Fig. 2. Single-particle tracking of wild-type GFP-Kv2.1-loopBAD channels. HEK cells expressing GFP-Kv2.1-loopBAD were enzymatically biotinylated and then tagged at low efficiency with streptavidin-labeled 605 quantum dots as described in the Materials and Methods. The two most common classes of single Qdot tracks observed are indicated. (A) Single-particle track of a channel trapped within a cluster perimeter along with the corresponding MSD analysis of channel diffusion. In this case, a diffusion coefficient of 0.04 µm²/second was calculated from the initial slope. Overall, MSD analysis of Kv2.1 channel movement within clusters yielded a mean diffusion coefficient of 0.03±0.02 µm²/second, n=21. (B) Single-particle track of a channel outside the cluster whose movement was indifferent to the cluster perimeter. Analysis of the corresponding MSD plot for this track yielded a diffusion coefficient of 0.06 µm²/second. Analysis of multiple tracks derived from non-clustered channels yielded a mean diffusion coefficient of 0.06±0.05 µm²/second, n=33. Cells were imaged every 0.8 seconds.