| Abstract
| - Single-molecule fluorescence spectroscopy addresses biologicalmechanisms and enables ultrasensitive diagnostics. We describea new family of single-molecule fluorescence methods that usesalternating-laser excitation (ALEX) of diffusing or immobilized biomolecules to study their structure, interactions, and dynamics. Thisis accomplished using ratios that report on the distance betweenand the stoichiometry of fluorophores attached to the moleculesof interest. The principle of alternation is compatible with severaltime scales, allowing monitoring of fast dynamics or simultaneousmonitoring of a large number of individual molecules.
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