Date of Award

2009

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physics

Advisor

Samuel T. Hess

Second Committee Member

R. Dean Astumian

Third Committee Member

Michael D. Mason

Abstract

The phenomenon of diffraction limits the resolution in fluorescence imaging. While biological structures span many orders in length scales, visualizing structures smaller than hundreds of nanometers in size requires techniques that are not limited by the diffraction barrier. A renaissance in the field of super resolution imaging is pushing the resolution of fluorescence microscopy toward the molecular level. For example, using repeated cycles of activation, localization, and bleaching of single photoactivatable fluorescent molecules, fluorescence photoactivation localization microscopy (FPALM) and related methods have achieved sub-diffraction resolution. By exploiting the fact that the position of a single fluorescent molecule can be determined with a precision that is in principle limited only by the number of detected photons and by controlling the number of fluorescent molecules emitting light at once, such that the images of single molecules are distinguishable, these methods have demonstrated resolution in the tens of nanometers. The clustering of the influenza protein hemagglutinin (HA) in the viral membrane is necessary for membrane fusion and entry of the virus into host cells. Because HA is also associated with lipid rafts, controversial membrane structures which are involved in a variety of normal cellular functions, the mechanism by which HA "hijacks" normal cell membrane rafts for its own purposes is of great interest. However, due to the limitations imposed by diffraction on spatial resolution in light microscopy, the properties and even the existence of rafts have remained elusive. Using FPALM it has been possible to obtain super-resolution images of the distribution of HA in living and fixed fibroblast cell membranes with resolution nearly an order of magnitude better than conventional fluorescence microscopy. In combination with two-color FPALM and quantitative analysis of HA diffusion and clustering, FPALM results for HA in fibroblast membranes are interpreted in terms of several current models of membrane raft organization. Rapid progress in this field of imaging has led to extensions of FPALM to three-dimensional and polarization imaging. Using simple modifications of the FPALM detection path to produce multi-channel detection on the same camera chip enables sub 100 nm axial resolution and nanoscale imaging of single molecule anisotropics. These advances in FPALM show great promise for advancing our understanding of biological systems.

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