[
International Worm Meeting,
2021]
Advances in deep learning and computer vision have revolutionized analysis of neuron activity and behavioral data. Here we present a deep learning (DL) toolbox - a collection of three deep learning methods to solve challenging problems in C. elegans whole-brain imaging. First, accurate segmentation of densely packed nuclei in fluorescence channels is critical for downstream tasks such as cell tracking, signal extraction and identity annotation. While many deep learning methods are available for 2D images, 3D segmentation methods for highly anisotropic images are not available. We combined a well-known DL framework for instance segmentation in 2D images with optimal transport based clustering to produce 3D segmentations in anisotropic images. Comparison against other methods on experimental and synthetic datasets show that our method is more accurate (5-8% higher F1 score) and more robust across a range of baseline cell signals and image noise levels (6-17% higher F1 score). Further DL method is 3.7 times faster than previous method. Second, whole-brain imaging in freely moving worms is currently not widespread because of the requirement of custom designed microscopes with low-magnification behavior tracking and high-magnification fluorescence imaging channels. We developed and optimized a fast DL framework (30-644 times smaller model size and 5-8 times faster than previous methods) to directly predict the worm pose (skeleton) from fluorescence channels. Fast inference of pose using only fluorescence channel enables worm-tracking thus eliminating the need of separate behavior channel. We show that predicted worm pose can be used for behavior analysis and cell-tracking in videos. Further, eliminating the need of custom microscopes will enable more labs to do whole-brain imaging in freely moving animals. Third, we developed a DL framework for restoring low signal-to-noise ratio (SNR) fluorescence images (acquired at low laser power/low exposure time) to high SNR images. Low laser power imaging eliminates photo-bleaching of fluorophores, light damage to worms and enables long-term neuron activity imaging across days. Image restoration can be performed on both GCaMP and RFP channels. We show that restored images provide cleaner calcium signals compared to traditional de-noising methods (13-30% smaller mean-absolute error compared to clean trace). Additionally, restored images also improve cell detection, tracking and identity annotation tasks. Methods in our toolbox can be easily adapted for similar tasks in other organisms.
[
International Worm Meeting,
2017]
Artificial light at night (ALAN) has many broad-scale and global implications for ecosystems and wildlife that have evolved under a 24-h circadian cycle. With increased urbanization, artificial light at night has directly altered natural photoperiods and nocturnal light intensity. Artificial light at night can disrupt behavioral patterns such as foraging activity and mating in animals. Disturbances in natural light and dark cycles also affect melatonin-regulated circadian and seasonal rhythms in Drosophila. We investigated the impact of ecologically relevant levels of light pollution on an important invertebrate model, Caenorhabditis elegans, as the impact of night lighting at these light levels is currently unknown. In this study, we exposed worms to artificial light at four intensities: 10-4 lx (control, comparable to natural nocturnal darkness), 10-2 lx (comparable to full-moon lighting and a low level of light pollution), 1 lx (comparable to dawn/dusk or intense light pollution), and 100 lx (dim daylight level comparable to extreme light pollution) on a 12L:12D photoperiod (100 lx treatments experienced constant light). We measured the impact of these light treatments on offspring production in hermaphroditic C. elegans. We grew worms for 2 generations in each light treatment, and then recorded the lifespan and counted the number of hatched offspring produced in the F3 generation. Our data show no significant differences among light levels for lifespan or offspring production suggesting that at least for these life history traits, ALAN does not affect these soil nematodes. Future directions include measuring additional life history traits and circadian gene expression for worms exposed to ALAN.
[
International Worm Meeting,
2007]
Multi-generational growth will be essential for any hope of long term human colonization of the cosmos. However, there is a lack of information about any species in space beyond three generations. In addition, trips to the Moon or Mars will result in greater exposure to space radiation, although little is known about cumulative biological effects. C. elegans provides a simple model system in which to study multi-generational growth and radiation exposure in space. Cultures of C. elegans (wt CC1 and balancer eT1 strains) were maintained on-board the ISS for periods well in excess of 3 months. Worms were grown through 10+ generations on the ISS using an automated culturing system employing defined liquid medium, commercial growth chambers, peristaltic pumps to passage worms and control instrumentation. The culturing system, the C. elegans Habitat, was housed in a temperature controlled incubator located in the ISS module Destiny. Integrated video cameras with micro lenses, combined with data downlink, were utilized to image worms for real-time assessment of larval stages, population density and movement behavior. Data analysis was performed by students at 35+ middle and high schools across the United States, Canada and Malaysia, allowing students to learn about the benefits of C. elegans research while gaining experience in the scientific method. While preliminary, data confirm what was inferred from past shorter duration spaceflight missions: growth, development, and behavior of worms are grossly unaltered during spaceflight. Changes in worm muscle that were previously observed (decreased myosin heavy chain and MyoD expression, movement defect) may reflect adaptive changes in muscle in space or may be artifacts of past culturing techniques. Planned post-flight analyses should distinguish these two possibilities. Post flight analysis of eT1 worms will determine if increased rates of genetic mutation occur with long-term exposure to space radiation. Further insights should be gained into radiation concerns for future planned interplanetary human exploration missions. In conclusion, there is no major gravity-dependent process associated with spaceflight that precludes essentially normal animal growth and development for at least ten generations in C. elegans. This work was sponsored by the National Space Agency of Malaysia (ANGKASA).