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Nanoscopic Approach to Quantification of Equilibrium and Rate Constants of Complex Formation at Single-Molecule Level
journal contribution
posted on 2017-11-13, 00:00 authored by Xuzhu Zhang, Evangelos Sisamakis, Krzysztof Sozanski, Robert HolystEquilibrium and rate constants are
key descriptors of complex-formation
processes in a variety of chemical and biological reactions. However,
these parameters are difficult to quantify, especially in the locally
confined, heterogeneous, and dynamically changing living matter. Herein,
we address this challenge by combining stimulated emission depletion
(STED) nanoscopy with fluorescence correlation spectroscopy (FCS).
STED reduces the length-scale of observation to tens of nanometres
(2D)/attoliters (3D) and the time-scale to microseconds, with direct,
gradual control. This allows one to distinguish diffusional and binding
processes of complex-formation, even at reaction rates higher by an
order of magnitude than in confocal FCS. We provide analytical autocorrelation
formulas for probes undergoing diffusion-reaction processes under
STED condition. We support the theoretical analysis of experimental
STED-FCS data on a model system of dye–micelle, where we retrieve
the equilibrium and rates constants. Our work paves a promising way
toward quantitative characterization of molecular interactions in vivo.
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Keywords
fluorescence correlation spectroscopyemission depletioncomplex-formation processesreaction ratesautocorrelation formulasdiffusion-reaction processesrate constantsSTED conditionComplex Formationbinding processesmodel systemNanoscopic ApproachSTED-FCS dataconfocal FCSRate ConstantsSingle-Molecule Level Equilibriumrates constants
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