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

Flavia S. Papini, Mona Seifert, David Dulin

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

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

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

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

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

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

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

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

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

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

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

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

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

Interaction of Axonal Chondrolectin with Collagen XIXa1 Is Necessary for Precise Neuromuscular Junction Formation

Ana-Maria Oprisoreanu, Hannah L. Smith, Sukrat Arya, Richard Webster, Zhen Zhong, Daniel Wehner, Marcos J. Cardozo, Thomas Becker, Kevin Talbot, et al.

Chondrolectin (Chodl) is needed for motor axon extension in zebrafish and is dysregulated in mouse models of spinal muscular atrophy (SMA). However, the mechanistic basis of Chodl function is not known. Here, we use Chodl-deficient zebrafish and mouse mutants to show that the absence of Chodl leads to anatomical and functional defects of the neuromuscular synapse. In zebrafish, the growth of an identified motor axon beyond an "en passant'' synapse and later axon branching from synaptic points are impaired, leading to functional deficits. Mechanistically, motor-neuron-autonomous Chodl function depends on its intracellular domain and on binding muscle-derived collagen XIXa1 by its extracellular C-type lectin domain. Our data support evolutionarily conserved roles of Chodl in synaptogenesis and provide evidence for a "synapse-first'' scenario of motor axon growth in zebrafish.

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

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

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

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

Katharina Blessing, Shivani Sharma, Alexander Gumann, Kanwarpal Singh

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

Rectification of Bacterial Diffusion in Microfluidic Labyrinths

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

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

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

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

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

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

Gonzalo Rosso, Jochen Guck

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

Some thoughts on the future of cell mechanics

Jochen Guck

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

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

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

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

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

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

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

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

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

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

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

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

Interferometric Scattering (iSCAT) Microscopy & Related Techniques

Richard W. Taylor, Vahid Sandoghdar

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

Coherent nonlinear optics of quantum emitters in nanophotonic waveguides

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

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

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

Richard W. Taylor, Vahid Sandoghdar

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

High-Throughput Microfluidic Characterization of Erythrocyte Shapes and Mechanical Variability

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

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

Coherent coupling of single molecules to on-chip ring resonators

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

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

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

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

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

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

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

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

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

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

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

How bacterial cells and colonies move on solid substrates

Wolfram Poenisch, Christoph A. Weber, Vasily Zaburdaev

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

Nanoprinting organic molecules at the quantum level

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

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

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

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

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

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

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

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

Turning a molecule into a coherent two-level quantum system

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

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

Identifying the mechanism for superdiffusivity in mouse fibroblast motility

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

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

The shape of pinned forced polymer loops

Wenwen Huang, Vasily Zaburdaev

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

High throughput magnetic tweezers to characterize inhibitors of RNA virus replication

David Dulin