The Investigation Of Human Peripheral Blood Lymphocyte And Cell Surface Molecules's Visualization And Quantitation By Scanning Probe Microscope(SPM)

AFM(atomic force microscopy) and NSOM(near field scanning microscopy) possess very high resolution and many advantages in exploring the micro world and life science. In this work, AFM and NSOM were used to investigate the morphology and ultrastructure of human peripheral blood lymphocytes before and after activation. Human resting lymphocytes were separated and activated by PDB and Ion, the cells before activation, activated for 12h and 24h were all scanned by AFM and NSOM in air. The result shows that the activated lymphocytes were obvious enlarged and have a more complicated surface after activation, small granules and immunological synapse were found on the activated lymphocytes. The NSOM images shows abundant structure informations inside the cells.Besides morphology and ultrastructure of human peripheral blood lymphocytes were studied, we demonstrate the optical investigation of molecule on human peripheral T lymphocytes by NSOM (scanning near field microscope) combined with quantum dots(QD) as fluorescent probe. The T lymphocytes membrane molecules CD3 and early activation molecules CD69, CD25 were labeled with QD, the location, distribution and variation of these molecules were imaged by an high-resolve NSOM. The result shows that CD3 were obviously located in different sites on the resting T cells membrane, distribute on the whole cell surface separately. When the resting T cells is activated, CD3 were found aggregated and CD69, CD25 began to express sequently. CD69 was found located at the same site with CD3 and CD25 located at different sites compare with the other two molecules. The aggregation of those membrane molecules probably shows the formation of immunological synapse or membrane raft. Which is very important to the T cells biological functions. And when the T cells is activated by different agent, the aggregated molecules has different performance in sort and location. Further more, the space oriental information of CD3 molecules were obtained by NSOM combined with polarization imaging technology.This work provide a novel and useful method for the quantification and visualization of molecules on cellular or subcellular level, NSOM combines QD shows great potential in biology research.