Study On Real-time Multifunctional Fluorescence Microscopy With High Sensitivity For Single Living Cells

Real-time detection in single living cells is of great importance in both theory and practical application. Fluorescence microscopic imaging techniques possess high sensitivity and good specificity, as well as easy realization of"visualization", and become the most potential approaches in this field. However, applicable methods are still facing challenges because of the complexity of living cells. In this thesis, great efforts have been made to establish a real-time multifunctional fluorescence microscopic imaging system with high sensitivity for real-time detection in single living cells and studies on single biomolecules, and to explore its application to specific biological research.First, a real-time multifunctional fluorescence microscopic imaging system with high sensitivity was set up and relevant image processing methods were developed. An intensified charge-coupled device was employed to realize real-time fluorescence imaging, which was suitable for detecting molecular and ionic distributions and their variations in living cells in real-time with the advantages of high sensitivity, fast acquisition speed and little photodamage of cells. Objective-type total internal reflection fluorescence imaging was introduced into the system to meet the need of tracking single molecules within, on or around cell membranes in real-time, for it greatly improved the vertical resolution, signal to noise ratio in images and sensitivity. To study molecular interactions or molecular conformational changes in living cells, fluorescence resonance energy transfer imaging was realized to break through the restriction of diffraction limit and reach a resolution of nanometer order. Other real-time imaging functions for living cells were also extended, such as fluorescent ratio detection, double-labeling with two- or three-channel detection, fluorescence with differential interference contrast imaging and three-dimensional optical sectioning. Each function mentioned above could be used separately or combined with other functions for complementary advantages, providing multifunctional techniques for biological studies. Then specific applications of the system were explored, taking studies on thymocyte apoptosis as an example, and relevant quantitative analysis methods were developed. The start-up processes of thymocyte apoptosis induced by S-nitrosoglutathione were recorded in real-time in the level of single living cells for the first time, with the apoptosis initiating time points determined. Real-time studies of the cooperation processes of the inducer with different inhibitors suggested different mechanisms of action. During the apoptosis-inducing course, the decrease of intracellular pH, the rapid increase of intracellular free calcium ion concentration and the gradual loss of mitochondrial membrane potential were also detected in real-time and quantitatively analyzed. The results revealed some regularity, providing valuable clues for apoptotic signaling pathway studies.Furthermore, some fundamental researches on single biomolecules were carried out based on the system, in which experimental conditions were optimized and relevant data analysis methods were developed, laying a foundation for further application to studies on single biomolecules within living cells. Single stretched DNA molecules were clearly imaged, and experimental conditions for long-term imaging were optimized. The motion trajectories of single nano-scaled particles were recorded in real-time and tracked in three-dimension, and a diffusion coefficient of constrained Brownian motion, which was consistent with theoretical calculation, was obtained by analyzing the trajectories. Fluorescence resonance energy transfer between quantum dots and fluorescent molecules was also detected.This thesis is supposed to result in, to some extent, promotion of the application of fluorescence microscopic imaging techniques to studies on living cells.