This thesis studies optical microscopy based high order autocorrelation approaches for measuring molecular aggregation of fluorescently labeled particles in fluid systems. As the particles randomly diffuse into and out of the volume defined by the focus of a confocal laser beam illumination, the collected fluorescence intensity fluctuates. Fluorescence Correlation Spectroscopy (FCS) and Image Correlation Spectroscopy (ICS) have been used as methods which analyse temporal and spatial intensity fluctuations, and provide quantitative information of the molecular transport processes. Theoretical expressions for the high order autocorrelation function magnitudes for a non-interactive model are derived as well as their fitting equations for single- and multicomponent diffusion. / We present an experimental verification of the model applied to simple systems. Solutions of fluorescent microspheres of well-defined size have been imaged using confocal laser scanning microscopy. It has been shown that translational diffusion coefficients were not very sensitive to molecular size dispersion, which made a first order autocorrelation approach to be somewhat ineffective for dealing with multicomponent systems. We demonstrate that the number densities of a mixture of two fluorescent particles can be determined analyzing the higher order autocorrelation function magnitudes. Numerical simulations have been analyzed for testing the experimental tools we use. The technique outlined may be developed to detect and characterize aggregates of fluorescently labeled biological molecules such as membrane proteins and cell surface receptors. Such quantitative aggregation measurements, therefore, can provide information about the mechanism of intercellular signaling which is believed to depend on the oligomerization of cell membrane protein receptors.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.81437 |
| Date | January 2004 |
| Creators | Sergeev, Mikhail. |
| 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 | Master of Science (Department of Physics.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002182956, proquestno: AAIMR06452, Theses scanned by UMI/ProQuest. |
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