Fluid flow simulations meet high-speed video: Computer vision comparison of droplet dynamics.

HYPOTHESIS: While multiphase flows, particularly droplet dynamics, are ordinary in nature as well as in industrial processes, their mathematical and computational modelling continue to pose challenging research tasks - patent approaches for tackling them are yet to be found. The lack of analytical flow field solutions for non-trivial droplet dynamics hinders validation of computer simulations and, hence, their application in research problems. High-speed videos and computer vision algorithms can provide a viable approach to validate simulations directly against experiments. EXPERIMENTS: Droplets of water (or glycerol-water mixtures) impacting on both hydrophobic and superhydrophobic surfaces were imaged with a high-speed camera. The corresponding configurations were simulated using a lattice-Boltzmann multiphase scheme. Video frames from experiments and simulations were compared, by means of computer vision, over entire droplet impact events. FINDINGS: The proposed experimental validation procedure provides a detailed, dynamic one-on-one comparison of a droplet impact. The procedure relies on high-speed video recording of the experiments, computer vision, and on a software package for the analyzation routines. The procedure is able to quantitatively validate computer simulations against experiments and it is widely applicable to multiphase flow systems in general.

Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing.

The isolation and analysis of single prokaryotic cells down to 1 µm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 µm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry.

Enhanced single-cell printing by acoustophoretic cell focusing.

Recent years have witnessed a strong trend towards analysis of single-cells. To access and handle single-cells, many new tools are needed and have partly been developed. Here, we present an improved version of a single-cell printer which is able to deliver individual single cells and beads encapsulated in free-flying picoliter droplets at a single-bead efficiency of 96% and with a throughput of more than 10 beads per minute. By integration of acoustophoretic focusing, the cells could be focused in x and y direction. This way, the cells were lined-up in front of a 40 µm nozzle, where they were analyzed individually by an optical system prior to printing. In agreement with acoustic simulations, the focusing of 10 µm beads and Raji cells has been achieved with an efficiency of 99% (beads) and 86% (Raji cells) to a 40 µm wide center region in the 1 mm wide microfluidic channel. This enabled improved optical analysis and reduced bead losses. The loss of beads that ended up in the waste (because printing them as single beads arrangements could not be ensured) was reduced from 52% ± 6% to 28% ± 1%. The piezoelectric transducer employed for cell focusing could be positioned on an outer part of the device, which proves the acoustophoretic focusing to be versatile and adaptable.

Single-cell PCR of genomic DNA enabled by automated single-cell printing for cell isolation.

Single-cell analysis has developed into a key topic in cell biology with future applications in personalized medicine, tumor identification as well as tumor discovery (Editorial, 2013). Here we employ inkjet-like printing to isolate individual living single human B cells (Raji cell line) and load them directly into standard PCR tubes. Single cells are optically detected in the nozzle of the microfluidic piezoelectric dispenser chip to ensure printing of droplets with single cells only. The printing process has been characterized by using microbeads (10µm diameter) resulting in a single bead delivery in 27 out of 28 cases and relative positional precision of ±350µm at a printing distance of 6mm between nozzle and tube lid. Process-integrated optical imaging enabled to identify the printing failure as void droplet and to exclude it from downstream processing. PCR of truly single-cell DNA was performed without pre-amplification directly from single Raji cells with 33% success rate (N=197) and Cq values of 36.3±2.5. Additionally single cell whole genome amplification (WGA) was employed to pre-amplify the single-cell DNA by a factor of >1000. This facilitated subsequent PCR for the same gene yielding a success rate of 64% (N=33) which will allow more sophisticated downstream analysis like sequencing, electrophoresis or multiplexing.

Single-cell printing based on impedance detection.

Label-free isolation of single cells is essential for the growing field of single-cell analysis. Here, we present a device which prints single living cells encapsulated in free-flying picoliter droplets. It combines inkjet printing and impedance flow cytometry. Droplet volume can be controlled in the range of 500 pl-800 pl by piezo actuator displacement. Two sets of parallel facing electrodes in a 50 µm × 55 µm channel are applied to measure the presence and velocity of a single cell in real-time. Polystyrene beads with <5% variation in diameter generated signal variations of 12%-17% coefficients of variation. Single bead efficiency (i.e., printing events with single beads vs. total number of printing events) was 73% ± 11% at a throughput of approximately 9 events/min. Viability of printed HeLa cells and human primary fibroblasts was demonstrated by culturing cells for at least eight days.