The diffusivity of a biological molecule depends not only on its structure and size, but also on its environment. Thus, diffusion has been used extensively as a physical probe in a wide variety of biological applications. However, diffusion measurements based on ensemble methods are unable to reveal the underlying heterogeneity in molecular diffusivity, for example, that caused by conformational distribution or environmental changes. In this thesis, we demonstrate by specific examples that diffusion measurements by Fluorescence Correlation Spectroscopy (FCS) at the single-molecule level are very useful in this regard.

First, we show that the diffusion of a fluorescent molecule trapped inside a lipid tubule, which has been shown to be a potential drug delivery vehicle, is position dependent and sometimes deviates significantly from Brownian motion. Second, we show, using a multi domain protein IgG as an example, that FCS is capable of revealing the existence of conformational species populated during the course of chemically induced protein denaturation, and that charge-charge interactions play an understated role in determining the hydrodynamic radius of the denatured protein. Third, we demonstrate that diffusion measurements at the single-molecule level are surprisingly useful in investigating the conformational distribution of membrane-bound peptides, as well as, peptide binding induced membrane domain formation. Moreover, we show that diffusion measurements can be used to study protein-ligand interactions. Finally, we demonstrate that confinement provided by microfabricated PDMS micro-chambers can be used to increase the sensitivity of FCS and suppress the photobleaching probability of single fluorescent molecules.