Linear Behavior of the Phase Lifetime in Frequency-Domain Fluorescence Lifetime Imaging of FRET Constructs

We utilize a cost-effective frequency-domain fluorescence lifetime imaging microscope to measure the phase lifetime of mTFP1 in mTFP1-mVenus fluorescence resonance energy transfer (FRET) constructs relevant to the VinTS molecular tension probe. Our data were collected at 15 modulation frequencies omega/2 pi selected between 14 and 70 MHz. The lifetime of mTFP1 was tau(D) = 3.11 +/- 0.02 ns in the absence of acceptor. For modulation frequencies, omega, such that (omega center dot tau(D)) < 1.1, the phase lifetime of mTFP1in the presence of acceptor (mVenus), tau(DA)(phi), was directly related to the amplitude-weighted lifetime tau(DA)(ave) inferred from the known FRET efficiency (E-FRET(true)) of the constructs. A linear fit to a plot of (omega center dot tau(DA)(phi)) vs. (omega center dot tau(DA)(ave)) yielded a slope of 0.79 +/- 0.05 and intercept of 0.095 +/- 0.029 (R-2 = 0.952). Thus, our results suggest that a linear relationship exists between the apparent E-FRET(app) based on the measured phase lifetime and E-FRET(true) for frequencies such that (omega center dot tau(D)) < 1.1. We had previously reported a similar relationship between E-FRET(app) and E-FRET(true) at 42 MHz. Our current results provide additional evidence in support of this observation, but further investigation is still required to fully characterize these results. A direct relationship between tau(DA)(phi) and tau(DA)(ave) has the potential to simplify significantly data acquisition and interpretation in fluorescence lifetime measurements of FRET constructs.