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Title: 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 ω/2π selected between 14 and 70 MHz. The lifetime of mTFP1 was τ D = 3.11 ± 0.02 ns in the absence of acceptor. For modulation frequencies, ω, such that (ω · τ D ) < 1.1, the phase lifetime of mTFP1in the presence of acceptor (mVenus), τ ϕ D A , was directly related to the amplitude-weighted lifetime τ a v e D A inferred from the known FRET efficiency ( E FRET true ) of the constructs. A linear fit to a plot of ( ω · τ ϕ D A )   v s .   ( ω · τ a v e D A )   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 (ω · τ 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 τ ϕ D A and τ a v e D A has the potential to simplify significantly data acquisition and interpretation in fluorescence lifetime measurements of FRET constructs.  more » « less
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Frontiers in Physics
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National Science Foundation
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