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. 2006 Nov;128(5):569–581. doi: 10.1085/jgp.200609638

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Copyright © 2006, The Rockefeller University Press

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Figure 1. — Principle of lifetime measurements in the frequency domain. (A) If a fluorophore absorbs a photon (hν) it is excited to the S1 state. Each fluorophore has a characteristic dwell-time τ in the S1 state before it relaxes back to the ground state S0. This can occur by emitting a photon (red) by nonradiative deexcitation (k_nr) or by quenching (q). (B) If the amplitude of the excitation light (blue) is sinusoidally modulated, the emission will follow this time course with a time delay (due to the dwell time in S1), resulting in the phase shift ΔΦ. Due to a low-pass filtering effect, the amplitude of the emission is reduced (ΔM). (C) Labeling efficiency: comparison of labeling of single-cysteine mutant and WT channel. The labeling ratio of mutant to WT is ∼13.