The nematode worm Caenorhabditis elegans has emerged as an important model organism to study the molecular mechanisms of protein misfolding diseases associated with amyloid formation because of its small size, ease of genetic manipulation and optical transparency. Obtaining a reliable and quantitative read-out of protein aggregation in this system, however, remains a challenge. To address this problem, we here present a fast time-gated fluorescence lifetime imaging (TG-FLIM) method and show that it provides functional insights into the process of protein aggregation in living animals by enabling the rapid characterisation of different types of aggregates. More specifically, in longitudinal studies of C. elegans models of Parkinson's and Huntington's diseases, we observed marked differences in the aggregation kinetics and the nature of the protein inclusions formed by α-synuclein and polyglutamine.In particular, we found that α-synuclein inclusions do not display amyloid-like features until late in the life of the worms, whereas polyglutamine forms amyloid characteristics rapidly in early adulthood.Furthermore, we show that the TG-FLIM method is capable of imaging live and non-anaesthetised worms moving in specially designed agarose micro-chambers. Taken together, our results show that the TG-FLIM method enables high-throughput functional imaging of living C. elegans that can be used to study in vivo mechanisms of aggregation and that has the potential to aid the search for therapeutic modifiers of protein aggregation and toxicity.
A variety of human diseases, including neurodegenerative disorders such as Parkinson's andAlzheimer's diseases, are characterised by the misfolding of protein species and their subsequent aggregation into amyloid fibrils 1,2 . The nematode Caenorhabditis elegans is a particularly useful model organism through which to study these diseases 3-8 and to screen for small molecule inhibitors of the protein aggregation process 9,10 . C. elegans has a simple body plan of 959 somatic cells, its genetics are well-characterised, and at least 40% of its genes have known human homologs 11 . Furthermore, it has a relatively short lifespan of only 2-3 weeks and is optically transparent, making it a highly suitable system for longitudinal imaging studies of protein aggregation. Despite these advantages, obtaining quantitative read-outs for amyloidogenic protein aggregation in vivo remains challenging 12 .Fluorescence intensity measurements are prone to artefacts, and classifying aggregates by counting protein inclusions relies on arbitrary choices for intensity and size cut-offs. Furthermore, reliable