Digital particle image velocimetry (DPIV) is a non-intrusive analysis technique that is very popular for mapping flows quantitatively. To get accurate results, in particular in complex flow fields, a number of challenges have to be faced and solved: The quality of the flow measurements is affected by computational details such as image pre-conditioning, sub-pixel peak estimators, data validation procedures, interpolation algorithms and smoothing methods. The accuracy of several algorithms was determined and the best performing methods were implemented in a user-friendly open-source tool for performing DPIV flow analysis in Matlab.

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PIVlab – Towards User-friendly, Affordable and Accurate Digital Particle Image Velocimetry in MATLAB

To improve the performance of particle image velocimetry in measuring instantaneous velocity fields, direct cross-correlation of image fields can be used in place of auto-correlation methods of interrogation of double- or multiple-exposure recordings. With improved speed of photographic recording and increased resolution of video array detectors, cross-correlation methods of interrogation of successive single-exposure frames can be used to measure the separation of pairs of particle images between successive frames. By knowing the extent of image shifting used in a multiple-exposure and by a priori knowledge of the mean flow-field, the cross-correlation of different sized interrogation spots with known separation can be optimized in terms of spatial resolution, detection rate, accuracy and reliability.

Theory of cross-correlation analysis of PIV images : Image analysis as measuring technique in flows

Systemic absorption and metabolism of drugs in the small intestine, metabolism by the liver as well as excretion by the kidney are key determinants of efficacy and safety for therapeutic candidates. However, these systemic responses of applied substances lack in most in vitro assays. In this study, a microphysiological system maintaining the functionality of four organs over 28 days in co-culture has been established at a minute but standardized microsystem scale. Preformed human intestine and skin models have been integrated into the four-organ-chip on standard cell culture inserts at a size 100 000-fold smaller than their human counterpart organs. A 3D-based spheroid, equivalent to ten liver lobules, mimics liver function. Finally, a barrier segregating the media flow through the organs from fluids excreted by the kidney has been generated by a polymeric membrane covered by a monolayer of human proximal tubule epithelial cells. A peristaltic on-chip micropump ensures pulsatile media flow interconnecting the four tissue culture compartments through microfluidic channels. A second microfluidic circuit ensures drainage of the fluid excreted through the kidney epithelial cell layer. This four-organ-chip system assures near to physiological fluid-to-tissue ratios. In-depth metabolic and gene analysis revealed the establishment of reproducible homeostasis among the co-cultures within two to four days, sustainable over at least 28 days independent of the individual human cell line or tissue donor background used for each organ equivalent. Lastly, 3D imaging two-photon microscopy visualised details of spatiotemporal segregation of the two microfluidic flows by proximal tubule epithelia. To our knowledge, this study is the first approach to establish a system for in vitro microfluidic ADME profiling and repeated dose systemic toxicity testing of drug candidates over 28 days.

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A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents

Epithelial tissues (epithelia) remove excess cells through extrusion, preventing the accumulation of unnecessary or pathological cells. The extrusion process can be triggered by apoptotic signalling, oncogenic transformation and overcrowding of cells. Despite the important linkage of cell extrusion to developmental, homeostatic and pathological processes such as cancer metastasis, its underlying mechanism and connections to the intrinsic mechanics of the epithelium are largely unexplored. We approach this problem by modelling the epithelium as an active nematic liquid crystal (that has a long range directional order), and comparing numerical simulations to strain rate and stress measurements within monolayers of MDCK (Madin Darby canine kidney) cells. Here we show that apoptotic cell extrusion is provoked by singularities in cell alignments in the form of comet-shaped topological defects. We find a universal correlation between extrusion sites and positions of nematic defects in the cell orientation field in different epithelium types. The results confirm the active nematic nature of epithelia, and demonstrate that defect-induced isotropic stresses are the primary precursors of mechanotransductive responses in cells, including YAP (Yes-associated protein) transcription factor activity, caspase-3-mediated cell death, and extrusions. Importantly, the defect-driven extrusion mechanism depends on intercellular junctions, because the weakening of cell-cell interactions in an α-catenin knockdown monolayer reduces the defect size and increases both the number of defects and extrusion rates, as is also predicted by our model. We further demonstrate the ability to control extrusion hotspots by geometrically inducing defects through microcontact printing of patterned monolayers. On the basis of these results, we propose a mechanism for apoptotic cell extrusion: spontaneously formed topological defects in epithelia govern cell fate. This will be important in predicting extrusion hotspots and dynamics in vivo, with potential applications to tissue regeneration and the suppression of metastasis. Moreover, we anticipate that the analogy between the epithelium and active nematic liquid crystals will trigger further investigations of the link between cellular processes and the material properties of epithelia.

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Topological defects in epithelia govern cell death and extrusion.

Inspired by the relatively simple morphological blueprint provided by batoid fish such as stingrays and skates, we created a biohybrid system that enables an artificial animal—a tissue-engineered ray—to swim and phototactically follow a light cue. By patterning dissociated rat cardiomyocytes on an elastomeric body enclosing a microfabricated gold skeleton, we replicated fish morphology at 1 10 scale and captured basic fin deflection patterns of batoid fish. Optogenetics allows for phototactic guidance, steering, and turning maneuvers. Optical stimulation induced sequential muscle activation via serpentine-patterned muscle circuits, leading to coordinated undulatory swimming. The speed and direction of the ray was controlled by modulating light frequency and by independently eliciting right and left fins, allowing the biohybrid machine to maneuver through an obstacle course.

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Phototactic guidance of a tissue-engineered soft-robotic ray.

PIVlab is a free toolbox and app for MATLAB®. It is used to perform Particle Image Velocimetry (PIV) with image data: A light sheet illuminates particles that are suspended in a fluid. A digital camera records a series of images of the illuminated particles. The input images are divided into sub-images (interrogation areas), and for each of these, a cross-correlation is performed. The resulting correlation matrix is used to estimate the most probable displacement within each interrogation area. PIV is extensively used for flow analyses where a thin laser sheet illuminates suspended particles in the fluid, but also for other moving textures, like cell migration or ultrasonic images. This paper presents several improvements that were implemented in PIVlab, enhancing the robustness of displacement estimates. The benefit of these improvements is evaluated using experimental images and synthetic images of particle and non-particle textures. Linear correlation and repeated correlation increase the robustness and decrease bias and root-mean-square (RMS) error of the displacement estimates. Particle images have a significantly lower bias and RMS error than non-particle images.

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Particle Image Velocimetry for MATLAB: Accuracy and enhanced algorithms in PIVlab

PIVlab - Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB

De aerodynamica van slagvlucht bij vogels is eigenlijk helemaal niet zo duidelijk en bekend als vaak wordt gedacht, zoals tijdens recente studies aan het licht kwam. De aerodynamische mechanismen tijdens actieve slagvlucht verschillen namelijk behoorlijk van de aerodynamica van bijv. vliegtuigen. Tijdens langzame slagvlucht treden mechanismen op die veel gelijkenis vertonen met insectenvlucht waarvan de slagvlucht zich kenmerkt door relatief hoge opwaartse krachten en grote manoeuvreerbaarheid: Bovenop de vleugels ontwikkelen zich meelopende wervels tijdens iedere vleugelslag. Deze zgn. voorkantwervels maken het insecten en dus ook vogels mogelijk om zeer hoge opwaartse krachten op te wekken. Dergelijke hoge krachten zijn nodig om in de lucht te blijven bij lage snelheid en ook nog nauwkeurig te kunnen manoevreren. Bij de analyses in dit proefschrift is gebruik gemaakt van continue 3D-stromingsmetingen aan modellen van vleugels die de slagvlucht exact nadoen, om zo na te gaan hoe de wervels zich ontwikkelen tijdens een vleugelslag en hoe dat proces beheerst of beinvloed kan worden. Onze inzichten zijn zowel verklarend als ook zeer geschikt voor toepassingen op bijv. relatief kleine onbemande vliegtuigen: Door de bijzondere aerodynamica tijdens slagvlucht combineren vogels de hoge manoevreerbaarheid van een helicopter met de efficientie van een zweefvliegtuig. Windstunnel-tests met een prototype wat eveneens beide kenmerken combineerde lieten zien dat onbemande kleine vliegtuigen die slagvlucht met zweefvlucht combineren inderdaad hoge manoevreerbaarheid aan hoge aerodynamische efficientie koppelen, precies zoals onze inspiratoren, de vogels. Deze combinatie kan een grote stimulans zijn voor verdere ontwikkleing en toepassing van kleine ombemande vliegtuigen voor velerlei doelen.

The flapping flight of birds: Analysis and application

The effect of airfoil design parameters, such as airfoil thickness and camber, are well understood in steady-state aerodynamics. But this knowledge cannot be readily applied to the flapping flight in insects and birds: flow visualizations and computational analyses of flapping flight have identified that in many cases, a leading-edge vortex (LEV) contributes substantially to the generation of aerodynamic force. In flapping flight, very high angles of attack and partly separated flow are common features. Therefore, it is expected that airfoil design parameters affect flapping wing aerodynamics differently. Existing studies have focused on force measurements, which do not provide sufficient insight into the dominant flow features. To analyse the influence of wing morphology in slow-speed bird flight, the time-resolved three-dimensional flow field around different flapping wing models in translational motion at a Reynolds number of 22 000 <Re <26 000 was studied. The effect of several Strouhal numbers (0.2 <St <0.4), camber and thickness on the flow morphology and on the circulation was analysed. A strong LEV was found on all wing types at high St. The vortex is stronger on thin wings and enhances the total circulation. Airfoil camber decreases the strength of the LEV, but increases the total bound circulation at the same time, due to an increase of the 'conventional' bound circulation at the inner half of the wing. The results provide new insights into the influence of airfoil shape on the LEV and force generation at low Re. They contribute to a better understanding of the geometry of vertebrate wings, which seem to be optimized to benefit from LEVs in slow-speed flight.

William Thielicke

Papers

PIVlab – Towards User-friendly, Affordable and Accurate Digital Particle Image Velocimetry in MATLAB

Particle Image Velocimetry for MATLAB: Accuracy and enhanced algorithms in PIVlab

PIVlab - Time-Resolved Digital Particle Image Velocimetry Tool for MATLAB

The flapping flight of birds: Analysis and application

The influence of wing morphology on the three-dimensional flow patterns of a flapping wing at bird scale