The innate immune system utilises a set of receptors, called pattern recognition receptors (PRRs), in order to recognise specific molecular patterns or motifs called pathogen-associated molecular patterns (PAMPs) on invading pathogens. The toll-like receptor (TLR) family of proteins is an integral part of the mammalian innate immune system. We are now beginning to decipher which TLRs are involved in the recognition of particular microbial patterns, but questions remain as to the homo- and heterotypic associations that TLRs form and how these associations affect their activation. Technical advances in fluorescence microscopy has enabled us to investigate the functional associations of TLRs and other PPRs in living cells in response to different pathogens using non-invasive fluorescence imaging methods. In this chapter, we will describe some of the fluorescent imaging techniques, such as FRET and FRAP, that we employ in order to study PAMP-PRR associations.