Study Of High Resolution Scanning Electron Microscopy Of Tumor Cell Membrane Proteins

Breast cancer is a malignant tumor with a high incidence of women,accounting for the second of the female malignant tumors.The mortality rate in China is on the rise.Triple negative breast cancer?TNBC?is a special type of breast cancer which is negative for estrogen receptor,progesterone receptor and human epidermal growth factor receptor,accounting for about from 15%to 20%of breast cancer.Due to the lack of effective targeted therapy,the prognosis of the disease is poor and the mortality rate is high.Epidermal growth factor receptor?EGFR,HER1?and receptor tyrosine kinase?c-Met?are highly expressed in TNBC.The high expression of membrane proteins is closely related to the proliferation,migration and invasion,and drug resistance of tumor cells.Through the formation of homodimer and heterodimer,EGFR and c-Met activate tyrosine protein kinase activity and induce downstream protein phosphorylation to initiate downstream signaling pathway to promote and regulate tumor cell proliferation,angiogenesis and tumor metastasis.HER1 and c-Met are important drug targets for the treatment of TNBC,but their distribution and aggregation on TNBC cell membrane are not clear.Biological imaging plays an important role in cell biology,molecular biology,molecular genetics,oncology,pathology,immunology and other fields.However,the ordinary optical microscope and fluorescence microscope are limited by the diffraction limit of light and can not recognize single membrane protein.Electron microscopy has nano-atomic scale spatial resolution and is a high-resolution imaging technique for identifying biological ultrastructures.In this paper,the field emission environment scanning electron microscope?ESEM?and the observation platform based on ESEM are used to realize the specificity of single-molecule level,accurately locate and quantify the specificity of nanoprobe and fluorescent probe.The spatial distribution,aggregation state,and quantitative expression of the labeled EGFR?HER1?and c-Met membrane proteins of TNBC cells were obtained as follows.1.Liquid environment ESEM observation platform mainly includes:homemade scanning transmission imaging device?STEM-in-SEM?and liquid chip device.In addition,combined with high-resolution cathodoluminescence?CL?spectrometers,and a variety of imaging modes,including scanning transmission?STEM?electronic images,secondary electron?SE?images,backscattered electron?BSE?images,and cathodoluminescence?CL?)images.Using these techniques,as well as a variety of electron and photon imaging modes,the high expression characteristics of membrane proteins in tumor cells was studied.2.A high resolution image of TNBC cells in a liquid environment scanning electron microscope was obtained for the first time.The membrane protein HER1specifically calibrated by ZnS@CdSe quantum dots?QDs?was precisely identified.The preferred distribution of HER1,and the aggregation state,including monomers,dimers,and multimers,were studied.The liquid-state STEM image has a resolution of approximately 7 nm and a magnification of 400,000×.Compared with the traditional scanning electron microscope imaging method,the liquid environment STEM imaging method improves the image resolution,image contrast and SNR,and reduces the radiation damage of the high energy electron beam to the biological sample.3.The high-resolution,cross-scale observation of the location,distribution,and aggregation state of HER1 and c-Met specifically labeled by nano-Au particles?Au NPs?on TNBC whole cells were realized.Secondary electron?SE?images and backscattered electron?BSE?images have resolutions better than 10 nm and magnifications from 50×to 200,000×.4.The whole cell high expression profile of HER1 and c-Met was obtained for the first time,and the quantitative expression of membrane proteins?for more than 20cells,more than 3,000 high-resolution images,and statistics of about one million proteins.1?The expression levels of HER1 and c-Met at the cell edge increased;2)the expression level of HER1 in the cell edge fold region was significantly higher than that in the marginal flat region;3)HER1 monomer,dimer and multimer in whole cells The expression levels are approximately 40%,12%,and 48%,respectively.The preferential distribution characteristics and quantitative data of the obtained membrane protein dimer provide accurate localization information and drug targets for studying the downstream function and intracellular signaling mechanism of the dimer.5.Applying ligand to induce TNBC cells,regulating the aggregation state of TNBC membrane protein c-Met,and promoting high expression of c-Met dimer.This provides a precise visualization of the downstream functions of the dimer,the interactions between the proteins,and the response of the tumor cells to the drug.6.High-resolution cathodoluminescence?CL?images of TNBC cells transfected with fluorescent probes in a liquid environment were obtained for the first time.It was detected that the edge of TNBC cells specifically transfected with the red fluorescent probe?Cy5?was at a position where the fluorescence quantum yield was high.This is consistent with the high expression of HER1 identified by QDs and Au NPs probes in electronic images?STEM,BSE,and SE images?.In addition,the CL image clearly shows the tumor cell substructure transfected with the enhanced green fluorescent protein?EGFP?probe and the blue fluorescent?DAPI?probe.The CL image has a resolution of about 10 nm and is a high resolution fluorescence imaging technique that recognizes cellular substructures to a single membrane protein.6.Different types of materials were used as substrates for incubating cell.The high resolution STEM images of membrane proteins were obtained on the Si₃N₄ thin window substrate in liquid cell.The charging effect of cells is eliminated,and the imaging quality is improved on the ITO.The hybrid SiO₂-Au@Si film with surface plasmon resonance?SPR?enhancement effect was used to enhance the quantum yield of CL.7.Different types of materials are used as substrates for cell climbing.Including:the high resolution STEM image of membrane protein was obtained in solution by using Si3N4 thin window of liquid chip.the charging effect of biological samples such as cells was eliminated and imaging quality was improved by using ITO conductive glass substrate.The quantum yield of cathodoluminescence?CL?was increased by using the SiO2-Au@Si composite film with surface plasmon resonance?SPR?.In this paper,high-resolution environmental scanning electron microscopy is used to study the whole tumor cells as the research object.It is of practical significance to understand the occurrence and development of membrane protein high expression tumor cells,to study its lesion characteristics and mechanism,and to develop targeted drugs.The liquid environment STEM-in-SEM imaging technology developed in this paper provides a high-resolution visualization research method and observation platform for solving scientific problems in the field of cell biology.At the same time,it also lays a foundation for further development of high-resolution imaging technology of tumor cells in their natural state and/or near physiological environment.