Photophysical image analysis: Unsupervised probabilistic thresholding for images from electron-multiplying charge-coupled devices.

We introduce the concept photophysical image analysis (PIA) and an associated pipeline for unsupervised probabilistic image thresholding for images recorded by electron-multiplying charge-coupled device (EMCCD) cameras. We base our approach on a closed-form analytic expression for the characteristic function (Fourier-transform of the probability mass function) for the image counts recorded in an EMCCD camera, which takes into account both stochasticity in the arrival of photons at the imaging camera and subsequent noise induced by the detection system of the camera. The only assumption in our method is that the background photon arrival to the imaging system is described by a stationary Poisson process (we make no assumption about the photon statistics for the signal).

Geometry-Dependent Elastic Flow Dynamics in Micropillar Arrays.

Regular device-scale DNA waves for high DNA concentrations and flow velocities have been shown to emerge in quadratic micropillar arrays with potentially strong relevance for a wide range of microfluidic applications. Hexagonal arrays constitute another geometry that is especially relevant for the microfluidic pulsed-field separation of DNA. Here, we report on the differences at the micro and macroscopic scales between the resulting wave patterns for these two regular array geometries and one disordered array geometry.

Quantification of growth and nutrient consumption of bacterial and fungal cultures in microfluidic microhabitat models.

Understanding microbes in nature requires consideration of their microenvironment. Here, we present a protocol for quantifying biomass and nutrient degradation of bacterial and fungal cultures (Pseudomonas putida and Coprinopsis cinerea, respectively) in microfluidics. We describe steps for mask design and fabrication, master printing, polydimethylsiloxane chip fabrication, and chip inoculation and imaging using fluorescence microscopy.

Using symmetry to control viscoelastic waves in pillar arrays.

Solutions of macromolecules exhibit viscoelastic properties and unlike Newtonian fluids, they may break time-reversal symmetry at low Reynolds numbers resulting in elastic turbulence. Furthermore, under some conditions, instead of the chaotic turbulence, the result is large-scale waves in the form of cyclic spatial and temporal concentration variations, as has been shown for macromolecular DNA flowing in microfluidic pillar arrays. We here demonstrate how altering the symmetry of the individual pillars can be used to influence the symmetry of these waves.

Fluorescence Microscopy of Nanochannel-Confined DNA.

Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level, and the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments, and analyze the data.

Exposure to polystyrene nanoplastics reduces bacterial and fungal biomass in microfabricated soil models.

Nanoplastics have been proven to induce toxicity in diverse organisms, yet their effect on soil microbes like bacteria and fungi remains largely unexplored. In this paper, we used micro-engineered soil models to investigate the effect of polystyrene (PS) nanospheres on Pseudomonas putida and Coprinopsis cinerea. Specifically, we explored the effects of increasing concentrations of 60 nm carboxylated bovine serum albumin (BSA) coated nanospheres (0, 0.

Habitat complexity affects microbial growth in fractal maze.

The great variety of earth's microorganisms and their functions are attributed to the heterogeneity of their habitats, but our understanding of the impact of this heterogeneity on microbes is limited at the microscale. In this study, we tested how a gradient of spatial habitat complexity in the form of fractal mazes influenced the growth, substrate degradation, and interactions of the bacterial strain Pseudomonas putida and the fungal strain Coprinopsis cinerea. These strains responded in opposite ways: complex habitats strongly reduced fungal growth but, in contrast, increased the abundance of bacteria.

Short and long-range cyclic patterns in flows of DNA solutions in microfluidic obstacle arrays.

We observe regular patterns emerging across multiple length scales with high-concentration DNA solutions in microfluidic pillar arrays at low Reynolds numbers and high Deborah numbers. Interacting vortices between pillars lead to long-range order in the form of large travelling waves consisting of DNA at high concentration and extension. Waves are formed in quadratic arrays of pillars, while randomizing the position of the pillar in each unit cell of a quadratic array leads to suppression of the long-range patterns.

Use of dielectrophoresis for directing T cells to microwells before nanostraw transfection: modelling and experiments.

Nanostraw substrates have great potential for achieving minimally invasive cell transfection. Cells located on the nanostraw substrate are subjected to mild DC electric pulses applied across the nanostraw substrate, which open pores in the cell membrane on top of the nanostraws and drives charged cargo through these pores electrophoresis. However, with this method, the current may leak through uncovered nanostraws, thereby decreasing the desired effect in the cell-covered nanostraws.

High-Throughput Separation of Long DNA in Deterministic Lateral Displacement Arrays.

Length-based separation of DNA remains as relevant today as when gel electrophoresis was introduced almost 100 years ago. While new, long-read genomics technologies have revolutionised accessibility to powerful genomic data, the preparation of samples has not proceeded at the same pace, with sample preparation often constituting a considerable bottleneck, both in time and difficulty. Microfluidics holds great potential for automated, sample-to-answer analysis via the integration of preparatory and analytical steps, but for this to be fully realised, more versatile, powerful and integrable unit operations, such as separation, are essential.

Nanoscale binding site localization by molecular distance estimation on native cell surfaces using topological image averaging.

Antibody binding to cell surface proteins plays a crucial role in immunity, and the location of an epitope can altogether determine the immunological outcome of a host-target interaction. Techniques available today for epitope identification are costly, time-consuming, and unsuited for high-throughput analysis. Fast and efficient screening of epitope location can be useful for the development of therapeutic monoclonal antibodies and vaccines.

Habitat geometry in artificial microstructure affects bacterial and fungal growth, interactions, and substrate degradation.

Microhabitat conditions determine the magnitude and speed of microbial processes but have been challenging to investigate. In this study we used microfluidic devices to determine the effect of the spatial distortion of a pore space on fungal and bacterial growth, interactions, and substrate degradation. The devices contained channels differing in bending angles and order.

Microfluidic Obstacle Arrays Induce Large Reversible Shape Change in Red Blood Cells.

Red blood cell (RBC) shape change under static and dynamic shear stress has been a source of interest for at least 50 years. High-speed time-lapse microscopy was used to observe the rate of deformation and relaxation when RBCs are subjected to periodic shear stress and deformation forces as they pass through an obstacle. We show that red blood cells are reversibly deformed and take on characteristic shapes not previously seen in physiological buffers when the maximum shear stress was between 2.

Cell Sorting Using Electrokinetic Deterministic Lateral Displacement.

We show that by combining deterministic lateral displacement (DLD) with electrokinetics, it is possible to sort cells based on differences in their membrane and/or internal structures. Using heat to deactivate cells, which change their viability and structure, we then demonstrate sorting of a mixture of viable and non-viable cells for two different cell types. For the size change due to deactivation is insufficient to allow size-based DLD separation.

Charge-Based Separation of Micro- and Nanoparticles.

Deterministic Lateral Displacement (DLD) is a label-free particle sorting method that separates by size continuously and with high resolution. By combining DLD with electric fields (eDLD), we show separation of a variety of nano and micro-sized particles primarily by their zeta potential. Zeta potential is an indicator of electrokinetic charge-the charge corresponding to the electric field at the shear plane-an important property of micro- and nanoparticles in colloidal or separation science.

Deterministic Lateral Displacement: Challenges and Perspectives.

The advent of microfluidics in the 1990s promised a revolution in multiple industries from healthcare to chemical processing. Deterministic lateral displacement (DLD) is a continuous-flow microfluidic particle separation method discovered in 2004 that has been applied successfully and widely to the separation of blood cells, yeast, spores, bacteria, viruses, DNA, droplets, and more. Deterministic lateral displacement is conceptually simple and can deliver consistent performance over a wide range of flow rates and particle concentrations.

Embedded sacrificial AlAs segments in GaAs nanowires for substrate reuse.

We report on the use of a sacrificial AlAs segment to enable substrate reuse for nanowire synthesis. A silicon nitride template was deposited on a p-type GaAs substrate. Then a pattern was transferred to the substrate by nanoimprint lithography and reactive ion etching.

Label-free enrichment of primary human skeletal progenitor cells using deterministic lateral displacement.

Skeletal stem cells (SSCs) are present in bone marrow (BM) and offer great potential for bone regenerative therapies. However, in the absence of a unique marker, current sorting approaches remain challenging in the quest for simple strategies to deliver SSCs with consistent regeneration and differentiation capacities. Microfluidics offers the possibility to sort cells marker-free, based on intrinsic biophysical properties.

Separation of pathogenic bacteria by chain length.

Using Deterministic Lateral Displacement devices optimized for sensitivity to particle length, we separate subpopulations of bacteria depending on known properties that affect their capability to cause disease (virulence). For the human bacterial pathogen Streptococcus pneumoniae, bacterial chain length and the presence of a capsule are known virulence factors contributing to its ability to cause severe disease. Separation of cultured pneumococci into subpopulations based on morphological type (single cocci, diplococci and chains) will enable more detailed studies of the role they play in virulence.

RhoB controls the Rab11-mediated recycling and surface reappearance of LFA-1 in migrating T lymphocytes.

The regulation of cell adhesion and motility is complex and requires the intracellular trafficking of integrins to and from sites of cell adhesion, especially in fast-moving cells such as leukocytes. The Rab family of guanosine triphosphatases (GTPases) is essential for vesicle transport, and vesicles mediate intracellular integrin trafficking. We showed that RhoB regulates the vesicular transport of the integrin LFA-1 along the microtubule network in migrating T lymphocytes.

Microfluidics-Based Approaches to the Isolation of African Trypanosomes.

African trypanosomes are responsible for significant levels of disease in both humans and animals. The protozoan parasites are free-living flagellates, usually transmitted by arthropod vectors, including the tsetse fly. In the mammalian host they live in the bloodstream and, in the case of human-infectious species, later invade the central nervous system.

Open channel deterministic lateral displacement for particle and cell sorting.

We present the use of capillary driven flow over patterned surfaces to achieve cheap and simple, but powerful separation of biologically relevant particle systems. The wide use of microfluidics is often hampered by the propensity for devices to clog due to the small channel sizes and the inability to access the interior of devices for cleaning. Often the devices can only be used for a limited duration and most frequently only once.

Fluorescence Microscopy of Nanochannel-Confined DNA.

Stretching of DNA in nanoscale confinement allows for several important studies. The genetic contents of the DNA can be visualized on the single DNA molecule level and both the polymer physics of confined DNA and also DNA/protein and other DNA/DNA-binding molecule interactions can be explored. This chapter describes the basic steps to fabricate the nanostructures, perform the experiments and analyze the data.

Applications of optical DNA mapping in microbiology.

Optical mapping (OM) has been used in microbiology for the past 20 years, initially as a technique to facilitate DNA sequence-based studies; however, with decreases in DNA sequencing costs and increases in sequence output from automated sequencing platforms, OM has grown into an important auxiliary tool for genome assembly and comparison. Currently, there are a number of new and exciting applications for OM in the field of microbiology, including investigation of disease outbreaks, identification of specific genes of clinical and/or epidemiological relevance, and the possibility of single-cell analysis when combined with cell-sorting approaches. In addition, designing lab-on-a-chip systems based on OM is now feasible and will allow the integrated and automated microbiological analysis of biological fluids.

Sorting cells by their dynamical properties.

Recent advances in cell sorting aim at the development of novel methods that are sensitive to various mechanical properties of cells. Microfluidic technologies have a great potential for cell sorting; however, the design of many micro-devices is based on theories developed for rigid spherical particles with size as a separation parameter. Clearly, most bioparticles are non-spherical and deformable and therefore exhibit a much more intricate behavior in fluid flow than rigid spheres.