Study on lipid droplet dynamics in live cells and fluidity changes in model bacterial membranes using optical microscopy techniques
Wong, Christine Shiang Yee
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In this thesis optical microscopy techniques are used to consider aspects of viral and bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model bacterial membranes are studied. The optical microscopy techniques used to study the effects of murine-cytomegalovirus (mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are coherent anti- Stokes Raman scattering (CARS), two-photon fluorescence (TPF) and differential interference contrast (DIC) microscopies. Using a multimodal CARS and TPF imaging system, the infection process was monitored by imaging the TPF signal caused by a green fluorescent protein (GFP)-expressing strain of mCMV, where the amount of TPF detected allowed distinct stages of infection to be identified. Meanwhile, changes to lipid droplet configuration were observed using CARS microscopy. Quantitative analysis of lipid droplet numbers and size distributions were obtained from live cells, which showed significant perturbations as the infection progressed. The CARS and TPF images were acquired simultaneously and the experimental design allowed incorporation of an environmental control chamber to maintain cell viability. Photodamage to the live cell population was also assessed, which indicated that alternative imaging methods must be adopted to study a single cell over longer periods of time. To this end, DIC microscopy was used to study the lipid droplet dynamics, allowing lipid droplet motion to be tracked during infection. In this way, the effects of viral infection on the mobility and arrangement of the lipid droplets were analysed and quantified. It was found that the diffusion coefficient of the lipid droplets undergoing diffusive motion increased, and the droplets undergoing directed motion tended to move at greater speeds as the infection progressed. In addition, the droplets were found to accumulate and cluster in infected cells. The second part of this thesis presents a study on the effects of an antimicrobial peptide on model bacterial membranes. Giant unilamellar vesicles (GUVs) were produced as a simple model of E. Coli membrane using a 3:1 mixture of DPPC and POPG lipids. Incorporating Laurdan fluorescent dye into the lipid membrane of the GUVs allowed the membrane fluidity to be probed and visualised using TPF microscopy, whereby the fluidity was quantified by determining the general polarization (GP) values. Studying GUVs comprising single lipid and mixed lipid compositions over a temperature range from 25 C to 55 C enabled the lipid phase bands to be identified on the basis of GP value as gel phase and liquid crystalline phase. As such, the changes in lipid phase as a result of interaction with AMP were quantified, and phase domains were identified. It was found that the amount of liquid crystalline phase domains increased significantly as a result of AMP interaction.