Study On Lipid Rafts Structures Based On Single Molecule/Particle Localization With Nano-resolution

Lipid rafts are sphingolipid-and cholesterol-enriched nanoscale domains floating within the plasma membrane and fluctuating in size and composition.The classic biochemical method for lipid rafts study is membrane extraction,which is complicated and might change properties of rafts.The conventional optical microscope provides a spatial resolution(about 250 nm)insufficient for observing the details of rafts with a diameter of(10~200 nm).In recent years,with the development of super-resolution fluorescence microscopy,there are several microscopy approaches that break the diffraction-limit,achieving lateral(x,y)spatial resolution of 10~20 nm and 20~50 nm in the axial(z),hence becoming a powerful research tool for people to observe and study the lipid rafts.In this paper,experiments and methods of lipid rafts structures imaging are studied.Based on the single molecule / particle localization nanometer resolution microscopic imaging technique,the super-resolution nanometer imaging of the lipid rafts structures was realized.The results of nanometer images were compared with those of different cell lines.The differences of nano-images between normal cells and abnormal cells was obtained.In addition,in order to obtain the three-dimensional dynamic nanostructures on the lipid rafts and to realize the dynamic tracing of the rafts in the whole cell,a dynamic tracking method of large depth of field and multi-particle is proposed.It is proved that the method can realize multi-particle simultaneous three-dimensional nano-resolution localization and tracking in 10 ?m depth of field.The main work of this paper is as follows:1.The three-dimensional resolution of the single molecule / particle localization superresolution imaging system built by the laboratory was measured.The experimental results show that the three-dimensional spatial resolution of the system is 22 nm in the lateral and 52 nm in the axial,which satisfies the scale of the lipid rafts structures.2.For the application of lipid rafts structures nanometer imaging,the relevant experimental conditions were optimized.The optimal experimental conditions of PH value,medium solution,exposure time and laser power were determined,and the super-resolution nanometer imaging of the lipid rafts structures was realized after the drift correction.The nanometer images of lipid rafts structures of different cell lines were analyzed and discussed.The differences of normal and abnormal cell lipid rafts structures were studied.3.A large depth of field 3D dynamic tracking method is proposed.The theoretical analysis of distorted grating multi-stage imaging is carried out.How to realize three-dimensional nano localization by dual-objective detection of Bi-focal imaging method is introduced.Based on Matlab,the numerical simulation is carried out,and the feasibility of the method is verified.The innovation point of this paper is:1.The nanometer images of lipid rafts structures of different cell lines were achieved,and their nanotic characteristics were obtained.And the difference of the nanostructures of normal and abnormal cell rafts was obtained.These findings will provide the basis for research in related fields.2.We proposed a dynamic tracking method of large depth of field and multi-particle.Based on this method,it is expected to achieve simultaneous multi-target and three-dimensional nanoresolution localization and tracking in living cells.