FRET-enabled optical modulation for high sensitivity fluorescence imaging

Chris I. Richards, Jung Cheng Hsiang, Andrew M. Khalil, Nathan P. Hull, Robert M. Dickson

Research output: Contribution to journalArticlepeer-review

30 Scopus citations


Fluorescence resonance energy transfer is utilized to engineer donor photophysics for facile signal amplification and selective fluorescence recovery from high background. This is generalized such that many different fluorophores can be used in optical modulation schemes to drastically improve fluorescence imaging sensitivity. Dynamic, simultaneous, and direct excitation of the acceptor brightens and optically modulates higher energy donor emission. The externally imposed modulation waveform enables selective donor fluorescence extraction through demodulation. By incorporating an acceptor with significant, spectrally shifted, dark-state population, necessary excitation intensities are quite low and agree well with simulated enhancements. Enhancement versus modulation frequency directly yields dark-state lifetimes in a simple ensemble measurement. Using the long-lived Cy5 dark state in conjunction with Cy3 donors, we demonstrate image extraction from a large background to yield ≫10-fold sensitivity improvements through synchronously amplified fluorescence image recovery (SAFIRe).

Original languageEnglish
Pages (from-to)6318-6323
Number of pages6
JournalJournal of the American Chemical Society
Issue number18
StatePublished - May 12 2010

ASJC Scopus subject areas

  • Catalysis
  • General Chemistry
  • Biochemistry
  • Colloid and Surface Chemistry


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