| Life science focuses on the phenomenon of life, the nature, characteristics and developmental rules of life activities. It has close relation to human existence, health, and development of society, and attracts most worldwide attentions in basic sciences. Cell biology, which aims to study the cells, is one of the basic topics in life sciences, and it researches on the structure and function of cell and organelle from the cellular, sub-micron and molecular level. The metabolism, trans-membrane transport and signaling of live cells are all the results of the interaction of cell and other macromolecules such as protein, polysaccharides, and nucleic acid. In these interactions, the mechanical property of cell and molecule may change, and this change reflects the function and rules of cell activities. Traditional cell biological and molecular biological researches, which are based on the variation of concentration of some molecules in biochemical and pharmacological experiments, are the average results of the activities of many cells and molecules. The traditional research cannot reveal the performance of a single molecule; therefore, study of activities of a single molecule in cellular and molecular level is of great importance.Biology not only relies on physical and chemical rules, but also relies on the experimental apparatus and research methods, such as protein electrophoresis, optical microscopy, electron microscopy, section scanner etc. Optical tweezers is a proper technique in cell biological research in cellular and molecular level. Factually, since its invention, optical tweezers has played important roles in biology and because of its technical uniqueness. Optical tweezers has attracted wide attention from biologists. Recently, optical tweezers becomes an important tool in cell biological and molecular biological researches.Optical tweezers is able to trap and manipulate particles ranging in tens of nanometers to tens of microns, and can be used as probes to detect the forces of protein interaction or cellular forces in the range of pico-Newton to subpico-Newton. Furthermore, results from optical tweezers can be used to reveal life activity in cellular level. In this thesis, we expand the application of optical tweezers into the areas of cell biology. The detailed researches lie in the measurements of the mechanical property of cells, specific identification of immune cell to target cell, interaction of antigen and antibody, theoretical and experimental research on the influence of osmotic pressure to the formation of cell membrane, and developed novel optical tweezers'technique to further study the mechanical property of cell membrane.Immune is a physiological function of human body, which sustains healthy by identifying foreign antigen and generating specific immunological response. Natural Killer cells (NK cells) are one kind of immunological cells. Since its discovery, it gradually becomes hot topic in immunology. The activation of NK cell is very complex, and a simple description may goes as follows surface molecule on effective cell identifies ligand on target cell, forming immune synapse, and finally kill target cell. K562 cell is the sensitive cell of NK92, but the mechanism of identification and kill of this pair of cells hasn't clear. We utilize optical tweezers manipulate NK cell contact target cell, observing the kill effect of NK cell to target cell in real time. Experimental results show that K562 cell deforms with a vesicle on the surface, and the cell membrane hasn't broken. Our results help to reveal the kill mechanism of NK92 to K562.The activation of NK cells has close relation with the surface receptors. Lymphocyte function associated antigen-1 (CD11a/CD18, LFA-1) is a member of the integrin family of cell surface receptors. It is possible one of the early actors in process of the recognition and killing of NK cells. LFA-1 was found in the killing experiment mediated by the CTLs and was likely to be involved in some immunological diseases and tumour recurrence. To study the activation mode of LFA-1 on the NK cell surfuse, a platform based on optical tweezers was constructed to observe the molecular behavior of LFA-1. According to the results, the adhesion force between an NK cell and a polystyrene microsphere coated with anti-LFA-1 antibody was confirmed to be time-dependent. And the force was increased with the time of contact between the cell and sphere which indicated the LFA-1 on cell surface has clustered induced by anti-LFA-1 antibody. So, a new method is developed in our works to study the interaction between two cells in real time and to observe the single molecular behavior. Our results may provide a clue to explore the activation mechanism of LFA-1 on NK cells.The human red blood cell has a relatively simple structure, and it does not contain a nucleus. Consequently, it has often been regarded as'model system'in the study of single living cells. The formation of RBC greatly influences the flow of blood, and relates with some diseases. Naturally, the RBC is in a surroundings with many physical parameters such as osmotic pressure, pH etc. We adopt optical tweezers to study the formation characteristics of RBC under different osmotic pressure. The cell has large deformation ability under isotonic pressure; while the cell has low deformation ability under hypotonic environment because of change of cell shape; and the cell also has low deformation ability under hypertonic pressure due to the exponential increase of concentration of intracellular liquid. Additionally, a simulation adopting ABAQUS software to simulate the deformation of RBC with different shapes is conducted, and the results agree well with our experimental results.Traditional method adopting optical tweezers to measure the elasticity of cell is to use micro bead handle to stretch cell. In fact, the form of cell suffers tiny changes under the trap. When a cell exposed in a jumping optical tweezers, this change may be larger that can be easily measured. We realized time-sharing multiple optical tweezers utilizing tilt rotating glass plate to further manipulate multiple particles and to study the formation characteristic of red blood cells. Meanwhile, we utilize this novel technique to investigate the effective stiffness under different trap switching frequencies both theoretically and experimentallyThe main researches of our lab focus on the optical tweezers'technique. In biological applications, we adopt the means of providing instruments by physicist and sample prepared by biologists. In many of the applications, because of the limitation of sample preparation, many researches cannot go in deep. Therefore, we setup our own cell cultivation room to independently cultivate cells in our lab. Recently, we can cultivate Hela cells and perform relative experiments.The highlights of this thesis lie in research of the mechanical property of biological cell in cellular level utilizing optical tweezers. The main results include formation of cell membrane, specific identification of immunological cells and interaction of antigen and antibody in cell membrane. Our results help us understand the basic rules of life activities of cells. Additionally, we developed novel methods to generate multiple optical tweezers, which will find applications in the researches of mechanics of cells. |
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