Fluorescence Time-lapse Imaging of the Complete S. venezuelae Life Cycle Using a Microfluidic Device

Live-cell imaging of biological processes at the single cell level has been instrumental to our current understanding of the subcellular organization of bacterial cells. However, the application of time-lapse microscopy to study the cell biological processes underpinning development in the sporulating filamentous bacteria Streptomyces has been hampered by technical difficulties. Here we present a protocol to overcome these limitations by growing the new model species, Streptomyces venezuelae, in a commercially available microfluidic device which is connected to an inverted fluorescence widefield microscope. Unlike the classical model species, Streptomyces coelicolor, S. venezuelae sporulates in liquid, allowing the application of microfluidic growth chambers to cultivate and microscopically monitor the cellular development and differentiation of S. venezuelae over long time periods. In addition to monitoring morphological changes, the spatio-temporal distribution of fluorescently labeled target proteins can also be visualized by time-lapse microscopy. Moreover, the microfluidic platform offers the experimental flexibility to exchange the culture medium, which is used in the detailed protocol to stimulate sporulation of S. venezuelae in the microfluidic chamber. Images of the entire S. venezuelae life cycle are acquired at specific intervals and processed in the open-source software Fiji to produce movies of the recorded time-series.     

An Easy-To-Handle Microfluidic Device Suitable for Immunohistochemical Procedures in Mammalian Cells Grown Under Flow Conditions

Microfluidics, the technology that manipulates small amount of fluids in microscale complex devices, has undergone a remarkable development during the last decade, by targeting a significant range of applications, including biological tests and single-cell analysis, and by displaying many advantages such as reduced reagent consumption, decreased costs and faster analysis. Furthermore, the introduction of microfluidic tools has revolutionized the study of vascular functions, because the controlled three-dimensional environment and the continuous perfusion provided by the microdevice allow simulating the physiological characteristics of the circulatory system. Researchers interested in the study of vascular physiology, however, are often hampered by the difficulty in handling reduced number of cells after growth in these devices. This work shows how to apply different protocols commonly used in biology, such as the immunofluorescence technique, to cells grown in reversibly-bound microfluidic devices, obtaining results comparable to those retrieved under static conditions in multiwells. In this way, we are able to combine the advantages of microfluidic, i.e., application of continuous flow and shear stress, with classical protocols for the study of endothelial cells.     

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.     

Ultrasonic Welding of Thermoplastic Composite Coupons for Mechanical Characterization of Welded Joints through Single Lap Shear Testing

This paper presents a novel straightforward method for ultrasonic welding of thermoplastic-composite coupons in optimum processing conditions. The ultrasonic welding process described in this paper is based on three main pillars. Firstly, flat energy directors are used for preferential heat generation at the joining interface during the welding process. A flat energy director is a neat thermoplastic resin film that is placed between the parts to be joined prior to the welding process and heats up preferentially owing to its lower compressive stiffness relative to the composite substrates. Consequently, flat energy directors provide a simple solution that does not require molding of resin protrusions on the surfaces of the composite substrates, as opposed to ultrasonic welding of unreinforced plastics. Secondly, the process data provided by the ultrasonic welder is used to rapidly define the optimum welding parameters for any thermoplastic composite material combination. Thirdly, displacement control is used in the welding process to ensure consistent quality of the welded joints. According to this method, thermoplastic-composite flat coupons are individually welded in a single lap configuration. Mechanical testing of the welded coupons allows determining the apparent lap shear strength of the joints, which is one of the properties most commonly used to quantify the strength of thermoplastic composite welded joints.     

Three-dimensional Particle Tracking Velocimetry for Turbulence Applications: Case of a Jet Flow

3D-PTV is a quantitative flow measurement technique that aims to track the Lagrangian paths of a set of particles in three dimensions using stereoscopic recording of image sequences. The basic components, features, constraints and optimization tips of a 3D-PTV topology consisting of a high-speed camera with a four-view splitter are described and discussed in this article. The technique is applied to the intermediate flow field (5 <x/d <25) of a circular jet at Re ≈ 7,000. Lagrangian flow features and turbulence quantities in an Eulerian frame are estimated around ten diameters downstream of the jet origin and at various radial distances from the jet core. Lagrangian properties include trajectory, velocity and acceleration of selected particles as well as curvature of the flow path, which are obtained from the Frenet-Serret equation. Estimation of the 3D velocity and turbulence fields around the jet core axis at a cross-plane located at ten diameters downstream of the jet is compared with literature, and the power spectrum of the large-scale streamwise velocity motions is obtained at various radial distances from the jet core.     

Procedure for Decellularization of Rat Livers in an Oscillating-pressure Perfusion Device

Decellularization and recellularization of parenchymal organs may enable the generation of functional organs in vitro, and several protocols for rodent liver decellularization have already been published. We aimed to improve the decellularization process by construction of a proprietary perfusion device enabling selective perfusion via the portal vein and/or the hepatic artery. Furthermore, we sought to perform perfusion under oscillating surrounding pressure conditions to improve the homogeneity of decellularization. The homogeneity of perfusion decellularization has been an underestimated factor to date. During decellularization, areas within the organ that are poorly perfused may still contain cells, whereas the extracellular matrix (ECM) in well-perfused areas may already be affected by alkaline detergents. Oscillating pressure changes can mimic the intraabdominal pressure changes that occur during respiration to optimize microperfusion inside the liver. In the study presented here, decellularized rat liver matrices were analyzed by histological staining, DNA content analysis and corrosion casting. Perfusion via the hepatic artery showed more homogenous results than portal venous perfusion did. The application of oscillating pressure conditions improved the effectiveness of perfusion decellularization. Livers perfused via the hepatic artery and under oscillating pressure conditions showed the best results. The presented techniques for liver harvesting, cannulation and perfusion using our proprietary device enable sophisticated perfusion set-ups to improve decellularization and recellularization experiments in rat livers.