Presegmentation procedure generates smooth-sided microfluidic devices: unlocking multiangle imaging for everyone?

We present a simple procedure to create smooth-sided, transparent polymer-based microfluidic devices by presegmentation with hydrophobized glass slides. We study the hypothesis that the smooth side planes permit rapid multiangle imaging of microfluidic systems in contrast to the turbid side planes that result from cutting the polymer. We compare the compatibility of the entire approach with the conventional widefield microscopy, confocal and 2-photon microscopy, as well as three-dimensional (3D) rendering and discuss limitations and potential applications

Microfluidic devices for drug assays

In this review, we give an overview of the current state of microfluidic-based high-throughput drug assays. In this highly interdisciplinary research field, various approaches have been applied to high-throughput drug screening, including microtiter plate, droplets microfluidics as well as continuous flow, diffusion and concentration gradients-based microfluidic drug assays. Therefore, we reviewed over 100 recent publications in the field and sorted them according to their microfluidic approach. As a result, we are showcasing, comparing and discussing broadly applied approaches as well as singular promising ones that might contribute to shaping the future of this field

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. Diagnosis of the disease requires the positive identification of parasites in the bloodstream. This can be particularly challenging where parasite numbers are low, as is often the case in peripheral blood. Enriching parasites from body fluids is an important part of the diagnostic pathway. As more is learned about the physicochemical properties of trypanosomes, this information can be exploited through use of different microfluidic-based approaches to isolate the parasites from blood or other fluids. Here, we discuss recent advances in the use of microfluidics to separate trypanosomes from blood and to isolate single trypanosomes for analyses including drug screening

The fate of lipid-coated and uncoated fluorescent nanodiamonds during cell division in yeast

Fluorescent nanodiamonds are frequently used as biolabels. They have also recently been established for magnetic resonance and temperature sensing at the nanoscale level. To properly use them in cell biology, we first have to understand their intracellular fate. Here, we investigated, for the first time, what happens to diamond particles during and after cell division in yeast (Saccharomyces cerevisiae) cells. More concretely, our goal was to answer the question of whether nanodiamonds remain in the mother cells or end up in the daughter cells. Yeast cells are widely used as a model organism in aging and biotechnology research, and they are particularly interesting because their asymmetric cell division leads to morphologically different mother and daughter cells. Although yeast cells have a mechanism to prevent potentially harmful substances from entering the daughter cells, we found an increased number of diamond particles in daughter cells. Additionally, we found substantial excretion of particles, which has not been reported for mammalian cells. We also investigated what types of movement diamond particles undergo in the cells. Finally, we also compared bare nanodiamonds with lipid-coated diamonds, and there were no significant differences in respect to either movement or intracellular fate

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. Despite wide use and in-depth study, DLD has not yet been fully elucidated or optimized, with different approaches to the same problem yielding varying results. We endeavor here to provide up-to-date expert opinion on the state-of-art and current fundamental, practical, and commercial challenges with DLD as well as describe experimental and modeling opportunities. Because these challenges and opportunities arise from constraints on hydrodynamics, fabrication, and operation at the micro- and nanoscale, we expect this Perspective to serve as a guide for the broader micro- and nanofluidic community to identify and to address open questions in the field

Motility, manipulation and controlling of unicellular organisms

Introduction: Motility is a measure for vitality of unicellular organisms By using a microfluidic setup it is possible to analyse single-cell organisms and their motility. Thus it is possible to achieve several goals, from characterising the way of movement and the forces thereby generated to analysing drug effects and controlling pathogen displacement and spatial concentration to facilitate diagnosis. In order to do so, the microfluidic device as well as the manipulation and analysis tools have to be calibrated and adapted to the varying parameters as determined by the matter under study. Methods: Using microfluidics in combination with optical trapping of unicellular organisms and high-speed microscopy, displacement trajectories were recorded and subsequently analysed using computer aided image analysis to characterise the flagellar propulsion of Trypanosoma brucei brucei and Caulobacter crescentus. Additionally, changes in the motility of T. b. brucei under the influence of drugs and different environments were determined and holdfast formation in C.crescentis was induced. The calibration parameters of the optical trap and the microfluidic devices were determined for different experimental setups in order to minimise phototoxic effects and maximise retention time of the organisms in the device. Results: Swimming, Caulobacter crescentus generate an average force of 0.3 pN while being capable of a maximal force of 2.6 pN. C.crescentis and Trypanosoma brucei brucei rotate when they are inside an optical trap but for the trypanosomes this depends on the type of movement they were exhibiting directly before being trapped. The movement of T. b. brucei around the trap has a frequency of 15 Hz for the flagellar beat and a frequency of 1.5 Hz for the rotation itself. The hydrodynamic interaction between swimming trypanosomes and the environment shows characteristic flow patters around the trypanosome that reveal it to be a pusher and not a puller. Their random-walk like migration can be directed by the geometry of the microfluidic device in order to contain them inside the device. In our experimental setup, Caulobacter crescentus exhibits a phototoxic reaction when trapped with a laser of the wavelength of 808 nm. The combination of optical traps and microfluidic devices can be furthermore used as a versatile methodology to study the impact of drugs and chemicals on motile unicellular organisms. Due to diffusion driven drug control, dosage-dependent effects can be determined through a motility factor. Conclusion: Microfluidics in combination with optical trapping of cells and high speed microscopy can be used to analyse, manipulate, and control the motility of unicellular organisms, thus providing us with an interdisciplinary toolset to study living soft matter in a complex fluidic environment

Lab-on-a-Chip Technologies for the Single Cell Level: Separation, Analysis, and Diagnostics

In the last three decades, microfluidics and its applications have been on an exponential rise, including approaches to isolate rare cells and diagnose diseases on the single-cell level. The techniques mentioned herein have already had significant impacts in our lives, from in-the-field diagnosis of disease and parasitic infections, through home fertility tests, to uncovering the interactions between SARS-CoV-2 and their host cells. This review gives an overview of the field in general and the most notable developments of the last five years, in three parts: 1. What can we detect? 2. Which detection technologies are used in which setting? 3. How do these techniques work? Finally, this review discusses potentials, shortfalls, and an outlook on future developments, especially in respect to the funding landscape and the field-application of these chips

Optical trapping reveals propulsion forces, power generation and motility efficiency of the unicellular parasites Trypanosoma brucei brucei

Unicellular parasites have developed sophisticated swimming mechanisms to survive in a wide range of environments. Cell motility of African trypanosomes, parasites responsible for fatal illness in humans and animals, is crucial both in the insect vector and the mammalian host. Using millisecond-scale imaging in a microfluidics platform along with a custom made optical trap, we are able to confine single cells to study trypanosome motility. From the trapping characteristics of the cells, we determine the propulsion force generated by cells with a single flagellum as well as of dividing trypanosomes with two fully developed flagella. Estimates of the dissipative energy and the power generation of single cells obtained from the motility patterns of the trypanosomes within the optical trap indicate that specific motility characteristics, in addition to locomotion, may be required for antibody clearance. Introducing a steerable second optical trap we could further measure the force, which is generated at the flagellar tip. Differences in the cellular structure of the trypanosomes are correlated with the trapping and motility characteristics and in consequence with their propulsion force, dissipative energy and power generation

Motility, Force Generation, and Energy Consumption of Unicellular Parasites

Motility is a key factor for pathogenicity of unicellular parasites, enabling them to infiltrate and evade host cells, and perform several of their life-cycle events. State-of-the-art methods of motility analysis rely on a combination of optical tweezers with high-resolution microscopy and microfluidics. With this technology, propulsion forces, energies, and power generation can be determined so as to shed light on the motion mechanisms, chemotactic behavior, and specific survival strategies of unicellular parasites. With these new tools in hand, we can elucidate the mechanisms of motility and force generation of unicellular parasites, and identify ways to manipulate and eventually inhibit them

Matrix-masking to balance nonuniform illumination in microscopy

With a perfectly uniform illumination, the amount and concentration of fluorophores in any (biological) sample can be read directly from fluorescence micrographs. However, non-uniform illumination in optical micrographs is a common, yet avoidable artefact, often caused by the setup of the microscope, or by inherent properties caused by the nature of the sample. In this paper, we demonstrate simple matrix-based methods using the common computing environments MATLAB and Python to correct nonuniform illumination, using either a background image or extracting illumination information directly from the sample image, together with subsequent image processing. We compare the processes, algorithms, and results obtained from both MATLAB (commercially available) and Python (freeware). Additionally, we validate our method by evaluating commonly used alternative approaches, demonstrating that the best nonuniform illumination correction can be achieved when a separate background image is available