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

Abstract

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 ω/2π selected between 14 and 70 MHz. The lifetime of mTFP1 was τD = 3.11 ± 0.02 ns in the absence of acceptor. For modulation frequencies, ω, such that (ω · τD) < 1.1, the phase lifetime of mTFP1in the presence of acceptor (mVenus), (Formula presented.), was directly related to the amplitude-weighted lifetime (Formula presented.) inferred from the known FRET efficiency ((Formula presented.)) of the constructs. A linear fit to a plot of (Formula presented.) yielded a slope of 0.79 ± 0.05 and intercept of 0.095 ± 0.029 (R2 = 0.952). Thus, our results suggest that a linear relationship exists between the apparent (Formula presented.) based on the measured phase lifetime and (Formula presented.) for frequencies such that (ω · τD) < 1.1. We had previously reported a similar relationship between (Formula presented.) and (Formula presented.) 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 (Formula presented.) and (Formula presented.) has the potential to simplify significantly data acquisition and interpretation in fluorescence lifetime measurements of FRET constructs.