Analysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity.

Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.

Active topolectrical circuits.

The transfer of topological concepts from the quantum world to classical mechanical and electronic systems has opened fundamentally different approaches to protected information transmission and wave guidance. A particularly promising emergent technology is based on recently discovered topolectrical circuits that achieve robust electric signal transduction by mimicking edge currents in quantum Hall systems. In parallel, modern active matter research has shown how autonomous units driven by internal energy reservoirs can spontaneously self-organize into collective coherent dynamics. Here, we unify key ideas from these two previously disparate fields to develop design principles for active topolectrical circuits (ATCs) that can self-excite topologically protected global signal patterns. Realizing autonomous active units through nonlinear Chua diode circuits, we theoretically predict and experimentally confirm the emergence of self-organized protected edge oscillations in one- and two-dimensional ATCs. The close agreement between theory, simulations, and experiments implies that nonlinear ATCs provide a robust and versatile platform for developing high-dimensional autonomous electrical circuits with topologically protected functionalities.

Topological Defect Engineering and PT Symmetry in Non-Hermitian Electrical Circuits.

We employ electric circuit networks to study topological states of matter in non-Hermitian systems enriched by parity-time symmetry PT and chiral symmetry anti-PT (APT). The topological structure manifests itself in the complex admittance bands which yields excellent measurability and signal to noise ratio. We analyze the impact of PT-symmetric gain and loss on localized edge and defect states in a non-Hermitian Su-Schrieffer-Heeger (SSH) circuit. We realize all three symmetry phases of the system, including the APT-symmetric regime that occurs at large gain and loss. We measure the admittance spectrum and eigenstates for arbitrary boundary conditions, which allows us to resolve not only topological edge states, but also a novel PT-symmetric Z_{2} invariant of the bulk. We discover the distinct properties of topological edge states and defect states in the phase diagram. In the regime that is not PT symmetric, the topological defect state disappears and only reemerges when APT symmetry is reached, while the topological edge states always prevail and only experience a shift in eigenvalue. Our findings unveil a future route for topological defect engineering and tuning in non-Hermitian systems of arbitrary dimension.

Whole tissue and single cell mechanics are correlated in human brain tumors.

Biomechanical changes are critical for cancer progression. However, the relationship between the rheology of single cells measured ex-vivo and the living tumor is not yet understood. Here, we combined single-cell rheology of cells isolated from primary tumors with in vivo bulk tumor rheology in patients with brain tumors. Eight brain tumors (3 glioblastoma, 3 meningioma, 1 astrocytoma, 1 metastasis) were investigated in vivo by magnetic resonance elastography (MRE), and after surgery by the optical stretcher (OS). MRE was performed in a 3-Tesla clinical MRI scanner and magnitude modulus |G*|, loss angle φ, storage modulus G', and loss modulus G'' were derived. OS experiments measured cellular creep deformation in response to laser-induced step stresses. We used a Kelvin-Voigt model to deduce two parameters related to cellular stiffness (µKV) and cellular viscosity (ηKV) from OS measurements in a time regimen that overlaps with that of MRE. We found that single-cell µKV was correlated with |G*| (R = 0.962, p < 0.001) and G'' (R = 0.883, p = 0.004) but not G' of the bulk tissue. These results suggest that single-cell stiffness affects tissue viscosity in brain tumors. The observation that viscosity parameters of individual cells and bulk tissue were not correlated suggests that collective mechanical interactions (i.e. emergent effects or cellular unjamming) of many cancer cells, which depend on cellular stiffness, influence the mechanical dissipation behavior of the bulk tissue. Our results are important to understand the emergent rheology of active multiscale compound materials such as brain tumors and its role in disease progression.

Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro.

Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell-cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.

The lensing effect of trapped particles in a dual-beam optical trap.

In dual-beam optical traps, two counterpropagating, divergent laser beams emitted from opposing laser fibers trap and manipulate dielectric particles. We investigate the lensing effect that trapped particles have on the beams. Our approach makes use of the intrinsic coupling of a beam to the opposing fiber after having passed the trapped particle. We present measurements of this coupling signal for PDMS particles, as well as a model for its dependence on size and refractive index of the trapped particle. As a more complex sample, the coupling of inhomogeneous biological cells is measured and discussed. We show that the lensing effect is well captured by the simple ray optics approximation. The measurements reveal intricate details, such as the thermal lens effect of the beam propagation in a dual-beam trap. For a particle of known size, the model further allows to infer its refractive index simply from the coupling signal.

Active contractions in single suspended epithelial cells.

Investigations of active contractions in tissue cells to date have been focused on cells that exert forces via adhesion sites to substrates or to other cells. In this study we show that also suspended epithelial cells exhibit contractility, revealing that contractions can occur independently of focal adhesions. We employ the Optical Stretcher to measure adhesion-independent mechanical properties of an epithelial cell line transfected with a heat-sensitive cation channel. During stretching the heat transferred to the ion channel causes a pronounced Ca(2+) influx through the plasma membrane that can be blocked by adequate drugs. This way the contractile forces in suspended cells are shown to be partially triggered by Ca(2+) signaling. A phenomenological mathematical model is presented, incorporating a term accounting for the active stress exerted by the cell, which is both necessary and sufficient to describe the observed increase in strain when the Ca(2+) influx is blocked. The median and the shape of the strain distributions depend on the activity of the cells. Hence, it is unlikely that they can be described by a simple Gaussian or log normal distribution, but depend on specific cellular properties such as active contractions. Our results underline the importance of considering activity when measuring cellular mechanical properties even in the absence of measurable contractions. Thus, the presented method to quantify active contractions of suspended cells offers new perspectives for a better understanding of cellular force generation with possible implications for medical diagnosis and therapy.

Hyperbolic matter in electrical circuits with tunable complex phases.

Curved spaces play a fundamental role in many areas of modern physics, from cosmological length scales to subatomic structures related to quantum information and quantum gravity. In tabletop experiments, negatively curved spaces can be simulated with hyperbolic lattices. Here we introduce and experimentally realize hyperbolic matter as a paradigm for topological states through topolectrical circuit networks relying on a complex-phase circuit element. The experiment is based on hyperbolic band theory that we confirm here in an unprecedented numerical survey of finite hyperbolic lattices. We implement hyperbolic graphene as an example of topologically nontrivial hyperbolic matter. Our work sets the stage to realize more complex forms of hyperbolic matter to challenge our established theories of physics in curved space, while the tunable complex-phase element developed here can be a key ingredient for future experimental simulation of various Hamiltonians with topological ground states.

Simulating hyperbolic space on a circuit board.

The Laplace operator encodes the behavior of physical systems at vastly different scales, describing heat flow, fluids, as well as electric, gravitational, and quantum fields. A key input for the Laplace equation is the curvature of space. Here we discuss and experimentally demonstrate that the spectral ordering of Laplacian eigenstates for hyperbolic (negatively curved) and flat two-dimensional spaces has a universally different structure. We use a lattice regularization of hyperbolic space in an electric-circuit network to measure the eigenstates of a 'hyperbolic drum', and in a time-resolved experiment we verify signal propagation along the curved geodesics. Our experiments showcase both a versatile platform to emulate hyperbolic lattices in tabletop experiments, and a set of methods to verify the effective hyperbolic metric in this and other platforms. The presented techniques can be utilized to explore novel aspects of both classical and quantum dynamics in negatively curved spaces, and to realise the emerging models of topological hyperbolic matter.

Milliwatt terahertz harmonic generation from topological insulator metamaterials.

Achieving efficient, high-power harmonic generation in the terahertz spectral domain has technological applications, for example, in sixth generation (6G) communication networks. Massless Dirac fermions possess extremely large terahertz nonlinear susceptibilities and harmonic conversion efficiencies. However, the observed maximum generated harmonic power is limited, because of saturation effects at increasing incident powers, as shown recently for graphene. Here, we demonstrate room-temperature terahertz harmonic generation in a Bi2Se3 topological insulator and topological-insulator-grating metamaterial structures with surface-selective terahertz field enhancement. We obtain a third-harmonic power approaching the milliwatt range for an incident power of 75 mW-an improvement by two orders of magnitude compared to a benchmarked graphene sample. We establish a framework in which this exceptional performance is the result of thermodynamic harmonic generation by the massless topological surface states, benefiting from ultrafast dissipation of electronic heat via surface-bulk Coulomb interactions. These results are an important step towards on-chip terahertz (opto)electronic applications.

Calcium imaging in the optical stretcher.

The Microfluidic Optical Stretcher (MOS) has previously been shown to be a versatile tool to measure mechanical properties of single suspended cells. In this study we combine optical stretching and fluorescent calcium imaging. A cell line transfected with a heat sensitive cation channel was used as a model system to show the versatility of the setup. The cells were loaded with the Ca(2+) dye Fluo-4 and imaged with confocal laser scanning microscopy while being stretched. During optical stretching heat is transferred to the cell causing a pronounced Ca(2+) influx through the cation channel. The technique opens new perspectives for investigating the role of Ca(2+) in regulating cell mechanical behavior.

Automated Morphological Feature Assessment for Zebrafish Embryo Developmental Toxicity Screens.

Detection of developmental phenotypes in zebrafish embryos typically involves a visual assessment and scoring of morphological features by an individual researcher. Subjective scoring could impact results and be of particular concern when phenotypic effect patterns are also used as a diagnostic tool to classify compounds. Here we introduce a quantitative morphometric approach based on image analysis of zebrafish embryos. A software called FishInspector was developed to detect morphological features from images collected using an automated system to position zebrafish embryos. The analysis was verified and compared with visual assessments of 3 participating laboratories using 3 known developmental toxicants (methotrexate, dexamethasone, and topiramate) and 2 negative compounds (loratadine and glibenclamide). The quantitative approach exhibited higher sensitivity and made it possible to compare patterns of effects with the potential to establish a grouping and classification of developmental toxicants. Our approach improves the robustness of phenotype scoring and reliability of assay performance and, hence, is anticipated to improve the predictivity of developmental toxicity screening using the zebrafish embryo.

The optical cell rotator.

The optical cell rotator (OCR) is a modified dual-beam laser trap for the holding and controlled rotation of suspended dielectric microparticles, such as cells. In contrast to optical tweezers, OCR uses two counter-propagating divergent laser beams, which are shaped and delivered by optical fibers. The rotation of a trapped specimen is carried out by the rotation of a dual-mode fiber, emitting an asymmetric laser beam. Experiments were performed on human erythrocytes, promyelocytic leukemia cells (HL60), and cell clusters (MCF-7). Since OCR permits the rotation of cells around an axis perpendicular to the optical axis of any microscope and is fully decoupled from imaging optics, it could be a suitable and expedient tool for tomographic microscopy.