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Numerical Correction of Detector Channel Cross-Talk Using Full-Spectrum Fluorescence Correlation Spectroscopy

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Abstract:

Fluorescence correlation spectroscopy (FCS) uses fluctuations in the fluorescence collected from a small illuminated volume to measure dynamic processes of fluorophores. In traditional FCS, spectral overlap produces cross-talk in dedicated detector channels, undermining the accuracy of measurements of molecular interactions. Here, the experimental realization of full-spectrum fluorescence correlation spectroscopy is described and coupled with multivariate data analysis to numerically correct detector cross-talk, isolating spectra and fluctuation traces of mixture components in spite of overlap. Application of this methodology is illustrated using the measurement of the diffusion constant of labeled polystyrene in hydroxypropyl cellulose in the presence of a persistent dye. Additionally, the results show that full-spectrum FCS with multivariate analysis can isolate and characterize signals from unanticipated sample components.

Keywords: CHEMOMETRICS; DIFFUSION CONSTANT; FCS; FLUORESCENCE CORRELATION SPECTROSCOPY; LABELED POLYSTYRENE; MULTIVARIATE DATA ANALYSIS; POLYMER; SPECTRAL RESOLUTION; VISCOSITY

Document Type: Research Article

DOI: http://dx.doi.org/10.1366/000370210792973479

Affiliations: University of Delaware, Dept. of Chemistry and Biochemistry, Brown Laboratory, 163 The Green, Newark, Delaware 19716, USA

Publication date: October 1, 2010

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