TIRFM recording of a single CHO cell, transfected with M₁ muscarinic receptors and labeled with the fluorescent antagonist, Cy3B–telenzepine. (A) Structural formula of Cy3B–telenzepine. (B) A single video frame (33-ms exposure) at the start of a recording. The insert shows a 5 × 5-μm² expanded region containing 15 objects, 4 having intensity consistent with two fluorophores (attributed to receptor dimers) and 11 with intensity similar to a single fluorophore. A pseudocolor look-up table is shown inset. The first 100 frames of the recording are shown in Movie S1. (C) A total of 2,659 tracks of moving M₁ receptors identified from the entire recording of the cell shown in B. The insert shows nine trajectories that illustrate the random nature of the diffusive process. Movie S2 shows the identification of tracks by the tracking algorithm. (D) Plot of average mean square displacement (MSD) versus the time interval (δt) for the trajectories shown in C. The plot is linear (r ² = 0.99), over 1 μm in length, with a 5-s timescale, which is consistent with receptor movement following a random walk, and shows no evidence for anomalous diffusive behavior. (E) Histogram of the intensity of 911 objects identified in the single video frame shown in B. The shape of the distribution is due to the presence of two populations, attributed to receptor dimers (red arrowhead, intensity ~120 counts/pixel) and monomers (black arrowhead, ~60 counts/pixel). The spread of the data arises from a combination of photon noise and intensity variation between spots that are located at different regions within the specimen.

Representative examples of some of the different intensity changes shown by individual tracks of Cy3B-labeled M₁ receptors. (A) Intensity trajectory corresponding to a single molecule (upper trace), compared to the background intensity of a neighboring region containing no fluorescently labeled receptors (lower trace). (B) Intensity trajectory of a single moving object with an intensity corresponding to two molecules of Cy3B–telenzepine, i.e., a dimer. (C) Intensity trajectory of a single moving object with an intensity corresponding to two Cy3B fluorophores (level 2), which changes level abruptly to that of a single fluorophore (level 1), which is due to either photobleaching or dissociation of the dimer into two monomers. (D) Intensity trajectory that changes abruptly from level 1 to level 2 (i.e., 1 → 2). This might arise if the initially tracked monomer forms a dimer with another Cy3B-labeled receptor. (E) Intensity trajectory that exhibits abrupt changes from level 1 → 2 → 1. (monomer → dimer → monomer). (F) A rare and more complex intensity track that switches from 2 → 1 → 2 (dimer → monomer → dimer). This track could be interpreted as the transient dissociation of a dimer. This behavior was confirmed by dual-color imaging (see Fig. 4G: record 3). Automatic assignment of intensity changes is described in the SI Text and Fig. S5. The signal fluctuations observed in each record are mainly due to photon noise whereas the differences in average intensity level attributed to one or two fluorophores found for the different objects are caused by spatial heterogeneity in the laser illumination and object position within the depth of the evanescent field.

Illustration of two molecules of Cy3B-labeled M₁ receptors reversibly forming a dimer. One hundred sequential frames (30-ms exposure time/frame) of two molecules of Cy3B-labeled M₁ receptors diffusing on the surface of a CHO cell. These molecules are initially monomers (about the first 21 frames), form a dimer (single red spot) for ∼40 frames (∼1 s), and then dissociate into monomers, possibly for the rest of the recording. There may be a transient reformation of short lifetime dimers for about three frames in the latter part of the recording, but it is not possible to exclude this being a chance overlap of the images of two molecules that are not interacting but diffusing independently close to each other. (Lower) A color look-up table. Two-color imaging (see text and Fig. 4) was used to study the details of M₁ receptor dimerization.

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.