Fluorescence Imaging For The Study Of Yeast Cells

Cell, as a fundamental unit of life movement, is the minimal unit to put up the entire characteristics of living life. All the key problems of life sciences can be traced back to cell problems. However, cells are so tiny, the compounds in cells are so infinitesimal and complicated, and chemical reactions in cells are so fleet that the cell research, especially the single living cell research is very difficult. Yeast cells as one of the perfect models for single cell research, have many similar facts with human cells and are relatively ease to be operated. So many scientific findings in human cells are first investigated in yeast cells. In this paper, some life movements in yeast cells were investigated mainly using fluorescence microscopy, combined with AFM, SEM, laser scanning confocal microscopy and some biological techniques. A new and simple fixation method was developed to study the dynamic process in a single living yeast cell. The main contents in this thesis can be summarized as follows:·The effect of Li~+ and PEG on genetic transformation in yeast cellsFor the transformation of intact yeast cells, a most-commonly-used efficient protocol makes use of PEG and Li~+. However, until now it is still unclear how plasmid DNA binds to the yeast cells and what the roles of Li~+ and PEG play in the transformation of the intact yeast cells. In this thesis, the effect of Li~+ and PEG was firstly visualized by using fluorescence microscopy, AFM, SEM, coupled with transformation frequency experiment, and the direct evidence for relating the DNA binding to yeast cell surfaces with DNA transforming into yeast cell was firstly obtained. This research offered a new method for yeast transformation study.·The application of polysaccharide biofunctionalized QDs in yeast cellsQDs, for their extraordinary fluorescence properties, have being used as a new kind of biomedical fluorescent nanomaterials applied in biological cell imaging research. In this thesis, the interactions of yeast cells with QDs modified respectively with three kinds of polysaccharide, i.e. chitosan, carboxyl methyl chitosan and carboxyl methylchitin, were investigated. The electric charges of QDs modified with different polysaccharide are different. The concentration of NaCl in solution influences the interaction of the QDs with yeast cells. The quantities of QDs modified with polysaccharide binding to yeast cell surface and penetrating into yeast cells could be controlled by the concentration of NaCl in solution.·Fluorescence labeling in single living yeast cell researchSingle living cell research is very important in the whole cell science. In this thesis, a new and simple fixation method was used to fix a single yeast cell on a self-made glass slide, keeping the cell living in the normal condition. Some life processes were dynamically investigated in situ using fluorescence imaging methods. The apoptotic process induced by H2O2 in a single living yeast cell was investigated, and the quick movement of an unknown object in the big vacuole in a single living yeast cell was firstly imaging, and the trajectory of a negative charged QDs modified by methacarboxyl chitosan interacting with a single living yeast cell was traced in situ. These researches would provide a solid foundation for imaging research of single living cell, especially single living microbe cell.