Total Internal Reflection Fluorescence Microscopy To Detect The Transferrin Receptor Molecules On Gastric Cancer Cell Membrane

This thesis includes three parts: I . Determination of single biomolecules. II. Assay of transferrin receptors on human stomach cancer cell member by total internal reflection fluorescence microscopy. III. Formation of single endosomes on human stomach cancer cell was determinated by total internal reflection fluorescence microscopy.In Chaper I .of this thesis, first of all, the research of single molecue detection was introduced briefly. Secondly, the detail review of analysis of single molecule using laser-induced fluorescence detection was delivered. The analysis and control technique of single molecule, application of single molecule detection, key technique of single molecule detection and evidence of single molecule, fluorescent probe and prospect. Emphases of this chaper is total internal reflection fluorescence microscopy and it's application in detection of single biomolecules, including principle of microscopy imaging , detection of single biomolecules in vitro and in vivo.99 references have been referred.In Chaper II., a method for determination of transferrin receptors on human stomach cancer cell member by total internal reflection fluorescence microscopy was developed. Several parameters including background reduce, time of cell attached, density of the cells, temporation of transferrin incubating, concentration, time,sequence of adding was optimized. The following conditions were suitable for the determination of transferrin receptors on cell member: density of the cells: 5 X 105 个mL; time of cell attached: 10 h; temporation of transferrin incubating: 4℃; concentration: 1.25 X 10~-8 mol/L; time: 30 min; sequence of adding: cell attached on cover glass after mediaing transferrin through centrifuging. FITC conjugated transferrin plus 100 times of biotin conjugated transferrin incubating on cell was indicated specific of transferrin receptors binding transferrin.In Chaper III., a method for determination of formation of single endosomes on human stomach cancer cell was developed based on total internal reflection fluorescence microscopy. Formation of single endosomes on cell membrane was analysised by total internal reflection fluorescence microscopy at first . Alexa488 conjugated transferrin was binded on transferrin receptors on cell membrane on 4℃, after centrifuging to remove unbinded transferrin, cell attached to cover glass was 10 h. cells was washed by PBS twice in order to removing the unattached cells. Transferrin was formed endosomes by endocytosis on 37℃,...