Two -Photon Dual Channel Fluctuation Correlation Spectroscopy: Theory and Application
Eid, John Safwat
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https://hdl.handle.net/2142/85426
Description
Title
Two -Photon Dual Channel Fluctuation Correlation Spectroscopy: Theory and Application
Author(s)
Eid, John Safwat
Issue Date
2002
Doctoral Committee Chair(s)
Gratton, Enrico
Department of Study
Biophysics and Computational Biology
Discipline
Biophysics and Computational Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
Language
eng
Abstract
Fluctuation correlation spectroscopy (FCS) is a non-perturbative method used to gain molecular information from stochastic processes such as Brownian motion (Magde, Elson et al. 1972). Dual channel FCS allows for the investigation of static (molecular associations) and kinetic (protein dynamics) parameters of interaction between two species. This technique is able to quantitate in vivo molecular interactions that are critical for cellular study. Data acquisition hardware was built which allows 25 ns time resolution and records the entire photon sequence using a hardware data compression technique. This allows multiple analysis to be performed (i.e. photon counting histogram (Muller, Chen et al. 1999), moment analysis, etc.) The basic theory and simulation of oligomer size effect on dual channel FCS data was done to allow interpretation of an in vivo study involving the amount of aggregation between different somatostatin membrane receptors. The cross-correlation, in combination with Forster resonance energy transfer (FRET), provides a more elegant method to study protein dynamics than the equivalent quenching experiment involving only one channel of detection (Haas and Steinberg 1984). The extra relaxation is present as an extra hump in the autocorrelation and as an anti-correlation for the cross-correlation. This signature anti-correlation differentiates this process from others such as triplet state, rotational diffusion, quenching, etc. In vitro confirmation is provided through studies of ribosome and cameleon proteins.
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