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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.  Analytically challenging scenarios are ubiquitous, such as diffusion in or around vesicles, micelles, and nanoscale corrals on polymer-coated surfaces.

The third year of activity brought development of the simulator to more fully incorporate the dynamics of vesicles and micelles as they encapsulate other compounds.  We published a paper in Analytical Chemistry dealing with how to accurately measure entrapment and are now seeking to use the simulator to help quantify partitioning between the interior and exterior of vesicles and micelles.

We have also completed work on the potential limitations that arise whenever the optical detection volume (DV) in FCS does not match the ideal Gaussian geometry ubiquitously assumed by FCS theory.  In practice, a lack of correspondence between theory and experiment is commonplace and can lead to misinterpretation of data, particularly for analyses where multiple properties are assessed simultaneously.  In a manuscript submitted for publication, we describe an application of the simulator that closely mimics the measurement conditions established in an FCS microscope. In this study, we employ numerical simulation to systematically explore the influence of DV geometry.  Using different levels of DV distortion, we delineate a range of conditions that are necessary for obtaining accurate FCS results and show experimental circumstances that should be generally avoided.