The random walker's toolbox for analyzing single-particle tracking data.

Technological advances and a burst of new microscopy methods have boosted the use of quantitative tracking experiments, in Soft Matter and Biological Physics but also in the Life Sciences. However, in contrast to highly advanced measurement techniques and tracking tools, subsequent analyses of trajectories frequently do not exploit the data's full potential. Aiming especially at experimental laboratories and early-career scientists, we introduce, discuss, and apply in this Tutorial Review a large set of versatile measures that have proven to be useful for analyzing trajectories from single-particle tracking experiments, beyond a simple extraction of diffusion constants from mean squared displacements. To support a direct test and application of these measures, we supplement the text with a download package that comprises a low-threshold toolbox of ready-to-use routines and training data sets, hence relaxing the need to develop home-brewed solutions and/or to create suitable benchmark data.

DNA damage alters nuclear mechanics through chromatin reorganization.

DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

Thermoresponsive Water Transportation in Dually Electrostatically Crosslinked Nanocomposite Hydrogels.

Controlling water transportation within hydrogels makes hydrogels attractive for diverse applications, but it is still a very challenging task. Herein, a novel type of dually electrostatically crosslinked nanocomposite hydrogel showing thermoresponsive water absorption, distribution, and dehydration processes are developed. The nanocomposite hydrogels are stabilized via electrostatic interactions between negatively charged poly(acrylic acid) and positively charged layered double hydroxide (LDH) nanosheets as well as poly(3-acrylamidopropyltrimethylammonium chloride). Both LDH nanosheets as crosslinkers and the surrounding temperatures played pivotal roles in tuning the water transportation within these nanocomposite hydrogels. By changing the surrounding temperature from 60 to 4 °C, these hydrogels showed widely adjustable swelling times between 2 and 45 days, while the dehydration process lasted between 7 and 27 days. A swift temperature decrease, for example, from 60 to 25 °C, generated supersaturation within these nanocomposite hydrogels, which further retarded the water transportation and distribution in hydrogel networks. Benefiting from modified water transportation and rapidly alternating water uptake capability during temperature change, pre-loaded compounds can be used to track and visualize these processes within nanocomposite hydrogels. At the same time, the discharge of water and loaded compounds from the interior of hydrogels demonstrates a thermoresponsive sustained release process.

Topology Counts: Force Distributions in Circular Spring Networks.

Filamentous polymer networks govern the mechanical properties of many biological materials. Force distributions within these networks are typically highly inhomogeneous, and, although the importance of force distributions for structural properties is well recognized, they are far from being understood quantitatively. Using a combination of probabilistic and graph-theoretical techniques, we derive force distributions in a model system consisting of ensembles of random linear spring networks on a circle. We show that characteristic quantities, such as the mean and variance of the force supported by individual springs, can be derived explicitly in terms of only two parameters: (i) average connectivity and (ii) number of nodes. Our analysis shows that a classical mean-field approach fails to capture these characteristic quantities correctly. In contrast, we demonstrate that network topology is a crucial determinant of force distributions in an elastic spring network. Our results for 1D linear spring networks readily generalize to arbitrary dimensions.

The 2018 correlative microscopy techniques roadmap.

Developments in microscopy have been instrumental to progress in the life sciences, and many new techniques have been introduced and led to new discoveries throughout the last century. A wide and diverse range of methodologies is now available, including electron microscopy, atomic force microscopy, magnetic resonance imaging, small-angle x-ray scattering and multiple super-resolution fluorescence techniques, and each of these methods provides valuable read-outs to meet the demands set by the samples under study. Yet, the investigation of cell development requires a multi-parametric approach to address both the structure and spatio-temporal organization of organelles, and also the transduction of chemical signals and forces involved in cell-cell interactions. Although the microscopy technologies for observing each of these characteristics are well developed, none of them can offer read-out of all characteristics simultaneously, which limits the information content of a measurement. For example, while electron microscopy is able to disclose the structural layout of cells and the macromolecular arrangement of proteins, it cannot directly follow dynamics in living cells. The latter can be achieved with fluorescence microscopy which, however, requires labelling and lacks spatial resolution. A remedy is to combine and correlate different readouts from the same specimen, which opens new avenues to understand structure-function relations in biomedical research. At the same time, such correlative approaches pose new challenges concerning sample preparation, instrument stability, region of interest retrieval, and data analysis. Because the field of correlative microscopy is relatively young, the capabilities of the various approaches have yet to be fully explored, and uncertainties remain when considering the best choice of strategy and workflow for the correlative experiment. With this in mind, the Journal of Physics D: Applied Physics presents a special roadmap on the correlative microscopy techniques, giving a comprehensive overview from various leading scientists in this field, via a collection of multiple short viewpoints.

Robust Heterogeneous Hydrogels with Dynamic Nanocrystal-Polymer Interface.

A kind of novel heterogeneous composite hydrogel with dynamic nanocrosslinkers is designed, which is built via the preorganized host-guest interaction on the surface of cellulose nanocrystals. The reversible ß-cyclodextrin/adamantane conjunctions and their gradual dissociation on the nanocrystal-polymer interface guarantee the compressibility and stretchability of the composite hydrogels. While the sacrificed toughening mechanism can be rebuilt in the as-prepared hydrogels, it fails to be regenerated in the swollen hydrogels. This fact is originally due to the extreme mechanical contrast between rigid nanocrystals and the flexible polymer phase. This heterogeneity is largely amplified by the swelling process: polymer chains are prestretched between nanocrosslinkers and generate residual stress on the dynamic nanocrystal-polymer interface. Thus, this swelling-induced heterogeneity resists the reassociation of the sacrificed ß-cyclodextrin/adamantane complexes. Furthermore, the unstable nanocrystal-polymer interface induces the crack propagate along the nanocrosslinker surface, which remarkably retards the crack propagation during the stretch.

X-Ray Micro- and Nanodiffraction Imaging on Human Mesenchymal Stem Cells and Differentiated Cells.

Adult human mesenchymal stem cells show structural rearrangements of their cytoskeletal network during mechanically induced differentiation toward various cell types. In particular, the alignment of acto-myosin fibers is cell fate-dependent and can serve as an early morphological marker of differentiation. Quantification of such nanostructures on a mesoscopic scale requires high-resolution imaging techniques. Here, we use small- angle x-ray scattering with a spot size in the micro- and submicrometer range as a high-resolution and label-free imaging technique to reveal structural details of stem cells and differentiated cell types. We include principal component analysis into an automated empirical analysis scheme that allows the local characterization of oriented structures. Results on freeze-dried samples lead to quantitative structural information for all cell lines tested: differentiated cells reveal pronounced structural orientation and a relatively intense overall diffraction signal, whereas naive human mesenchymal stem cells lack these features. Our data support the hypothesis of stem cells establishing ordered structures along their differentiation process.

Mechanotransduction: use the force(s).

Mechanotransduction - how cells sense physical forces and translate them into biochemical and biological responses - is a vibrant and rapidly-progressing field, and is important for a broad range of biological phenomena. This forum explores the role of mechanotransduction in a variety of cellular activities and highlights intriguing questions that deserve further attention.

Elasticity of 3D networks with rigid filaments and compliant crosslinks.

Disordered filamentous networks with compliant crosslinks exhibit a low linear elastic shear modulus at small strains, but stiffen dramatically at high strains. Experiments have shown that the elastic modulus can increase by up to three orders of magnitude while the networks withstand relatively large stresses without rupturing. Here, we perform an analytical and numerical study on model networks in three dimensions. Our model consists of a collection of randomly oriented rigid filaments connected by flexible crosslinks that are modeled as wormlike chains. Due to zero probability of filament intersection in three dimensions, our model networks are by construction prestressed in terms of initial tension in the crosslinks. We demonstrate how the linear elastic modulus can be related to the prestress in these networks. Under the assumption of affine deformations in the limit of infinite crosslink density, we show analytically that the nonlinear elastic regime in 1- and 2-dimensional networks is characterized by power-law scaling of the elastic modulus with the stress. In contrast, 3-dimensional networks show an exponential dependence of the modulus on stress. Independent of dimensionality, if the crosslink density is finite, we show that the only persistent scaling exponent is that of the single wormlike chain. We further show that there is no qualitative change in the stiffening behavior of filamentous networks even if the filaments are bending-compliant. Consequently, unlike suggested in prior work, the model system studied here cannot provide an explanation for the experimentally observed linear scaling of the modulus with the stress in filamentous networks.

Novel growth regime of MDCK II model tissues on soft substrates.

It is well established that MDCK II cells grow in circular colonies that densify until contact inhibition takes place. Here, we show that this behavior is only typical for colonies developing on hard substrates and report a new growth phase of MDCK II cells on soft gels. At the onset, the new phase is characterized by small, three-dimensional droplets of cells attached to the substrate. When the contact area between the agglomerate and the substrate becomes sufficiently large, a very dense monolayer nucleates in the center of the colony. This monolayer, surrounded by a belt of three-dimensionally packed cells, has a well-defined structure, independent of time and cluster size, as well as a density that is twice the steady-state density found on hard substrates. To release stress in such dense packing, extrusions of viable cells take place several days after seeding. The extruded cells create second-generation clusters, as evidenced by an archipelago of aggregates found in a vicinity of mother colonies, which points to a mechanically regulated migratory behavior.

Micro-topography influences blood platelet spreading.

Injuries in blood vessels are accompanied by disrupted endothelial cell layers. Missing or destroyed endothelial cells lead to rough, structured surfaces on the micrometer scale. The first cells to arrive at the site of injury and to cover the wound are platelets, which subsequently drive blood clot formation. Therefore, investigating the interactions of platelets with structured surfaces is essential for the understanding of blood clotting. Here, we study the effects of underlying topography on platelet spreading using microstructured model substrates with varying area fractions of protein coating. We thereby distinguish the effects of (physical) topography and of (biochemical) protein availability. By analyzing the cell area and morphology, we find that the extent of protrusion formation - but not the total spread area - is determined by the area fractions of coating. The extent of filopodia formation is influenced by the availability of binding sites and the reaction of cells to the substrate's topography. The cells react to the structured substrate by avoiding topographic holes at the cell periphery and thus adapting their outer shape. This finding leads us to the conclusion that both chemically blocked and fibrinogen-coated holes represent "energetic obstacles" to the cells. Thus, the shape of the cell is governed by the interplay between spreading to an optimized area and adaption to the substrate topography.

FilamentSensor 2.0: An open-source modular toolbox for 2D/3D cytoskeletal filament tracking.

Cytoskeletal pattern formation and structural dynamics are key to a variety of biological functions and a detailed and quantitative analysis yields insight into finely tuned and well-balanced homeostasis and potential pathological alterations. High content life cell imaging of fluorescently labeled cytoskeletal elements under physiological conditions is nowadays state-of-the-art and can record time lapse data for detailed experimental studies. However, systematic quantification of structures and in particular the dynamics (i.e. frame-to-frame tracking) are essential. Here, an unbiased, quantitative, and robust analysis workflow that can be highly automatized is needed. For this purpose we upgraded and expanded our fiber detection algorithm FilamentSensor (FS) to the FilamentSensor 2.0 (FS2.0) toolbox, allowing for automatic detection and segmentation of fibrous structures and the extraction of relevant data (center of mass, length, width, orientation, curvature) in real-time as well as tracking of these objects over time and cell event monitoring.

Metal-Induced Energy Transfer (MIET) for Live-Cell Imaging with Fluorescent Proteins.

Metal-induced energy transfer (MIET) imaging is an easy-to-implement super-resolution modality that achieves nanometer resolution along the optical axis of a microscope. Although its capability in numerous biological and biophysical studies has been demonstrated, its implementation for live-cell imaging with fluorescent proteins is still lacking. Here, we present its applicability and capabilities for live-cell imaging with fluorescent proteins in diverse cell types (adult human stem cells, human osteo-sarcoma cells, and Dictyostelium discoideum cells), and with various fluorescent proteins (GFP, mScarlet, RFP, YPet). We show that MIET imaging achieves nanometer axial mapping of living cellular and subcellular components across multiple time scales, from a few milliseconds to hours, with negligible phototoxic effects.

Measuring Nuclear Mechanics with Atomic Force Microscopy.

Atomic force microscopy is an ideal tool to map topography and mechanical properties of materials on the micro- and nanoscale. Here, we describe its application to measure and analyze the mechanics, in particular the effective Young's elastic modulus E* of the mammalian nucleus in live cells. We present three approaches which enable the mechanics to be probed under varying conditions. This includes fully adhered cells, initially adhered cells which lack an established cytoskeleton, and purified nuclei to study their isolated response.

High internal phase Pickering emulsions stabilized by dialdehyde amylopectin/chitosan complex nanoparticles.

High internal phase Pickering emulsions (HIPPEs) have attracted intensive interest for their great potential in foods, cosmetics, and biomedical applications. However, the relatively poor biodegradability and biocompatibility of inorganic and synthetic particulate emulsifiers greatly limit their practical applications. Here, a kind of biobased nanoparticles, namely dialdehyde amylopectin/chitosan complex nanoparticles (DAPCNPs), were fabricated by Schiff base reaction between dialdehyde amylopectin and chitosan with the assistance of ultrasonication treatment. The resultant DAPCNPs were employed to stabilize O/W HIPPEs with various oils, such as toluene, cyclohexane, styrene and edible rapeseed oil. Moreover, the resultant DAPCNPs-stabilized HIPPEs showed high stability under various environmental stresses (80 °C; 20 mM and 100 mM aqueous NaCl solutions). Furthermore, porous scaffolds were also fabricated by freeze-drying cyclohexane-in-water HIPPEs stabilized by DAPCNPs after the introduction of polyvinyl alcohol (PVA) into the continuous phase. These findings would give inspiration for designing polysaccharides-based nanoparticles to stabilize HIPPEs and improve their practical applications.

A Focal Adhesion Filament Cross-correlation Kit for fast, automated segmentation and correlation of focal adhesions and actin stress fibers in cells.

Focal adhesions (FAs) and associated actin stress fibers (SFs) form a complex mechanical system that mediates bidirectional interactions between cells and their environment. This linked network is essential for mechanosensing, force production and force transduction, thus directly governing cellular processes like polarization, migration and extracellular matrix remodeling. We introduce a tool for fast and robust coupled analysis of both FAs and SFs named the Focal Adhesion Filament Cross-correlation Kit (FAFCK). Our software can detect and record location, axes lengths, area, orientation, and aspect ratio of focal adhesion structures as well as the location, length, width and orientation of actin stress fibers. This enables users to automate analysis of the correlation of FAs and SFs and study the stress fiber system in a higher degree, pivotal to accurately evaluate transmission of mechanocellular forces between a cell and its surroundings. The FAFCK is particularly suited for unbiased and systematic quantitative analysis of FAs and SFs necessary for novel approaches of traction force microscopy that uses the additional data from the cellular side to calculate the stress distribution in the substrate. For validation and comparison with other tools, we provide datasets of cells of varying quality that are labelled by a human expert. Datasets and FAFCK are freely available as open source under the GNU General Public License.

Metasurface-based total internal reflection microscopy.

Recent years have seen a tremendous progress in the development of dielectric metasurfaces for visible light applications. Such metasurfaces are ultra-thin optical devices that can manipulate optical wavefronts in an arbitrary manner. Here, we present a newly developed metasurface which allows for coupling light into a microscopy coverslip to achieve total internal reflection (TIR) excitation. TIR fluorescence microscopy (TIRFM) is an important bioimaging technique used specifically to image cellular membranes or surface-localized molecules with high contrast and low background. Its most commonly used modality is objective-type TIRFM where one couples a focused excitation laser beam at the edge of the back focal aperture of an oil-immersion objective with high numerical aperture (N.A.) to realize a high incident-angle plane wave excitation above the critical TIR angle in sample space. However, this requires bulky and expensive objectives with a limited field-of-view (FOV). The metasurface which we describe here represents a low cost and easy-to-use alternative for TIRFM. It consists of periodic 2D arrays of asymmetric structures fabricated in TiO2 on borosilicate glass. It couples up to 70% of the incident non-reflected light into the first diffraction order at an angle of 65° in glass, which is above the critical TIR angle for a glass-water interface. Only ∼7% of the light leaks into propagating modes traversing the glass surface, thus minimizing any spurious background fluorescence originating far outside the glass substrate. We describe in detail design and fabrication of the metasurface, and validate is applicability for TIRFM by imaging immunostained human mesenchymal stem cells over a FOV of 200 µm x 200 µm. We envision that these kinds of metasurfaces can become a valuable tool for low-cost and TIRFM, offering high contrast, low photodamage, and high surface selectivity in fluorescence excitation and detection.

Liquid-Behaviors-Assisted Fabrication of Multidimensional Birefringent Materials from Dynamic Hybrid Hydrogels.

Liquid-solid transition is a widely used strategy to shape polymeric materials and encode their microstructures. However, it is still challenging to fully exploit liquid behaviors of material precursors. In particular, the dynamic and static liquid behaviors naturally conflict with each other, which makes it difficult to integrate their advantages in the same materials. Here, by utilizing a shear-thinning phenomenon in the dynamic hybrid hydrogels, we achieve a hydrodynamic alignment of cellulose nanocrystals (CNC) and preserve it in the relaxed hydrogel networks due to the much faster relaxation of polymer networks (within 500 s) than CNC after the unloading of external force. During the following drying process, the surface tension of hydrogels further enhances the orientation index of CNC up to 0.872 in confined geometry, and these anisotropic microstructures demonstrate highly tunable birefringence (up to 0.004 14). Due to the presence of the boundaries of dynamic hydrogels, diverse xerogels including fibers, films, and even complex three-dimensional structures with variable anisotropic microstructures can be fabricated without any external molds.

Effect of Adhesion and Substrate Elasticity on Neutrophil Extracellular Trap Formation.

Neutrophils are the most abundant type of white blood cells. Upon stimulation, they are able to decondense and release their chromatin as neutrophil extracellular traps (NETs). This process (NETosis) is part of immune defense mechanisms but also plays an important role in many chronic and inflammatory diseases such as atherosclerosis, rheumatoid arthritis, diabetes, and cancer. For this reason, much effort has been invested into understanding biochemical signaling pathways in NETosis. However, the impact of the mechanical micro-environment and adhesion on NETosis is not well-understood. Here, we studied how adhesion and especially substrate elasticity affect NETosis. We employed polyacrylamide (PAA) gels with distinctly defined elasticities (Young's modulus E) within the physiologically relevant range from 1 to 128 kPa and coated the gels with integrin ligands (collagen I, fibrinogen). Neutrophils were cultured on these substrates and stimulated with potent inducers of NETosis: phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS). Interestingly, PMA-induced NETosis was neither affected by substrate elasticity nor by different integrin ligands. In contrast, for LPS stimulation, NETosis rates increased with increasing substrate elasticity (E > 20 kPa). LPS-induced NETosis increased with increasing cell contact area, while PMA-induced NETosis did not require adhesion at all. Furthermore, inhibition of phosphatidylinositide 3 kinase (PI3K), which is involved in adhesion signaling, completely abolished LPS-induced NETosis but only slightly decreased PMA-induced NETosis. In summary, we show that LPS-induced NETosis depends on adhesion and substrate elasticity while PMA-induced NETosis is completely independent of adhesion.

Lipid Emulsion-Based OCT Angiography for Ex Vivo Imaging of the Aqueous Outflow Tract.

Purpose: Contrast agents applicable for optical coherence tomography (OCT) imaging are rare. The intrascleral aqueous drainage system would be a potential application for a contrast agent, because the aqueous veins are of small diameter and located deep inside the highly scattering sclera. We tested lipid emulsions (LEs) as candidate OCT contrast agents in vitro and ex vivo, including milk and the anesthetic substance Propofol. Methods: Commercial OCT and OCT angiography (OCTA) devices were used. Maximum reflectivity and signal transmission of LE were determined in tube phantoms. Absorption spectra and light scattering was analyzed. The anterior chamber of enucleated porcine eyes was perfused with LEs, and OCTA imaging of the LEs drained via the aqueous outflow tract was performed. Results: All LEs showed a significantly higher reflectivity than water (P < 0.001). Higher milk lipid content was positively correlated with maximum reflectivity and negatively with signal transmission. Propofol exhibited the best overall performance. Due to a high degree of signal fluctuation, OCTA could be applied for detection of LE. Compared with blood, the OCTA signal of Propofol was significantly stronger (P = 0.001). As a proof of concept, time-resolved aqueous angiography of porcine eyes was performed. The three-dimensional (3D) structure and dynamics of the aqueous outflow were significantly different from humans. Conclusions: LEs induced a strong signal in OCT and OCTA. LE-based OCTA allowed the ability to obtain time-resolved 3D datasets of aqueous outflow. Possible interactions of LE with inner eye's structures need to be further investigated before in vivo application.