Kinetics model for the wavelength-dependence of excited-state dynamics of hetero-FRET sensors

Jacob Schwarz, Ryan Leighton, Hannah J. Leopold, Megan Currie, Arnold J. Boersma, Erin Sheets, Ahmed A Heikal

Research output: Chapter in Book/Report/Conference proceedingConference contribution

3 Scopus citations


Foerster (or fluorescence) resonance energy transfer (FRET) is a powerful tool for investigating protein-protein interactions, in both living cells and in controlled environments. A typical hetero-FRET pair consists of a donor and acceptor tethered together with a linker. The corresponding energy transfer efficiency of a hetero-FRET pair probe depends upon the donor-acceptor distance, relative dipole orientation, and spectral overlap. Because of the sensitivity of the energy transfer efficiency on the donor-acceptor distance, FRET is often referred to as a "molecular ruler". Time-resolved fluorescence approach for measuring the excited-state lifetime of the donor and acceptor emissions is one of the most reliable approaches for quantitative assessment of the energy transfer efficiency in hetero-FRET pairs. In this contribution, we provide an analytical kinetics model that describes the excited-state depopulation of a FRET probe as a means to predicts the time-resolved fluorescence profile as a function of excitation and detection wavelengths. In addition, we used this developed kinetics model to simulate the time-dependence of the excited-state population of both the donor and acceptor. These results should serve as a guide for our ongoing studies of newly developed hetero-FRET sensors (mCerulean3-linker-mCitrine) that are designed specifically for in vivo studies of macromolecular crowding. The same model is applicable to other FRET pairs with the careful consideration of their steady-state spectroscopy and the experimental design for wavelength- dependence of the fluorescence lifetime measurements.

Original languageEnglish (US)
Title of host publicationUltrafast Nonlinear Imaging and Spectroscopy V
EditorsZhiwen Liu, Kebin Shi, Iam Choon Khoo, Demetri Psaltis
ISBN (Electronic)9781510612174
StatePublished - Jan 1 2017
EventUltrafast Nonlinear Imaging and Spectroscopy V 2017 - San Diego, United States
Duration: Aug 6 2017Aug 7 2017

Publication series

NameProceedings of SPIE - The International Society for Optical Engineering
ISSN (Print)0277-786X
ISSN (Electronic)1996-756X


ConferenceUltrafast Nonlinear Imaging and Spectroscopy V 2017
CountryUnited States
CitySan Diego


  • FRET
  • excited-state dynamics
  • fluorescence lifetime
  • kinetics model

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