Multipair Förster Resonance Energy Transfer via Spectrally Resolved Single-Molecule Detection

Carey Phelps, Tao Huang, Jing Wang, Xiaolin Nan

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)5765-5771
Number of pages7
JournalJournal of Physical Chemistry B
Issue number31
StatePublished - Aug 11 2022

ASJC Scopus subject areas

  • Physical and Theoretical Chemistry
  • Surfaces, Coatings and Films
  • Materials Chemistry


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