Higher order transient membrane protein structures

Yuxi Zhang, Hisham Mazal, Venkata Shiva Mandala, Gonzalo Perez-Mitta, Vahid Sandoghdar, Christoph A. Haselwandter, Roderick McKinnon

This study shows that five membrane proteins—three GPCRs, an ion channel, and an enzyme—form self-clusters under natural expression levels in a cardiac-derived cell line. The cluster size distributions imply that these proteins self-oligomerize reversibly through weak interactions. When the concentration of the proteins is increased through heterologous expression, the cluster size distributions approach a critical distribution at which point a phase transition occurs, yielding larger bulk phase clusters. A thermodynamic model like that explaining micellization of amphiphiles and lipid membrane formation accounts for this behavior. We propose that many membrane proteins exist as oligomers that form through weak interactions, which we call higher-order transient structures (HOTS). The key characteristics of HOTS are transience, molecular specificity, and a monotonically decreasing size distribution that may become critical at high concentrations. Because molecular specificity invokes self-recognition through protein sequence and structure, we propose that HOTS are genetically encoded supramolecular units.

Cryogenic light microscopy with Ångstrom precision deciphers structural conformations of PIEZO1

Hisham Mazal, Alexandra Schambony, Vahid Sandoghdar

Despite the impressive progress in molecular biochemistry and biophysics, many questions regarding the conformational states of large (transmembrane) protein complexes persist. In the case of the PIEZO protein, investigations by cryogenic electron microscopy (Cryo-EM) and atomic force microscopy (AFM) have established a symmetric trimer structure with three long-bladed domains in a propeller-like configuration. A transition of PIEZO protein from curved to flat conformation is hypothesized to actuate closed and open channels for the flow of ions. However, conclusive high-resolution data on the molecular organization of PIEZO in its native form are lacking. To address this shortcoming, we exploit single-particle cryogenic light microscopy (spCryo-LM) to decipher the conformational states of the mouse PIEZO1 protein (mPIEZO1) in the cell membrane. Here, we implement a high-vacuum cryogenic shuttle to transfer shock-frozen unroofed cell membranes in and out of a cryostat for super-resolution microscopy at liquid helium temperature. By localizing fluorescent labels placed at the extremities of the three blades with Ångstrom precision, we ascertain three configurations of the protein with radii of 6, 12, and 20 nm as projected onto the membrane plane. Our data suggest that in the smallest configuration, the blades form a nano-dome structure that is more strongly curved than previously observed and predicted by AlphaFold-3. In the largest conformation, we believe the structure must fully unbend in an anticlockwise manner to form a flat extended state. We attribute the 12 nm conformation, the most frequently occupied state, to an intermediate state and discuss our results in the context of the findings from other groups. Combination of spCryo-LM and Cryo-EM measurements together with in situ photothermal stimulation promises to provide quantitative insight into the interplay between structure and function of PIEZO and other biomolecular complexes in their native environments.

Supported Lipid Bilayers as Stochastic Conveyor Belt for Delivery to the Near Field of Nanoscopic Structures

Yazgan Tuna, Vahid Sandoghdar

Placement of nanoscopic entities in the optical near field of a structure such as a plasmonic nanoantenna or the aperture of a scanning near-field optical microscopy (SNOM) remains a nontrivial task, which often requires sophisticated nanofabrication techniques. Here, we show that the fluidity and diffusion of lipid molecules in bilayer geometries can be exploited for facile delivery of nano-objects such as organic dye molecules, semiconductor quantum dots, and gold nanoparticles to the near field of well-defined surface structures. We demonstrate this in three different scenarios with substantial plasmonic enhancement of fluorescence.

Combined selective plane illumination microscopy (SPIM) and full-field optical coherence tomography (FF-OCT) for in vivo imaging

Rui Ma, Olga Lyraki, Daniel Wehner, Jochen Guck

Selective plane illumination microscopy (SPIM), also known as light sheet fluorescence microscopy, provides high specificity through fluorescence labeling. However, it lacks complementary structural information from the surrounding context, which is essential for the comprehensive analysis of biological samples. Here, we present a high-resolution, multimodal imaging system that integrates SPIM with full-field optical coherence tomography (FF-OCT), without requiring modifications to the existing SPIM setup. Both SPIM and FF-OCT offer low phototoxicity and intrinsic optical sectioning, making them well-suited for in vivo imaging. Their shared detection path enables seamless and efficient co-registration of fluorescence and structural data. We demonstrate the unctionality of this combined system by performing in vivo imaging of zebrafish larvae.

Rapid Stiffness Mapping in Soft Biologic Tissues With Micrometer Resolution Using Optical Multifrequency Time‐Harmonic Elastography

Jakob Jordan, Noah Jaitner, Tom Meyer, Luca Bramè, Mnar Ghrayeb, Julia Köppke, Oliver Böhm, Stefan Klemmer Chandia, Vasily Zaburdaev, et al.

Rapid mapping of the mechanical properties of soft biological tissues from light microscopy to macroscopic imaging can transform fundamental biophysical research by providing clinical biomarkers to complement in vivo elastography. This work introduces superfast optical multifrequency time-harmonic elastography (OMTHE) to remotely encode surface and subsurface shear wave fields for generating maps of tissue stiffness with unprecedented detail resolution. OMTHE rigorously exploits the space-time propagation characteristics of multifrequency time-harmonic waves to address current limitations of biomechanical imaging and elastography. Key solutions are presented for stimulation, wave decoding, and stiffness reconstruction of shear waves at multiple harmonic frequencies, all tuned to provide consistent stiffness values across resolutions from microns to millimeters. OMTHE's versatility is demonstrated by simulations, phantoms, Bacillus subtilis biofilms, zebrafish embryos and adult zebrafish, reflecting the diversity of biological systems from a mechanics perspective. By zooming in on stiffness details from coarse to finer scales, OMTHE has the potential to advance mechanobiology and offers a way to perform biomechanics-based tissue histology that consistently matches in vivo time-harmonic elastography in patients.

Optical characterization of molecular interaction strength in protein condensates

Timon Beck, Lize-Mari van der Linden, Wade M. Borcherds, Kyoohyun Kim, Raimund Schlüßler, Paul Müller, Titus M. Franzmann, Conrad Möckel, Ruchi Goswami, et al.

Biomolecular condensates have been identified as a ubiquitous means of intracellular organization, exhibiting very diverse material properties. However, techniques to characterize these material properties and their underlying molecular interactions are scarce. Here, we introduce two optical techniques—Brillouin microscopy and quantitative phase imaging (QPI)—to address this scarcity. We establish Brillouin shift and linewidth as measures for average molecular interaction and dissipation strength, respectively, and we used QPI to obtain the protein concentration within the condensates. We monitored the response of condensates formed by fused in sarcoma (FUS) and by the low-complexity domain of hnRNPA1 (A1-LCD) to altering temperature and ion concentration. Conditions favoring phase separation increased Brillouin shift, linewidth, and protein concentration. In comparison to solidification by chemical cross-linking, the ion-dependent aging of FUS condensates had a small effect on the molecular interaction strength inside. Finally, we investigated how sequence variations of A1-LCD, that change the driving force for phase separation, alter the physical properties of the respective condensates. Our results provide a new experimental perspective on the material properties of protein condensates. Robust and quantitative experimental approaches such as the presented ones will be crucial for understanding how the physical properties of biological condensates determine their function and dysfunction.

Mechanical stresses govern myoblast fusion and myotube growth

Yoann Le Toquin, Sushil Dubey, Aleksandra Ardaševa, Lakshmi Balasubramaniam, Emilie Delaune, Valérie Morin, Amin Doostmohammadi, Christophe Marcelle, Benoît Ladoux

Myoblast fusion into myotubes is critical for muscle formation, growth and repair. While the cellular and molecular mechanisms regulating myoblast fusion are increasingly understood, the role of biomechanics in this process remains largely unexplored. Here, we reveal that a dynamic feedback loop between evolving cell mechanics and cell-generated stresses shape the fusion of primary myoblasts in vitro. Applying principles from active nematics, we show that myoblast and myotube patterning follows physical rules similar to liquid crystal organization. Remarkably, fusion predominantly occurs at comet-shaped topological defects in cellular alignment, which we identified as regions of high compressive stress. We further find that this stress-driven organization depends on extracellular matrix (ECM) deposition, which mirrors the nematic order of the cell population. Our integrated data, supported by active nematics-based mathematical modeling, accurately predict self-organization patterns and mechanical stresses that regulate myoblast fusion. By revealing the essential role of biomechanics and ECM interplay in myogenesis, this work establishes a foundational framework for understanding biomechanical principles in morphogenesis.

Consensus Statement on Brillouin Light Scattering Microscopy of Biological Materials

Pierre Bouvet, Carlo Bevilacqua, Yogeshwari Ambekar, Giuseppe Antonacci, Joshua Au, Silvia Caponi, Sophie Chagnon-Lessard, Juergen Czarske, Thomas Dehoux, et al.

Brillouin Light Scattering (BLS) spectroscopy is a non-invasive, non-contact, label-free optical technique that can provide information on the mechanical properties of a material on the sub-micron scale. Over the last decade it has seen increased applications in the life sciences, driven by the observed significance of mechanical properties in biological processes, the realization of more sensitive BLS spectrometers and its extension to an imaging modality. As with other spectroscopic techniques, BLS measurements not only detect signals characteristic of the investigated sample, but also of the experimental apparatus, and can be significantly affected by measurement conditions. The aim of this consensus statement is to improve the comparability of BLS studies by providing reporting recommendations for the measured parameters and detailing common artifacts. Given that most BLS studies of biological matter are still at proof-of-concept stages and use different--often self-built--spectrometers, a consensus statement is particularly timely to assure unified advancement.

Tailored Bisacylphosphane Oxides for Precise Induction of Oxidative Stress-Mediated Cell Death in Biological Systems

Karim Almahayni, Jana Bachir Salvador, Riccardo Conti, Anna Widera, Malte Spiekermann, Daniel Wehner, Hansjörg Grützmacher, Leonhard Möckl

Precise cell elimination within intricate cellular populations is hampered by issues arising from the multifaceted biological properties of cells and the expansive reactivity of chemical agents. Current chemical platforms are often limited by their complexity, toxicity, and poor physical/chemical properties. Here, we report on the synthesis of a structurally versatile library of chemically tunable bisacylphosphane oxides (BAPOs), which harnesses the spatiotemporal precision of light delivery, thereby establishing a universal strategy for on-demand, precise cellular ablation in vitro and in vivo.

S96 Circulating neutrophils in idiopathic pulmonary fibrosis have a distinct biomechanical phenotype of systemic activation that correlates with disease severity

Katherina Lodge, Sara Nakanashi, Leda Yazbeck, Jochen Guck, Philip L. Molyneaux , Andrew S. Cowburn

Background Idiopathic Pulmonary Fibrosis (IPF) is a chronic interstitial lung disease associated with impaired gas transfer and systemic hypoxia. Elevated peripheral neutrophil counts correlate with increased morbidity and mortality in IPF. Real-Time Deformability Cytometry (RT-DC) is a novel, sensitive high-throughput approach that measures quantitative biomechanical parameters of single cells with minimal manipulation and maintenance of physiological normoxia, thus enabling indirect determination of basal cellular activity. We hypothesized that biomechanical profiling can identify phenotypic diversity in patients with IPF.

Sensing the force in living embryos

Kristian Franze

During morphogenesis, a key process of embryonic development, cells undergo massive rearrangements to give rise to tissue shapes and ultimately organ systems. Shape changes of tissues are naturally driven by forces arising from mechanical interactions between cells and their environment. These forces generate cell movements, mechanical stresses and strains at the tissue level. Abnormalities in these stresses can lead to malformations and developmental disorders, and in mature organisms, cancer can be considered an example of pathological tissue morphogenesis. Knowledge about the forces and stresses generated by cells and tissues is crucial to fully understand embryonic development and related pathological processes. Now, writing in Nature Materials, Maniou et al. 1 present a method to quantify tissue-level morphogenetic forces based on the deformation of three-dimensional soft force sensors …

Transport in cellular aggregates described by fluctuating hydrodynamics

Subhadip Chakraborti, Vasily Zaburdaev

Biological functionality of cellular aggregates is largely influenced by the activity and displacements of individual constituent cells. From a theoretical perspective this activity can be characterized by hydrodynamic transport coefficients of diffusivity and conductivity. Motivated by the clustering dynamics of bacterial microcolonies we propose a model of active multicellular aggregates and use recently developed macroscopic fluctuation theory to derive a fluctuating hydrodynamics for this model system. Both semianalytic theory and microscopic simulations show that the hydrodynamic transport coefficients are affected by nonequilibrium microscopic parameters and significantly decrease inside of the clusters. We further find that the Einstein relation connecting the transport coefficients and fluctuations breaks down in the parameter regime where the detailed balance is not satisfied. This study offers valuable tools for experimental investigation of hydrodynamic transport in other systems of cellular aggregates such as tumor spheroids and organoids.

Scale-free flocking and giant fluctuations in epithelial active solids

Yuan Shen, Jérémy O’Byrne, Andreas Schoenit, Ananyo Maitra, Rene-Marc Mege, Raphael Voituriez, Benoît Ladoux

The collective motion of epithelial cells is a fundamental biological process which plays a significant role in embryogenesis, wound healing and tumor metastasis. While it has been broadly investigated for over a decade both in vivo and in vitro, large scale coherent flocking phases remain underexplored and have so far been mostly described as fluid. In this work, we report a mode of large-scale collective motion for different epithelial cell types in vitro with distinctive new features. By tracking individual cells, we show that cells move over long time scales coherently not as a fluid, but as a polar elastic solid with negligible cell rearrangements. Our analysis reveals that this solid flocking phase exhibits signatures of long-range polar order, unprecedented in cellular systems, with scale-free correlations, anomalously large density fluctuations, and shear waves. Based on a general theory of active polar solids, we argue that these features result from massless Goldstone modes, which, in contrast to polar fluids where they are generic, require the decoupling of global rotations of the polarity and in-plane elastic deformations in polar solids. We theoretically show and consistently observe in experiments that the fluctuations of elastic deformations diverge for large system size in such polar active solid phases, leading eventually to rupture and thus potentially loss of tissue integrity at large scales.

Long-Range Three-Dimensional Tracking of Nanoparticles Using Interferometric Scattering Microscopy

Kiarash Kasaian, Mahdi Mazaheri, Vahid Sandoghdar

Tracking nanoparticle movement is highly desirable in many scientific areas, and various imaging methods have been employed to achieve this goal. Interferometric scattering (iSCAT) microscopy has been particularly successful in combining very high spatial and temporal resolution for tracking small nanoparticles in all three dimensions. However, previous works have been limited to an axial range of only a few hundred nanometers. Here, we present a robust and efficient measurement and analysis strategy for three-dimensional tracking of nanoparticles at high speed and with nanometer precision. After discussing the principle of our approach using synthetic data, we showcase the performance of the method by tracking gold nanoparticles with diameters ranging from 10 to 80 nm in water, demonstrating an axial tracking range from 4 μm for the smallest particles up to over 30 μm for the larger ones. We point out the limitations and robustness of our system across various noise levels and discuss its promise for applications in cell biology and material science, where the three-dimensional motion of nanoparticles in complex media is of interest.

Microglia are essential for tissue contraction in wound closure after brain injury in zebrafish larvae

Francois El-Daher, Stephen J. Enos, Louisa K. Drake, Daniel Wehner, Markus Westphal, Nicola J. Porter, Catherine G. Becker, Thomas Becker

Wound closure after brain injury is crucial for tissue restoration but remains poorly understood at the tissue level. We investigated this process using in vivo observations of larval zebrafish brain injury. Our findings show that wound closure occurs within the first 24 h through global tissue contraction, as evidenced by live-imaging and drug inhibition studies. Microglia accumulate at the wound site before closure, and computational models suggest that their physical traction could drive this process. Depleting microglia genetically or pharmacologically impairs tissue repair. At the cellular level, live imaging reveals centripetal deformation of astrocytic processes contacted by migrating microglia. Laser severing of these contacts causes rapid retraction of microglial processes and slower retraction of astrocytic processes, indicating tension. Disrupting the lcp1 gene, which encodes the F-actin–stabilising protein L-plastin, in microglia results in failed wound closure. These findings support a mechanical role of microglia in wound contraction and suggest that targeting microglial mechanics could offer new strategies for treating traumatic brain injury.

Dynamic traction force measurements of migrating immune cells in 3D biopolymer matrices

David Böhringer, Mar Cóndor, Lars Bischof, Tina Czerwinski, Niklas Gampl, Phuong Anh Ngo, Andreas Bauer, Caroline Voskens, Rocío López-Posadas, et al.

Immune cells, such as natural killer cells, migrate with high speeds of several micrometres per minute through dense tissue. However, the magnitude of the traction forces during this migration is unknown. Here we present a method to measure dynamic traction forces of fast migrating cells in biopolymer matrices from the observed matrix deformations. Our method accounts for the mechanical nonlinearity of the three-dimensional tissue matrix and can be applied to time series of confocal or bright-field image stacks. It allows for precise force reconstruction over a wide range of force magnitudes and object sizes—even when the imaged volume captures only a small part of the matrix deformation field. We demonstrate the broad applicability of our method by measuring forces from around 1 nN for axon growth cones up to around 10 μN for mouse intestinal organoids. We find that natural killer cells show bursts of large traction forces around 50 nN that increase with matrix stiffness. These force bursts are driven by myosin II contractility, mediated by integrin β1 adhesions, focal adhesion kinase and Rho-kinase activity, and occur predominantly when the cells migrate through narrow matrix pores.

Different Biomechanical Cell Behaviors in an Epithelium Drive Collective Epithelial Cell Extrusion

Lakshmi Balasubramaniam, Shreyansh Jain, Tien Dang, Emilie Lagoutte, René Marc Mège, Philippe Chavrier, Benoît Ladoux, Carine Rossé

In vertebrates, many organs, such as the kidney and the mammary gland form ductal structures based on the folding of epithelial sheets. The development of these organs relies on coordinated sorting of different cell lineages in both time and space, through mechanisms that remain largely unclear. Tissues are composed of several cell types with distinct biomechanical properties, particularly at cell-cell and cell-substrate boundaries. One hypothesis is that adjacent epithelial layers work in a coordinated manner to shape the tissue. Using in vitro experiments on model epithelial cells, differential expression of atypical Protein Kinase C iota (aPKCi), a key junctional polarity protein, is shown to reinforce cell epithelialization and trigger sorting by tuning cell mechanical properties at the tissue level. In a broader perspective, it is shown that in a heterogeneous epithelial monolayer, in which cell sorting occurs, forces arising from epithelial cell growth under confinement by surrounding cells with different biomechanical properties are sufficient to promote collective cell extrusion and generate emerging 3D organization related to spheroids and buds. Overall, this research sheds light on the role of aPKCi and the biomechanical interplay between distinct epithelial cell lineages in shaping tissue organization, providing insights into the understanding of tissue and organ development.

Longitudinal associations between depressive symptoms and cell deformability: do glucocorticoids play a role?

Julian Eder, Martin Kräter, Clemens Kirschbaum, Wei Gao, Magdalena Wekenborg, Marlene Penz, Nicole Rothe, Jochen Guck, Lucas Daniel Wittwer, et al.

Cell deformability of all major blood cell types is increased in depressive disorders (DD). Furthermore, impaired glucocorticoid secretion is associated with DD, as well as depressive symptoms in general and known to alter cell mechanical properties. Nevertheless, there are no longitudinal studies examining accumulated glucocorticoid output and depressive symptoms regarding cell deformability. The aim of the present study was to investigate, whether depressive symptoms predict cell deformability one year later and whether accumulated hair glucocorticoids mediate this relationship. In 136 individuals (nfemale = 100; Mage = 46.72, SD = 11.28; age range = 20-65), depressive symptoms (PHQ-9) and hair glucocorticoids (cortisol and cortisone) were measured at time point one (T1), while one year later (T2) both depressive symptoms and hair glucocorticoids were reassessed. Additionally, cell deformability of peripheral blood cells was assessed at T2. Depression severity at T1 predicted higher cell deformability in monocytes and lymphocytes at T2. Accumulated hair cortisol and cortisone concentrations from T1 and T2 were not associated with higher cell deformability and further did not mediate the relationship between depressive symptoms and cell deformability. Elevated depressive symptomatology in a population based sample is longitudinally associated with higher immune cell deformability, while long-term integrated glucocorticoid levels seem not to be implicated in the underlying mechanism.

How bacteria actively use passive physics to make biofilms

Liraz Chai, Vasily Zaburdaev, Roberto Kolter

Modern molecular microbiology elucidates the organizational principles of bacterial biofilms via detailed examination of the interplay between signaling and gene regulation. A complementary biophysical approach studies the mesoscopic dependencies at the cellular and multicellular levels with a distinct focus on intercellular forces and mechanical properties of whole biofilms. Here, motivated by recent advances in biofilm research and in other, seemingly unrelated fields of biology and physics, we propose a perspective that links the biofilm, a dynamic multicellular organism, with the physical processes occurring in the extracellular milieu. Using Bacillus subtilis as an illustrative model organism, we specifically demonstrate how such a rationale explains biofilm architecture, differentiation, communication, and stress responses such as desiccation tolerance, metabolism, and physiology across multiple scales—from matrix proteins and polysaccharides to macroscopic wrinkles and water-filled channels.

Environmental stiffness regulates neuronal maturation via Piezo1-mediated TTR activity

Eva Kreysing, Hélène Gautier, Robert Humphrey, Katrin Mooslehner, Leila Muresan, Daniel Haarhoff, Sudipta Mukherjee, Xiaohui Zhao, Alexander Winkel, et al.

During brain development, neurons extend axons to connect to their target cells while initiating a maturation process, during which neurons start expressing voltage-gated ion channels, form synapses, express synaptic transmitters and receptors, and start communicating via action potentials. Little is known about external factors regulating this process. Here, we identified environmental mechanics as an important regulator of neuronal maturation, and a molecular pathway linking tissue stiffness to this process. Using patch clamp electrophysiology, calcium imaging and immunofluorescence, we found that neurons cultured on stiffer substrates showed a delay in voltage-gated ion channel activity, spontaneous and evoked action potentials, and synapse formation. RNA sequencing and CRISPR/Cas9 knockdown strategies revealed that the mechanosensitive ion channel Piezo1 supresses transthyretin (TTR) expression on stiffer substrates, slowing down synaptic receptor expression and consequently electrical maturation. Stiffening of brain tissue in Xenopus laevis embryos also resulted in a significant delay of synaptic activity in vivo. Our data indicate that environmental stiffness represents a fundamental regulator of neuronal maturation, which is important for the development of normal circuitry in the brain, and potentially for neurodevelopmental disorders.

Fluctuating Hydrodynamics Describes Transport in Cellular Aggregates

Subhadip Chakraborti, Vasily Zaburdaev

Biological functionality of cellular aggregates is largely influenced by the activity and displacements of individual constituent cells. From a theoretical perspective this activity can be characterized by hydrodynamic transport coefficients of diffusivity and conductivity. Motivated by the clustering dynamics of bacterial microcolonies we propose a model of active multicellular aggregates and use recently developed macroscopic fluctuation theory to derive a fluctuating hydrodynamics for this model system. Both semi-analytic theory and microscopic simulations show that the hydrodynamic transport coefficients are affected by non-equilibrium microscopic parameters and significantly decrease inside of the clusters. We further find that the Einstein relation connecting the transport coefficients and fluctuations breaks down in the parameter regime where the detailed balance is not satisfied. This study offers valuable tools for experimental investigation of hydrodynamic transport in other systems of cellular aggregates such as tumor spheroids and organoids.

Omega-3 supplementation changes the physical properties of leukocytes but not erythrocytes in healthy individuals: An exploratory trial

Jan Philipp Schuchardt, Martin Kräter, Maximilian Schlögel, Jochen Guck, Brigitte A. van Oirschot-Hermans, Jennifer Bos, Richard van Wijk, Nathan L. Tintle, Jason Westra, et al.

n3-PUFA impact health in several ways, including cardiovascular protection and anti-inflammatory effects, but the underlying mechanisms are not fully understood. In this exploratory study involving 31 healthy subjects, we aimed to investigate the effects of 12 weeks of fish-oil supplementation (1500 mg EPA+DHA/day) on the physical properties of multiple blood cell types. We used deformability cytometry (DC) for all cell types and Laser-assisted Optical Rotational Red Cell Analysis (Lorrca) to assess red blood cell (RBC) deformability. We also investigated the correlation between changes in the physical properties of blood cells and changes in the Omega-3 Index (O3I), defined as the relative content of EPA+DHA in RBCs. Following supplementation, the mean±SD O3I increased from 5.3%±1.5% to 8.3%±1.4% (p<0.001). No significant changes in RBC properties were found by both techniques. However, by DC we observed a consistent pattern of physical changes in lymphocytes, neutrophils and monocytes. Among these were significant increases in metrics correlated with the cells’ deformability resulting in less stiff cells. The results suggest that leukocytes become softer and have an increased ability to deform under induced short-term physical stress such as hydrodynamic force in the circulation. These changes could impact immune function since softer leukocytes can potentially circulate more easily and could facilitate a more rapid response to systemic inflammation or infection. In conclusion, fish-oil supplementation modulates some physical properties of leukocyte-subfractions, potentially enhancing their biological function. Further studies are warranted to explore the impact of n3-PUFA on blood cell biology, particularly in disease states associated with leukocyte dysregulation.

Long-range chemical signalling in vivo is regulated by mechanical signals

Eva K. Pillai, Sudipta Mukherjee, Niklas Gampl, Ross J. McGinn, Katrin A. Mooslehner, Julia M. Becker, Amelia J. Thompson, Kristian Franze

Biological processes are regulated by chemical and mechanical signals, yet the interaction between these signalling modalities remains unclear. Using the developing Xenopus laevis brain as a model system, we identified a critical crosstalk between tissue stiffness and chemical signalling in vivo. Targeted knockdown of the mechanosensitive ion channel Piezo1 in retinal ganglion cells (RGCs) led to pathfinding errors in vivo. However, pathfinding errors were also observed in RGCs expressing Piezo1, when Piezo1 was downregulated in the surrounding brain tissue. Depleting Piezo1 in brain parenchyma led to decreases in the expression of the long-range chemical guidance cues, Semaphorin3A and Slit1, and markedly reduced tissue stiffness. While tissue softening was independent of Sema3A depletion, Slit1 and Sema3A expression increased significantly in stiffer environments in vitro. Moreover, stiffening soft brain regions in vivo induced ectopic Sema3A production via a Piezo1-dependent mechanism. Our results demonstrate that brain tissue mechanics modulates the expression of key chemical signals, a likely phenomenon across diverse biological systems.

The Holotomography

Geon Kim, Herve Hugonnet, Kyoohyun Kim, Chungha Lee, Jae-Hyuk Lee, Seongsoo Lee, Sung Sik Lee, Gabor Csucs, Jeongmin Ha, et al.

Holotomography (HT) represents a 3D, label-free optical imaging methodology that leverages refractive index as an inherent quantitative contrast for imaging. This technique has recently seen notable advancements, creating novel opportunities for the comprehensive visualization and analysis of living cells and their subcellular organelles. It has manifested wide-ranging applications spanning cell biology, biophysics, microbiology and biotechnology, substantiating its vast potential. In this Primer, we elucidate the foundational physical principles underpinning HT, detailing its experimental implementations and providing case studies of representative research employing this methodology. We also venture into interdisciplinary territories, exploring how HT harmonizes with emergent technologies, such as regenerative medicine, 3D biology and organoid-based drug discovery and screening. Looking ahead, we engage in a prospective analysis of potential future trajectories for HT, discussing innovation-focused initiatives that may further elevate this field. We also propose possible future applications of HT, identifying opportunities for its integration into diverse realms of scientific research and technological development.

Cell State-Specific Cytoplasmic Material Properties Control Spindle Architecture and Scaling

Tobias Kletter, Omar Muñoz, Sebastian Reusch, Abin Biswas, Aliaksandr Halavatyi, Beate Neumann, Benno Kuropka, Vasily Zaburdaev, Simone Reber

Mitotic spindles are dynamically intertwined with the cytoplasm they assemble in. How the physicochemical properties of the cytoplasm affect spindle architecture and size remains largely unknown. Using quantitative biochemistry in combination with adaptive feedback microscopy, we investigated mitotic cell and spindle morphology during neural differentiation of embryonic stem cells. While tubulin biochemistry and microtubule dynamics remained unchanged, spindles changed their scaling behaviour: in differentiating cells, spindles were significantly smaller than those in equally-sized undifferentiated stem cells. Integrating quantitative phase imaging, biophysical perturbations and theory, we found that as cells differentiated, their cytoplasm became more dilute. The concomitant decrease in free tubulin activated CPAP (centrosomal P4.1-associated protein) to enhance the centrosomal nucleation capacity. As a consequence, in differentiating cells, microtubule mass shifted towards spindle poles at the expense of the spindle bulk, explaining the differentiation-associated switch in spindle architecture. This study shows that cell state-specific cytoplasmic density tunes mitotic spindle architecture. Thus, we reveal physical properties of the cytoplasm as a major determinant in organelle size control.

iSCAT microscopy and particle tracking with tailored spatial coherence

Mahdi Mazaheri, Kiarash Kasaian, David Albrecht, Jan Renger, Tobias Utikal, Cornelia Holler, Vahid Sandoghdar

Interferometric scattering (iSCAT) microscopy has demonstrated unparalleled performance among label-free optical methods for detecting and imaging isolated nanoparticles and molecules. However, when imaging complex structures such as biological cells, the superposition of the scattering fields from different locations of the sample leads to a speckle-like background, posing a significant challenge in deciphering fine features. Here, we show that by controlling the spatial coherence of the illumination, one can eliminate the spurious speckle without sacrificing sensitivity. We demonstrate this approach by positioning a rotating diffuser coupled with an adjustable lens and an iris in the illumination path. We report on imaging at a high frame rate of 25 kHz and across a large field of view of 100µm×100µm, while maintaining diffraction-limited resolution. We showcase the advantages of these features by three-dimensional (3D) tracking over 1000 vesicles in a single COS-7 cell and by imaging the dynamics of the endoplasmic reticulum (ER) network. Our approach opens the door to the combination of label-free imaging, sensitive detection, and 3D high-speed tracking using wide-field iSCAT microscopy.

High-Resolution Cryogenic Spectroscopy of Single Molecules in Nanoprinted Crystals

Mohammad Musavinezhad, Jan Renger, Johannes Zirkelbach , Tobias Utikal, Claudio U. Hail, Thomas Basché, Dimos Poulikakos, Stephan Götzinger, Vahid Sandoghdar

We perform laser spectroscopy at liquid helium temperatures (T = 2 K) to investigate single dibenzoterrylene (DBT) molecules doped in anthracene crystals of nanoscopic height fabricated by electrohydrodynamic dripping. Using high-resolution fluorescence excitation spectroscopy, we show that zero-phonon lines of single molecules in printed nanocrystals are nearly as narrow as the Fourier-limited transitions observed for the same guest–host system in the bulk. Moreover, the spectral instabilities are comparable to or less than one line width. By recording super-resolution images of DBT molecules and varying the polarization of the excitation beam, we determine the dimensions of the printed crystals and the orientation of the crystals’ axes. Electrohydrodynamic printing of organic nano- and microcrystals is of interest for a series of applications, where controlled positioning of quantum emitters with narrow optical transitions is desirable.

Measuring Concentration of Nanoparticles in Polydisperse Mixtures Using Interferometric Nanoparticle Tracking Analysis

Anna D. Kashkanova, David Albrecht, Michelle Küppers, Martin Blessing, Vahid Sandoghdar

Quantitative measurements of nanoparticle concentration in liquid suspensions are in high demand, for example, in the medical and food industries. Conventional methods remain unsatisfactory, especially for polydisperse samples with overlapping size ranges. Recently, we introduced interferometric nanoparticle tracking analysis (iNTA) for high-precision measurement of nanoparticle size and refractive index. Here, we show that by counting the number of trajectories that cross the focal plane, iNTA can measure concentrations of subpopulations in a polydisperse mixture in a quantitative manner and without the need for a calibration sample. We evaluate our method on both monodisperse samples and mixtures of known concentrations. Furthermore, we assess the concentration of SARS-CoV-2 in supernatant samples obtained from infected cells.

Mutation of the ALS-/FTD-Associated RNA-Binding Protein FUS Affects Axonal Development

Francesca W. van Tartwijk, Lucia C. S. Wunderlich, Ioanna Mela, Stanislaw Makarchuk, Maximilian A. H. Jakobs, Seema Qamar, Kristian Franze, Gabriele S. Kaminski Schierle, Peter H. St George-Hyslop, et al.

Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.

Beyond comparison: Brillouin microscopy and AFM-based indentation reveal divergent insights into the mechanical profile of the murine retina

Marcus Gutmann, Jana Bachir Salvador, Paul Müller, Kyoohyun Kim, Martin Schicht, Serhii Aif, Friedrich Paulsen, Lorenz Meinel, Jochen Guck, et al.

Mechanical tissue properties increasingly serve as pivotal phenotypic characteristics that are subject to change during development or pathological progression. The quantification of such material properties often relies on physical contact between a load-applying probe and an exposed sample surface. For most tissues, meeting these requirements entails an invasive preparation, which poses the risk of yielding mechanical properties that do not portray the physiological state of a tissue within a functioning organism. Brillouin microscopy has emerged as a non-invasive, optical technique that enables the assessment of mechanical cell and tissue properties with high spatio-temporal resolution. In optically transparent specimens, it does not require animal sacrifice, tissue dissection or sectioning. However, the extent to which results obtained from Brillouin microscopy allow to infer conclusions about potential results obtained with a contact-based technique, and vice versa, is unclear. Sources for discrepancies include the varying characteristic temporal and spatial scales, the directionality of measurement, environmental factors, and mechanical moduli probed. In this work, we addressed those aspects by quantifying the mechanical properties of acutely dissected murine retinae using Brillouin microscopy and atomic force microscopy (AFM)-based indentation measurements. Our results show a distinct mechanical profile of the retinal layers with respect to the Brillouin frequency shift, the Brillouin linewidth and the apparent Young's modulus. Contrary to previous reports, our findings do not support a simple correlative relationship between Brillouin frequency shift and apparent Young's modulus. Additionally, the divergent sensitivities of Brillouin microscopy and AFM-indentation measurements to structural features, as visualized by transmission electron microscopy, to cross-linking or changes post mortem underscore the dangers of assuming interchangeability between the two methods. In conclusion, our study advocates for viewing Brillouin microscopy and AFM-based indentation measurements as complementary tools, discouraging direct comparisons a priori and suggesting their combined use for a more comprehensive understanding of tissue mechanical properties.

Non-equilibrium structure and relaxation in active microemulsions

Rakesh Chatterjee, Hui-Shun Kuan, Frank Julicher, Vasily Zaburdaev

Microphase separation is common in active biological systems as exemplified by the separation of RNA and DNA-rich phases in the cell nucleus driven by the transcriptional activity of polymerase enzymes acting similarly to amphiphiles in a microemulsion. Here we propose an analytically tractable model of an active microemulsion to investigate how the activity affects its structure and relaxation dynamics. Continuum theory derived from a lattice model exhibits two distinct regimes of the relaxation dynamics and is linked to the broken detailed balance due to intermittent activity of the amphiphiles.

Nonlinear dynamics of femtosecond laser interaction with the central nervous system in zebrafish

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

Understanding the photodamage mechanism underlying the highly nonlinear dynamic of femtosecond laser pulses at the second transparent window of tissue is crucial for label-free microscopy. Here, we report the identification of two cavitation regimes from 1030 nm pulses when interacting with the central nervous system in zebrafish. We show that at low repetition rates, the damage is confined due to plasma-based ablation and sudden local temperature rise. At high repetition rates, the damage becomes collateral due to plasma-mediated photochemistry. Furthermore, we investigate the role of fluorescence labels with linear and nonlinear absorption pathways in optical breakdown. To verify our findings, we examined cell death and cellular responses to tissue damage, including the recruitment of fibroblasts and immune cells after irradiation. These findings contribute to advancing the emerging nonlinear optical microscopy techniques and provide a strategy for inducing precise, and localized injuries using near-infrared femtosecond laser pulses.

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

Conrad Möckel, Timon Beck, Sara Kaliman, Shada Abuhattum Hofemeier, 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. One of the basic biophysical properties is the mass density (MD). It affects the dynamics in sub-cellular compartments and 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 a methodology on how to account for assumptions about the biochemical composition of the sample and respective RI measurements. To this 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 and 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 and employ it to estimate the MD of living zebrafish (Danio rerio) larvae trunk tissue. Our results illustrate the importance of implementing this methodology not only for MD estimations but for many other related biophysical problems, such as 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 (CRC) 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 poorly understood. Using CRC patient derived organoids (PDOs), we find that compared to LGR5- cells, 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 single cell RNA-sequencing (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 dissemination from the primary tumor whereas LGR5+ cells display a mechanically stable and resilient phenotype suitable for extravasation and metastatic growth.

High-throughput viscoelastic characterization of cells in hyperbolic microchannels

Felix Reichel, Ruchi Goswami, Salvatore Girardo, Jochen Guck

Extensive research has demonstrated the potential of cell viscoelastic properties as intrinsic indicators of cell state, functionality, and disease. For this, several microfluidic techniques have been developed to measure cell viscoelasticity with high-throughput. However, current microchannel designs introduce complex stress distributions on cells, leading to inaccuracies in determining the stress-strain relationship and, consequently, the viscoelastic properties. Here, we introduce a novel approach using hyperbolic microchannels that enable precise measurements under a constant extensional stress and offer a straightforward stress-strain relationship, while operating at a measurement rate of up to 100 cells per second. We quantified the stresses acting in the channels using mechanical calibration particles made from polyacrylamide (PAAm) and found that the measurement buffer, a solution of methyl cellulose and phosphate buffered saline, has a constant extensional viscosity of 0.5 Pa s up to 200 s-1. By measuring oil droplets with varying viscosities, we successfully detected changes in the relaxation time of the droplets and our approach could be used to get the interfacial tension and viscosity of liquid-liquid droplet systems from the same measurement. We further applied this methodology to PAAm microgel beads, demonstrating the accurate recovery of Young’s moduli and the near-ideal elastic behavior of the beads. To explore the influence of altered cell viscoelasticity, we treated HL60 human leukemia cells with Latrunculin B and Nocodazole, resulting in clear changes in cell stiffness while relaxation times were only minimally affected. In conclusion, our approach offers a streamlined and time-efficient solution for assessing the viscoelastic properties of large cell populations and other microscale soft particles.

Cell viscosity influences hematogenous dissemination and metastatic extravasation of tumor cells

Valentin Gensbittel , Gautier Follain, Louis Bochler, Klemens Uhlmann, Olivier Lefèbvre, Annabel Larnicol, Sébastien Harlepp, Ruchi Goswami, Salvatore Girardo, et al.

Metastases arise from a multi-step process during which tumor cells change their mechanics in response to microenvironmental cues. While such mechanical adaptability could influence metastatic success, how tumor cell mechanics directly impacts intravascular behavior of circulating tumor cells (CTCs) remains poorly understood. In the present study, we demonstrate how the deformability of CTCs affects hematogenous dissemination and identify the mechanical profiles that favor metastatic extravasation. Combining intravital microscopy with CTC-mimicking elastic beads and mechanically-tuned tumor cells, we demonstrate that the inherent properties of circulating objects dictate their ability to enter constraining vessels. We identify cellular viscosity as the key property that governs CTC circulation and arrest patterns. We further demonstrate that cellular viscosity is required for efficient extravasation and find that properties that favor extravasation and subsequent metastatic outgrowth can be opposite. Altogether, we identify CTC viscosity as a key biomechanical parameter that shapes several steps of metastasis.

An optofluidic antenna for enhancing the sensitivity of single-emitter measurements

Luis Morales-Inostroza, Julian Folz, Ralf Kühnemuth, Suren Felekyan, Franz Wieser, Claus A.M. Seidel, Stephan Götzinger, Vahid Sandoghdar

Many single-molecule investigations are performed in fluidic environments, e.g., to avoid unwanted consequences of contact with surfaces. Diffusion of molecules in this arrangement limits the observation time and the number of collected photons, thus, compromising studies of processes with fast or slow dynamics. Here, we introduce a planar optofluidic antenna (OFA), which enhances the fluorescence signal from molecules by about 5 times per passage, leads to about 7-fold more frequent returns to the observation volume, and significantly lengthens the diffusion time within one passage. We use single-molecule multi-parameter fluorescence detection (sm-MFD), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) measurements to characterize our OFAs. The antenna advantages are showcased by examining both the slow (ms) and fast (50μs) dynamics of DNA four-way (Holliday) junctions with real-time resolution. The FRET trajectories provide evidence for the absence of an intermediate conformational state and introduce an upper bound for its lifetime. The ease of implementation and compatibility with various microscopy modalities make OFAs broadly applicable to a diverse range of studies.

A deep‐learning workflow to predict upper tract urothelial carcinoma protein‐based subtypes fromH&Eslides supporting the prioritization of patients for molecular testing

Miriam Angeloni, Thomas van Doeveren, Sebastian Lindner, Patrick Volland, Jorina Schmelmer, Sebastian Foersch, Christian Matek, Robert Stoehr, Carol I Geppert, et al.

Upper tract urothelial carcinoma (UTUC) is a rare and aggressive, yet understudied, urothelial carcinoma (UC). The more frequent UC of the bladder comprises several molecular subtypes, associated with different targeted therapies and overlapping with protein-based subtypes. However, if and how these findings extend to UTUC remains unclear. Artificial intelligence-based approaches could help elucidate UTUC's biology and extend access to targeted treatments to a wider patient audience. Here, UTUC protein-based subtypes were identified, and a deep-learning (DL) workflow was developed to predict them directly from routine histopathological H&E slides. Protein-based subtypes in a retrospective cohort of 163 invasive tumors were assigned by hierarchical clustering of the immunohistochemical expression of three luminal (FOXA1, GATA3, and CK20) and three basal (CD44, CK5, and CK14) markers. Cluster analysis identified distinctive luminal (N = 80) and basal (N = 42) subtypes. The luminal subtype mostly included pushing, papillary tumors, whereas the basal subtype diffusely infiltrating, non-papillary tumors. DL model building relied on a transfer-learning approach by fine-tuning a pre-trained ResNet50. Classification performance was measured via three-fold repeated cross-validation. A mean area under the receiver operating characteristic curve of 0.83 (95% CI: 0.67–0.99), 0.8 (95% CI: 0.62–0.99), and 0.81 (95% CI: 0.65–0.96) was reached in the three repetitions. High-confidence DL-based predicted subtypes showed significant associations (p < 0.001) with morphological features, i.e. tumor type, histological subtypes, and infiltration type. Furthermore, a significant association was found with programmed cell death ligand 1 (PD-L1) combined positive score (p < 0.001) and FGFR3 mutational status (p = 0.002), with high-confidence basal predictions containing a higher proportion of PD-L1 positive samples and high-confidence luminal predictions a higher proportion of FGFR3-mutated samples. Testing of the DL model on an independent cohort highlighted the importance to accommodate histological subtypes. Taken together, our DL workflow can predict protein-based UTUC subtypes, associated with the presence of targetable alterations, directly from H&E slides.

Exploring the Physics of Basic Medical Research

Vahid Sandoghdar

The 20th century witnessed the emergence of many paradigm-shifting technologies from the physics community, which have revolutionized medical diagnostics and patient care. However, fundamental medical research has been mostly guided by methods from areas such as cell biology, biochemistry, and genetics, with fairly small contributions from physicists. In this Essay, I outline some key phenomena in the human body that are based on physical principles and yet govern our health over a vast range of length and time scales. I advocate that research in life sciences can greatly benefit from the methodology, know-how, and mindset of the physics community and that the pursuit of basic research in medicine is compatible with the mission of physics.<br><br>

invited essay

Single-Cell Mechanics: Structural Determinants and Functional Relevance

Marta Urbanska, Jochen Guck

The mechanical phenotype of a cell determines its ability to deform under force and is therefore relevant to cellular functions that require changes in cell shape, such as migration or circulation through the microvasculature. On the practical level, the mechanical phenotype can be used as a global readout of the cell's functional state, a marker for disease diagnostics, or an input for tissue modeling. We focus our review on the current knowledge of structural components that contribute to the determination of the cellular mechanical properties and highlight the physiological processes in which the mechanical phenotype of the cells is of critical relevance. The ongoing efforts to understand how to efficiently measure and control the mechanical properties of cells will define the progress in the field and drive mechanical phenotyping toward clinical applications.

Eph/ephrin signalling in the developing brain is regulated by tissue stiffness

Jana Sipkova, Kristian Franze

Eph receptors and their membrane-bound ligands, ephrins, provide key signals in many biological processes, such as cell proliferation, cell motility and cell sorting at tissue boundaries. However, despite immense progress in our understanding of Eph/ephrin signalling, there are still discrepancies between in vitro and in vivo work, and the regulation of Eph/ephrin signalling remains incompletely understood. Since a major difference between in vivo and most in vitro experiments is the stiffness of the cellular environment, we here investigated the interplay between tissue mechanics and Eph/ephrin signalling using the Xenopus laevis optic pathway as a model system. Xenopus retinal neurons cultured on soft substrates mechanically resembling brain tissue showed the opposite response to ephrinB1 compared to those cultured on glass. In vivo atomic force microscopy (AFM)-based stiffness mapping revealed that the visual area of the Xenopus brain, the optic tectum, becomes mechanically heterogeneous during its innervation by axons of retinal neurons. The resulting stiffness gradient correlated with both a cell density gradient and expression patterns of EphB and ephrinB family members. Exposing ex vivo brains to stiffer matrices or locally stiffening the optic tectum in vivo led to an increase in EphB2 expression in the optic tectum, indicating that tissue mechanics is an important regulator of Eph/ephrin signalling. Similar mechanisms are likely to be involved in the development and diseases of many other organ systems.

A paintbrush for delivery of nanoparticles and molecules to live cells with precise spatiotemporal control

Cornelia Holler, Richard W. Taylor, Alexandra Schambony, Leonhard Möckl, Vahid Sandoghdar

Delivery of very small amounts of reagents to the near-field of cells with micrometer spatial precision and millisecond time resolution is currently out of reach. Here we present μkiss as a micropipette-based scheme for brushing a layer of small molecules and nanoparticles onto the live cell membrane from a subfemtoliter confined volume of a perfusion flow. We characterize our system through both experiments and modeling, and find excellent agreement. We demonstrate several applications that benefit from a controlled brush delivery, such as a direct means to quantify local and long-range membrane mobility and organization as well as dynamical probing of intercellular force signaling.

A buoyant nucleus is a universal characteristic of eukaryotic cells

Abin Biswas, Kyoohyun Kim, Omar Munoz, Vasily Zaburdaev, Simone Reber, Jochen Guck

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. Rather unexpectedly, we had discovered previously that the nucleus has a lower density than the cytoplasm in some cells and that this was robust against various perturbations. Here, we generalize this finding and show that living systems establish and maintain a constant density ratio between the nucleus and the cytoplasm across 10 model organisms: the nucleus is always 20% less dense than the cytoplasm. Using optical diffraction tomography and fluorescence microscopy, various biochemical and cell biological perturbations, together with theoretical modelling, we show that nuclear density is set by a pressure balance across the nuclear envelope in vitro (Xenopus egg extracts), in vivo (cell lines), and during early development (C. elegans embryos). The nuclear proteome exerts a colloid osmotic pressure, which, assisted by entropic chromatin pressure, draws water into the nucleus, while keeping osmotically inactive but heavy and large components excluded. This study reveals a previously unidentified homeostatic coupling of macromolecular densities that drives cellular organization with implications for pathophysiologies such as senescence and cancer.

Mean zero artificial diffusion for stable finite element approximation of convection in cellular aggregate formation

Soheil Firooz, B. Daya Reddy, Vasily Zaburdaev, Paul Steinmann

We develop and implement finite element approximations for the coupled problem of cellular aggregate formation. The equation governing evolution of cell density is convective in nature, necessitating a modification of standard approaches to circumvent the instabilities associated with standard finite element approximations. To this end, a novel mean zero artificial diffusion approach is proposed, in which the classical artificial diffusion term is replaced by one that is orthogonal to its projection on to continuous functions. The resulting approach for the convective equation is shown to be well-posed. A range of numerical results illustrate the stability and accuracy of the new approach, and its behaviour in comparison with an alternative approach using Taylor–Hood elements. The results also provide insights into the behaviour of cellular aggregates in the context of the model studied here.

Brillouin microscopy

Irina Kabakova, Jitao Zhang, Yuchen Xiang, Silvia Caponi, Alberto Bilenca, Jochen Guck, Guiliano Scarcelli

The field of Brillouin microscopy and imaging was established approximately 20 years ago, thanks to the development of non-scanning high-resolution optical spectrometers. Since then, the field has experienced rapid expansion, incorporating technologies from telecommunications, astrophotonics, multiplexed microscopy, quantum optics and machine learning. Consequently, these advancements have led to much-needed improvements in imaging speed, spectral resolution and sensitivity. The progress in Brillouin microscopy is driven by a strong demand for label-free and contact-free methods to characterize the mechanical properties of biomaterials at the cellular and subcellular scales. Understanding the local biomechanics of cells and tissues has become crucial in predicting cellular fate and tissue pathogenesis. This Primer aims to provide a comprehensive overview of the methods and applications of Brillouin microscopy. It includes key demonstrations of Brillouin microscopy and imaging that can serve as a reference for the existing research community and new adopters of this technology. The article concludes with an outlook, presenting the authors’ vision for future developments in this vibrant field. The Primer also highlights specific examples where Brillouin microscopy can have a transformative impact on biology and biomedicine.

p21 Prevents the Exhaustion of CD4+ T Cells Within the Antitumor Immune Response Against Colorectal Cancer

Oana-Maria Thoma, Elisabeth Naschberger, Markéta Kubánková, Imen Larafa, Viktoria Kramer, Bianca Menchicchi, Susanne Merkel, Nathalie Britzen-Laurent, André Jefremow, et al.

BACKGROUND & AIMS: T cells are crucial for the antitumor response against colorectal cancer (CRC). T-cell reactivity to CRC is nevertheless limited by T-cell exhaustion. However, molecular mechanisms regulating T-cell exhaustion are only poorly understood. METHODS: We investigated the functional role of cyclin-dependent kinase 1a (Cdkn1a or p21) in cluster of differentiation (CD) 4+ T cells using murine CRC models. Furthermore, we evaluated the expression of p21 in patients with stage I to IV CRC. In vitro coculture models were used to understand the effector function of p21-deficient CD4+ T cells. RESULTS: We observed that the activation of cell cycle regulator p21 is crucial for CD4+ T-cell cytotoxic function and that p21 deficiency in type 1 helper T cells (Th1) leads to increased tumor growth in murine CRC. Similarly, low p21 expression in CD4+ T cells infiltrated into tumors of CRC patients is associated with reduced cancer-related survival. In mouse models of CRC, p21-deficient Th1 cells show signs of exhaustion, where an accumulation of effector/effector memory T cells and CD27/CD28 loss are predominant. Immune reconstitution of tumor-bearing Rag1−/− mice using ex vivo-treated p21-deficient T cells with palbociclib, an inhibitor of cyclin-dependent kinase 4/6, restored cytotoxic function and prevented exhaustion of p21-deficient CD4+ T cells as a possible concept for future immunotherapy of human disease. CONCLUSIONS: Our data reveal the importance of p21 in controlling the cell cycle and preventing exhaustion of Th1 cells. Furthermore, we unveil the therapeutic potential of cyclin-dependent kinase inhibitors such as palbociclib to reduce T-cell exhaustion for future treatment of patients with colorectal cancer.

A universal strategy to induce oxidative stress-mediated cell death in biological systems

Leonhard Möckl, Karim Almahayni, Jana Bachir Salvador, Riccardo Conti, Anna Widera, Malte Spiekermann, Daniel Wehner, Hansjörg Grützmacher

Precise cell elimination within intricate cellular populations is hampered by issues arising from the multifaceted biological properties of cells and the expansive reactivity of chemical agents. Current platforms are often limited by their complexity, toxicity, and poor physical/chemical properties. Here, we integrate the spatio-temporal precision of light delivery and the structural versatility of bisacylphosphane oxides (BAPOs), establishing a universal strategy for on-demand, precise cellular ablation in vitro and in vivo.

Where bacteria and eukaryotes meet

Liraz Chai, Elizabeth A. Shank, Vasily Zaburdaev, Mohamed Y. El-Naggar

The international workshop “Interdisciplinary life of microbes: from single cells to multicellular aggregates,” following a virtual preassembly in November 2021, was held in person in Dresden, from 9 to 13 November 2022. It attracted not only prominent experts in biofilm research but also researchers from broadly neighboring disciplines, such as medicine, chemistry, and theoretical and experimental biophysics, both eukaryotic and prokaryotic. Focused brainstorming sessions were the special feature of the event and are at the heart of this commentary.

Cell morphology as a quantifier for functional states of resident tissue macrophages

Miriam Schnitzerlein, Anja Wegner, Oumaima Ben Brahim, Stefan Uderhardt, Vasily Zaburdaev

Resident tissue macrophages (RTMs) are essential for maintaining homeostasis in a physiological tissue state. They monitor interstitial fluids, contain acute damage while actively preventing inflammation, and remove dead cells and debris. All these cellular functions are accompanied by characteristic changes in cell morphology, the expression of which can provide information about the functional status of the cells. What is currently known about morphological patterns and dynamic behavior of macrophages is derived primarily from experimental ex vivo cell cultures. However, how macrophages operate in living organisms is in many ways fundamentally different from how they do in a cell culture system. In this work, we employed an intravital imaging platform to generate dynamic data from peritoneal RTMs in vivo in mice under various conditions induced either chemically or physically. Using this data, we built an image processing pipeline and defined a set of human-interpretable cell size and shape features which allowed us to quantify RTM morphodynamics over time. We used those features to quantitatively differentiate cells in various functional states - when macrophages are activated, for instance, or when they “shut down” due to detrimental changes in the environment. The qualitative morphology changes associated with these functional states could be inferred directly from the quantitative measurements. Finally we used the set of cell morphology features monitoring the health of RTMs to improve a setup for explanted tissues. Thus, the proposed method is a versatile tool to provide insights into the dynamic behavior of bona fide macrophages in vivo and helps distinguish between physiological and pathological cell states.

AI-driven projection tomography with multicore fibre-optic cell rotation

Jiawei Sun, Bin Yang, Nektarios Koukourakis, Jochen Guck, Juergen W. Czarske

Optical tomography has emerged as a non-invasive imaging method, providing three-dimensional insights into subcellular structures and thereby enabling a deeper understanding of cellular functions, interactions, and processes. Conventional optical tomography methods are constrained by a limited illumination scanning range, leading to anisotropic resolution and incomplete imaging of cellular structures. To overcome this problem, we employ a compact multi-core fibre-optic cell rotator system that facilitates precise optical manipulation of cells within a microfluidic chip, achieving full-angle projection tomography with isotropic resolution. Moreover, we demonstrate an AI-driven tomographic reconstruction workflow, which can be a paradigm shift from conventional computational methods, often demanding manual processing, to a fully autonomous process. The performance of the proposed cell rotation tomography approach is validated through the three-dimensional reconstruction of cell phantoms and HL60 human cancer cells. The versatility of this learning-based tomographic reconstruction workflow paves the way for its broad application across diverse tomographic imaging modalities, including but not limited to flow cytometry tomography and acoustic rotation tomography. Therefore, this AI-driven approach can propel advancements in cell biology, aiding in the inception of pioneering therapeutics, and augmenting early-stage cancer diagnostics.

Tensed axons are on fire

Kristian Franze