Figure 2.
Figure 2. Single-molecule analysis of CD9 in PC3/CD9 cells. (A) ADC distribution and mean value (±SD) for CD9 molecules at 100-ms acquisition time. Note that all determinations were made by linear fitting in the MSD-τ plot using points two to four of the total curve, and 261 trajectories were analyzed. As delineated by the dashed line, two populations can be identified. Trajectories with the lower diffusion coefficient correspond to pure confined molecules, whereas the other trajectories correspond to molecules with Brownian or mixed (Brownian with a transient confinement in the same trajectory) behavior. D is the mean value of the ADC. (B) Superposition of ensemble CD9 labeling achieved with anti-CD9 Cy3B-labeled antibodies (green) and several fluorescent CD9 single-molecule trajectories obtained after tracking Atto647N-conjugated Fab fragments of SYB-1 (white thin lines). The letters in red correspond to the different diffusion modes (shown in C). (C, top to bottom) Trajectories of various single CD9 molecules exhibiting different diffusion modes: Brownian (B), confined (C), and mixed (M), a combination of Brownian and confined diffusion. The purple ring in trajectory M corresponds to transient confinement, and the blue numbers correspond to the duration of the trajectories. Video 1 corresponds to this experiment (available). Note that the scale bar is different for trajectory C. (D) Histograms (open boxes) representing the percentage of each CD9 diffusion mode relative to the total number of trajectories. The gray part corresponds to the proportion of trajectories associated with TEAs (identified with the ensemble membrane labeling) for each diffusion mode. (E) Time lapse of a mixed trajectory. The red spots correspond to a single CD9 molecule entering and exiting from a TEA (green). The purple rings in the last micrograph correspond to the confined areas in which CD9 ADC was decreased (see F). The duration of Video 1 is 21 s. (F) Plot of the instantaneous diffusion coefficient of the particle analyzed in the time lapse of E as a function of time (total duration of the time lapse). The green shaded part corresponds to the time period when the position of the single CD9 molecule overlaps with the ensemble CD9 labeling. A clear decrease in the instantaneous ADC of the particle is detected during this period.

Single-molecule analysis of CD9 in PC3/CD9 cells. (A) ADC distribution and mean value (±SD) for CD9 molecules at 100-ms acquisition time. Note that all determinations were made by linear fitting in the MSD-τ plot using points two to four of the total curve, and 261 trajectories were analyzed. As delineated by the dashed line, two populations can be identified. Trajectories with the lower diffusion coefficient correspond to pure confined molecules, whereas the other trajectories correspond to molecules with Brownian or mixed (Brownian with a transient confinement in the same trajectory) behavior. D is the mean value of the ADC. (B) Superposition of ensemble CD9 labeling achieved with anti-CD9 Cy3B-labeled antibodies (green) and several fluorescent CD9 single-molecule trajectories obtained after tracking Atto647N-conjugated Fab fragments of SYB-1 (white thin lines). The letters in red correspond to the different diffusion modes (shown in C). (C, top to bottom) Trajectories of various single CD9 molecules exhibiting different diffusion modes: Brownian (B), confined (C), and mixed (M), a combination of Brownian and confined diffusion. The purple ring in trajectory M corresponds to transient confinement, and the blue numbers correspond to the duration of the trajectories. Video 1 corresponds to this experiment . Note that the scale bar is different for trajectory C. (D) Histograms (open boxes) representing the percentage of each CD9 diffusion mode relative to the total number of trajectories. The gray part corresponds to the proportion of trajectories associated with TEAs (identified with the ensemble membrane labeling) for each diffusion mode. (E) Time lapse of a mixed trajectory. The red spots correspond to a single CD9 molecule entering and exiting from a TEA (green). The purple rings in the last micrograph correspond to the confined areas in which CD9 ADC was decreased (see F). The duration of Video 1 is 21 s. (F) Plot of the instantaneous diffusion coefficient of the particle analyzed in the time lapse of E as a function of time (total duration of the time lapse). The green shaded part corresponds to the time period when the position of the single CD9 molecule overlaps with the ensemble CD9 labeling. A clear decrease in the instantaneous ADC of the particle is detected during this period.

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