Counting individual bacteria during high-throughput growth in microfluidic droplets

Bacterial growth in microfluidic droplets is relevant in biotechnology, in microbial ecology, and in understanding stochastic population dynamics in small populations. However, it has proved challenging to automate measurement of absolute bacterial numbers within droplets, forcing the use of proxy measures for population size. Here we present a microfluidic device and imaging protocol that allows high-resolution imaging of thousands of droplets, such that individual bacteria stay in the focal plane and can be counted automatically. We demonstrate the use of this approach to track stochastic growth of multiple replicate Escherichia coli populations within droplets.

Precision ejection of microfluidic droplets into air with a superhydrophobic outlet

We describe a general approach to controllably and precisely eject droplets of tunable composition from microfluidic devices using superhydrophobic patterning.

A photofabricated honeycomb micropillar array for loss-free trapping of microfluidic droplets and application to digital PCR

Droplet microfluidics is a promising platform for various biological and biomedical applications. Among which, droplet-based digital PCR (ddPCR) is one of the most challenging examples, with practical issues involving possible...

Thermo-responsive fluorinated surfactant for on-demand demulsification of microfluidic droplets

Thermo-responsive fluorinated surfactant can lead to destabilization of droplets and subsequently cause droplet coalescence. Thus, the encapsulated cargoes can be retrieved on-demand from the droplets without complicated processing.

Method for Passive Droplet Sorting after Photo-Tagging

We present a method to photo-tag individual microfluidic droplets for latter selection by passive sorting. The use of a specific surfactant leads to the interfacial tension to be very sensitive to droplet pH. The photoexcitation of droplets containing a photoacid, pyranine, leads to a decrease in droplet pH. The concurrent increase in droplet interfacial tension enables the passive selection of irradiated droplets. The technique is used to select individual droplets within a droplet array as illuminated droplets remain in the wells while other droplets are eluted by the flow of the external oil. This method was used to select droplets in an array containing cells at a specific stage of apoptosis. The technique is also adaptable to continuous-flow sorting. By passing confined droplets over a microfabricated trench positioned diagonally in relation to the direction of flow, photo-tagged droplets were directed toward a different chip exit based on their lateral movement. The technique can be performed on a conventional fluorescence microscope and uncouples the observation and selection of droplets, thus enabling the selection on a large variety of signals, or based on qualitative user-defined features.

Co-cultivation of microbial sub-communities in microfluidic droplets facilitates high-resolution genomic dissection of microbial ‘dark matter’

While the ‘unculturable’ majority of the bacterial world is accessible with culture-independent tools, the inability to study these bacteria using culture-dependent approaches has severely limited our understanding of their ecological roles and interactions. To circumvent cultivation barriers, we utilize microfluidic droplets as localized, nanoliter-size bioreactors to co-cultivate subsets of microbial communities. This co-localization can support ecological interactions between a reduced number of encapsulated cells. We demonstrated the utility of this approach in the encapsulation and co-cultivation of droplet sub-communities from a fecal sample collected from a healthy human subject. With the whole genome amplification and metagenomic shotgun sequencing of co-cultivated sub-communities from 22 droplets, we observed that this approach provides accessibility to uncharacterized gut commensals for study. The recovery of metagenome-assembled genomes from one droplet sub-community demonstrated the capability to dissect the sub-communities with high-genomic resolution. In particular, genomic characterization of one novel member of the family Neisseriaceae revealed implications regarding its participation in fatty acid degradation and production of atherogenic intermediates in the human gut. The demonstrated genomic resolution and accessibility to the microbial ‘dark matter’ with this methodology can be applied to study the interactions of rare or previously uncultivated members of microbial communities.