Polarization-Encoded Colocalization Microscopy at Cryogenic Temperatures

Daniel Böning, Franz Wieser, Vahid Sandoghdar

Super-resolution localization microscopy is based on determining the positions of individual fluorescent markers in a sample. The major challenge in reaching an ever higher localization precision lies in the limited number of collected photons from single emitters. To tackle this issue, it has been shown that one can exploit the increased photostability at low temperatures, reaching localization precisions in the sub-nanometer range. Another crucial ingredient of single-molecule super-resolution imaging is the ability to activate individual emitter within a diffraction-limited spot. Here, we report on photoblinking behavior of organic dyes at low temperature and elaborate on the limitations of this ubiquitous phenomenon for selecting single molecules. We then show that recording the emission polarization not only provides access to the molecular orientation, but it also facilitates the assignment of photons to individual blinking molecules. Furthermore, we employ periodical modulation of the excitation polarization as a robust method to effectively switch fluorophores. We bench mark each approach by resolving two emitters on different DNA origami structures.

Kerker effect, superscattering, and scattering dark states in atomic antennas

Rasoul Alaee Khanghah, Akbar Safari, Vahid Sandoghdar, Robert W. Boyd

We study scattering phenomena such as the Kerker effect, superscattering, and scattering dark states in a subwavelength atomic antenna consisting of atoms with only electric dipole transitions. We show that an atomic antenna can exhibit arbitrarily large or small scattering cross sections depending on the geometry of the structure and the direction of the impinging light. We also demonstrate that atoms with only an electric dipole transition can exhibit a directional radiation pattern with zero backscattering when placed in a certain configuration. This is a special case of a phenomenon known as the Kerker effect, which typically occurs in the presence of both electric and magnetic transitions. Our findings open a pathway to design highly directional emitters, nonradiating sources, and highly scattering objects based on individually controlled atoms.

Differential diffusional properties in loose and tight docking prior to membrane fusion

Agata Witkowska, Susann Spindler, Reza Gholami Mahmoodabadi, Vahid Sandoghdar, Reinhard Jahn

Fusion of biological membranes, although mediated by divergent proteins, is believed to follow a common pathway. It proceeds through distinct steps including docking, merger of proximal leaflets (stalk formation), and formation of a fusion pore. However, the structure of these intermediates is difficult to study due to their short lifetime. Previously, we observed a loosely and tightly docked state preceding leaflet merger using arresting point mutations in SNARE proteins, but the nature of these states remained elusive. Here we used interferometric scattering (iSCAT) microscopy to monitor diffusion of single vesicles across the surface of giant unilamellar vesicles (GUVs). We observed that the diffusion coefficients of arrested vesicles decreased during progression through the intermediate states. Modeling allowed for predicting the number of tethering SNARE complexes upon loose docking and the size of the interacting membrane patches upon tight docking. These results shed new light on the nature of membrane-membrane interactions immediately before fusion.

Reactive oligodendrocyte progenitor cells (re-)myelinate the regenerating zebrafish spinal cord

Vasiliki Tsata, Volker Kroehne, Daniel Wehner, Fabian Rost, Christian Lange, Cornelia Hoppe, Thomas Kurth, Susanne Reinhardt, Andreas Petzold, et al.

Spinal cord injury (SCI) results in loss of neurons, oligodendrocytes and myelin sheaths, all of which are not efficiently restored. The scarcity of oligodendrocytes in the lesion site impairs re-myelination of spared fibres, which leaves axons denuded, impedes signal transduction and contributes to permanent functional deficits. In contrast to mammals, zebrafish can functionally regenerate the spinal cord. Yet, little is known about oligodendroglial lineage biology and re-myelination capacity after SCI in a regeneration-permissive context. Here, we report that, in adult zebrafish, SCI results in axonal, oligodendrocyte and myelin sheath loss. We find that OPCs, the oligodendrocyte progenitor cells, survive the injury, enter a reactive state, proliferate and differentiate into oligodendrocytes. Concomitantly, the oligodendrocyte population is reestablished to pre-injury levels within 2 weeks. Transcriptional profiling revealed that reactive OPCs upregulate the expression of several myelination-related genes. Interestingly, global reduction of axonal tracts and partial re-myelination, relative to pre-injury levels, persist at later stages of regeneration, yet are sufficient for functional recovery. Taken together, these findings imply that, in the zebrafish spinal cord, OPCs replace lost oligodendrocytes and, thus, re-establish myelination during regeneration.

Maturation of Monocyte-Derived DCs Leads to Increased Cellular Stiffness, Higher Membrane Fluidity, and Changed Lipid Composition

Jennifer J. Lühr, Nils Alex, Lukas Amon, Martin Kräter, Markéta Kubánková, Erdinc Sezgin, Christian H. K. Lehmann, Lukas Heger, Gordon F. Heidkamp, et al.

Dendritic cells (DCs) are professional antigen-presenting cells of the immune system. Upon sensing pathogenic material in their environment, DCs start to mature, which includes cellular processes, such as antigen uptake, processing and presentation, as well as upregulation of costimulatory molecules and cytokine secretion. During maturation, DCs detach from peripheral tissues, migrate to the nearest lymph node, and find their way into the correct position in the net of the lymph node microenvironment to meet and interact with the respective T cells. We hypothesize that the maturation of DCs is well prepared and optimized leading to processes that alter various cellular characteristics from mechanics and metabolism to membrane properties. Here, we investigated the mechanical properties of monocyte-derived dendritic cells (moDCs) using real-time deformability cytometry to measure cytoskeletal changes and found that mature moDCs were stiffer compared to immature moDCs. These cellular changes likely play an important role in the processes of cell migration and T cell activation. As lipids constitute the building blocks of the plasma membrane, which, during maturation, need to adapt to the environment for migration and DC-T cell interaction, we performed an unbiased high-throughput lipidomics screening to identify the lipidome of moDCs. These analyses revealed that the overall lipid composition was significantly changed during moDC maturation, even implying an increase of storage lipids and differences of the relative abundance of membrane lipids upon maturation. Further, metadata analyses demonstrated that lipid changes were associated with the serum low-density lipoprotein (LDL) and cholesterol levels in the blood of the donors. Finally, using lipid packing imaging we found that the membrane of mature moDCs revealed a higher fluidity compared to immature moDCs. This comprehensive and quantitative characterization of maturation associated changes in moDCs sets the stage for improving their use in clinical application.

Soft Polydimethylsiloxane-Supported Lipid Bilayers for Studying T Cell Interactions

Anna H. Lippert, Ivan B. Dimov, Alexander K. Winkel, Jane Humphrey, James McColl, Kevin Y. Chen, Ana M. Santos, Edward Jenkins, Kristian Franze, et al.

Much of what we know about the early stages of T cell activation has been obtained from studies of T cells interacting with glass-supported lipid bilayers that favor imaging but are orders of magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing "soft bilayers" with physiological levels of mechanical resistance (Young's modulus of 4 kPa). Comparisons of T cell behavior on soft and glass-supported bilayers revealed that whereas late stages of T cell activation are thought to be substrate-stiffness dependent, early calcium signaling was unaffected by substrate rigidity, implying that early steps in T cell receptor triggering are not mechanosensitive. The exclusion of large receptor-type phosphatases was observed on the soft bilayers, however, even though it is yet to be demonstrated at authentic cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

Mechanical Adaptability of Tumor Cells in Metastasis

Valentin Gensbittel, Martin Kräter, Sébastien Harlepp, Ignacio Busnelli, Jochen Guck, Jacky G. Goetz

The most dangerous aspect of cancer lies in metastatic progression. Tumor cells will successfully form life-threatening metastases when they undergo sequential steps along a journey from the primary tumor to distant organs. From a biomechanics standpoint, growth, invasion, intravasation, circulation, arrest/adhesion, and extravasation of tumor cells demand particular cell-mechanical properties in order to survive and complete the metastatic cascade. With metastatic cells usually being softer than their non-malignant counterparts, high deformability for both the cell and its nucleus is thought to offer a significant advantage for metastatic potential. However, it is still unclear whether there is a finely tuned but fixed mechanical state that accommodates all mechanical features required for survival throughout the cascade or whether tumor cells need to dynamically refine their properties and intracellular components at each new step encountered. Here, we review the various mechanical requirements successful cancer cells might need to fulfill along their journey and speculate on the possibility that they dynamically adapt their properties accordingly. The mechanical signature of a successful cancer cell might actually be its ability to adapt to the successive microenvironmental constraints along the different steps of the journey.

Optical quantification of intracellular mass density and cell mechanics in 3D mechanical confinement

Sadra Bakhshandeh, Hubert Taïeb, Raimund Schlüßler, Kyoohyun Kim, Timon Beck, Anna Taubenberger, Jochen Guck, Amaia Cipitria

Biophysical properties of cells such as intracellular mass density and cell mechanics are known to be involved in a wide range of homeostatic functions and pathological alterations. An optical readout that can be used to quantify such properties is the refractive index (RI) distribution. It has been recently reported that the nucleus, initially presumed to be the organelle with the highest dry mass density (ρ) within the cell, has in fact a lower RI and ρ than its surrounding cytoplasm. These studies have either been conducted in suspended cells, or cells adhered on 2D substrates, neither of which reflects the situation in vivo where cells are surrounded by the extracellular matrix (ECM). To better approximate the 3D situation, we encapsulated cells in 3D covalently-crosslinked alginate hydrogels with varying stiffness, and imaged the 3D RI distribution of cells, using a combined optical diffraction tomography (ODT)-epifluorescence microscope. Unexpectedly, the nuclei of cells in 3D displayed a higher ρ than the cytoplasm, in contrast to 2D cultures. Using a Brillouin-epifluorescence microscope we subsequently showed that in addition to higher ρ, the nuclei also had a higher longitudinal modulus (M) and viscosity (η) compared to the cytoplasm. Furthermore, increasing the stiffness of the hydrogel resulted in higher M for both the nuclei and cytoplasm of cells in stiff 3D alginate compared to cells in compliant 3D alginate. The ability to quantify intracellular biophysical properties with non-invasive techniques will improve our understanding of biological processes such as dormancy, apoptosis, cell growth or stem cell differentiation.

Estrogens Determine Adherens Junction Organization and E-Cadherin Clustering in Breast Cancer Cells via Amphiregulin

Philip Bischoff, Marja Kornhuber, Sebastian Dunst, Jakob Zell, Beatrix Fauler, Thorsten Mielke, Anna V. Taubenberger, Jochen Guck, Michael Oelgeschlaeger, et al.

Estrogens play an important role in the development and progression of human cancers, particularly in breast cancer. Breast cancer progression depends on the malignant destabilization of adherens junctions (AJs) and disruption of tissue integrity. We found that estrogen receptor alpha (ER alpha) inhibition led to a striking spatial reorganization of AJs and microclustering of E-Cadherin (E-Cad) in the cell membrane of breast cancer cells. This resulted in increased stability of AJs and cell stiffness and a reduction of cell motility. These effects were actomyosindependent and reversible by estrogens. Detailed investigations showed that the ERa target gene and epidermal growth factor receptor (EGFR) ligand Amphiregulin (AREG) essentially regulates AJ reorganization and E-Cad microclustering. Our results not only describe a biological mechanism for the organization of AJs and the modulation of mechanical properties of cells but also provide a new perspective on how estrogens and anti-estrogens might influence the formation of breast tumors.

The Relative Densities of Cytoplasm and Nuclear Compartments Are Robust against Strong Perturbation

Kyoohyun Kim, Jochen Guck

The cell nucleus is a compartment in which essential processes such as gene transcription and DNA replication occur. Although the large amount of chromatin confined in the finite nuclear space could install the picture of a particularly dense organelle surrounded by less dense cytoplasm, recent studies have begun to report the opposite. However, the generality of this newly emerging, opposite picture has so far not been tested. Here, we used combined optical diffraction tomography and epi-fluorescence microscopy to systematically quantify the mass densities of cytoplasm, nucleoplasm, and nucleoli of human cell lines, challenged by various perturbations. We found that the nucleoplasm maintains a lower mass density than cytoplasm during cell cycle progression by scaling its volume to match the increase of dry mass during cell growth. At the same time, nucleoli exhibited a significantly higher mass density than the cytoplasm. Moreover, actin and microtubule depolymerization and changing chromatin condensation altered volume, shape, and dry mass of those compartments, whereas the relative distribution of mass densities was generally unchanged. Our findings suggest that the relative mass densities across membrane-bound and membraneless compartments are robustly conserved, likely by different as-of-yet unknown mechanisms, which hints at an underlying functional relevance. This surprising robustness of mass densities contributes to an increasing recognition of the importance of physico-chemical properties in determining cellular characteristics and compartments.

Grain Dependent Growth of Bright Quantum Emitters in Hexagonal Boron Nitride

Noah Mendelson, Luis Morales-Inostroza, Chi Li, Ritika Ritika, Minh Anh Phan Nguyen, Jacqueline Loyola-Echeverria, Sejeong Kim, Stephan Götzinger, Milos Toth, et al.

Point defects in hexagonal boron nitride have emerged as a promising quantum light source due to their bright and photostable room temperature emission. In this work, the incorporation of quantum emitters during chemical vapor deposition growth on a nickel substrate is studied. Combining a range of characterization techniques, it is demonstrated that the incorporation of quantum emitters is limited to (001) oriented nickel grains. Such emitters display improved emission properties in terms of brightness and stability. These emitters are further utilized and integrated with a compact optical antenna enhancing light collection from the sources. The hybrid device yields average saturation count rates of ≈2.9 × 106 cps and an average photon purity of g(2)(0) ≈ 0.1. The results advance the understanding of single photon emitter incorporation during chemical vapor deposition growth and demonstrate a key step towards compact devices for achieving maximum collection efficiency.

High-precision protein-tracking with interferometric scattering microscopy

Richard W. Taylor, Cornelia Holler, Reza Gholami Mahmoodabadi, Michelle Küppers, Houman Mirzaalian Dastjerdi, Vasily Zaburdaev, Alexandra Schambony, Vahid Sandoghdar

The mobility of proteins and lipids within the cell, sculpted oftentimes by the organisation of the membrane, reveals a great wealth of information on the function and interaction of these molecules as well as the membrane itself. Single particle tracking has proven to be a vital tool to study the mobility of individual molecules and unravel details of their behaviour. Interferometric scattering (iSCAT) microscopy is an emerging technique well suited for visualising the diffusion of gold nanoparticle-labelled membrane proteins to a spatial and temporal resolution beyond the means of traditional fluorescent labels. We discuss the applicability of interferometric single particle tracking (iSPT) microscopy to investigate the minutia in the motion of a protein through measurements visualising the mobility of the epidermal growth factor receptor in various biological scenarios on the live cell.

Smartphone‐based multimodal tethered capsule endoscopic platform for white‐light, narrow‐band, and fluorescence/autofluorescence imaging

Gargi Sharma, Oana-Maria Thoma, Katharina Blessing, Robert Gall, Maximilian Waldner, Kanwarpal Singh

Multimodal low‐cost endoscopy is highly desirable in poor resource settings such as in developing nations. In this work, we developed a smartphone‐based low‐cost, reusable tethered capsule endoscopic platform that allows white‐light, narrowband, and fluorescence/autofluorescence imaging of the esophagus. The ex‐vivo studies of swine esophagus were performed and compared with a commercial endoscope to test the white‐light imaging capabilities of the endoscope. The efficacy of the capsule for narrow‐band imaging was tested by imaging the vascularization of the tongue. To determine the autofluorescence/fluorescence capability of the endoscope, fluorescein dye with different concentrations was imaged. Furthermore, swine esophagus injected with fluorescein dye was imaged using the fluorescence/autofluorescence and the white‐light imaging modules, ex‐vivo. The overall cost of the capsules is approximately 12 €, 15 €, and 42 € for the white light imaging, the narrow‐band imaging, and the fluorescence/autofluorescence imaging respectively. In addition, the cost of the laser source module required for the narrow‐band imaging and the fluorescence/autofluorescence imaging is approximately 218 €. This device will open the possibility of imaging the esophagus in underprivileged areas.

Acquired demyelination but not genetic developmental defects in myelination leads to brain tissue stiffness changes

Dominic Eberle, Georgia Fodelianaki, Thomas Kurth, Anna Jagielska, Stephanie Möllmert, Elke Ulbricht, Katrin Wagner, Anna V. Taubenberger, Nicolas Träber, et al.

Changes in axonal myelination are an important hallmark of aging and a number of neurological diseases. Demyelinated axons are impaired in their function and degenerate over time. Oligodendrocytes, the cells responsible for myelination of axons, are sensitive to mechanical properties of their environment. Growing evidence indicates that mechanical properties of demyelinating lesions are different from the healthy state and thus have the potential to affect myelinating potential of oligodendrocytes. We performed a high-resolution spatial mapping of the mechanical heterogeneity of demyelinating lesions using atomic force microscope-enabled indentation. Our results indicate that the stiffness of specific regions of mouse brain tissue is influenced by age and degree of myelination. Here we specifically demonstrate that acquired acute but not genetic demyelination leads to decreased tissue stiffness, which could influence the remyelination potential of oligodendrocytes. We also demonstrate that specific brain regions have unique ranges of stiffness in white and grey matter. Our ex vivo findings may help the design of future in vitro models to mimic the mechanical environment of the brain in healthy and diseased states. The mechanical properties of demyelinating lesions reported here may facilitate novel approaches in treating demyelinating diseases such as multiple sclerosis.

Combined fluorescence, optical diffraction tomography and Brillouin microscopy

Raimund Schlüßler, Kyoohyun Kim, Martin Nötzel, Anna Taubenberger, Shada Abuhattum Hofemeier, Timon Beck, Paul Müller, Shovamayee Maharana, Gheorghe Cojoc, et al.

Quantitative measurements of physical parameters become increasingly important for understanding biological processes. Brillouin microscopy (BM) has recently emerged as one technique providing the 3D distribution of viscoelastic properties inside biological samples — so far relying on the implicit assumption that refractive index (RI) and density can be neglected. Here, we present a novel method (FOB microscopy) combining BM with optical diffraction tomography and epi-fluorescence imaging for explicitly measuring the Brillouin shift, RI and absolute density with molecular specificity. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the cell nucleus, we find that it has lower density but higher longitudinal modulus. Thus, the longitudinal modulus is not merely sensitive to the water content of the sample — a postulate vividly discussed in the field. We demonstrate the further utility of FOB on various biological systems including adipocytes and intracellular membraneless compartments. FOB microscopy can provide unexpected scientific discoveries and shed quantitative light on processes such as phase separation and transition inside living cells.

Quantitative phase microscopy enables precise and efficient determination of biomolecular condensate composition

Patrick M. McCall, Kyoohyun Kim, Anatol W. Fritsch, J.M. Iglesias-Artola, L.M. Jawerth, Jie Wang, M. Ruer, J. Peychl, Andrey Poznyakovskiy, et al.

Many compartments in eukaryotic cells are protein-rich biomolecular condensates demixed from the cyto- or nucleoplasm. Although much has been learned in recent years about the integral roles condensates play in many cellular processes as well as the biophysical properties of reconstituted condensates, an understanding of their most basic feature, their composition, remains elusive. Here we combined quantitative phase microscopy (QPM) and the physics of sessile droplets to develop a precise method to measure the shape and composition of individual model condensates. This technique does not rely on fluorescent dyes or tags, which we show can significantly alter protein phase behavior, and requires 1000-fold less material than traditional label-free technologies. We further show that this QPM method measures the protein concentration in condensates to a 3-fold higher precision than the next best label-free approach, and that commonly employed strategies based on fluorescence intensity dramatically underestimate these concentrations by as much as 50-fold. Interestingly, we find that condensed-phase protein concentrations can span a broad range, with PGL3, TAF15(RBD) and FUS condensates falling between 80 and 500 mg/ml under typical in vitro conditions. This points to a natural diversity in condensate composition specified by protein sequence. We were also able to measure temperature-dependent phase equilibria with QPM, an essential step towards relating phase behavior to the underlying physics and chemistry. Finally, time-resolved QPM reveals that PGL3 condensates undergo a contraction-like process during aging which leads to doubling of the internal protein concentration coupled to condensate shrinkage. We anticipate that this new approach will enable understanding the physical properties of biomolecular condensates and their function.

Liquid Phase Separation Controlled by pH

Omar Adame-Arana, Christoph A. Weber, Vasily Zaburdaev, Jacques Prost, Frank Julicher

We present a minimal model to study the effects of pH on liquid phase separation of macromolecules. Our model describes a mixture composed of water and macromolecules that exist in three different charge states and have a tendency to phase separate. This phase separation is affected by pH via a set of chemical reactions describing protonation and deprotonation of macromolecules, as well as self-ionization of water. We consider the simple case in which interactions are captured by Flory-Huggins interaction parameters corresponding to Debye screening lengths shorter than a nanometer, which is relevant to proteins inside biological cells under physiological conditions. We identify the conjugate thermodynamic variables at chemical equilibrium and discuss the effective free energy at fixed pH. First, we study phase diagrams as a function of macromolecule concentration and temperature at the isoelectric point of the macromolecules. We find a rich variety of phase diagram topologies, including multiple critical points, triple points, and first-order transition points. Second, we change the pH relative to the isoelectric point of the macromolecules and study how phase diagrams depend on pH. We find that these phase diagrams as a function of pH strongly depend on whether oppositely charged macromolecules or neutral macromolecules have a stronger tendency to phase separate. One key finding is that we predict the existence of a reentrant behavior as a function of pH. In addition, our model predicts that the region of phase separation is typically broader at the isoelectric point. This model could account for both in vitro phase separation of proteins as a function of pH and protein phase separation in yeast cells for pH values close to the isoelectric point of many cytosolic proteins.

Proteomic, biomechanical and functional analyses define neutrophil heterogeneity in systemic lupus erythematosus

Kathleen R. Bashant, Angel M. Aponte, Davide Randazzo, Paniz Rezvan Sangsari, Alexander J. T. Wood, Jack A. Bibby, Erin E. West, Arlette Vassallo, Zerai G. Manna, et al.

Objectives <br>Low-density granulocytes (LDGs) are a distinct subset of proinflammatory and vasculopathic neutrophils expanded in systemic lupus erythematosus (SLE). Neutrophil trafficking and immune function are intimately linked to cellular biophysical properties. This study used proteomic, biomechanical and functional analyses to further define neutrophil heterogeneity in the context of SLE.<br><br>Methods <br>Proteomic/phosphoproteomic analyses were performed in healthy control (HC) normal density neutrophils (NDNs), SLE NDNs and autologous SLE LDGs. The biophysical properties of these neutrophil subsets were analysed by real-time deformability cytometry and lattice light-sheet microscopy. A two-dimensional endothelial flow system and a three-dimensional microfluidic microvasculature mimetic (MMM) were used to decouple the contributions of cell surface mediators and biophysical properties to neutrophil trafficking, respectively.<br><br>Results <br>Proteomic and phosphoproteomic differences were detected between HC and SLE neutrophils and between SLE NDNs and LDGs. Increased abundance of type 1 interferon-regulated proteins and differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs. The cell surface of SLE LDGs was rougher than in SLE and HC NDNs, suggesting membrane perturbances. While SLE LDGs did not display increased binding to endothelial cells in the two-dimensional assay, they were increasingly retained/trapped in the narrow channels of the lung MMM.<br><br>Conclusions <br>Modulation of the neutrophil proteome and distinct changes in biophysical properties are observed alongside differences in neutrophil trafficking. SLE LDGs may be increasingly retained in microvasculature networks, which has important pathogenic implications in the context of lupus organ damage and small vessel vasculopathy.

Nanostructured alkali-metal vapor cells

Tom F. Cutler, William J. Hamlyn, Jan Renger, Kate A. Whittaker, Danielle Pizzey, Ifan G. Hughes, Vahid Sandoghdar, Charles S. Adams

Atom-light interactions in nano-scale systems hold great promise for novel technologies based on integrated emitters and optical modes. We present the design architecture, construction method,<br>and characterization of an all-glass alkali-metal vapor cell with nanometer-scale internal structure. Our cell has a glue-free design which allows versatile optical access, in particular with high numerical aperture optics. By performing spectroscopy in different illumination and detection schemes, we investigate atomic densities and velocity distributions in various nanoscopic landscapes. We apply a two-photon excitation scheme to atoms confined in one dimension within our cells, achieving a resonance line-width of 32 MHz in a counter-propagating geometry, and 57.5 MHz in a co-propagating geometry. Both of these are considerably narrower than the Doppler width (GHz), and are limited<br>by transit time broadening and velocity selection. We also demonstrate sub-Doppler line-widths for atoms confined in two dimensions to micron-sized channels. Furthermore, we illustrate control over vapor density within our cells through nano-scale confinement alone, which could offer a scalable route towards room-temperature devices with single atoms within an interaction volume. Our design offers a robust platform for miniaturized devices that could easily be combined with integrated<br>photonic circuits.

suggested by editors

Exogenous ethanol induces a metabolic switch that prolongs the survival of Caenorhabditis elegansdauer larva and enhances its resistance to desiccation

Damla Kaptan, Sider Penkov, Xingyu Zhang, Vamshidhar R. Gade, Bharath Kumar Raghuraman, Roberta Galli, Julio L. Sampaio, Robert Haase, Edmund Koch, et al.

The dauer larva ofCaenorhabditis elegans, destined to survive long periods of food scarcity and harsh environment, does not feed and has a very limited exchange of matter with the exterior. It was assumed that the survival time is determined by internal energy stores. Here, we show that ethanol can provide a potentially unlimited energy source for dauers by inducing a controlled metabolic shift that allows it to be metabolized into carbohydrates, amino acids, and lipids. Dauer larvae provided with ethanol survive much longer and have greater desiccation tolerance. On the cellular level, ethanol prevents the deterioration of mitochondria caused by energy depletion. By modeling the metabolism of dauers of wild-type and mutant strains with and without ethanol, we suggest that the mitochondrial health and survival of an organism provided with an unlimited source of carbon depends on the balance between energy production and toxic product(s) of lipid metabolism.

Buckling of an Epithelium Growing under Spherical Confinement

Anastasiya Trushko, Ilaria Di Meglio, Aziza Merzouki, Carles Blanch-Mercader, Shada Abuhattum, Jochen Guck, Kevin Alessandri, Pierre Nassoy, Karsten Kruse, et al.

Many organs are formed through folding of an epithelium. This change in shape is usually attributed to tissue heterogeneities, for example, local apical contraction. In contrast, compressive stresses have been proposed to fold a homogeneous epithelium by buckling. While buckling is an appealing mechanism, demonstrating that it underlies folding requires measurement of the stress field and the material properties of the tissue, which are currently inaccessible in vivo. Here, we show that monolayers of identical cells proliferating on the inner surface of elastic spherical shells can spontaneously fold. By measuring the elastic deformation of the shell, we infer the forces acting within the monolayer and its elastic modulus. Using analytical and numerical theories linking forces to shape, we find that buckling quantitatively accounts for the shape changes of our monolayers. Our study shows that forces arising from epithelial growth in three-dimensional confinement are sufficient to drive folding by buckling.

Partial cloaking of a gold particle by a single molecule

Johannes Zirkelbach, Benjamin Gmeiner, Jan Renger, Pierre Türschmann, Tobias Utikal, Stephan Götzinger, Vahid Sandoghdar

Extinction of light by material particles stems from losses incurred by absorption or scattering. The extinction cross section is usually treated as an additive quantity, leading to the exponential laws that govern the macroscopic attenuation of light. In this work, we demonstrate that the extinction cross section of a large gold nanoparticle can be substantially reduced, i.e., the particle becomes<br>more transparent, if a single molecule is placed in its near field. This partial cloaking eect results from a coherent plasmonic interaction between the molecule and the nanoparticle, whereby each of them acts as a nano-antenna to modify the radiative properties of the other.

suggested by editors

Quantum metamaterials with magnetic response at optical frequencies

Rasoul Alaee Khanghah, Burak Gürlek, Mohammad Albooyeh, Diego-Martin Cano, Vahid Sandoghdar

We propose novel quantum antennas and metamaterials with strong magnetic response at optical frequencies. Our design is based on the arrangement of natural atoms with only electric dipole transition moments at distances smaller than a wavelength of light but much larger than their physical size. In particular, we show that an atomic dimer can serve as a magnetic antenna at its antisymmetric mode to enhance the decay rate of a magnetic transition in its vicinity by several orders of magnitude. Furthermore, we study metasurfaces composed of atomic bilayers with and without cavities and show that they can fully reflect the electric and magnetic fields of light, thus, forming nearly perfect electric/magnetic mirrors. The proposed quantum metamaterials can be fabricated with available state-of-the-art technologies and promise several applications both in classical optics and quantum engineering.

suggested by editors

Point spread function in interferometric scattering microscopy (iSCAT). Part I: aberrations in defocusing and axial localization

Reza Gholami Mahmoodabadi, Richard W. Taylor, Martin Kaller, Susann Spindler, Mahdi Mazaheri, Kiarash Kasaian, Vahid Sandoghdar

Interferometric scattering (iSCAT) microscopy is an emerging label-free technique optimized for the sensitive detection of nano-matter. Previous iSCAT studies have approximated the point spread function in iSCAT by a Gaussian intensity distribution. However, recent efforts to track the mobility of nanoparticles in challenging speckle environments and over extended axial ranges has necessitated a quantitative description of the interferometric point spread function (iPSF). We present a robust vectorial diffraction model for the iPSF in tandem with experimental measurements and rigorous FDTD simulations. We examine the iPSF under various imaging scenarios to understand how aberrations due to the experimental configuration encode information about the nanoparticle. We show that the lateral shape of the iPSF can be used to achieve nanometric three-dimensional localization over an extended axial range on the order of 10 µm either by means of a fit to an analytical model or calibration-free unsupervised machine learning. Our results have immediate implications for three-dimensional single particle tracking in complex scattering media.

Truncated Metallo-Dielectric Omnidirectional Reflector: Collecting Single Photons in the Fundamental Gaussian Mode with 95% Efficiency

Wancong Li, Luis Morales-Inostroza, Weiwang Xu, Pu Zhang, Stephan Götzinger, Xue-Wen Chen

We propose a novel antenna structure that funnelssingle photons from a single emitter with unprecedented efficiencyinto a low-divergence fundamental Gaussian mode. Our devicerelies on the concept of creating an omnidirectional photonicbandgap to inhibit unwanted large-angle emission and to enhancesmall-angle defect-guided-mode emission. The new photoncollection strategy is intuitively illustrated, rigorously verified,and optimized by implementing an efficient, body-of-revolution,finite-difference, time-domain method for in-plane dipole emitters.We investigate a few antenna designs to cover various boundaryconditions posed by fabrication processes or material restrictions and theoretically demonstrate that collection efficiencies into thefundamental Gaussian mode exceeding 95% are achievable. Our antennas are broadband, insensitive to fabrication imperfections andcompatible with a variety of solid-state emitters such as organic molecules, quantum dots, and defect centers in diamond.Unidirectional and low-divergence Gaussian-mode emission from a single emitter may enable the realization of a variety of photonicquantum computer architectures as well as highly efficient light−matter interfaces.

Molecule-photon interactions in phononic environments

Michael Reitz, Christian Sommer, Burak Gürlek, Vahid Sandoghdar, Diego-Martin Cano, Claudiu Genes

Molecules constitute compact hybrid quantum optical systems that can interface photons, electronic degrees of freedom, localized mechanical vibrations, and phonons. In particular, the strong vibronic interaction between electrons and nuclear motion in a molecule resembles the optomechanical radiation pressure Hamiltonian. While molecular vibrations are often in the ground state even at elevated temperatures, one still needs to get a handle on decoherence channels associated with phonons before an efficient quantum optical network based on optovibrational interactions in solid-state molecular systems could be realized. As a step towards a better understanding of decoherence in phononic environments, we take here an open quantum system approach to the nonequilibrium dynamics of guest molecules embedded in a crystal, identifying regimes of Markovian versus non-Markovian vibrational relaxation. A stochastic treatment, based on quantum Langevin equations, predicts collective vibron-vibron dynamics that resembles processes of sub- and super-radiance for radiative transitions. This in turn leads to the possibility of decoupling intramolecular vibrations from the phononic bath, allowing for enhanced coherence times of collective vibrations. For molecular polaritonics in strongly confined geometries, we also show that the imprint of optovibrational couplings onto the emerging output field results in effective polariton cross-talk rates for finite bath occupancies.

Point spread function in interferometric scattering microscopy (iSCAT). Part I: aberrations in defocusing and axial localization

Reza Gholami Mahmoodabadi, Richard W. Taylor, Martin Kaller, Susann Spindler, Mahdi Mazaheri, Kiarash Kasaian, Vahid Sandoghdar

Interferometric scattering (iSCAT) microscopy is an emerging label-free technique optimized for the sensitive detection of nano-matter. Previous iSCAT studies have approximated the point spread function in iSCAT by a Gaussian intensity distribution. However, recent efforts to track the mobility of nanoparticles in challenging speckle environments and over extended axial ranges has necessitated a quantitative description of the interferometric point spread function (iPSF). We present a robust vectorial diffraction model for the iPSF in tandem with experimental measurements and rigorous FDTD simulations. We examine the iPSF under various imaging scenarios to understand how aberrations due to the experimental configuration encode information about the nanoparticle. We show that the lateral shape of the iPSF can be used to achieve nanometric three-dimensional localization over an extended axial range on the order of 10 µm either by means of a fit to an analytical model or calibration-free unsupervised machine learning. Our results have immediate implications for three-dimensional single particle tracking in complex scattering media.

Stretching and heating cells with light-nonlinear photothermal cell rheology

Constantin Huster, Devavrat Rekhade, Adina Hausch, Saeed Ahmed, Nicolas Hauck, Julian Thiele, Jochen Guck, Klaus Kroy, Gheorghe Cojoc

Stretching and heating are everyday experiences for skin and tissue cells. They are also standard procedures to reduce the risk for injuries in physical exercise and to relieve muscle spasms in physiotherapy. Here, we ask which immediate and long-term mechanical effects of such treatments are quantitatively detectable on the level of individual living cells. Combining versatile optical stretcher techniques with a well-tested mathematical model for viscoelastic polymer networks, we investigate the thermomechanical properties of suspended cells with a photothermal rheometric protocol that can disentangle fast transient and slow 'inelastic' components in the nonlinear mechanical response. We find that a certain minimum strength and duration of combined stretching and heating is required to induce long-lived alterations of the mechanical state of the cells, which then respond qualitatively differently to mechanical tests than after weaker/shorter treatments or merely mechanical preconditioning alone. Our results suggest a viable protocol to search for intracellular biomolecular signatures of the mathematically detected dissimilar mechanical response modes.

Sub-nanometer resolution in single-molecule photoluminescence imaging

Ben Yang, Gong Chen, Atif Ghafoor, Yufan Zhang, Yao Zhang, Yang Zhang, Yi Luo, Jinlong Yang, Vahid Sandoghdar, et al.

Ambitions to reach atomic resolution with light have been a major force in shaping nano-optics, whereby a central challenge is achieving highly localized optical fields. A promising approach employs plasmonic nanoantennas, but fluorescence quenching in the vicinity of metallic structures often imposes a strict limit on the attainable spatial resolution, and previous studies have reached only 8 nm resolution in fluorescence mapping. Here, we demonstrate spatially and spectrally resolved photolumines-cence imaging of a single phthalocyanine molecule coupled to nanocavity plasmons in a tunnelling junction with a spatial reso-lution down to ∼8 Å and locally map the molecular exciton energy and linewidth at sub-molecular resolution. This remarkable resolution is achieved through an exquisite nanocavity control, including tip-apex engineering with an atomistic protrusion, quenching management through emitter–metal decoupling and sub-nanometre positioning precision. Our findings provide new routes to optical imaging, spectroscopy and engineering of light–matter interactions at sub-nanometre scales.

Novel input polarisation independent endoscopic cross‐polarised optical coherence tomography probe

Katharina Blessing, Judith Schirmer, Gargi Sharma, Kanwarpal Singh

Lead by the original idea to perform noninvasive optical biopsies of various tissues, optical coherence tomography<br>found numerous medical applications<br>within the last two decades. The interference<br>based imaging technique opens the<br>possibility to visualise subcellular morphology up to an imaging depth of 3 mm<br>and up to micron level axial and lateral<br>resolution. The birefringence properties<br>of the tissue are visualisedwith enhanced<br>contrast using polarisation sensitive or<br>cross-polarised optical coherence tomography (OCT) techniques. Although, it<br>requires strict control over the polarisation states, resulting in several polarisation<br>controlling elements. In this work, we propose a novel input-polarisation independent endoscopic system based on cross-polarised OCT. We tested the feasibility of our approach by measuring the polarisation change from a quarter-wave plate for different rotational angles. Further performance tests reveal a lateral resolution of 30 μm and a sensitivity of 103 dB. Images of the human nail bed and cow muscle tissue demonstrate the potential of the system to measure structural and birefringence properties of the tissue endoscopically.

Mechanochemical Crosstalk Produces Cell-Intrinsic Patterning of the Cortex to Orient the Mitotic Spindle

Andrea Dimitracopoulos, Pragya Srivastava, Agathe Chaigne, Zaw Win, Roie Shlomovitz, Oscar M. Lancaster, Maël Le Berre, Matthieu Piel, Kristian Franze, et al.

Proliferating animal cells are able to orient their mitotic spindles along their interphase cell axis, setting up the axis of cell division, despite rounding up as they enter mitosis. This has previously been attributed to molecular memory and, more specifically, to the maintenance of adhesions and retraction fibers in mitosis [1-6], which are thought to act as local cues that pattern cortical Gαi, LGN, and nuclear mitotic apparatus protein (NuMA) [3, 7-18]. This cortical machinery then recruits and activates Dynein motors, which pull on astral microtubules to position the mitotic spindle. Here, we reveal a dynamic two-way crosstalk between the spindle and cortical motor complexes that depends on a Ran-guanosine triphosphate (GTP) signal [12], which is sufficient to drive continuous monopolar spindle motion independently of adhesive cues in flattened human cells in culture. Building on previous work [1, 12, 19-23], we implemented a physical model of the system that recapitulates the observed spindle-cortex interactions. Strikingly, when this model was used to study spindle dynamics in cells entering mitosis, the chromatin-based signal was found to preferentially clear force generators from the short cell axis, so that cortical motors pulling on astral microtubules align bipolar spindles with the interphase long cell axis, without requiring a fixed cue or a physical memory of interphase shape. Thus, our analysis shows that the ability of chromatin to pattern the cortex during the process of mitotic rounding is sufficient to translate interphase shape into a cortical pattern that can be read by the spindle, which then guides the axis of cell division.

Ultrahigh-speed imaging of rotational diffusion on a lipid bilayer

Mahdi Mazaheri, Jens Ehrig, Alexey Shkarin, Vasily Zaburdaev, Vahid Sandoghdar

We studied the rotational and translational diffusion of a single gold nanorod linked to a supported lipid bilayer with ultrahigh temporal resolution of two microseconds. By using a home-built polarization-sensitive dark-field microscope, we recorded particle trajectories with lateral precision of three nanometers and rotational precision of four degrees. The large number of trajectory points in our measurements allows us to characterize the statistics of rotational diffusion with unprecedented detail. Our data show apparent signatures of anomalous diffusion such as sublinear scaling of the mean-squared angular displacement and negative values of angular correlation function at small lag times. However, a careful analysis reveals that these effect stem from the residual noise contributions and confirms normal diffusion. Our experimental approach and observations can be extended to investigate diffusive processes of anisotropic nanoparticles in other fundamental systems such as cellular membranes or other two-dimensional fluids.

Prevalence of IgG and IgM antibodies to SARS-CoV-2 among clinic staff and patients

Marcus Inyama Asuquo, Emmanuel Effa, Akaninyene Otu, Okokon Ita, Ubong Udoh, Victor Umoh, Oluwabukola Gbotosho, Anthonia Ikpeme, Soter Ameh, et al.

The coronavirus disease 2019 (COVID-19) is now a pandemic with devastating social and economic consequences. The extent of the spread of COVID-19 within populations is uncertain since diagnostic tests have not been carried out on all eligible persons and doing such diagnostic tests on everyone is much less feasible in developing countries such as Nigeria. Tests for antibodies to SARS-CoV-2, the virus that causes COVID-19, are more affordable, readily available, and require minimal training than current diagnostic tests. Employing a seroepidemiological strategy, serological tests were conducted on 66 volunteering staff and patients at the University of Calabar Teaching Hospital (UCTH), a Federal Government owned tertiary healthcare facility, to determine the extent of exposure to SARS-CoV-2, from 17th to 25th June 2020. Using a COVID-19 IgG/IgM Rapid Test Cassette with emergency use authorization (EUA) from the Food and Drug Administration (FDA) of the United States, it was observed that of the 66 samples tested, 5 (7.6%) were both IgG and IgM positive and 17 (26%) were IgG positive. Moreover, for 44 of the 66 participants, simultaneous tests were carried out using a rapid test kit from a different manufacturer but without FDA-EUA and all the results completely matched with the FDA-EUA kit, except one case where the FDA-EUA kit showed positive for both IgG and IgM while the other kit was positive only for IgM. The 26% positive IgG indicates a high exposure rate for the hospital staff and patients and points to community transmission where the facility is situated. Hence, immediate activation of WHO guidelines for controlling community transmission is called for. These results can further serve as a pilot study to guide public health policies in response to COVID-19 pandemic in both the general population and in healthcare settings.<br>It is made available under a CC-BY-NC-ND 4.0 International license .<br>(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.<br>medRxiv preprint doi: https://doi.org/10.1101/2020.07.02.20145441; this version posted July 24, 2020. The copyright holder for this preprint

Towards brain-tissue-like biomaterials

Eneko Axpe, Gorka Orive, Kristian Franze, Eric A. Appel

Many biomaterials have been developed which aim to match the elastic modulus of the brain for improved interfacing. However, other properties such as ultimate toughness, tensile strength, poroviscoelastic responses, energy dissipation, conductivity, and mass diffusivity also need to be considered.

Spatial heterogeneity of cell-matrix adhesive forces predicts human glioblastoma migration

Rasha Rezk, Bill Zong Jia, Astrid Wendler, Ivan Dimov, Colin Watts, Athina E. Markaki, Kristian Franze, Alexandre J. Kabla

BACKGROUND: Glioblastoma (GBM) is a highly aggressive incurable brain tumor. The main cause of mortality in GBM patients is the invasive rim of cells migrating away from the main tumor mass and invading healthy parts of the brain. Although the motion is driven by forces, our current understanding of the physical factors involved in glioma infiltration remains limited. This study aims to investigate the adhesion properties within and between patients' tumors on a cellular level and test whether these properties correlate with cell migration.<br>METHODS: Six tissue samples were taken from spatially separated sections during 5-aminolevulinic acid (5-ALA) fluorescence-guided surgery. Navigated biopsy samples were collected from strongly fluorescent tumor cores, a weak fluorescent tumor rim, and nonfluorescent tumor margins. A microfluidics device was built to induce controlled shear forces to detach cells from monolayer cultures. Cells were cultured on low modulus polydimethylsiloxane representative of the stiffness of brain tissue. Cell migration and morphology were then obtained using time-lapse microscopy.<br>RESULTS: GBM cell populations from different tumor fractions of the same patient exhibited different migratory and adhesive behaviors. These differences were associated with sampling location and amount of 5-ALA fluorescence. Cells derived from weak- and nonfluorescent tumor tissue were smaller, adhered less well, and migrated quicker than cells derived from strongly fluorescent tumor mass.<br>CONCLUSIONS: GBM tumors are biomechanically heterogeneous. Selecting multiple populations and broad location sampling are therefore important to consider for drug testing.

Integrating Chemistry and Mechanics: The Forces Driving Axon Growth

Kristian Franze

The brain is our most complex organ. During development, neurons extend axons, which may grow over long distances along well-defined pathways to connect to distant targets. Our current understanding of axon pathfinding is largely based on chemical signaling by attractive and repulsive guidance cues. These cues instruct motile growth cones, the leading tips of growing axons, where to turn and where to stop. However, it is not chemical signals that cause motion-motion is driven by forces. Yet our current understanding of the mechanical regulation of axon growth is very limited. In this review, I discuss the origin of the cellular forces controlling axon growth and pathfinding, and how mechanical signals encountered by growing axons may be integrated with chemical signals. This mechanochemical cross talk is an important but often overlooked aspect of cell motility that has major implications for many physiological and pathological processes involving neuronal growth.

Nano-Optics in 2020 ± 20

Vahid Sandoghdar

Paclitaxel Drug-Coated Balloon Angioplasty Suppresses Progression and Inflammation of Experimental Atherosclerosis in Rabbits

Mohammed M. Chowdhury, Kanwarpal Singh, Mazen S. Albaghdadi, Haitham Khraishah, Adam Mauskapf, Chase W. Kessinger, Eric A. Osborn, Stephan Kellnberger, Zhonglie Piao, et al.

Paclitaxel drug-coated balloons (DCBs) reduce restenosis, but their overall safety has recently raised concerns. This study hypothesized that DCBs could lessen inflammation and reduce plaque progression. Using 25 rabbits with cholesterol feeding- and balloon injury-induced lesions, DCB-percutaneous transluminal angioplasty (PTA), plain PTA, or sham-PTA (balloon insertion without inflation) was investigated using serial intravascular near-infrared fluorescence−optical coherence tomography and serial intravascular ultrasound. In these experiments, DCB-PTA reduced inflammation and plaque burden in nonobstructive lesions compared with PTA or sham-PTA. These findings indicated the potential for DCBs to serve safely as regional anti-atherosclerosis therapy.

Temperature controlled high-throughput magnetic tweezers show striking difference in activation energies of replicating viral RNA-dependent RNA polymerases

Mona Seifert, Pauline van Nies, Flávia S. Papini, Jamie J. Arnold, Minna M. Poranen, Craig E. Cameron, Martin Depken, David Dulin

RNA virus survival depends on efficient viral genome replication, which is performed by the viral RNA dependent RNA polymerase (RdRp). The recent development of high throughput magnetic tweezers has enabled the simultaneous observation of dozens of viral RdRp elongation traces on kilobases long templates, and this has shown that RdRp nucleotide addition kinetics is stochastically interrupted by rare pauses of 1–1000 s duration, of which the short-lived ones (1–10 s) are the temporal signature of a low fidelity catalytic pathway. We present a simple and precise temperature controlled system for magnetic tweezers to characterize the replication kinetics temperature dependence between 25°C and 45°C of RdRps from three RNA viruses, i.e. the double-stranded RNA bacteriophage Φ6, and the positive-sense single-stranded RNA poliovirus (PV) and human rhinovirus C (HRV-C). We found that Φ6 RdRp is largely temperature insensitive, while PV and HRV-C RdRps replication kinetics are activated by temperature. Furthermore, the activation energies we measured for PV RdRp catalytic state corroborate previous estimations from ensemble pre-steady state kinetic studies, further confirming the catalytic origin of the short pauses and their link to temperature independent RdRp fidelity. This work will enable future temperature controlled study of biomolecular complex at the single molecule level.

DryMass: handling and analyzing quantitative phase microscopy images of spherical, cell-sized objects

Paul Müller, Gheorghe Cojoc, Jochen Guck

Quantitative phase imaging (QPI) is an established tool for the marker-free classification and quantitative characterization of biological samples. For spherical objects, such as cells in suspension, microgel beads, or liquid droplets, a single QPI image is sufficient to extract the radius and the average refractive index. This technique is invaluable, as it allows the characterization of large sample populations at high measurement rates. However, until now, no universal software existed that could perform this type of analysis. Besides the choice of imaging modality and the variety in imaging software, the main difficulty has been to automate the entire analysis pipeline from raw data to ensemble statistics.

Intelligent image-based deformation-assisted cell sorting with molecular specificity

Ahmad Ahsan Nawaz, Marta Urbanska, Maik Herbig, Martin Nötzel, Martin Kräter, Philipp Rosendahl, Christoph Herold, Nicole Töpfner, Markéta Kubánková, et al.

Although label-free cell sorting is desirable for providing pristine cells for further analysis or use, current approaches lack molecular specificity and speed. Here, we combine real-time fluorescence and deformability cytometry with sorting based on standing surface acoustic waves and transfer molecular specificity to image-based sorting using an efficient deep neural network. In addition to general performance, we demonstrate the utility of this method by sorting neutrophils from whole blood without labels.<br> Sorting RT-FDC combines real-time fluorescence and deformability cytometry with sorting based on standing surface acoustic waves to transfer molecular specificity to label-free, image-based cell sorting using an efficient deep neural network.

A comparison of microfluidic methods for high-throughput cell deformability measurements

Marta Urbanska, Hector E. Munoz, Josephine Shaw Bagnall, Oliver Otto, Scott R. Manalis, Dino Di Carlo, Jochen Guck

The mechanical phenotype of a cell is an inherent biophysical marker of its state and function, with many applications in basic and applied biological research. Microfluidics-based methods have enabled single-cell mechanophenotyping at throughputs comparable to those of flow cytometry. Here, we present a standardized cross-laboratory study comparing three microfluidics-based approaches for measuring cell mechanical phenotype: constriction-based deformability cytometry (cDC), shear flow deformability cytometry (sDC) and extensional flow deformability cytometry (xDC). All three methods detect cell deformability changes induced by exposure to altered osmolarity. However, a dose-dependent deformability increase upon latrunculin B-induced actin disassembly was detected only with cDC and sDC, which suggests that when exposing cells to the higher strain rate imposed by xDC, cellular components other than the actin cytoskeleton dominate the response. The direct comparison presented here furthers our understanding of the applicability of the different deformability cytometry methods and provides context for the interpretation of deformability measurements performed using different platforms.<br> This Analysis compares microfluidics-based methods for assessing mechanical properties of cells in high throughput.

High spatiotemporal resolution data from a custom magnetic tweezers instrument

Eugeniu Ostrofet, Flavia S. Papini, David Dulin

Gene expression is achieved by enzymes as RNA polymerases that translocate along nucleic acids with steps as small as a single base pair, i.e., 0.34 nm for DNA. Deciphering the complex biochemical pathway that describes the activity of such enzymes requires an exquisite spatiotemporal resolution. Magnetic tweezers are a powerful single molecule force spectroscopy technique that uses a camera-based detection to enable the simultaneous observation of hundreds of nucleic acid tethered magnetic beads at a constant force with subnanometer resolution [1,2]. High spatiotemporal resolution magnetic tweezers have recently been reported [3-5]. We present data acquired using a bespoke magnetic tweezers instrument that is able to perform either in high throughput or at high resolution. The data reports on the best achievable resolution for surface-attached polystyrene beads and DNA tethered magnetic beads, and highlights the influence of mechanical stability for such assay. We also present data where we are able to detect 0.3 nm steps along the z-axis using DNA tethered magnetic beads. Because the data presented here are in agreement with the best resolution obtained with magnetic tweezers, they provide a useful benchmark comparison for setup adjustment and optimization. (C) 2020 The Author(s). Published by Elsevier Inc.

Recent progress and current opinions in Brillouin Microscopy for life science application

Giuseppe Antonacci, Timon Beck, Alberto Bilenca, Jürgen Czarske, Kareem Elsayad, Jochen Guck, Kyoohyun Kim, Benedikt Krug, Francesca Palombo, et al.

Many important biological functions and processes are reflected in cell and tissue mechanical properties such as elasticity and viscosity. However, current techniques used for measuring these properties have major limitations, such as that they can often not measure inside intact cells and/or require physical contact—which cells can react to and change. Brillouin light scattering offers the ability to measure mechanical properties in a non-contact and label-free manner inside of objects with high spatial resolution using light, and hence has emerged as an attractive method during the past decade. This new approach, coined “Brillouin microscopy,” which integrates highly interdisciplinary concepts from physics, engineering, and mechanobiology, has led to a vibrant new community that has organized itself via a European funded (COST Action) network. Here we share our current assessment and opinion of the field, as emerged from a recent dedicated workshop. In particular, we discuss the prospects towards improved and more bio-compatible instrumentation, novel strategies to infer more accurate and quantitative mechanical measurements, as well as our current view on the biomechanical interpretation of the Brillouin spectra.

Cortical cell stiffness is independent of substrate mechanics

Johannes Rheinlaender, Andrea Dimitracopoulos, Bernhard Wallmeyer, Nils M. Kronenberg, Kevin J. Chalut, Malte C. Gather, Timo Betz, Guillaume Charras, Kristian Franze

Cortical stiffness is an important cellular property that changes during migration, adhesion and growth. Previous atomic force microscopy (AFM) indentation measurements of cells cultured on deformable substrates have suggested that cells adapt their stiffness to that of their surroundings. Here we show that the force applied by AFM to a cell results in a significant deformation of the underlying substrate if this substrate is softer than the cell. This 'soft substrate effect' leads to an underestimation of a cell's elastic modulus when analysing data using a standard Hertz model, as confirmed by finite element modelling and AFM measurements of calibrated polyacrylamide beads, microglial cells and fibroblasts. To account for this substrate deformation, we developed a 'composite cell-substrate model'. Correcting for the substrate indentation revealed that cortical cell stiffness is largely independent of substrate mechanics, which has major implications for our interpretation of many physiological and pathological processes.

RNA-Induced Conformational Switching and Clustering of G3BP Drive Stress Granule Assembly by Condensation

Jordina Guillén Boixet , Andrii Kopach , Alex S. Holehouse, Sina Wittmann , Marcus Jahnel, Raimund Schlüssler, Kyoohyun Kim, Irmela Trussina , Jie Wang , et al.

Stressed cells shut down translation, release mRNA molecules from polysomes, and form stress granules (SGs) via a network of interactions that involve G3BP. Here we focus on the mechanistic underpinnings of SG assembly. We show that, under non-stress conditions, G3BP adopts a compact auto-inhibited state stabilized by electrostatic intramolecular interactions between the intrinsically disordered acidic tracts and the positively charged arginine-rich region. Upon release from polysomes, unfolded mRNAs outcompete G3BP auto-inhibitory interactions, engendering a conformational transition that facilitates clustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP clusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins that promote SG maturation or induce a liquid-to-solid transition that may underlie disease. We propose that condensation coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule assembly.

Input polarization-independent polarization-sensitive optical coherence tomography using a depolarizer

Shivani Sharma, Georg Hartl, Sheeza K. Naveed, Katharina Blessing, Gargi Sharma, Kanwarpal Singh

Polarization-sensitive optical coherence tomography is gaining attention because of its ability to diagnose certain pathological conditions at an early stage. The majority of polarization-sensitive optical coherence tomography systems require a polarization controller and a polarizer to obtain the optimal polarization state of the light at the sample. Such systems are prone to misalignment since any movement of the optical fiber normally coupled to the light source will change the polarization state of the incident beam. We propose and demonstrate an input polarization-independent polarization-sensitive optical coherence tomography system using a depolarizer that works for any input polarization state of the light source. The change in the optical power at the sample for arbitrary input polarized light for the standard polarization-sensitive optical coherence tomography system was found to be approximately 84% compared to 9% for our proposed method. The developed system was used to measure the retardance and optical axis orientation of a quarter-wave plate and the obtained values matched closely to the expectation. To further demonstrate the capability of measuring the birefringent properties of biological samples, we also imaged the nail bed. We believe that the proposed system is a robust polarization-sensitive optical coherence tomography system and that it will improve the diagnostic capabilities in clinical settings.

The mechanics of myeloid cells

Kathleen R. Bashant, Nicole Toepfner, Christopher J. Day, Nehal N. Mehta, Mariana J. Kaplan, Charlotte Summers, Jochen Guck, Edwin R. Chilvers

The effects of cell size, shape and deformability on cellular function have long been a topic of interest. Recently, mechanical phenotyping technologies capable of analysing large numbers of cells in real time have become available. This has important implications for biology and medicine, especially haemato-oncology and immunology, as immune cell mechanical phenotyping, immunologic function, and malignant cell transformation are closely linked and potentially exploitable to develop new diagnostics and therapeutics. In this review, we introduce the technologies used to analyse cellular mechanical properties and review emerging findings following the advent of high throughput deformability cytometry. We largely focus on cells from the myeloid lineage, which are derived from the bone marrow and include macrophages, granulocytes and erythrocytes. We highlight advances in mechanical phenotyping of cells in suspension that are revealing novel signatures of human blood diseases and providing new insights into pathogenesis of these diseases. The contributions of mechanical phenotyping of cells in suspension to our understanding of drug mechanisms, identification of novel therapeutics and monitoring of treatment efficacy particularly in instances of haematologic diseases are reviewed, and we suggest emerging topics of study to explore as high throughput deformability cytometers become prevalent in laboratories across the globe.

Ensemble-induced strong light-matter coupling of a single quantum emitter

Stefan Schütz, Johannes Schachenmayer, David Hagenmüller, Gavin K. Brennen, Thomas Volz, Vahid Sandoghdar, Thomas W. Ebbesen, Claudiu Genes, Guido Pupillo

We discuss a technique to strongly couple a single target quantum emitter to a cavity mode, which is enabled by virtual excitations of a nearby mesoscopic ensemble of emitters. A collective coupling of the latter to both the cavity and the target emitter induces strong photon nonlinearities in addition to polariton formation, in contrast to common schemes for ensemble strong coupling. We demonstrate that strong coupling at the level of a single emitter can be engineered via coherent and dissipative dipolar interactions with the ensemble, and provide realistic parameters for a possible implementation with <br>SiV− defects in diamond. Our scheme can find applications, amongst others, in quantum information processing or in the field of cavity-assisted quantum chemistry.

Roadmap on quantum light spectroscopy

Shaul Mukamel, Matthias Freyberger, Wolfgang Schleich, Marco Bellini, Alessandro Zavatta, Gerd Leuchs, Christine Silberhorn, Robert W. Boyd, Luis Lorenzo Sánchez-Soto, et al.

Conventional spectroscopy uses classical light to detect matter properties through the variation<br>of its response with frequencies or time delays. Quantum light opens up new avenues for<br>spectroscopy by utilizing parameters of the quantum state of light as novel control knobs and<br>through the variation of photon statistics by coupling to matter. This Roadmap article focuses on<br>using quantum light as a powerful sensing and spectroscopic tool to reveal novel information<br>about complex molecules that is not accessible by classical light. It aims at bridging the quantum<br>optics and spectroscopy communities which normally have opposite goals: manipulating<br>complex light states with simple matter e.g. qubits versus studying complex molecules with<br>simple classical light, respectively. Articles cover advances in the generation and manipulation<br>of state-of-the-art quantum light sources along with applications to sensing, spectroscopy,<br>imaging and interferometry.

Mechanical Regulation of Neurite Polarization and Growth: A Computational Study

Maximilian A. H. Jakobs, Kristian Franze, Assaf Zemel

The densely packed microtubule (MT) array found in neuronal cell projections (neurites) serves two fundamental functions simultaneously: it provides a mechanically stable track for molecular motor-based transport and produces forces that drive neurite growth. The local pattern of MT polarity along the neurite shaft has been found to differ between axons and dendrites. In axons, the neurons' dominating long projections, roughly 90% of the MTs orient with their rapidly growing plus end away from the cell body, whereas in vertebrate dendrites, their orientations are locally mixed. Molecular motors are known to be responsible for cytoskeletal ordering and force generation, but their collective function in the dense MT cytoskeleton of neurites remains elusive. We here hypothesized that both the polarity pattern of MTs along the neurite shaft and the shaft's global extension are simultaneously driven by molecular motor forces and should thus be regulated by the mechanical load acting on the MT array as a whole. To investigate this, we simulated cylindrical bundles of MTs that are cross-linked and powered by molecular motors by iteratively solving a set of force-balance equations. The bundles were subjected to a fixed load arising from actively generated tension in the actomyosin cortex enveloping the MTs. The magnitude of the load and the level of motor-induced connectivity between the MTs have been varied systematically. With an increasing load and decreasing motor-induced connectivity between MTs, the bundles became wider in cross section and extended more slowly, and the local MT orientational order was reduced. These results reveal two, to our knowledge, novel mechanical factors that may underlie the distinctive development of the MT cytoskeleton in axons and dendrites: the cross-linking level of MTs by motors and the load acting on this cytoskeleton during growth.

Oncogenic signaling alters cell shape and mechanics to facilitate cell division under confinement

Helen K Matthews, Sushila Ganguli, Katarzyna Plak, Anna V. Taubenberger, Zaw Win, Max Williamson, Matthieu Piel, Jochen Guck, Buzz Baum

To divide in a tissue, both normal and cancer cells become spherical and mechanically stiffen as they enter mitosis. We investigated the effect of oncogene activation on this process in normal epithelial cells. We found that short-term induction of oncogenic RasV12 activates downstream mitogen-activated protein kinase (MEK-ERK) signaling to alter cell mechanics and enhance mitotic rounding, so that RasV12-expressing cells are softer in interphase but stiffen more upon entry into mitosis. These RasV12-dependent changes allow cells to round up and divide faithfully when confined underneath a stiff hydrogel, conditions in which normal cells and cells with reduced levels of Ras-ERK signaling suffer multiple spindle assembly and chromosome segregation errors. Thus, by promoting cell rounding and stiffening in mitosis, oncogenic RasV12 enables cells to proliferate under conditions of mechanical confinement like those experienced by cells in crowded tumors.

Swept source cross-polarized optical coherence tomography for any input polarized light

Gargi Sharma, Shivani Sharma, Katharina Blessing, Georg Hartl, Maximilian Waldner, Kanwarpal Singh

Cross polarized optical coherence tomography offers enhanced contrast in certain<br>pathological conditions. Traditional cross-polarized optical coherence tomography systems<br>require a defined input polarization and thus require several polarization controlling elements<br>increasing the overall complexity of the system. Our proposed system requires a single<br>quarter wave plate as a polarization controller thus simplifying the system significantly.<br>Majority of Cross-polarized optical coherence tomography systems are spectrometer based<br>which suffers from slow speed and low signal to noise ratio. In this work, we present a swept<br>source based cross-polarized optical coherence tomography system that works for any input<br>polarization state. The system was tested against known birefringent materials such as quarter<br>wave plate. Furthermore, biological samples such as finger, nail and chicken breast were<br>imaged to demonstrate the potential of our technique.

Defining the Adult Neural Stem Cell Niche Proteome Identifies Key Regulators of Adult Neurogenesis

Jacob Kjell, Judith Fischer-Sternjak, Amelia J. Thompson, Christian Friess, Matthew J. Sticco, Favio Salinas, Jürgen Cox, David C. Martinelli, Jovica Ninkovic, et al.

The mammalian brain contains few niches for neural stem cells (NSCs) capable of generating new neurons, whereas other regions are primarily gliogenic. Here we leverage the spatial separation of the sub-ependymal zone NSC niche and the olfactory bulb, the region to which newly generated neurons from the sub-ependymal zone migrate and integrate, and present a comprehensive proteomic characterization of these regions in comparison to the cerebral cortex, which is not conducive to neurogenesis and integration of new neurons. We find differing compositions of regulatory extracellular matrix (ECM) components in the neurogenic niche. We further show that quiescent NSCs are the main source of their local ECM, including the multi-functional enzyme transglutaminase 2, which we show is crucial for neurogenesis. Atomic force microscopy corroborated indications from the proteomic analyses that neurogenic niches are significantly stiffer than non-neurogenic parenchyma. Together these findings provide a powerful resource for unraveling unique compositions of neurogenic niches.

The Costs of Close Contacts: Visualizing the Energy Landscape of Cell Contacts at the Nanoscale

Klara Kulenkampff, Anna H. Lippert, James McColl, Ana Mafalda Santos, Aleks Ponjavic, Edward Jenkins, Jane Humphrey, Alexander Winkel, Kristian Franze, et al.

Cell-cell contacts often underpin signaling between cells. For immunology, the binding of a T cell receptor to an antigen-presenting pMHC initiates downstream signaling and an immune response. Although this contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm, many, often contradictory observations have been made regarding the influence of the contact on parameters such as the binding kinetics, spatial distribution, and diffusion of signaling proteins within the contact. Understanding the basic physical constraints on probes inside this crowded environment will help inform studies on binding kinetics and dynamics of signaling of relevant proteins in the synapse. By tracking quantum dots of different dimensions for extended periods of time, we have shown that it is possible to obtain the probability of a molecule entering the contact, the change in its diffusion upon entry, and the impact of spatial heterogeneity of adhesion protein density in the contact. By analyzing the contacts formed by a T cell interacting with adhesion proteins anchored to a supported lipid bilayer, we find that probes are excluded from contact entry in a size-dependent manner for gap-to-probe differences of 4.1 nm. We also observed probes being trapped inside the contact and a decrease in diffusion of up to 85% in dense adhesion protein contacts. This approach provides new, to our knowledge, insights into the nature of cell-cell contacts, revealing that cell contacts are highly heterogeneous because of topography- and protein-density-related processes. These effects are likely to profoundly influence signaling between cells.

Zebrafish spinal cord repair is accompanied by transient tissue stiffening

Stephanie Möllmert, Maria A. Kharlamova, Tobias Hoche, Anna V. Taubenberger, Shada Abuhattum, Veronika Kuscha, Thomas Kurth, Michael Brand, Jochen Guck

Severe injury to the mammalian spinal cord results in permanent loss of function due to the formation of a glial-fibrotic scar. Both the chemical composition and the mechanical properties of the scar tissue have been implicated to inhibit neuronal regrowth and functional recovery. By contrast, adult zebrafish are able to repair spinal cord tissue and restore motor function after complete spinal cord transection owing to a complex cellular response that includes neurogenesis and axon regrowth. The mechanical mechanisms contributing to successful spinal cord repair in adult zebrafish are, however, currently unknown. Here, we employ AFM-enabled nano-indentation to determine the spatial distributions of apparent elastic moduli of living spinal cord tissue sections obtained from uninjured zebrafish and at distinct time points after complete spinal cord transection. In uninjured specimens, spinal gray matter regions were stiffer than white matter regions. During regeneration after transection, the spinal cord tissues displayed a significant increase of the respective apparent elastic moduli that transiently obliterated the mechanical difference between the two types of matter, before returning to baseline values after completion of repair. Tissue stiffness correlated variably with cell number density, oligodendrocyte interconnectivity, axonal orientation, and vascularization. The presented work constitutes the first quantitative mapping of the spatio-temporal changes of spinal cord tissue stiffness in regenerating adult zebrafish and provides the tissue mechanical basis for future studies into the role of mechanosensing in spinal cord repair.

The mechanics of myeloid cells

Kathleen R. Bashant, Nicole Toepfner, Christopher J. Day, Nehal N. Mehta, Mariana J. Kaplan, Charlotte Summers, Jochen Guck, Edwin A Chilvers

The effects of cell size, shape and deformability on cellular function have long been a topic of interest. Recently, mechanical phenotyping technologies capable of analysing large numbers of cells in real time have become available. This has important implications for biology and medicine, especially haemato‐oncology and immunology, as immune cell mechanical phenotyping, immunologic function, and malignant cell transformation are closely linked and potentially exploitable to develop new diagnostics and therapeutics. In this review, we introduce the technologies used to analyse cellular mechanical properties and review emerging findings following the advent of high throughput deformability cytometry. We largely focus on cells from the myeloid lineage, which are derived from the bone marrow and include macrophages, granulocytes and erythrocytes. We highlight advances in mechanical phenotyping of cells in suspension that are revealing novel signatures of human blood diseases and providing new insights into pathogenesis of these diseases. The contributions of mechanical phenotyping of cells in suspension to our understanding of drug mechanisms, identification of novel therapeutics and monitoring of treatment efficacy particularly in instances of haematologic diseases are reviewed, and we suggest emerging topics of study to explore as high throughput deformability cytometers become prevalent in laboratories across the globe.