Single-Molecule Three-Color FRET with Both Negligible Spectral Overlap and Long Observation Time
نویسندگان
چکیده
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.
منابع مشابه
Detecting buried channels using linear least square RGB color stacking method based on deconvolutive short time Fourier transform
Buried channels are one of the stratigraphic hydrocarbon traps. They are often filled with a variety of porous and permeable sediments so they are important in the exploration of oil and gas reservoirs. In reflection seismic data, high-frequency components are sensitive to the channel thickness, whereas, low-frequency components are sensitive to the channel infill materials. Therefore, decompos...
متن کاملThree-color alternating-laser excitation of single molecules: monitoring multiple interactions and distances.
We introduce three-color alternating-laser excitation (3c-ALEX), a fluorescence resonance energy transfer (FRET) method that measures up to three intramolecular distances and complex interaction stoichiometries of single molecules in solution. This tool extends substantially the capabilities of two-color ALEX, which employs two alternating lasers to study molecular interactions (through probe s...
متن کاملSimultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light
Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential ...
متن کاملSingle-molecule three-color FRET.
Fluorescence resonance energy transfer (FRET) measured at the single-molecule level can reveal conformational changes of biomolecules and intermolecular interactions in physiologically relevant conditions. Thus far single-molecule FRET has been measured only between two fluorophores. However, for many complex systems, the ability to observe changes in more than one distance is desired and FRET ...
متن کاملSingle-Molecule Imaging With One Color Fluorescence.
Single-molecule fluorescence imaging is a powerful tool that enables real-time observation of DNA-protein or RNA-protein interactions with a nanometer precision. Here, we provide a detailed procedure for a previously developed single-molecule fluorescence method, termed "single-molecule protein-induced fluorescence enhancement" (smPIFE). While smFRET (Förster resonance energy transfer) requires...
متن کامل