Characterization of Flipper,
NR12S, and NR12A lifetimes in different
lipid environments. Spectral fluorescence lifetime measurements of
the probes in LUVs were carried out within 500–700 nm in intervals
of 20 nm. Multiexponential curve fitting was performed for the fluorescence
decays. (A) Normalized intensity spectra of Flipper (left), NR12S
(middle), and NR12A (right) in varying lipid environments. (B) Normalized
fluorescence decays (full spectrum, 500–700 nm) of Flipper
(left), NR12S (middle), and NR12A (right) in Δ9cis DOPC or DPPC:Chol 50:50. (C) Normalized fluorescence decays at 570
nm vs 650 nm of Flipper (left), NR12S (middle), and NR12A (right)
in POPC:Chol 80:20. (D) Spectrally resolved intensity-weighted lifetime
of Flipper, NR12S, and NR12A in Δ9cis DOPC and the control AF488
in water. The line corresponds to the median of individual biological
replicates (n ≥ 3). The band corresponds to
the standard deviation. (E) Spectrally resolved intensity-weighted
lifetime of Flipper (left), NR12S (middle), and NR12A (right) in different
lipid environments with varying saturation indices. The line corresponds
to the median of individual biological replicates shown with different
symbols (n ≥ 3). The band corresponds to the
standard deviation.