Please use this identifier to cite or link to this item: http://scholarbank.nus.edu.sg/handle/10635/17109
Title: Quantification of biomolecule dynamics and interactions in living zebrafish embryos by fluorescence correlation spectroscopy
Authors: SHI XIANKE
Keywords: fluorescence, fluorescence correlation spectroscopy, zebrafish, in vivo, dissociate constant, Cdc42
Issue Date: 20-Jul-2009
Source: SHI XIANKE (2009-07-20). Quantification of biomolecule dynamics and interactions in living zebrafish embryos by fluorescence correlation spectroscopy. ScholarBank@NUS Repository.
Abstract: Recent advances in light microscopy and spectroscopy make it possible to study single molecule behaviors in an intact 3D multicelluar organism. Taking advantage of the transparent tissues of zebrafish embryos as well as the established molecular and genetic approaches in this animal model, we present in this thesis the measurements of molecular dynamics and interactions in living zebrafish embryos by fluorescence correlation spectroscopy (FCS) and single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS). FCS/SW-FCCS are ultra-sensitive experimental techniques that analyze fluorescence fluctuations from a static observation volume and provide information about the concentration, diffusion constant and biophysical properties of the fluorescent particle. We first examined how and to what extent zebrafish embryos can be studied using FCS. Then the applicability of FCS to study molecular processes in embryo was demonstrated by the determination of blood flow velocities with high spatial resolution, and the determination of diffusion coefficients of cytoplasmic and membrane-bound enhanced green fluorescence protein (EGFP) labeled proteins in different subcellular compartments as well as in different cell types. Lastly, we show, for the first time to our knowledge, the quantification of protein-protein interactions in single cells in living zebrafish embryo with SW-FCCS. The extension of this biophysical tool into living organism allows answering developmental biology question directly on a molecular basis, and provides a platform to address the potential artifacts arising from Petri dish-based in vitro studies.
URI: http://scholarbank.nus.edu.sg/handle/10635/17109
Appears in Collections:Ph.D Theses (Open)

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