| The single cell or particle suspended in the liquid flows one by one at a high speed could be analyzed by a multi-channel flow cytometry system. The scatted light and fluorescent light signals are detected. Multi-parameter information of the light pulse signals could be acquired for the fast-quantitative analysis of a large number of cells.Multi-channel flow cytometry system is a high-throughput clinical instrument for single cell analysis. It is widely applied in areas of cell biology, clinical medicine, cell genetics,procreatics, pharmaceutics, immunology, foundation medicine and clinical examination.At present, the key technology is mainly controlled by a few of foreign companies. It is significant to research the key technology of each module of the multi-channel flow cytometry system.Effort of this dissertation has been focused on the laminar flow formation, laser beam shaping, excitation of scattered light, multi-color fluorescent light collection, photovoltaic conversion, and so on. In this research, the multi-channel flow cytometry system is divided into air-liquid module, optical module, electrical module and software module.The width and velocity stability of core flow, the method of scattered light detection, the pulse signal restoration, the representation of fluorescent signal and fluorescent compensation are analyzed separated. Based on the analysis, the main errors in cytometric measurement are demonstrated. Further, a serial of optimizing methods are proposed to decrease the errors. The main works of this dissertation are as follows:1) A flow chamber with two sheath flows injected parallelly is designed, and the dissymmetry of core flow with single sheath flow is settled. The focused results are simulated and experimentally verified with the sheath flows injected at angle 0°, 60° and 90° respectively. The results show that, the mean values of core flow width at "low","medium" and "high" flow rate are 8.38?m (maximum error 1.42?m,standard deviation 0.70?m), 13.73?m (maximum error 1.27?m, standard deviation 0.71 ?m) and 20.11?m(maximum error 1.09?m, standard deviation 0.59?m).2) In order to evaluate the stability of the liquid path of the flow cytometer directly, a method of high-speed particle image velocimetry via high-speed particle image-capturing is used. The velocity stability of the particles in the flow chamber is used to represent the flow stability of the fluid path of the flow cytometer. A high-speed particle image-capturing system (HPICS) is used to record the images of the particles in the flow chamber,and the following classes of images are identified using a grey clustering analysis algorithm (GCA) according to the exposure condition of particles: blank, inadequate,normal, and overlapped. The trajectory boundaries of the normal images are selected and the standard deviation of the particle velocity is calculated to represent the stability of the liquid path system. Finally, the relative flow stability of the fluid path system of the flow cytometer is evaluated at three flow rates using microspheres with different diameters.3) A new method based on array forward light detection is proposed. A model is built base on Mie scattered theory. And then, the property weights on different types of scattered light are determined based on the simulation result. Finally, the experiments are executed with microsphere with different diameters and refractive index. The results are analyzed with the of algorithm K-modes clustering. Experimental results indicate that,cells with different diameters could be discriminated, the maximum error is 2.55%.Meanwhile, cells with same diameter but different refractive indexes could be clustered,the maximum error is 4.51%.4) The measurement of fluorescence lifetimes emerged in flow cytometry because it is not impacted by the non-linearity, which occurs in fluorescence intensity measurements.However, this significantly increases the cost and complexity of a traditional flow cytometer. A simple method of fluorescence lifetime measurement of a flow cytometer based on the cytometric fluorescence pulse time-delay estimation and hardware time-delay calibration is proposed. The modified chirp Z-transform (MCZT) algorithm,combined with the algorithm of fine interpolation of correlation peak (FICP), is applied to improve the temporal resolution of the cross-correlation function of the scattering and fluorescence signals, which in turn improves the time-delay estimation accuracy. The estimation accuracy is verified by Gauss fitting. Cells that were labeled simultaneously with three-color reagents are measured; the statistical results of 5,000 cells are compared with reference values, and are verified with the pulse width variation. The results show the potential of fluorescence lifetime measurements in the traditional flow cytometer.5) In multicolor analysis, it is impossible to discriminate between fluorophores that spectrally overlap; this influences the accuracy of the fluorescence pulse signal representation. Here, we focus on spectral overlap in two-color analysis, and assume that the fluorescence follows the single exponential decay model. We overcome these problems by analyzing the influence of the spectral overlap quantitatively, which enables us to propose a method of fluorescence pulse signal representation based on time-delay estimation (between fluorescence and scattered pulse signals). Time-delay estimation simulation and fluorescence signal representation experiments are conducted on fluorescently labeled cells. The results show that the calculated lifetimes with spectral overlap can be rectified from 8.28 and 4.86 ns to 8.51 and 4.63 ns, respectively, using the comprehensive approach presented in this work. These values agree well with the lifetimes (8.48 and 4.67 ns) acquired for cells stained with single-color fluorochrome. |
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