Estimation of the mass density of biological matter from refractive index measurements

Conrad Möckel, Timon Beck, Sara Kaliman, Shada Abuhattum, Kyoohyun Kim, Julia Kolb, Daniel Wehner, Vasily Zaburdaev, Jochen Guck

The quantification of physical properties of biological matter gives rise to novel ways of understanding functional mechanisms by utilizing models that explicitly depend on physical observables. One of the basic biophysical properties is the mass density (MD), which determines the degree of<br>crowdedness. It impacts the dynamics in subcellular compartments and further plays a major role in defining the opto-acoustical properties of cells and tissues. As such, the MD can be connected to the refractive index (RI) via the well known Lorentz-Lorenz relation, which takes into account the polarizability of matter. However, computing the MD based on RI measurements poses a challenge as it requires detailed knowledge of the biochemical composition of the sample. Here we propose<br>a methodology on how to account for a priori and a posteriori assumptions about the biochemical composition of the sample as well as respective RI measurements. To that aim, we employ the Biot mixing rule of RIs alongside the assumption of volume additivity to find an approximate relation of MD and RI. We use Monte-Carlo simulations as well as Gaussian propagation of uncertainty to obtain approximate analytical solutions for the respective uncertainties of MD and RI. We validate this approach by applying it to a set of well characterized complex mixtures given by bovine milk and intralipid emulsion. Further, we employ it to estimate the mass density of trunk tissue of living zebrafish (Danio rerio) larvae. Our results enable quantifying changes of mass density estimates based on variations in the a priori assumptions. This illustrates the importance of implementing this methodology not only for MD estimations but for many other related biophysical problems, such as<br>mechanical measurements using Brillouin microscopy and transient optical coherence elastography.

Membrane to cortex attachment determines different mechanical phenotypes in LGR5+ and LGR5- colorectal cancer cells

Sefora Conti, Valeria Venturini, Adrià Cañellas-Socias, Carmen Cortina, Juan F. Abenza, Camille Stephan-Otto Attolini, Emily Middendorp Guerra, Catherine Xu, Jia Hui Li, et al.

Colorectal cancer tumors are composed of heterogeneous and plastic cell populations, including a pool of cancer stem cells that express LGR5. Whether these distinct cell populations display different mechanical properties, and how these properties might contribute to metastasis is unknown. Using CRC patient derived organoids (PDOs), we found that compared to LGR5- cells,<br>LGR5+ cancer stem cells are stiffer, adhere better to the extracellular matrix (ECM), move slower both as single cells and clusters, display higher nuclear YAP, show a higher survival rate in response to mechanical confinement, and form larger transendothelial gaps. These differences are largely explained by the downregulation of the membrane to cortex attachment proteins Ezrin/Radixin/Moesin (ERMs) in the LGR5+ cells. By analyzing scRNA-seq expression patterns from a patient cohort, we show that this downregulation is a robust signature of colorectal tumors. Our results show that LGR5- cells display a mechanically dynamic phenotype suitable for<br>dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and<br>resilient phenotype suitable for extravasation and metastatic growth.

Insights into protein structure using cryogenic light microscopy

Hisham Mazal, Franz Wieser, Vahid Sandoghdar

Fluorescence microscopy has witnessed many clever innovations in the last two decades, leading to new methods such as structured illumination and super-resolution microscopies. The attainable resolution in biological samples is, however, ultimately limited by residual motion within the sample or in the microscope setup. Thus, such experiments are typically performed on chemically fixed samples. Cryogenic light microscopy (Cryo-LM) has been investigated as an alternative, drawing on various preservation techniques developed for cryogenic electron microscopy (Cryo-EM). Moreover, this approach offers a powerful platform for correlative microscopy. Another key advantage of Cryo-LM is the strong reduction in photobleaching at low temperatures, facilitating the collection of orders of magnitude more photons from a single fluorophore. This results in much higher localization precision, leading to Angstrom resolution. In this review, we discuss the general development and progress of Cryo-LM with an emphasis on its application in harnessing structural information on proteins and protein complexes.<br>

Bile Is a Selective Elevator for Mucosal Mechanics and Transport

Simon Hanio, Stephanie Möllmert, Conrad Möckel, Susobhan Choudhury, Andreas I. Höpfel, Theresa Zorn, Sebastian Endres, Jonas Schlauersbach, Lena Scheller, et al.

Mucus mechanically protects the intestinal epithelium and impacts the absorption of drugs, with a largely unknown role for bile. We explored the impacts of bile on mucosal biomechanics and drug transport within mucus. Bile diffused with square-root-of-time kinetics and interplayed with mucus, leading to transient stiffening captured in Brillouin images and a concentration-dependent change from subdiffusive to Brownian-like diffusion kinetics within the mucus demonstrated by differential dynamic microscopy. Bile-interacting drugs, Fluphenazine and Perphenazine, diffused faster through mucus in the presence of bile, while Metoprolol, a drug with no bile interaction, displayed consistent diffusion. Our findings were corroborated by rat studies, where co-dosing of a bile acid sequestrant substantially reduced the bioavailability of Perphenazine but not Metoprolol. We clustered over 50 drugs based on their interactions with bile and mucin. Drugs that interacted with bile also interacted with mucin but not vice versa. This study detailed the dynamics of mucus biomechanics under bile exposure and linked the ability of a drug to interact with bile to its abbility to interact with mucus.

Small leucine-rich proteoglycans inhibit CNS regeneration by modifying the structural and mechanical properties of the lesion environment

Julia Kolb, Vasiliki Tsata, Nora John, Kyoohyun Kim, Conrad Möckel, Gonzalo Rosso, Veronika Kurbel, Asha Parmar, Gargi Sharma, et al.

Extracellular matrix (ECM) deposition after central nervous system (CNS) injury leads to inhibitory scarring in humans and other mammals, whereas it facilitates axon regeneration in the zebrafish. However, the molecular basis of these different fates is not understood. Here, we identify small leucine-rich proteoglycans (SLRPs) as a contributing factor to regeneration failure in mammals. We demonstrate that the SLRPs chondroadherin, fibromodulin, lumican, and prolargin are enriched in rodent and human but not zebrafish CNS lesions. Targeting SLRPs to the zebrafish injury ECM inhibits axon regeneration and functional recovery. Mechanistically, we find that SLRPs confer mechano-structural properties to the lesion environment that are adverse to axon growth. Our study reveals SLRPs as inhibitory ECM factors that impair axon regeneration by modifying tissue mechanics and structure, and identifies their enrichment as a feature of human brain and spinal cord lesions. These findings imply that SLRPs may be targets for therapeutic strategies to promote CNS regeneration.

Varying the Stiffness and Diffusivity of Rod-Shaped Microgels Independently through Their Molecular Building Blocks

Yonca Kittel, Luis P. B. Guerzoni, Carolina Itzin, Dirk Rommel, Matthias Mork, Céline Bastard, Bernhard Häßel, Abdolrahman Omidinia-Anarkoli, Silvia P. Centeno, et al.

Microgels are water-swollen, crosslinked polymers that are widely used as colloidal building blocks in scaffold materials for tissue engineering and regenerative medicine. Microgels can be controlled in their stiffness, degree of swelling, and mesh size depending on their polymer architecture, crosslink density, and fabrication method – all of which influence their function and interaction with the environment. Currently, there is a lack of understanding of how the polymer composition influences the internal structure of soft microgels and how this morphology affects specific biomedical applications. In this report, we systematically vary the architecture and molar mass of polyethylene glycol-acrylate (PEG-Ac) precursors, as well as their concentration and combination, to gain insight in the different parameters that affect the internal structure of rod-shaped microgels. We characterize the mechanical properties and diffusivity, as well as the conversion of acrylate groups during photopolymerization, in both bulk hydrogels and microgels produced from the PEG-Ac precursors. Furthermore, we investigate cell-microgel interaction, and we observe improved cell spreading on microgels with more accessible RGD peptide and with a stiffness in a range of 20 kPa to 50 kPa lead to better cell growth.

Conserved nucleocytoplasmic density homeostasis drives cellular organization across eukaryotes

Abin Biswas, Omar Munos, Kyoohyun Kim, Carsten Hoege, Benjamin M. Lorton, David Shechter, Jochen Guck, Vasily Zaburdaev, Simone Reber

The packing and confinement of macromolecules in the cytoplasm and nucleoplasm has profound implications for cellular biochemistry. How intracellular density distributions vary and affect cellular physiology remains largely unknown. Here, we show that the nucleus is less dense than the cytoplasm and that living systems establish and maintain a constant density ratio between these compartments. Using label-free biophotonics and theory, we show that nuclear density is set by a pressure balance across the nuclear envelope in vitro, in vivo and during early development. Nuclear transport establishes a specific nuclear proteome that exerts a colloid osmotic pressure, which, assisted by entropic chromatin pressure, draws water into the nucleus. Using C. elegans, we show that while nuclear-to-cytoplasmic (N/C) volume ratios change during early development, the N/C density ratio is robustly maintained. We propose that the maintenance of a constant N/C density ratio is the biophysical driver of one of the oldest tenets of cell biology: the N/C volume ratio. In summary, this study reveals a previously unidentified homeostatic coupling of macromolecular densities that drives cellular organization with implications for pathophysiologies such as senescence and cancer.

Label-free composition determination for biomolecular condensates with an arbitrarily large number of components

Patrick McCall, Kyoohyun Kim, Martine Ruer-Gruß, Jan Peychl, Jochen Guck, Anthony A. Hyman, Jan Brugués

Biomolecular condensates are membrane-less organelles made of multiple components, often including several distinct proteins and nucleic acids. However, current tools to measure condensate composition are limited and cannot capture this complexity quantitatively, as they either require fluorescent labels, which we show can perturb composition, or can distinguish only 1-2 components. Here, we describe a label-free method based on quantitative phase microscopy to measure the composition of condensates with an arbitrarily large number of components. We first validate the method empirically in binary mixtures, revealing sequence-encoded density variation and complex aging dynamics for condensates composed of full-length proteins. In simplified multi-component protein/RNA condensates, we uncover a regime of constant condensate density and a large range of protein:RNA stoichiometry when varying average composition. The unexpected decoupling of density and composition highlights the need to determine molecular stoichiometry in multi-component condensates. We foresee this approach enabling the study of compositional regulation of condensate properties and function.

Human T cells loaded with superparamagnetic iron oxide nanoparticles retain antigen-specific TCR functionality

Felix Pfister, Jan Dörrie, Niels Schaft, Vera Buchele, Harald Unterweger, Lucas R. Carnell, Patrick Schreier, Rene Stein, Markéta Kubánková, et al.

BACKGROUND: Immunotherapy of cancer is an emerging field with the potential to improve long-term survival. Thus far, adoptive transfer of tumor-specific T cells represents an effective treatment option for tumors of the hematological system such as lymphoma, leukemia or myeloma. However, in solid tumors, treatment efficacy is low owing to the immunosuppressive microenvironment, on-target/off-tumor toxicity, limited extravasation out of the blood vessel, or ineffective trafficking of T cells into the tumor region. Superparamagnetic iron oxide nanoparticles (SPIONs) can make cells magnetically controllable for the site-specific enrichment. METHODS: In this study, we investigated the influence of SPION-loading on primary human T cells for the magnetically targeted adoptive T cell therapy. For this, we analyzed cellular mechanics and the T cell response after stimulation via an exogenous T cell receptor (TCR) specific for the melanoma antigen MelanA or the endogenous TCR specific for the cytomegalovirus antigen pp65 and compared them to T cells that had not received SPIONs. RESULTS: SPION-loading of human T cells showed no influence on cellular mechanics, therefore retaining their ability to deform to external pressure. Additionally, SPION-loading did not impair the T cell proliferation, expression of activation markers, cytokine secretion, and tumor cell killing after antigen-specific activation mediated by the TCR. CONCLUSION: In summary, we demonstrated that SPION-loading of T cells did not affect cellular mechanics or the functionality of the endogenous or an exogenous TCR, which allows future approaches using SPIONs for the magnetically enrichment of T cells in solid tumors.

Highly Nonlinear Dynamics of In Vivo Deep-Tissue Interaction with Femtosecond Laser Pulses at 1030 nm

Soyeon Jun, Andreas Herbst, Kilian Scheffter, Nora John, Julia Kolb, Daniel Wehner, Hanieh Fattahi

We report on the highly nonlinear behavior observed in the central nervous system tissue of zebrafish (Danio rerio) when exposed to femtosecond pulses at 1030 nm. At this irradiation wavelength, photo damage becomes detectable only after exceeding a specific peak intensity threshold, which is independent of the photon flux and irradiation time, distinguishing it from irradiation at shorter wavelengths. Furthermore, we investigate and quantify the role of excessive heat in reducing the damage threshold, particularly during high-repetition-rate operations, which are desirable for label-free and multi-dimensional microscopy techniques. To verify our findings, we examined cellular responses to tissue damage, including apoptosis and the recruitment of macrophages and fibroblasts at different time points post-irradiation. These findings substantially contribute to advancing the emerging nonlinear optical microscopy techniques and provide a strategy for inducing deep-tissue, precise and localized injuries using near-infrared femtosecond laser pulses.

IL-3 receptor signalling suppresses chronic intestinal inflammation by controlling mechanobiology and tissue egress of regulatory T cells

Karen Anne-Marie Ullrich, Julia Derdau, Carsten Baltes, Alice Battistella, Gonzalo Rosso, Stefan Uderhardt, Lisa Lou Schulze, Li-Juan Liu, Mark Dedden, et al.

IL-3 has been reported to be involved in various inflammatory disorders, but its role in inflammatory bowel disease (IBD) has not been addressed so far. Here, we determined IL-3 expression in samples from patients with IBD and studied the impact of Il3 or Il3r deficiency on T cell-dependent experimental colitis. We explored the mechanical, cytoskeletal and migratory properties of Il3r −/− and Il3r +/+ T cells using real-time deformability cytometry, atomic force microscopy, scanning electron microscopy, fluorescence recovery after photobleaching and in vitro and in vivo cell trafficking assays. We observed that, in patients with IBD, the levels of IL-3 in the inflamed mucosa were increased. In vivo, experimental chronic colitis on T cell transfer was exacerbated in the absence of Il-3 or Il-3r signalling. This was attributable to Il-3r signalling-induced changes in kinase phosphorylation and actin cytoskeleton structure, resulting in increased mechanical deformability and enhanced egress of Tregs from the inflamed colon mucosa. Similarly, IL-3 controlled mechanobiology in human Tregs and was associated with increased mucosal Treg abundance in patients with IBD. Collectively, our data reveal that IL-3 signaling exerts an important regulatory role at the interface of biophysical and migratory T cell features in intestinal inflammation and suggest that this might be an interesting target for future intervention.

Label-free discrimination of extracellular vesicles from large lipoproteins

Anna D. Kashkanova, Martin Blessing, Marie Reischke, Jan-Ole Baur, Andreas S. Baur, Vahid Sandoghdar, Jan Van Deun

Extracellular vesicles (EVs) are increasingly gaining interest as biomarkers and therapeutics. Accurate sizing and quantification of EVs remain problematic, given their nanometre size range and small scattering cross-sections. This is compounded by the fact that common EV isolation methods result in co-isolation of particles with comparable features. Especially in blood plasma, similarly-sized lipoproteins outnumber EVs to a great extent. Recently, interferometric nanoparticle tracking analysis (iNTA) was introduced as a particle analysis method that enables determining the size and refractive index of nanoparticles with high sensitivity and precision. In this work, we apply iNTA to differentiate between EVs and lipoproteins, and compare its performance to conventional nanoparticle tracking analysis (NTA). We show that iNTA can accurately quantify EVs in artificial EV-lipoprotein mixtures and in plasma-derived EV samples of varying complexity. Conventional NTA could not report on EV numbers, as it was not able to distinguish EVs from lipoproteins. iNTA has the potential to become a new standard for label-free EV characterization in suspension.

Revolutionizing microfluidics with artificial intelligence: a new dawn for lab-on-a-chip technologies

Keisuke Goda, Hang Lu, Peng Fei, Jochen Guck

De novo identification of universal cell mechanics gene signatures

Marta Urbanska, Yan Ge, Maria Winzi, Shada Abuhattum Hofemeier, Syed Shafat Ali, Maik Herbig, Martin Kräter, Nicole Toepfner, Joanna Durgan, et al.

Cell mechanical properties determine many physiological functions, such as cell fate specification, migration, or circulation through vasculature. Identifying factors that govern the mechanical properties is therefore a subject of great interest. Here we present a mechanomics approach for establishing links between single-cell mechanical phenotype changes and the genes involved in driving them. We combine mechanical characterization of cells across a variety of mouse and human systems with machine learning-based discriminative network analysis of associated transcriptomic profiles to infer a conserved network module of five genes with putative roles in cell mechanics regulation. We validate in silico that the identified gene markers are universal, trustworthy and specific to the mechanical phenotype, and demonstrate experimentally that a selected target, CAV1, changes the mechanical phenotype of cells accordingly when silenced or overexpressed. Our data-driven approach paves the way towards engineering cell mechanical properties on demand to explore their impact on physiological and pathological cell functions.

Alcohol-sourced acetate impairs T cell function by promoting cortactin acetylation

Vugar Azizov, Michael Hübner, Michael Frech, Jörg Hofmann, Markéta Kubánková, Dennis Lapuente, Matthias Tenbusch, Jochen Guck, Georg Schett, et al.

Alcohol is among the most widely consumed dietary substances. Excessive alcohol consumption damages the liver, heart, and brain. Alcohol also has strong immunoregulatory properties. Here, we report how alcohol impairs T cell function via acetylation of cortactin, a protein that binds filamentous actin and facilitates branching. Upon alcohol consumption, acetate, the metabolite of alcohol, accumulates in lymphoid organs. T cells exposed to acetate, exhibit increased acetylation of cortactin. Acetylation of cortactin inhibits filamentous actin binding and hence reduces T cell migration, immune synapse formation and activation. While mutated, acetylation-resistant cortactin rescues the acetate-induced inhibition of T cell migration, primary mouse cortactin knockout T cells exhibited impaired migration. Acetate-induced cytoskeletal changes effectively inhibited activation, proliferation, and immune synapse formation in T cells in vitro and in vivo in an influenza infection model in mice. Together these findings reveal cortactin as a possible target for mitigation of T cell driven autoimmune diseases.

Identification of a Distinct Monocyte-Driven Signature in Systemic Sclerosis Using Biophysical Phenotyping of Circulating Immune Cells

Alexandru-Emil Matei, Markéta Kubánková, Liyan Xu, Andrea-Hermina Györfi, Evgenia Boxberger, Despina Soteriou, Maria Papava, Julia Prater, Xuezhi Hong, et al.

OBJECTIVE: Pathologically activated circulating immune cells, including monocytes, play major roles in systemic sclerosis (SSc). Their functional characterization can provide crucial information with direct clinical relevance. However, tools for the evaluation of pathologic immune cell activation and, in general, of clinical outcomes in SSc are scarce. Biophysical phenotyping (including characterization of cell mechanics and morphology) provides access to a novel, mostly unexplored layer of information regarding pathophysiologic immune cell activation. We hypothesized that the biophysical phenotyping of circulating immune cells, reflecting their pathologic activation, can be used as a clinical tool for the evaluation and risk stratification of patients with SSc. METHODS: We performed biophysical phenotyping of circulating immune cells by real-time fluorescence and deformability cytometry (RT-FDC) in 63 SSc patients, 59 rheumatoid arthritis (RA) patients, 28 antineutrophil cytoplasmic antibody–associated vasculitis (AAV) patients, and 22 age- and sex-matched healthy donors. RESULTS: We identified a specific signature of biophysical properties of circulating immune cells in SSc patients that was mainly driven by monocytes. Since it is absent in RA and AAV, this signature reflects an SSc-specific monocyte activation rather than general inflammation. The biophysical properties of monocytes indicate current disease activity, the extent of skin or lung fibrosis, and the severity of manifestations of microvascular damage, as well as the risk of disease progression in SSc patients. CONCLUSION: Changes in the biophysical properties of circulating immune cells reflect their pathologic activation in SSc patients and are associated with clinical outcomes. As a high-throughput approach that requires minimal preparations, RT-FDC–based biophysical phenotyping of monocytes can serve as a tool for the evaluation and risk stratification of patients with SSc.

Dynamics of cell rounding during detachment

Agata Nyga, Katarzyna Plak, Martin Kräter, Marta Urbanska, Kyoohyun Kim, Jochen Guck, Buzz Baum

Animal cells undergo repeated shape changes, for example by rounding up and respreading as they divide. Cell rounding can be also observed in interphase cells, for example when cancer cells switch from a mesenchymal to an ameboid mode of cell migration. Nevertheless, it remains unclear how interphase cells round up. In this article, we demonstrate that a partial loss of substrate adhesion triggers actomyosin-dependent cortical remodeling and ERM activation, which facilitates further adhesion loss causing cells to round. Although the path of rounding in this case superficially resembles mitotic rounding in involving ERM phosphorylation, retraction fiber formation, and cortical remodeling downstream of ROCK, it does not require Ect2. This work provides insights into the way partial loss of adhesion actives cortical remodeling to drive cell detachment from the substrate. This is important to consider when studying the mechanics of cells in suspension, for example using methods like real-time deformability cytometry (RT-DC).

Rapid single-cell physical phenotyping of mechanically dissociated tissue biopsies

Despina Soteriou, Markéta Kubánková, Christine Schweitzer, Rocío López-Posadas, Rahmita Pradhan, Oana-Maria Thoma, Andrea-Hermina Györfi, Alexandru-Emil Matei, Maximilian Waldner, et al.

During surgery, rapid and accurate histopathological diagnosis is essential for clinical decision making. Yet the prevalent method of intra-operative consultation pathology is intensive in time, labour and costs, and requires the expertise of trained pathologists. Here we show that biopsy samples can be analysed within 30 min by sequentially assessing the physical phenotypes of singularized suspended cells dissociated from the tissues. The diagnostic method combines the enzyme-free mechanical dissociation of tissues, real-time deformability cytometry at rates of 100–1,000 cells s−1 and data analysis by unsupervised dimensionality reduction and logistic regression. Physical phenotype parameters extracted from brightfield images of single cells distinguished cell subpopulations in various tissues, enhancing or even substituting measurements of molecular markers. We used the method to quantify the degree of colon inflammation and to accurately discriminate healthy and tumorous tissue in biopsy samples of mouse and human colons. This fast and label-free approach may aid the intra-operative detection of pathological changes in solid biopsies.

Confocal Interferometric Scattering Microscopy Reveals 3D Nanoscopic Structure and Dynamics in Live Cells

Michelle Küppers, David Albrecht, Anna D. Kashkanova, Jennifer Lühr, Vahid Sandoghdar

Bright-field light microscopy and related techniques continue to play a key role in life sciences because they provide a facile and label-free insight into biological specimen. However, lack of three-dimensional imaging and low sensitivity to nanoscopic features hamper their application in high-end quantitative studies. Here, we remedy these shortcomings by employing confocal interferometric scattering (iSCAT) microscopy. We demonstrate the performance of this label-free technique in a selection of case studies in live cells and benchmark our findings against simultaneously acquired fluorescence images. We reveal the nanometric topography of the nuclear envelope, quantify the dynamics of the endoplasmic reticulum, detect single microtubules, and map nanoscopic diffusion of clathrin-coated pits undergoing endocytosis. Furthermore, we introduce the combination of confocal and wide-field iSCAT modalities for simultaneous imaging of cellular structures and high-speed tracking of nanoscopic entities such as single SARS-CoV2 virions. Confocal iSCAT can be readily implemented as an additional contrast mechanism in existing laser scanning microscopes.

Impact of crowding on the diversity of expanding populations

Carl F. Schreck, Diana Fusco, Yuya Karita, Stephen Martis, Jona Kayser, Marie-Cécilia Duvernoy, Oskar Hallatschek

Crowding effects critically impact the self-organization of densely packed cellular assemblies, such as biofilms, solid tumors, and developing tissues. When cells grow and divide, they push each other apart, remodeling the structure and extent of the population’s range. Recent work has shown that crowding has a strong impact on the strength of natural selection. However, the impact of crowding on neutral processes, which controls the fate of new variants as long as they are rare, remains unclear. Here, we quantify the genetic diversity of expanding microbial colonies and uncover signatures of crowding in the site frequency spectrum. By combining Luria–Delbrück fluctuation tests, lineage tracing in a novel microfluidic incubator, cell-based simulations, and theoretical modeling, we find that the majority of mutations arise behind the expanding frontier, giving rise to clones that are mechanically “pushed out” of the growing region by the proliferating cells in front. These excluded-volume interactions result in a clone-size distribution that solely depends on where the mutation first arose relative to the front and is characterized by a simple power law for low-frequency clones. Our model predicts that the distribution depends on a single parameter—the characteristic growth layer thickness—and hence allows estimation of the mutation rate in a variety of crowded cellular populations. Combined with previous studies on high-frequency mutations, our finding provides a unified picture of the genetic diversity in expanding populations over the whole frequency range and suggests a practical method to assess growth dynamics by sequencing populations across spatial scales.

Self-supervised machine learning pushes the sensitivity limit in label-free detection of single proteins below 10 kDa

Mahyar Dahmardeh, Houman Mirzaalian Dastjerdi, Hisham Mazal, Harald Köstler, Vahid Sandoghdar

Interferometric scattering (iSCAT) microscopy is a label-free optical method capable of detecting single proteins, localizing<br>their binding positions with nanometer precision, and measuring their mass. In the ideal case, iSCAT is limited by shot noise<br>so that collection of more photons should allow its detection sensitivity to biomolecules of arbitrarily low mass. However, a<br>number of technical noise sources combined with speckle-like background fluctuations have restricted the detection limit in<br>iSCAT. Here, we show that an unsupervised machine learning isolation forest algorithm for anomaly detection pushes the<br>mass sensitivity limit by a factor of four to below 10 kDa. We implement this scheme both with a user-defined feature matrix<br>and a self-supervised FastDVDNet and validate our results with correlative fluorescence images recorded in total internal<br>reflection mode. Our work opens the door to the optical detection of small traces of disease markers such as alpha-synuclein,<br>chemokines, and cytokines.

A new hyperelastic lookup table for RT-DC

Lukas Daniel Wittwer, Felix Reichel, Paul Mueller, Jochen Guck, Sebastian Aland

Real-time deformability cytometry (RT-DC) is an established method that quantifies features like size, shape, and stiffness for whole cell populations on a single-cell level in real-time. A lookup table (LUT) disentangles the experimentally derived steady-state cell deformation and the projected area to extract the cell stiffness in the form of the Young's modulus. So far, two lookup tables exist but are limited to simple linear material models and cylindrical channel geometries. Here, we present two new lookup tables for RT-DC based on a neo-Hookean hyperelastic material numerically derived by simulations based on the finite element method in square and cylindrical channel geometries. At the same time, we quantify the influence of the shear-thinning behavior of the surrounding medium on the stationary deformation of cells in RT-DC and discuss the applicability and impact of the proposed LUTs regarding past and future RT-DC data analysis. Additionally, we provide insights about the cell strain and stresses, as well as the influence resulting from the rotational symmetric assumption on the cell deformation and volume estimation. The new lookup tables and the numerical cell shapes are made freely available.

Shear rheology of methyl cellulose based solutions for cell mechanical measurements at high shear rates

Beyza Büyükurganci, Santanu Kumar Basu, Markus Neuner, Jochen Guck, Andreas Wierschem, Felix Reichel

Methyl cellulose (MC) is a widely used material in various microfluidic applications in biology. Due to its biocompatibility, it has become a popular crowding agent for microfluidic cell deformability measurements, which usually operate at high shear rates (>10 000 s−1). However, a full rheological characterization of methyl cellulose solutions under these conditions has not yet been reported. With this study, we provide a full shear-rheological description for solutions of up to 1% MC dissolved in phosphate-buffered saline (PBS) that are commonly used in real-time deformability cytometry (RT-DC). We characterized three different MC-PBS solutions used for cell mechanical measurements in RT-DC with three different shear rheometer setups to cover a range of shear rates from 0.1–150 000 s−1. We report viscosities and normal stress differences in this regime. Viscosity functions can be well described using a Carreau–Yasuda model. Furthermore, we present the temperature dependency of shear viscosity and first normal stress difference of these solutions. Our results show that methyl cellulose solutions behave like power-law liquids in viscosity and exhibit first normal stress difference at shear rates between 5000–150 000 s−1. We construct a general viscosity equation for each MC solution at a certain shear rate and temperature. Furthermore, we investigated how MC concentration influences the rheology of the solutions and found the entanglement concentration at around 0.64 w/w%. Our results help to better understand the viscoelastic behavior of MC solutions, which can now be considered when modelling stresses in microfluidic channels.

Embracing the diversity of model systems to deconstruct the basis of regeneration and tissue repair

Aldine Amiel, Stephanie Tsai, Daniel Wehner

The eighth EMBO conference in the series ‘The Molecular and Cellular Basis of Regeneration and Tissue Repair’ took place in Barcelona (Spain) in September 2022. A total of 173 researchers from across the globe shared their latest advances in deciphering the molecular and cellular basis of wound healing, tissue repair and regeneration, as well as their implications for future clinical applications. The conference showcased an ever-expanding diversity of model organisms used to identify mechanisms that promote regeneration. Over 25 species were discussed, ranging from invertebrates to humans. Here, we provide an overview of the exciting topics presented at the conference, highlighting novel discoveries in regeneration and perspectives for regenerative medicine.

Image-based cell sorting using focused travelling surface acoustic waves

Ahmad Ahsan Nawaz, Despina Soteriou, Catherine Xu, Ruchi Goswami, Maik Herbig, Jochen Guck, Salvatore Girardo

Sorting cells is an essential primary step in many biological and clinical applications such as high-throughput drug screening, cancer research and cell transplantation. Cell sorting based on their mechanical properties has long been considered as a promising label-free biomarker that could revolutionize the isolation of cells from heterogeneous populations. Recent advances in microfluidic image-based cell analysis combined with subsequent label-free sorting by on-chip actuators demonstrated the possibility of sorting cells based on their physical properties. However, the high purity of sorting is achieved at the expense of a sorting rate that lags behind the analysis throughput. Furthermore, stable and reliable system operation is an important feature in enabling the sorting of small cell fractions from a concentrated heterogeneous population. Here, we present a label-free cell sorting method, based on the use of focused travelling surface acoustic wave (FTSAW) in combination with real-time deformability cytometry (RT-DC). We demonstrate the flexibility and applicability of the method by sorting distinct blood cell types, cell lines and particles based on different physical parameters. Finally, we present a new strategy to sort cells based on their mechanical properties. Our system enables the sorting of up to 400 particles per s. Sorting is therefore possible at high cell concentrations (up to 36 million per ml) while retaining high purity (>92%) for cells with diverse sizes and mechanical properties moving in a highly viscous buffer. Sorting of small cell fraction from a heterogeneous population prepared by processing of small sample volume (10 μl) is also possible and here demonstrated by the 667-fold enrichment of white blood cells (WBCs) from raw diluted whole blood in a continuous 10-hour sorting experiment. The real-time analysis of multiple parameters together with the high sensitivity and high-throughput of our method thus enables new biological and therapeutic applications in the future.

Epithelial RAC1-dependent cytoskeleton dynamics controls cell mechanics, cell shedding and barrier integrity in intestinal inflammation

Luz del Carmen Martínez-Sánchez, Phuong Anh Ngo, Rashmita Pradhan, Lukas-Sebastian Becker, David Boehringer, Despina Soteriou, Markéta Kubánková, Christine Schweitzer, Tatyana Koch, et al.

OBJECTIVE: Increased apoptotic shedding has been linked to intestinal barrier dysfunction and development of inflammatory bowel diseases (IBD). In contrast, physiological cell shedding allows the renewal of the epithelial monolayer without compromising the barrier function. Here, we investigated the role of live cell extrusion in epithelial barrier alterations in IBD. DESIGN: Taking advantage of conditional GGTase and RAC1 knockout mice in intestinal epithelial cells (Pggt1biΔIEC and Rac1iΔIEC mice), intravital microscopy, immunostaining, mechanobiology, organoid techniques and RNA sequencing, we analysed cell shedding alterations within the intestinal epithelium. Moreover, we examined human gut tissue and intestinal organoids from patients with IBD for cell shedding alterations and RAC1 function. RESULTS: Epithelial Pggt1b deletion led to cytoskeleton rearrangement and tight junction redistribution, causing cell overcrowding due to arresting of cell shedding that finally resulted in epithelial leakage and spontaneous mucosal inflammation in the small and to a lesser extent in the large intestine. Both in vivo and in vitro studies (knockout mice, organoids) identified RAC1 as a GGTase target critically involved in prenylation-dependent cytoskeleton dynamics, cell mechanics and epithelial cell shedding. Moreover, inflamed areas of gut tissue from patients with IBD exhibited funnel-like structures, signs of arrested cell shedding and impaired RAC1 function. RAC1 inhibition in human intestinal organoids caused actin alterations compatible with arresting of cell shedding. CONCLUSION: Impaired epithelial RAC1 function causes cell overcrowding and epithelial leakage thus inducing chronic intestinal inflammation. Epithelial RAC1 emerges as key regulator of cytoskeletal dynamics, cell mechanics and intestinal cell shedding. Modulation of RAC1 might be exploited for restoration of epithelial integrity in the gut of patients with IBD.

Evolutionary rescue of resistant mutants is governed by a balance between radial expansion and selection in compact populations

Serhii Aif, Nico Appold, Lucas Kampman, Oskar Hallatschek, Jona Kayser

Mutation-mediated treatment resistance is one of the primary challenges for modern antibiotic and anti-cancer therapy. Yet, many resistance mutations have a substantial fitness cost and are subject to purifying selection. How emerging resistant lineages may escape purifying selection via subsequent compensatory mutations is still unclear due to the difficulty of tracking such evolutionary rescue dynamics in space and time. Here, we introduce a system of fluorescence-coupled synthetic mutations to show that the probability of evolutionary rescue, and the resulting long-term persistence of drug resistant mutant lineages, is dramatically increased in dense microbial populations. By tracking the entire evolutionary trajectory of thousands of resistant lineages in expanding yeast colonies we uncover an underlying quasi-stable equilibrium between the opposing forces of radial expansion and natural selection, a phenomenon we term inflation-selection balance. Tailored computational models and agent-based simulations corroborate the fundamental nature of the observed effects and demonstrate the potential impact on drug resistance evolution in cancer. The described phenomena should be considered when predicting multi-step evolutionary dynamics in any mechanically compact cellular population, including pathogenic microbial biofilms and solid tumors. The insights gained will be especially valuable for the quantitative understanding of response to treatment, including emerging evolution-based therapy strategies.

Caveolin-1 dolines form a distinct and rapid caveolae-independent mechanoadaptation system

Fidel-Nicolás Lolo, Nikhil Walani, Eric Seemann, Dobryna Zalvidea, Dácil María Pavón, Gheorghe Cojoc, Moreno Zamai, Christine Varis de Lesegno, Fernando Martínez de Benito, et al.

In response to different types and intensities of mechanical force, cells modulate their physical properties and adapt their plasma membrane (PM). Caveolae are PM nano-invaginations that contribute to mechanoadaptation, buffering tension changes. However, whether core caveolar proteins contribute to PM tension accommodation independently from the caveolar assembly is unknown. Here we provide experimental and computational evidence supporting that caveolin-1 confers deformability and mechanoprotection independently from caveolae, through modulation of PM curvature. Freeze-fracture electron microscopy reveals that caveolin-1 stabilizes non-caveolar invaginations—dolines—capable of responding to low-medium mechanical forces, impacting downstream mechanotransduction and conferring mechanoprotection to cells devoid of caveolae. Upon cavin-1/PTRF binding, doline size is restricted and membrane buffering is limited to relatively high forces, capable of flattening caveolae. Thus, caveolae and dolines constitute two distinct albeit complementary components of a buffering system that allows cells to adapt efficiently to a broad range of mechanical stimuli.

Ultralong Imaging Range Chromatic Confocal Microscopy

Gargi Sharma, Kanwarpal Singh

Confocal microscopy is regularly used in cellular research but unfortunately, the imaging is restricted to a single plane. Chromatic confocal microscopy (CCM) offers the possibility to image multiple planes simultaneously, thus providing a manifold increase in the imaging speed, whereas eliminating the need for z-axis scanning. Standard chromatic confocal systems have a limited imaging range of the order of a few hundreds of micrometers which limits their applications. Herein, using a single zinc selenide lens, a CCM system that has an imaging range of 18 mm (±68 nm) with an average spatial resolution of 2.46 μm (±44 nm) and another system with a 1.55 mm (±14 nm) imaging range with 0.86 μm (±30 nm) average lateral spatial resolution is demonstrated. In doing so, sevenfold increase in the imaging range for the system with 1.55 mm imaging when compared with previously reported systems with similar lateral spatial resolution is achieved. The proposed approach can be a powerful tool for confocal imaging of biological samples or surface profiling of industrial samples.

Immune Cell Deformability in Depressive Disorders: Longitudinal Associations Between Depression, Glucocorticoids and Cell Deformabilit

Andreas Walter, Martin Kräter, Clemens Kirschbaum, Wei Gao, Magdalena Wekenborg, Marlene Penz, Nicole Rothe, Jochen Guck, Lukas Daniel Wittwer, et al.

Background: Cell deformability of all major blood cell types is increased in depressive disorders (DD).<br>Furthermore, impaired glucocorticoid secretion is causally related to DD. Nevertheless, there are no longitudinal studies examining changes in glucocorticoid output and depressive symptoms regarding cell deformability in DD.<br>Aim: To investigate, whether changes in depressive symptoms or hair glucocorticoids predict cell deformability in DD.<br>Methods: In 136 individuals, depressive symptoms (PHQ-9) and hair glucocorticoids (cortisol and cortisone) were measured at timepoint one (T1), while one year later (T2) depressive symptoms and hair glucocorticoids were remeasured and additionally cell deformability of peripheral blood cells was assessed and DD status was determined by clinical interview.<br>Results: Depression severity at T1 predicted higher cell deformability in monocytes and lymphocytes over the entire sample. Subjects with continuously high depressive symptoms at T1 and T2 showed elevated monocyte<br>deformability as compared to subjects with low depressive symptoms. Depression severity at T1 of subjects with a lifetime persistent depressive disorder (PDD) was associated with elevated monocyte, neutrophil, and granulo-<br>monocyte deformability. Depression severity at T1 of subjects with a 12-month PDD was positively associated with monocyte deformability. Furthermore, increases in glucocorticoid concentrations from T1 to T2 tended to be associated with higher immune cell deformability, while strongest associations emerged for the increase in cortisone with elevated neutrophil and granulo-monocyte deformability in the 12-month PDD group.<br>Conclusion: Continuously elevated depressive symptomatology as well as an increase in glucocorticoid levels over one year are associated with higher immune cell deformability, particularly in PDD. These findings suggest, that persistent depressive symptomatology associated with increased glucocorticoid secretion may lead to<br>increased immune cell deformability thereby compromising immune cell function and likely contributing to the perpetuation of PDD.

Viscoelastic properties of suspended cells measured with shear flow deformation cytometry

Richard Gerum, Elham Mirzahossein, Mar Eroles, Jennifer Elsterer, Astrid Mainka, Andreas Bauer, Selina Sonntag, Alexander Winterl, Johannes Bartl, et al.

Numerous cell functions are accompanied by phenotypic changes in viscoelastic properties, and measuring them can help elucidate higher level cellular functions in health and disease. We present a high-throughput, simple and low-cost microfluidic method for quantitatively measuring the elastic (storage) and viscous (loss) modulus of individual cells. Cells are suspended in a high-viscosity fluid and are pumped with high pressure through a 5.8 cm long and 200 µm wide microfluidic channel. The fluid shear stress induces large, ear ellipsoidal cell deformations. In addition, the flow profile in the channel causes the cells to rotate in a tank-treading manner. From the cell deformation and tank treading frequency, we extract the frequency-dependent viscoelastic cell properties based on a theoretical framework developed by R. Roscoe [1] that describes the deformation of a viscoelastic sphere in a viscous fluid under steady laminar flow. We confirm the accuracy of the method using atomic force microscopy-calibrated polyacrylamide beads and cells. Our measurements demonstrate that suspended cells exhibit power-law, soft glassy rheological behavior that is cell-cycle-dependent and mediated by the physical interplay between the actin filament and intermediate filament networks.

PNIPAAm microgels with defined network architecture as temperature sensors in optical stretchers

Nicolas Hauck, Timon Beck, Gheorghe Cojoc, Raimund Schlüßler, Saeed Ahmed, Ivan Raguzin, Martin Mayer, Jonas Schubert, Paul Müller, et al.

Stretching individual living cells with light is a standard method to assess their mechanical properties. Yet, heat introduced by the laser light of optical stretchers may unwittingly change the mechanical properties of cells therein. To estimate the temperature induced by an optical trap, we introduce cell-sized, elastic poly(N-isopropylacrylamide) (PNIPAAm) microgels that relate temperature changes to hydrogel swelling. For their usage as a standardized calibration tool, we analyze the effect of free-radical chain-growth gelation (FCG) and polymer-analogous photogelation (PAG) on hydrogel network heterogeneity, micromechanics, and temperature response by Brillouin microscopy and optical diffraction tomography. Using a combination of tailor-made PNIPAAm macromers, PAG, and microfluidic processing, we obtain microgels with homogeneous network architecture. With that, we expand the capability of standardized microgels in calibrating and validating cell mechanics analysis, not only considering cell and microgel elasticity but also providing stimuli-responsiveness to consider dynamic changes that cells may undergo during characterization.

Quantitative phase imaging through an ultra-thin lensless fiber endoscope

Jiawei Sun, Jiachen Wu, Ruchi Goswami, Salvatore Girardo, Liangcai Cao, Jochen Guck, Nektarios Koukourakis, Jürgen W. Czarske

Quantitative phase imaging (QPI) is a label-free technique providing both morphology and quantitative biophysical information in biomedicine. However, applying such a powerful technique to in vivo pathological diagnosis remains challenging. Multi-core fiber bundles (MCFs) enable ultra-thin probes for in vivo imaging, but current MCF imaging techniques are limited to amplitude imaging modalities. We demonstrate a computational lensless microendoscope that uses an ultra-thin bare MCF to perform quantitative phase imaging with microscale lateral resolution and nanoscale axial sensitivity of the optical path length. The incident complex light field at the measurement side is precisely reconstructed from the far-field speckle pattern at the detection side, enabling digital refocusing in a multi-layer sample without any mechanical movement. The accuracy of the quantitative phase reconstruction is validated by imaging the phase target and hydrogel beads through the MCF. With the proposed imaging modality, three-dimensional imaging of human cancer cells is achieved through the ultra-thin fiber endoscope, promising widespread clinical applications.

Long COVID: Association of Functional Autoantibodies against G-Protein-Coupled Receptors with an Impaired Retinal Microcirculation

Charlotte Szewczykowski, Christian Mardin, Marianna Lucio, Gerd Wallukat, Jakob Hoffmanns, Thora Schröder, Franziska Raith, Lennart Rogge, Felix Heltmann, et al.

Long COVID (LC) describes the clinical phenotype of symptoms after infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Diagnostic and therapeutic options are limited, as the pathomechanism of LC is elusive. As the number of acute SARS-CoV-2 infections was and is large, LC will be a challenge for the healthcare system. Previous studies revealed an impaired blood flow, the formation of microclots, and autoimmune mechanisms as potential factors in this complex interplay. Since functionally active autoantibodies against G-protein-coupled receptors (GPCR-AAbs) were observed in patients after SARS-CoV-2 infection, this study aimed to correlate the appearance of GPCR-AAbs with capillary microcirculation. The seropositivity of GPCR-AAbs was measured by an established cardiomyocyte bioassay in 42 patients with LC and 6 controls. Retinal microcirculation was measured by OCT–angiography and quantified as macula and peripapillary vessel density (VD) by the Erlangen-Angio Tool. A statistical analysis yielded impaired VD in patients with LC compared to the controls, which was accentuated in female persons. A significant decrease in macula and peripapillary VD for AAbs targeting adrenergic β2-receptor, MAS-receptor angiotensin-II-type-1 receptor, and adrenergic α1-receptor were observed. The present study might suggest that a seropositivity of GPCR-AAbs can be linked to an impaired retinal capillary microcirculation, potentially mirroring the systemic microcirculation with consecutive clinical symptoms.

In vivo assessment of mechanical properties during axolotl development and regeneration using confocal Brillouin microscopy

Camilo Riquelme-Guzmán, Timon Beck, Sandra Edwards-Jorquera, Raimund Schlüßler, Paul Müller, Jochen Guck, Stephanie Möllmert, Tatiana Sandoval-Guzmán

In processes such as development and regeneration, where large cellular and tissue rearrangements occur, cell fate and behaviour are strongly influenced by tissue mechanics. While most well-established tools probing mechanical properties require an invasive sample preparation, confocal Brillouin microscopy captures mechanical parameters optically with high resolution in a contact-free and label-free fashion. In this work, we took advantage of this tool and the transparency of the highly regenerative axolotl to probe its mechanical properties in vivo for the first time. We mapped the Brillouin frequency shift with high resolution in developing limbs and regenerating digits, the most studied structures in the axolotl. We detected a gradual increase in the cartilage Brillouin frequency shift, suggesting decreasing tissue compressibility during both development and regeneration. Moreover, we were able to correlate such an increase with the regeneration stage, which was undetected with fluorescence microscopy imaging. The present work evidences the potential of Brillouin microscopy to unravel the mechanical changes occurring in vivo in axolotls, setting the basis to apply this technique in the growing field of epimorphic regeneration.

Adipose cells and tissues soften with lipid accumulation while in diabetes adipose tissue stiffens

Shada Abuhattum, Petra Kotzbeck, Raimund Schlüßler, Alexandra Harger, Angela Ariza de Schellenberger, Kyoohyun Kim, Joan-Carles Escolano, Torsten Müller, Jürgen Braun, et al.

Adipose tissue expansion involves both differentiation of new precursors and size increase of mature adipocytes. While the two processes are well balanced in healthy tissues, obesity and diabetes type II are associated with abnormally enlarged adipocytes and excess lipid accumulation. Previous studies suggested a link between cell stiffness, volume and stem cell differentiation, although in the context of preadipocytes, there have been contradictory results regarding stiffness changes with differentiation. Thus, we set out to quantitatively monitor adipocyte shape and size changes with differentiation and lipid accumulation. We quantified by optical diffraction tomography that differentiating preadipocytes increased their volumes drastically. Atomic force microscopy (AFM)-indentation and -microrheology revealed that during the early phase of differentiation, human preadipocytes became more compliant and more fluid-like, concomitant with ROCK-mediated F-actin remodelling. Adipocytes that had accumulated large lipid droplets were more compliant, and further promoting lipid accumulation led to an even more compliant phenotype. In line with that, high fat diet-induced obesity was associated with more compliant adipose tissue compared to lean animals, both for drosophila fat bodies and murine gonadal adipose tissue. In contrast, adipose tissue of diabetic mice became significantly stiffer as shown not only by AFM but also magnetic resonance elastography. Altogether, we dissect relative contributions of the cytoskeleton and lipid droplets to cell and tissue mechanical changes across different functional states, such as differentiation, nutritional state and disease. Our work therefore sets the basis for future explorations on how tissue mechanical changes influence the behaviour of mechanosensitive tissue-resident cells in metabolic disorders.

Amoeboid-like migration ensures correct horizontal cell layer formation in the developing vertebrate retina

Rana Amini, Archit Bhatnagar, Raimund Schlüßler, Stephanie Möllmert, Jochen Guck, Caren Norden

Migration of cells in the developing brain is integral for the establishment of neural circuits and function of the central nervous system. While migration modes during which neurons employ predetermined directional guidance of either preexisting neuronal processes or underlying cells have been well explored, less is known about how cells featuring multipolar morphology migrate in the dense environment of the developing brain. To address this, we here investigated multipolar migration of horizontal cells in the zebrafish retina. We found that these cells feature several hallmarks of amoeboid-like migration that enable them to tailor their movements to the spatial constraints of the crowded retina. These hallmarks include cell and nuclear shape changes, as well as persistent rearward polarization of stable F-actin. Interference with the organization of the developing retina by changing nuclear properties or overall tissue architecture hampers efficient horizontal cell migration and layer formation showing that cell-tissue interplay is crucial for this process. In view of the high proportion of multipolar migration phenomena observed in brain development, the here uncovered amoeboid-like migration mode might be conserved in other areas of the developing nervous system.

Deciphering a hexameric protein complex with Angstrom optical resolution

Hisham Mazal, Franz Wieser, Vahid Sandoghdar

Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases, where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and the hexamer geometry of Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic, environmental and dynamic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.<br><br>Significance statement Fluorescence super-resolution microscopy has witnessed many clever innovations in the last two decades. Here, we advance the frontiers of this field of research by combining partial labeling and 2D image classification schemes with polarization-encoded single-molecule localization at liquid helium temperature to reach Angstrom resolution in three dimensions. We demonstrate the performance of the method by applying it to trimer and hexamer protein complexes. Our approach holds great promise for examining membrane protein structural assemblies and conformations in challenging native environments. The methodology closes the gap between electron and optical microscopy and offers an ideal ground for correlating the two modalities at the single-particle level. Indeed, correlative light and electron microscopy is an emerging technique that will provide new insight into cell biology.

Depth of focus extension in optical coherence tomography using ultrahigh chromatic dispersion of zinc selenide

Maria N. Romodina, Kanwarpal Singh

We report a novel technique to overcome<br>the depth-of-focus limitation in optical coherence tomography (OCT) using chromatic<br>dispersion of zinc selenide lens.<br>OCT is an established method of optical<br>imaging, which found numerous biomedical<br>applications. However, the depth scanning range of high-resolution OCT is limited by its depth of focus. Chromatic dispersion of zinc selenide lens allows to get high lateral resolution along extended depth of focus, because the different spectral components are focused at a different position along axes of light propagation. Test measurements with nanoparticle phantom show 2.8 times extension of the depth of focus compare to the system with a standard achromatic lens. The feasibility of biomedical applications was demonstrated by ex vivo imaging of the pig cornea and chicken fat tissue.

Precision size and refractive index analysis of weakly scattering nanoparticles in polydispersions

Anna D. Kashkanova, Martin Blessing, André Gemeinhardt, Didier Soulat, Vahid Sandoghdar

Characterization of the size and material properties of particles in liquid suspensions is in very high demand, for example, in the analysis of colloidal samples or of bodily fluids such as urine or blood plasma. However, existing methods are limited in their ability to decipher the constituents of realistic samples. Here we introduce iNTA as a new method that combines interferometric detection of scattering with nanoparticle tracking analysis to reach unprecedented sensitivity and precision in determining the size and refractive index distributions of nanoparticles in suspensions. After benchmarking iNTA with samples of colloidal gold, we present its remarkable ability to resolve the constituents of various multicomponent and polydisperse samples of known origin. Furthermore, we showcase the method by elucidating the refractive index and size distributions of extracellular vesicles from Leishmania parasites and human urine. The current performance of iNTA already enables advances in several important applications, but we also discuss possible improvements.

Best practices for reporting throughput in biomedical research

Maik Herbig, Akihiro Isozaki, Dino Di Carlo, Jochen Guck, Nao Nitta, Robert Damoiseaux, Shogo Kamikawaji, Eigo Suyama, Hirofumi Shintaku, et al.

mRNA Subtype of Cancer-Associated Fibroblasts Significantly Affects Key Characteristics of Head and Neck Cancer Cells

Barbora Peltanová, Hana Holcová Polanská, Martina Raudenská, Jan Balvan, Jiri Navrátil, Tomás Vicar, Jaromir Gumulec, Barbora Cechová, Martin Kräter, et al.

Head and neck squamous cell carcinomas (HNSCC) belong among severe and highly complex malignant diseases showing a high level of heterogeneity and consequently also a variance in therapeutic response, regardless of clinical stage. Our study implies that the progression of HNSCC may be supported by cancer-associated fibroblasts (CAFs) in the tumour microenvironment (TME) and the heterogeneity of this disease may lie in the level of cooperation between CAFs and epithelial cancer cells, as communication between CAFs and epithelial cancer cells seems to be a key factor for the sustained growth of the tumour mass. In this study, we investigated how CAFs derived from tumours of different mRNA subtypes influence the proliferation of cancer cells and their metabolic and biomechanical reprogramming. We also investigated the clinicopathological significance of the expression of these metabolism-related genes in tissue samples of HNSCC patients to identify a possible gene signature typical for HNSCC progression. We found that the right kind of cooperation between cancer cells and CAFs is needed for tumour growth and progression, and only specific mRNA subtypes can support the growth of primary cancer cells or metastases. Specifically, during coculture, cancer cell colony supporting effect and effect of CAFs on cell stiffness of cancer cells are driven by the mRNA subtype of the tumour from which the CAFs are derived. The degree of colony-forming support is reflected in cancer cell glycolysis levels and lactate shuttle-related transporters.

Wie mikrobielle Modellsysteme helfen, Tumorevolution zu entschlüsseln

Maximilian Eiche, Jona Kayser

While cellular evolution is one of the most fundamental concepts of<br>life, its consequences are among the most pressing issues of modern<br>health care, including cancer and the emergence of therapy resistance.<br>We currently still lack the ability to accurately predict evolutionary trajectories,<br>especially in spatially dense, pathogenic cellular populations<br>such as microbial biofi lms or solid tumors. Here, we discuss the conceptual<br>framework of evolution in dense populations and the potential<br>of tailored microbial model systems to systematically study the underlying<br>mechanisms.

A Proposal to Perform High Contrast Imaging of Human Palatine Tonsil with Cross Polarized Optical Coherence Tomography

Gargi Sharma, Asha Parmar, Franziska Hoffmann, Katharina Geißler, Ferdinand von Eggeling, Orlando Guntinas-Lichius, Kanwarpal Singh

The palatine tonsils provide the first line of immune defense against foreign pathogens<br>inhaled or ingested. However, a disruption in the epithelial layer within the tonsil crypts can lead to recurrent acute tonsillitis (RAT). Current imaging techniques suffer from poor resolution and contrast and do not allow a classification of the severity of RAT. We have developed a cross-polarized optical coherence tomography system. The system can detect a change in the polarization of the light after the light-tissue interaction. We demonstrate improved resolution and contrast in tonsil imaging with the developed method. Intensity, as well as retardance images of the excised tonsil tissue, were acquired. Features such as crypt epithelium, lymphoid follicles, and dense connective tissue were observed with improved contrast. Cross polarized optical coherence tomography can be a valuable tool in the clinic to evaluate palatine tonsils as it would allow visualizing common tonsil features without the need for any external contrast agent.

Depressive disorders are associated with increased peripheral blood cell deformability: a cross-sectional case-control study (Mood-Morph)

Andreas Walther, Anne Mackens-Kiani, Julian Eder, Maik Herbig, Christoph Herold, Clemens Kirschbaum, Jochen Guck, Lucas Wittwer, Katja Beesdo-Baum, et al.

Pathophysiological landmarks of depressive disorders are chronic low-grade inflammation and elevated glucocorticoid output. Both can potentially interfere with cytoskeleton organization, cell membrane bending and cell function, suggesting altered cell morpho-rheological properties like cell deformability and other cell mechanical features in depressive disorders. We performed a cross-sectional case-control study using the image-based morpho-rheological characterization of unmanipulated blood samples facilitating real-time deformability cytometry (RT-DC). Sixty-nine pre-screened individuals at high risk for depressive disorders and 70 matched healthy controls were included and clinically evaluated by Composite International Diagnostic Interview leading to lifetime and 12-month diagnoses. Facilitating deep learning on blood cell images, major blood cell types were classified and morpho-rheological parameters such as cell size and cell deformability of every individual cell was quantified. We found peripheral blood cells to be more deformable in patients with depressive disorders compared to controls, while cell size was not affected. Lifetime persistent depressive disorder was associated with increased cell deformability in monocytes and neutrophils, while in 12-month persistent depressive disorder erythrocytes deformed more. Lymphocytes were more deformable in 12-month major depressive disorder, while for lifetime major depressive disorder no differences could be identified. After correction for multiple testing, only associations for lifetime persistent depressive disorder remained significant. This is the first study analyzing morpho-rheological properties of entire blood cells and highlighting depressive disorders and in particular persistent depressive disorders to be associated with increased blood cell deformability. While all major blood cells tend to be more deformable, lymphocytes, monocytes, and neutrophils are mostly affected. This indicates that immune cell mechanical changes occur in depressive disorders, which might be predictive of persistent immune response.

Changes in Blood Cell Deformability in Chorea-Acanthocytosis and Effects of Treatment With Dasatinib or Lithium

Felix Reichel, Martin Kräter, Kevin Peikert, Hannes Glaß, Philipp Rosendahl, Maik Herbig, Alejandro Rivera Prieto, Alexander Kihm, Giel Bosman, et al.

Misshaped red blood cells (RBCs), characterized by thorn-like protrusions known as acanthocytes, are a key diagnostic feature in Chorea-Acanthocytosis (ChAc), a rare neurodegenerative disorder. The altered RBC morphology likely influences their biomechanical properties which are crucial for the cells to pass the microvasculature. Here, we investigated blood cell deformability of five ChAc patients compared to healthy controls during up to 1-year individual off-label treatment with the tyrosine kinase inhibitor dasatinib or several weeks with lithium. Measurements with two microfluidic techniques allowed us to assess RBC deformability under different shear stresses. Furthermore, we characterized leukocyte stiffness at high shear stresses. The results showed that blood cell deformability–including both RBCs and leukocytes - in general was altered in ChAc patients compared to healthy donors. Therefore, this study shows for the first time an impairment of leukocyte properties in ChAc. During treatment with dasatinib or lithium, we observed alterations in RBC deformability and a stiffness increase for leukocytes. The hematological phenotype of ChAc patients hinted at a reorganization of the cytoskeleton in blood cells which partly explains the altered mechanical properties observed here. These findings highlight the need for a systematic assessment of the contribution of impaired blood cell mechanics to the clinical manifestation of ChAc.

Unbiased retrieval of frequency-dependent mechanical properties from noisy time-dependent signals

Shada Abuhattum, Hui-Shun Kuan, Paul Mueller, Jochen Guck, Vasily Zaburdaev

The mechanical response of materials to dynamic loading is often quantified by the frequency-dependent complex modulus. Probing materials directly in the frequency domain faces technical challenges such as a limited range of frequencies, long measurement times, or small sample sizes. Furthermore, many biological samples, such as cells or tissues, can change their properties upon repetitive probing at different frequencies. Therefore, it is common practice to extract the material properties by fitting predefined mechanical models to measurements performed in the time domain. This practice, however, precludes the probing of unique and yet unexplored material properties. In this report, we demonstrate that the frequency-dependent complex modulus can be robustly retrieved in a model-independent manner directly from time-dependent stress-strain measurements. While applying a rolling average eliminates random noise and leads to a reliable complex modulus in the lower frequency range, a Fourier transform with a complex frequency helps to recover the material properties at high frequencies. Finally, by properly designing the probing procedure, the recovery of reliable mechanical properties can be extended to an even wider frequency range. Our approach can be used with many state-of-the-art experimental methods to interrogate the mechanical properties of biological and other complex materials.

PiSCAT: A Python Package for Interferometric Scattering Microscopy

Houman Mirzaalian Dastjerdi, Reza Gholami Mahmoodabadi, Matthias Bär, Vahid Sandoghdar, Harald Köstler

Interferometric scattering (iSCAT) microscopy allows one to image and track nano-objects with a nanometer spatial and microsecond temporal resolution over arbitrarily long measurement times (Lindfors et al., 2004; Taylor & Sandoghdar, 2019b, 2019a). A key advantage of this technique over the well-established fluorescence methods is the indefinite photostability of the scattering phenomenon in contrast to the photobleaching of fluorophores. This means that one can perform very long measurements. Moreover, scattering processes are linear and thus do not saturate. This leads to larger signals than is possible from a single fluorophore. As a result, one can image at a much faster rate than in fluorescence microscopy. Furthermore, the higher signal makes it possible to localize a nano-object with much better spatial precision.<br>The remarkable sensitivity of iSCAT, however, also brings about the drawback that one obtains a rich speckle-like background from other nano-objects in the field of view.

An explicit model to extract viscoelastic properties of cells from AFM force-indentation curves

Shada Abuhattum Hofemeier, Dominic Mokbel, Paul Müller, Despina Soteriou, Jochen Guck, Sebastian Aland

Atomic force microscopy (AFM) is widely used for quantifying the mechanical properties of soft materials such as cells. AFM force-indentation curves are conventionally fitted with a Hertzian model to extract elastic properties. These properties solely are, however, insufficient to describe the mechanical properties of cells. Here, we expand the analysis capabilities to describe the viscoelastic behavior while using the same force-indentation curves. Our model gives an explicit relation of force and indentation and extracts physically meaningful mechanical parameters. We first validated the model on simulated force-indentation curves. Then, we applied the fitting model to the force-indentation curves of two hydrogels with different crosslinking mechanisms. Finally, we characterized HeLa cells in two cell cycle phases, interphase and mitosis, and showed that mitotic cells have a higher apparent elasticity and a lower apparent viscosity. Our study provides a simple method, which can be directly integrated into the standard AFM framework for extracting the viscoelastic properties of materials.

Polarization Independent Optical Coherence Tomography

Asha Parmar, Gargi Sharma, Kanwarpal Singh

Optical coherence tomography (OCT) is a well established imaging modality for high-resolution three-dimensional imaging in clinical settings. While imaging, care must be taken to minimize the imaging artifacts related to the polarization differences between the sample and the reference signals. Current OCT systems adopt complicated mechanisms, such as the use of multiple detectors, polarization-maintaining fibers, polarization controllers to achieve polarization artifacts free sample images.<br>Often the polarization controllers need readjustment which is not suitable for clinical settings. In this work, we demonstrate a simple approach that can minimize the polarization-related artifacts in the OCT systems. Polarization artifact-free images are acquired using two orthogonally polarized reference signals where the orthogonal polarization is achieved using a Faraday mirror. In the current approach, only a single detector is required which makes the current approach compatiblewith swept-source or camera-basedOCT systems. Furthermore, no polarization controllers are used in the system which increases the system stability while minimizing the artifacts related to the sample birefringence, polarization change due to the sample scattering, and polarization change due to the optical fiber movements present in the system.

An exception to the rule? Regeneration of the injured spinal cord in the spiny mouse

Daniel Wehner, Catherina G. Becker

The capacity for long-distance axon regeneration and functional recovery after spinal cord injury in the adult has long been thought to be a unique feature of certain non-mammalian vertebrates. However, in this issue of Developmental Cell, Nogueira-Rodrigues et al. report an astonishingly high regenerative ability in the spiny mouse.

Quantitative imaging of Caenorhabditis elegans dauer larvae during cryptobiotic transition

Kyoohyun Kim, Vamshidhar Gade, Teymuras V. Kurzchalia, Jochen Guck

Upon starvation or overcrowding, the nematode Caenorhabditis elegans enters diapause by forming a dauer larva, which can then further survive harsh desiccation in an anhydrobiotic state. We have previously identified the genetic and biochemical pathways essential for survival—but without detailed knowledge of their material properties, the mechanistic understanding of this intriguing phenomenon remains incomplete. Here we employed optical diffraction tomography (ODT) to quantitatively assess the internal mass density distribution of living larvae in the reproductive and diapause stages. ODT revealed that the properties of the dauer larvae undergo a dramatic transition upon harsh desiccation. Moreover, mutants that are sensitive to desiccation displayed structural abnormalities in the anhydrobiotic stage that could not be observed by conventional microscopy. Our advance opens a door to quantitatively assessing the transitions in material properties and structure necessary to fully understand an organism on the verge of life and death.

Nonlinear microscopy using impulsive stimulated Brillouin scattering for high-speed elastography

Benedikt Krug, Nektarios Koukourakis, Jochen Guck, Jürgen Czarske

The impulsive stimulated Brillouin microscopy promises fast, non-contact measurements of the elastic properties of biological samples. The used pump-probe approach employs an ultra-short pulse laser and a cw laser to generate Brillouin signals. Modeling of the microscopy technique has already been carried out partially, but not for biomedical applications. The nonlinear relationship between pulse energy and Brillouin signal amplitude is proven with both simulations and experiments. Tayloring of the excitation parameters on the biologically relevant polyacrylamide hydrogels outline sub-ms temporal resolutions at a relative precision of <1%. Brillouin microscopy using the impulsive stimulated scattering therefore exhibits high potential for the measurements of viscoelastic properties of cells and tissues.

Cross-Polarized Optical Coherence Tomography System with Unpolarized Light

Georg R. Hartl, Asha Parmar, Gargi Sharma, Kanwarpal Singh

Cross-polarized optical coherence tomography offers improved contrast for samples which<br>can alter the polarization of light when it interacts with the sample. This property has been utilized to screen pathological conditions in several organs. Existing cross-polarized optical coherence tomography systems require several polarization-controlling elements to minimize the optical fiber movement-related image artifacts. In this work, we demonstrate a cross-polarized optical coherence tomography system using unpolarized light and only two quarter-wave plates, which is free from fiber-induced image artifacts. The simplicity of the approach will find many applications in clinical settings.

Label-free imaging flow cytometry for analysis and sorting of enzymatically dissociated tissues

Maik Herbig, Karen Tessmer, Martin Nötzel, Ahmad Ahsan Nawaz, Tiago Santos-Ferreira, Oliver Borsch, Sylvia J. Gasparini, Jochen Guck, Marius Ader

Biomedical research relies on identification and isolation of specific cell types using molecular biomarkers and sorting methods such as fluorescence or magnetic activated cell sorting. Labelling processes potentially alter the cells’ properties and should be avoided, especially when purifying cells for clinical applications. A promising alternative is the label-free identification of cells based on physical properties. Sorting real-time deformability cytometry (soRT-DC) is a microfluidic technique for label-free analysis and sorting of single cells. In soRT-FDC, bright-field images of cells are analyzed by a deep neural net (DNN) to obtain a sorting decision, but sorting was so far only demonstrated for blood cells which show clear morphological differences and are naturally in suspension. Most cells, however, grow in tissues, requiring dissociation before cell sorting which is associated with challenges including changes in morphology, or presence of aggregates. Here, we introduce methods to improve robustness of analysis and sorting of single cells from nervous tissue and provide DNNs which can distinguish visually similar cells. We employ the DNN for image-based sorting to enrich photoreceptor cells from dissociated retina for transplantation into the mouse eye.

Machine learning assisted real-time deformability cytometry of CD34+ cells allows to identify patients with myelodysplastic syndromes

Maik Herbig, Angela Jacobi, Manja Wobus, Heike Weidner, Anna Mies, Martin Kräter, Oliver Otto, Christian Thiede, Marie-Theresa Weickert, et al.

Diagnosis of myelodysplastic syndrome (MDS) mainly relies on a manual assessment of the peripheral blood and bone marrow cell morphology. The WHO guidelines suggest a visual screening of 200 to 500 cells which inevitably turns the assessor blind to rare cell populations and leads to low reproducibility. Moreover, the human eye is not suited to detect shifts of cellular properties of entire populations. Hence, quantitative image analysis could improve the accuracy and reproducibility of MDS diagnosis. We used real-time deformability cytometry (RT-DC) to measure bone marrow biopsy samples of MDS patients and age-matched healthy individuals. RT-DC is a high-throughput (1000 cells/s) imaging flow cytometer capable of recording morphological and mechanical properties of single cells. Properties of single cells were quantified using automated image analysis, and machine learning was employed to discover morpho-mechanical patterns in thousands of individual cells that allow to distinguish healthy vs. MDS samples. We found that distribution properties of cell sizes differ between healthy and MDS, with MDS showing a narrower distribution of cell sizes. Furthermore, we found a strong correlation between the mechanical properties of cells and the number of disease-determining mutations, inaccessible with current diagnostic approaches. Hence, machine-learning assisted RT-DC could be a promising tool to automate sample analysis to assist experts during diagnosis or provide a scalable solution for MDS diagnosis to regions lacking sufficient medical experts.

Mechanical spinal cord transection in larval zebrafish and subsequent whole-mount histological processing

Nora John, Julia Kolb, Daniel Wehner

Zebrafish regenerate their spinal cord after injury, both at larval and adult stages. Larval zebrafish have emerged as a powerful model system to study spinal cord injury and regeneration due to their high optical transparency for in vivo imaging, amenability to high-throughput analysis, and rapid regeneration time. Here, we describe a protocol for the mechanical transection of the larval zebrafish spinal cord, followed by whole-mount tissue processing for in situ hybridization and immunohistochemistry to elucidate principles of regeneration.

Correlative all-optical quantification of mass density and mechanics of subcellular compartments with fluorescence specificity

Raimund Schlüßler, Kyoohyun Kim, Martin Nötzel, Anna Taubenberger, Shada Abuhattum, Timon Beck, Paul Müller, Shovamaye 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 epifluorescence imaging for explicitly measuring the Brillouin shift, RI, and absolute density with specificity to fluorescently labeled structures. We show that neglecting the RI and density might lead to erroneous conclusions. Investigating the nucleoplasm of wild-type HeLa cells, we find that it has lower density but higher longitudinal modulus than the cytoplasm. 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.

Real-Time Deformability Cytometry Detects Leukocyte Stiffening After Gadolinium-Based Contrast Agent Exposure

Angela Jacobi, Angela Ariza de Schellenberger, Yavuz Oguz Uca, Maik Herbig, Jochen Guck, Ingolf Sack

OBJECTIVES: Reports on gadolinium (Gd) retention in soft tissues after administration of Gd-based contrast agents (GBCAs) raise concerns about Gd-induced changes in the biophysical properties of cells and tissues. Here, we investigate if clinical GBCAs of both classes of linear and macrocyclic structure cause changes in the mechanical properties of leukocytes in human blood samples. MATERIAL AND METHODS: Real-time deformability cytometry was applied to human blood samples from 6 donors. The samples were treated with 1 mM gadoteric acid (Dotarem), gadopentetic acid (Magnevist), gadobutrol (Gadovist), or Gd trichloride at 37°C for 1 hour to mimic clinical doses of GBCAs and exposure times. Leukocyte subtypes—lymphocytes, monocytes, and neutrophils—were identified based on their size and brightness and analyzed for deformability, which is inversely correlated with cellular stiffness. RESULTS: We observed significant stiffening (3%–13%, P < 0.01) of all investigated leukocyte subtypes, which was most pronounced for lymphocytes, followed by neutrophils and monocytes, and the effects were independent of the charge and steric structure of the GBCA applied. In contrast, no changes in cell size and brightness were observed, suggesting that deformability and cell stiffness measured by real-time deformability cytometry are sensitive to changes in the physical phenotypes of leukocytes after GBCA exposure. CONCLUSIONS: Real-time deformability cytometry might provide a quantitative blood marker for critical changes in the physical properties of blood cells in patients undergoing GBCA-enhanced magnetic resonance imaging.

Passive coupling of membrane tension and cell volume during active response of cells to osmosis

Chloé Roffay, Guillaume Molinard, Kyoohyun Kim, Marta Urbanska, Virginia Andrade, Victoria Barbarasa, Paulina Nowak, Vincent Mercier, José García-Calvo, et al.

During osmotic changes of their environment, cells actively regulate their volume and plasma membrane tension that can passively change through osmosis. How tension and volume are coupled during osmotic adaptation remains unknown, as their quantitative characterization is lacking. Here, we performed dynamic membrane tension and cell volume measurements during osmotic shocks. During the first few seconds following the shock, cell volume varied to equilibrate osmotic pressures inside and outside the cell, and membrane tension dynamically followed these changes. A theoretical model based on the passive, reversible unfolding of the membrane as it detaches from the actin cortex during volume increase quantitatively describes our data. After the initial response, tension and volume recovered from hypoosmotic shocks but not from hyperosmotic shocks. Using a fluorescent membrane tension probe (fluorescent lipid tension reporter [Flipper-TR]), we investigated the coupling between tension and volume during these asymmetric recoveries. Caveolae depletion and pharmacological inhibition of ion transporters and channels, mTORCs, and the cytoskeleton all affected tension and volume responses. Treatments targeting mTORC2 and specific downstream effectors caused identical changes to both tension and volume responses, their coupling remaining the same. This supports that the coupling of tension and volume responses to osmotic shocks is primarily regulated by mTORC2.

Mapping Tumor Spheroid Mechanics in Dependence of 3D Microenvironment Stiffness and Degradability by Brillouin Microscopy

Vaibhav Mahajan, Timon Beck, Paulina Gregorczyk, André Ruland, Simon Alberti, Jochen Guck, Carsten Werner, Raimund Schlüßler, Anna V. Taubenberger

Altered biophysical properties of cancer cells and of their microenvironment contribute to cancer progression. While the relationship between microenvironmental stiffness and cancer cell mechanical properties and responses has been previously studied using two-dimensional (2D) systems, much less is known about it in a physiologically more relevant 3D context and in particular for multicellular systems. To investigate the influence of microenvironment stiffness on tumor spheroid mechanics, we first generated MCF-7 tumor spheroids within matrix metalloproteinase (MMP)-degradable 3D polyethylene glycol (PEG)-heparin hydrogels, where spheroids showed reduced growth in stiffer hydrogels. We then quantitatively mapped the mechanical properties of tumor spheroids in situ using Brillouin microscopy. Maps acquired for tumor spheroids grown within stiff hydrogels showed elevated Brillouin frequency shifts (hence increased longitudinal elastic moduli) with increasing hydrogel stiffness. Maps furthermore revealed spatial variations of the mechanical properties across the spheroids’ cross-sections. When hydrogel degradability was blocked, comparable Brillouin frequency shifts of the MCF-7 spheroids were found in both compliant and stiff hydrogels, along with similar levels of growth-induced compressive stress. Under low compressive stress, single cells or free multicellular aggregates showed consistently lower Brillouin frequency shifts compared to spheroids growing within hydrogels. Thus, the spheroids’ mechanical properties were modulated by matrix stiffness and degradability as well as multicellularity, and also to the associated level of compressive stress felt by tumor spheroids. Spheroids generated from a panel of invasive breast, prostate and pancreatic cancer cell lines within degradable stiff hydrogels, showed higher Brillouin frequency shifts and less cell invasion compared to those in compliant hydrogels. Taken together, our findings contribute to a better understanding of the interplay between cancer cells and microenvironment mechanics and degradability, which is relevant to better understand cancer progression.

Single-molecule vacuum Rabi splitting: four-wave mixing and optical switching at the single-photon level

André Pscherer, Manuel Meierhofer, Daqing Wang, Hrishikesh Kelkar, Diego-Martin Cano, Tobias Utikal, Stephan Götzinger, Vahid Sandoghdar

A single quantum emitter can possess a very strong intrinsic nonlinearity, but its overall promise for nonlinear effects is hampered by the challenge of efficient coupling to incident photons. Common nonlinear optical materials, on the other hand, are easy to couple to but are bulky, imposing a severe limitation on the miniaturization of photonic systems. In this work, we show that a single organic molecule acts as an extremely efficient nonlinear optical element in the strong coupling regime of cavity quantum electrodynamics. We report on single-photon sensitivity in nonlinear signal generation and all-optical switching. Our work promotes the use of molecules for applications such as integrated photonic circuits, operating at very low powers.

suggested by editors

Engineering long-lived vibrational states for an organic molecule

Burak Gürlek, Vahid Sandoghdar, Diego-Martin Cano

The optomechanical character of molecules was discovered by Raman about one century ago. Today, molecules are promising contenders for high-performance quantum optomechanical platforms because their small size and large energy-level separations make them intrinsically robust against thermal agitations. Moreover, the precision and throughput of chemical synthesis can ensure a viable route to quantum technological applications. The challenge, however, is that the coupling of molecular vibrations to environmental phonons limits their coherence to picosecond time scales. Here, we improve the optomechanical quality of a molecule by several orders of magnitude through phononic engineering of its surrounding. By dressing a molecule with long-lived high-frequency phonon modes of its nanoscopic environment, we achieve storage and retrieval of photons at millisecond time scales and allow for the emergence of single-photon strong coupling in optomechanics. Our strategy can be extended to the realization of molecular optomechanical networks.

Matrix stiffness mechanosensing modulates the expression and distribution of transcription factors in Schwann cells

Gonzalo Rosso, Daniel Wehner, Christine Schweitzer, Stephanie Möllmert, Elisabeth Sock, Jochen Guck, Victor Shahin

After peripheral nerve injury, mature Schwann cells (SCs) de-differentiate and undergo cell reprogramming to convert into a specialized cell repair phenotype that promotes nerve regeneration. Reprogramming of SCs into the repair phenotype is tightly controlled at the genome level and includes downregulation of pro-myelinating genes and activation of nerve repair-associated genes. Nerve injuries induce not only biochemical but also mechanical changes in the tissue architecture which impact SCs. Recently, we showed that SCs mechanically sense the stiffness of the extracellular matrix and that SC mechanosensitivity modulates their morphology and migratory behavior. Here, we explore the expression levels of key transcription factors and myelin-associated genes in SCs, and the outgrowth of primary dorsal root ganglion (DRG) neurites, in response to changes in the stiffness of generated matrices. The selected stiffness range matches the physiological conditions of both utilized cell types as determined in our previous investigations. We find that stiffer matrices induce upregulation of the expression of transcription factors Sox2, Oct6, and Krox20, and concomitantly reduce the expression of the repair-associated transcription factor c-Jun, suggesting a link between SC substrate mechanosensing and gene expression regulation. Likewise, DRG neurite outgrowth correlates with substrate stiffness. The remarkable intrinsic physiological plasticity of SCs, and the mechanosensitivity of SCs and neurites, may be exploited in the design of bioengineered scaffolds that promote nerve regeneration upon injury.

Physical phenotype of blood cells is altered in COVID-19

Markéta Kubánková, Bettina Hohberger, Jakob Hoffmanns, Julia Fürst, Martin Herrmann, Jochen Guck, Martin Kräter

Clinical syndrome coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 is characterized by rapid spreading and high mortality worldwide. Although the pathology is not yet fully understood, hyperinflammatory response and coagulation disorders leading to congestions of microvessels are considered to be key drivers of the still-increasing death toll. Until now, physical changes of blood cells have not been considered to play a role in COVID-19 related vascular occlusion and organ damage. Here, we report an evaluation of multiple physical parameters including the mechanical features of five frequent blood cell types, namely erythrocytes, lymphocytes, monocytes, neutrophils, and eosinophils. More than four million blood cells of 17 COVID-19 patients at different levels of severity, 24 volunteers free from infectious or inflammatory diseases, and 14 recovered COVID-19 patients were analyzed. We found significant changes in lymphocyte stiffness, monocyte size, neutrophil size and deformability, and heterogeneity of erythrocyte deformation and size. Although some of these changes recovered to normal values after hospitalization, others persisted for months after hospital discharge, evidencing the long-term imprint of COVID-19 on the body.

HIF2α is a Direct Regulator of Neutrophil Motility

Sundary Sormendi, Mathieu Deygas, Anupam Sinha, Anja Krüger, Ioannis Kourtzelis, Gregoire Le Lay, Mathilde Bernard, Pablo J. Sáez, Michael Gerlach, et al.

Orchestrated recruitment of neutrophils to inflamed tissue is essential during initiation of inflammation. Inflamed areas are usually hypoxic, and adaptation to reduced oxygen pressure is typically mediated by hypoxia pathway proteins. However, it is still unclear how these factors influence the migration of neutrophils to and at the site of inflammation either during their transmigration through the blood-endothelial cell barrier, or their motility in the interstitial space. Here, we reveal that activation of the Hypoxia Inducible Factor-2 (HIF2α) due to deficiency of HIF-prolyl hydroxylase domain protein-2 (PHD2) boosts neutrophil migration specifically through highly confined microenvironments. In vivo, the increased migratory capacity of PHD2-deficient neutrophils resulted in massive tissue accumulation in models of acute local inflammation. Using systematic RNAseq analyses and mechanistic approaches, we identified RhoA, a cytoskeleton organizer, as the central downstream factor that mediates HIF2α-dependent neutrophil motility. Thus, we propose that the here identified novel PHD2-HIF2α-RhoA axis is vital to the initial stages of inflammation as it promotes neutrophil movement through highly confined tissue landscapes.

A unique macrophage subpopulation signals directly to progenitor cells to promote regenerative neurogenesis in the zebrafish spinal cord

Leonardo Cavone, Tess McCann, Louisa K. Drake, Erika A. Aguzzi, Ana-Maria Oprisoreanu, Elisa Pedersen, Soe Sandi, Jathurshan Selvarajah, Themistoklis M. Tsarouchas, et al.

Central nervous system injury re-initiates neurogenesis in anamniotes (amphibians and fishes), but not in mammals. Activation of the innate immune system promotes regenerative neurogenesis, but it is fundamentally unknown whether this is indirect through the activation of known developmental signaling pathways or whether immune cells directly signal to progenitor cells using mechanisms that are unique to regeneration. Using single-cell RNA-seq of progenitor cells and macrophages, as well as cell-type-specific manipulations, we provide evidence for a direct signaling axis from specific lesion-activated macrophages to spinal progenitor cells to promote regenerative neurogenesis in zebrafish. Mechanistically, TNFa from pro-regenerative macrophages induces Tnfrsf1a-mediated AP-1 activity in progenitors to increase regeneration-promoting expression of hdac1 and neurogenesis. This establishes the principle that macrophages directly communicate to spinal progenitor cells via non-developmental signals after injury, providing potential targets for future interventions in the regeneration-deficient spinal cord of mammals.

Know How to Regrow-Axon Regeneration in the Zebrafish Spinal Cord

Vasiliki Tsata, Daniel Wehner

The capacity for long-distance axon regeneration and functional recovery after spinal cord injury is poor in mammals but remarkable in some vertebrates, including fish and salamanders. The cellular and molecular basis of this interspecies difference is beginning to emerge. This includes the identification of target cells that react to the injury and the cues directing their pro-regenerative responses. Among existing models of successful spinal cord regeneration, the zebrafish is arguably the most understood at a mechanistic level to date. Here, we review the spinal cord injury paradigms used in zebrafish, and summarize the breadth of neuron-intrinsic and -extrinsic factors that have been identified to play pivotal roles in the ability of zebrafish to regenerate central nervous system axons and recover function.

Efficient and gentle delivery of molecules into cells with different elasticity via Progressive Mechanoporation

Alena Uvizl, Ruchi Goswami, Shanil Durgeshkumar Gandhi, Martina Augsburg, Frank Buchholz, Jochen Guck, Jörg Mansfeld, Salvatore Girardo

On Quantum Efficiency Measurements and Plasmonic Nano-Antennas

Korenobu Matsuzaki, Hsuan-Wei Liu, Stephan Götzinger, Vahid Sandoghdar

Quantum efficiency is a key quantity that describes the performance of light-emitting materials and is, thus, an important metric for assessing novel nanophotonic systems. This Perspective provides a concise discussion of the difficulties encountered in the characterization of quantum efficiencies, especially for studies that involve single emitters. In particular, we review various approaches that have been recently used for determining quantum efficiencies of emitters coupled to plasmonic antennas and highlight the subtleties and challenges that hinder precise measurements.

Rapid computational cell-rotation around arbitrary axes in 3D with multi-core fiber

Jiawei Sun, Nektarios Koukourakis, Jochen Guck, Jürgen W. Czarske

Optical trapping is a vital tool in biology, allowing precise optical manipulation of nanoparticles, micro-robots, and cells. Due to the low risk of photodamage and high trap stiffness, fiber-based dual-beam traps are widely used for optical manipulation of large cells. Besides trapping, advanced applications like 3D refractive index tomography need a rotation of cells, which requires precise control of the forces, for example, the acting-point of the forces and the intensities in the region of interest (ROI). A precise rotation of large cells in 3D about arbitrary axes has not been reported yet in dual-beam traps. We introduce a novel dual-beam optical trap in which a multi-core fiber (MCF) is transformed to a phased array, using wavefront shaping and computationally programmable light. The light-field distribution in the trapping region is holographically controlled within 0.1 s, which determines the orientation and the rotation axis of the cell with small retardation. We demonstrate real-time controlled rotation of HL60 cells about all 3D axes with a very high degree of freedom by holographic controlled light through an MCF with a resolution close to the diffraction limit. For the first time, the orientation of the cell can be precisely controlled about all 3D axes in a dual-beam trap. MCFs provide much higher flexibility beyond the bulky optics, enabling lab-on-a-chip applications and can be easily integrated for applications like contactless cell surgery, refractive index tomography, cell-elasticity measurement, which require precise 3D manipulation of cells.

Single organic molecules for photonic quantum technologies

C. Toninelli, I. Gerhardt, A.S. Clark, A. Reserbat-Plantey, Stephan Götzinger, Z. Ristanovic, M. Colautti, P. Lombardi, K.D. Major, et al.

Isolating single molecules in the solid state has allowed fundamental experiments in basic and applied sciences. When cooled down to liquid helium temperature, certain molecules show transition lines, that are tens of megahertz wide, limited only by the excited state lifetime. The extreme flexibility in the synthesis of organic materials provides, at low costs, a wide palette of emission wavelengths and supporting matrices for such single chromophores. In the last decades, the controlled coupling to photonic structures has led to an optimized interaction efficiency with light. Molecules can hence be operated as single photon sources and as non-linear elements with competitive performance in terms of coherence, scalability and compatibility with diverse integrated platforms. Moreover, they can be used as transducers for the optical read-out of fields and material properties, with the promise of single-quanta resolution in the sensing of charges and motion. We show that quantum emitters based on single molecules hold promise to play a key role in the development of quantum science and technologies.

Depth encoded input polarisation independent swept source cross-polarised optical coherence tomography probe

Katharina Blessing, Judith Schirmer, Asha Parmar, Kanwarpal Singh

Within the last decades, several studies have been published that prove the benefit of polarisation sensitive optical coherence (psOCT) tomography for the field of biomedical diagnostics. However, polarisation sensitive imaging typically requires careful control of the polarisation state of the input illumination, which leads to bulky and delicate systems. While psOCT provides quantitative information, it is mostly sufficient to analyse the images qualitatively in the field of biomedical diagnostics. Therefore, a reduced form of this technique, cross-polarised optical coherence tomography (cpOCT), moves into the focus of interest that serves to visualise the birefringence properties of a sample. Despite the low requirements for the illumination's polarisation, most of the proposed systems still include complex illumination control mechanisms. Here, we propose a common path probe based endoscopic system with an lateral resolution of 30 µm and a sensitivity of 103 dB comprising a commercially available swept-source OCT system and a free-space module which does not require any polarisation controlling elements. A Faraday mirror substitutes the complex polarisation control apparatus. We demonstrate the independence of the approach from the polarisation state of the light source by monitoring the illumination power in the orthogonal channels while varying the source polarisation. Furthermore, we validate the ability of the system to reveal the birefringence properties of different samples, starting from a quarter-wave plate, since its properties are fully characterised. Additionally, we present imaging results from several tissues to demonstrate its feasibility for the field of biomedical diagnostics.

Toward deep biophysical cytometry: prospects and challenges

Kelvin C.M. Lee, Jochen Guck, Keisuke Goda, Kevin K. Tsia

Recent advances in biophysical cytometry now make it possible to recapitulate cellular heterogeneity at the levels of throughput, precision, specificity, and sensitivity that were once inconceivable. Technological developments in state-of-the-art biophysical cytometry include single-cell mass assays, cell traction force assays, deformability and impedance cytometry, and label-free imaging cytometry. Next-generation biophysical cytometry could be more comprehensive and information-rich by exploring multimodal integration, such as simultaneous read-out of cell mass, stiffness, and morphology. How molecular signatures translate into cellular biophysical properties is not fully understood. Advanced techniques involving microfluidics, imaging, and deep learning could investigate this link. Standardizing the protocols and datasets of biophysical cytometry will be crucial to ensure wide dissemination.

Portable Optical Coherence Elastography System With Flexible and Phase Stable Common Path Optical Fiber Probe

Asha Parmar, Gargi Sharma, Shivani Sharma, Kanwarpal Singh

Biomechanical properties drive the functioning of cells and tissue. Measurement of such properties in the clinic is quite challenging, however. Optical coherence elastography is an emerging technique in this field that can measure the biomechanical properties of the tissue. Unfortunately, such systems have been limited to benchtop configuration with limited clinical applications. A truly portable system with a flexible probe that could probe different sample sites with ease is still missing. In this work, we report a portable optical coherence elastography system based on a flexible common path optical fiber probe. The common path approach allows us to reduce the undesired phase noise in the system by an order of magnitude less than the standard non-common path systems. The flexible catheter makes it possible to probe different parts of the body with ease. Being portable, our system can be easily transported to and from the clinic. We tested the efficacy of the system by measuring the mechanical properties of the agar-based tissue phantoms. We also measured the mechanical properties (Young’s Modulus) of the human skin at different sites. The measured values for the agar phantom and the skin were found to be comparable with the previously reported studies. Ultra-high phase stability and flexibility of the probe along with the portability of the whole system makes an ideal combination for the faster clinical adoption of the optical coherence elastography technique.

The Xenopus spindle is as dense as the surrounding cytoplasm

Abin Biswas, Kyoohyun Kim, Gheorghe Cojoc, Jochen Guck, Simone Reber

The mitotic spindle is a self-organizing molecular machine, where hundreds of different molecules continuously interact to maintain a dynamic steady state. While our understanding of key molecular players in spindle assembly is significant, it is still largely unknown how the spindle’s material properties emerge from molecular interactions. Here, we use correlative fluorescence imaging and label-free three-dimensional optical diffraction tomography (ODT) to measure the Xenopus spindle’s mass density distribution. While the spindle has been commonly referred to as a denser phase of the cytoplasm, we find that it has the same density as its surrounding, which makes it neutrally buoyant. Molecular perturbations suggest that spindle mass density can be modulated by tuning microtubule nucleation and dynamics. Together, ODT provides direct, unbiased, and quantitative information of the spindle’s emergent physical properties—essential to advance predictive frameworks of spindle assembly and function.

Nanoscopic charge fluctuations in a gallium phosphide waveguide measured by single molecules

Alexey Shkarin, Dominik Rattenbacher, Jan Renger, Simon Hönl, Tobias Utikal, Paul Seidler, Stephan Götzinger, Vahid Sandoghdar

We present efficient coupling of single organic molecules to a gallium phosphide subwavelengthwaveguide (nanoguide). By examining and correlating the temporal dynamics of various single-molecule resonances at different locations along the nanoguide, we reveal light-induced fluctuationsof their Stark shifts. Our observations are consistent with the predictions of a simple model basedon the optical activation of a small number of charges in the GaP nanostructure.

Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response

Joan-Carles Escolano, Anna V. Taubenberger, Shada Abuhattum, Christine Schweitzer, Aleeza Farrukh, Aránzazu del Campo, Clare E. Bryant, Jochen Guck

Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1β and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1β and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses.

Precision single-particle localization using radial variance transform

Anna D. Kashkanova, Alexey Shkarin, Reza Gholami Mahmoodabadi, Martin Blessing, Yazgan Tuna, André Gemeinhardt, Vahid Sandoghdar

We introduce an image transform designed to highlight features with high degree of radial symmetry for identification and subpixel localization of particles in microscopy images. The transform is based on analyzing pixel value variations in radial and angular directions. We compare the subpixel localization performance of this algorithm to other common methods based on radial or mirror symmetry (such as fast radial symmetry transform, orientation alignment transform, XCorr, and quadrant interpolation), using both synthetic and experimentally obtained data. We find that in all cases it achieves the same or lower localization error, frequently reaching the theoretical limit.

Chromatic Dispersion Based Wide-Band, Fiber-Coupled, Tunable Light Source for Hyperspectral Imaging

Gargi Sharma, Sheeza Kainat Naveed, Asha Parmar, Kanwarpal Singh

Hyperspectral imaging is a powerful label-free imaging technique that provides topological and spectral information at once. In this work, we have designed and characterized a hyperspectral source based on the chromatic dispersion property of off-the-shelf lenses and converted a supercontinuum laser light source into a hyperspectral imaging light source for 490 nm to 900 nm wavelength range with a spectral resolution of 3.5 nm to 18 nm respectively. The potential of the source was demonstrated by imaging two color dots with different absorption bands. Further, we generated the hypercube of the lily ovary and dense connective tissue and measured their spectral signature as a function of wavelength. We also imaged the lower tongue of a healthy volunteer at 540 nm, 630 nm, and white light. Our simple hyperspectral light source design can easily be incorporated in a standard endoscope or microscope to perform hyperspectral imaging.

AIDeveloper: deep learning image classification in life science and beyond

Martin Kräter, Shada Abuhattum Hofemeier, Despina Soteriou, Angela Jacobi, Thomas Krüger, Jochen Guck, Maik Herbig

Artificial intelligence (AI)‐based image analysis has increased drastically in recent years. However, all applications use individual solutions, highly specialized for a particular task. Here, an easy‐to‐use, adaptable, and open source software, called AIDeveloper (AID) to train neural nets (NN) for image classification without the need for programming is presented. AID provides a variety of NN‐architectures, allowing to apply trained models on new data, obtain performance metrics, and export final models to different formats. AID is benchmarked on large image datasets (CIFAR‐10 and Fashion‐MNIST). Furthermore, models are trained to distinguish areas of differentiated stem cells in images of cell culture. A conventional blood cell count and a blood count obtained using an NN are compared, trained on >1.2 million images, and demonstrated how AID can be used for label‐free classification of B‐ and T‐cells. All models are generated by non‐programmers on generic computers, allowing for an interdisciplinary use.

Mechanical properties of cell- and microgel bead-laden oxidized alginate-gelatin hydrogels

Thomas Distler, Lena Kretzschmar, Dominik Schneidereit, Salvatore Girardo, Ruchi Goswami, Oliver Friedrich, Rainer Detsch, Jochen Guck, Aldo R. Boccaccini, et al.

3D-printing technologies, such as biofabrication, capitalize on the homogeneous distribution and growth of cells inside biomaterial hydrogels, ultimately aiming to allow for cell differentiation, matrix remodeling, and functional tissue analogues. However, commonly, only the mechanical properties of the bioinks or matrix materials are assessed, while the detailed influence of cells on the resulting mechanical properties of hydrogels remains insufficiently understood. Here, we investigate the properties of hydrogels containing cells and spherical PAAm microgel beads through multi-modal complex mechanical analyses in the small- and large-strain regimes. We evaluate the individual contributions of different filler concentrations and a non-fibrous oxidized alginate-gelatin hydrogel matrix on the overall mechanical behavior in compression, tension, and shear. Through material modeling, we quantify parameters that describe the highly nonlinear mechanical response of soft composite materials. Our results show that the stiffness significantly drops for cell- and bead concentrations exceeding four million per milliliter hydrogel. In addition, hydrogels with high cell concentrations (≥6 mio ml−1) show more pronounced material nonlinearity for larger strains and faster stress relaxation. Our findings highlight cell concentration as a crucial parameter influencing the final hydrogel mechanics, with implications for microgel bead drug carrier-laden hydrogels, biofabrication, and tissue engineering.

A switch in pdgfrb+ cell-derived ECM composition prevents inhibitory scarring and promotes axon regeneration in the zebrafish spinal cord

Vasiliki Tsata, Stephanie Möllmert, Christine Schweitzer, Julia Kolb, Conrad Möckel, Benjamin Böhm, Gonzalo Rosso, Christian Lange, Mathias Lesche, et al.

In mammals, perivascular cell-derived scarring after spinal cord injury impedes axonal regrowth. In contrast, the extracellular matrix (ECM) in the spinal lesion site of zebrafish is permissive and required for axon regeneration. However, the cellular mechanisms underlying this interspecies difference have not been investigated. Here, we show that an injury to the zebrafish spinal cord triggers recruitment of pdgfrb+ myoseptal and perivascular cells in a PDGFR signaling-dependent manner. Interference with pdgfrb+ cell recruitment or depletion of pdgfrb+ cells inhibits axonal regrowth and recovery of locomotor function. Transcriptional profiling and functional experiments reveal that pdgfrb+ cells upregulate expression of axon growth-promoting ECM genes (cthrc1a and col12a1a/b) and concomitantly reduce synthesis of matrix molecules that are detrimental to regeneration (lum and mfap2). Our data demonstrate that a switch in ECM composition is critical for axon regeneration after spinal cord injury and identify the cellular source and components of the growth-promoting lesion ECM.

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.

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.

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.

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.

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.

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

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

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

High-yield fabrication of DNA and RNA constructs for single molecule force and torque spectroscopy experiments

Flavia S. Papini, Mona Seifert, David Dulin

Single molecule biophysics experiments have enabled the observation of biomolecules with a great deal of precision in space and time, e.g. nucleic acids mechanical properties and protein-nucleic acids interactions using force and torque spectroscopy techniques. The success of these experiments strongly depends on the capacity of the researcher to design and fabricate complex nucleic acid structures, as the outcome and the yield of the experiment also strongly depend on the high quality and purity of the final construct. Though the molecular biology techniques involved are well known, the fabrication of nucleic acid constructs for singlemolecule experiments still remains a difficult task. Here, we present new protocols to generate high quality coilable double-stranded DNA and RNA, as well as DNA and RNA hairpins with similar to 500-1000 bp long stems. Importantly, we present a new approach based on single-stranded DNA (ssDNA) annealing and we use magnetic tweezers to show that this approach simplifies the fabrication of complex DNA constructs, such as hairpins, and converts more efficiently the input DNA into construct than the standard PCR-digestion-ligation approach. The protocols we describe here enable the design of a large range of nucleic acid construct for single molecule biophysics experiments.

All fiber polarization insensitive detection for spectrometer based optical coherence tomography using optical switch

David Odeke Otuya, Gargi Sharma, Guillermo J. Tearney, Kanwarpal Singh

Polarization dependent image artifacts are common in optical coherence tomography imaging. Polarization insensitive detection scheme for swept source based optical coherence tomography systems is well established but is yet to be demonstrated for all fiber spectrometer-based Fourier domain optical coherence tomography systems. In this work, we present an all fiber polarization insensitive detection scheme for spectrometer based optical coherence tomography systems. Images from chicken breast muscle tissue were acquired to demonstrate the effectiveness of this scheme for the conventional Fourier domain optical coherence tomography system.

Polyacrylamide Bead Sensors for in vivo Quantification of Cell-Scale Stress in Zebrafish Development

Nicole Träber, Klemens Uhlmann, Salvatore Girardo, Gokul Kesavan, Katrin Wagner, Jens Friedrichs, Ruchi Goswami, K Bai, Michael Brand, et al.

Mechanical stress exerted and experienced by cells during tissue morphogenesis and organ formation plays an important role in embryonic development. While techniques to quantify mechanical stresses in vitro are available, few methods exist for studying stresses in living organisms. Here, we describe and characterize cell-like polyacrylamide (PAAm) bead sensors with well-defined elastic properties and size for in vivo quantification of cell-scale stresses. The beads were injected into developing zebrafish embryos and their deformations were computationally analyzed to delineate spatio-temporal local acting stresses. With this computational analysis-based cell-scale stress sensing (COMPAX) we are able to detect pulsatile pressure propagation in the developing neural rod potentially originating from polarized midline cell divisions and continuous tissue flow. COMPAX is expected to provide novel spatio-temporal insight into developmental processes at the local tissue level and to facilitate quantitative investigation and a better understanding of morphogenetic processes.

Colloidal crystals of compliant microgel beads to study cell migration and mechanosensitivity in 3D

Katrin Wagner, Salvatore Girardo, Ruchi Goswami, Gonzalo Rosso, Elke Ulbricht, Paul Müller, Despina Soteriou, Nicole Träber, Jochen Guck

Tissues are defined not only by their biochemical composition, but also by their distinct mechanical properties. It is now widely accepted that cells sense their mechanical environment and respond to it. However, studying the effects of mechanics in in vitro 3D environments is challenging since current 3D hydrogel assays convolve mechanics with gel porosity and adhesion. Here, we present novel colloidal crystals as modular 3D scaffolds where these parameters are principally decoupled by using monodisperse, protein-coated PAAm microgel beads as building blocks, so that variable stiffness regions can be achieved within one 3D colloidal crystal. Characterization of the colloidal crystal and oxygen diffusion simulations suggested the suitability of the scaffold to support cell survival and growth. This was confirmed by live-cell imaging and fibroblast culture over a period of four days. Moreover, we demonstrate unambiguous durotactic fibroblast migration and mechanosensitive neurite outgrowth of dorsal root ganglion neurons in 3D. This modular approach of assembling 3D scaffolds from mechanically and biochemically well-defined building blocks allows the spatial patterning of stiffness decoupled from porosity and adhesion sites in principle and provides a platform to investigate mechanosensitivity in 3D environments approximating tissues in vitro.

CASP1 variants influence subcellular caspase-1 localization, pyroptosome formation, pro-inflammatory cell death and macrophage deformability

Franz Kapplusch, Felix Schulze, Sabrina Rabe-Matschewsky, Susanne Russ, Maik Herbig, Michael Christian Heymann, Katharina Schoepf , Robert Stein, Ursula Range, et al.

CASP1 variants result in reduced enzymatic activity of procaspase-1 and impaired IL-1β release. Despite this, affected individuals can develop systemic autoinflammatory disease. These seemingly contradictory observations have only partially been explained by increased NF-κB activation through prolonged interaction of variant procaspase-1 with RIP2. To identify further disease underlying pathomechanisms, we established an in vitro model using shRNA-directed knock-down of procaspase-1 followed by viral transduction of human monocytes (THP-1) with plasmids encoding for wild-type procaspase-1, disease-associated CASP1 variants (p.L265S, p.R240Q) or a missense mutation in the active center of procaspase-1 (p.C285A). THP1-derived macrophages carrying CASP1 variants exhibited mutation-specific molecular alterations. We here provide in vitro evidence for abnormal pyroptosome formation (p.C285A, p.240Q, p.L265S), impaired nuclear (pro)caspase-1 localization (p.L265S), reduced pro-inflammatory cell death (p.C285A) and changes in macrophage deformability that may contribute to disease pathophysiology of patients with CASP1 variants. This offers previously unknown molecular pathomechanisms in patients with systemic autoinflammatory disease.

Cell Mechanics Based Computational Classification of Red Blood Cells Via Unsupervised Machine Intelligence Applied to Morpho-Rheological Markers

Yan Ge, Philipp Rosenddahl, Claudio Duran, Sara Ciucci, Nicole Töpfner, Jochen Guck, Carlo Vittorio Cannistraci

Despite fluorescent cell-labelling being widely employed in biomedical studies, some of its drawbacks are inevitable, with unsuitable fluorescent probes or probes inducing a functional change being the main limitations. Consequently, the demand for and development of label-free methodologies to classify cells is strong and its impact on precision medicine is relevant. Towards this end, high-throughput techniques for cell mechanical phenotyping have been proposed to get a multidimensional biophysical characterization of single cells. With this motivation, our goal here is to investigate the extent to which an unsupervised machine learning methodology, which is applied exclusively on morpho-rheological markers obtained by real-time deformability and fluorescence cytometry (RT-FDC), can address the difficult task of providing label-free discrimination of reticulocytes from mature red blood cells. We focused on this problem, since the characterization of reticulocytes (their percentage and cellular features) in the blood is vital in multiple human disease conditions, especially bone-marrow disorders such as anemia and leukemia. Our approach reports promising label-free results in the classification of reticulocytes from mature red blood cells, and it represents a step forward in the development of high-throughput morpho-rheological-based methodologies for the computational categorization of single cells. Besides, our methodology can be an alternative but also a complementary method to integrate with existing cell-labelling techniques.<br>

Low cost scalable monolithic common path probe design for the application in endoscopic optical coherence tomography

Katharina Blessing, Shivani Sharma, Alexander Gumann, Kanwarpal Singh

Endoscopic optical coherence tomography is an interference based imaging technique which due to its micron level resolution ability found several applications in medical diagnostics. However, the standard image performance suffers from artefacts caused by dispersion imbalance and polarisation mismatches between reference and sample arm. Such artefacts can be minimised with the use of a special class of probes called common path probes where the reference surface is placed in the vicinity of the sample. Previously reported common path probes suffered from a compromise between sensitivity and resolution. In most cases, proposed probes were not scalable for industrial applications and required sophisticated machines for fabrication, thus limiting their mass production for clinical use. We propose and demonstrate a simple fabrication procedure which would allow small laboratories and industries to mass produce common path probes. Our probe design is based on a thin gold layer within the probe which acts as a reference surface. Low-cost ball lenses were used to focus the signal on the sample. We achieved a sensitivity of 104 dB with the designed probes which is comparable to previously reported common path and non-common path probes. Imaging of biological samples such as pig's oesophagus and pig's coronary artery is also presented.

Rectification of Bacterial Diffusion in Microfluidic Labyrinths

Ariane Weber, Marco Bahrs, Zahra Alirezai, Xingyu Zhang, Carsten Beta, Vasily Zaburdaev

In nature as well as in the context of infection and medical applications, bacteria often have to move in highly complex environments such as soil or tissues. Previous studies have shown that bacteria strongly interact with their surroundings and are often guided by confinements. Here, we investigate theoretically how the dispersal of swimming bacteria can be augmented by microfluidic environments and validate our theoretical predictions experimentally. We consider a system of bacteria performing the prototypical run-and-tumble motion inside a labyrinth with square lattice geometry. Narrow channels between the square obstacles limit the possibility of bacteria to reorient during tumbling events to an area where channels cross. Thus, by varying the geometry of the lattice it might be possible to control the dispersal of cells. We present a theoretical model quantifying diffusive spreading of a run-and-tumble random walker in a square lattice. Numerical simulations validate our theoretical predictions for the dependence of the diffusion coefficient on the lattice geometry. We show that bacteria moving in square labyrinths exhibit enhanced dispersal as compared to unconfined cells. Importantly, confinement significantly extends the duration of the phase with strongly non-Gaussian diffusion, when the geometry of channels is imprinted in the density profiles of spreading cells. Finally, in good agreement with our theoretical findings, we observe the predicted behaviors in experiments with E. coli bacteria swimming in a square lattice labyrinth created in a microfluidic device. Altogether, our comprehensive understanding of bacterial dispersal in a simple two-dimensional labyrinth makes the first step toward the analysis of more complex geometries relevant for real world applications.

Histone H3K27 acetylation precedes active transcription during zebrafish zygotic genome activation as revealed by live-cell analysis

Yuko Sato, Lennart Hilbert, Haruka Oda, Yinan Wan, John M. Heddleston, Teng-Leong Chew, Vasily Zaburdaev, Philipp Keller, Timothee Lionnet, et al.

Histone post-translational modifications are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We applied a Fab-based live endogenous modification labeling technique to monitor the changes in histone modification levels during zygotic genome activation (ZGA) in living zebrafish embryos. Among various histone modifications, H3 Lys27 acetylation (H3K27ac) exhibited most drastic changes, accumulating in two nuclear foci in the 64- to 1k-cell-stage embryos. The elongating form of RNA polymerase II, which is phosphorylated at Ser2 in heptad repeats within the C-terminal domain (RNAP2 Ser2ph), and miR-430 transcripts were also concentrated in foci closely associated with H3K27ac. When treated with alpha-amanitin to inhibit transcription or JQ-1 to inhibit binding of acetyl-reader proteins, H3K27ac foci still appeared but RNAP2 Ser2ph and miR-430 morpholino were not concentrated in foci, suggesting that H3K27ac precedes active transcription during ZGA. We anticipate that the method presented here could be applied to a variety of developmental processes in any model and non-model organisms.

Mechanical changes of peripheral nerve tissue microenvironment and their structural basis during development

Gonzalo Rosso, Jochen Guck

Peripheral nerves are constantly exposed to mechanical stresses associated with body growth and limb movements. Although some aspects of these nerves' biomechanical properties are known, the link between nerve biomechanics and tissue microstructures during development is poorly understood. Here, we used atomic force microscopy to comprehensively investigate the elastic modulus of living peripheral nerve tissue cross sections ex vivo at distinct stages of development and correlated these elastic moduli with various cellular and extracellular aspects of the underlying histological microstructure. We found that local nerve tissue stiffness is spatially heterogeneous and evolves biphasically during maturation. Furthermore, we found the intracellular microtubule network and the extracellular matrix collagens type I and type IV as major contributors to the nerves' biomechanical properties, but surprisingly not cellular density and myelin content as previously shown for the central nervous system. Overall, these findings characterize the mechanical microenvironment that surrounds Schwann cells and neurons and will further our understanding of their mechanosensing mechanisms during nerve development. These data also provide the design of artificial nerve scaffolds to promote biomedical nerve regeneration therapies by considering mechanical properties that better reflect the nerve microenvironment.

Some thoughts on the future of cell mechanics

Jochen Guck

Targeting Mechanoresponsive Proteins in Pancreatic Cancer: 4-Hydroxyacetophenone Blocks Dissemination and Invasion by Activating MYH14

Alexandra Surcel, Eric S. Schiffhauer, Dustin G. Thomas, Qingfeng Zhu, Kathleen T. DiNapoli, Maik Herbig, Oliver Otto, Hoku West-Foyle, Angela Jacobi, et al.

Metastasis is complex, involving multiple genetic, epigenetic, biochemical, and physical changes in the cancer cell and its microenvironment. Cells with metastatic potential are often characterized by altered cellular contractility and deformability, lending them the flexibility to disseminate and navigate through different microenvironments. We demonstrate that mechanoresponsiveness is a hallmark of pancreatic cancer cells. Key mechanoresponsive proteins, those that accumulate in response to mechanical stress, specifically nonmuscle myosin IIA (MYH9) and IIC (MYH14), alpha-actinin 4, and filamin B, were highly expressed in pancreatic cancer as compared with healthy ductal epithelia. Their less responsive sister paralogs-myosin IIB (MYH10), alpha-actinin 1, and filamin A-had lower expression differential or disappeared with cancer progression. We demonstrate that proteins whose cellular contributions are often overlooked because of their low abundance can have profound impact on cell architecture, behavior, and mechanics. Here, the low abundant protein MYH14 promoted metastatic behavior and could be exploited with 4-hydroxyacetophenone (4-HAP), which increased MYH14 assembly, stiffening cells. As a result, 4-HAP decreased dissemination, induced cortical actin belts in spheroids, and slowed retrograde actin flow. 4-HAP also reduced liver metastases in human pancreatic cancer-bearing nude mice. Thus, increasing MYH14 assembly overwhelms the ability of cells to polarize and invade, suggesting targeting the mechanoresponsive proteins of the actin cytoskeleton as a new strategy to improve the survival of patients with pancreatic cancer.<br> Significance: This study demonstrates that mechanoresponsive proteins become upregulated with pancreatic cancer progression and that this system of proteins can be pharmacologically targeted to inhibit the metastatic potential of pancreatic cancer cells.

nanite: using machine learning to assess the quality of atomic force microscopy-enabled nano-indentation data

Paul Müller, Shada Abuhattum Hofemeier, Stephanie Möllmert, Elke Ulbricht, Anna V. Taubenberger, Jochen Guck

Atomic force microscopy (AFM) allows the mechanical characterization of single cells and live tissue by quantifying force-distance (FD) data in nano-indentation experiments. One of the main problems when dealing with biological tissue is the fact that the measured FD curves can be disturbed. These disturbances are caused, for instance, by passive cell movement, adhesive forces between the AFM probe and the cell, or insufficient attachment of the tissue to the supporting cover slide. In practice, the resulting artifacts are easily spotted by an experimenter who then manually sorts out curves before proceeding with data evaluation. However, this manual sorting step becomes increasingly cumbersome for studies that involve numerous measurements or for quantitative imaging based on FD maps.

3D Microenvironment Stiffness Regulates Tumor Spheroid Growth and Mechanics via p21 and ROCK

Anna V. Taubenberger, Salvatore Girardo, Nicole Träber, Elisabeth Fischer-Friedrich, Martin Kräter, Katrin Wagner, Thomas Kurth, Isabel Richter, Barbara Haller, et al.

The mechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using 2D substrates, much less is known about it in a physiologically more relevant 3D context. Here it is demonstrated that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor sphe-roid growth, compressive stresses of up to 2 kPa build up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy reveals that tumor spheroid stiffness increases with hydrogel stiffness. Also, constituent cell stiffness increases in a Rho associated kinase (ROCK)- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase, and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition not only reverses cell stiffening upon culture in stiff hydrogels but also increases tumor spheroid growth. Taken together, a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoen-vironment is revealed here. Thus, the findings contribute to a better under-standing of how cancer cells react to compressive stress when growing under confinement in stiff environments.

Interferometric Scattering (iSCAT) Microscopy & Related Techniques

Richard W. Taylor, Vahid Sandoghdar

Interferometric scattering (iSCAT) microscopy is a powerful tool for label-free sensitive detection and imaging of nanoparticles to high spatiotemporal resolution. As it was born out of detection principles central to conventional microscopy, we begin by surveying the historical development of the microscope to examine how the exciting possibility for interferometric scattering microscopy with sensitivities sufficient to observe single molecules has become a reality. We discuss the theory of interferometric detection and also issues relevant to achieving a high detection sensitivity and speed. A showcase of numerous applications and avenues of novel research across various disciplines that iSCAT microscopy has opened up is also presented.

Effects of rigosertib on the osteo-hematopoietic niche in myelodysplastic syndromes

Ekaterina Balaian, Heike Weidner, Manja Wobus, Ulrike Baschant, Angela Jacobi, Anna Mies, Martin Bornhäuser, Jochen Guck, Lorenz C Hofbauer, et al.

Rigosertib is a novel multi-kinase inhibitor, which has clinical activity towards leukemic progenitor cells of patients with high-risk myelodysplastic syndromes (MDS) after failure or progression on hypomethylating agents. Since the bone marrow microenvironment plays an important role in MDS pathogenesis, we investigated the impact of rigosertib on cellular compartments within the osteo-hematopoietic niche. Healthy C57BL/6J mice treated with rigosertib for 3 weeks showed a mild suppression of hematopoiesis (hemoglobin and red blood cells, both - 16%, p < 0.01; white blood cells, - 34%, p < 0.05; platelets, - 38%, p < 0.05), whereas there was no difference in the number of hematopoietic stem cells in the bone marrow. Trabecular bone mass of the spine was reduced by rigosertib (- 16%, p = 0.05). This was accompanied by a lower trabecular number and thickness (- 6% and - 10%, respectively, p < 0.05), partly explained by the increase in osteoclast number and surface (p < 0.01). Milder effects of rigosertib on bone mass were detected in an MDS mouse model system (NHD13). However, rigosertib did not further aggravate MDS-associated cytopenia in NHD13 mice. Finally, we tested the effects of rigosertib on human mesenchymal stromal cells (MSC) in vitro and demonstrated reduced cell viability at nanomolar concentrations. Deterioration of the hematopoietic supportive capacity of MDS-MSC after rigosertib pretreatment demonstrated by decreased number of colony-forming units, especially in the monocytic lineage, further supports the idea of disturbed crosstalk within the osteo-hematopoietic niche mediated by rigosertib. Thus, rigosertib exerts inhibitory effects on the stromal components of the osteo-hematopoietic niche which may explain the dissociation between anti-leukemic activity and the absence of hematological improvement.

Coherent nonlinear optics of quantum emitters in nanophotonic waveguides

Pierre Türschmann, Hanna Le Jeannic, Signe F. Simonsen, Harald Haakh, Stephan Götzinger, Vahid Sandoghdar, Peter Lodahl, Nir Rotenberg

Coherent quantum optics, where the phase of a photon is not scrambled as it interacts with an emitter, lies at the heart of many quantum optical effects and emerging technologies. Solid-state emitters coupled to nanophotonic waveguides are a promising platform for quantum devices, as this element can be integrated into complex photonic chips. Yet, preserving the full coherence properties of the coupled emitter-waveguide system is challenging because of the complex and dynamic electromagnetic landscape found in the solid state. Here, we review progress toward coherent light-matter interactions with solid-state quantum emitters coupled to nanophotonic waveguides. We first lay down the theoretical foundation for coherent and nonlinear light-matter interactions of a two-level system in a quasi-one-dimensional system, and then benchmark experimental realizations. We discuss higher order nonlinearities that arise as a result of the addition of photons of different frequencies, more complex energy level schemes of the emitters, and the coupling of multiple emitters via a shared photonic mode. Throughout, we highlight protocols for applications and novel effects that are based on these coherent interactions, the steps taken toward their realization, and the challenges that remain to be overcome.

Interferometric Scattering Microscopy: Seeing Single Nanoparticles and Molecules via Rayleigh Scattering

Richard W. Taylor, Vahid Sandoghdar

Fluorescence microscopy has been the workhorse for investigating optical phenomena at the nanometer scale but this approach confronts several fundamental limits. As a result, there have been a growing number of activities toward the development of fluorescent-free imaging methods. In this Mini Review, we demonstrate that elastic scattering, the most ubiquitous and oldest optical contrast mechanism, offers excellent opportunities for sensitive detection and imaging of nanoparticles and molecules at very high spatiotemporal resolution. We present interferometric scattering (iSCAT) microscopy as the method of choice, explain its theoretical foundation, discuss its experimental nuances, elaborate on its deep connection to bright-field imaging and other established microscopies, and discuss its promise as well as challenges. A showcase of numerous applications and avenues made possible by iSCAT demonstrates its rapidly growing impact on various disciplines concerned with nanoscopic phenomena.

High-Throughput Microfluidic Characterization of Erythrocyte Shapes and Mechanical Variability

Felix Reichel, Johannes Mauer, Ahmad Ahsan Nawaz, Gerhard Gompper, Jochen Guck, Dmitry A. Fedosov

The motion of red blood cells (RBCs) in microchannels is important for microvascular blood flow and biomedical applications such as blood analysis in microfluidics. The current understanding of the complexity of RBC shapes and dynamics in microchannels is mainly based on several simulation studies, but there are a few systematic experimental investigations. Here, we present a combined study that systematically characterizes RBC behavior for a wide range of flow rates and channel sizes. Even though simulations and experiments generally show good agreement, experimental observations demonstrate that there is no single well-defined RBC state for fixed flow conditions but rather a broad distribution of states. This result can be attributed to the inherent variability in RBC mechanical properties, which is confirmed by a model that takes the variation in RBC shear elasticity into account This represents a significant step toward a quantitative connection between RBC behavior in microfluidic devices and their mechanical properties, which is essential for a high-throughput characterization of diseased cells.

Coherent coupling of single molecules to on-chip ring resonators

Dominik Rattenbacher, Alexey Shkarin, Jan Renger, Tobias Utikal, Stephan Götzinger, Vahid Sandoghdar

We report on cryogenic coupling of organic molecules to ring microresonators obtained by looping subwavelength waveguides (nanoguides). We discuss fabrication and characterization of the chip-based nanophotonic elements which yield a resonator finesse in the order of 20 when covered by molecular crystals. Our observed extinction dips from single molecules reach 22%, consistent with an expected enhancement factor of up to 11 for the molecular emission into the nanoguide. Future efforts will aim at efficient coupling of a handful of molecules via their interaction with a ring microresonator mode, setting the ground for the realization of quantum optical cooperative effects.

Analysis of biomechanical properties of hematopoietic stem and progenitor cells with Real-Time Deformability Cytometry

Angela Jacobi, Philipp Rosendahl, Martin Kräter, Marta Urbanska, Maik Herbig, Jochen Guck

Stem cell mechanics, determined predominantly by the cell's cytoskeleton, plays an important role in different biological processes such as stem cell differentiation or migration. Several methods to measure mechanical properties of cells are currently available, but most of them are limited in the ability to screen large heterogeneous populations in a robust and efficient manner-a feature required for successful translational applications. With real-time fluorescence and deformability cytometry (RT-FDC), mechanical properties of cells in suspension can be screened continuously at rates of up to 1,000 cells/s-similar to conventional flow cytometers-which makes it a suitable method not only for basic research but also for a clinical setting. In parallel to mechanical characterization, RT-FDC allows to measure specific molecular markers using standard fluorescence labeling. In this chapter, we provide a detailed protocol for the characterization of hematopoietic stem and progenitor cells (HSPCs) in heterogeneous mobilized peripheral blood using RT-FDC and present a specific morpho-rheological fingerprint of HSPCs that allows to distinguish them from all other blood cell types.

Electrically driven single-photon superradiance from molecular chains in a plasmonic nanocavity

Yang Luo, Gong Chen, Yang Zhang, Li Zhang, Yunjie Yu, Fanfang Kong, Xiaojun Tian, Yao Zhang, Chongxin Shan, et al.

We demonstrate single-photon superradiance from artificially constructed nonbonded zinc-phthalocyanine molecular chains of up to 12 molecules. We excite the system via electron tunneling in a plasmonic nanocavity and quantitatively investigate the interaction of the localized plasmon with single-exciton superradiant states resulting from dipole-dipole coupling. Dumbbell-like patterns obtained by subnanometer resolved spectroscopic imaging disclose the coherent nature of the coupling associated with superradiant states while second-order photon correlation measurements demonstrate single-photon emission. The combination of spatially resolved spectral measurements with theoretical considerations reveals that nanocavity plasmons dramatically modify the linewidth and intensity of emission from the molecular chains, but they do not dictate the intrinsic coherence of the superradiant states. Our studies shed light on the optical properties of molecular collective states and their interaction with nanoscopically localized plasmons.

Morpho-Rheological Fingerprinting of Rod Photoreceptors Using Real-Time Deformability Cytometry

Tiago Santos-Ferreira, Maik Herbig, Oliver Otto, Madalena Carido, Mike O. Karl, Stylianos Michalakis, Jochen Guck, Marius Ader

Distinct cell-types within the retina are mainly specified by morphological and molecular parameters, however, physical properties are increasingly recognized as a valuable tool to characterize and distinguish cells in diverse tissues. High-throughput analysis of morpho-rheological features has recently been introduced using real-time deformability cytometry (RT-DC) providing new insights into the properties of different cell-types. Rod photoreceptors represent the main light sensing cells in the mouse retina that during development forms apically the densely packed outer nuclear layer. Currently, enrichment and isolation of photoreceptors from retinal primary tissue or pluripotent stem cell-derived organoids for analysis, molecular profiling, or transplantation is achieved using flow cytometry or magnetic activated cell sorting approaches. However, such purification methods require genetic modification or identification of cell surface binding antibody panels. Using primary retina and embryonic stem cell-derived retinal organoids, we characterized the inherent morpho-mechanical properties of mouse rod photoreceptors during development based on RT-DC. We demonstrate that rods become smaller and more compliant throughout development and that these features are suitable to distinguish rods within heterogenous retinal tissues. Hence, physical properties should be considered as additional factors that might affect photoreceptor differentiation and retinal development besides representing potential parameters for label-free sorting of photoreceptors.

How bacterial cells and colonies move on solid substrates

Wolfram Poenisch, Christoph A. Weber, Vasily Zaburdaev

Many bacteria rely on active cell appendages, such as type IV pili, to move over substrates and interact with neighboring cells. Here, we study the motion of individual cells and bacterial colonies, mediated by the collective interactions of multiple pili. It was shown experimentally that the substrate motility of Neisseria gonorrhoeae cells can be described as a persistent random walk with a persistence length that exceeds the mean pili length. Moreover, the persistence length increases for a higher number of pili per cell. With the help of a simple, tractable stochastic model, we test whether a tug of war without directional memory can explain the persistent motion of single Neisseria gonorrhoeae cells. While persistent motion of single cells indeed emerges naturally in the model, a tug of war alone is not capable of explaining the motility of microcolonies, which becomes weaker with increasing colony size. We suggest sliding friction between the microcolonies and the substrate as the missing ingredient. While such friction almost does not affect the general mechanism of single cell motility, it has a strong effect on colony motility. We validate the theoretical predictions by using a three-dimensional computational model that includes explicit details of the pili dynamics, force generation, and geometry of cells.

Nanoprinting organic molecules at the quantum level

Claudio U. Hail, Christian Höller, Korenobu Matsuzaki, Patrik Rohner, Jan Renger, Vahid Sandoghdar, Dimos Poulikakos, Hadi Eghlidi

Organic compounds present a powerful platform for nanotechnological applications. In particular, molecules suitable for optical functionalities such as single photon generation and energy transfer have great promise for complex nanophotonic circuitry due to their large variety of spectral properties, efficient absorption and emission, and ease of synthesis. Optimal integration, however, calls for control over position and orientation of individual molecules. While various methods have been explored for reaching this regime in the past, none satisfies requirements necessary for practical applications. Here, we present direct non-contact electrohydrodynamic nanoprinting of a countable number of photostable and oriented molecules in a nanocrystal host with subwavelength positioning accuracy. We demonstrate the power of our approach by writing arbitrary patterns and controlled coupling of single molecules to the near field of optical nanostructures. Placement precision, high yield and fabrication facility of our method open many doors for the realization of novel nanophotonic devices.

Spheroid Culture of Mesenchymal Stromal Cells Results in Morphorheological Properties Appropriate for Improved Microcirculation

Stefanie Tietze, Martin Kräter, Angela Jacobi, Anna Taubenberger, Maik Herbig, Rebekka Wehner, Marc Schmitz, Oliver Otto, Catrin List, et al.

Human bone marrow mesenchymal stromal cells (MSCs) are used in clinical trials for the treatment of systemic inflammatory diseases due to their regenerative and immunomodulatory properties. However, intravenous administration of MSCs is hampered by cell trapping within the pulmonary capillary networks. Here, it is hypothesized that traditional 2D plastic-adherent cell expansion fails to result in appropriate morphorheological properties required for successful cell circulation. To address this issue, a method to culture MSCs in nonadherent 3D spheroids (mesenspheres is adapted. The biological properties of mesensphere-cultured MSCs remain identical to conventional 2D cultures. However, morphorheological analyses reveal a smaller size and lower stiffness of mesensphere-derived MSCs compared to plastic-adherent MSCs, measured using real-time deformability cytometry and atomic force microscopy. These properties result in an increased ability to pass through microconstrictions in an ex vivo microcirculation assay. This ability is confirmed in vivo by comparison of cell accumulation in various organ capillary networks after intravenous injection of both types of MSCs in mouse. The findings generally identify cellular morphorheological properties as attractive targets for improving microcirculation and specifically suggest mesensphere culture as a promising approach for optimized MSC-based therapies.

Interferometric scattering microscopy reveals microsecond nanoscopic protein motion on a live cell membrane

Richard W. Taylor, Reza Gholami Mahmoodabadi, Verena Rauschenberger, Andreas Giessl, Alexandra Schambony, Vahid Sandoghdar

Much of the biological functions of a cell are dictated by the intricate motion of proteins within its membrane over a spatial range of nanometers to tens of micrometers and time intervals of microseconds to minutes. While this rich parameter space is not accessible to fluorescence microscopy, it can be within reach of interferometric scattering (iSCAT) particle tracking. Being sensitive even to single unlabeled proteins, however, iSCAT is easily accompanied by a large speckle-like background, which poses a substantial challenge for its application to cellular imaging. Here, we show that these difficulties can be overcome and demonstrate tracking of transmembrane epidermal growth factor receptors (EGFR) with nanometer precision in all three dimensions at up to microsecond speeds and tens of minutes duration. We provide unprecedented examples of nanoscale motion and confinement in ubiquitous processes such as diffusion in the plasma membrane, transport on filopodia, and endocytosis.

The shape of pinned forced polymer loops

Wenwen Huang, Vasily Zaburdaev

Loop geometry is a frequent encounter in synthetic and biological polymers. Here we provide an analytical theory to characterize the shapes of polymer loops subjected to an external force field. We show how to calculate the polymer density, gyration radius and its distribution. Interestingly, the distribution of the gyration radius shows a non-monotonic behavior as a function of the external force. Furthermore, we analyzed the gyration tensor of the polymer loop characterizing its overall shape. Two parameters called asphericity and the nature of asphericity derived from the gyration tensor, along with the gyration radius, can be used to quantify the shape of polymer loops in theory and experiments.

Turning a molecule into a coherent two-level quantum system

Daqing Wang, Hrishikesh Kelkar, Diego-Martin Cano, Dominik Rattenbacher, Alexey Shkarin, Tobias Utikal, Stephan Götzinger, Vahid Sandoghdar

The use of molecules in quantum optical applications has been hampered by incoherent internal vibrations and other phononic interactions with their environment. Here we show that an organic molecule placed into an optical microcavity behaves as a coherent two-level quantum system. This allows the observation of 99% extinction of a laser beam by a single molecule, saturation with less than 0.5 photons and non-classical generation of few-photons super-bunched light. Furthermore, we demonstrate efficient interaction of the molecule–microcavity system with single photons generated by a second molecule in a distant laboratory. Our achievements represent an important step towards linear and nonlinear quantum photonic circuits based on organic platforms.

Identifying the mechanism for superdiffusivity in mouse fibroblast motility

Giuseppe Passucci, Megan E. Brasch, James H. Henderson, Vasily Zaburdaev, M. Lisa Manning

We seek to characterize the motility of mouse fibroblasts on 2D substrates. Utilizing automated tracking techniques, we find that cell trajectories are super-diffusive, where displacements scale faster than t(1/2) in all directions. Two mechanisms have been proposed to explain such statistics in other cell types: run and tumble behavior with Levy-distributed run times, and ensembles of cells with heterogeneous speed and rotational noise. We develop an automated toolkit that directly compares cell trajectories to the predictions of each model and demonstrate that ensemble-averaged quantities such as the mean-squared displacements and velocity autocorrelation functions are equally well-fit by either model. However, neither model correctly captures the short-timescale behavior quantified by the displacement probability distribution or the turning angle distribution. We develop a hybrid model that includes both run and tumble behavior and heterogeneous noise during the runs, which correctly matches the short-timescale behaviors and indicates that the run times are not Levy distributed. The analysis tools developed here should be broadly useful for distinguishing between mechanisms for superdiffusivity in other cells types and environments.

High throughput magnetic tweezers to characterize inhibitors of RNA virus replication

David Dulin

Label-Free Imaging of Single Proteins Secreted from Living Cells via iSCAT Microscopy

André Gemeinhardt, Matthew Paul McDonald, Katharina König, Michael Aigner, Andreas Mackensen, Vahid Sandoghdar

We demonstrate interferometric scattering (iSCAT) microscopy, a method capable of detecting single unlabeled proteins secreted from individual living cells in real time. In this protocol, we cover the fundamental steps to realize an iSCAT microscope and complement it with additional imaging channels to monitor the viability of a cell under study. Following this, we use the method for real-time detection of single proteins as they are secreted from a living cell which we demonstrate with an immortalized B-cell line (Laz388). Necessary steps concerning the preparation of microscope and sample as well as the analysis of the recorded data are discussed. The video protocol demonstrates that iSCAT microscopy offers a straightforward method to study secretion at the single-molecule level.

Pili mediated intercellular forces shape heterogeneous bacterial microcolonies prior to multicellular differentiation

Wolfram Poenisch, Kelly B. Eckenrode, Khaled Alzurqa, Hadi Nasrollahi, Christoph Weber, Vasily Zaburdaev, Nicolas Biais

Microcolonies are aggregates of a few dozen to a few thousand cells exhibited by many bacteria. The formation of microcolonies is a crucial step towards the formation of more mature bacterial communities known as biofilms, but also marks a significant change in bacterial physiology. Within a microcolony, bacteria forgo a single cell lifestyle for a communal lifestyle hallmarked by high cell density and physical interactions between cells potentially altering their behaviour. It is thus crucial to understand how initially identical single cells start to behave differently while assembling in these tight communities. Here we show that cells in the microcolonies formed by the human pathogen Neisseria gonorrhoeae (Ng) present differential motility behaviors within an hour upon colony formation. Observation of merging microcolonies and tracking of single cells within microcolonies reveal a heterogeneous motility behavior: cells close to the surface of the microcolony exhibit a much higher motility compared to cells towards the center. Numerical simulations of a biophysical model for the microcolonies at the single cell level suggest that the emergence of differential behavior within a multicellular microcolony of otherwise identical cells is of mechanical origin. It could suggest a route toward further bacterial differentiation and ultimately mature biofilms.

Exactly solvable dynamics of forced polymer loops

Wenwen Huang, Yen Ting Lin, Daniela Froemberg, Jaeoh Shin, Frank Juelicher, Vasily Zaburdaev

Here, we show that a problem of forced polymer loops can be mapped to an asymmetric simple exclusion process with reflecting boundary conditions. The dynamics of the particle system can be solved exactly using the Bethe ansatz. We thus can fully describe the relaxation dynamics of forced polymer loops. In the steady state, the conformation of the loop can be approximated by a combination of Fermi-Dirac and Brownian bridge statistics, while the exact solution is found by using the fermion integer partition theory. With the theoretical framework presented here we establish a link between the physics of polymers and statistics of many-particle systems opening new paths of exploration in both research fields. Our result can be applied to the dynamics of the biopolymers which form closed loops. One such example is the active pulling of chromosomal loops during meiosis in yeast cells which helps to align chromosomes for recombination in the viscous environment of the cell nucleus.

Correction-free force calibration for magnetic tweezers experiments

Eugen Ostrofet, Flavia Stal Papini, David Dulin

Magnetic tweezers are a powerful technique to perform high-throughput and high-resolution force spectroscopy experiments at the single-molecule level. The camera-based detection of magnetic tweezers enables the observation of hundreds of magnetic beads in parallel, and therefore the characterization of the mechanochemical behavior of hundreds of nucleic acids and enzymes. However, magnetic tweezers experiments require an accurate force calibration to extract quantitative data, which is limited to low forces if the deleterious effect of the finite camera open shutter time (tau(sh)) is not corrected. Here, we provide a simple method to perform correction-free force calibration for high-throughput magnetic tweezers at low image acquisition frequency (f(ac)). By significantly reducing tau(sh) to at least 4-fold the characteristic times of the tethered magnetic bead, we accurately evaluated the variance of the magnetic bead position along the axis parallel to the magnetic field, estimating the force with a relative error of similar to 10% (standard deviation), being only limited by the bead-to-bead difference. We calibrated several magnets - magnetic beads configurations, covering a force range from similar to 50 fN to similar to 60 pN. In addition, for the presented configurations, we provide a table with the mathematical expressions that describe the force as a function of the magnets position.

High-Speed Microscopy of Diffusion in Pore-Spanning Lipid Membranes

Susann Spindler, Jeremias Sibold, Reza Gholami Mahmoodabadi, Claudia Steinem, Vahid Sandoghdar

Pore-spanning membranes (PSMs) provide a highly attractive model system for investigating fundamental processes in lipid bilayers. We measure and compare lipid diffusion in the supported and suspended regions of PSMs prepared on a microfabricated porous substrate. Although some properties of the suspended regions in PSMs have been characterized using fluorescence studies, it has not been possible to examine the mobility of membrane components on the supported membrane parts. Here, we resolve this issue by employing interferometric scattering microscopy (iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoylsn-glycero-3-phosphoethanolamine) lipids (DOPE) labeled with gold nanoparticles in (l,2-dioleoyl-sn-glycero-3-phosphocholine) (DOPC) bilayers prepared on holey silicon nitride substrates that were either (i) oxygen-plasma-treated or (ii) functionalized with gold and 6-mercapto-l-hexanol. For both substrate treatments, diffusion in regions suspended on pores with diameters of 5 mu m is found to be free. In the case of functionalization with gold and 6-mercapto-l-hexanol, similar diffusion coefficients are obtained for both the suspended and the supported regions, whereas for oxygen-plasma-treated surfaces, diffusion is almost 4 times slower in the supported parts of the membranes. We attribute this reduced diffusion on the supported parts in the case of oxygen-plasma-treated surfaces to larger membrane-substrate interactions, which lead to a higher membrane tension in the freestanding membrane parts. Furthermore, we find clear indications for a decrease of the diffusion constant in the freestanding regions away from the pore center. We provide a detailed characterization of the diffusion behavior in these membrane systems and discuss future directions.

Pausing controls branching between productive and non-productive pathways during initial transcription in bacteria

David Dulin, David L. V. Bauer, Anssi M. Malinen, Jacob J. W. Bakermans, Martin Kaller, Zakia Morichaud, Ivan Petushkov, Martin Depken, Konstantin Brodolin, et al.

Transcription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. It has been recently shown that initial RNA synthesis by the bacterial RNA polymerase (RNAP) is interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Using single-molecule FRET and biochemical analysis, here we show that the pause encountered by RNAP after the synthesis of a 6-nt RNA (ITC6) renders the promoter escape strongly dependent on the NTP concentration. Mechanistically, the paused ITC6 acts as a checkpoint that directs RNAP to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway. The cyclic unscrunching/scrunching of the promoter generates a long-lived, RNA-bound paused state; the abortive RNA release and DNA unscrunching are thus not as tightly linked as previously thought. Finally, our new model couples the pausing with the abortive and productive outcomes of initial transcription.

Manipulation of Quenching in Nanoantenna–Emitter Systems Enabled by External Detuned Cavities: A Path to Enhance Strong-Coupling

Burak Gürlek, Vahid Sandoghdar, Diego-Martin Cano

We show that a broadband Fabry Perot microcavity can assist an emitter coupled to an off-resonant plasmonic nanoantenna to inhibit the nonradiative channels that affect the quenching of fluorescence. We identify the interference mechanism that creates the necessary enhanced couplings and bandwidth narrowing of the hybrid resonance and show that it can assist entering into the strong coupling regime. Our results provide new possibilities for improving the efficiency of solid-state emitters and accessing diverse realms of photophysics with hybrid structures that can be fabricated using existing technologies.

Visualizing single-cell secretion dynamics with single protein sensitivity

Matthew Paul McDonald, André Gemeinhardt, Katharina König, Marek Piliarik, Stefanie Schaffer, Simon Völkl, Andreas Mackensen, Vahid Sandoghdar

Cellular secretion of proteins into the extracellular environment is an essential mediator of critical biological mechanisms, including cell-to-cell communication, immunological response, targeted delivery, and differentiation. Here, we report a novel methodology that allows for the real-time detection and imaging of single unlabeled proteins that are secreted from individual living cells. This is accomplished via interferometric detection of scattered light (iSCAT) and is demonstrated with Laz388 cells, an Epstein Barr virus (EBV)-transformed B cell line. We find that single Laz388 cells actively secrete IgG antibodies at a rate of the order of 100 molecules per second. Intriguingly, we also find that other proteins and particles spanning ca. 100 kDa-1 MDa are secreted from the Laz388 cells in tandem with IgG antibody release, likely arising from EBV-related viral proteins. The technique is general and, as we show, can also be applied to studying the lysate of a single cell. Our results establish label-free iSCAT imaging as a powerful tool for studying the real-time exchange between cells and their immediate environment with single-protein sensitivity.

Intracellular Mass Density Increase Is Accompanying but Not Sufficient for Stiffening and Growth Arrest of Yeast Cells

Shada Abuhattum, Kyoohyun Kim, Titus M. Franzmann, Anne Esslinger, Daniel Midtvedt, Raimund Schluessler, Stephanie Mollmert, Hui-Shun Kuan, Simon Alberti, et al.

Many organisms, including yeast cells, bacteria, nematodes, and tardigrades, endure harsh environmental conditions, such as nutrient scarcity, or lack of water and energy for a remarkably long time. The rescue programs that these organisms launch upon encountering these adverse conditions include reprogramming their metabolism in order to enter a quiescent or dormant state in a controlled fashion. Reprogramming coincides with changes in the macromolecular architecture and changes in the physical and mechanical properties of the cells. However, the cellular mechanisms underlying the physical-mechanical changes remain enigmatic. Here, we induce metabolic arrest of yeast cells by lowering their intracellular pH. We then determine the differences in the intracellular mass density and stiffness of active and metabolically arrested cells using optical diffraction tomography (ODT) and atomic force microscopy (AFM). We show that an increased intracellular mass density is associated with an increase in stiffness when the growth of yeast is arrested. However, increasing the intracellular mass density alone is not sufficient for maintenance of the growth-arrested state in yeast cells. Our data suggest that the cytoplasm of metabolically arrested yeast displays characteristics of a solid. Our findings constitute a bridge between the mechanical behavior of the cytoplasm and the physical and chemical mechanisms of metabolically arrested cells with the ultimate aim of understanding dormant organisms.

Kinetics of CrPV and HCV IRES-mediated eukaryotic translation using single-molecule fluorescence microscopy

Olivier Bugaud, Nathalie Barbier, Helene Chommy, Nicolas Fiszman, Antoine Le Gall, David Dulin, Matthieu Saguy, Nathalie Westbrook, Karen Perronet, et al.

Protein synthesis is a complex multistep process involving many factors that need to interact in a coordinated manner to properly translate the messenger RNA. As translating ribosomes cannot be synchronized over many elongation cycles, single-molecule studies have been introduced to bring a deeper understanding of prokaryotic translation dynamics. Extending this approach to eukaryotic translation is very appealing, but initiation and specific labeling of the ribosomes are much more complicated. Here, we use a noncanonical translation initiation based on internal ribosome entry sites (IRES), and we monitor the passage of individual, unmodified mammalian ribosomes at specific fluorescent milestones along mRNA. We explore initiation by two types of IRES, the intergenic IRES of cricket paralysis virus (CrPV) and the hepatitis C (HCV) IRES, and show that they both strongly limit the rate of the first elongation steps compared to the following ones, suggesting that those first elongation cycles do not correspond to a canonical elongation. This new system opens the possibility of studying both IRES-mediated initiation and elongation kinetics of eukaryotic translation and will undoubtedly be a valuable tool to investigate the role of translation machinery modifications in human diseases.

Signatures of Nucleotide Analog Incorporation by an RNA-Dependent RNA Polymerase Revealed Using High-Throughput Magnetic Tweezers

David Dulin, Jamie J. Arnold, Theo van Laar, Hyung-Suk Oh, Cheri Lee, Angela L. Perkins, Daniel A. Harki, Martin Depken, Craig E. Cameron, et al.

RNA viruses pose a threat to public health that is exacerbated by the dearth of antiviral therapeutics. The RNA-dependent RNA polymerase (RdRp) holds promise as a broad-spectrum, therapeutic target because of the conserved nature of the nucleotide-substrate-binding and catalytic sites. Conventional, quantitative, kinetic analysis of antiviral ribonucleotides monitors one or a few incorporation events. Here, we use a high-throughput magnetic tweezers platformto monitor the elongation dynamics of a prototypicalRdRpover thousands of nucleotide-addition cycles in the absence and presence of a suite of nucleotide analog inhibitors. We observe multiple RdRpRNA elongation complexes; only a subset of which are competent for analog utilization. Incorporation of a pyrazine-carboxamide nucleotide analog, T-1106, leads to RdRp backtracking. This analysis reveals a mechanism of action for this antiviral ribonucleotide that is corroborated by cellular studies. We propose that induced backtracking represents a distinct mechanistic class of antiviral ribonucleotides.

Probing the salt dependence of the torsional stiffness of DNA by multiplexed magnetic torque tweezers

Franziska Kriegel, Niklas Ermann, Ruaridh Forbes, David Dulin, Nynke H. Dekker, Jan Lipfert

The mechanical properties of DNA fundamentally constrain and enable the storage and transmission of genetic information and its use in DNA nanotechnology. Many properties of DNA depend on the ionic environment due to its highly charged backbone. In particular, both theoretical analyses and direct single-molecule experiments have shown its bending stiffness to depend on salt concentration. In contrast, the salt-dependence of the twist stiffness of DNA is much less explored. Here, we employ optimized multiplexed magnetic torque tweezers to study the torsional stiffness of DNA under varying salt conditions as a function of stretching force. At low forces (< 3 pN), the effective torsional stiffness is similar to 10% smaller for high salt conditions (500 mM NaCl or 10 mM MgCl2) compared to lower salt concentrations (20 mM NaCl and 100 mM NaCl). These differences, however, can be accounted for by taking into account the known salt dependence of the bending stiffness. In addition, the measured high-force (6.5 pN) torsional stiffness values of C = 103 +/- 4 nm are identical, within experimental errors, for all tested salt concentration, suggesting that the intrinsic torsional stiffness of DNA does not depend on salt.