Automated phenotyping of Caenorhabditis elegans embryos with a high-throughput-screening microfluidic platform

The nematode Caenorhabditis elegans has been extensively used as a model multicellular organism to study the influence of osmotic stress conditions and the toxicity of chemical compounds on developmental and motility-associated phenotypes. However, the several-day culture of nematodes needed for such studies has caused researchers to explore alternatives. In particular, C. elegans embryos, due to their shorter developmental time and immobile nature, could be exploited for this purpose, although usually their harvesting and handling is tedious. Here, we present a multiplexed, high-throughput and automated embryo phenotyping microfluidic approach to observe C. elegans embryogenesis after the application of different chemical compounds. After performing experiments with up to 800 embryos per chip and up to 12h of time-lapsed imaging per embryo, the individual phenotypic developmental data were collected and analyzed through machine learning and image processing approaches. Our proof-of-concept platform indicates developmental lag and the induction of mitochondrial stress in embryos exposed to high doses (200mM) of glucose and NaCl, while small doses of sucrose and glucose were shown to accelerate development. Overall, our new technique has potential for use in large-scale developmental biology studies and opens new avenues for very rapid high-throughput and high-content screening using C. elegans embryos. Microfluidics: High-throughput screening of embryosA new microfluidic approach enables the high-throughput and automated phenotyping of C. elegans embryos. Understanding the effect of osmotic stress-induced damage on cells is vital for understanding a number of biological processes. C. elegans embryos are considered ideal model systems for osmotic studies, but their phenotyping is traditionally a labor intensive and time-consuming process. Now, a team from EPFL in Switzerland led by Martin Gijs demonstrate a microfluidic platform for high-throughput phenotyping of C. elegans. Embryos are exposed to various osmotic compounds, followed by an automated phenotyping script to assess phenotypes via machine learning and image processing. Their proof of concept experiment demonstrates that their technique can be used for a systems-based approach for osmotic studies.

Quantitative single-cell analysis of S. cerevisiae using a microfluidic live-cell imaging platform

Johannes Becker

Genome-wide manipulations and measurements have made huge progress over the last decades. In Saccharomyces cerevisiae, a well-studied eukaryotic model organism, homologous recombination allows for systematic deletion or alteration of a majority of its genes. Important products of these manipulation techniques are two libraries of modified strains: A deletion library consisting of all viable knockout mutants, and a GFP library in which 4159 proteins are successfully tagged with GFP. In addition, the development of a method that allows for the systematic construction of double mutants led to a virtually infinite number of potential strains of interest. These advancements in combinatorial biology need to be matched by methods of data measurement and analysis. In order to simultaneously observe the spatio-temporal dynamics of thousands of strains from the GFP library, Dénervaud et al. developed a microfluidic platform that allows for parallel imaging of 1152 strains in a single experiment. On this platform, strains can be grown and monitored in a controllable environment for several days, which results in the imaging of several millions of cells during one experiment. To objectively and quantitatively analyze this immense amount of information, we implemented an image analysis pipeline, which can extract experiment-wide information on single-cell protein abundance and subcellular localization. The construction of a supervised classifier to quantify localization information on a single cell level is a new approach and was invaluable to detect dynamic localization changes within the proteome. Using five different stress conditions, we gained insight into temporal changes of abundance and localization of multiple proteins. For example, we found that while localization changes can often be fast and transient, long-term response of a cell is usually enabled by changes in abundance. This shows a well-orchestrated response of a cell to external stimuli. To extend knowledge about cellular mechanisms, we used our microfluidic platform for two separate screens, combining GFP-reporter with additional deletion mutants. The advantage of our platform in comparison to more common approaches lies in its simultaneous measurement of fluorescence and phenotypic information on cell size and growth. For each deletion, we can quantify not only its influence onto the respective GFP-reporter under changing conditions, but also its effect on cell growth and size. We showed that it is advantageous to combine this information, as it allows pointing out possible underlying mechanisms of gene network regulations. In a first screen we investigated the behavior of several gene networks upon UV irradiation damage. We were able to show that four gene deletions influenced the localization of ribonucleotide-diphosphate reductase (Rnr4p). A second screen was designed to find genes that influence the induction of the galactose network. This screen uses more than 500 deletions of genes mostly related to chromatin in combination with two different reporter strains. A main focus of this study was the inheritance of memory during galactose reinduction. We found several previously unknown genes that potentially influence either induction or reinduction and were picked as candidates for further inheritance studies. Our microfluidic platform allows for unprecedented studies of proteomes in flux. [...]

EPFL2015

Long-term culture and high-resolution imaging of C. elegans using an automated microfluidic platform

Johan Auwerx, Matteo Cornaglia, Martinus Gijs, Govindan Narayanan Krishnamani, Laurent Mouchiroud

The nematode Caernohabditis elegans is one of the most employed small model organisms in biology. Studies on transgenic animals often require the accurate observation of highly localized fluorescent signals inside the worms at high magnification, hence demanding the full immobilization of the animals. Moreover, in order to observe the dynamics of biological processes over the lifespan of a worm, the same worm has to be immobilized repeatedly, in a reversible manner and under normal physiological conditions. We describe a microfluidic platform for the automated culture, treatment and long-term high-resolution imaging of C. elegans. Our device features: (i) a microfluidic design tailored for the isolation of L4 larvae from a mixed larval population and for their successive culture and treatment; (ii) a worm immobilization method, based on the thermoreversible sol-gel transition of the biocompatible triblock copolymer Pluronic F127 inside the microfluidic chip, thereby enabling high-resolution imaging; (iii) an integrated temperature control system, both to ensure viable environmental conditions for C. elegans culture and to steer the worm immobilization/release process. We apply this device to observe mitochondrial dynamics in muscle cells during aging at single worm resolution. We expect our platform to enable the simultaneous study and repeated observation of multiple phenotypes in single worms or specific worm populations, such as lifespan and motility assays, in addition to high-resolution imaging.

2015

An automated microfluidic platform for C. elegans embryo arraying, phenotyping, and long-term live imaging

Johan Auwerx, Matteo Cornaglia, Martinus Gijs, Virginija Jovaisaite, Thomas Lehnert, Alexis Pierre Henri Marette, Laurent Mouchiroud, Shreya Narasimhan

Studies of the real-time dynamics of embryonic development require a gentle embryo handling method, the possibility of long-term live imaging during the complete embryogenesis, as well as of parallelization providing a population's statistics, while keeping single embryo resolution. We describe an automated approach that fully accomplishes these requirements for embryos of Caenorhabditis elegans, one of the most employed model organisms in biomedical research. We developed a microfluidic platform which makes use of pure passive hydrodynamics to run on-chip worm cultures, from which we obtain synchronized embryo populations, and to immobilize these embryos in incubator microarrays for long-term high-resolution optical imaging. We successfully employ our platform to investigate morphogenesis and mitochondrial biogenesis during the full embryonic development and elucidate the role of the mitochondrial unfolded protein response (UPR(mt)) within C. elegans embryogenesis. Our method can be generally used for protein expression and developmental studies at the embryonic level, but can also provide clues to understand the aging process and age-related diseases in particular.

Nature Publishing Group2015

Quantitative single-cell analysis of S. cerevisiae using a microfluidic live-cell imaging platform

Johannes Becker

EPFL2015