Real-Time Dynamic Observation&Quantitative Analysis For Living Cells And Application

Living cells are the basic structural units and functional units of organisms.Therefore, real-time dynamic observation in single living cells is of great importance inlife sciences and quantitative analysis the experiment data in depth is helpful to revealthe information of life sciences. In this thesis, a real-time multifunctional fluorescencemicroscopic imaging system as well as relevant image processing methods is improvedfor long term living cells observation. The studies of early apoptosis on thymocytes andthe spontaneous and synchronous intracellular Ca2+oscillations of cultured hippocampalneuronal networks are carried out in the system.First, the real-time multifunctional fluorescence microscopic imaging systembecame more suitable for long term living cells observation after the installation ofincubator and the development of relevant image processing methods.Secondly, a series of changes in thymocytes early apoptosis induced byS-nitrosoglutathione (GSNO) were studied. During the early apoptosis, the increase ofintracellular free calcium ion concentration ([Ca2+]i), the loss of mitochondrialmembrane potential (ΔΨm), the decrease of lysosomal pH, cytoplasmic acidification ofapoptosis cells were detected. The kinetic features were quantitatively analyzed andcompared by fitting the experiment data. The mathematical parameters, inflection pointand half-max effect point were proposed to analyze the fitting curve. The resultsrevealed that the inflection point of lysosomal pH always appeared prior to that of ΔΨmand posterior to that of [Ca2+]iin a certain GSNO concentration induced. Half-maxeffect point was also displayed the similar results. Such quantitative analyses ofreal-time observations are useful for interpreting the sequence of the biological eventsoperating in GSNO-induced thymocyte apoptosis and provide important clues forapoptotic signaling pathway studies.Finally, the modulation of different compatibilities of Chinese herbal medicine onsynchronized Ca2+oscillations in cultured hippocampal neuronal networks were carriedout. We investigated and quantitative analyzed the modulation effects of lowconcentration and different compatibilities on synchronized Ca2+oscillations of piperineand deoxyschizandrin, deoxyschizandrin and schisandrin B in cultured hippocampalneuronal networks. The results suggest that different compatibilities have different inhibition on the synchronized Ca2+oscillations. The inhibitory effect on the frequencyis irrelevantly to that on the amplitude. A calcium oscillation model of nerve cells wassetup and the effects of its seven parameters were analyzed.This thesis is a specific application of fluorescence microscopic imagingtechniques in living cells and quantitatively analyses and supposed to provideadvantageous techniques and methods to studies on living cells.