The phenomenon of diffusion was first observed by Robert Brown in 1827. It was recorded as uncharacteristic movement of particles which is now known as Brownian motion (Brown, 1828). Later Einstein elucidated the origins of this movement by describing it as the movement caused by a large particle being constantly impacted by a sea of smaller particles in which the larger particle resides (Einstein, 1956).
The mathematical solution that Einstein proposed gave rise to the concept of the diffusion coefficient of particles. It describes how particles move within a system when no external forces are applied. Although, at the time, this concept was primarily used in order to prove atomic theory (which describes that the constituents of mater are small elements called atoms) it has since been integrated into our understanding of diffusion for macromolecules such as proteins (Perrin & Soddy, 1910) (H. C. Berg, 1983).
Since then, the diffusion coefficient of proteins has been subject of much research. It allows for many significant insights into biological processes and can lead to innovation in medicine and chemistry. An example is the influence that the diffusion coefficient of transcription factor (TF) has, on the efficiency at which it can find its specific DNA binding site (O. G. Berg, Winter, & Von Hippel, 1981). This, in turn, directly influences many cellular functions such as transcription, replication, and recombination (Slutsky & Mirny, 2004). This is why the research field with relation to the diffusion coefficient of proteins has been well developed.
However, where this field has been heavily focused on 3 dimensional diffusion of cytosolic proteins, there hasn’t been nearly as much study of membrane protein diffusion (White, 2004). This is due to the intricate complexity of studying membrane proteins. Some of which are the dependence on in vivo experiments and the difficulty of creating functional recombinant membrane proteins which integrate successfully into the membrane (Seddon, Curnow, & Booth, 2004).
Recent advances in protein science, however, have opened up the possibilities for in depth study of membrane proteins. Such as a better understanding of probes for tagging membrane proteins and better techniques for creating functional fusion proteins that successfully integrate into the membrane (Yano & Matsuzaki, 2009) (Marreddy, Geertsma, & Poolman, 2011). These advances allow for the reliable in vivo expression of probe tagged functional membrane proteins.
Fluorescence microscopy then allows for the observation of the probe tagged proteins. For in vivo applications, this is usually limited to the imaging of a 2D plane of the sample due to the requirement to capture images at a high frequency. This high frequency is required in order to observe the fast movements of the tagged proteins.
SPT allows for the movements of the proteins to be precisely recorded. Using the coordinates of each movement and the time between each movement the diffusion coefficient can then be mathematically calculated.