Browse
Submit Data
Databases
API
Help

Dataset Information

23 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Monitoring single-cell bioenergetics via the coarsening of emulsion droplets.

ABSTRACT: Microorganisms are widely used to generate valuable products, and their efficiency is a major industrial focus. Bioreactors are typically composed of billions of cells, and available measurements only reflect the overall performance of the population. However, cells do not equally contribute, and process optimization would therefore benefit from monitoring this intrapopulation diversity. Such monitoring has so far remained difficult because of the inability to probe concentration changes at the single-cell level. Here, we unlock this limitation by taking advantage of the osmotically driven water flux between a droplet containing a living cell toward surrounding empty droplets, within a concentrated inverse emulsion. With proper formulation, excreted products are far more soluble within the continuous hydrophobic phase compared to initial nutrients (carbohydrates and salts). Fast diffusion of products induces an osmotic mismatch, which further relaxes due to slower diffusion of water through hydrophobic interfaces. By measuring droplet volume variations, we can deduce the metabolic activity down to isolated single cells. As a proof of concept, we present the first direct measurement of the maintenance energy of individual yeast cells. This method does not require any added probes and can in principle apply to any osmotically sensitive bioactivity, opening new routes for screening, and sorting large libraries of microorganisms and biomolecules.

SUBMITTER: Boitard L

PROVIDER: S-EPMC3358915 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Ultrasonically-guided flow focusing generates precise emulsion droplets for high-throughput single cell analyses.

Project description:Emulsion-based techniques have dramatically advanced our understanding of single-cell biology and complex single-cell features over the past two decades. Most approaches for precise single cell isolation rely on microfluidics, which has proven highly effective but requires substantial investment in equipment and expertise that can be difficult to access for researchers that specialize in other areas of bioengineering and molecular biotechnology. Inspired by the robust droplet generation technologies in modern flow cytometry instrumentation, here we established a new platform for high-throughput isolation of single cells within droplets of tunable sizes by combining flow focusing with ultrasonic vibration for rapid and effective droplet formation. Application of ultrasonic pressure waves to the flowing jet provided enhanced control of emulsion droplet size, permitting capture of 25,000 to 50,000 single cells per minute. As an example application, we applied this new droplet generation platform to sequence the antibody variable region heavy and light chain pairings (VH:VL) from large repertoires of single B cells. We demonstrated the recovery of > 40,000 paired CDRH3:CDRL3 antibody clusters from a single individual, validating that these droplet systems can enable the genetic analysis of very large single-cell populations. These accessible new technologies will allow rapid, large-scale, and precise single-cell analyses for a broad range of bioengineering and molecular biotechnology applications.

| S-EPMC6688500 | biostudies-literature

Multiphase water-in-oil emulsion droplets for cell-free transcription-translation.

Project description:The construction of genetically encoded cellular mimics in compartments containing organized synthetic cytosols is desirable for the development of artificial cells. Phase separated aqueous domains were placed within water-in-oil emulsion droplets in a manner compatible with transcription and translation machinery. Aqueous two-phase and three-phase systems (ATPS and A3PS) were assembled with dextran, poly(ethylene glycol), and Ficoll. Aqueous two-phase systems were capable of supporting the cell-free expression of protein within water droplets, whereas the aqueous three-phase-based system did not give rise to detectable protein synthesis. The expressed protein preferentially partitioned to the dextran-enriched phase. The system could serve as a foundation for building cellular mimics with liquid organelles.

| S-EPMC4038361 | biostudies-literature

Robust Bayesian inference via coarsening.

Project description:The standard approach to Bayesian inference is based on the assumption that the distribution of the data belongs to the chosen model class. However, even a small violation of this assumption can have a large impact on the outcome of a Bayesian procedure. We introduce a novel approach to Bayesian inference that improves robustness to small departures from the model: rather than conditioning on the event that the observed data are generated by the model, one conditions on the event that the model generates data close to the observed data, in a distributional sense. When closeness is defined in terms of relative entropy, the resulting "coarsened" posterior can be approximated by simply tempering the likelihood-that is, by raising the likelihood to a fractional power-thus, inference can usually be implemented via standard algorithms, and one can even obtain analytical solutions when using conjugate priors. Some theoretical properties are derived, and we illustrate the approach with real and simulated data using mixture models and autoregressive models of unknown order.

| S-EPMC6961963 | biostudies-literature

Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS.

Project description:In the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput screening via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying sizes and morphologies as well as a heterogeneous cell mixture of a whole dissociated flatworm (5-25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS screening of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional phenotyping.

| S-EPMC7670281 | biostudies-literature

On-Chip Quantitative Measurement of Mechanical Stresses During Cell Migration with Emulsion Droplets.

Project description:The ability of immune cells to migrate within narrow and crowded spaces is a critical feature involved in various physiological processes from immune response to metastasis. Several in-vitro techniques have been developed so far to study the behaviour of migrating cells, the most recent being based on the fabrication of microchannels within which cells move. To address the question of the mechanical stress a cell is able to produce during the encounter of an obstacle while migrating, we developed a hybrid microchip made of parallel PDMS channels in which oil droplets are sparsely distributed and serve as deformable obstacles. We thus show that cells strongly deform droplets while passing them. Then, we show that the microdevice can be used to study the influence of drugs on migration at the population level. Finally, we describe a quantitative analysis method of the droplet deformation that allows measuring in real-time the mechanical stress exerted by a single cell. The method presented herein thus constitutes a powerful analytical tool for cell migration studies under confinement.

| S-EPMC4931467 | biostudies-literature

One in a million: flow cytometric sorting of single cell-lysate assays in monodisperse picolitre double emulsion droplets for directed evolution.

Project description:Directed evolution relies on iterative cycles of randomization and selection. The outcome of an artificial evolution experiment is crucially dependent on (i) the numbers of variants that can be screened and (ii) the quality of the assessment of each clone that forms the basis for selection. Compartmentalization of screening assays in water-in-oil emulsion droplets provides an opportunity to screen vast numbers of individual assays with good signal quality. Microfluidic systems have been developed to make and sort droplets, but the operator skill required precludes their ready implementation in nonspecialist settings. We now establish a protocol for the creation of monodisperse double-emulsion droplets in two steps in microfluidic devices with different surface characteristics (first hydrophobic, then hydrophilic). The resulting double-emulsion droplets are suitable for quantitative analysis and sorting in a commercial flow cytometer. The power of this approach is demonstrated in a series of enrichment experiments, culminating in the successful recovery of catalytically active clones from a sea of 1?000?000-fold as many low-activity variants. The modular workflow allows integration of additional steps: the encapsulated lysate assay reactions can be stopped by heat inactivation (enabling ready control of selection stringency), the droplet size can be contracted (to concentrate its contents), and storage (at -80 °C) is possible for discontinuous workflows. The control that can be thus exerted on screening conditions will facilitate exploitation of the potential of protein libraries compartmentalized in droplets in a straightforward protocol that can be readily implemented and used by protein engineers.

| S-EPMC3952496 | biostudies-literature

A Multiplexed Cell-Free Assay to Screen for Antimicrobial Peptides in Double Emulsion Droplets.

Project description:The global surge in bacterial resistance against traditional antibiotics triggered intensive research for novel compounds, with antimicrobial peptides (AMPs) identified as a promising candidate. Automated methods to systematically generate and screen AMPs according to their membrane preference, however, are still lacking. We introduce a novel microfluidic system for the simultaneous cell-free production and screening of AMPs for their membrane specificity. On our device, AMPs are cell-free produced within water-in-oil-in-water double emulsion droplets, generated at high frequency. Within each droplet, the peptides can interact with different classes of co-encapsulated liposomes, generating a membrane-specific fluorescent signal. The double emulsions can be incubated and observed in a hydrodynamic trapping array or analyzed via flow cytometry. Our approach provides a valuable tool for the discovery and development of membrane-active antimicrobials.

| S-EPMC9303939 | biostudies-literature

Adsorption-Reaction Processes Between Gelatin and PDMS-E Emulsion Droplets.

Project description:In this work, the effects of droplet size and reaction time on the adsorption-reaction processes between gelatin and α-[3-(2,3-epoxypropoxy) propyl]-ω-butyl-polydimethylsiloxane (PDMS-E) emulsion droplets were studied. Gelatin molecules were only physically adsorbed on the surface of the PDMS-E droplet in the 0-75 min range, which was unrelated to the droplet size (100-1000 nm). For the small-size droplets (<410 nm), the physical adsorption proceeded over 75 min followed by agglomeration. For middle-size droplets (410-680 nm), the physical adsorption finished at 125 min; subsequently, the nucleophilic reaction between the primary amino group and the epoxy group began to happen, and globular-like or core-shell colloidal particles were formed. For large-size droplets (>680 nm), the nucleophilic reaction occurred at 75 min and produced core-shell or multi-layered colloidal particles. In a word, the physical absorption or nucleophilic reaction between gelatin and PDMS-E emulsion droplets could be controlled by controlling the droplet size and reaction time. Furthermore, the soft tissue paper coated with large-size droplets exhibited excellent resistance to water permeability and flame-resistant performance, which were carried out by water resistance and flammability tests.

| S-EPMC8173617 | biostudies-literature

Ion shuttling between emulsion droplets by crown ether modified gold nanoparticles.

Project description:Selective unidirectional transport of barium ions between droplets in a water-in-chloroform emulsion is demonstrated. Gold nanoparticles (GNPs) modified with a thiolated crown ether act as barium ion complexing shuttles that carry the ions from one population of droplets (source) to another (target). This process is driven by a steep barium ion concentration gradient between source and target droplets. The concentration of barium ions in the target droplets is kept low at all times by the precipitation of insoluble barium sulfate. A potential role of electrostatically coupled secondary processes that maintain the electroneutrality of the emulsion droplets is discussed. Charging of the GNP metal cores by electron transfer in the presence of the Fe(ii)/Fe(iii) redox couple appears to affect the partitioning of the GNPs between the water droplets and the chloroform phase. Processes have been monitored and studied by optical microscopy, Raman spectroscopy, cryogenic scanning electron microscopy (cryo-SEM) and zeta potential. The shuttle action of the GNPs has further been demonstrated electrochemically in a model system.

| S-EPMC8168925 | biostudies-literature

Generation of liquid metal double emulsion droplets using gravity-induced microfluidics.

Project description:Several microfluidic applications are available for liquid metal droplet generation, but the surface oxidation of liquid metal has placed limitations on its application. Multiphase microfluidics makes it possible to protect the inner droplets by producing the structure of double emulsion droplets. Thus, the generation of liquid metal double emulsion droplets has been developed to prevent the surface oxidation of Galinstan. However, the generation using common methods faces considerable challenges due to the gravity effect introduced from the high density of liquid metal, making it difficult for the shell phase to wrap the inner phase. To overcome this obstacle, we introduce an innovative method - a gravity-induced microfluidic device - to creatively generate controllable liquid metal double emulsion droplets, achieved by altering the measurable inclination angle of the plane. It is found that when the inclination angle ranges from 30° to 45°, the device manages to generate liquid metal double emulsion droplets with perfect double sphere-type configuration. Additionally, the core-shell liquid metal hydrogel capsules present potential applications as multifunctional materials for controlled release systems in drug delivery and biomedical applications. By regulating pH or imposing mechanical force, the hydrogel shell can be dissolved to recover the electrical conductivity of Galinstan for applications in flexible electronics, self-healing conductors, elastomer electronic skin, and tumor therapy.

| S-EPMC9295136 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data