%0 Journal Article
%T Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time
%A Sanghwa Lee
%A Jinwoo Lee
%A Sungchul Hohng
%J PLOS ONE
%D 2012
%I Public Library of Science (PLoS)
%R 10.1371/journal.pone.0012270
%X Full understanding of complex biological interactions frequently requires multi-color detection capability in doing single-molecule fluorescence resonance energy transfer (FRET) experiments. Existing single-molecule three-color FRET techniques, however, suffer from severe photobleaching of Alexa 488, or its alternative dyes, and have been limitedly used for kinetics studies. In this work, we developed a single-molecule three-color FRET technique based on the Cy3-Cy5-Cy7 dye trio, thus providing enhanced observation time and improved data quality. Because the absorption spectra of three fluorophores are well separated, real-time monitoring of three FRET efficiencies was possible by incorporating the alternating laser excitation (ALEX) technique both in confocal microscopy and in total-internal-reflection fluorescence (TIRF) microscopy.
%U http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0012270