TY - JOUR
T1 - Multipair Förster Resonance Energy Transfer via Spectrally Resolved Single-Molecule Detection
AU - Phelps, Carey
AU - Huang, Tao
AU - Wang, Jing
AU - Nan, Xiaolin
N1 - Funding Information:
We thank Drs. Steven Chu (Stanford), Joe W. Gray (OHSU), Malwina Szczepaniak (OHSU), and Philip Stork (OHSU) for helpful discussions on various aspects of the project. The project was funded by the OHSU Knight Cancer Institute (Hildegard Lamfrom Scholars Award), the Damon Runyon Cancer Research Foundation, the M. J. Murdock Charitable Trust, and the FEI Company (all to X.N.). X.N. is also supported by NIH grants R01 GM132322 and U54 CA209988. C.P. was supported by an NIH postdoctoral training fellowship.
Publisher Copyright:
© 2022 The Authors. Published by American Chemical Society.
PY - 2022/8/11
Y1 - 2022/8/11
N2 - Förster resonance energy transfer (FRET) is a powerful tool for studying molecular interactions. Its use for studying interactions involving more than two molecules, however, has been limited by spectral crosstalk among the fluorophores. Here, we report multispectral FRET (msFRET) for imaging multiple pairs of interactions in parallel by spectrally resolving single fluorescent molecules. By using a dual (positional and spectral) channel and wide-field imaging configuration, fluorophores with emission maxima as close as 6-10 nm could be reliably distinguished. We demonstrate msFRET by continuously monitoring the hybridization dynamics among 2 × 2 pairs of DNA oligos in parallel using Cy3 and Cy3.5 as donors and Cy5 and Cy5.5 as acceptors. Aside from studying molecular interactions, msFRET may also find applications in probing fluorophore photophysics during FRET and in multiplexed superresolution imaging.
AB - Förster resonance energy transfer (FRET) is a powerful tool for studying molecular interactions. Its use for studying interactions involving more than two molecules, however, has been limited by spectral crosstalk among the fluorophores. Here, we report multispectral FRET (msFRET) for imaging multiple pairs of interactions in parallel by spectrally resolving single fluorescent molecules. By using a dual (positional and spectral) channel and wide-field imaging configuration, fluorophores with emission maxima as close as 6-10 nm could be reliably distinguished. We demonstrate msFRET by continuously monitoring the hybridization dynamics among 2 × 2 pairs of DNA oligos in parallel using Cy3 and Cy3.5 as donors and Cy5 and Cy5.5 as acceptors. Aside from studying molecular interactions, msFRET may also find applications in probing fluorophore photophysics during FRET and in multiplexed superresolution imaging.
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U2 - 10.1021/acs.jpcb.2c03249
DO - 10.1021/acs.jpcb.2c03249
M3 - Article
C2 - 35897122
AN - SCOPUS:85136022518
SN - 1520-6106
VL - 126
SP - 5765
EP - 5771
JO - Journal of Physical Chemistry B
JF - Journal of Physical Chemistry B
IS - 31
ER -