Improving lifespan automation for Caenorhabditis elegans by using image processing and a post-processing adaptive data filter

Abstract

Automated lifespan determination for C. elegans cultured in standard Petri dishes is challenging. Problems include occlusions of Petri dish edges, aggregation of worms, and accumulation of dirt (dust spots on lids) during assays, etc. This work presents a protocol for a lifespan assay, with two image-processing pipelines applied to different plate zones, and a new data post-processing method to solve the aforementioned problems. Specifically, certain steps in the culture protocol were taken to alleviate aggregation, occlusions, contamination, and condensation problems. This method is based on an active illumination system and facilitates automated image sequence analysis, does not need human threshold adjustments, and simplifies the techniques required to extract lifespan curves. In addition, two image-processing pipelines, applied to different plate zones, were employed for automated lifespan determination. The first image-processing pipeline was applied to a wall zone and used only pixel level information because worm size or shape features were unavailable in this zone. However, the second image-processing pipeline, applied to the plate centre, fused information at worm and pixel levels. Simple death event detection was used to automatically obtain lifespan curves from the image sequences that were captured once daily throughout the assay. Finally, a new post-processing method was applied to the extracted lifespan curves to filter errors. The experimental results showed that the errors in automated counting of live worms followed the Gaussian distribution with a mean of 2.91% and a standard deviation of ±12.73% per Petri plate. Post-processing reduced this error to 0.54 ± 8.18% per plate. The automated survival curve incurred an error of 4.62 ± 2.01%, while the post-process method reduced the lifespan curve error to approximately 2.24 ± 0.55%.