An In-Vitro Study of the Flow Past a Transcatheter Aortic Valve Using Time-Resolved 3D Particle Tracking.

The performance of a transcatheter aortic valve (TAV) can be evaluated by analyzing the flow field downstream of the valve. However, three dimensional flow and pressure fields, and particle residence time, a quantity closely related to thrombosis risk, are challenging to obtain. This experimental study aims to provide a comprehensive 3D measurement of the flow field downstream of an Edwards SAPIEN 3 using time-resolved 3D particle tracking velocimetry (3D PTV) with Shake-the-Box (STB) algorithm. The valve was deployed in an idealized aorta model and tested in a left heart simulator under physiological conditions. Detailed 3D vortical structures, pressure distributions, and particle residence time were obtained by analyzing the 3D particle tracks. Results have shown large-scale retrograde flow entering the sinuses of the TAV at systole, reducing flow stasis there. However, the 3D particle tracks reveal that the retrograde flow has a high residence time and might have already experienced high shear stress near the main jet. Thus by only focusing on the flow in the sinus region is not sufficient to evaluate the leaflet thrombosis risk, and the flow downstream of the valve should be taken into consideration. The unique perspectives offered by 3D PTV are important when evaluating the performance of the TAVs.

Blood Protein Exclusion from Polymer Brushes.

We report interactions between adsorbed copolymers of poly(ethylene glycol) (PEG) in the presence of two abundant blood proteins, serum albumin and an immunoglobulin G, up to physiological blood concentrations. We directly and nonintrusively measure interactions between PEG triblock copolymers (PEG-PPO-PEG) adsorbed to hydrophobic colloids and surfaces using Total Internal Reflection Microscopy, which provides kT- and nanometer-scale resolution of interaction potentials (energy vs separation). In the absence of protein, adsorbed PEG copolymer repulsion is consistent with dimensions and architectures of PEG brushes on both colloids and surfaces. In the presence of proteins, we observe concentration dependent depletion attraction and no change to brush repulsion, indicating protein exclusion from PEG brushes. Because positive and negative protein adsorption are mutually exclusive, our observations of concentration dependent depletion attraction with no change to brush repulsion unambiguously indicate the absence of protein coronas at physiological protein concentrations. These findings demonstrate a direct sensitive approach to determine interactions between proteins and particle/surface coatings important to diverse biotechnology applications.

High speed two-photon laser scanning stereomicroscopy for three-dimension tracking multiple particles simultaneously in three-dimension.

In this paper, we will describe a video rate two-photon laser scanning stereomicroscopy for imaging-based three-dimensional particle tracking. Using a resonant galvanometer, we have now achieved 30 volumes per second (frame size 512 × 512) in volumetric imaging. Owing to the pulse multiplexing and demultiplexing techniques, the system does not suffer the speed loss for taking two parallax views of a volume. The switching time between left and right views is reduced to several nanoseconds. The extremely fast view switching and high volumetric imaging speed allow us to track fast transport processes of nanoparticles in deep light-scattering media. For instance, in 1%-intralipid solution and fibrillar scaffolds, the tracking penetration depth can be around 400 µm.

Dual Channel Microfluidics for Mimicking the Blood-Brain Barrier.

High-resolution imaging is essential for analysis of the steps and way stations of cargo transport in in vitro models of the endothelium. In this study, we demonstrate a microfluidic system consisting of two channels horizontally separated by a cell-growth-promoting membrane. Its design allows for high-resolution (down to single-molecule level) imaging using a high numerical aperture objective with a short working distance. To reduce optical aberrations and enable single-molecule-sensitive imaging, an observation window was constructed in the membrane via laser cutting with subsequent structuring using 3D multiphoton lithography for improved cell growth. The upper channel was loaded with endothelial cells under flow conditions, which showed polarization and junction formation. A coculture of human vascular endothelial cells with pericytes was developed that mimics the blood-brain barrier. Finally, this dual channel microfluidics system enabled 3D localization microscopy of the cytoskeleton and 3D single-molecule-sensitive tracing of lipoprotein particles.

A new approach to characterize firebrand showers using advanced 3D imaging techniques.

A new approach to characterize airborne firebrands during Wildland-Urban Interface (WUI) fires is detailed. The approach merges the following two imaging techniques in a single field-deployable diagnostic tool: (1) 3D Particle Tracking Velocimetry (3D-PTV), for time-resolved mapping of firebrand 3D trajectories, and (2) 3D Particle Shape Reconstruction (3D-PSR), to reconstruct 3D models of individual particles following the Visual Hull principle. This tool offers for the first time the possibility to simultaneously study time-resolved firebrand fluxes and firebrand size distribution to the full extent of their three-dimensional nature within a control volume. Methodologies used in the present work are presented and their technical implementation are discussed. Validation tests to confirm proper tracking/sizing of particles are detailed. The diagnostic tool is applied to a firebrand shower artificially generated at the NIST National Fire Research Laboratory. A novel graphic representation, that incorporates both the Cumulative Particle Count (CPC, particles m-2) and Particle Number Flux (PNF, particles m-2 s-1) as relevant exposure metrics, is presented and the exposure level is compared to that of an actual outdoor fire. Size distributions obtained for airborne firebrands are compared to those achieved through ground collection and strategies to improve the particle shape reconstruction method are discussed.

Genetically Encoded Phase Contrast Agents for Digital Holographic Microscopy.

Quantitative phase imaging and digital holographic microscopy have shown great promise for visualizing the motion, structure, and physiology of microorganisms and mammalian cells in three dimensions. However, these imaging techniques currently lack molecular contrast agents analogous to the fluorescent dyes and proteins that have revolutionized fluorescence microscopy. Here we introduce the first genetically encodable phase contrast agents based on gas vesicles. The relatively low index of refraction of the air-filled core of gas vesicles results in optical phase advancement relative to aqueous media, making them a "positive" phase contrast agent easily distinguished from organelles, dyes, or microminerals. We demonstrate this capability by identifying and tracking the motion of gas vesicles and gas vesicle-expressing bacteria using digital holographic microscopy, and by imaging the uptake of engineered gas vesicles by mammalian cells. These results give phase imaging a biomolecular contrast agent, expanding the capabilities of this powerful technology for three-dimensional biological imaging.

Three-Dimensional Two-Color Dual-Particle Tracking Microscope for Monitoring DNA Conformational Changes and Nanoparticle Landings on Live Cells.

Here, we present a three-dimensional two-color dual-particle tracking (3D-2C-DPT) technique that can simultaneously localize two spectrally distinct targets in three dimensions with a time resolution down to 5 ms. The dual-targets can be tracked with separation distances from 33 to 250 nm with tracking precisions of ∼15 nm (for static targets) and ∼35 nm (for freely diffusing targets). Since each target is individually localized, a wealth of data can be extracted, such as the relative 3D position, the 2D rotation, and the separation distance between the two targets. Using this technique, we turn a double-stranded DNA (dsDNA)-linked dumbbell-like dimer into a nanoscopic optical ruler to quantify the bending dynamics of nicked or gapped dsDNA molecules in free solution by manipulating the design of dsDNA linkers (1-nick, 3-nt, 6-nt, or 9-nt single-strand gap), and the results show the increase of kon (linear to bent) from 3.2 to 10.7 s-1. The 3D-2C-DPT is then applied to observe translational and rotational motions of the landing of an antibody-conjugated nanoparticle on the plasma membrane of living cells, revealing the reduction of rotations possibly due to interactions with membrane receptors. This study demonstrates that this 3D-2C-DPT technique is a new tool to shed light on the conformational changes of biomolecules and the intermolecular interactions on plasma membrane.

Comprehensive In Vitro Study of the Flow Past Two Transcatheter Aortic Valves: Comparison with a Severe Stenotic Case.

We investigate the flow past two transcatheter aortic valves (TAVs) and one severely calcified valve in an anatomically realistic aorta geometry to evaluate the ability of the TAVs to establish a healthier aortic flow compared to a diseased case. Velocity measurements of pulsatile flow are carried out using the 3D-particle tracking velocimetry technique. We present a novel approach based on the Smagorinsky model to assess the important subvoxel-scale (here smaller than 750 [Formula: see text]m) shear stress contribution that is usually unavailable in experiments. Both TAV models feature a small retrograde flow of about 5% of the stroke volume and a lower number of coherent vortical structures. Turbulence past the TAVs is strongly suppressed as evidenced by the lower levels of turbulent kinetic energy even though the newer generation TAV performs better than the old one. Also lysis indices are substantially reduced in both models. The new generation TAV displays a slightly higher risk for thrombogenicity due to longer exposure times. We anticipate that our new approach to include turbulence and shear stress related quantities may help to validate the design of cardiovascular devices.