Single-molecule three-color FRET with both negligible spectral overlap and long observation time.
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.
Lee
Sanghwa
S
Department of Physics and Astronomy, Seoul National University, Seoul, Korea.
Lee
Jinwoo
J
Hohng
Sungchul
S
D000042:Absorption; D001483:Base Sequence; D003116:Color; D004247:DNA / Q000737:chemistry / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D007834:Lasers; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010100:Oxygen / Q000737:chemistry; D038761:Photobleaching; D013997:Time Factors
D004247:DNA; D010100:Oxygen
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