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

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%.

Chemical Composition and Antioxidant Properties of Essential Oils from Peppermint, Native Spearmint and Scotch Spearmint.

Natural antioxidants have drawn growing interest for use in animal feed and the food industry. In the current study, essential oils (EOs) obtained from hydrodistillation of three mentha species, including Mentha piperita (peppermint), Mentha spicata (native spearmint) and Mentha gracilis (Scotch spearmint), harvested in the Midwest region in the United States, were analyzed for their chemical composition using gas chromatography-mass spectrometry, and their antioxidant properties were assessed through chemical assays, in vitro cell culture modeling and in Caenorhabditis elegans (C. elegans). The activity of ferric iron reduction and free-radical scavenging capacity were assessed through chemical-based assays, including the reducing power assay, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and Trolox equivalent antioxidant capacity assay (TEAC). Subsequently, the capacity of EOs to mitigate lipid peroxidation was analyzed at various doses using fresh liver homogenates from pigs. A porcine jejunum epithelial cell line (IPEC-J2) was employed as in vitro model to study the cellular antioxidant activity of the mint EOs. Finally, the effectiveness of mint EOs to alleviate acute systemic oxidative damage were evaluated in vivo using C. elegans. Data were analyzed by the MIXED procedure of SAS. Contrast statement was performed to assess linear or quadratic effects of mint EOs given at various doses. All three EOs are mostly composed of monoterpenes and their derivatives (76-90%), but differed in the major compounds, which are menthol and menthone (50%) in peppermint EO and carvone (70%) in spearmint EOs. Three mint EOs demonstrated prominent radical scavenging and Fe3+ reducing activity in chemical-based assays. In comparison with native and Scotch spearmint EOs, peppermint EO had the lowest (p < 0.05) half maximal effective concentration (EC50) in DPPH and TEAC assays and higher efficacy in the reducing power assay. All three EOs exhibited equivalent activity in mitigation of chemical-induced lipid peroxidation in liver tissues in a dose-dependent manner (linear, p < 0.001). The maximal cellular antioxidant activity (CAA) was observed at 5 µg/mL for peppermint, and 100 µg/mL for native and Scotch spearmint EOs. The addition of 25 µg/mL of both spearmint EOs increased (p < 0.05) cellular concentrations of glutathione in H2O2-treated IPEC-J2 cells, suggesting enhanced endogenous antioxidant defense. Supplementation of 100 µg/mL of peppermint or Scotch spearmint EO significantly increased (p < 0.05) the survival rate of C. elegans in response to H2O2-induced oxidative stress. The protective effect is comparable to that of supplementation of 10 µg/mL of ascorbic acid. However native spearmint EO failed to reduce the death rate within the same supplementation dose (10-200 µg/mL).

Active backlight for automating visual monitoring: An analysis of a lighting control technique for Caenorhabditis elegans cultured on standard Petri plates.

Lifespan and healthspan machines can undergo C. elegans image segmentation errors due to changes in lighting conditions, which produce non-uniform images. Most C. elegans monitoring machines use backlight techniques based on the transparency of both the container and media. Backlight illumination obtains high-contrast images with dark C. elegans and a bright background. However, changes in illumination or media transparency conditions can produce non-uniform images, which are currently alleviated by image processing techniques. Besides, these machines should avoid C. elegans exposure to light as much as possible because light stresses worms, and can even affect their lifespan, mainly when using (1) long exposure times, (2) high intensities or (3) wavelengths that come close to ultraviolet. However, if short exposure of worms to light is required for visual monitoring, then light can also be used as a movement stimulus. In this paper, an active backlight method is analysed. The proposed method consists of controlling the light intensities and wavelengths of an illumination dots matrix with PID regulators. These regulators adapt illumination to some changing conditions. The experimental results shows that this method simplifies the image segmentation problem because it is able to automatically compensate not only changes in media transparency throughout assay days, but also changes in ambient conditions, such as smooth condensation on the lid and light derivatives of the illumination source during its lifetime. In addition, the strategic application of wavelengths could be adapted for the requirements of each assay. For instance, a specific control strategy has been proposed to minimise stress to worms and trying to stimulate C. elegans movement in lifespan assays.