| Light scattering is a general physical process,and it has been an important information in reflecting particle properties.The scattering power of a particle depends on its size,material composition and shape.By the determination and analysis of scattering intensity,it is able to characterize the properties of particles by light scattering techniques.Particle size and size distribution characterization has been the focus for the applications of light scattering,yet the complicate dependence of light scattering intensity on particle size renders it challenging to the accurate characterization of particle size.In order to predict the scattering power of particles and explain the optical phenomenon,Mie scattering theory and related numerical methods were developed.According to the Mie scattering theory,the amount of Mie scattering is a function of numerous parameters:particle size,refractive indexes(RIs)of particle and surrounding medium,collection angle,particle shape and orientation,incident light wavelength,and the polarization angle of the incident light.As a kind of single-particle theoretical tool,the Mie scattering theory has been widely used in particle sizing,atmospheric modeling,optical-medical diagostics,and environmental monitoring,it is of great importance in the experimental validation of Mie theory via single-particle light scattering detection.Though there were several single-particle experiments reported on the impacts of refractive index(RI),polarization,or scattering angles on Mie scattering intensity,these validations of the Mie theory were done without the consideration of all the light scattering parameters and were limited to micron-size range.In contrary to the advanced theoretical and computational development of the Mie theory,experimental validation has been lagging mainly due to the lack of sensitive single-particle detection techniques and synthesis of monodisperse particles.The systematic study of the light-scattered intensity by single-particle analysis and theoretical comparison is of great importance in the accurate size measurement of nanospheres and the explaination of experimental results of light scattering.Flow cytometry(FCM)is a well-established technology for high-throughput,quantitative,and multiparameter analysis of individual cells or cell-sized particles,which employs light scattering to reveal the physicochemical features of cells.Limited to the instrumentation sensitivity and the background particles from the sheath fluid,the detection limit of biological particles is 300 nm-500 nm for most conventional flow cytometers.Adopting strategies for single-molecule fluorescence detection in a sheathed flow,our laboratory has developed a highly sensitive flow cytometer,which enables the detection of single silica nanoparticles and viruses with diameter down to 24 nm and 27 nm,respectively.Compared to conventional FCM,the dwell time,particles flow through the interrogating laser beam,is extended for enhanced photo generation.Besides,a reduced probe volume for background reduction and the high quantum yield of single-photon-counting avalanche photodiode(APD)are the key characteristics to the significantly improved scattering sensitivity for HSFCM.Silica nanospheres are adopted as the model system due to the method developed in present study for the precise synthesis of particle size from 20 nm to 1000 nm.Integrating the high-sensitivity of HSFCM for the light scattering detection of single nanospheres and the established numerical method for Mie theory calculation,we attempted to validate the Mie theory in the submicron size range.The dependences of scattering intensity on particle size,polarization angle,and refractive index of nanospheres were investigated for silica spheres and commercially available polystyrene(PS)beads.Besides,we developed a new method for the refractive index determination of biological nanoparticles.Based on the membrane-lumen RI model,we can quantify the inner composition of biological nanoparticles without lysing the membrane particles and without fluorescence labeling.Taking advantage of the high-sensitivity of HSFCM in fluorescent analysis,it is possible to investigate fluorescence polarization at the single-particle level to enable the accurate fluorescence quantification of nanospheres.Brief introduction of this dissertation is given as follows:In chapter one,an overview of the single-particle light scattering detection techniques and the principle of using light scattering intensity for particle sizing is given.Besides,the importance of RI matching for particle sizing is also discussed.Chapter two is the experimental validation of Mie scattering theory at the single-particle level.We developed a method for the synthesis of higly monodisperse silica nanospheres in the size range of 180 nm to 880 nm.Based on the Mie theory,we developed a model for spheroidal particles by strictly incorporating all the Mie parameters related to the theory.The experimental results are compared with the numerical calculations.The dependence of scattering intensity on particle size,polarization angle,and refractive index was investigated for both the silica nanospheres and commercially available polystyrene(PS)nanobeads.Based on the excellent agreements between theoretical calculations and experimental results,the numerical model was confirmed to be a powerful tool to predict the impact of some parameters on light scattering intensity,including RI and collection angle.Chapter three decribes accurate refractive index measurement and inner composition quantification of individual biological nanoparticles.We demonstrated the influence of RI in scattering intensity and the linear relationship between measured scattering intensity and theoretical calculation.Combining HSFCM scattering analysis and Mie theory calculation,we developed a method for the RI determination of individual biological nanoparticles.Based on the proposed membrane-lumen RI model,we measured the RI of composition inside the lumen and quantified their concentration at the single-particle level.Chapter four depicts the investigation of fluorescence polarization on the fluorescence intensity at the single-particle level.We developed a method to incorporate fluorochromes into silica particles and prepared a series of monodisperse fluorescent silica nanospheres with different molecules of equivalent soluble fluorochrome(MESF).The influence of polarization in the angular distribution of fluorescence intensity was studied by the polarization control and single-particle fluorescent analysis of HSFCM.Combining the fluorospectrophotometer measurement of both the standard solutions of fluorochromes and the fluorescent silica nanospheres with the particle concentration measurement of HSFCM,we achieved accurate quantification of MESF for single fluorescence silica nanospheres.In chapter five,the work of this thesis is summarized and the future prospect of the following work is discussed. |
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