Fig. 4.

Two-color TIRFM of labeled M₁ receptors moving on a single CHO cell. (A) A 33-ms frame of Alexa488–telenzepine-labeled M₁ receptors (green). (B) Image of the same cell recorded 33 ms later with the Cy3B–telenzepine-labeled receptors (red). Scale bar (5 μm) in B applies to A–F. (C) Superposition of images in A and B. Yellow spots indicate candidate dimers or chance colocalization. (D) A total of 825 trajectories of Alexa488–telenzepine-labeled M₁ receptors identified during the 44-s recording. (E) A total of 970 trajectories of Cy3B–telenzepine-labeled M₁ receptors. (F) A total of 241 trajectories of M₁ dimers labeled with both green and red fluorophores. (G) Different tracks showing dimer formation and dissociation. The x and y coordinates are shown for five examples in the upper and lower sections of each numbered plot. Coincidence of the tracks in both the x and y dimensions for a period >660 ms was taken as evidence of dimer formation (separation <160 nm). The green and red objects are not viewed simultaneously but 33 ms apart so the objects may move during that time. The five examples shown demonstrate different behaviors: (1) trajectory of a two-color dimer formation' (2) trajectory of a two-color dimer dissociation; (3) trajectory of a two-color dimer that dissociates after ∼1.5 s and then reforms a dimer; (4, 5) formation and dissociation of two two-color dimers. (Inset) A histogram of the lifetimes of 84 M₁ receptor dimers taken from trajectories similar to those in examples 4 and 5 and collected in 0.5-s bins. The solid line is a monoexponential fit for a mean lifetime of 0.5 s.