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Since its initial development in 1976, fluorescence recovery after photobleaching (FRAP) has been one of the most popular tools for studying diffusion and protein dynamics in living cells. Its popularity is derived from the widespread availability of confocal microscopes and the relative ease of the experiment and analysis. FRAP, however, is limited in its ability to resolve spatial heterogeneity. Here, we combine selective plane illumination microscopy (SPIM) and FRAP to create SPIM-FRAP, wherein we use a sheet of light to bleach a 2D plane and subsequently image the recovery of the same image plane. This provides simultaneous quantification of diffusion or protein recovery for every pixel in a given 2D slice, thus moving FRAP measurements beyond these previous limitations. We demonstrate this technique by mapping intranuclear diffusion of NLS-GFP in live MDA-MB-231 cells; SPIM-FRAP proves to be an order of magnitude faster than fluorescence correlation spectroscopy (FCS) based techniques for such measurements. We observe large length-scale (> ~500 nm) heterogeneity in the recovery times of NLS-GFP, which is validated against simulated data sets. 2D maps of recovery times were correlated with fluorescence images of H2B to address conflicting literature on the role of chromatin in diffusion of small molecules. We observed no correlation between histone density and diffusion. We developed a diffusion simulation for our SPIM-FRAP experiments to compare across techniques; our measured diffusion coefficients are on the order of previously reported results, thus validating the quantitative accuracy of SPIM-FRAP relative to well-established methods. With the recent rise of accessibility of SPIM systems, SPIM-FRAP is set to provide a simple and quick means of quantifying the spatial distribution of protein recovery or diffusion in living cells.Statement of SignificanceWe developed selective plane illumination microscopy combined with fluorescence recovery after photobleaching (SPIM-FRAP) to perform simultaneous FRAP measurements for each pixel in a 2D slice. This technique has the potential to be implemented on almost any light sheet microscope with minimal software development. FRAP studies were previously unable to resolve spatial heterogeneity and FCS techniques require minute-long acquisition times; SPIM-FRAP remedies both of these issues by generating FRAP-based diffusion maps in 4 seconds. This technique can easily be expanded to 3D by photobleaching a single plane and performing light sheet volumetric imaging, which has the benefits of minimal photobleaching and phototoxicity for studying long-term protein turnover. Furthermore, SPIM-FRAP of slowly-recovering structures enables characterization of spatial distortions to measure intracellular stresses.

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.

Nuclei are constantly under external stress – be it during migration through tight constrictions or compressive pressure by the actin cap – and the mechanical properties of nuclei govern their subsequent deformations. Both altered mechanical properties of nuclei and abnormal nuclear morphologies are hallmarks of a variety of disease states. Little work, however, has been done to link specific changes in nuclear shape to external forces. Here, we utilize a combined atomic force microscope and light sheet microscope (AFM-LS) to show SKOV3 nuclei exhibit a two-regime force response that correlates with changes in nuclear volume and surface area, allowing us to develop an empirical model of nuclear deformation. Our technique further decouples the roles of chromatin and lamin A/C in compression, showing they separately resist changes in nuclear volume and surface area respectively; this insight was not previously accessible by Hertzian analysis. A two-material finite element model supports our conclusions. We also observed that chromatin decompaction leads to lower nuclear curvature under compression, which is important for maintaining nuclear compartmentalization and function. The demonstrated link between specific types of nuclear morphological change and applied force will allow researchers to better understand the stress on nuclei throughout various biological processes.

Summary: At the cellular level, α-tubulin acetylation alters the structure of microtubules to render them mechanically resistant to compressive forces. How this biochemical property of microtubule acetylation relates to mechanosensation remains unknown, although prior studies have shown that microtubule acetylation influences touch perception. Here, we identify the major Drosophila α-tubulin acetylase (dTAT) and show that it plays key roles in several forms of mechanosensation. dTAT is highly expressed in the larval peripheral nervous system (PNS), but it is largely dispensable for neuronal morphogenesis. Mutation of the acetylase gene or the K40 acetylation site in α-tubulin impairs mechanical sensitivity in sensory neurons and behavioral responses to gentle touch, harsh touch, gravity, and vibration stimuli, but not noxious thermal stimulus. Finally, we show that dTAT is required for mechanically induced activation of NOMPC, a microtubule-associated transient receptor potential channel, and functions to maintain integrity of the microtubule cytoskeleton in response to mechanical stimulation. : Yan et al. identify the major microtubule acetylase in Drosophila and show that the enzyme and microtubule acetylation broadly control mechanosensation, but not other sensory modalities. Acetylation is required for mechanosensation by the TRP channel NOMPC, and possibly other channels, by virtue of its effects on microtubule mechanical stability and/or dynamics. Keywords: Drosophila, mechanosensation, microtubule acetylation, TRP channel, somatosensory neuron

It is increasingly appreciated that the cell nucleus is not only a home for DNA but also a complex material that resists physical deformations and dynamically responds to external mechanical cues. The molecules that confer mechanical properties to nuclei certainly contribute to laminopathies and possibly contribute to cellular mechanotransduction and physical processes in cancer such as metastasis. Studying nuclear mechanics and the downstream biochemical consequences or their modulation requires a suite of complex assays for applying, measuring, and visualizing mechanical forces across diverse length, time, and force scales. Here, we review the current methods in nuclear mechanics and mechanobiology, placing specific emphasis on each of their unique advantages and limitations. Furthermore, we explore important considerations in selecting a new methodology as are demonstrated by recent examples from the literature. We conclude by providing an outlook on the development of new methods and the judicious use of the current techniques for continued exploration into the role of nuclear mechanobiology.

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 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.

A novel chromosome simulator recapitulates the position and dynamics of centromeric chromatin in a model composed of cross-linked intramolecular loops. Simulations reveal that chromatin loops stiffen the centromere and dictate the distribution of pericentric cohesin., ChromoShake is a three-dimensional simulator designed to find the thermodynamically favored states for given chromosome geometries. The simulator has been applied to a geometric model based on experimentally determined positions and fluctuations of DNA and the distribution of cohesin and condensin in the budding yeast centromere. Simulations of chromatin in differing initial configurations reveal novel principles for understanding the structure and function of a eukaryotic centromere. The entropic position of DNA loops mirrors their experimental position, consistent with their radial displacement from the spindle axis. The barrel-like distribution of cohesin complexes surrounding the central spindle in metaphase is a consequence of the size of the DNA loops within the pericentromere to which cohesin is bound. Linkage between DNA loops of different centromeres is requisite to recapitulate experimentally determined correlations in DNA motion. The consequences of radial loops and cohesin and condensin binding are to stiffen the DNA along the spindle axis, imparting an active function to the centromere in mitosis.

Here we describe development of a microfluidic viscometer based on arrays of magnetically actuated micro-posts. Quantitative viscosities over a range of three orders of magnitude were determined for samples of less than 20 μL. This represents the first demonstration of quantitative viscometry using driven flexible micropost arrays. Critical to the success of our system is a comprehensive analytical model that includes the mechanical and magnetic properties of the actuating posts, the optical readout, and fluid-structure interactions. We found that alterations of the actuator beat shape as parameterized by the dimensionless "sperm number" must be taken into account to determine the fluid properties from the measured actuator dynamics. Beyond our particular system, the model described here can provide dynamics predictions for a broad class of flexible microactuator designs. We also show how the model can guide the design of new arrays that expand the accessible range of measurements.

The ability to measure dynamic structural changes within a cell under applied load is essential for developing more accurate models of cell mechanics and mechanotransduction. Atomic force microscopy is a powerful tool for evaluating cell mechanics, but the dominant applied forces and sample strains are in the vertical direction, perpendicular to the imaging plane of standard fluorescence imaging. Here we report on a combined sideways imaging and vertical light sheet illumination system integrated with AFM. Our system enables high frame rate, low background imaging of subcellular structural dynamics in the vertical plane synchronized with AFM force data. Using our system for cell compression measurements, we correlated stiffening features in the force indentation data with onset of nuclear deformation revealed in the imaging data. In adhesion studies we were able to correlate detailed features in the force data during adhesive release events with strain at the membrane and within the nucleus.

The geometry and arrangement of DNA loops in the pericentric region of the budding yeast centromere create a DNA-based molecular shock absorber that serves as the basis for how tension is generated between sister centromeres in mitosis., The centromere is the DNA locus that dictates kinetochore formation and is visibly apparent as heterochromatin that bridges sister kinetochores in metaphase. Sister centromeres are compacted and held together by cohesin, condensin, and topoisomerase-mediated entanglements until all sister chromosomes bi-orient along the spindle apparatus. The establishment of tension between sister chromatids is essential for quenching a checkpoint kinase signal generated from kinetochores lacking microtubule attachment or tension. How the centromere chromatin spring is organized and functions as a tensiometer is largely unexplored. We have discovered that centromere chromatin loops generate an extensional/poleward force sufficient to release nucleosomes proximal to the spindle axis. This study describes how the physical consequences of DNA looping directly underlie the biological mechanism for sister centromere separation and the spring-like properties of the centromere in mitosis.

We developed VIEW-MOD (Versatile Illumination Engine With a Modular Optical Design): a compact, multi-modality microscope, which accommodates multiple illumination schemes including variable angle total internal reflection, point scanning and vertical/horizontal light sheet. This system allows combining and flexibly switching between different illuminations and imaging modes by employing three electrically tunable lenses and two fast-steering mirrors. This versatile optics design provides control of 6 degrees of freedom of the illumination source (3 translation, 2 tilt, and beam shape) plus the axial position of the imaging plane. We also developed standalone software with an easy-to-use GUI to calibrate and control the microscope. We demonstrate the applications of this system and software in biosensor imaging, optogenetics and fast 3D volume imaging. This system is ready to fit into complex imaging circumstances requiring precise control of illumination and detection paths, and has a broad scope of usability for a myriad of biological applications.

Fibrin fibers form the structural scaffold of blood clots. Thus, their mechanical properties are of central importance to understanding hemostasis and thrombotic disease. Recent studies have revealed that fibrin fibers are elastomeric despite their high degree of molecular ordering. These results have inspired a variety of molecular models for fibrin’s elasticity, ranging from reversible protein unfolding to rubber-like elasticity. An important property that has not been explored is the timescale of elastic recoil, a parameter that is critical for fibrin’s mechanical function and places a temporal constraint on molecular models of fiber elasticity. Using high-frame-rate imaging and atomic force microscopy-based nanomanipulation, we measured the recoil dynamics of individual fibrin fibers and found that the recoil was orders of magnitude faster than anticipated from models involving protein refolding. We also performed steered discrete molecular-dynamics simulations to investigate the molecular origins of the observed recoil. Our results point to the unstructured αC regions of the otherwise structured fibrin molecule as being responsible for the elastic recoil of the fibers.

Nanotechnology has been touted as the next ‘industrial revolution’ of our modern age. In order for successful research, development, and social discourses to take place in this field, education research is needed to inform the development of standards, course development, and workforce preparation. In addition, there is a growing need to educate citizens and students about risks, benefits, and social and ethical issues related to nanotechnology. This position paper describes the advancements that have been made in nanoscale science and nanotechnology, and the challenges that exist to educate students and the public about critical nanoscience concepts. This paper reviews the current research on nanotechnology education including curricula, educational programs, informal education, and teacher education. Furthermore, the unique risks, benefits and ethics of these unusual technological applications are described in relation to nanoeducation goals. Finally, we outline needed future research in the areas of na...

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.

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.

When a pathogen enters the body, the immune system releases small proteins that opsonize the foreign entity. This serves as a signal to macrophages to engulf the pathogen, a process known as phagocytosis. Depending on the type of opsonin covering the invasive species, phagocytosis can occur in structurally different ways. My research aimed to examine how different opsonin types and densities affect phagocytosis both qualitatively and quantitatively. Microspheres were used to replicate pathogens, and IgG antibody alongside iC3b complement were the opsonins studied. First, a Dose-Response curve was made to show it was possible to cover the bead with different amounts of opsonin, i.e. change the density of antibody or complement on the bead surface. Then, the distinctly opsonized beads were fed to cells and the effectiveness of phagocytosis was analyzed using two different techniques: time-lapse movies and 3D image reconstruction. Through the former method, it was quantitatively found that more densely opsonized beads were more effectively phagocytosed, and that cells transitioned through phagocytosis quicker with IgG covered beads. This may suggest that the actin dynamics and cytoskeleton remodeling occur faster through the IgG signaling pathway than through that of iC3b. Progress was made through the 3D image reconstruction to better observe the phagocytic cup structure formed by the cell and to more efficiently note a difference between iC3b and IgG consumption in a qualitative sense. Phagocytic studies can supply useful information for drug treatments involving passage of the drug through the cell membrane to reach its activation site. My research showed that opsonin density and type affect phagocytosis tremendously.

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 μm, 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.

Fibrin polymerizes into the fibrous network that is the major structural component of blood clots and thrombi. We demonstrate that fibrin from three different species can also spontaneously polymerize into extensive, molecularly thin, 2D sheets. Sheet assembly occurs in physiologic buffers on both hydrophobic and hydrophilic surfaces, but is routinely observed only when polymerized using very low concentrations of fibrinogen and thrombin. Sheets may have been missed in previous studies because they may be very short-lived at higher concentrations of fibrinogen and thrombin, and their thinness makes them very difficult to detect. We were able to distinguish fluorescently labeled fibrin sheets by polymerizing fibrin onto micro-patterned structured surfaces that suspended polymers 10 μm above and parallel to the cover-glass surface. We used a combined fluorescence/atomic force microscope system to determine that sheets were ≈5 nm thick, flat, elastic and mechanically continuous. Video microscopy of assembling sheets showed that they could polymerize across 25-μm channels at hundreds of μm 2 /sec (≈10 13 subunits/s·M), an apparent rate constant many times greater than those of other protein polymers. Structural transitions from sheets to fibers were observed by fluorescence, transmission, and scanning electron microscopy. Sheets appeared to fold and roll up into larger fibers, and also to develop oval holes to form fiber networks that were “pre-attached” to the substrate and other fibers. We propose a model of fiber formation from sheets and compare it with current models of end-wise polymerization from protofibrils. Sheets could be an unanticipated factor in clot formation and adhesion in vivo , and are a unique material in their own right.

Nanoscale electronics seeks to decrease the critical dimension of devices in order to improve performance while reducing power consumption. Single-walled carbon nanotubes fit well with this strategy because, in addition to their molecular size, they demonstrate a number of unique electronic, mechanical and electromechanical properties. In particular, theory1,2,3,4,5,6,7,8 predicts that strain can have a large effect on the band structure of a nanotube, which, in turn, has an influence on its electron transport properties. This has been demonstrated in experiments where axial strain was applied by a scanning probe9,10,11,12. Theory also predicts that torsional strain can influence transport properties, which was observed recently in multiwalled nanotubes13. Here we present the first experimental evidence of an electromechanical effect from torsional strain in single-walled nanotubes, and also the first measurements of piezoresistive response in a self-contained nanotube-based nanoelectromechanical structure.

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.

We present a novel technology for microfluidic elastometry and demonstrate its ability to measure stiffness of blood clots as they form. A disposable micro-capillary strip draws small volumes (20 μL) of whole blood into a chamber containing a surface-mounted micropost array. The posts are magnetically actuated, thereby applying a shear stress to the blood clot. The posts' response to magnetic field changes as the blood clot forms; this response is measured by optical transmission. We show that a quasi-static model correctly predicts the torque applied to the microposts. We experimentally validate the ability of the system to measure clot stiffness by correlating our system with a commercial thromboelastograph. We conclude that actuated surface-attached post (ASAP) technology addresses a clinical need for point-of-care and small-volume elastic haemostatic assays.

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.

Fibrin fibers form the structural backbone of blood clots; fibrinolysis is the process in which plasmin digests fibrin fibers, effectively regulating the size and duration of a clot. To understand blood clot dissolution, the influence of clot structure and fiber properties must be separated from the effects of enzyme kinetics and perfusion rates into clots. Using an inverted optical microscope and fluorescently-labeled fibers suspended between micropatterned ridges, we have directly measured the lysis of individual fibrin fibers. We found that during lysis 64 ± 6% of fibers were transected at one point, but 29 ± 3% of fibers increase in length rather than dissolving or being transected. Thrombin and plasmin dose-response experiments showed that the elongation behavior was independent of plasmin concentration, but was instead dependent on the concentration of thrombin used during fiber polymerization, which correlated inversely with fiber diameter. Thinner fibers were more likely to lyse, while fibers greater than 200 ± 30 nm in diameter were more likely to elongate. Because lysis rates were greatly reduced in elongated fibers, we hypothesize that plasmin activity depends on fiber strain. Using polymer physics- and continuum mechanics-based mathematical models, we show that fibers polymerize in a strained state and that thicker fibers lose their prestrain more rapidly than thinner fibers during lysis, which may explain why thick fibers elongate and thin fibers lyse. These results highlight how subtle differences in the diameter and prestrain of fibers could lead to dramatically different lytic susceptibilities.

We present here the use of amino-terminated DNA strands in functionalizing the open ends and defect sites of oxidatively prepared single-walled carbon nanotubes, an important first step in realizing a DNA-guided self-assembly process for carbon nanotubes.

We report on experiments in which multiwall carbon nanotubes (CNTs) are manipulated with AFM on a graphite (HOPG) substrate. We find certain discrete orientations in which the lateral force of manipulation dramatically increases as we rotate the CNT in the plane of the HOPG surface with the AFM tip. The three-fold symmetry of these discrete orientations indicates commensurate contact of the hexagonal graphene surfaces of the HOPG and CNT. As the CNT moves into commensurate contact, we observe the motion change from sliding/rotating in-plane to stick–roll motion.

In this overview, we will give an introduction to experiments in which manipulation is used a means of uncovering the intrinsic response and dynamical behavior of small objects. Experiments done on individual particles reveal new and rich behaviors that are inaccessible to averaging methods. Experiments exploring the stiffness and toughness of carbon nanotubes will be presented showing that nanometer scale engineered materials can far outperform current engineering materials. Through AFM manipulation, imaging and force measurements, the stiffness of this material was found to equal or exceed diamond. Their toughness is also extraordinary. Due to their near crystalline perfection, carbon nanotubes are able to undergo strains exceeding 15% during bending without damage. Through AFM manipulation experiments, these large deformations have been shown to be highly reversible. Experiments in which the lateral force of manipulation of small objects across surfaces is measured show that friction at the nanometer scale occurs without wear processes and is an intrinsic property of the particular interface. Results are also presented showing anisotropic behavior in friction and movement due to commensurate lattice effects. At the nanometer scale, the contacting surfaces can be nearly perfect so that commensurate effects are not partially averaged out by many differently oriented domains. It has been shown that friction can very over an order of magnitude depending on the relative orientation of the contacting surfaces. The relative orientation of object and substrate lattices also can determine the modes of motion. In some cases the particle is confined to move in one direction. In other cases the relative orientation determines whether the particle rolls, rotates in-plane or slides. These effects may have implications on the fundamental mechanisms of friction. They provide a laboratory for testing different geometrical configurations of atoms sliding on atoms. The results may also have implications in the design of nanometer scale electromechanical mechanisms.

The mechanical properties of blood clots are of central importance to hemostasis, thrombosis and embolism. Fibrin fiber networks are the major structural constituent of clots, and numerous studies dating back several decades have characterized their macroscopic viscoelastic properties [1, 2]. The fiber-level and molecular details giving rise to these properties have not been established, however. The correlation between mechanical properties and amino acid sequence is critical for a predictive understanding of the role of genetic defects in clot pathologies. To address this issue, we have developed a nanomanipulation technique for evaluating individual fibrin fibers. It consists of a combination fluorescence/atomic force microscope system that permits viewing of fiber deformation simultaneous with quantitative strain data. Recently we and our colleagues reported on the high extensibility of individual human fibrin fibers [3], with extensibility (or strain at breaking) of some fibers exceeding 300%, and elastic recovery with strains of up to 180%. This places human fibrin among the most extensible protein polymers, exceeding elastin and resilin in extensibility [4]. Here we test the hypothesis that the majority of the strain is taken up by the tandem repeat segment of the flexible αC region of fibrin. Our study focused on this portion of the protein by mechanically evaluating fibrins with varying lengths of the tandem repeat segment. Using our integrated nanomanipulation system, we stretched individual fibrin fibers made of human, mouse and chicken fibrinogen which have long, intermediate and zero length tandem repeat segments respectively. We found that extensibility correlated with the lengths of the tandem repeat segments. The fibrin monomer is 45 nm long and consists of three pairs of polypeptides (α, β, γ) joined through over two dozen disulfide bridges (for review see [5]) . The monomer has two globular regions (D) on the ends of the molecule joined to a smaller central globular region (E) through a triple coiled-coil (Fig. 1A). The crystal structure for the majority of the fibrinogen molecule has been solved [6]. However, roughly 2/3 of the α chain on the C-terminal end known as the “αC region” (α221-610 in human), has defied definitive structural characterization. These αC regions have been shown to interact and to facilitate fiber formation, and have been implicated in the mechanical performance of clots [7, 8]. What has received less attention is the question of how this portion of the fibrin molecule operates as a mechanical element. The αC region has two distinct parts: the connector region (α221-391 in human) and the terminal region (α392-610 in human). Within the connector region are a series of tandem amino acid repeats. Both the number of repeats and the number of residues within the repeat differ greatly between species [9, 10]. We took advantage of this inherent variability to compare the extensibility of fibers with no tandem repeat segment (chicken), intermediate length (mouse) and long (human) repeat segment (Figure 1B). In human fibrinogen, the tandem repeat segment is 128 amino acids long (10 repeats of mostly 13), in mouse 60 (5 repeats of mostly 13) [10], and in chicken fibrinogen, is completely absent. Consequently, the contour length of the loosely tethered αC region is roughly 22 nm longer in mouse and 50 nm longer in human than in chicken fibrinogen. With two α chains per monomer, this would be a total difference in contour length of 44 nm and 100 nm per monomer. Figure 1 (A) Cartoon of fibrin molecule depicting released αC region. The thicker checked portion indicates the approximate location of the tandem repeat segment. (B) Schematics of the α chain for human, mouse and chicken. Solid black indicates ... To test the extensibility of individual fibrin fibers, we polymerized low concentrations of fibrinogen onto micropatterened corrugated surfaces of transparent ridges and channels such that fibrin fibers were suspended across the channels [3, 4]. We then used the combined AFM-fluorescence microscope to stretch the fibers to the point of failure while simultaneously monitoring deformation through video microscopy. Video of the extension was recorded for each pull, and analyzed to determine extensibility. The extensibility is simply the change in length of the fiber segment at the breaking point, divided by the original length (here presented as a percentage). An example stretching sequence is shown in panels C-F of Fig. 1. Our extensibility results are shown in panels G and H of Fig. 1. Extensibility for chicken fibers is 47 +/- 23 % (N=42); mouse: 187 +/- 44 % (N=89); human: 217 +/- 47 (N=75). The differences were significant for each pairing (mouse/human, p=0.0004; chicken/human, p < 0.0001; mouse/chicken, p< 0.0001). Thus the relatively low extensibility of chicken fibrin, and the intermediate values for mouse as compared to human fibers, correlate with the length of their corresponding connector regions. Along with the mechanical evidence, the primary structure of the repeat segment also suggests mechanical function. The segment is intriguingly reminiscent of repeat sequences in elastomeric proteins such as elastin, resilin, and spider-silk [11]. Though the sequence of the repeats varies in length and content among these proteins, they share some interesting similarities such an unusually high content of glycine and proline (21% and 11% respectively in human fibrin). These amino acids have been implicated as blockers of secondary structure and in high frequency promote amorphous or “natively unfolded” structure [12-14]. Natively unfolded protein domains have been shown to function as non-linear springs [15] as well as providing tethers for multiple interactions facilitated by lack of strict conformational constraints [16]. Fibrin is a very large protein (340 kDa) and has other domains that are candidates for conformational changes under strain. These include the two globular D regions at each end of the protein (the C terminal of the β and γ chains), and the coiled coil region. A recent force spectroscopy study on engineered oligomers of fibrinogen demonstrated that the unfolding of the coiled- coil region could account for a fraction of fibrin’s extensibility (up to strains of 100%) [17], while at the same time found no evidence for contribution from the globular D regions. Because that study used engineered, end-to-end linked oligomers, the contribution of the αC region was not addressed. Aside from the variant tandem repeat segment, there are other substantive differences in the primary structures of the three fibrins studied here, and these may influence the species-specific properties. Nevertheless, the similarity of the primary structure of the tandem repeat segment of the αC with other elastomeric proteins suggests it exhibits similar mechanical properties and may have a significant role in the extensibility of fibrin fibers, and ultimately the mechanics of fibrin clots.

In many cases in experimental science, the instrument interface becomes a limiting factor in the efficacy of carrying out unusual experiments or prevents the complete understanding of the acquired data. We have developed an advanced interface for scanning probe microscopy (SPM) that allows intuitive rendering of data sets and natural instrument control, all in real time. The interface, called the nanoManipulator, combines a high-performance graphics engine for real-time data rendering with a haptic interface that places the human operator directly into the feedback loop that controls surface manipulations. Using a hand-held stylus, the operator moves the stylus laterally, directing the movement of the SPM tip across the sample. The haptic interface enables the user to “feel” the surface by forcing the stylus to move up and down in response to the surface topography. In this way the user understands the immediate location of the tip on the sample and can quickly and precisely maneuver nanometer-scale objects. We have applied this interface to studies of the mechanical properties of nanotubes and to substrate-nanotube interactions. The mechanical properties of carbon nanotubes have been demonstrated to be extraordinary. They have an elastic modulus rivaling that of the stiffest material known, diamond, while maintaining a remarkable resistance to fracture. We have used atomic-force microscopy (AFM) to manipulate the nanotubes through a series of configuration that reveal buckling behavior and high-strain resilience. Nanotubes also serve as test objects for nanometer-scale contact mechanics. We have found that nanotubes will roll under certain conditions. This has been determined through changes in the images and through the acquisition of lateral force during manipulation. The lateral force data show periodic stick-slip behavior with a periodicity matching the perimeter of the nanotube.

We present our results on the manipulation of individual viruses using an advanced interface for atomic force microscopes (AFMs). We show that the viruses can be dissected, rotated, and translated with great facility. We interpret the behavior of tobacco mosaic virus with a mechanical model that makes explicit the competition between sample-substrate lateral friction and the flexural rigidity of the manipulated object. The manipulation behavior of tobacco mosaic virus on graphite is shown to be consistent with values of lateral friction observed on similar interfaces and the flexural rigidity expected for macromolecular assemblies. The ability to manipulate individual samples broadens the scope of possible studies by providing a means for positioning samples at specific binding sites or predefined measuring devices. The mechanical model provides a framework for interpreting quantitative measurements of virus binding and mechanical properties and for understanding the constraints on the successful, nondestructive AFM manipulation of delicate samples.

Motile cilia are hair-like organelles that beat to produce fluid flow. It is not fully understood how these incredibly complex molecular machines function at the protein level to produce complicated beat patterns. The mechanical properties of microtubule doublets, the main structural element of the cilium, have not been well studied. Here we show that doublet microtubules separated from human airway cilia curl up with an average radius of curvature of 1.39 +- 0.01 µm, determined from transmission electron and atomic force imaging. This result is surprising given that cytoplasmic microtubules have a persistence length on the order of millimeters, but is consistent with curvature seen in the literature. Doublet microtubules were isolated by digesting the axoneme with trypsin and inducing sliding by the addition of ATP. After sliding, transmission electron images showed that dynein motors were still attached, which is consistent with prior studies. We prepared samples with a fraction of the outer dynein arms removed using a 0.6M potassium chloride salt extraction. Removal of dynein was confirmed by SDS-PAGE. Samples with removed dynein had an increased radius of curvature, 2.3 +- 0.1 µm. The straightening of the doublet microtubules with the removal of dynein motors implies that the asymmetric distribution of dynein motors causes a stress that bends the doublet microtubule. Our results suggest an alternative source for curvature in the beating cilium. The distribution of dynein heads bound to a microtubule doublet is known to change dynamically during the cilium beat. Changing distribution of dynein heads bound to the microtubules could induce changes in the curvature of the cilium without the need for the motors to apply a shear force.

We present a new biosensing device which consists of an array of flexible high-aspect ratio magnetic structures cantilevered to a solid substrate. The structures are 25 x 2.5 micron cylindrical silicone rods which are encased with a shell of nickel; the silicone structure lends the rod flexibility while the nickel enables magnetic control. Fabrication begins in a 25-micron-thick porous polycarbonate track-etched membrane in which pore diameter is tuned by etching in a sodium hydroxide solution. A gold and aluminum working electrode is sputtercoated onto the back of the membrane, followed by electrodeposition of 100-200 nm of nickel onto the pore wall. This deposition forms a hollow nickel cylinder which is then filled with a poly(dimethyl siloxane) core. After curing, dichloromethane is used to dissolve the polycarbonate template, resulting in a magnetically active microrod array containing two million rods per square centimeter. Furthermore, we demonstrate that changes in the range of motion of the oscillation of the microrod array can be measured by monitoring optical transmission through the sample. This allows us to monitor the aggregation kinetics of the rod array or coagulation of an intervening fluid, leading to a generalized platform for biosensing applications.

We describe biophysical and ultrastructural differences in genome release from adeno-associated virus (AAV) capsids packaging wild-type DNA, recombinant single-stranded DNA (ssDNA), or dimeric, self-complementary DNA (scDNA) genomes. Atomic force microscopy and electron microscopy (EM) revealed that AAV particles release packaged genomes and undergo marked changes in capsid morphology upon heating in physiological buffer (pH 7.2). When different AAV capsids packaging ss/scDNA varying in length from 72 to 123% of wild-type DNA (3.4 to 5.8 kb) were incrementally heated, the proportion of uncoated AAV capsids decreased with genome length as observed by EM. Genome release was further characterized by a fluorimetric assay, which demonstrated that acidic pH and high osmotic pressure suppress genome release from AAV particles. In addition, fluorimetric analysis corroborated an inverse correlation between packaged genome length and the temperature needed to induce uncoating. Surprisingly, scAAV vectors required significantly higher temperatures to uncoat than their ssDNA-packaging counterparts. However, externalization of VP1 N termini appears to be unaffected by packaged genome length or self-complementarity. Further analysis by tungsten-shadowing EM revealed striking differences in the morphologies of ssDNA and scDNA genomes upon release from intact capsids. Computational modeling and molecular dynamics simulations suggest that the unusual thermal stability of scAAV vectors might arise from partial base pairing and optimal organization of packaged scDNA. Our work further defines the biophysical mechanisms underlying adeno-associated virus uncoating and genome release.

We present a quartz tuning-fork-based atomic force microscopy (AFM) setup that is capable of mapping the surface contact resistance while scanning topography. The tuning-fork setup allows us to use etched Pt/Ir tips, which have higher durability and better conductivity than probes used in earlier AFM conductance measurements. The performance of the method is demonstrated with contact resistance measurements of gold lines on silicon dioxide and carbon nanotubes on graphite.

We present a fast, high yield, low cost method for the production of scanning probes with aligned carbon nanotubes protruding from the ends. The procedure is described and images of undercut films are used to demonstrate the improved probe quality for topography measurements. A magnetophoretic model of the attachment and alignment processes is discussed.

We report on the precise placement of a single carbon nanotube (CNT) onto a microlectromechanial system (MEMS) structure. Using a hybrid atomic force microscope/scanning electron microscope (AFM/SEM) system, an individual multiwalled CNT was retrieved from a cartridge by the AFM tip, translated to a MEMS device, and then placed across a gap between an actuating and a stationary structure. Progress toward a resistance versus stress/strain measurement on a CNT will be discussed, including SEM images of a MEMS structure we have designed specifically for such a measurement. © 2002 American Institute of Physics.

Fibrin fibers, which form the mesh network structure of blood clots, display a hierarchical design. The fibrin molecule is a rotationally symmetric dimer consisting of two identical subunits, each composed of three nonidentical polypeptide chains: α, β, and γ. The central region (E), where the N-termini of all 6 chains are connected via disulfide bridges, is connected to two outer, beta-sheet rich regions (labeled the βC, and γC and collectively called the D region) by a triple-helix coiled coil. The C-terminal region of the alpha chain contains two distinct parts: the connector region (amino acids 221-391 in human) and the αC domain (a392-610 in human). Within the connector region are a series of tandem amino acid repeats, leading to a largely disordered structure, while the terminal region is thought to contain beta sheet structure. The fibrin monomer polymerizes into a half-staggered protofibril via specific interactions; these protofibrils then laterally aggregate to form a fiber.We recently published a report showing that the extensibility of fibrin fibers was directly related to the length of the alpha-C connector region across various species. Chicken fibers, which have no connector region, fail at strains of 47±23%, while human fibers fail at strains of >200%. To investigate this effect further, we have begun mechanical tests of two recombinant fibrin variants, Aα251, in which the αC connector region of the human fibrin molecule has been truncated at amino acid 251, and a human/chicken hybrid fibrin molecule consisting of the amino acids 1- 197 of the human Aα chain linked to residues 199-487 of chicken Aα chain. Preliminary results indicate that Aα251 has breaking strains similar to normal human fibers (185±46%) while the human/chicken hybrid has breaking strains similar to the chicken fibers (70±35%).

Fibrin fibers form the mechanical backbone of blood clots, stemming the flow of blood at the sights of vascular injury. The heart pumps blood at approximately 1Hz, requiring fibers within the vasculature to recover elastically within a second, or risk clot failure. Recent work on fibrin fiber mechanics showed that fibers are soft in stretching (1-10 MPa modulus), reversibly stretch to up to three times their original length and strain stiffen above 100% strain; however, fibrin fiber dynamics has received less attention. Using a combined AFM/Optical fluorescent microscope we have probed the recoil dynamics of fibrin fibers on the millisecond timescale. Fluorescently labeled fibers were suspended between channels and then stretched from the center with an AFM tip. Fibers were allowed to slip off the tip and fiber relaxation was measured optically at 3-11 ms time intervals. The data indicate two temporal regimes of fibrin dynamics: A fast initial regime (≤1ms) where the fiber snaps back into a loose conformation, and a slow regime on the order of 10's of ms, where the fiber regains a taut conformation. The two timescales of fiber relaxation indicate two different mechanisms. We find that the timescale of the slow regime is directly related to the strain of the fiber at the point of release. The fast timescale is likely governed by the entropic recoil of unstructured polypeptide chains, while the second timescale is likely governed by either protein folding or a macroscopic re-ordering of the fiber substructure. Further experiments will differentiate between these mechanisms, and the implications of these two regimes of fiber recovery will be discussed.