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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.

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.

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.

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.

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.

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.

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.

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.Whenhumanwhole 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.

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.

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 fibrinolysis

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.