reconstructing thousands of images, a biomolecular distribution map with nanoscale resolution can be obtained, providing an innovative technical pathway for the dynamic monitoring of electron transport-related membrane structures in Shewanella.
[0035] In some embodiments, in the single-molecule detection method for labeling Shewanella electron transport provided by the present invention, a Shewanella sample containing a fluorescent probe is placed on the stage of a single-molecule super-resolution fluorescence microscope equipped with a PAINT super-resolution module. An excitation light of 637 nm wavelength is selected, and appropriate filter detection channels, exposure time, and number of frames are set. The sample is observed to obtain a single-molecule fluorescence image of the binding site of the fluorescent probe on Shewanella cells. The present invention can accurately observe the binding site and dynamic changes of Nile blue on Shewanella cells through this detection method, providing nanometer-resolution visualization data for studying the cell electron transport process.
[0036] In the single-molecule detection method for labeling Shewanella electron transfer provided by this invention, the fluorescent probe used in this invention is a Nile blue fluorescent probe.
[0037] According to publicly available technology, Nile Blue, also known as Nile Blue sulfate or Nile Blue, is a benzophenoxazine compound.
[0038] In the single-molecule detection method for labeling Shewanella electron transfer provided by the present invention, the detection method specifically includes the following steps:
[0039] Step (1), preparation of Shewanella cell samples: Shewanella was inoculated into TSB medium and cultured to the mid-exponential growth stage. Then, 1 mL of bacterial suspension was taken, and the bacterial suspension was washed by centrifugation with PBS. The supernatant was discarded, and the bacterial suspension was washed by centrifugation with PBS three times. Then, PBS was added to the bacterial precipitate to obtain Shewanella cell suspension.
[0040] Step (2): Use ozone to clean the coverslip, spin-coat polylysine solution onto the coverslip, drop the Shewanella cell suspension obtained in step (1) onto the coverslip, incubate at room temperature for a period of time, then wash away unfixed bacteria with PBS, and then drop PBS again to maintain the normal physiological and metabolic state of the bacteria; then drop Nile blue fluorescent probe to obtain the coverslip sample.
[0041] Step (3): Place the coverslip sample obtained in step (2) into a single-molecule super-resolution fluorescence microscope, turn on the laser, and use excitation light with a wavelength of 637 nm to label the Nile blue fluorescent probe on the Shewanella sample with a suitable fluorescence density.
[0042] According to the present invention, the density of immobilized Shewanella can be adjusted by changing the concentration of the polylysine solution and the incubation time of Shewanella. In some embodiments, in step (2) above, the concentration of the polylysine solution is 0.05-0.15 mg / mL (e.g., 0.10 mg / mL), the spin-coating speed is 1000-2000 rpm (e.g., 1500 rpm), and the incubation time at room temperature (typically 30°C) is 1.5-2.5 hours, e.g., 2 hours.
[0043] In some embodiments, the concentration of the Nile blue fluorescent probe is 0.01-0.05 μM, preferably 0.01 μM, and the excitation light power of the exciter is 40 or 80 mW. According to the present invention, the fluorescent probe can be marked on the sample at a suitable density and flash at a certain frequency by controlling the concentration of the Nile blue fluorescent probe and the power of the excitation light.
[0044] In scenarios where increased or decreased fluorescence intensity is required, clearer super-resolution images can be achieved by adding electron transport promoters or inhibitors. The single-molecule detection method for labeling Shewanella electron transport provided by this invention further includes: adding an electron transport promoter or inhibitor to a Shewanella sample containing a Nile blue fluorescent probe, imaging with a single-molecule super-resolution fluorescence microscope, and dynamically monitoring the changes in the number of single-molecule fluorescent labels undergoing electron transport on Shewanella cells.
[0045] In some embodiments, the electron transport promoter used in this invention is riboflavin (Rbf), and the electron transport inhibitor used is carbonyl cyanide 3-chlorophenylhydrazone (CCCP).
[0046] In the single-molecule detection method for labeling Shewanella electron transport provided by this invention, after adding an electron transport promoter or an electron transport inhibitor in the above steps, images are taken at intervals using a single-molecule super-resolution fluorescence microscope, and the images are analyzed using the ThunderSTORM plugin of the open-source image analysis platform ImageJ.
[0047] In some embodiments, during the above steps, the images from the above steps are analyzed using the ThunderSTORM plugin of the open-source image analysis platform ImageJ. A two-dimensional Gaussian function is used to fit the experimentally obtained point spread function (PSF) pattern to obtain the corresponding single-molecule localization. Incorrect localization results are further removed based on the PSF's full width at half maximum (FWHM) and single-molecule brightness. Finally, single-molecule localization results are further filtered through visual perception. Quantitative analysis was performed on the number of photons per molecule and the experimental localization accuracy in each imaging frame. After fitting the initial single molecules with a spatiotemporal filter, density statistics were performed on the single-molecule count results to obtain a super-resolution image of the bacterial surface. Observation of the super-resolution image of the bacterial surface shows that after adding an electron transport promoter, the number of single-molecule fluorescent labels on the bacteria gradually increases over time; after adding an electron transport inhibitor, the number of single-molecule fluorescent labels on the bacteria gradually decreases over time, and the spatial distribution of the fluorescent labels exhibits uneven changes.
[0048] The present invention will be further illustrated below with reference to specific embodiments. The purpose of these embodiments is to provide a better understanding of the invention and to demonstrate its essential characteristics. Therefore, the examples given should not be considered as limitations on the scope of protection of the present invention. It is also specifically noted that, unless otherwise specified, the specific experimental methods and equipment involved in the embodiments are conventional methods or implemented according to the conditions recommended in the manufacturer's instructions, and the reagents involved are all commercially available unless otherwise specified. In the following embodiments, the coverslips used are made of glass, and the excitation light power of the exciter used is 80mW.
[0049] Example 1:
[0050] This embodiment provides a single-molecule detection method for labeling electron transfer in Shewanella, comprising the following steps:
[0051] Step (1), strain culture: Shewanella was inoculated into TSB medium and cultured with shaking at 30℃ and 200 rpm until the logarithmic growth phase; 1 mL of bacterial solution was taken and centrifuged at 4000×g for 3 minutes, the supernatant was discarded, and the solution was washed 3 times with PBS at pH 7.4; PBS was added to the bacterial pellet to obtain Shewanella cell suspension;
[0052] Step (2), sample preparation: After cleaning a 22mm×22mm coverslip with O3, spin-coat 20μL of 0.1mg / mL polylysine solution onto the coverslip at 1500rpm; add 20μL of Shewanella cell suspension to the coverslip and incubate at room temperature for 5min to fix the bacteria; wash the coverslip three times with PBS to remove unfixed bacteria; add 200μL of PBS to the sample to maintain the normal physiological and metabolic state of the bacteria;
[0053] Step (3), label the sample: add 2 μL of 1 μM Nile blue fluorescent probe to the sample obtained in step (2) to make the imaging concentration 0.01 μM;
[0054] Step (4), Microscopic observation: Place the sample slide on the stage of a single-molecule super-resolution fluorescence microscope equipped with a PAINT super-resolution module, select an excitation light of 637 nm wavelength, set an appropriate filter detection channel, exposure time and number of frames, observe the sample, and obtain a single-molecule fluorescence image of the binding site of Nile blue on Shewanella cells.
[0055] Example 2:
[0056] This embodiment provides a single-molecule detection method for labeling electron transfer in Shewanella, comprising the following steps:
[0057] Step (1), strain culture: Shewanella was inoculated into TSB medium and cultured with shaking at 30℃ and 200 rpm until the logarithmic growth phase; 1 mL of bacterial suspension was taken and centrifuged at 4000×g for 3 minutes, the supernatant was discarded, and the suspension was washed 3 times with PBS at pH 7.4; the bacterial cells were resuspended in PBS to obtain Shewanella cell suspension;
[0058] Step (2), sample preparation: After cleaning a 22mm×22mm coverslip with O3, spin-coat 20μL of 0.1mg / mL polylysine solution onto the coverslip at 1500rpm; add 20μL of Shewanella cell suspension to the coverslip and incubate at room temperature for 5min to fix the bacteria; wash the coverslip three times with PBS to remove unfixed bacteria; add 200μL of PBS to the sample to maintain the normal physiological and metabolic state of the bacteria;
[0059] Step (3), label the sample: Add 2 μL of 1 μM Nile blue fluorescent probe (imaging concentration of 0.01 μM) and 2 μL of 500 mM electron transport promoter riboflavin (Rbf) (imaging concentration of 5 mM) or 2 μL of 12.2 mM electron transport inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (imaging concentration of 0.122 mM) to the sample obtained in step (2);
[0060] Step (4), Microscopic observation: Place the sample slide on the stage of a single-molecule super-resolution fluorescence microscope equipped with a PAINT super-resolution module, select an excitation light of 637 nm wavelength, set an appropriate filter detection channel, exposure time and number of frames, observe the sample, and obtain a single-molecule fluorescence image of the binding site of Nile blue on Shewanella cells.
[0061] Step (5), Data Processing: The raw data acquired by the camera was analyzed using the ThunderSTORM plugin of the open-source image analysis platform ImageJ; the PSF pattern obtained from the experiment was fitted with a two-dimensional Gaussian function to obtain the corresponding single-molecule localization; erroneous localization results were further removed and filtered based on the full width at half maximum (FWHM) and single-molecule brightness of the PSF; finally, the single-molecule localization results were further filtered through visual perception. The number of photons and the experimental localization accuracy of single molecules in each imaging frame were quantitatively analyzed; after fitting the initial single molecules with a spatiotemporal filter, the density statistics of the single-molecule counting results were performed to obtain a super-resolution image of the bacterial surface.
[0062] like Figure 1 The image shows the bright field image of the Nile blue fluorescent probe used in Shewanella cells and the super-resolution localization images before and after the addition of the electron transport promoter riboflavin (Rbf) according to Example 2 of the present invention; wherein, (1) is the bright field image, (2) is before the addition of Rbf, and (3) and (4) are after 4 and 6 min of Rbf addition, respectively.
[0063] from Figure 1 It can be seen that after adding the electron transport promoter riboflavin (Rbf) in Example 2, the number of single fluorescent markers on bacteria gradually increased over time.
[0064] like Figure 2 The image shows the bright field image of the Nile blue fluorescent probe used in Shewanella cells in Example 2 of this invention and the super-resolution localization images before and after the addition of the electron transfer inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP); where (1) is the bright field image, (2) is before the addition of CCCP, and (3) and (4) are after 9 and 12 min of CCCP addition, respectively.
[0065] from Figure 2 It can be seen that after adding the electron transfer inhibitor carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in Example 2, the number of single fluorescent markers on bacteria gradually decreased over time.
[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A single-molecule detection method for labeling electron transfer in Shewanella, characterized in that, The procedure includes the following steps: using single-molecule super-resolution fluorescence microscopy, a fluorescent probe is added to a Shewanella sample. The fluorescent probe specifically binds to Shewanella cells and is excited to emit fluorescence, thereby labeling the Shewanella cells with single-molecule fluorescence.
2. The single-molecule detection method for labeling Shewanella electron transfer according to claim 1, characterized in that, The single-molecule super-resolution fluorescence microscopy technique employs PAINT super-resolution technology.
3. The single-molecule detection method for labeling Shewanella electron transfer according to claim 1, characterized in that, The fluorescent probe is a Nile blue fluorescent probe.
4. The single-molecule detection method for labeling Shewanella electron transfer according to claim 1, characterized in that, The excitation wavelength used was 637 nm, and the concentration of the Nile blue fluorescent probe was 0.01-0.05 μM.
5. The single-molecule detection method for labeling Shewanella electron transfer according to claim 1, characterized in that, The process includes the following steps: After cleaning the coverslip, spin-coat a polylysine solution, then add Shewanella cell suspension and a fluorescent probe. Excite the fluorescent probe with excitation light using a single-molecule super-resolution fluorescence microscope, collect the fluorescence signal, and generate a super-resolution image based on the single-molecule localization method.
6. The single-molecule detection method for labeling Shewanella electron transfer according to claim 5, characterized in that, The concentration of the polylysine solution is 0.05-0.15 mg / mL, and the spin coating speed is 1000-2000 rpm.
7. The single-molecule detection method for labeling Shewanella electron transfer according to claim 5, characterized in that, include: Step (1), preparation of Shewanella cell samples: Shewanella was inoculated into TSB medium and cultured to the mid-exponential growth stage. Then, the bacterial suspension was taken, and the bacterial suspension was washed by centrifugation with PBS. The supernatant was discarded, and the bacterial suspension was washed by centrifugation with PBS three times. Then, PBS was added to the bacterial precipitate to obtain Shewanella cell suspension. Step (2): Use ozone to clean the coverslip, spin-coat polylysine solution onto the coverslip, drop the Shewanella cell suspension obtained in step (1) onto the coverslip, incubate at room temperature for a period of time, then wash away unfixed bacteria with PBS, and then drop PBS again to maintain the normal physiological and metabolic state of the bacteria; then drop Nile blue fluorescent probe to obtain the coverslip sample. Step (3) Place the coverslip sample obtained in step (2) into a single-molecule super-resolution fluorescence microscope, turn on the laser, and use excitation light with a wavelength of 637 nm to excite the Nile blue fluorescent probe to emit fluorescence, thereby marking the Shewanella sample.
8. The single-molecule detection method for labeling Shewanella electron transfer according to claim 1, characterized in that, Also includes: Electron transport promoters or inhibitors were added to Shewanella samples containing Nile blue fluorescent probes, and the changes in the number of single-molecule fluorescent labels undergoing electron transport on Shewanella cells were dynamically monitored by imaging with single-molecule super-resolution fluorescence microscopy.
9. The single-molecule detection method for labeling Shewanella electron transfer according to claim 1 or 8, characterized in that, Images were captured at regular intervals using a single-molecule super-resolution fluorescence microscope, and the images were analyzed using the ThunderSTORM plugin of the open-source image analysis platform ImageJ.
10. The application of the single-molecule detection method for labeling Shewanella electron transport according to any one of claims 1-9 in environmental monitoring, bioremediation or bioenergy production.