Search for: All records

We report a label-free acoustic microfluidic method to confine single, cilia-driven swimming cells in space without limiting their rotational degrees of freedom. Our platform integrates a surface acoustic wave (SAW) actuator and bulk acoustic wave (BAW) trapping array to enable multiplexed analysis with high spatial resolution and trapping forces that are strong enough to hold individual microswimmers. The hybrid BAW/SAW acoustic tweezers employ high-efficiency mode conversion to achieve submicron image resolution while compensating for parasitic system losses to immersion oil in contact with the microfluidic chip. We use the platform to quantify cilia and cell body motion for wildtype biciliate cells, investigating effects of environmental variables like temperature and viscosity on ciliary beating, synchronization, and three-dimensional helical swimming. We confirm and expand upon the existing understanding of these phenomena, for example determining that increasing viscosity promotes asynchronous beating. Motile cilia are subcellular organelles that propel microorganisms or direct fluid and particulate flow. Thus, cilia are critical to cell survival and human health. The unicellular algaChlamydomonas reinhardtiiis widely used to investigate the mechanisms underlying ciliary beating and coordination. However, freely swimming cells are difficult to image with sufficient resolution to capture cilia motion, necessitating that the cell body be held during experiments. Acoustic confinement is a compelling alternative to use of a micropipette, or to magnetic, electrical, and optical trapping that may modify the cells and affect their behavior. Beyond establishing our approach to studying microswimmers, we demonstrate a unique ability to mechanically perturb cells via rapid acoustic positioning. 

The structure of the axoneme in motile cilia and flagella is emerging with increasing detail from high-resolution imaging, but the mechanism by which the axoneme creates oscillatory, propulsive motion remains mysterious. It has recently been proposed that this motion may be caused by a dynamic ‘flutter’ instability that can occur under steady dynein loading, and not by switching or modulation of dynein motor activity (as commonly assumed). In the current work, we have built an improved multi-filament mathematical model of the axoneme and implemented it as a system of discrete equations using the finite-element method. The eigenvalues and eigenvectors of this model predict the emergence of oscillatory, wave-like solutions in the absence of dynein regulation and specify the associated frequencies and waveforms of beating. Time-domain simulations with this model illustrate the behaviour predicted by the system's eigenvalues. This model and analysis allow us to efficiently explore the potential effects of difficult to measure biophysical parameters, such as elasticity of radial spokes and inter-doublet links, on the ciliary waveform. These results support the idea that dynamic instability without dynamic dynein regulation is a plausible and robust mechanism for generating ciliary beating. 

Motile cilia beat with an asymmetric waveform consisting of a power stroke that generates a propulsive force and a recovery stroke that returns the cilium back to the start. Cilia are anchored to the cell cortex by basal bodies (BBs) that are directly coupled to the ciliary doublet microtubules (MTs). We find that, consistent with ciliary forces imposing on BBs, bending patterns in BB triplet MTs are responsive to ciliary beating. BB bending varies as environmental conditions change the ciliary waveform. Bending occurs where striated fibers (SFs) attach to BBs and mutants with short SFs that fail to connect to adjacent BBs exhibit abnormal BB bending, supporting a model in which SFs couple ciliary forces between BBs. Finally, loss of the BB stability protein Poc1, which helps interconnect BB triplet MTs, prevents the normal distributed BB and ciliary bending patterns. Collectively, BBs experience ciliary forces and manage mechanical coupling of these forces to their surrounding cellular architecture for normal ciliary beating. 

Motile primary cilia in pancreatic islets control insulin secretion through glucose-controlled movement. 

Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell’s cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light (DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces. 

Acoustic microfluidics has emerged as a versatile solution for particle manipulation in medicine and biology. However, current technologies are largely confined to specialized research laboratories. The translation of acoustofluidics from research to clinical and industrial settings requires improved consistency and repeatability across different platforms. Performance comparisons will require straightforward experimental assessment tools that are not yet available. We introduce a method for characterizing acoustofluidic devices in real-time by exploiting the capacity of swimming microorganisms to respond to changes in their environment. The unicellular alga, Chlamydomonas reinhardtii , is used as an active probe to visualize the evolving acoustic pressure field within microfluidic channels and chambers. In contrast to more familiar mammalian cells, C. reinhardtii are simple to prepare and maintain, and exhibit a relatively uniform size distribution that more closely resembles calibration particles; however, unlike passive particles, these motile cells naturally fill complex chamber geometries and redistribute when the acoustic field changes or is turned off. In this way, C. reinhardtii cells offer greater flexibility than conventional polymer or glass calibration beads for in situ determination of device operating characteristics. To illustrate the technique, the varying spatial density and distribution of swimming cells are correlated to the acoustic potential to automatically locate device resonances within a specified frequency range. Peaks in the correlation coefficient of successive images not only identify the resonant frequencies for various geometries, but the peak shape can be related to the relative strength of the resonances. Qualitative mapping of the acoustic field strength with increasing voltage amplitude is also shown. Thus, we demonstrate that dynamically responsive C. reinhardtii enable real-time measurement and continuous monitoring of acoustofluidic device performance. 

Abstract This paper describes the propagation of shear waves in a Holzapfel–Gasser–Ogden (HGO) material and investigates the potential of magnetic resonance elastography (MRE) for estimating parameters of the HGO material model from experimental data. In most MRE studies the behavior of the material is assumed to be governed by linear, isotropic elasticity or viscoelasticity. In contrast, biological tissue is often nonlinear and anisotropic with a fibrous structure. In such materials, application of a quasi-static deformation (predeformation) plays an important role in shear wave propagation. Closed form expressions for shear wave speeds in an HGO material with a single family of fibers were found in a reference (undeformed) configuration and after imposed predeformations. These analytical expressions show that shear wave speeds are affected by the parameters (μ0, k1, k2, κ) of the HGO model and by the direction and amplitude of the predeformations. Simulations of corresponding finite element (FE) models confirm the predicted influence of HGO model parameters on speeds of shear waves with specific polarization and propagation directions. Importantly, the dependence of wave speeds on the parameters of the HGO model and imposed deformations could ultimately allow the noninvasive estimation of material parameters in vivo from experimental shear wave image data. 

Abstract Magnetic resonance elastography (MRE) has emerged as a sensitive imaging technique capable of providing a quantitative understanding of neural microstructural integrity. However, a reliable method for the quantification of the anisotropic mechanical properties of human white matter is currently lacking, despite the potential to illuminate the pathophysiology behind neurological disorders and traumatic brain injury. In this study, we examine the use of multiple excitations in MRE to generate wave displacement data sufficient for anisotropic inversion in white matter. We show the presence of multiple unique waves from each excitation which we combine to solve for parameters of an incompressible, transversely isotropic (ITI) material: shear modulus, μ, shear anisotropy, ϕ, and tensile anisotropy, ζ. We calculate these anisotropic parameters in the corpus callosum body and find the mean values as μ = 3.78 kPa, ϕ = 0.151, and ζ = 0.099 (at 50 Hz vibration frequency). This study demonstrates that multi-excitation MRE provides displacement data sufficient for the evaluation of the anisotropic properties of white matter. 

Abstract This paper describes a new method for estimating anisotropic mechanical properties of fibrous soft tissue by imaging shear waves induced by focused ultrasound (FUS) and analyzing their direction-dependent speeds. Fibrous materials with a single, dominant fiber direction may exhibit anisotropy in both shear and tensile moduli, reflecting differences in the response of the material when loads are applied in different directions. The speeds of shear waves in such materials depend on the propagation and polarization directions of the waves relative to the dominant fiber direction. In this study, shear waves were induced in muscle tissue (chicken breast) ex vivo by harmonically oscillating the amplitude of an ultrasound beam focused in a cylindrical tissue sample. The orientation of the fiber direction relative to the excitation direction was varied by rotating the sample. Magnetic resonance elastography (MRE) was used to visualize and measure the full 3D displacement field due to the ultrasound-induced shear waves. The phase gradient (PG) of radially propagating “slow” and “fast” shear waves provided local estimates of their respective wave speeds and directions. The equations for the speeds of these waves in an incompressible, transversely isotropic (TI), linear elastic material were fitted to measurements to estimate the shear and tensile moduli of the material. The combination of focused ultrasound and MR imaging allows noninvasive, but comprehensive, characterization of anisotropic soft tissue.