Kinetics of M₁R activation. (A) Cartoon of the double-labeled M₁R construct, M₁R-YFP-CFP. (B) Confocal images of three resting cells, only one of which expresses the M₁R. The transfected cell shows the distributions of cyan and yellow fluorescence. Bar, 10 µm. (C) FRETr photometry time course for a single cell. The top panel shows corrected CFP_C fluorescence (blue trace, left axis) and YFP_C fluorescence (yellow trace, right axis), and the bottom panel shows the corrected ratio, YFP_C/CFP_C (black), for a 5-s exposure to 10 µM oxo-M. Sampling frequency: 1 Hz during baseline and 10 Hz during agonist. (D) Normalized mean time course for five 5-s exposures to oxo-M in a single cell (same cell as C). Black line is a single-exponential fit with τ_off = 180 ms.