Force microscopy of the Caenorhabditis elegans embryonic eggshell

Assays focusing on emerging biological phenomena in an animal’s life can be performed during embryogenesis.While the embryo of Caenorhabditis elegans has been extensively studied,its biomechanical properties are largely unknown.Here,we demonstrate that cellular force microscopy(CFM),a recently developed technique that combines micro-indentation with high resolution force sensing approaching that of atomic force microscopy,can be successfully applied to C.elegans embryos.We performed,for the first time,a quantitative study of the mechanical properties of the eggshell of living C.elegans embryos and demonstrate the capability of the system to detect alterations of its mechanical parameters and shell defects upon chemical treatments.In addition to investigating natural eggshells,we applied different eggshell treatments,i.e.,exposure to sodium hypochlorite and chitinase solutions,respectively,that selectively modified the multilayer eggshell structure,in order to evaluate the impact of the different layers on the mechanical integrity of the embryo.Finite element method simulations based on a simple embryo model were used to extract characteristic eggshell parameters from the experimental micro-indentation force-displacement curves.We found a strong correlation between the severity of the chemical treatment and the rigidity of the shell.Furthermore,our results showed,in contrast to previous assumptions,that short bleach treatments not only selectively remove the outermost vitelline layer of the eggshell,but also significantly degenerate the underlying chitin layer,which is primarily responsible for the mechanical stability of the egg.

Microfluidics-enabled phenotyping of a whole population of C.elegans worms over their embryonic and post-embryonic development at single-organism resolution

The organism Caenorhabditis elegans is a performant model system for studying human biological processes and diseases,but until now all phenome data are produced as population-averaged read-outs.Monitoring of individual responses to drug treatments would however be more informative.Here,a new strategy to track different phenotypic traits of individual C.elegans nematodes throughout their full life-cycle-i.e.,embryonic and post-embryonic development,until adulthood onset,differently from life-span-is presented.In an automated fashion,single worms were synchronized,isolated,and cultured from egg to adulthood in a microfluidic device,where their identity was preserved during their whole development.Several phenotypes were monitored and quantified for each animal,resulting in high-content phenome data.Specifically,the method was validated by analyzing the response of C.elegans to doxycycline,an antibiotic fairly well-known to prolong the development and activate mitochondrial stress-response pathways in different species.Interestingly,the obtained extensive single-worm phenome not only confirmed the dramatic doxycycline effect on the worm developmental delay,but more importantly revealed subtle yet severe treatment-dependent phenotypes that are representative of minority subgroups and would have otherwise stayed hidden in an averaged dataset.Such heterogeneous response started during the embryonic development,which makes essential having a dedicated chip that allows including this early developmental stage in the drug assay.Our approach would therefore allow elucidating pharmaceutical or therapeutic responses that so far were still being overlooked.

Effect of inoculum size and antibiotics on bacterial traveling bands in a thin microchannel defined by optical adhesive

Phenotypic diversity in bacterial flagella-induced motility leads to complex collective swimming patterns,appearing as traveling bands with transient locally enhanced cell densities.Traveling bands are known to be a bacterial chemotactic response to self-generated nutrient gradients during growth in resource-limited microenvironments.In this work,we studied different parameters of Escherichia coli(E.coli)collective migration,in particular the quantity of bacteria introduced initially in a microfluidic chip(inoculum size)and their exposure to antibiotics(ampicillin,ciprofloxacin,and gentamicin).We developed a hybrid polymer-glass chip with an intermediate optical adhesive layer featuring the microfluidic channel,enabling high-content imaging of the migration dynamics in a single bacterial layer,i.ev bacteria are confined in a quasi-2D space that is fully observable with a high-magnification microscope objective.On-chip bacterial motility and traveling band analysis was performed based on individual bacterial trajectories by means of custom-developed algorithms.Quantifications of swimming speed,tumble bias and effective diffusion properties allowed the assessment of phenotypic heterogeneity,resulting in variations in transient cell density distributions and swimming performance.We found that incubation of isogeneic£coli with different inoculum sizes eventually generated different swimming phenotype distributions.Interestingly,incubation with antimicrobials promoted bacterial chemotaxis in specific cases,despite growth inhibition.Moreover,E.coli filamentation in the presence of antibiotics was assessed,and the impact on motility was evaluated.We propose that the observation of traveling bands can be explored as an alternative for fast antimicrobial susceptibility testing.

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-assisted multiplexed biomarker detection for in situ mapping of immune cells in tumor sections

Because of the close interaction between tumors and the immune system,immunotherapies are nowadays considered as the most promising treatment against cancer.In order to define the diagnosis and the subsequent therapy,crucial information about the immune cells at the tumor site is needed.Indeed,different types or activation status of cells may be indicative for specific and personalized treatments.Here,we present a quantitative method to identify ten different immuno-markers in the same tumor cut section,thereby saving precious samples and enabling correlative analysis on several cell families and their activation status in a tumor microenvironment context.We designed and fabricated a microfluidic chip with optimal thermomechanical and optical properties for fast delivery of reagents on tissue slides and for fully automatic imaging by integration with an optical microscope.The multiplexing capability of the system is enabled by an optimized cyclic immunofluorescence protocol,with which we demonstrated quantitative sequential immunostaining of up to ten biomarkers on the same tissue section.Furthermore,we developed high-quality image-processing algorithms to map each cell in the entire tissue.As proofof-concept analyses,we identified coexpression and colocalization patterns of biomarkers to classify the immune cells and their activation status.Thanks to the quantitativeness and the automation of both the experimental and analytical methods,we believe that this multiplexing approach will meet the increasing clinical need of personalized diagnostics and therapy in cancer pathology.