Research On Lymphocyte Morphology And Biomechanics Based On Scanning Probe Microscopy

This dissertation includes two parts:(1) Using the high-resolution and force spectrum properties of the atomic force microscopy(AFM),detect the changes of the morphology,adhesion force and Young's modulus properties of the lymphocyte by different stimulation;(2) Visualization of the cell surface receptors recognition was performed by AFM and scanning near-field optical microscope(SNOM).In the first part,based on the high-resolution and force spectrum properties of the AFM,Hertz model was used to detect lymphocyte adhesion force and Young's modulus.The morphology,ultrastructure and adhesion force properties of the resting, LPS or ConA activated human periphery lymphocyte were investigated.The AFM images revealed that the surface of the lymphocyte treated with LPS or ConA were rougher than that of resting lymphocyte,and coated with an outer layer of extracellular polymers.High-spatially resolved force-distance curves indicated that the adhesion force values of lymphocyte activated by LPS or ConA were approximately two to three times stronger than that of resting lymphocyte.Using high-sensitivity force-distance curves,the Young's modulus of the resting,activated and apoptotic lymphocyte were detected.We found that the value of the activated lymphocyte is about two to three times stiffer(Young's modulus of～20 kPa) than those of the two other morphotypes(5～11 kPa).These results can improve our understanding the mechanical properties of cells during growth and differentiation.The second part of this paper,based on AFM and tip modification technology, combined with SNOM,fluorescent quantum dots(QDs) mark,fluorescence microscope,confocal microscope and flow cytometry,the structure and function or the immune recognition of the biological molecules were being studied.A method involving the antibody functionalized modified AFM tip to detect specific cell surface receptor molecule interaction.To determine the nanoscale distribution of molecules on cell-membrane,Ab-functionalized AFM tip was tested for its atomic force binding to molecules potentially expressed on the cell membrane.The AFM tip was first treated with the strepavidin.The strepavidin-labeled AFM tip was then attached to biotinylated Ab.We have found that SNOM/QD-based nanoscale fluorescence imaging is a powerful system in which to visualize nano-spatial distribution and organization of cell-surface immune molecules.The distributive patterns and force properties of CD₄ and CD₆₉ molecules on human Peripheral Blood CD₄⁺ T cell surface were compared by AFM and SNOM.The main results are the following:(1) Using a functionalized AFM tip,the strength of the specific binding force of the CD₄ antigen-antibody interaction was found to be approximately three times that of the unspecific force.The adhesion forces were not randomly distributed over the surface of a single activated CD₄⁺ T cell indicated that the CD₄ molecules concentrated into nanodomains;(2) Multiple bonds involved in the CD₄ antigen-antibody interaction were measured at different activation times;(3) Using the same method,quantitative measurement of the CD₆₉ antigen-antibody interaction,about 276±71 pN;(4) SNOM/QD-based nanoscale imaging showed that CD₆₉ were non-uniformly distributed as 80-200 nm nanoclusters on cell-membrane of activated CD₄⁺ T cell.