Application Of Expansion Microscopy In The Study Of Cell Membrane Protein Distribution

In recent years,super-resolution fluorescence technology can not only specifically label proteins on ell membranes,but also achieve single-molecule precision and nano-scale imaging,which is vital of studying the protein distribution characteristics on cell membranes.However,the tight distribution of proteins on cell membranes is a new challenges for protein labeling.Expansion microscopy amplifies biological samples and breaks optical diffraction,which increases the distance of closely aligned biomolecules.In this paper,Expansion microscopy and direct stochastic optical reconstruction microscopy（dSTORM）were combined to study the distribution of proteins on the cell membrane.The main research progress is as follows:A549-GFP cells were adhered to the gel by Expansion microscopy.The cells before and after expansion were imaged by confocal microscope.The results showed that A549-GFP cells increased with the expansion of the gel.The expansion ratio of cells is related to expansion time and ion content in the expasnion solution.In deionized water,the cells can be expanded by a maximum multiple,up to 2.51±0.47 times in all directions.In order to observe the distribution of proteins on the cell membrane,this paper analyzed and contrasted the different ways.Finally,the expanded cell membrane on the expanded gel.after cell membranes was covalently linked to the gel could be tore.This work provides support for further study of protein distribution on cell membranes.The expanded cell membrane prepared above was specifically labeled with antibodies and imaged using dSTORM.It was confirmed that the distribution of Band III protein on the expanded cell membrane was more dispersed,and the density of localizations decreased from 475.4±27.73μm^-2 to 386.9±40.86μm^-2,the cluster density decreased from 1.373±0.0898μm^-2 to 1.126±0.1066μm^-2.The average area of protein clusters on the cells decreased from 11866±649.8 nm² to 5749±546.4 nm².The average number of localizations in the cluster was decreased from 140.1±9.927 to 116.1±6.737.The distance between individual Band III proteins increased from 47.61±2.62 nm to77.81±5.21 nm.The imaging of the outer membrane EpCAM protein by dSTORM further confirmed that the diameter of the protein cluster on the cell membrane became smaller as the gel expanded,the number of protein localizations decreased,and the distance between individual proteins increased.It increased from 39.53±3.53 nm before expansion to 92.11±6.75 nm after expansion about 2.3 times.Compared with antibodies,RNA aptamer is smaller in size,higher in labeling efficiency,and the distance after the expansion of the fluorescent probe was more increased,which is more favorable for imaging.The results of this study confirmed the combination of Expansion microscopy and single-molecular localization microscopy.It was important to study the distribution characteristics of proteins on cell membranes and to elucidate the morphological structure of protein clusters.