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Analyst. 2001 Aug;126(8):1229-39.

Dynamic two-dimensional fluorescence correlation spectroscopy. Generalized correlation and experimental factors.

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  • 1Department of Chemistry, University of Iowa, Iowa City 52242, USA.


Dynamic two-dimensional fluorescence correlation spectroscopy (2D FCS) is presented in the general form. Dynamic 2D FCS evaluates the time correlation function between two wavelength axes when an external perturbation is applied to the sample. It displays the vibronic features with similar time response functions in the synchronous correlation spectrum and the features with different time responses in the asynchronous correlation spectrum. The correlation analysis allows detailed assignments of the vibronic spectra of multicomponent samples. The emission-emission 2D FCS has proven to be able to resolve spectra with substantial overlaps, of species in equilibrium with each other, and of reacting species whose kinetic constants are linked and multiexponential. Similarly, the correlation analysis between excitation wavelengths allows the assignment of the excitation bands to fluorescent components. When a sinusoidal light source is used to excite the sample, the excitation-emission correlation requires the collection of only four spectra, two in-phase and two quadrature. The two-dimensional excitation-emission correlation analysis uncovers the association between the excitation and the emission vibronic features, enabling the complete assignment of the component spectra. The band associations and spectral assignments are facilitated by the two-dimensional phase map that is constructed from the synchronous and asynchronous correlation spectra. Spectral resolution can be optimized by varying the frequency of excitation and is not influenced by the detector phase angle used to collect the spectra. The resolution power of the 2D FCS is demonstrated with the retrieval of the anthracene emission spectrum from a pyrene-anthracene mixture when it contributes only 4% to the total fluorescence intensity.

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