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Abstract
A general strategy to identify and quantify sample molecules in dilute solution employing
a new spectroscopic method for data registration and specific burst analysis denoted
as multi-parameter fluorescence detection (MFD) was recently developed. While keeping
the experimental advantage of monitoring single molecules diffusing through the microscopic
open volume element of a confocal epi-illuminated set-up as in experiments of fluorescence
correlation spectroscopy, MFD uses pulsed excitation and time-correlated single-photon
counting to simultaneously monitor the evolution of the four-dimensional fluorescence
information (intensity, F; lifetime, tau; anisotropy, r; and spectral range, lambda(r))
in real time and allows for exclusion of extraneous events for subsequent analysis.
In this review, the versatility of this technique in confocal fluorescence spectroscopy
will be presented by identifying freely diffusing single dyes via their characteristic
fluorescence properties in homogenous assays, resulting in significantly reduced misclassification
probabilities. Major improvements in background suppression are demonstrated by time-gated
autocorrelation analysis of fluorescence intensity traces extracted from MFD data.
Finally, applications of MFD to real-time conformational dynamics studies of fluorescence
labeled oligonucleotides will be presented.