Fluorescence microscopy provides an efficient and convenient approach to study biological processes because of its versatility, specificity, and high sensitivity. By detecting the fluorescence signal emitted from the labeled molecules in biological samples, a fluorescence microscope can gather a series of information, including position, interaction, and deformation, to help us visualize and analyze the complex dynamic events in cells. In recent years, various advanced fluorescence imaging techniques have been developed. The fluorescent tracking technique is ideal for obtaining the high resolution of spatio-temporal information of the particles. Furthermore, Förster (or Fluorescence) Resonance Energy Transfer (FRET), which is unique in generating fluorescence signals sensitive t...[ Read more ]

Fluorescence microscopy provides an efficient and convenient approach to study biological processes because of its versatility, specificity, and high sensitivity. By detecting the fluorescence signal emitted from the labeled molecules in biological samples, a fluorescence microscope can gather a series of information, including position, interaction, and deformation, to help us visualize and analyze the complex dynamic events in cells. In recent years, various advanced fluorescence imaging techniques have been developed. The fluorescent tracking technique is ideal for obtaining the high resolution of spatio-temporal information of the particles. Furthermore, Förster (or Fluorescence) Resonance Energy Transfer (FRET), which is unique in generating fluorescence signals sensitive to molecular conformation, association, and separation in the 1-10 nm range, has been widely used in studying molecular interactions inside living cells. In this thesis, we introduced a new total internal reflection fluorescence microscope to achieve the spatio-temporal information and FRET efficiency change at the same time. Moreover, we applied this system to investigate the vesicle fusion process. By measuring the position and FRET efficiency of two vesicles simultaneously, we obtained more detailed information to understand how two vesicles undergo fusions.

Single particle tracking is a powerful tool to investigate the dynamic process in the biology system. In this thesis, we used single particle tracking to investigate the binding process of two important proteins, STIM1 and Orai1. STIM1 is a calcium ion sensor in the endoplasmic reticulum (ER) membrane, and Orai1 is the subunit of calcium release-activated calcium (CRAC) channel in the plasma membrane. After depletion of calcium store in the ER, STIM1 molecules move from the ER membrane into the ER-PM junction and bind with Orai1 to trigger the formation of the CRAC channel. By analyzing the trajectory of STIM1 and Orai1, we found that the motion of single STIM1 and Orai1 particles exhibits anomalous diffusion both before and after store depletion. We also found that the motion of these two proteins exhibit increased confinement after store depletion.

Magnetic tweezers is a widely used tool to investigate the fore transduction in living cells. In this thesis, we used magnetic tweezers to apply mechanical force to cells expressing LifeAct-GFP to investigate the mechanical responses of living cells, especially in the actin stress fibre. By quantifying the lateral displacement of stress fibres with different positions in cells, two clear force-dependent responses were observed: an external force directly induced deformation and a long-term force-induced remodeling deformation at the cell edges, far from the point of contact. The unexpected deformation model demonstrates that force stimulus does not result in simplistic isotropic deformation of the stress fibre network, but rather sophisticated and localized responses.