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 language||English (US)|
|Title of host publication||Ultrafast Nonlinear Imaging and Spectroscopy V|
|Editors||Zhiwen Liu, Kebin Shi, Iam Choon Khoo, Demetri Psaltis|
|State||Published - 2017|
|Event||Ultrafast Nonlinear Imaging and Spectroscopy V 2017 - San Diego, United States|
Duration: Aug 6 2017 → Aug 7 2017
|Name||Proceedings of SPIE - The International Society for Optical Engineering|
|Conference||Ultrafast Nonlinear Imaging and Spectroscopy V 2017|
|Period||8/6/17 → 8/7/17|
Bibliographical noteFunding Information:
E.D.S. and A.A.H. acknowledge the financial support provided by the University of Minnesota Grant-in-Aid. A.J.B. also acknowledges the financial support of the Netherlands Organization for Scientific Research Vidi grant. R.L. was supported by the Swenson Family Foundation. Additionally, the financial support provided by a Chancellor’s Small Grant, the Department of Chemistry and Biochemistry, the Swenson College of Science and Engineering, all of which are from the University of Minnesota Duluth. M.C., J.S. and H.L. were supported by teaching fellowships from the University of Minnesota Duluth Department of Chemistry and Biochemistry. The authors also acknowledge the Minnesota Supercomputing Institute (MSI) at the University of Minnesota for providing resources that contributed to the research results reported within this paper.
© 2017 SPIE.
- excited-state dynamics
- fluorescence lifetime
- kinetics model