The object of this thesis is to develop a new extension of Image Correlation Spectroscopy (ICS) that can measure velocity vectors for flowing protein populations in living cells. This new technique, called Spatio-Temporal Image Correlation Spectroscopy (STICS), allows measurement of both diffusion coefficients and velocity vectors (magnitude and direction) from fluorescence microscopy image time series of fluorescently labeled cellular proteins via monitoring of the time evolution of the full space-time correlation function of the intensity fluctuations. By using filtering in Fourier space to remove frequencies associated with immobile or slow components, it is possible to measure the protein transport even in the presence of a large fraction of immobile species that are static in the image series. The STICS method can generate complete transport maps of proteins within sub-regions of the basal membrane even if the protein concentration is too high to perform single particle tracking measurements, and it can be applied to any type of fluorescence microscopy image time series. This thesis presents the background theory, computer simulations, and analysis of measurements on fluorescent microspheres and fixed cell samples to demonstrate proof of principle, capabilities, and limitations of the method. Visible fluorescent proteins (VFPs) were used to label a variety of the proteins involved in cell-to-extra-cellular-matrix adhesions, including focal adhesion kinase, paxillin, alpha-actinin, alpha5-integrin, talin, vinculin and actin. Various fusion protein pairs were transfected in living cells and imaged using both laser scanning microscopy and total internal reflection microscopes. Using STICS analysis, co-transport maps of proteins were generated within protruding sub-regions of the basal membrane. The new space time image correlation method can probe the mechanistic details of the hypothesized molecular clutch that regulates the extra cellular matrix/cytoskeletal interactions during migration. The technique was also applied to mapping fluid flow in migrating keratocytes in order to elucidate the role that fluid flow plays in migrating cells.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.102507 |
| Date | January 2006 |
| Creators | Hébert, Benedict. |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Physics.) |
| Rights | © Benedict Hébert, 2006 |
| Relation | alephsysno: 002573453, proquestno: AAINR27788, Theses scanned by UMI/ProQuest. |
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