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.

Thermal fluctuations assist mechanical signal propagation in coiled-coil proteins

Judit Clopes, Jaeoh Shin, Marcus Jahnel, Stephan W. Grill, Vasily Zaburdaev

Recently, it has been shown that the long coiled-coil membrane tether protein early endosome antigen 1 (EEA1) switches from a rigid to a flexible conformation upon binding of a signaling protein to its free end. This flexibility switch represents a motorlike activity, allowing EEA1 to generate a force that moves vesicles closer to the membrane they will fuse with. It was hypothesized that the binding-induced signal could propagate along the coiled coil and lead to conformational changes through the localized domains of the protein chain that deviate from a perfect coiled-coil structure. To elucidate, if upon binding of a single protein the corresponding mechanical signal could propagate through the whole 200-nm-long chain, we propose a simplified description of the coiled coil as a one-dimensional Frenkel-Kontorova chain. Using numerical simulations, we find that an initial perturbation of the chain can propagate along its whole length in the presence of thermal fluctuations. This may enable the change of the configuration of the entire molecule and thereby affect its stiffness. Our work sheds light on intramolecular communication and force generation in long coiled-coil proteins.

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.

Optimized analysis for sensitive detection and analysis of single proteins via interferometric scattering microscopy

Houman Mirzaalian Dastjerdi, Mahyar Dahmardeh, André Gemeinhardt, Reza Gholami Mahmoodabadi, Harald Köstler, Vahid Sandoghdar

It has been shown that interferometric detection of Rayleigh scattering (iSCAT) can reach an exquisite sensitivity for label-free detection of nano-matter, down to single proteins. The sensitivity of iSCAT detection is intrinsically limited by shot noise, which can be indefinitely improved by employing higher illumination power or longer integration times. In practice, however, a large speckle-like background and technical issues in the experimental setup limit the attainable signal-to-noise ratio. Strategies and algorithms in data analysis are, thus, crucial for extracting quantitative results from weak signals, e.g. regarding the mass (size) of the detected nano-objects or their positions. In this article, we elaborate on some algorithms for processing iSCAT data and identify some key technical as well as conceptual issues that have to be considered when recording and interpreting the data. The discussed methods and analyses are made available in the extensive python-based platform, PiSCAT.

StemBond hydrogels control the mechanical microenvironment for pluripotent stem cells

Céline Labouesse, Bao Xiu Tan, Chibeza C. Agley, Moritz Hofer, Alexander K. Winkel, Giuliano G. Stirparo, Hannah T. Stuart, Christophe M. Verstreken, Carla Mulas, et al.

Studies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and extracellular matrix tethering to substrates, making matrix tethering a potentially confounding variable in mechanical signalling investigations. Moreover, unstable matrix tethering can lead to poor cell attachment and weak engagement of cell adhesions. To address this, we developed StemBond hydrogels, a hydrogel in which matrix tethering is robust and can be varied independently of stiffness. We validate StemBond hydrogels by showing that they provide an optimal system for culturing mouse and human pluripotent stem cells. We further show how soft StemBond hydrogels modulate stem cell function, partly through stiffness-sensitive ERK signalling. Our findings underline how substrate mechanics impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

Vinculin is required for neuronal mechanosensing but not for axon outgrowth

De-Yao Wang, Cristina Melero, Ashwaq Albaraky, Paul Atherton, Karin A. Jansen, Andrea Dimitracopoulos, Federico Dajas-Bailador, Adam Reid, Kristian Franze, et al.

Integrin receptors are transmembrane proteins that bind to the extracellular matrix (ECM). In most animal cell types integrins cluster together with adaptor proteins at focal adhesions that sense and respond to external mechanical signals. In the central nervous system (CNS), ECM proteins are sparsely distributed, the tissue is comparatively soft and neurons do not form focal adhesions. Thus, how neurons sense tissue stiffness is currently poorly understood. Here, we found that integrins and the integrin-associated proteins talin and focal adhesion kinase (FAK) are required for the outgrowth of neuronal processes. Vinculin, however, whilst not required for neurite outgrowth was a key regulator of integrin-mediated mechanosensing of neurons. During growth, growth cones of axons of CNS derived cells exerted dynamic stresses of around 10-12 Pa on their environment, and axons grew significantly longer on soft (0.4 kPa) compared to stiff (8 kPa) substrates. Depletion of vinculin blocked this ability of growth cones to distinguish between soft and stiff substrates. These data suggest that vinculin in neurons acts as a key mechanosensor, involved in the regulation of growth cone motility.

RNA polymerase II clusters form in line with surface condensation on regulatory chromatin

Agnieszka Pancholi, Tim Klingberg, Weichun Zhang, Roshan Prizak, Irina Mamontova, Amra Noa, Marcel Sobucki, Andrei Yu Kobitski, Gerd Ulrich Nienhaus, et al.

It is essential for cells to control which genes are transcribed into RNA. In eukaryotes, two major control points are recruitment of RNA polymerase II (Pol II) into a paused state, and subsequent pause release toward transcription. Pol II recruitment and pause release occur in association with macromolecular clusters, which were proposed to be formed by a liquid-liquid phase separation mechanism. How such a phase separation mechanism relates to the interaction of Pol II with DNA during recruitment and transcription, however, remains poorly understood. Here, we use live and super-resolution microscopy in zebrafish embryos to reveal Pol II clusters with a large variety of shapes, which can be explained by a theoretical model in which regulatory chromatin regions provide surfaces for liquid-phase condensation at concentrations that are too low for canonical liquid-liquid phase separation. Model simulations and chemical perturbation experiments indicate that recruited Pol II contributes to the formation of these surface-associated condensates, whereas elongating Pol II is excluded from these condensates and thereby drives their unfolding.

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.

The Potential of OMICs Technologies for the Treatment of Immune-Mediated Inflammatory Diseases

Charles Gwellem Anchang, Cong Xu, Maria Gabriella Raimondo, Raja Atreya, Andreas Maier, Georg Schett, Vasily Zaburdaev, Simon Rauber, Andreas Ramming

Immune-mediated inflammatory diseases (IMIDs), such as inflammatory bowel diseases and inflammatory arthritis (e.g., rheumatoid arthritis, psoriatic arthritis), are marked by increasing worldwide incidence rates. Apart from irreversible damage of the affected tissue, the systemic nature of these diseases heightens the incidence of cardiovascular insults and colitis-associated neoplasia. Only 40-60% of patients respond to currently used standard-of-care immunotherapies. In addition to this limited long-term effectiveness, all current therapies have to be given on a lifelong basis as they are unable to specifically reprogram the inflammatory process and thus achieve a true cure of the disease. On the other hand, the development of various OMICs technologies is considered as "the great hope" for improving the treatment of IMIDs. This review sheds light on the progressive development and the numerous approaches from basic science that gradually lead to the transfer from "bench to bedside" and the implementation into general patient care procedures.

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

The hierarchical packing of euchromatin domains can be described as multiplicative cascades

Amra Noa, Hui-Shun Kuan, Vera Aschmann, Vasily Zaburdaev, Lennart Hilbert

The genome is packed into the cell nucleus in the form of chromatin. Biochemical approaches have revealed that chromatin is packed within domains, which group into larger domains, and so forth. Such hierarchical packing is equally visible in super-resolution microscopy images of large-scale chromatin organization. While previous work has suggested that chromatin is partitioned into distinct domains via microphase separation, it is unclear how these domains organize into this hierarchical packing. A particular challenge is to find an image analysis approach that fully incorporates such hierarchical packing, so that hypothetical governing mechanisms of euchromatin packing can be compared against the results of such an analysis. Here, we obtain 3D STED super-resolution images from pluripotent zebrafish embryos labeled with improved DNA fluorescence stains, and demonstrate how the hierarchical packing of euchromatin in these images can be described as multiplicative cascades. Multiplicative cascades are an established theoretical concept to describe the placement of ever-smaller structures within bigger structures. Importantly, these cascades can generate artificial image data by applying a single rule again and again, and can be fully specified using only four parameters. Here, we show how the typical patterns of euchromatin organization are reflected in the values of these four parameters. Specifically, we can pinpoint the values required to mimic a microphase-separated state of euchromatin. We suggest that the concept of multiplicative cascades can also be applied to images of other types of chromatin. Here, cascade parameters could serve as test quantities to assess whether microphase separation or other theoretical models accurately reproduce the hierarchical packing of chromatin.<br> Author summary<br> DNA is stored inside the cell nucleus in the form of chromatin. Chromatin exhibits a striking three-dimensional organization, where small domains group into larger domains, which again group into larger domains, and so forth. While this hierarchical domain packing is obvious from microscopy images, it is still not entirely clear how it is established, or how it should be properly characterized. Here, we demonstrate that multiplicative cascades-a concept from theoretical physics used to characterize for example cloud patterns, galaxy locations, or soil patterns-are also ideally suited to describe the domain-within-domain organization of chromatin. This description is rather simple, using only four numbers, and can thus facilitate testing of competing theories for the packing of chromatin domains.

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.

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.

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.

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.

Transcription organizes euchromatin via microphase separation

Lennart Hilbert, Yuko Sato, Ksenia Kuznetsova, Tommaso Bianucci, Hiroshi Kimura, Frank Jülicher, Alf Honigmann, Vasily Zaburdaev, Nadine L. Vastenhouw

In eukaryotes, DNA is packed inside the cell nucleus in the form of chromatin, which consists of DNA, proteins such as histones, and RNA. Euchromatin, which is permissive for transcription, is spatially organized into transcriptionally inactive domains interspersed with pockets of transcriptional activity. While transcription and RNA have been implicated in euchromatin organization, it remains unclear how their interplay forms and maintains transcription pockets. Here we combine theory and experiment to analyze the dynamics of euchromatin organization as pluripotent zebrafish cells exit mitosis and begin transcription. We show that accumulation of RNA induces formation of transcription pockets which displace transcriptionally inactive chromatin. We propose that the accumulating RNA recruits RNA-binding proteins that together tend to separate from transcriptionally inactive euchromatin. Full phase separation is prevented because RNA remains tethered to transcribed euchromatin through RNA polymerases. Instead, smaller scale microphases emerge that do not grow further and form the typical pattern of euchromatin organization.

Theory for Durotactic Axon Guidance

Hadrien Oliveri, Kristian Franze, Alain Goriely

During the development of the nervous system, neurons extend bundles of axons that grow and meet other neurons to form the neuronal network. Robust guidance mechanisms are needed for these bundles to migrate and reach their functional target. Directional information depends on external cues such as chemical or mechanical gradients. Unlike chemotaxis that has been extensively studied, the role and mechanism of durotaxis, the directed response to variations in substrate rigidity, remain unclear. We model bundle migration and guidance by rigidity gradients by using the theory of morphoelastic rods. We show that, at a rigidity interface, the motion of axon bundles follows a simple behavior analogous to optic ray theory and obeys Snell's law for refraction and reflection. We use this powerful analogy to demonstrate that axons can be guided by the equivalent of optical lenses and fibers created by regions of different stiffnesses.

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.

Continuum Theory of Active Phase Separation in Cellular Aggregates

Hui-Shun Kuan, Wolfram Poenisch, Frank Juelicher, Vasily Zaburdaev

Dense cellular aggregates are common in biology, ranging from bacterial biofilms to organoids, cell spheroids, and tumors. Their dynamics, driven by intercellular forces, is intrinsically out of equilibrium. Motivated by bacterial colonies as a model system, we present a continuum theory to study dense, active, cellular aggregates. We describe the process of aggregate formation as an active phase separation phenomenon, while the merging of aggregates is rationalized as a coalescence of viscoelastic droplets where the key timescales are linked to the turnover of the active force. Our theory provides a general framework for studying the rheology and nonequilibrium dynamics of dense cellular aggregates.