Long-term imaging, automated high-content phenotyping and sorting of C. elegans larvae and embryos using microfluidic platforms
Hüseyin Baris Atakan
2019
The model organism C. elegans is frequently studied to address fundamental biological questions motivated by its short life cycle, small size, hermaphrodite behavior and well-annotated genome. In this work, we present novel microfluidic platforms that support long-term culture, automated high-content phenotyping, high-resolution imaging and multiplexing, as well as a hand-held device for sorting of larvae and embryos of C. elegans. In the microfluidics-enabled studies of C. elegans, in vivo
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... tode culturing platforms that allow high-content phenotyping during worms' life cycle in an automated fashion have been rare so far. In our first study, we present a multiplexed polydimethylsiloxane (PDMS)-based microfluidic platform for the rapid high-content phenotyping of populations of C. elegans down to single animal resolution. We reversibly and frequently (every 35 minutes) confined nematodes during their life cycle inside tapered channels for stable fluorescence imaging and extraction of growth parameters, in combination with a determination of motility phenotypes obtained from video-recording (every 7 hours) of freely moving nematodes. We extracted 19 phenotypic parameters and proved that dietary restriction (DR) plays a crucial role in the disease regression of a worm model of Huntington's disease. In a second work, we demonstrate a multiplexed, high-throughput and automated embryo phenotyping microfluidic platform to analyze embryogenesis of C. elegans under application of different chemical compounds. We performed up to 800-embryo experiments within a preparation time of only 35 minutes and executed 12 hours of imaging (with 5 and 10 minute intervals), which was followed by an automated phenotyping using machine learning and image processing approaches. Our validation study of osmosensation on the embryos indicated a developmental lag and an induction of mitochondrial stress in embryos exposed to high doses (200 mM) of glucose and NaCl, while small doses of sucrose and glucose accelerated development. We also realized that microfluidic technologies could further be exploited for nematode studies at single organism resolution, providing more of a 'personalized' phenotyping while also collecting population-averaged information like in conventional bio-assays. In particular, we have realized a multiplexed, potentially high-throughput and high-resolution microfluidic platform to culture C. elegans from embryo to the adult stage at single-animal resolution. We first placed single embryos in growth chambers, observing the main embryonic stages (image-captured every 5 minutes) and continued with C. elegans larvae developmental studies (video-recorded every 4 hours). As a proof-of-concept, we exposed the nematodes to 8 different concentrations, from 1 nM to 1 mM, to the anthelmintic drug tetramisole diluted in iii Abstract an E. coli feeding solution, and we observed from our automated phenotyping results that towards the late stages of the nematodes' life, tetramisole influence on the development and motility became more prominent. We thereby showed that our device offers high-precision phenotyping of the impact of chemicals on C. elegans. C. elegans is also an exceptionally valuable model organism to target epigenetic studies but most of these have focused so far on phenotypic variations over multiple generations, omitting the evaluation of epigenetic inheritance in the early stages of the nematodes' life. We have also realized an epigenetics-oriented microfluidic platform to observe mother-to-progeny heritable transmission in the nematode C. elegans at high-resolution, under significant automation and enabling a high degree of parallelization. After a 24 hours culture of nematodes starting from L4 larvae under application of various concentrations and durations of the drug doxycycline, we quantified the mitochondrial unfolded protein response (UPR mt ) of the first released hsp-6::gfp embryos by computing the average green fluorescent protein (GFP) intensity as an indicator to mitochondrial stress inheritance. After analyzing the different phenotypes with our automated and customized phenotyping algorithms, we noticed that a minimum doxycycline concentration of 30 µg/mL and a drug exposure time of 15 hours applied to the mothers could induce mitochondrial stress in embryos. C. elegans-based assays require age-synchronized populations prior to experimentation to avoid any kind of outliers in the resulting data. There has been many proposed approaches to synchronize populations of C. elegans at certain larval stages; however, such methods often utilize tedious, complex and low-throughput handling procedures of the nematodes. In the last part of this thesis, we demonstrate a PDMS microfluidic device for high-throughput, efficient and extremely rapid sorting of C. elegans at various life stages. Our device, which consists of three plasma-activated and bonded PDMS parts, empowers sorting of two consecutive larval stages in two minutes with a sorting purity of 73 -100%. In parallel, we could retain the remaining population in the chip, providing collection of two sorted larval stages at both outputs of the device. In addition, we employed the equivalent of a standard bleaching procedure for embryo harvesting on-chip and showed that egg extraction and a synchronized L1 population could also be easily obtained. We think that our device can potentially find further use as a tool for C. elegans synchronization and rapid egg extraction.
doi:10.5075/epfl-thesis-7509
fatcat:pi6ibf4xhnfdzgjscecop72xge