Distilling Information from Noisy Data: Examples from Microscopy
Description:... In Part I, I describe theoretical, experimental, and computational developments in two-color fluorescence fluctuation microscopy (TCFFM). In TCFFM, intensity fluctuations of fluorescently-labeled molecules within a small optical volume are analyzed to glean information about the dynamics of system components. Focusing on bimolecular reactive systems of the form (A + B & rlhar2; C), I present a general TCFFM theory and discuss how the analysis of fluctuation correlations can be used to measure absolute particle numbers and kinetic rates of binding. I also discuss the influence of Forester resonance energy transfer, reaction rates and reactant concentrations, diffusion, and component visibility on correlation analyses. After describing a two-photon microscope system that I assembled for TCFFM measurements, I present experimental proof-of-principle results in a discussion of how TCFFM can be used to measure the equilibrium constant of two fluorescently-labeled interacting molecules. Complementing these experimental TCFFM studies, I describe a molecular dynamics/Monte Carlo-based simulation tool and demonstrate how it can be used to study more complex systems and experimental conditions that cannot be accounted for with the current theory.
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