The Construction Of Multicellular Real-time DIC Microscopy Imaging System And Its Application

Multicellular real-time differential interference contrast microscopy imaging technique using light interference and diffraction principle makes phase difference be converted into light intensity difference in the cells, differential interference contrast microscopy makes cellular structure bring out the different colors of the image and structure can be clearly seen. Differential interference contrast microscope is of high sensitivity and is not affected by the thickness of the sample, it can real-time monitor the large samples in the natural environment. Samples under the microscope present a clear picture of the three dimensional structure. In recent years, as a new analysis method, differential interference contrast microscopy imaging technology has important value of application in the field of physics, chemistry, material and life science. Due to the laser optical tweezers Raman microscopy combined with optical trap control and the advantage of rapidly and non-destructice Raman spectroscopy, a single cell can be captured and fixed in the solution for a long time. Moreover, because of the high spatial resolution of Raman spectra, one can obtain precisely a lot of information on the Raman spectra of the micro samples observed, such as, the chemical composition, molecular structure, molecular orientation, and molecular interaction without the interference of other material.Spore is bacillus genus, clostridium genera lactobacillus and bacillus bacterial cells in response to the adverse growing conditions and formation of dormancy body in a cell. As spores from dormancy state key starting to vegetative growth, spore germination is the key node to know spore resistance and to find the way to kill spores of bacteria. Therefore, optical technology and single cell analysis method play an extremely important role in understanding the mechanism of spore germination, inhibiting or promoting the study of spore germination.Our work combined multicellular differential interference contrast microscopy imaging with the laser optical tweezers Raman spectroscopy and related experimental methods for the following experiments:1. Bacillus thuringiensis (Bt) is the most widely used and most successful microbial pesticide in the word, it can generate spores and spore crystal in intracellular. Dormant spores have a strong ability to resist adversity, and they germinate rapidly once meeting the right conditions. Germination of spores is a special kind of development process. In this thesis we paper established a imaging method based on differential interference contrast (DIC) microscopy to analyze immediately Bt spores germination process in nutrition germination and in non-nutrition germination with high-output, and compared whit the outcome of using Raman spectroscopy. The results shown:(1) the dynamic change of DIC image brightness in the process of Bt spore germination and the released process of the intracellular pyridine dicarboxylic acid (DPA) detected by Raman spectroscopy have correspondence; (2) temporal resolution in 6 seconds can better present the dynamic state of spore germination; (3) in the 10 mM alanine, the germination process of Bt spores in Tris-HC1 buffer (pH8.3) is similar to the Hepes buffer (pH 7.3); (4) Bt spore germinate in the non-nutrition agent of dodecylamine is very slow, but extremely rapid in exogenous CaDPA. It is found that the application of differential interference contrast microscopy imaging technology can observe immediately a large number of single Bt spore germination dynamic state with high-output, and get the rule of spore germinateion and heterogeneity.2. Physical factors have great effects on Bt spore and spore germination. Through laser tweezers Raman spectroscopy and differential interference contrast microscope to observe, using single-cell analysis synchronous to analyze single Bt spore germination process after ultraviolet irradiation. According to the physical changes inside the spore of real-time dynamic information, it is revealed the influence of ultraviolet on Bt spore germination. The result shows that:Ultraviolet rays will make protein of spore denaturation, and the longer the irradiation, the lower the spore survival rate, the greater volatility the peak of Bt spore Raman spectrum characteristic; In Alanine nutrition agent, ultraviolet rays extended the every stage of Bt spore germination, but the irradiation time have no effect on time of CaDPA releasing and cortex dissolving; In CaDPA non-nutrition agent, ultraviolet radiation increases the Bt spore germination of parameter values, and the germination rate declined slightly. According to the above, Bt spores have certain resistance to ultraviolet ray, but ultraviolet ray also can damage the Bt spore.