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

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

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

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

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

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

Mechanical behavior of the hippocampus and corpus callosum: An attempt to reconcile ex vivo with in vivo and micro with macro properties

Gergerly Bertalan, Julia Becker, Heiko Tzschätzsch, Anna Morr, Helge Herthum, Mehrgan Shahryari, Ryan D. Greenhalgh, Jing Guo, Leif Schröder, et al.

Mechanical properties of brain tissue are very complex and vary with the species, region, method, and dynamic range, and between in vivo and ex vivo measurements. To reconcile this variability, we investigated in vivo and ex vivo stiffness properties of two distinct regions in the human and mouse brain - the hippocampus (HP) and the corpus callosum (CC) - using different methods. Under quasi-static conditions, we examined ex vivo murine HP and CC by atomic force microscopy (AFM). Between 16 and 40Hz, we investigated the in vivo brains of healthy volunteers by magnetic resonance elastography (MRE) in a 3-T clinical scanner. At high-frequency stimulation between 1000 and 1400Hz, we investigated the murine HP and CC ex vivo and in vivo with MRE in a 7-T preclinical system. HP and CC showed pronounced stiffness dispersion, as reflected by a factor of 32-36 increase in shear modulus from AFM to low-frequency human MRE and a 25-fold higher shear wave velocity in murine MRE than in human MRE. At low frequencies, HP was softer than CC, in both ex vivo mouse specimens (p < 0.05) and in vivo human brains (p < 0.01) while, at high frequencies, CC was softer than HP under in vivo (p < 0.01) and ex vivo (p < 0.05) conditions. The standard linear solid model comprising three elements reproduced the observed HP and CC stiffness dispersions, while other two- and three-element models failed. Our results indicate a remarkable consistency of brain stiffness across species, ex vivo and in vivo states, and different measurement techniques when marked viscoelastic dispersion properties combining equilibrium and non-equilibrium mechanical elements are considered.

Retinal regions shape human and murine Müller cell proteome profile and functionality

Lew Kaplan, Corinne Drexler, Anna M. Pfaller, Santra Brenna, Kirsten A. Wunderlich, Andrea Dimitracopoulos, Juliane Merl-Pham, Maria-Theresa Perez, Ursula Schlötzer-Schrehardt, et al.

The human macula is a highly specialized retinal region with pit-like morphology and rich in cones. How Müller cells, the principal glial cell type in the retina, are adapted to this environment is still poorly understood. We compared proteomic data from cone- and rod-rich retinae from human and mice and identified different expression profiles of cone- and rod-associated Müller cells that converged on pathways representing extracellular matrix and cell adhesion. In particular, epiplakin (EPPK1), which is thought to play a role in intermediate filament organization, was highly expressed in macular Müller cells. Furthermore, EPPK1 knockout in a human Müller cell-derived cell line led to a decrease in traction forces as well as to changes in cell size, shape, and filopodia characteristics. We here identified EPPK1 as a central molecular player in the region-specific architecture of the human retina, which likely enables specific functions under the immense mechanical loads in vivo.

Ultralong Imaging Range Chromatic Confocal Microscopy

Gargi Sharma, Kanwarpal Singh

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

Unrestrained growth of correctly oriented microtubules instructs axonal microtubule orientation

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

In many eukaryotic cells, directed molecular transport occurs along microtubules. Within neuronal axons, transport over vast distances particularly relies on uniformly oriented microtubules, whose plus-ends point towards the distal axon tip (anterogradely polymerizing, or plus-end-out). However, axonal microtubules initially have mixed orientations, and how they orient during development is not yet fully understood. Using live imaging of primary Drosophila melanogaster neurons, we found that, in the distal part of the axon, catastrophe rates of plus-end-out microtubules were significantly reduced compared to those of minus-end-out microtubules. Physical modelling revealed that plus-end-out microtubules should therefore exhibit persistent long-term growth, while growth of minus-end-out microtubules should be limited, leading to a bias in overall axonal microtubule orientation. Using chemical and physical perturbations of microtubule growth and genetic perturbations of the anti -catastrophe factor p150, which was enriched in the distal axon tip, we confirmed that the enhanced growth of plus-end-out microtubules is critical for achieving uniform microtubule orientation. Computer simulations of axon development integrating the enhanced plus-end-out microtubule growth identified here with previously suggested mechanisms, that is, dynein-based microtubule sliding and augmin-mediated templating, correctly predicted the long-term evolution of axonal microtubule orientation as found in our experiments. Our study thus leads to a holistic explanation of how axonal microtubules orient uniformly, a prerequisite for efficient long-range transport essential for neuronal functioning.

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

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

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

Viscoelastic properties of suspended cells measured with shear flow deformation cytometry

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

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

A DNA Segregation Module for Synthetic Cells

Mai P. Tran, Rakesh Chatterjee, Yannik Dreher, Julius Fichtler, Kevin Jahnke, Lennart Hilbert, Vasily Zaburdaev, Kerstin Göpfrich

The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made toward reliable compartment division, yet mechanisms to segregate the DNA-encoded informational content are still in their infancy. Herein, droplets of DNA Y-motifs are formed by liquid–liquid phase separation. DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y-motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled DNA segregation in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice-based theoretical model which mimics the interactions between the DNA Y-motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, can represent a strategy toward a DNA segregation module within bottom-up assembled synthetic cells.

Robust Tipless Positioning Device for Near-Field Investigations: Press and Roll Scan (PROscan)

Hsuan-Wei Liu, Michael A. Becker, Korenobu Matsuzaki, Randhir Kumar, Stephan Götzinger, Vahid Sandoghdar

Scanning probe microscopes scan and manipulate a sharp tip in the immediate vicinity of a sample surface. The limited bandwidth of the feedback mechanism used for stabilizing the separation between the tip and the sample makes the fragile nanoscopic tip very susceptible to mechanical instabilities. We propose, demonstrate, and characterize an alternative device based on bulging a thin substrate against a second substrate and rolling them with respect to each other. We showcase the power of this method by placing gold nanoparticles and semiconductor quantum dots on the two opposite substrates and positioning them with nanometer precision to enhance the fluorescence intensity and emission rate. Furthermore, we exhibit the passive mechanical stability of the system over more than 1 h. Our design concept finds applications in a variety of other scientific and technological contexts, where nanoscopic features have to be positioned and kept near contact with each other. a thin substrate against a second substrate and rolling them with respect each other. We showcase the power of this method by placing gold nanoparticles and semiconductor quantum dots on the two opposite substrates and positioning them with nanometer precision to enhance the fluorescence intensity and emission rate. We exhibit the passive mechanical stability of the system over more than one hour. The design concept presented in this work holds promise in a variety of other contexts, where nanoscopic features have to be positioned and kept near contact with each other.

On continuum modeling of cell aggregation phenomena

Soheil Firooz, Stefan Kaessmair, Vasily Zaburdaev, Ali Javili, Paul Steinmann

Cellular aggregates play a significant role in the evolution of biological systems such as tumor growth, tissue spreading, wound healing, and biofilm formation. Analysis of such biological systems, in principle, includes examining the interplay of cell–cell interactions together with the cell–matrix interaction. These two interaction types mainly drive the dynamics of cellular aggregates which is intrinsically out of equilibrium. Here we propose a non-linear continuum mechanics formulation and the corresponding finite element simulation framework to model the physics of cellular aggregate formation. As an example, we focus in particular on the process of bacterial colony formation as recently studied by Kuan et al. (2021). Thereby we describe the aggregation process as an active phase separation phenomenon. We develop a Lagrangian continuum description of the problem which yields a substantial simplification to the formulations of the governing equations. Due to the presence of spatial Hessian and Laplacian operators, a gradient-enhanced approach is required to incorporate continuity. In addition, a robust and efficient finite element formulation of the problem is provided. Taylor–Hood finite elements are utilized for the implementation to avoid instabilities related to the LBB condition. Finally, through a set of numerical examples, the influence of various parameters on the dynamics of the cellular aggregate formation is investigated. Our proposed methodology furnishes a general framework for the investigation of the rheology and non-equilibrium dynamics of cellular aggregates.

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

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

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

Quantitative phase imaging through an ultra-thin lensless fiber endoscope

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Deciphering a hexameric protein complex with Angstrom optical resolution

Hisham Mazal, Franz Wieser, Vahid Sandoghdar

Cryogenic optical localization in three dimensions (COLD) was recently shown to resolve up to four binding sites on a single protein. However, because COLD relies on intensity fluctuations that result from the blinking behavior of fluorophores, it is limited to cases where individual emitters show different brightness. This significantly lowers the measurement yield. To extend the number of resolved sites as well as the measurement yield, we employ partial labeling and combine it with polarization encoding in order to identify single fluorophores during their stochastic blinking. We then use a particle classification scheme to identify and resolve heterogenous subsets and combine them to reconstruct the three-dimensional arrangement of large molecular complexes. We showcase this method (polarCOLD) by resolving the trimer arrangement of proliferating cell nuclear antigen (PCNA) and six different sites of the hexamer protein Caseinolytic Peptidase B (ClpB) of Thermus thermophilus in its quaternary structure, both with Angstrom resolution. The combination of polarCOLD and single-particle cryogenic electron microscopy (cryoEM) promises to provide crucial insight into intrinsic heterogeneities of biomolecular structures. Furthermore, our approach is fully compatible with fluorescent protein labeling and can, thus, be used in a wide range of studies in cell and membrane biology.

Wie mikrobielle Modellsysteme helfen, Tumorevolution zu entschlüsseln

Maximilian Eiche, Jona Kayser

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

A Pili-Driven Bacterial Turbine

Wolfram Pönisch, Vasily Zaburdaev

Work generated by self-propelled bacteria can be harnessed with the help of microdevices. Such nanofabricated microdevices, immersed in a bacterial bath, may exhibit unidirectional rotational or translational motion. Swimming bacteria that propel with the help of actively rotating flagella are a prototypical example of active agents that can power such microdevices. In this work, we propose a computational model of a micron-sized turbine powered by bacteria that rely on active type IV pili appendages for surface-associated motility. We find that the turbine can rotate persistently over a time scale that significantly exceeds the characteristic times of the single cell motility. The persistent rotation is explained by the collective dynamics of multiple pili of groups of cells attaching to and pulling on turbine. Furthermore, we show that the turbine can rotate permanently in the same direction by altering the pili binding to the turbine surface in an asymmetric fashion. We thus can show that by changing the adhesive properties of the turbine while keeping its symmetric geometry, we can still break the symmetry of its rotation. Altogether, this study widely expands the range of bacteria that can be used to power nanofabricated microdevices, and, due to high pili forces generated by pili retraction, promises to push the harnessed work by several orders of magnitude.

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

Maria N. Romodina, Kanwarpal Singh

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

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

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

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

Best practices for reporting throughput in biomedical research

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

PiSCAT: A Python Package for Interferometric Scattering Microscopy

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

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

High-resolution vibronic spectroscopy of a single molecule embedded in a crystal

Johannes Zirkelbach, Masoud Mirzaei, Irena Deperasińska, Boleslaw Kozankiewicz, Burak Gürlek, Alexey Shkarin, Tobias Utikal, Stephan Götzinger, Vahid Sandoghdar

Vibrational levels of the electronic ground states in dye molecules have not been previously explored at a high resolution in solid matrices. We present new spectroscopic measurements on single polycyclic aromatic molecules of dibenzoterrylene embedded in an organic crystal made of para-dichlorobenzene. To do this, we use narrow-band continuous-wave lasers and combine spectroscopy methods based on fluorescence excitation and stimulated emission depletion to assess individual vibrational linewidths in the electronic ground state at a resolution of ∼30 MHz dictated by the linewidth of the electronic excited state. In this fashion, we identify several exceptionally narrow vibronic levels with linewidths down to values around 2 GHz. Additionally, we sample the distribution of vibronic wavenumbers, relaxation rates, and Franck–Condon factors, in both the electronic ground and excited states for a handful of individual molecules. We discuss various noteworthy experimental findings and compare them with the outcome of density functional theory calculations. The highly detailed vibronic spectra obtained in our work pave the way for studying the nanoscopic local environment of single molecules. The approach also provides an improved understanding of the vibrational relaxation mechanisms in the electronic ground state, which may help create long-lived vibrational states for applications in quantum technology.

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

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

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

Polarization Independent Optical Coherence Tomography

Asha Parmar, Gargi Sharma, Kanwarpal Singh

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

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

Daniel Wehner, Catherina G. Becker

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

Cell surface fluctuations regulate early embryonic lineage sorting

Ayaka Yanagida, Elena Corujo-Simon, Christopher K. Revell, Preeti Sahu, Giuliano G. Stirparo, Irene M. Aspalter, Alex K. Winkel, Ruby Peters, Henry De Belly, et al.

In development, lineage segregation is coordinated in time and space. An important example is the mammalian inner cell mass, in which the primitive endoderm (PrE, founder of the yolk sac) physically segregates from the epiblast (EPI, founder of the fetus). While the molecular requirements have been well studied, the physical mechanisms determining spatial segregation between EPI and PrE remain elusive. Here, we investigate the mechanical basis of EPI and PrE sorting. We find that rather than the differences in static cell surface mechanical parameters as in classical sorting models, it is the differences in surface fluctuations that robustly ensure physical lineage sorting. These differential surface fluctuations systematically correlate with differential cellular fluidity, which we propose together constitute a non-equilibrium sorting mechanism for EPI and PrE lineages. By combining experiments and modeling, we identify cell surface dynamics as a key factor orchestrating the correct spatial segregation of the founder embryonic lineages.

Quantitative imaging of Caenorhabditis elegans dauer larvae during cryptobiotic transition

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

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

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

Benedikt Krug, Nektarios Koukourakis, Jochen Guck, Jürgen Czarske

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

Cross-Polarized Optical Coherence Tomography System with Unpolarized Light

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

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

Effective cell membrane tension is independent of polyacrylamide substrate stiffness

Eva Kreysing, Jeffrey Mc Hugh, Sarah K. Foster, Kurt Andresen, Ryan D. Greenhalgh, Eva K. Pillai, Andrea Dimitracopoulos, Ulrich F. Keyser, Kristian Franze

Most animal cells are surrounded by a cell membrane and an underlying actomyosin cortex. Both structures are linked, and they are under tension. In-plane membrane tension and cortical tension both influence many cellular processes, including cell migration, division, and endocytosis. However, while actomyosin tension is regulated by substrate stiffness, how membrane tension responds to mechanical substrate properties is currently poorly understood. Here, we probed the effective membrane tension of neurons and fibroblasts cultured on glass and polyacrylamide substrates of varying stiffness using optical tweezers. In contrast to actomyosin-based traction forces, both peak forces and steady-state tether forces of cells cultured on hydrogels were independent of substrate stiffness and did not change after blocking myosin II activity using blebbistatin, indicating that tether and traction forces are not directly linked. Peak forces in fibroblasts on hydrogels were about twice as high as those in neurons, indicating stronger membrane-cortex adhesion in fibroblasts. Steady-state tether forces were generally higher in cells cultured on hydrogels than on glass, which we explain by a mechanical model. Our results provide new insights into the complex regulation of effective membrane tension and pave the way for a deeper understanding of the biological processes it instructs.

Single photon sources for quantum radiometry: a brief review about the current state‑of‑the‑art

Stefan Kück, Marco López, Helmuth Hofer, Hristina Georgieva, Justus Christinck, Beatrice Rodiek, Geiland Porrovecchio, Marek Smid, Stephan Götzinger, et al.

Single-photon sources have a variety of applications. One of these is quantum radiometry, which is reported on in this paper in the form of an overview, specifically of the current state of the art in the application of deterministic single photon sources to the calibration of single photon detectors. To optimize single-photon sources for this purpose, extensive research is currently carried out at the European National Metrology Institutes (NMIs), in collaboration with partners from universities. Single-photon sources of different types are currently under investigation, including sources based on defect centres in (nano-)diamonds, on molecules and on semiconductor quantum dots. We will present, summarise, and compare the current results obtained at European NMIs for single-photon sources in terms of photon flux, single-photon purity, and spectral power distribution as well as the results of single-photon detector calibrations carried out with this type of light sources.

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

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

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

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

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

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

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

Nora John, Julia Kolb, Daniel Wehner

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

Prevention of the foreign body response to implantable medical devices by inflammasome inhibition

Damiano G. Barone, Alejandro Carnicer-Lombarte, Panagiotis Tourlomousis, Russell S. Hamilton, Malwina Prater, Alexandra L. Rutz, Ivan B. Dimov, George G. Malliaras, Stephanie P. Lacour, et al.

SignificanceImplantable electronic medical devices (IEMDs) are used for some clinical applications, representing an exciting prospect for the transformative treatment of intractable conditions such Parkinson's disease, deafness, and paralysis. The use of IEMDs is limited at the moment because, over time, a foreign body reaction (FBR) develops at the device-neural interface such that ultimately the IEMD fails and needs to be removed. Here, we show that macrophage nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activity drives the FBR in a nerve injury model yet integration of an NLRP3 inhibitor into the device prevents FBR while allowing full healing of damaged neural tissue to occur.

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

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

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

Comparison of back focal plane imaging of nitrogen vacancy centers in nanodiamond and core-shell CdSe/CdS quantum dots

Justus Christinck, Beatrice Rodiek, Marco Lopez , Hristina Georgieva, Stephan Götzinger, Stefan Kück

We report on the characterization of the angular-dependent emission of two different single-photon emitters based on nitrogen-vacancy centers in nanodiamond and on core-shell CdSe/CdS quantum dot nanoparticles. The emitters were characterized in a confocal microscope setup by spectroscopy and Hanbury-Brown and Twiss interferometry. The angular-dependent emission is measured using a back focal plane imaging technique. A theoretical model of the angular emission patterns of the 2D dipoles of the emitters is developed to determine their orientation. Experiment and model agree well with each other.

Axons in the Chick Embryo Follow Soft Pathways Through Developing Somite Segments

Julia Schaeffer, Isabell P. Weber, Amelia J. Thompson, Roger J. Keynes, Kristian Franze

During patterning of the peripheral nervous system, motor axons grow sequentially out of the neural tube in a segmented fashion to ensure functional integration of the motor roots between the surrounding cartilage and bones of the developing vertebrae. This segmented outgrowth is regulated by the intrinsic properties of each segment (somite) adjacent to the neural tube, and in particular by chemical repulsive guidance cues expressed in the posterior half. Yet, knockout models for such repulsive cues still display initial segmentation of outgrowing motor axons, suggesting the existence of additional, yet unknown regulatory mechanisms of axon growth segmentation. As neuronal growth is not only regulated by chemical but also by mechanical signals, we here characterized the mechanical environment of outgrowing motor axons. Using atomic force microscopy-based indentation measurements on chick embryo somite strips, we identified stiffness gradients in each segment, which precedes motor axon growth. Axon growth was restricted to the anterior, softer tissue, which showed lower cell body densities than the repulsive stiffer posterior parts at later stages. As tissue stiffness is known to regulate axon growth during development, our results suggest that motor axons also respond to periodic stiffness gradients imposed by the intrinsic mechanical properties of somites.