High-throughput single-molecule spectroscopy in free solution

A high-speed high-throughput single-molecule imaging technique for identify ing molecules in free solution based on differences in their fluorescence e mission spectra is presented. Unlike previous reports, the entire spectrum, rather than selected wavelengths through optical filters, is recorded. Fur thermore, the millisecond data acquisition time means that the molecules do not need to be immobilized or spatially confined. In one example, individu al lambda DNA molecules labeled with YOYO-I, POPO-III, or a combination of the two dyes can be distinguished from one another. In another example, bio tinylated 2.1-kb DNA labeled with YOYO-I was reacted with avidin-conjugated R-phycoerythrin. The two different reactant molecules and the product mole cule can be simultaneously imaged and identified by their spectroscopic cha racteristics. This technique can therefore be used for screening single mol ecules for disease markers and for monitoring individual molecular interact ions at a rate of thousands of molecules per second.