A survey of physical methods for studying nuclear mechanics and mechanobiology

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

Precollege nanotechnology education: A different kind of thinking

The introduction of nanotechnology education into K-12 education has happened so quickly that there has been little time to evaluate the approaches and knowledge goals that are most effective to teach precollege students. This review of nanotechnology education examines the instructional approaches and types of knowledge that frame nanotechnology precollege education. Methods used to teach different forms of knowledge are examined in light of the goal of creating effective and meaningful instruction. The developmental components needed to understand concepts such as surface area to volume relationships as well as the counterintuitive behavior of nanoscale materials are described. Instructional methods used in precollege nanotechnology education and the levels at which different nanoscale topics are introduced is presented and critiqued. Suggestions are made for the development of new nanotechnology educational programs that are developmental, sequenced, and meaningful

Lithographically Defined Micropost Arrays for Programmable Actuation and Interfacial Hydrodynamics

Magnetically actuating surface-attached post (ASAP) arrays have great potential in microfluidic flow control, including mixing and pumping. Both passive (nonactuating) and active (actuating) micropillar arrays can also be used to control pressure-driven flow and the motion of microscopic particles carried by the fluid through microfluidic channels. Molding techniques are popular for generating these microstructures. However, fabricating high aspect ratio elastomeric microstructures over large surface areas suffers from practical problems such as damage incurred in the demolding process. Here, we report on a fabrication protocol that generates ASAP with an aspect ratio as high as 23:1 and a cross-sectional area less than 1 μm2 using straightforward photolithography processes. We generated 50 unique ASAP arrays, each occupying an area of 1 mm2 on a silicon mold; these arrays have varied cross-sectional shape and size, aspect ratio, and spacings between neighboring posts. Our protocol also controls the level of magnetic material in the ASAP tips with a centrifugation step. Using a herringbone pattern ASAP array, we have demonstrated control over the relative phase of actuation between neighboring posts. Such ASAP serve as an experimental platform to test current models predicting that reciprocal actuators in close proximity can successfully drive flow in a low Reynolds (Re) number environment

Blister resistant targets for nuclear reaction experiments with α-particle beams

Solid targets for nuclear measurements that use α-particle beams commonly experience a form of degradation known as blistering. The effect can prevent the use of solid targets for high intensity α-particle experiments, often necessitating complex gas target systems. To combat this problem, three different blister resistant target backings were designed for use in direct reaction measurements with high intensity α-particle beams. The blister resistant target designs utilize gas diffusive properties of fused silica, sintered metal, and porous evaporated metal. Each target was implanted with 22 Ne ions and bombarded with α-particle beam to test blister resistance. Targets were characterized and monitored using the 22 Ne(p,γ) 23 Na reaction to determine the degradation of implanted material, and compare them to typical implanted noble gas targets. We find that all targets studied exhibit resistance to blistering, with the porous evaporated metal targets displaying the least amount of target material degradation

Combined Selective Plane Illumination Microscopy and FRAP Maps Intranuclear Diffusion of NLS-GFP

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 two-dimensional (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 both intranuclear diffusion of NLS-GFP and recovery of 53BP1-mCherry, a marker for DNA damage, in live MDA-MB-231 cells. SPIM-FRAP proves to be an order of magnitude faster than fluorescence-correlation-spectroscopy-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 NLS-GFP recovery times showed no pixel-by-pixel correlation with histone density, although slower diffusion was observed in nucleoli. Additionally, recovery of 53BP1-mCherry was observed to be slowed at sites of DNA damage. We finally 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 straightforward means of quantifying the spatial distribution of protein recovery or diffusion in living cells

Gearlike rolling motion mediated by commensurate contact: Carbon nanotubes on HOPG

We report on experiments in which multiwall carbon nanotubes (CNT's) are manipulated with atomic force microscopy (AFM) on a graphite highly oriented pyrolytic 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 threefold 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

Thermally actuated untethered impact-driven locomotive microdevices

The authors have developed steerable locomotive devices as small as 30 μm using the inertial impact drive as the thrust method. The devices consist of three-legged, thin-metal-film bimorphs designed to rest on three sharp tips with the device body curved up off the surface. Rapid, thermally induced curvature of one leg leads to stepwise translation on a low friction surface. A focused laser was used to supply energy and its parameters controlled the velocity and direction of motion of the device

Flow profoundly influences fibrin network structure: Implications for fibrin formation and clot stability in haemostasis

Dear Sirs, Haemostasis requires fibrinogen conversion to fibrin and formation of a stable fibrin network. Fibrin network properties, including fibre thickness, branchpoint density, fibre density, mechanical stability, porosity, and resistance to lysis can differentiate plasma clots of healthy individuals from those with haemostatic or thrombotic disorders. Plasma from patients with a bleeding history produces thick, minimally-branched fibres in coarse, deformable networks that are highly susceptible to lysis, whereas plasma from patients with a personal or family history of thrombosis produces thin, highly-branched fibres in impermeable, rigid networks that are relatively resistant to fibrinolysi

Length of tandem repeats in fibrin's αC region correlates with fiber extensibility

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. 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, 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. 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

Correlating nuclear morphology and external force with combined atomic force microscopy and light sheet imaging separates roles of chromatin and lamin A/C in nuclear mechanics

Nuclei are often 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 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