Tracking single proteins within cells

We present experiments in which single proteins were imaged and tracked wit hin mammalian cells. Single proteins of R-phycoerythrin (RPE) were imaged b y epifluorescence microscopy in the nucleoplasm and cytoplasm at 71 frames/ s. We acquired two-dimensional trajectories of proteins (corresponding to t he projection of three-dimensional trajectories onto the plane of focus) fo r an average of 17 frames in the cytoplasm and 16 frames in the nucleus. Di ffusion constants were determined from linear fits to the mean square displ acement and from the mean displacement squared per frame. We find that the distribution of diffusion constants for RPE within cells is broader than th e distributions obtained from RPE in a glycerol solution, from a Monte Cart e simulation, and from the theoretical distribution for simple diffusion. T his suggests that on the time scales of our measurements, the motion of sin gle RPE proteins in the cytoplasm and nucleoplasm cannot be modeled by simp le diffusion with a unique diffusion constant. Our results demonstrate that it is possible to follow the motion of single proteins within cells and th at the technique of single molecule tracking can be used to probe the dynam ics of intracellular macromolecules.