| Cell is the fundamental structural and functional group of living organisms.Every cell is an independent,self-controlled and highly ordered metabolic system.Cells are also the basic units for the functioning of each tissue and the metabolism activity of the whole organism.So,the investigation of various life processes of cells can help us better understand the rule of the growth of organisms and the interactions between the different species in the environment,as well as discover the mechanism for disease to guide the cure of them.However,due to the complex,dynamic and active features of cells,it is of great both academic and application significances to develop noninvasive and reliable analytical methods for the investigation of cell systems.Raman spectroscopy is very suitable for the study of biological,especially cell system,since it is an noninvasive method that can reflect the inherent fingerprint information of samples and suffer little interference from auto-fluorescence and Raman signal of water and carbon dioxide.Surface-enhanced Raman spectroscopy(SERS)can overcome the shortcoming of low sensitivity of normal Raman benefited from the highly enhanced signal.The narrow bandwidth of SERS is also another advantage for multiplexed analysis.Nowadays,Raman spectroscopy is playing an important role in the investigation of biological system as an emerging optical method.However,it is still a big challenge to maintain the activity and normal cellular functions during experiments to get the information which can truly reflect cellular activities.Even though people have realized this problem and paid more and more attention to in-situ cell experiments,most of the reported in-situ conditions are still not perfect.Moreover,since the concentrations,distributions and structures of bio-molecules in cells are complex,the acquired normal Raman and SERS spectra are usually complex as well.This phenomenon requires us pay special attention to further investigate and analyze the spectral data to get meaningful biological information.Concerning the questions and challenges above,this thesis systematically studied the appropriate condition of in-situ cell Raman experiments.We also applied several chemometric methods for the investigation of cell apoptosis and cell endocytosis process.The main content and results are listed as follows:1.Based on the light induced cell damage phenomenon under in-situ cell experiments,we investigated the influence of various physical and chemical conditions on the cell viability,and established the relationship between light energy doze and cell viability.We also confirmed that 660nm laser is suitable for in-situ normal Raman cell experiments and 785nm for in-situ SERS experiments.2.By combining K-means clustering and MCR analysis with traditional single variate analysis,we obtained the merge of cytochrome c,accumulation of saturated lipid and the vague boundary between cells and background rule for CaSki cells after drug induced apoptosis under ex-situ condition.Compared with traditional data analysis methods,K-means can clearly distinguish the morphology of cells and MCR can acquire higher contrast images of pure components.3.By combining PLS-LDA and statistic methods,we identified the adsorption of proteins,amino acids and RNA in early endocytosis and the desorption of these species in late endocytosis.We also found that nanoparticles at the later period are mostly localized in lysosomes compared with nanoparticles in early endocytosis.Moreover,modification of nanoparticles with cell penetrating peptide can both accelerate the uptake and intracellular transport of nanoparticles,and the modification of PEG can increase the reproducibility of SERS spectra in cells. |
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