Small proteinaceous oligomeric species contribute to the formation of larger aggregates, a phenomenon that is of direct relevance to the characterization of protein aggregation in biopharmaceuticals and understanding the underlying processes contributing to neurodegenerative diseases.The ability to monitor in situ oligomerization and aggregation processes renders imaging and image analysis an attractive approach for gaining a mechanistic insight into early processes contributing to the formation of larger aggregates in disease models and biologics. The combination of image analysis tools enables the detection of both oligomeric and larger aggregate subtype in contrast to conventional kinetic-based approaches that lack the ability to resolve dimers from monomeric moieties in samples containing mixed populations.In this chapter, we describe the process for confocal time series image acquisition for monitoring the in situ loss of monomers, and the subsequent analysis pipeline using spatial intensity distribution analysis (SpIDA) to evaluate oligomer content.
Keywords: Image analysis; Light scattering; Microscopy; Monomer loss; Protein aggregation; SpIDA.