Nanoscopic Approach to Quantification of Equilibrium and Rate Constants of Complex Formation at Single-Molecule Level
J. Phys. Chem. Lett. 2017, 8, 5785−5791
Xuzhu Zhang, Evangelos Sisamakis, Krzysztof Sozanski, and Robert Holyst
Equilibrium and rate constants are key descriptors of complex-formation processes in a variety of chemical and biological reactions. However, these parameters are dicult to quantify, especially in the locally conned, heterogeneous, and dynamically changing living matter. Herein, we address this challenge by combining stimulated emission depletion (STED) nanoscopy with uorescence 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 to distinguish diusional 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 diusion-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 towards quantitative characterization of molecular interactions in vivo.
This document is the Accepted Manuscript version of a Published Work that appeared in final form in the Journal of Physical Chemistry Letter, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see https://pubs.acs.org/doi/abs/10.1021/acs.jpclett.7b02742 .