Abstract
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.
Highlights
Many fundamental processes in biology occur in nanometer scale, a range in which conventional optical microscopy does not work
We developed a single-molecule three-color fluorescence resonance energy transfer (FRET) technique based on cyanine dyes– Cy3, Cy5, and Cy7
To compare the FRET ranges of the three FRET pairs, a Forster distance, R0, for each FRET pair was calculated from the following equation: R06 = 0.529 k2 WD J(l)/ NAn4 [16], where R0 and l are in the unit of centimeter, WD is the donor quantum yield, J(l) is the spectral overlap of the donor emission and the acceptor absorption, NA is the Avogadro number, n is the refractive index of the medium, and k2 is determined by the relative orientation of the two dyes
Summary
Many fundamental processes in biology occur in nanometer scale, a range in which conventional optical microscopy does not work. Single-molecule fluorescence resonance energy transfer (FRET) emerged as a tool for studying molecular interactions and dynamics with sub-nanometer sensitivity in unprecedented detail [1,2]. Due to its unique flexibility and adaptability, single-molecule FRET is rapidly expanding its application area in biological studies. As our research interest turns into more realistic situations inside the cell, interactions tend to become more complex, and the capability of acquiring multi-dimensional information is required. To cope with the challenge, researchers are trying to develop more versatile single-molecule FRET techniques, by combining FRET with optical tweezers [3,4], or magnetic tweezers [5,6]. Adding multi-color capabilities to single-molecule FRET experiments is on the same line of technical development
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