  Diffusion coefficient measurements are important for many biological and
material investigations, such as particle dynamics, kinetics, and size
determinations. Amongst current measurement methods, single particle tracking
(SPT) offers the unique capability of providing location and diffusion
information of a molecule simultaneously while using only femptomoles of
sample. However, the temporal resolution of SPT is limited to seconds for
single-color labeled samples. By directly imaging three dimensional (3D)
diffusing fluorescent proteins and studying the widths of their intensity
profiles, we determine the proteins' diffusion coefficients using single
protein images of sub-millisecond exposure times. This simple method improves
the temporal resolution of diffusion coefficient measurements to
sub-millisecond, and can be readily applied to a range of particle sizes in SPT
investigations and applications where diffusion coefficient measurements are
needed, such as reaction kinetics and particle size determinations.
