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46865-B6
Dissecting the Dynamics of Surfactant Assemblies with Numerical Fluorescence Correlation Spectroscopy

Daniel L. Burden, Wheaton College

Numerical Fluorescence Correlation Spectroscopy (NFCS) follows the general protocol established for traditional Fluorescence Correlation Spectroscopy (FCS), but replaces analytical modeling with two numerical features: (1) a numerical map of the detection volume; (2) a computer simulation that produces correlation curves for scenarios that are difficult or impossible to model analytically. 

Diffusion within and around vesicles and micelles is difficult to interpret with traditional FCS due to the nanoscale geometric constraints imposed on the fluorescent tracers. The approach under development in our lab allows investigation of nearly any geometric scenario by building obstacles or other diffusion constraints into the simulation code.  Thus, the simulator is able to distinguish diffusive components in FCS data that would not be interpretable otherwise.  Example components include: A, macroscopic motion of the vesicle; B) diffusion of tracers within the vesicle; C) diffusion on the vesicle surface; D) free tracers moving in between structures.

The first year of activity has involved developing mapping procedures for NFCS and writing appropriate algorithms for the numerical simulation.

(1) The production of numeric maps involves raster scanning a small light-generating particle fixed to a microscope slide through the detection volume.  We have been using fluorospheres purchased from Invitrogen.  Our work indicates that 3D maps with adequate signal-to-noise ratio require 30-60 minutes for acquisition.  A subsequent 3D deconvolution step is required to extract the correct size and shape from the raw data. 

(2) We have implemented a random-walk diffusion algorithm for simulating free molecular motion through a 3D confocal detection volume that is defined the numeric map.  The software generates a record of photon-bursts over time for molecules diffusing according to user-specified diffusion coefficients and emission intensities.  The simulator has facilities to store the photon-burst record, perform autocorrelations, and fit the autocorrelated data with a specified analytical model.  By comparing the diffusion constant entered into the simulator with the diffusion constant produced by fitting the resulting autocorrelation curve, the simulation parameters required to produce accurate diffusion results have been defined. 

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