High-throughput single-molecule spectroscopy in free solution.

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