(A) Time course of population-averaged (black; upper panel) and typical single-cell (colors; lower panel) measurements of the FRET ratio for ΔcheRΔcheB strain expressing TarQEEE as the sole receptor. Measurements were performed as in Figure 1. Cells were first equilibrated in buffer (red) and subsequently stimulated by addition (blue) and subsequent removal of 30 µM MeAsp, saturating stimulus for this receptor. (B) Same as (A) but for ΔcheRΔcheB strain expressing TarQEQE as the sole receptor and upon stimulation with 30 µM (blue) and then 100 µM (green) MeAsp. Note that for this receptor, 30 µM MeAsp is the sub-saturating stimulus whereas 100 µM MeAsp is the saturating stimulus. The measurement traces for single cells in (A) and (B) have been shifted along the y-axis to facilitate visualization. (C) PSD of the single-cell FRET ratio for TarQEEE in buffer (blue) or in 30 µM MeAsp (cyan), TarQEQE in buffer (orange), in 30 µM MeAsp (red) or in 100 µM MeAsp (green). (D) Corresponding time autocorrelation functions of the single-cell FRET ratio for indicated strains/conditions. Dashed lines show fits by single exponential decay. Error bars represent standard errors of the mean (SEM), and the sample sizes are 153 (TarQEEE, buffer), 65 (TarQEEE, 30 µM), 471 (TarQEQE, buffer), 404 (TarQEQE, 30 µM) and 136 (TarQEQE, 100 µM) single cells coming from at least three independent experiments in each case.