30 March 2005 Membrane protein dynamics measured by two-photon ring correlation spectroscopy: theory and application to living cells
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Proceedings Volume 5700, Multiphoton Microscopy in the Biomedical Sciences V; (2005); doi: 10.1117/12.593894
Event: SPIE BiOS, 2005, San Jose, CA, United States
Abstract
Many biochemical reactions and processes are regulated by proteins associated with cellular membranes. Trans-membrane proteins play an important role in many aspects of cellular development, cellular migration and signaling, and many diseases. Quantitative measurement of protein dynamics under various experimental conditions can give insights into the mechanisms of interaction and the functionality of the protein. Fluctuation techniques, such as fluorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS), have been used for such dynamic measurements in membranes. However, FCS is limited to fast dynamics, and ICS works best on a flat 2-dimensional area. We present an alternative way to measure protein transport in spherical (non flat) living cells that combines laser scanning microscopy and image correlation methods: ring correlation spectroscopy (RCS). The RCS analysis is performed on CLSM or two-photon cross-sectional images of labeled proteins in the cell membrane, where the optical sectioning gives a “ring” of fluorescence in the images. We present computer simulations of two dimensional diffusion confined to the surface of a spherical shell, where the RCS analysis can extract the set input parameters from the simulation. As well, we present RCS analysis of two-photon microscopy images of Pre-B leukocytes cells expressing CD44 labeled with EGFP.
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Benedict Hebert, S. Elizabeth Hulme, Paul W. Wiseman, "Membrane protein dynamics measured by two-photon ring correlation spectroscopy: theory and application to living cells", Proc. SPIE 5700, Multiphoton Microscopy in the Biomedical Sciences V, (30 March 2005); doi: 10.1117/12.593894; http://dx.doi.org/10.1117/12.593894
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KEYWORDS
Diffusion

Proteins

Luminescence

Spectroscopy

Fluorescence correlation spectroscopy

Spherical lenses

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