Advanced fluorescence methods for the investigation of biological membranes

This thesis explores the use of advanced fluorescence imaging and spectroscopic methods for investigating various properties of biological membranes. The cell membrane is a complex environment comprised of a variety of important molecules all necessary for maintaining cellular function. The dynamics of processes involved in membranes are typically over very short time and spatial frames. Advanced fluorescence imaging and spectroscopic methods present an opportunity for probing the dynamic nature of this environment due to their high levels of both spatial and temporal resolution. The following thesis consists of three biological problems centred on the cellular membrane, investigated through high resolution techniques. The first area of investigation focusses on the insulin regulated metabolism of glucose in fat and muscle tissue. Traditional experiments are performed using either isolated rat adipocytes or differentiated fibroblasts which both required lengthy and expensive culturing procedures. A new modified HeLa cell line was investigated to determine its efficacy as a homologue to the well characterised adipocyte model as a method for investigating factors affecting glucose metabolism. A direct comparison of the dynamic recruitment of the molecule Glucose Transporter 4 (GLUT4) to the plasma membrane was undertaken using a custom built Total Internal Reflection Fluorescence Microscopy (TIRFM) system. Utilising TIRFM the time dependant translocation of GLUT4 to the plasma membrane under insulin stimulation was investigated in the two cell lines. This was achieved through analysis of the increase in normalised fluorescence signal found within the 110 nm illuminated region of the TIRFM system. It was found that in the HeLa cell line responsiveness to insulin stimulation was present but with a significant difference in GLUT4 levels to the imaged adipocytes. It was also seen that this observed response occurred over a significantly longer time frame than in adipocyte cells with a half rise time in fluorescence intensity taking, on average, 5 minutes longer. In addition, the dynamic mobility of GLUT4 Storage Vesicles (GSVs) within the vicinity of the membrane was assessed through image analysis techniques. The abundance of mobile and stationary vesicles was assessed. In the adipocyte cells a sharp increase in mobile GSVs was observed over the initial 5 minutes after insulin stimulation. The amount of immobilised GSVs was seen to increase at a constant rate over the time course of experimentation. In the HeLa cell line, a similar rate of mobile GSV activity was observed, however, a decline in stationary GSVs was found. The increased accumulation of mobile vesicles at the plasma membrane is in accordance with previously proposed models of GSV recruitment. However, the reduction in stationary vesicles at the membrane surface in the HeLa cell line suggested differences in the machinery associated with vesicle fusion. The second area of study focussed on the analysis of the environmentally sensitive class of fluorophores known as molecular rotors, in particular the meso-substituted BODIPY rotor. Molecular rotors are said to report on the viscosity of the environment in which they reside but questions still remained over their efficacy of assessing viscosity in complex environments such as lipid bilayers. A combined Fluorescence Correlation Spectroscopy (FCS) and fluorescence lifetime system was optimised to simultaneously probe the lateral mobility and viscosity sensitive fluorescent lifetime of the dye in artificial bilayer systems. The diffusion coefficients measured directly through FCS were compared with those inferred from the lifetime values by conversion through the Saffman-Delbruck model. Those measured by FCS were found to be similar to previously simulated values suggesting a well working experimental system. The values found through lifetime analysis were of the same order to those measured by FCS but differed by as much as a factor of 2 in some cases. The reasons for this most likely lie through the inherent assumptions made using the Saffman-Delbruck model. In addition, the probes were assessed in bilayers of differing degrees of phospholipid saturation. It was observed that the viscosity of the environment increased with decreasing saturation in the hydrocarbon tail regions of the lipids. This was noted through the diffusion coefficients measured with both methods. The final chapter focussed on the creation of a system to increase the resolution of Fluorescence Lifetime Imaging Microscopy (FLIM) by implementing a TIRFM illumination scheme. The focus of this work was to increase resolution for imaging of membrane viscosity through the use of molecular rotors. Molecular rotors in cellular systems are susceptible to endocytosis over certain time frames, limiting their use in physiologically relevant studies in vitro. A gated FLIM system was constructed through the combination of pulsed laser diodes and a gated Intensified Charge Coupled Device (ICCD) camera. To investigate the abilities of the system to selectively image a surface localised signal relating to membrane viscosity, Supported Lipid Bilayers (SLBs) were used. It was found, through both lifetime and FCS, that the substrate on which the bilayer was deposited reduced the mobility of the probe and the measured fluorescence lifetime. The effect was a change in diffusion coefficient by a factor or 2-3 which was taken into account when assessing the viscosity measured through FLIM of SLBs. The TIRF-FLIM principle was then demonstrated through imaging of SLBs containing the molecular rotor BODIPY against a highly fluorescent background of the fluorophore FITC. FITC provided a background with a distinctly longer lifetime to that of the rotor in the bilayer. The system was able to resolve the surface localised lifetime signal over a range of concentrations of background signal from nano-molar to micro-molar. The critical point, where the bilayer lifetime became indistinguishable from the background, came at a fluorophore ratio of 2:1 BODIPY to FITC