Method for observing cyanobacteria cell communication by using FRAP technology

Abstract

The invention discloses a method for observing cyanobacteria cell communication by using an FRAP technology, and relates to the technical field of biological research, and the method comprises the following steps: culturing cyanobacteria; pretreating the cells; performing fluorescent dyeing and washing; fixing the sample; carrying out sample adaptation treatment; carrying out FRAP experiment observation; controlling an observation program; extracting and preprocessing data; processing and analyzing data; and verifying and applying a result. The problem of sample observation displacement is thoroughly solved through an innovative agar back-off fixation method, background fluorescence interference is eliminated by combining cell synchronization enrichment and multiple rounds of mild washing, and imaging and bleaching parameters adaptive to cyanobacteria light sensitivity characteristics are customized to balance signal quality and cell activity; meanwhile, a complete analysis process from sample adaptation and program automation control to data noise reduction calibration and nonlinear fitting is established, core parameters of communication dynamics are accurately extracted, and stable, accurate and quantitative observation of the communication dynamic process between cyanobacteria cells is achieved.

Description

Technical Field

[0001] This invention relates to the field of biological research technology, specifically to a method for observing cyanobacterial cell communication using FRAP technology. Background Technology

[0002] Cyanobacteria, as the earliest organisms to adopt oxygen-containing photosynthesis, played a central role in the early evolution of life on Earth. Their multicellular filamentous strains exchange substances and transmit signals through specific intercellular channels. This cell communication mechanism directly regulates their ability to adapt to environmental changes and the coordination of physiological functions. Currently, the technology for studying cyanobacterial cell communication mostly relies on static structural observation methods such as cryo-electron microscopy or computational modeling methods. These methods can only obtain structural information about intercellular channels and cannot achieve real-time, dynamic monitoring of communication processes within living cells, making it difficult to reveal the dynamic characteristics and functional regulatory mechanisms of communication.

[0003] Fluorescent recovery after bleaching (FRAP) technology, a classic method for studying the diffusion dynamics of biological macromolecules, has been widely used in animal cell research. However, its direct application to cyanobacteria faces several technical bottlenecks: First, cyanobacterial cells are rich in chlorophyll, phycocyanin, and other substances, which produce strong autofluorescence at the excitation wavelength of fluorescent dyes, overlapping with the target fluorescence signal and creating high background noise, severely interfering with the accuracy of signal detection. Second, the filamentous structure of cyanobacteria is fragile, and conventional sample preparation methods cannot achieve stable fixation during observation. Even slight displacement can lead to misalignment between the bleached and collected areas, resulting in experimental failure. Third, the physiological state of cyanobacteria is crucial for dye loading and retention. Inappropriate culture or staining conditions can easily lead to low staining efficiency and decreased cell viability, thereby interfering with the normal presentation of channel functions. Fourth, existing FRAP imaging and bleaching parameters are mostly designed for animal cells and are not adapted to the photosensitivity characteristics of cyanobacteria, making it difficult to avoid cell photodamage while ensuring the signal-to-noise ratio, thus limiting the reliable application of the technology.

[0004] Therefore, it is urgent to develop a FRAP method optimized for the characteristics of cyanobacteria to overcome the above-mentioned technical challenges and achieve stable and accurate observation of the dynamic process of intercellular communication in cyanobacteria. Summary of the Invention

[0005] To address the aforementioned technical problems, a method for observing cyanobacterial cell communication using FRAP technology is provided. This technical solution solves the problems described above.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for observing cyanobacterial cell communication using FRAP technology, the specific procedure of which is as follows: S1. Cyanobacterial culture: Construct a cyanobacterial culture system under normal physiological conditions to ensure that cell viability meets the requirements for subsequent staining and observation. S2. Cell pretreatment: Synchronize and enrich the cultured cyanobacteria to provide a uniform cell population for fluorescent staining. S3. Fluorescent staining and washing: Calcein acetylated methyl ester was used to specifically stain cyanobacteria and remove free dye to reduce background fluorescence interference. S4. Sample fixation: The stained cyanobacteria samples are stabilized and fixed using the agar inverting method to prevent displacement during observation. S5. Sample adaptation treatment: The fixed sample is moisturized and adapted to the observation environment to maintain cell physiological activity until the end of the observation. S6 and FRAP experiments were conducted by using a confocal microscope to select target cell regions and performing time-series imaging of the laser bleaching and fluorescence recovery process. S7. Observation program control, completes continuous imaging of baseline acquisition before bleaching, target area bleaching and recovery process after bleaching according to preset procedures; S8. Data extraction and preprocessing: Extract fluorescence intensity data of the target area from the time series image and perform noise reduction and standardization preprocessing. S9. Data processing and analysis: Normalize the preprocessed fluorescence intensity data and obtain the kinetic parameters of intercellular communication through nonlinear fitting. S10. Result verification and application: Verify the reliability of the kinetic parameters through repeated experiments and control analysis, and determine the cyanobacterial cell communication patterns and capabilities based on the parameters.

[0007] Preferably, the S1 cyanobacterium culture process is as follows: Filamentous cyanobacteria were selected as the core observation target, with priority given to model strains exhibiting typical intercellular communication characteristics to ensure the representativeness and reproducibility of experimental results. The selected cyanobacteria were inoculated into suitable liquid culture media and placed in a light incubator for directional culture. Appropriate light conditions, constant temperature, and gas environment were set according to the physiological characteristics of the strains to ensure stable cellular metabolic activity. During culture, samples were taken periodically to observe cell morphology and growth status. Cell density, filament integrity, and the absence of abnormal aggregation were observed under a microscope to determine whether cells had entered the logarithmic growth phase. When cells reached the logarithmic growth phase and their physiological state was uniform, the culture was stopped, and the bacterial solution was stored in a suitable environment for short-term preservation. This provided uniformly active and stable cell material for subsequent cell pretreatment and staining steps, mitigating experimental bias caused by differences in cell activity from the outset.

[0008] Preferably, the S2 cell pretreatment process is as follows: Transfer the cyanobacterial culture to the logarithmic growth phase into sterile centrifuge tubes and centrifuge at low speed. Strictly control the centrifugation force to avoid mechanical damage to the cyanobacterial filaments. After centrifugation, carefully remove the supernatant and collect the cell population at the bottom. Gently resuspend the cells in fresh culture medium consistent with the culture system, ensuring uniform cell dispersion without aggregation. Repeat the resuspension-centrifugation process 2-3 times. This process effectively removes metabolic waste, aged cell debris, and residual culture medium impurities generated during culture. Simultaneously, the synergistic effect of centrifugation and resuspension synchronizes the cell population, ensuring that the vast majority of cells are at the same growth stage. This reduces uneven staining efficiency caused by cell cycle differences, providing a highly uniform and pure cell population for subsequent fluorescence staining and improving the stability of experimental data.

[0009] Preferably, the S3 fluorescent staining and washing process is as follows: A high-purity methyl calcein stock solution was prepared using a solvent with good compatibility with cyanobacterial cells and no cytotoxicity. Strict aseptic techniques were followed during preparation to avoid solvent contamination and dye degradation. Based on cell concentration and staining requirements, an appropriate amount of the stock solution was slowly added to the enriched cyanobacterial culture while gently stirring to ensure thorough and uniform mixing of the dye and culture. The mixed culture was then incubated in an environment consistent with the culture conditions, with the shaking speed carefully controlled to avoid damaging the cell structure. Shaking promoted contact between the dye and the cell surface, accelerating dye entry into the cells. After incubation, the cells were washed by centrifugation using fresh culture medium pre-warmed to culture temperature. After centrifugation, the supernatant containing free dye was removed, and the cells were resuspended in fresh culture medium. This washing process was repeated at least three times. After each wash, fluorescence detection was used to assess the removal of free dye until no obvious fluorescence signal was observed in the supernatant, completely eliminating background fluorescence interference from free dye and ensuring that subsequent fluorescence signals originated from dye molecules within the cells.

[0010] Preferably, the S4 sample fixation process is as follows: Based on a liquid culture medium suitable for cyanobacteria, a solid culture medium was prepared by adding an appropriate amount of agar powder. The amount of agar powder was adjusted to ensure that the medium had a certain firmness without affecting cell viability. After preparation, the medium was autoclaved. The sterilized solid culture medium was poured into sterile petri dishes while still hot and allowed to cool and solidify naturally to form uniform and flat agar plates. The solidified agar plates were then cut into appropriately sized square agar blocks using a sterile scalpel, taking care to avoid breaking or contaminating the edges of the blocks during the cutting process. The stained cyanobacterial culture was then slowly added dropwise to the agar plates using a sterile pipette. For the agar block surface, the amount of agar added should be just enough to cover the surface without overflowing. Place the agar block containing the bacterial solution in a sterile laminar flow hood and let it air dry. The drying time should be based on the partial evaporation of water from the bacterial solution and the initial adsorption of cyanobacterial filaments onto the agar surface, ensuring stable adsorption between the cells and the agar. Finally, carefully invert the agar block so that the bacterial solution side down adheres to the center of the confocal microscope's glass bottom dish. Utilize the weight of the agar block itself and the adsorption force generated by water evaporation to stably fix the cyanobacterial cells on the glass bottom surface, preventing misalignment between the bleached area and the collection area due to sample displacement during subsequent observations.

[0011] Preferably, the S5 sample adaptation process is as follows: After sample fixation, a small amount of fresh culture medium is added around the agar block in the glass dish. The amount of culture medium should just wet the edge of the agar block without submerging it, creating a localized moist environment to prevent cell dehydration and inactivation due to rapid evaporation of water during observation. The glass dish containing the sample is then placed in a constant temperature environment consistent with the cyanobacterial culture temperature for equilibration. The equilibration time is adjusted according to the ambient temperature difference to ensure that the sample temperature is completely consistent with the culture temperature, avoiding the impact of temperature changes on cell physiological function. At the same time, the light in the observation environment is controlled to avoid strong direct light on the sample, which could cause photodamage to cells and fluorescence quenching, thus maintaining the stability of the observation environment. During the equilibration process, the cell status is observed periodically through a microscope to confirm that the cells are morphologically intact and that there is no abnormal shrinkage or death, ensuring that the cells maintain normal physiological function and intercellular communication ability throughout the observation period, thus providing a guarantee for the successful conduct of the FRAP experiment.

[0012] Preferably, the S6FRAP experimental observation process is as follows: A high-resolution, high-sensitivity confocal microscope is used, paired with a high numerical aperture oil immersion microscope. The oil immersion magnification is adjusted to ensure clear observation of the structure of a single cyanobacterial cell, ensuring efficient acquisition of fluorescence signals. Based on the fluorescence characteristics of calcein, the corresponding excitation wavelength is selected, and the imaging parameters of the microscope, including laser intensity, scanning speed, and pinhole size, are adjusted. The core principle of parameter adjustment is to obtain clear fluorescence images without damaging cells, avoiding weak fluorescence signals and cell photodamage due to improper parameter settings. The prepared sample is then placed in the microscope. On the microscope stage, adjust the focus using the coarse and fine adjustment knobs to select observation targets in the field of view that are morphologically intact, have appropriate filament length, and are evenly distributed without overlap. After determining the observation target, use the microscope's built-in area selection function to precisely select a single target cell as the bleaching and observation area. The selection range should ideally completely cover the target cell and not include adjacent cells. Activate the microscope's time-series imaging function and set a reasonable imaging interval to ensure that the signal changes before, after, and during the recovery process of fluorescence bleaching can be completely recorded, providing a continuous and complete image sequence for subsequent data extraction.

[0013] Preferably, the S7 observation program control process is as follows: Before starting the observation program, the laser intensity of the microscope is calibrated to ensure that the low-intensity laser will not cause photodamage to the cells, while ensuring stable acquisition of fluorescence signals; firstly, multiple frames of images are continuously acquired using the low-intensity laser as the fluorescence baseline, with the number of frames acquired being sufficient to calculate a stable average fluorescence intensity, and the initial fluorescence level of the target area is determined through the baseline images; after the baseline acquisition is completed, the program automatically switches to the high-intensity laser mode to perform rapid bleaching on the pre-selected target area, with the bleaching time ensuring that the fluorescence in the target area is completely quenched without affecting the fluorescence of surrounding cells, avoiding excessive bleaching that could damage the cell structure; after bleaching, the program immediately returns to the low-intensity laser acquisition mode, continuously capturing multiple frames of images at preset imaging intervals, with the acquisition time sufficient to observe that the fluorescence intensity has reached a stable recovery state; throughout the observation process, the image acquisition status is observed through the microscope's real-time monitoring function, promptly eliminating sudden problems such as vibration and light interference, ensuring a stable baseline before bleaching, an efficient bleaching process, and complete recording of the recovery process, thus guaranteeing the accuracy of experimental data.

[0014] Preferably, the S8 data extraction and preprocessing process is as follows: Open the data processing software supporting the confocal microscope, import the collected time-series images, accurately locate and select the target area through the region analysis function of the software, extract the average fluorescence intensity data of the target area in each frame of image, and record the corresponding acquisition time points at the same time; conduct a preliminary screening of the extracted fluorescence intensity data, set reasonable outlier judgment criteria, and eliminate abnormal data points caused by image interference and instrument fluctuations to ensure the authenticity of the data; adopt an appropriate smoothing processing method to denoise the screened data, reduce the influence of random noise on the data trend, and retain the true change law of the fluorescence intensity at the same time; when extracting the data of the target area, select an adjacent non-bleached cell area as the control area, extract the fluorescence intensity data of this area, and judge whether there is overall drift and attenuation of the fluorescence signal during the whole observation process through the control area data; associate and calibrate the target area data with the control area data, and perform standardized preprocessing on the target area data to provide a high-quality and low-noise data basis for subsequent normalization analysis and non-linear fitting.

[0015] Preferably, the S10 result verification and application process is as follows: Conduct multiple independent repeated experiments on cyanobacteria of the same type and under the same treatment conditions. Each experiment strictly follows the same operation process to ensure the repeatability of the experiment, and obtain multiple groups of kinetic parameters of cell-to-cell communication; conduct statistical analysis on the parameters of multiple groups of repeated experiments, calculate the mean, standard deviation and coefficient of variation of the parameters, and judge the consistency between groups of data through statistical tests to verify the reliability and stability of the kinetic parameters, and eliminate abnormal repeated group data; set wild-type cyanobacteria as the positive control, and at the same time set mutant cyanobacteria with communication-related gene deletions as the negative control, and conduct FRAP experiments under the same experimental conditions to obtain the kinetic parameters of the control strains; compare and analyze the parameters of the test strains and the control strains, and judge the strength of the cell-to-cell communication ability of the test strains through the parameter differences; combine the morphological characteristics of the fluorescence recovery curve to interpret the communication mode between cyanobacteria cells, such as fast normal communication, slow communication, one-way communication and communication block; apply the kinetic parameters and communication mode analysis results obtained from the experiment to the research on the cyanobacteria cell communication mechanism, and provide direct experimental evidence for exploring the function of cell-to-cell channels, screening compounds that regulate communication, and analyzing the influence of environmental factors on communication.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Break through the limitations of traditional static observation techniques. Through an optimized FRAP experimental process, directly capture the diffusion and recovery process of fluorescent molecules between cyanobacteria cells in real time, visually present the dynamic characteristics of communication, and provide direct kinetic evidence for analyzing the communication mechanism.

[0017] 2. An innovative agar inverted fixation method is adopted, which uses the weight and adsorption force of the agar block to achieve stable fixation of cyanobacterial cells, completely avoiding the bleaching-collection area misalignment problem caused by sample drift during the observation process, and greatly improving the success rate and repeatability of the experiment.

[0018] 3. By synchronizing and enriching cells through pretreatment, and removing free dyes through multiple rounds of centrifugation and washing, the background interference caused by cyanobacterial autofluorescence and free dyes is effectively eliminated, ensuring that the observed fluorescence signals all come from dye molecules involved in intracellular communication, thus improving data accuracy.

[0019] 4. The entire process emphasizes environmental adaptation, from precise control of culture conditions and gentle operation during staining and washing to humidification and temperature balance during sample adaptation, to always maintain the normal physiological state and communication ability of cyanobacteria and avoid cell damage or abnormal communication function caused by improper operation.

[0020] 5. By optimizing the imaging parameters and observation program control of the confocal microscope, a mode of low-intensity laser acquisition for baseline and image recovery and high-intensity laser for rapid bleaching is adopted. This ensures clear fluorescence signals and efficient bleaching while minimizing light damage and photoinhibition effects, thus ensuring that cells maintain normal function during observation.

[0021] 6. Through standardized data extraction, preprocessing, and nonlinear fitting processes, kinetic parameters such as fluorescence recovery rate constant and recovery plateau height can be accurately obtained, providing objective evidence for quantitatively comparing the communication capabilities of cyanobacteria under different strains and environmental conditions.

[0022] 7. The entire method, from cyanobacterial culture, staining, fixation to observation and data analysis, has established a standardized process. Through multiple independent repeated experiments and control verifications, the experimental results are ensured to be stable and reliable, avoiding experimental deviations caused by differences in operation.

[0023] 8. It is not only applicable to wild-type cyanobacteria, but also to the assessment of the communication capabilities of mutant strains. It can also be adapted to different experimental scenarios such as environmental stress and compound treatment, providing a powerful tool for research on the impact of environmental factors on communication, screening communication-regulating compounds, and elucidating the function of communication-related genes.

[0024] 9. The optimized experimental procedure does not require complex special equipment and can be carried out using a conventional confocal microscope. The steps are clear and highly operable, making it easy for different laboratories to learn from and apply, thus promoting the popularization and in-depth study of cyanobacterial cell communication.

[0025] 10. By accurately interpreting communication patterns, key experimental support is provided for revealing the functional characteristics and regulatory networks of intercellular channels, while also laying the foundation for the development of related microbial technologies. Attached Figure Description

[0026] Figure 1 This is a flowchart illustrating the steps of the present invention. Figure 2 This is a schematic diagram of a typical time-series fluorescence image of the FRAP process; Figure 3 This is a fluorescence recovery curve obtained after normalizing and nonlinearly fitting the FRAP experimental data of wild-type cyanobacteria in an embodiment of the present invention. Figure 4 This is a fluorescence recovery curve fitting diagram obtained after performing a FRAP experiment on a mutant strain with weakened communication ability according to an embodiment of the present invention; Figure 5 This is a schematic diagram illustrating abnormal communication patterns that may occur in mutant strains as shown in an embodiment of the present invention. Detailed Implementation

[0027] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0028] Reference Figure 1-5 As shown: S1. Cyanobacterial culture, the specific implementation is as follows: Strains were selected and prepared: Anabaena sp. PCC 7120 was selected as the core observation target. This strain has typical intercellular communication characteristics, and the experimental results are highly representative and reproducible. The strain was stored in a low light environment at 30 degrees Celsius, and the plates were streaked regularly. The revival process was carried out by streaking the plates into a liquid.

[0029] Directional expansion culture: The revived cyanobacterial bacterial suspension was inoculated into fresh BG11 liquid medium at a ratio of 1:10 and placed in a light incubator for directional culture; the culture parameters were set according to the physiological characteristics of the strain: light intensity 3000 Lux, constant temperature 30℃, continuous light environment, and shaking speed 180 rpm to ensure stable cell metabolic activity; sterile Erlenmeyer flasks were used as culture containers, and the liquid volume did not exceed 1 / 3 of the volume of the Erlenmeyer flask to ensure sufficient gas exchange.

[0030] Growth status monitoring: Samples were taken daily during the culture process, and cell morphology and growth status were observed using an optical microscope, with a focus on cell density, filament integrity, and the presence of abnormal aggregation; simultaneously, the absorbance (OD) of the bacterial culture at 750 nm was measured using a spectrophotometer. 750 ), record the growth curve; when OD 750 When the value reaches around 0.2, and under a microscope, the cell filaments are observed to be intact, evenly distributed, and without obvious aggregation or aging debris, the cells are considered to have entered the logarithmic growth phase.

[0031] Short-term cell preservation: When the cells reach the logarithmic growth phase and are in a homogeneous physiological state, stop the expansion culture; transfer the bacterial culture to sterile centrifuge tubes and preserve them by plate for one month.

[0032] S2, Cell Pretreatment This step involves synchronization and enrichment to remove impurities and ensure the cell population is at a uniform growth stage, providing a highly homogeneous and pure cell population for fluorescence staining. The specific implementation is as follows: Cell enrichment: The cyanobacterial culture in the logarithmic phase was transferred to a sterile centrifuge tube and centrifuged using a low-speed centrifuge at 3000 rpm for 5 minutes. The centrifugation force was strictly controlled to avoid mechanical damage to the cyanobacterial filaments caused by high-speed centrifugation. After centrifugation, the supernatant was carefully removed using a sterile pipette, and the cell population at the bottom was retained.

[0033] Resuspension and washing: Add fresh BG11 liquid culture medium, consistent with the culture system, to the centrifuge tube. Gently pipette the resuspended cells to ensure that the cells are evenly dispersed and do not clump together. Centrifuge again (with the same parameters as before). Repeat the resuspension-centrifugation process 2-3 times. This process effectively removes metabolic waste, aging cell debris, and residual culture medium impurities generated by the cells during culture.

[0034] Cell synchronization: After the final centrifugation, resuspend the cells in a small amount of fresh BG11 medium and adjust the bacterial concentration to OD. 750 =Approximately 0.3; Place the resuspended bacterial solution in a light incubator and continue culturing for 12 hours under the original culture conditions. With the synergistic effect of centrifugation and resuscitation, most cells are in the same growth cycle stage, reducing the problem of uneven staining efficiency caused by cell cycle differences. Finally, a cell population with strong uniformity and high purity is obtained, which improves the stability of subsequent experimental data.

[0035] S3, Fluorescent staining and washing This step involves specifically fluorescently labeling intracellular diffusing molecules while thoroughly removing free dye and reducing background fluorescence interference. The specific implementation is as follows: Dye preparation: Dimethyl sulfoxide (DMSO) was selected as the solvent, which has good compatibility with cyanobacterial cells and is non-cytotoxic. An appropriate amount of calcein acetylated methyl ester powder was weighed to prepare a high-purity stock solution with a concentration of 1 mg / mL. The preparation process was carried out in a sterile laminar flow hood, strictly following aseptic operation procedures to avoid solvent contamination and dye degradation. After the stock solution was prepared, it was stored in a -20℃ refrigerator in the dark. Before use, it was thawed at room temperature and gently mixed.

[0036] Fluorescent staining: Based on cell concentration and staining requirements, take 10 μL of calcein methyl ester stock solution and slowly add it to 250 μL of enriched cyanobacterial culture. While adding, gently stir with a sterile pipette to ensure that the dye and the culture are fully and evenly mixed. Place the mixed culture in an environment consistent with the culture conditions (temperature 30℃, light intensity 3000 Lux, shaking speed 180 rpm) and incubate with shaking. The shaking speed should be such that it does not damage the cell structure. Shaking promotes the contact between the dye and the cell surface and accelerates the dye's entry into the cell. The incubation time is strictly controlled to 1 hour.

[0037] Washing to remove free dye: After incubation, transfer the bacterial culture to a sterile centrifuge tube and centrifuge at 3000 rpm for 5 minutes to remove the supernatant containing free dye. Add fresh BG11 medium pre-warmed to 30°C to the centrifuge tube, gently resuspend the cell pellet, and centrifuge again. Repeat this washing process at least 3 times. After each wash, take a small amount of supernatant and drop it onto a fluorescence microscope slide. Preliminarily detect the fluorescence signal in the supernatant using a fluorescence microscope until there is no obvious fluorescence signal in the supernatant. This ensures that the background fluorescence interference caused by free dye is completely eliminated, and the fluorescence signals observed subsequently all come from dye molecules inside the cells.

[0038] S4. Sample Fixation This step employs an innovative agar inversion method to achieve stable fixation of cyanobacterial samples, avoiding misalignment between the bleaching area and the collection area due to sample displacement during observation. The specific implementation is as follows: Preparation of solid culture medium: Based on BG11 liquid culture medium adapted to cyanobacteria, add agar powder to achieve a final concentration of 1.2% (w / v), stir evenly, and then autoclave (121℃, 20 minutes); after sterilization, when the temperature of the culture medium drops to 50-60℃, pour it into sterile petri dishes while it is still hot, about 20mL per petri dish, place them horizontally on a sterile operating table, and let them cool and solidify naturally to form a uniform and flat agar plate.

[0039] Agar block cutting and sample loading: Use a sterile scalpel to cut the solidified agar plate into 2cm×2cm square agar blocks. During the cutting process, avoid breaking or contaminating the edges of the agar blocks to ensure the integrity of the agar block structure. Use a sterile pipette to draw 20μL of stained and washed cyanobacterial bacterial solution and slowly drop it onto the surface of the agar block. The amount of solution added should just cover the surface of the agar block without overflowing. Place the agar block containing the bacterial solution in a sterile laminar flow hood and let it stand and dry for about 20 minutes, until some of the water in the bacterial solution evaporates and the cyanobacterial filaments are initially adsorbed onto the agar surface, ensuring that a stable adsorption is formed between the cells and the agar.

[0040] Inverted fixation: Carefully pick up the agar block containing the sample with sterile tweezers and invert it (bacterial liquid side down) to fit it into the center of the confocal microscope's special glass bottom dish, ensuring that the agar block and the glass bottom dish are tightly attached without any air bubbles. Utilize the weight of the agar block itself and the adsorption force generated by water evaporation to stably fix the cyanobacterial cells to the glass bottom surface. After fixation, place the glass bottom dish in a sterile laminar flow hood for later use to avoid external contamination.

[0041] S5. Sample Adaptation Processing This step maintains cell physiological activity until the end of the observation by moisturizing and balancing the environment, ensuring normal cell communication function. The specific implementation is as follows: Moisturizing treatment: After the sample is fixed, use a sterile pipette to drop a small amount of fresh BG11 liquid culture medium around the agar block in the glass dish. The amount of culture medium should be just enough to wet the edge of the agar block without submerging it, so as to create a local moisturizing environment and prevent the cells from dehydrating and becoming inactive due to rapid evaporation of water during the observation process.

[0042] Temperature equilibration: Place the glass dish containing the sample in a constant temperature incubator that is the same as the cyanobacterial culture temperature (30℃) for equilibration. The equilibration time is adjusted according to the difference in ambient temperature, generally 30 minutes, to ensure that the sample temperature is completely consistent with the culture temperature and to avoid the impact of temperature changes on cell physiological function.

[0043] Observation environment control: After equilibration, the glass-bottomed dish is transferred to the sample stage of the confocal microscope. The light in the observation environment is controlled to avoid direct strong light on the sample, which could cause photodamage to cells and fluorescence quenching, thus maintaining the stability of the observation environment. During the equilibration process, the cell status is observed through the microscope every 10 minutes to confirm that the cells are morphologically intact and there is no abnormal shrinkage or death. This ensures that the cells maintain normal physiological functions and intercellular communication capabilities throughout the observation period, providing a guarantee for the smooth conduct of the FRAP experiment.

[0044] S6, FRAP experimental observations This step utilizes confocal microscopy to achieve time-series imaging of laser bleaching and fluorescence recovery in the target cell region, obtaining clear and continuous fluorescence signal change data. The specific implementation is as follows: Microscope preparation: Select a high-resolution, high-sensitivity confocal microscope (such as Zeiss LSM900) with a 63x / 1.4NA oil immersion lens. This oil immersion lens magnification allows for clear observation of the structure of individual cyanobacterial cells, ensuring efficient acquisition of fluorescence signals. Based on the fluorescence characteristics of calcein, select 488nm as the excitation wavelength. Adjust the imaging parameters of the microscope, such as laser intensity, scanning speed, and pinhole size. The initial parameters are set as follows: laser intensity 0.4%, scanning speed 8, pinhole size 191μm, image resolution 512×512, 16-bit image format, and unidirectional scanning direction. The core principle of parameter adjustment is to obtain clear fluorescence images without damaging cells, avoiding weak fluorescence signals and cell photodamage due to improper parameter settings.

[0045] Target selection: Place the prepared sample on the microscope stage, adjust the stage height using the coarse adjustment knob, find the sample focusing surface, and then switch to the fine adjustment knob for precise focusing; slowly move the stage in the field of view to select cyanobacterial filaments with intact morphology, appropriate filament length (5-10 cells connected), and uniform cell distribution without overlap as observation targets, avoiding areas with broken filaments, deformed cells, or severe aggregation.

[0046] Target area selection and imaging initiation: After determining the observation target, use the microscope's built-in area selection function to precisely select a single target cell on the screen as the bleaching and observation area through software operation. The selection range should ideally completely cover the target cell and not include adjacent cells. The selected area should be circular in shape, with a diameter approximately 1.2 times the cell diameter. Initiate the microscope's time-series imaging function, setting the imaging interval to 1 second and the total acquisition time to 60 seconds. This ensures that the signal changes before, after, and during the recovery process of fluorescence bleaching can be completely recorded, providing a continuous and complete image sequence for subsequent data extraction.

[0047] S7, Observation Procedure Control This step automates baseline acquisition, target bleaching, and reconstruction imaging through a pre-programmed procedure, ensuring standardized experimental procedures and reliable data. The specific implementation is as follows: Laser intensity calibration: Before starting the observation program, the laser intensity of the microscope is calibrated. The low-intensity laser output is adjusted through the microscope's software to ensure that the 0.4% laser intensity will not cause photodamage to the cells, while also ensuring stable acquisition of fluorescence signals. After calibration, the parameter settings are saved to avoid parameter drift in subsequent operations.

[0048] Baseline acquisition: First, five frames of images were continuously acquired using a low-intensity laser of 0.4% as the fluorescence baseline. The acquisition interval for each frame was 1 second. The number of frames acquired was based on the ability to calculate a stable average fluorescence intensity. The initial fluorescence level of the target area was determined by the baseline images, providing benchmark data for subsequent calculation of fluorescence recovery percentage.

[0049] Target area bleaching: After baseline acquisition, the program automatically switches to high-intensity laser mode, with the laser intensity adjusted to 10%, to rapidly bleach the pre-selected target area. During the bleaching process, 40 frames are continuously scanned at the current fastest scanning speed, with the total bleaching time controlled within 2-3 seconds to ensure that the fluorescence in the target area is completely quenched, while avoiding excessive bleaching that could damage cell structures. During the bleaching process, real-time monitoring confirms that the fluorescence intensity in the target area has decreased significantly, and that the fluorescence of surrounding cells has not been significantly affected.

[0050] Imaging during the recovery process: After bleaching, the program immediately switches to a low-intensity laser acquisition mode of 0.4%, continuously capturing 95 frames of images at a preset 1-second imaging interval. Combined with the previous 5 baseline frames, the total number of frames acquired is 100, with an acquisition time of 60 seconds, ensuring that the fluorescence intensity reaches a stable recovery state. Throughout the observation process, the image acquisition is monitored in real-time using the microscope's monitoring function to promptly eliminate unexpected problems such as vibration and light interference. If image blurring or signal drift occurs, the experiment is immediately stopped and the sample is prepared again to ensure a stable baseline before bleaching, an efficient bleaching process, and complete recording of the recovery process, thus guaranteeing the accuracy of the experimental data.

[0051] S8. Data Extraction and Preprocessing This step extracts fluorescence intensity data from time-series images and performs noise reduction and calibration preprocessing to provide high-quality data for subsequent analysis. The specific implementation is as follows: Data extraction: Open the data processing software (such as Zen3.0) that comes with the confocal microscope and import the 100 time-series images acquired; use the software’s region analysis function to accurately locate and reselect the previously selected target cell region; the software automatically extracts the average fluorescence intensity data of the target region in each frame of the image and records the corresponding acquisition time point (timing from the baseline acquisition, in seconds); export the extracted data as an Excel file for subsequent processing.

[0052] Outlier handling: The exported fluorescence intensity data is initially screened, and an outlier judgment standard is set: when the fluorescence intensity value of a certain frame exceeds ±30% of the average intensity of the adjacent 5 frames, it is judged as an outlier; outliers are manually removed. If there is data missing after removal, linear interpolation is used to supplement it to ensure the continuity of data; outliers mainly come from factors such as image interference and instrument fluctuations. Removing them can ensure the authenticity of the data.

[0053] Noise reduction: The filtered data were denoised using the moving average method. The moving window size was set to 3 frames, meaning that the fluorescence intensity value at each time point was replaced with the average of the data at that point and the data in the frames before and after it. This reduced the impact of random noise on the data trend while preserving the true variation pattern of fluorescence intensity. After noise reduction, the original fluorescence intensity variation curve was plotted to observe the recovery trend.

[0054] Control calibration: While extracting data from the target region, adjacent unbleached cell regions within the same field of view are selected as control regions using software (these regions are at a suitable distance from the target region to avoid the influence of bleaching, and the cell states are consistent). Fluorescence intensity data of these regions are extracted. The control region data is used to determine whether there is overall drift or attenuation of the fluorescence signal throughout the observation process. If drift exists, the fluorescence intensity values ​​of the target region at each time point are divided by the fluorescence intensity values ​​of the control region at the corresponding time point for correlation calibration. This standardizes and preprocesses the target region data, providing a high-quality, low-noise data foundation for subsequent normalization analysis and nonlinear fitting.

[0055] S9. Data Processing and Analysis This step obtains the dynamic parameters of intercellular communication through normalization and nonlinear fitting, enabling quantitative analysis. The specific implementation is as follows: Data normalization: Open the Excel file, calculate the average fluorescence intensity value of the baseline images of the 5 frames before bleaching, and set the average value to 1.0 (i.e. 100%); divide the fluorescence intensity value of the target area at each time point after bleaching (after calibration) by the baseline average value to obtain the normalized fluorescence recovery percentage sequence; the normalized data eliminates the influence of the initial fluorescence intensity difference, which is convenient for comparison between different experiments.

[0056] Nonlinear fitting: Import the normalized "time-fluorescence recovery percentage" data into GraphPadPrism 9.3.0 software and select the "One phase association" nonlinear regression model. The fluorescence recovery curve was generated by fitting the data, where... It is fluorescence in bleached cells. It occurs immediately after bleaching and tends to occur after full recovery. E is the dye exchange coefficient between cells, usually measured in s⁻¹, which reflects the speed at which fluorescent molecules diffuse through intercellular channels and is a key kinetic parameter for measuring communication efficiency. t is time.

[0057] Dynamic parameter extraction: Key dynamic parameters are extracted from the fitting results, including the recovery rate constant, the height of the recovery plateau, and the goodness of fit. The recovery rate constant E is a core parameter for measuring the efficiency of intercellular communication; a larger value indicates higher communication efficiency. Goodness of fit... The closer the result is to 1, the better the data fit and the more reliable the result. The extracted parameters are organized and recorded to form a quantitative analysis report.

[0058] S10. Result Verification and Application This step verifies the reliability of the data through repeated experiments and control analysis, and determines the communication mode and capability based on the parameters, providing a basis for related research. The specific implementation is as follows: Reproducible experiments were conducted at least 10 times for the same type of cyanobacteria under the same treatment conditions. Each experiment strictly followed the same operating procedures to ensure reproducibility. Statistical analysis was performed on the kinetic parameters of the 10 replicate experiments, calculating the mean, standard deviation, and coefficient of variation. One-way ANOVA was used to assess the consistency between the data groups. If the coefficient of variation was less than 15% and the ANOVA analysis showed no significant difference between groups (P>0.05), the reliability and stability of the kinetic parameters were verified. Abnormal replicate data with a coefficient of variation greater than 20% were removed, and the valid data groups were retained for subsequent analysis.

[0059] Control Experiment Setup and Analysis: Two control experiments were set up: one as a positive control (wild-type cyanobacterium Anabaena sp. PCC 7120) and the other as a negative control (a mutant cyanobacterium lacking the communication-related gene SepJ). FRAP experiments were conducted under the same experimental conditions to obtain the kinetic parameters of the two control strains. The parameters of the test strain (e.g., cyanobacterium after environmental stress treatment) were compared and analyzed with those of the positive and negative controls. If the recovery rate constant E of the test strain was close to that of the positive control (difference less than 20%), and the recovery plateau phase was highly significant... ≥80% is considered rapid normal communication; if E is 30%–70% of the positive control, it is considered slow communication; if only adjacent cells on one side show fluorescence recovery, it is considered unidirectional communication; if E is close to the negative control (difference less than 15%), and If the percentage is ≤30%, it is considered a communication blockage.

[0060] Results Applications: Combining the morphological characteristics and kinetic parameters of fluorescence recovery curves, we interpreted the communication patterns between cyanobacterial cells. The experimental results were applied to the study of cyanobacterial cell communication mechanisms: First, we explored the function of intercellular channels by comparing parameter differences between wild-type and mutant strains to verify the function of communication-related genes; second, we screened compounds that regulate communication by adding test compounds to the culture system and judging their promoting or inhibiting effects on communication through parameter changes; third, we analyzed the influence of environmental factors on communication by simulating environmental conditions such as temperature stress and nutrient deprivation to study their regulatory mechanisms on cyanobacterial communication efficiency, providing direct experimental evidence for a deeper understanding of the multicellular cooperation mechanism of cyanobacteria.

[0061] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A method for observing cyanobacterial cell communication using FRAP technique, characterized in that, The specific method and process are as follows: S1. Cyanobacterial culture: Construct a cyanobacterial culture system under normal physiological conditions to ensure that cell viability meets the requirements for subsequent staining and observation. S2. Cell pretreatment: Synchronize and enrich the cultured cyanobacteria to provide a uniform cell population for fluorescent staining. S3. Fluorescent staining and washing: Calcein acetylated methyl ester was used to specifically stain cyanobacteria and remove free dye to reduce background fluorescence interference. S4. Sample fixation: The stained cyanobacteria samples are stabilized and fixed using the agar inverting method to prevent displacement during observation. S5. Sample adaptation treatment: The fixed sample is moisturized and adapted to the observation environment to maintain cell physiological activity until the end of the observation. S6 and FRAP experiments were conducted by using a confocal microscope to select target cell regions and performing time-series imaging of the laser bleaching and fluorescence recovery process. S7. Observation program control, completes continuous imaging of baseline acquisition before bleaching, target area bleaching and recovery process after bleaching according to preset procedures; S8. Data extraction and preprocessing: Extract fluorescence intensity data of the target area from the time series image and perform noise reduction and standardization preprocessing. S9. Data processing and analysis: Normalize the preprocessed fluorescence intensity data and obtain the kinetic parameters of intercellular communication through nonlinear fitting. S10. Result verification and application: Verify the reliability of the kinetic parameters through repeated experiments and control analysis, and determine the cyanobacterial cell communication patterns and capabilities based on the parameters.

2. The method of observing cyanobacterial cell communication using FRAP technique according to claim 1, wherein, The S1 cyanobacterial culture process is as follows: Filamentous cyanobacteria were selected as the core observation target, with priority given to model strains exhibiting typical intercellular communication characteristics to ensure the representativeness and reproducibility of experimental results. The selected cyanobacteria were inoculated into suitable liquid culture media and placed in a light incubator for directional culture. Light conditions, constant temperature, and gas environment were set according to the physiological characteristics of the strains to ensure stable cellular metabolic activity. During the culture process, samples were taken periodically to observe cell morphology and growth status. Cell density, filament integrity, and the absence of abnormal aggregation were observed under a microscope to determine whether the cells had entered the logarithmic growth phase. When the cells reached the logarithmic growth phase and their physiological state was uniform, the culture was stopped, and the bacterial culture was preserved.

3. The method of observing cyanobacterial cell communication using FRAP technique as claimed in claim 1, wherein, The S2 cell pretreatment process is as follows: The cyanobacterial culture in the logarithmic growth phase was transferred to a sterile centrifuge tube and centrifuged. After centrifugation, the supernatant was removed, and the cell population at the bottom was collected. The cells were gently resuspended in fresh culture medium consistent with the culture system to ensure that the cells were evenly dispersed without aggregation. The resuspension-centrifugation process was repeated 2-3 times to remove metabolic waste, aging cell debris, and residual culture medium impurities generated by the cells during culture. At the same time, the cell population was synchronized based on the synergistic effect of centrifugation and resuspension.

4. The method of observing cyanobacterial cell communication using FRAP technique as claimed in claim 1, wherein, The S3 fluorescent staining and washing process is as follows: A high-purity methyl calcein stock solution was prepared using a solvent with good compatibility with cyanobacterial cells and no cytotoxicity. Aseptic techniques were followed during preparation to avoid solvent contamination and dye degradation. Based on cell concentration and staining requirements, the stock solution was slowly added to the enriched cyanobacterial culture while stirring continuously to ensure thorough and uniform mixing of the dye and culture. The mixed culture was then incubated in an environment consistent with the culture conditions, with the shaking speed carefully controlled to avoid damaging the cell structure. Shaking promoted contact between the dye and the cell surface, accelerating dye entry into the cells. After incubation, the cells were washed by centrifugation using fresh culture medium pre-warmed to culture temperature. After centrifugation, the supernatant containing free dye was removed, and the cells were resuspended in fresh culture medium. This washing process was repeated at least three times. Fluorescence detection was used after each wash to assess the removal of free dye until no fluorescence signal was detected in the supernatant.

5. A method for observing cyanobacterial cell communication using FRAP technology according to claim 1, characterized in that, The S4 sample fixation process is as follows: Based on a liquid culture medium suitable for cyanobacteria, agar powder was added to prepare a solid culture medium, which was then autoclaved after preparation. The sterilized solid culture medium was poured into sterile petri dishes while still hot and placed horizontally to cool and solidify naturally, forming a uniform and flat agar plate. The solidified agar plate was cut into square agar blocks using a sterile scalpel. The stained cyanobacterial bacterial solution was aspirated with a sterile pipette and dropped onto the surface of the agar block. The agar block containing the bacterial solution was placed in a sterile workbench to dry. Finally, the agar block was inverted so that the bacterial solution side down was attached to the center of a small glass dish for confocal microscopes.

6. A method for observing cyanobacterial cell communication using FRAP technology according to claim 1, characterized in that, The S5 sample adaptation process is as follows: After sample fixation, fresh culture medium is added around the agar block in the glass dish, ensuring the medium covers the edge of the agar block but does not submerge it, creating a locally moist environment. The glass dish containing the sample is then placed in a constant temperature environment consistent with the cyanobacterial culture temperature for equilibration. Simultaneously, the light in the observation environment is controlled, and the cell state is periodically observed through a microscope during the equilibration process to confirm that the cells are intact and show no abnormal shrinkage or death.

7. A method for observing cyanobacterial cell communication using FRAP technology according to claim 1, characterized in that, The observation process of the FRAP experiment in S6 is as follows: A confocal microscope with an oil immersion lens of appropriate magnification was used to excite fluorescence at the corresponding excitation wavelength. The imaging parameters of the microscope were adjusted to ensure clear images without damaging the cells. Cyanobacterial filaments with intact morphology and uniform distribution were selected in the field of view. A single target cell was selected as the observation area, and the time-series imaging function was activated to record the dynamic changes of fluorescence signals.

8. A method for observing cyanobacterial cell communication using FRAP technology according to claim 1, characterized in that, The S7 observation program control process is as follows: First, multiple frames of images are acquired using a low-intensity laser as the fluorescence baseline; then, the high-intensity laser is switched to quickly bleach the selected target area to ensure that the fluorescence in the area is effectively quenched; after bleaching, the low-intensity laser acquisition mode is immediately restored, and multiple frames of images are continuously captured to fully record the entire process of fluorescence in the target area from quenching to gradual recovery.

9. A method for observing cyanobacterial cell communication using FRAP technology according to claim 1, characterized in that, The S8 data extraction and preprocessing process is as follows: using microscope software, the average fluorescence intensity data of the target area is extracted from the acquired time-series images; outliers in the data are removed, and noise interference is reduced through smoothing; at the same time, fluorescence intensity data of adjacent unbleached areas are extracted as a control to provide a reference benchmark for subsequent normalization processing.

10. A method for observing cyanobacterial cell communication using FRAP technology according to claim 1, characterized in that, The verification and application process of S10 results is as follows: Multiple independent replicate experiments were conducted on the same type of cyanobacteria to obtain multiple sets of kinetic parameters. The consistency and reliability of the parameters were verified by statistical analysis. Wild-type and mutant cyanobacteria were set as controls to compare the differences in parameters among the groups and determine the strength of cyanobacterial cell communication under different conditions. Based on the kinetic parameters and fluorescence recovery curves, the communication patterns between cyanobacterial cells were interpreted.

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