Fig. 5.

Time-resolved EEM measurements on double-labeled dsDNA. (a) Modulation and phase of quenched Alexa488 (donor EEM channel ê₁₁), in comparison with unquenched Alexa488. The fluorescence lifetime of Alexa488 decreased from 4.1 ± 0.1 ns to 3.0 ± 0.1 ns due to FRET. (b) Modulation and phase of Aelxa546 (acceptor EEM channel ê₂₂). The fluorescence lifetime of Alexa546 was 3.4 ± 0.1 ns, same as pure Alexa546. The acceptor EEM channel is unrelated to FRET, thus the lifetime remains constant. (c) Modulation and phase of the Alexa488-Alexa546 FRET EEM channel ê₁₂. Experimental results in (c) were overlaid with the theoretical model (Eq. (8)). The phase delay in the FRET EEM channel exceeded π/2, which was a signature of FRET. (d) Spectral configuration of the EEM measurement. Error bars in (a~c) represent standard deviations of multiple 46-μs frequency sweeps.