Buffer drains and mucus is transported upward in a tilted mucus clearance assay.

Mucociliary clearance (MCC) plays an essential role in maintaining airway sterility and health. Conversely, mucociliary dysfunction is implicated across many airway obstructive diseases. Understanding the necessary requirements for successful MCC is imperative to establish the pathology of disease, as well as to develop therapeutic strategies. Although postural, that is, gravitational, drainage is used clinically to aid mucus clearance, it is ignored in both animal and cell culture models of MCC. In this study, we develop a novel mucus clearance assay that enables the first particle image velocimetry of human bronchial epithelial cell cultures tilted relative to the gravitational field. This tilting system makes it possible to observe drainage of the airway surface liquid and, thus, reveals the effect gravity has on mucociliary clearance. First, we use this assay to demonstrate that beating cilia alone cannot transport buffer upward against gravity. Next, we show the same cilia successfully transporting mucus upward. These results indicate that the biophysical and biochemical properties of mucus enable vertical clearance and that current assay systems are not equipped to determine which properties are required for physiologically relevant vertical mucociliary clearance.

Analysis-aware microscopy video compression.

This article introduces an analysis-aware microscopy video compression method designed for microscopy videos that are consumed by analysis algorithms rather than by the human visual system. We define the quality of a microscopy video based on the level of preservation of analysis results. We evaluated our method with a bead tracking analysis program. For the same error level in the analysis result, our method can achieve 1,000× compression on certain test microscopy videos. Compared with a previous technique that yields exactly the exact same results by analysis algorithms, our method gives more flexibility for a user to control the quality. A modification to the new method also provides faster compression speed.

Elevated hematocrit enhances platelet accumulation following vascular injury.

Red blood cells (RBCs) demonstrate procoagulant properties in vitro, and elevated hematocrit is associated with reduced bleeding and increased thrombosis risk in humans. These observations suggest RBCs contribute to thrombus formation. However, effects of RBCs on thrombosis are difficult to assess because humans and mice with elevated hematocrit typically have coexisting pathologies. Using an experimental model of elevated hematocrit in healthy mice, we measured effects of hematocrit in 2 in vivo clot formation models. We also assessed thrombin generation, platelet-thrombus interactions, and platelet accumulation in thrombi ex vivo, in vitro, and in silico. Compared with controls, mice with elevated hematocrit (RBCHIGH) formed thrombi at a faster rate and had a shortened vessel occlusion time. Thrombi in control and RBCHIGH mice did not differ in size or fibrin content, and there was no difference in levels of circulating thrombin-antithrombin complexes. In vitro, increasing the hematocrit increased thrombin generation in the absence of platelets; however, this effect was reduced in the presence of platelets. In silico, direct numerical simulations of whole blood predicted elevated hematocrit increases the frequency and duration of interactions between platelets and a thrombus. When human whole blood was perfused over collagen at arterial shear rates, elevating the hematocrit increased the rate of platelet deposition and thrombus growth. These data suggest RBCs promote arterial thrombosis by enhancing platelet accumulation at the site of vessel injury. Maintaining a normal hematocrit may reduce arterial thrombosis risk in humans.

An Automated High-throughput Array Microscope for Cancer Cell Mechanics.

Changes in cellular mechanical properties correlate with the progression of metastatic cancer along the epithelial-to-mesenchymal transition (EMT). Few high-throughput methodologies exist that measure cell compliance, which can be used to understand the impact of genetic alterations or to screen the efficacy of chemotherapeutic agents. We have developed a novel array high-throughput microscope (AHTM) system that combines the convenience of the standard 96-well plate with the ability to image cultured cells and membrane-bound microbeads in twelve independently-focusing channels simultaneously, visiting all wells in eight steps. We use the AHTM and passive bead rheology techniques to determine the relative compliance of human pancreatic ductal epithelial (HPDE) cells, h-TERT transformed HPDE cells (HPNE), and four gain-of-function constructs related to EMT. The AHTM found HPNE, H-ras, Myr-AKT, and Bcl2 transfected cells more compliant relative to controls, consistent with parallel tests using atomic force microscopy and invasion assays, proving the AHTM capable of screening for changes in mechanical phenotype.

Analysis-preserving video microscopy compression via correlation and mathematical morphology.

The large amount video data produced by multi-channel, high-resolution microscopy system drives the need for a new high-performance domain-specific video compression technique. We describe a novel compression method for video microscopy data. The method is based on Pearson's correlation and mathematical morphology. The method makes use of the point-spread function (PSF) in the microscopy video acquisition phase. We compare our method to other lossless compression methods and to lossy JPEG, JPEG2000, and H.264 compression for various kinds of video microscopy data including fluorescence video and brightfield video. We find that for certain data sets, the new method compresses much better than lossless compression with no impact on analysis results. It achieved a best compressed size of 0.77% of the original size, 25× smaller than the best lossless technique (which yields 20% for the same video). The compressed size scales with the video's scientific data content. Further testing showed that existing lossy algorithms greatly impacted data analysis at similar compression sizes.

A high throughput array microscope for the mechanical characterization of biomaterials.

In the last decade, the emergence of high throughput screening has enabled the development of novel drug therapies and elucidated many complex cellular processes. Concurrently, the mechanobiology community has developed tools and methods to show that the dysregulation of biophysical properties and the biochemical mechanisms controlling those properties contribute significantly to many human diseases. Despite these advances, a complete understanding of the connection between biomechanics and disease will require advances in instrumentation that enable parallelized, high throughput assays capable of probing complex signaling pathways, studying biology in physiologically relevant conditions, and capturing specimen and mechanical heterogeneity. Traditional biophysical instruments are unable to meet this need. To address the challenge of large-scale, parallelized biophysical measurements, we have developed an automated array high-throughput microscope system that utilizes passive microbead diffusion to characterize mechanical properties of biomaterials. The instrument is capable of acquiring data on twelve-channels simultaneously, where each channel in the system can independently drive two-channel fluorescence imaging at up to 50 frames per second. We employ this system to measure the concentration-dependent apparent viscosity of hyaluronan, an essential polymer found in connective tissue and whose expression has been implicated in cancer progression.

Cylinders vs. spheres: biofluid shear thinning in driven nanoparticle transport.

Increasingly, the research community applies magnetophoresis to micro and nanoscale particles for drug delivery applications and the nanoscale rheological characterization of complex biological materials. Of particular interest is the design and transport of these magnetic particles through entangled polymeric fluids commonly found in biological systems. We report the magnetophoretic transport of spherical and rod-shaped particles through viscoelastic, entangled solutions using lambda-phage DNA (λ-DNA) as a model system. In order to understand and predict the observed phenomena, we fully characterize three fundamental components: the magnetic field and field gradient, the shape and magnetic properties of the probe particles, and the macroscopic rheology of the solution. Particle velocities obtained in Newtonian solutions correspond to macroscale rheology, with forces calculated via Stokes Law. In λ-DNA solutions, nanorod velocities are 100 times larger than predicted by measured zero-shear viscosity. These results are consistent with particles experiencing transport through a shear thinning fluid, indicating magnetically driven transport in shear thinning may be especially effective and favor narrow diameter, high aspect ratio particles. A complete framework for designing single-particle magnetic-based delivery systems results when we combine a quantified magnetic system with qualified particles embedded in a characterized viscoelastic medium.

Force generation and dynamics of individual cilia under external loading.

Motile cilia are unique multimotor systems that display coordination and periodicity while imparting forces to biological fluids. They play important roles in normal physiology, and ciliopathies are implicated in a growing number of human diseases. In this work we measure the response of individual human airway cilia to calibrated forces transmitted via spot-labeled magnetic microbeads. Cilia respond to applied forces by 1), a reduction in beat amplitude (up to an 85% reduction by 160-170 pN of force); 2), a decreased tip velocity proportionate to applied force; and 3), no significant change in beat frequency. Tip velocity reduction occurred in each beat direction, independently of the direction of applied force, indicating that the cilium is "driven" in both directions at all times. By applying a quasistatic force model, we deduce that axoneme stiffness is dominated by the rigidity of the microtubules, and that cilia can exert 62 +/- 18 pN of force at the tip via the generation of 5.6 +/- 1.6 pN/dynein head.

High throughput system for magnetic manipulation of cells, polymers, and biomaterials.

In the past decade, high throughput screening (HTS) has changed the way biochemical assays are performed, but manipulation and mechanical measurement of micro- and nanoscale systems have not benefited from this trend. Techniques using microbeads (particles approximately 0.1-10 mum) show promise for enabling high throughput mechanical measurements of microscopic systems. We demonstrate instrumentation to magnetically drive microbeads in a biocompatible, multiwell magnetic force system. It is based on commercial HTS standards and is scalable to 96 wells. Cells can be cultured in this magnetic high throughput system (MHTS). The MHTS can apply independently controlled forces to 16 specimen wells. Force calibrations demonstrate forces in excess of 1 nN, predicted force saturation as a function of pole material, and powerlaw dependence of F approximately r(-2.7+/-0.1). We employ this system to measure the stiffness of SR2+ Drosophila cells. MHTS technology is a key step toward a high throughput screening system for micro- and nanoscale biophysical experiments.

Chapter 16: Magnetic manipulation for force measurements in cell biology.

Life is a mechanical process. Cells, tissues, and bodies must act within their environments to grow, divide, move, communicate, and defend themselves. The stiffness and viscosity of cells and biologic materials will vary depending upon a wide variety of variables including for example environmental conditions, activation of signaling pathways, stage of development, gene expression. By pushing and pulling cells or materials such as mucus or extracellular matrix, one can learn about their mechanical properties. By varying the conditions, signaling pathways or genetic background, one can also assess how the response of the cell or material is modulated by that pathway. Magnetic particles are available commercially in many useful sizes, magnetic contents, and surface chemistries. The variety of surface chemistries allow forces to be applied to a specimen through specific linkages such as receptors or particular proteins, allowing the biologist to ask fundamental questions about the role of those linkages in the transduction of force or motion. In this chapter, we discuss the use of a magnetic system designed to apply a wide range of forces and force patterns fully integrated into a high numerical aperture inverted fluorescence microscope. Fine, thin and flat magnetic poles allow the use of high magnification microscope objectives, and flexible software to control the direction and pattern of applied forces supports a variety of experimental situations. The system can be coupled with simple video acquisition for medium-bandwidth, two-dimensional particle tracking. Alternatively, the system can be coupled with a laser tracking and position feedback system for higher resolution, high bandwidth, three-dimensional tracking.