Artifact-Free and Detection-Profile-Independent Higher-Order Fluorescence Correlation Spectroscopy for Microsecond-Resolved Kinetics. 1. Multidetector and Sub-Binning Approach

Fluorescence correlation spectroscopy
(FCS) is a powerful tool
in the time-resolved analysis of nonreacting or reacting molecules
in solution, based on fluorescence intensity fluctuations. However,
conventional (second-order) FCS alone is insufficient to measure all
parameters needed to describe a reaction or mixture, including concentrations,
fluorescence brightnesses, and forward and reverse rate constants.
For this purpose, correlations of higher powers of fluorescence intensity
fluctuations can be calculated to yield additional information from
the single-photon data stream collected in an FCS experiment. To describe
systems of diffusing and reacting molecules, considering cumulants
of fluorescence intensity results in simple expressions in which the
reaction and diffusion parts factorize. The computation of higher-order
correlations in experiments is hindered by shot-noise and common detector
artifacts, the effects of which become worse with increasing order.
In this article, we introduce a technique to calculate artifact-free
higher-order correlation functions with improved time resolution,
and without any need for modeling and calibration of detector artifacts.
The technique is formulated for general multidetector experiments
and verified in both two-detector and single-detector configurations.
Good signal-to-noise ratio is achieved down to 1 μs in correlation
curves up to order (2, 2). This capability makes possible a variety
of new measurements including multicomponent analysis and fast reaction
kinetics, as demonstrated in a companion article (10.1021/acs.jpcb.7b00408).