Data registration and selective single-molecule analysis using multi-parameter fluorescence detection

A general strategy to identify and quantify sample molecules in dilute solu tion employing a new spectroscopic method for data registration and specifi c burst analysis denoted as multi-parameter fluorescence detection (MFD) wa s recently developed. While keeping the experimental advantage of monitorin g single molecules diffusing through the microscopic open volume element of a confocal epi-illuminated set-up as in experiments of fluorescence correl ation spectroscopy, MFD uses pulsed excitation and time-correlated single-p hoton counting to simultaneously monitor the evolution of the four-dimensio nal fluorescence information (intensity, F; lifetime, tau; anisotropy, r; a nd spectral range, lambda (T)) in real time and allows for exclusion of ext raneous events for subsequent analysis. In this review, the versatility of this technique in confocal fluorescence spectroscopy will be presented by i dentifying freely diffusing single dyes via their characteristic fluorescen ce properties in homogenous assays, resulting in significantly reduced misc lassification probabilities. Major improvements in background suppression a re demonstrated by time-gated autocorrelation analysis of fluorescence inte nsity traces extracted from MFD data. Finally, applications of MFD to real- time conformational dynamics studies of fluorescence labeled oligonucleotid es will be presented. (C) 2001 Elsevier science B.V. All rights reserved.