Molecular chaperones maintain protein homeostasis in the cell by catalysing a wide variety of processes throughout a protein’s life cycle. Their functions include protein folding, remodelling of protein assemblies, protection from denaturation and resolubilisation of proteins from aggregates that accumulate during cell stress. Broad substrate specificity and the capacity to play this range of functional roles means chaperones are pivotal in mediating a plethora of neurodegenerative and protein conformational diseases. The inability to understand these biological processes at a molecular level substantially limits our current understanding. The aim of our research is to illuminate the molecular mechanisms of how chaperones from the Hsp70 family interact with proteins that have assembled into oligomers or are on the pathway towards aggregate formation. Utilisation of single-molecule fluorescence microscopy techniques will allow us to observe individual molecules in the dynamic chaperone-substrate interactions and thus elucidate reaction pathways that are obscured in traditional ensemble approaches. Here we present our initial results on developing strategies for design of the fluorescence imaging assays, protein labelling and assessing appropriate target substrates. We will use surface chemistry on glass cover-slips combined with microfluidic devices on a TIRF microscope set-up to study the dynamics of subunit exchanges, conformational rearrangements and membrane binding. This research has implications for a range of neurodegenerative diseases that are characterised by the accumulation of insoluble protein aggregates (amyloid fibrils). Whilst the presence of amyloid deposits in neuronal regions has canonically been the hallmark of amyloidoses, current research trends implicate soluble pre-fibrillar intermediates as the neurotoxic species. A more in-depth understanding of the structural and conformational pathways in the assembly of disease proteins and their interactions with molecular chaperones is critical. The answers will both shed light on the possible mechanism for chaperone mediated disease modulation as well as give insight into potential therapeutic approaches to prevent disease, rather than just ameliorate the symptoms.