| The function of these proteins is determined by the structure of proteins.At the same time,information exchange and transmission between proteins and proteins plays an important role in the signal transduction system,coordinate the function between cells and regulate cell growth and metabolism.Therefore,cell research has attracted extensive attention,and super-resolution technology has made great contributions in this regard.Some of these technologies depend on advanced optical settings,while others use the properties of fluorescent proteins or dyes to break through optical diffraction and improve resolution.But this does not mean that it is fully capable of resolving the structural distribution of the protein.In recent years,an expansion microscopy(ExM)technique that physically expands samples has indirectly improved resolution,enabling structures that cannot be distinguished before expansion to be distinguished after expansion.Therefore,this research combined expansion microscopy with super-resolution technology direct Stochastic Optical Reconstruction Microscopy to analyze the protein distribution on cell membrane.The contents of the study are as follows:1.First,in order to select the best staining method,by confocal imaging of different labeling methods of EGFR protein,the conclusion is that the best staining method is pre gel staining.In order to determine the effect of this method on the dye,the average fluorescence intensity of the 100 groups before and after gel was calculated to proved that this labeling method does not quench the fluorescent dye.Using the expansion microscopy technology,the integrity of the cell membrane can be ensured.After the expansion of A549 cells,the distribution of EGFR protein was observed by dSTORM,and the gel of the imaging base was expanded to ensure the shape of the gel.When EGFR on unexpanded cells was used as the control group,there is almost no difference between the results obtained by sealing operation and open system imaging.Therefore,a simple sealing operation was chosen for pre-expanded cell imaging.By comparing the distribution of EGFR protein on A459 basal membrane before and after expansion,it find that the expanded cluster information changes significantly.Cluster information includes the cluster diameter,the cluster localization density and the cluster density,localizations per cluster and the cluster proportion,because the protein molecules on the cell membrane are pulled further after the cells expand.The distance between the proteins before and after expansion was measured.The data again proved that the distance between the extended proteins increases after expansion.The farther the molecular distance is,the easier it is to be resolved.The molecules between the protein clusters can be more clearly located.2.In order to explore whether the same protein on different cells can be expanded to the same extent by expansion microscopy,this research combined dSTORM with dSTORM to observe the distribution of EGFR on Hela cell membrane under the above optimal conditions,and compared the results with those of A549.The conclusion is as follows:(1)The cluster information after expansion are significantly smaller than those of the clusters before expansion,and the distance between proteins and proteins are increased,which is consistent with the results on the A549 cell membrane.(2)The expansion degree of EGFR on Hela cell membrane is different from that on A549 cell membrane,and the expansion degree of EGFR on Hela cell membrane is large.The results show that the larger the cluster diameter of protein clusters is,the more dense the clusters is,the greater expansion the expansion microscopy displays.Expansion depends on clustering before expansion.Then the distribution of GLUT proteins on Hela cell membrane of the same cell was explored,and compared with EGFR on Hela cell membrane,the conclusion is similar to that above.In the end,other ways of expanding the cell were explored,with no satisfactory results. |
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