Microfabricated near-field optics for live cell imaging

Biochemical and biophysical evidence has suggested that nanometric lateral heterogeneities exist within the plane of the cellular membrane termed lipid rafts. These structures have been implicated in a number of important cellular processes including signal transduction and viral infection. The biochemical evidence is based on detergent resistance and allows the molecular constituents of the lipid rafts to be determined while the biophysical techniques suggest that lipid rafts exist on the order of 25–700 nm in diameter. Both the biochemical and biophysical evidence is indirect which has fueled debate over the actual existence of these elusive structures. The data suggest that rafts exist on the nanometric dimension which is smaller than what conventional microscopy is capable of resolving. Therefore, to directly probe lipid rafts in vivo, an optical technique with increased spatial resolution is needed. Near-field scanning optical microscopy (NSOM) allows an order of magnitude increase in spatial resolution over conventional microscopy making it capable of directly probing lipid rafts. However, to date NSOM has not been successful at imaging living cells due to the imaging forces that occur between the near-field probe and the cellular membrane which tends to damage the fragile cell. These forces arise from the delicate nature of the cellular membrane in combination with the inherent rigidity of conventional NSOM probes, thus limiting their application to fixed tissues. Therefore efforts in this thesis are directed at reducing the probe-sample interactions to allow live cell imaging with NSOM. A number of approaches have been taken to lessen the imaging forces associated with NSOM, including altering the mechanical characteristics of the fiber optic probe, changing the feedback mechanism to maintain the probe near the sample surface, and designing alternative NSOM probes. The latter has finally led to a NSOM probe that is capable of probing the cellular membrane of living cells under physiological conditions. This opens many exciting opportunities for NSOM in the biological sciences including the ability to image cellular membrane dynamics in living cells, including lipid rafts, with increased optical resolution over conventional optical microscopy.