Determination of the quantum yield of singlet-triplet conversion in complex organic compounds by the method of laser fluorimetry

FRET-Enabled Optical Modulation for High Sensitivity Fluorescence Imaging

“…3A). These observations, coupled with the excellent agreement with the rate-based model of FRET inhibition using only experimentally determined values,32–34 clearly demonstrate that donor fluorescence enhancement results from significant steady-state excited acceptor populations (Fig. 3A).…”

“…Measured enhancement values at varied 561 nm excitation nicely mirror the predicted intensity-dependent enhancements (Figure A). These observations, coupled with the excellent agreement with the rate-based model of FRET inhibition using only experimentally determined values, − clearly demonstrate that donor fluorescence enhancement results from significant steady-state excited acceptor populations (Figure A). Any spectral shift or decrease in acceptor absorption cross section resulting from excited-state occupation is likely to reduce FRET rates relative to those with optimized acceptor ground state overlap with donor emission (∼90% for 6-Fam-TAMRA).…”

“…Corresponding to the known and measured photophysics of the 6-Fam-TAMRA [32][33][34][35] and Cy3-Cy5 36-39 systems with FRET-…”

“…Corresponding to the known and measured photophysics of the 6-Fam-TAMRA − and Cy3-Cy5 − systems with FRET-coupled excited states, a 6-level model was constructed depicting the relevant states (Figure A). Rates for excitation and relaxation combined with dark-state transitions were utilized to yield steady-state populations of all levels when illuminated with known primary and secondary excitation intensities.…”

“…The photophysical parameters used for determining transitions between states for Cy3-Cy5 − were λ D = 496 nm, λ A = 633 nm, Φ dark = 0.0003, τ D = 3.5 ns, τ A = 3.0 ns, E FRET = 0.47, τ dark - = 500 μs, ε D (496 nm) = 30,000 M −1 cm −1 , ε A (633 nm) = 250,000 M −1 cm −1 . The photophysical parameters used for determining transitions between states for 6-Fam-TAMRA − were λ D = 476 nm, λ A = 561 nm, Φ dark = 0.001, τ D = 3.5 ns, τ A = 3.0 ns, E FRET = 0.90, τ dark - = 10 μs, ε D (476 nm) = 60,000 M −1 cm −1 , ε A (561 nm) = 50,000 M −1 cm −1 .…”