The Spectroscopic Basis
of Fluorescence Triple Correlation Spectroscopy
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Abstract
We have developed fluorescence triple correlation spectroscopy
(F3CS) as an extension of the widely used fluorescence microscopy
technique fluorescence correlation spectroscopy. F3CS correlates three
signals at once and provides additional capabilities for the study
of systems with complex stoichiometry, kinetic processes, and irreversible
reactions. A general theory of F3CS was developed to describe the
interplay of molecular dynamics and microscope optics, leading to
an analytical function to predict experimental triple correlations
of molecules that freely diffuse through the tight focus of the microscope.
Experimental correlations were calculated from raw fluorescence data
using triple correlation integrals that extend multiple-tau correlation
theory to delay times in two dimensions. The quality of experimental
data was improved by tuning specific spectroscopic parameters and
employing multiple independent detectors to minimize optoelectronic
artifacts. Experiments with the reversible system of freely diffusing
16S rRNA revealed that triple correlation functions contain symmetries
predicted from time-reversal arguments. Irreversible systems are shown
to break these symmetries, and correlation strategies were developed
to detect time-reversal asymmetries in a comprehensive way with respect
to two delay times, each spanning many orders of magnitude in time.
The correlation strategies, experimental approaches, and theory developed
here enable studies of the composition and dynamics of complex systems
using F3CS