Combined Atomic Force Microscope and Volumetric Light Sheet System for Correlative Force and Fluorescence Mechanobiology Studies.

The central goals of mechanobiology are to understand how cells generate force and how they respond to environmental mechanical stimuli. A full picture of these processes requires high-resolution, volumetric imaging with time-correlated force measurements. Here we present an instrument that combines an open-top, single-objective light sheet fluorescence microscope with an atomic force microscope (AFM), providing simultaneous volumetric imaging with high spatiotemporal resolution and high dynamic range force capability (10 pN - 100 nN). With this system we have captured lysosome trafficking, vimentin nuclear caging, and actin dynamics on the order of one second per single-cell volume. To showcase the unique advantages of combining Line Bessel light sheet imaging with AFM, we measured the forces exerted by a macrophage during FcɣR-mediated phagocytosis while performing both sequential two-color, fixed plane and volumetric imaging of F-actin. This unique instrument allows for a myriad of novel studies investigating the coupling of cellular dynamics and mechanical forces.

Highly responsive core-shell microactuator arrays for use in viscous and viscoelastic fluids.

We present a new fabrication method to produce arrays of highly responsive polymer-metal core-shell magnetic microactuators. The core-shell fabrication method decouples the elastic and magnetic structural components such that the actuator response can be optimized by adjusting the core-shell geometry. Our microstructures are 10 µm long, 550 nm in diameter, and electrochemically fabricated in particle track-etched membranes, comprising a poly(dimethylsiloxane) core with a 100 nm Ni shell surrounding the upper 3-8 µm. The structures can achieve deflections of nearly 90° with moderate magnetic fields and are capable of driving fluid flow in a fluid 550 times more viscous than water.

Single particle tracking reveals biphasic transport during nanorod magnetophoresis through extracellular matrix.

Magnetic drug targeting has been proposed as a means of efficiently targeting drugs to tumors. However, the extracellular matrix (ECM) remains a significant barrier to long-range magnetophoretic transport through the tumor volume. While ensemble measurements of nanoparticle magnetophoresis have been reported, a single particle level understanding of magnetophoretic transport remains at large. We quantify nanorod magnetophoresis through ECM based on single particle observations. We find that smaller diameter particles achieve larger velocities through ECM despite experiencing smaller magnetic forces. Additionally, two interesting dynamics are elucidated. First, 18 nm diameter nanorods experience bimodal stick-slip motion through ECM during static field magnetophoresis, while similar bimodal transport is not observed for 55 nm nor 200 nm diameter nanorods. Second, smaller particles experience larger deviations in their orientation angle with respect to the magnetic field. This work elucidates important dynamics of nanoparticle transport through complex, porous biomaterials that may go unnoticed during ensemble measurements.

Nonlinear signatures of entangled polymer solutions in active microbead rheology.

We present experimental data and numerical modeling of a nonlinear phenomenon in active magnetic microbead rheology that appears to be common to entangled polymer solutions (EPS). Dynamic experiments in a modest range of magnetic forces show: 1. a short-lived high viscosity plateau, followed by 2. a bead acceleration phase with a sharp drop in apparent viscosity, and 3. a terminal steady state that we show resides on the shear-thinning slope of the steady-state flow curve from cone and plate data. This latter feature implies a new protocol to access the nonlinear steady-state flow curve for many biological EPS only available in microliter-scale volumes. We solve the moment-closure form of the Rolie-Poly kinetic model for EPS hydrodynamics, together with a decoupling approximation that obviates the need for a full 3D flow solver, and show that the model qualitatively reproduces the dynamic experimental sequence above. In this way, we explain the phenomenon in terms of entangled polymer physics, and show how the nonlinear event (acceleration and termination on the shear-thinning response curve) is tunable by the interplay between molecular-scale mechanisms (relaxation via reptation and chain retraction) and magnetic force controls. The experimental conditions mimic movement of cilia tips, bacteria, and sperm in mucus barriers, implying a physiological relevance of the phenomenon, and compelling further development of the fully coupled, 3D flow-microstructure model to achieve quantitative accuracy.

A Highly Tunable Silicone-Based Magnetic Elastomer with Nanoscale Homogeneity.

Magnetic elastomers have been widely pursued for sensing and actuation applications. Silicone-based magnetic elastomers have a number of advantages over other materials such as hydrogels, but aggregation of magnetic nanoparticles within silicones is difficult to prevent. Aggregation inherently limits the minimum size of fabricated structures and leads to non-uniform response from structure to structure. We have developed a novel material which is a complex of a silicone polymer (polydimethylsiloxane-co-aminopropylmethylsiloxane) adsorbed onto the surface of magnetite (γ-Fe(2)0(3)) nanoparticles 7-10 nm in diameter. The material is homogenous at very small length scales (< 100 nm) and can be crosslinked to form a flexible, magnetic material which is ideally suited for the fabrication of micro- to nanoscale magnetic actuators. The loading fraction of magnetic nanoparticles in the composite can be varied smoothly from 0 - 50% wt. without loss of homogeneity, providing a simple mechanism for tuning actuator response. We evaluate the material properties of the composite across a range of nanoparticle loading, and demonstrate a magnetic-field-induced increase in compressive modulus as high as 300%. Furthermore, we implement a strategy for predicting the optimal nanoparticle loading for magnetic actuation applications, and show that our predictions correlate well with experimental findings.

High accuracy FIONA-AFM hybrid imaging.

Multi-protein complexes are ubiquitous and play essential roles in many biological mechanisms. Single molecule imaging techniques such as electron microscopy (EM) and atomic force microscopy (AFM) are powerful methods for characterizing the structural properties of multi-protein and multi-protein-DNA complexes. However, a significant limitation to these techniques is the ability to distinguish different proteins from one another. Here, we combine high resolution fluorescence microscopy and AFM (FIONA-AFM) to allow the identification of different proteins in such complexes. Using quantum dots as fiducial markers in addition to fluorescently labeled proteins, we are able to align fluorescence and AFM information to ≥8nm accuracy. This accuracy is sufficient to identify individual fluorescently labeled proteins in most multi-protein complexes. We investigate the limitations of localization precision and accuracy in fluorescence and AFM images separately and their effects on the overall registration accuracy of FIONA-AFM hybrid images. This combination of the two orthogonal techniques (FIONA and AFM) opens a wide spectrum of possible applications to the study of protein interactions, because AFM can yield high resolution (5-10nm) information about the conformational properties of multi-protein complexes and the fluorescence can indicate spatial relationships of the proteins in the complexes.

Biomimetic cilia arrays generate simultaneous pumping and mixing regimes.

Living systems employ cilia to control and to sense the flow of fluids for many purposes, such as pumping, locomotion, feeding, and tissue morphogenesis. Beyond their use in biology, functional arrays of artificial cilia have been envisaged as a potential biomimetic strategy for inducing fluid flow and mixing in lab-on-a-chip devices. Here we report on fluid transport produced by magnetically actuated arrays of biomimetic cilia whose size approaches that of their biological counterparts, a scale at which advection and diffusion compete to determine mass transport. Our biomimetic cilia recreate the beat shape of embryonic nodal cilia, simultaneously generating two sharply segregated regimes of fluid flow: Above the cilia tips their motion causes directed, long-range fluid transport, whereas below the tips we show that the cilia beat generates an enhanced diffusivity capable of producing increased mixing rates. These two distinct types of flow occur simultaneously and are separated in space by less than 5 microm, approximately 20% of the biomimetic cilium length. While this suggests that our system may have applications as a versatile microfluidics device, we also focus on the biological implications of our findings. Our statistical analysis of particle transport identifying an enhanced diffusion regime provides novel evidence for the existence of mixing in ciliated systems, and we demonstrate that the directed transport regime is Poiseuille-Couette flow, the first analytical model consistent with biological measurements of fluid flow in the embryonic node.

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.

DNA relaxation dynamics as a probe for the intracellular environment.

Investigations into the biophysical properties of single molecules traditionally involve well defined in vitro systems where parameters such as solvent viscosity and applied forces are known a priori. These systems provide means to develop models describing the polymers response to a variety of conditions, including the entropically driven relaxation of a stretched biopolymer upon release of the tension inducing force. While these techniques have proven instrumental for recent advancements in the fields of polymer physics and biophysics, how applicable they are to life inside the cell remains poorly understood. Here we report an investigation of in vivo stretched polymer relaxation dynamics using chromatin relaxation following the breakage of a dicentric chromosome subjected to microtubule-based spindle forces. Additionally, we have developed an in vitro system used to verify the conformations observed during the in vivo relaxation, including the predicted but previously unidentified taut conformation. These observations motivate our use of existing polymer models to determine both the in vivo viscosity as seen by the relaxing chromatin and the tension force applied by the microtubule-based spindle in vivo. As a result, the technique described herein may be used as a biophysical strategy to probe the intranuclear environment.

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.

A comparison of the mechanical and structural properties of fibrin fibers with other protein fibers.

In the past few years a great deal of progress has been made in studying the mechanical and structural properties of biological protein fibers. Here, we compare and review the stiffness (Young's modulus, E) and breaking strain (also called rupture strain or extensibility, epsilon(max)) of numerous biological protein fibers in light of the recently reported mechanical properties of fibrin fibers. Emphasis is also placed on the structural features and molecular mechanisms that endow biological protein fibers with their respective mechanical properties. Generally, stiff biological protein fibers have a Young's modulus on the order of a few Gigapascal and are not very extensible (epsilon(max) < 20%). They also display a very regular arrangement of their monomeric units. Soft biological protein fibers have a Young's modulus on the order of a few Megapascal and are very extensible (epsilon(max) > 100%). These soft, extensible fibers employ a variety of molecular mechanisms, such as extending amorphous regions or unfolding protein domains, to accommodate large strains. We conclude our review by proposing a novel model of how fibrin fibers might achieve their extremely large extensibility, despite the regular arrangement of the monomeric fibrin units within a fiber. We propose that fibrin fibers accommodate large strains by two major mechanisms: (1) an alpha-helix to beta-strand conversion of the coiled coils; (2) a partial unfolding of the globular C-terminal domain of the gamma-chain.

Magnetically actuated nanorod arrays as biomimetic cilia.

We present a procedure for producing high-aspect-ratio cantilevered micro- and nanorod arrays of a PDMS-ferrofluid composite material. The rods have been produced with diameters ranging from 200 nm to 1 mum and aspect ratios as high as 125. We demonstrate actuation of these superparamagnetic rod arrays with an externally applied magnetic field from a permanent magnet and compare this actuation with a theoretical energy-minimization model. The structures produced by these methods may be useful in microfluidics, photonic, and sensing applications.

Experimental measurement of single-wall carbon nanotube torsional properties.

We report on the characterization of nanometer-scale torsional devices based on individual single-walled carbon nanotubes as the spring elements. The axial shear moduli of the nanotubes are obtained through modeling of device reaction to various amounts of applied electrostatic force and are compared to theoretical values.

Fibrin fibers have extraordinary extensibility and elasticity.

Blood clots perform an essential mechanical task, yet the mechanical behavior of fibrin fibers, which form the structural framework of a clot, is largely unknown. By using combined atomic force-fluorescence microscopy, we determined the elastic limit and extensibility of individual fibers. Fibrin fibers can be strained 180% (2.8-fold extension) without sustaining permanent lengthening, and they can be strained up to 525% (average 330%) before rupturing. This is the largest extensibility observed for protein fibers. The data imply that fibrin monomers must be able to undergo sizeable, reversible structural changes and that deformations in clots can be accommodated by individual fiber stretching.

Thin-foil magnetic force system for high-numerical-aperture microscopy.

Forces play a key role in a wide range of biological phenomena from single-protein conformational dynamics to transcription and cell division, to name a few. The majority of existing microbiological force application methods can be divided into two categories: those that can apply relatively high forces through the use of a physical connection to a probe and those that apply smaller forces with a detached probe. Existing magnetic manipulators utilizing high fields and high field gradients have been able to reduce this gap in maximum applicable force, but the size of such devices has limited their use in applications where high force and high-numerical-aperture (NA) microscopy must be combined. We have developed a magnetic manipulation system that is capable of applying forces in excess of 700 pN on a 1 mum paramagnetic particle and 13 nN on a 4.5 mum paramagnetic particle, forces over the full 4pi sr, and a bandwidth in excess of 3 kHz while remaining compatible with a commercially available high-NA microscope objective. Our system design separates the pole tips from the flux coils so that the magnetic-field geometry at the sample is determined by removable thin-foil pole plates, allowing easy change from experiment to experiment. In addition, we have combined the magnetic manipulator with a feedback-enhanced, high-resolution (2.4 nm), high-bandwidth (10 kHz), long-range (100 mum xyz range) laser tracking system. We demonstrate the usefulness of this system in a study of the role of forces in higher-order chromosome structure and function.

Resonant oscillators with carbon-nanotube torsion springs.

We report on the characterization of nanometer-scale resonators. Each device incorporates one multiwalled carbon nanotube (MWNT) as a torsional spring. The devices are actuated electrostatically, and their deflections, both low frequency and on resonance, are detected optically. These are some of the smallest electromechanical devices ever created and are a demonstration of practical integrated MWNT-based oscillators. The results also show surprising intershell mechanical coupling behavior in the MWNTs.

Visualization and mechanical manipulations of individual fibrin fibers suggest that fiber cross section has fractal dimension 1.3.

We report protocols and techniques to image and mechanically manipulate individual fibrin fibers, which are key structural components of blood clots. Using atomic force microscopy-based lateral force manipulations we determined the rupture force, FR, f fibrin fibers as a function of their diameter, D, in ambient conditions. As expected, the rupture force increases with increasing diameter; however, somewhat unexpectedly, it increases as FR approximately D1.30+/-0.06. Moreover, using a combined atomic force microscopy-fluorescence microscopy instrument, we determined the light intensity, I, of single fibers, that were formed with fluorescently labeled fibrinogen, as a function of their diameter, D. Similar to the force data, we found that the light intensity, and thus the number of molecules per cross section, increases as I approximately D1.25+/-0.11. Based on these findings we propose that fibrin fibers are fractals for which the number of molecules per cross section increases as about D1.3. This implies that the molecule density varies as rhoD approximately D -0.7, i.e., thinner fibers are denser than thicker fibers. Such a model would be consistent with the observation that fibrin fibers consist of 70-80% water and only 20-30% protein, which also suggests that fibrin fibers are very porous.

Torsional response and stiffening of individual multiwalled carbon nanotubes.

We report on the characterization of torsional oscillators which use multiwalled carbon nanotubes as the spring elements. Through atomic-force-microscope force-distance measurements we are able to apply torsional strains to the nanotubes and measure their torsional spring constants, and estimate their effective shear moduli. The data show that the nanotubes are stiffened by repeated flexing. We speculate that changes in the intershell mechanical coupling are responsible for the stiffening.

Tunable resistance of a carbon nanotube-graphite interface.

The transfer of electrons from one material to another is usually described in terms of energy conservation, with no attention being paid to momentum conservation. Here we present results on the junction resistance between a carbon nanotube and a graphite substrate and show that details of momentum conservation also can change the contact resistance. By changing the angular alignment of the atomic lattices, we found that contact resistance varied by more than an order of magnitude in a controlled and reproducible fashion, indicating that momentum conservation, in addition to energy conservation, can dictate the junction resistance in graphene systems such as carbon nanotube junctions and devices.