Quantitative Evaluation of the Cellular Uptake of Nanodiamonds by Monocytes and Macrophages.

Fluorescent nanodiamonds (FNDs) with negative nitrogen-vacancy (NV- ) defect centers are great probes for biosensing applications, with potential to act as biomarkers for cell differentiation. To explore this concept, uptake of FNDs (≈120 nm) by THP-1 monocytes and monocyte-derived M0-macrophages is studied. The time course analysis of FND uptake by monocytes confirms differing FND-cell interactions and a positive time-dependence. No effect on cell viability, proliferation, and differentiation potential into macrophages is observed, while cells saturated with FNDs, unload the FNDs completely by 25 cell divisions and subsequently take up a second dose effectively. FND uptake variations by THP-1 cells at early exposure-times indicate differing phagocytic capability. The cell fraction that exhibits relatively enhanced FND uptake is associated to a macrophage phenotype which derives from spontaneous monocyte differentiation. In accordance, chemical-differentiation of the THP-1 cells into M0-macrophages triggers increased and homogeneous FND uptake, depleting the fraction of cells that were non-responsive to FNDs. These observations imply that FND uptake allows for distinction between the two cell subtypes based on phagocytic capacity. Overall, FNDs demonstrate effective cell labeling of monocytes and macrophages, and are promising candidates for sensing biological processes that involve cell differentiation.

Classification of fetal and adult red blood cells based on hydrodynamic deformation and deep video recognition.

Flow based deformation cytometry has shown potential for cell classification. We demonstrate the principle with an injection moulded microfluidic chip from which we capture videos of adult and fetal red blood cells, as they are being deformed in a microfluidic chip. Using a deep neural network - SlowFast - that takes the temporal behavior into account, we are able to discriminate between the cells with high accuracy. The accuracy was larger for adult blood cells than for fetal blood cells. However, no significant difference was observed between donors of the two types.

Manipulation and Motion of Organelles and Single Molecules in Living Cells.

The biomolecule is among the most important building blocks of biological systems, and a full understanding of its function forms the scaffold for describing the mechanisms of higher order structures as organelles and cells. Force is a fundamental regulatory mechanism of biomolecular interactions driving many cellular processes. The forces on a molecular scale are exactly in the range that can be manipulated and probed with single molecule force spectroscopy. The natural environment of a biomolecule is inside a living cell, hence, this is the most relevant environment for probing their function. In vivo studies are, however, challenged by the complexity of the cell. In this review, we start with presenting relevant theoretical tools for analyzing single molecule data obtained in intracellular environments followed by a description of state-of-the art visualization techniques. The most commonly used force spectroscopy techniques, namely optical tweezers, magnetic tweezers, and atomic force microscopy, are described in detail, and their strength and limitations related to in vivo experiments are discussed. Finally, recent exciting discoveries within the field of in vivo manipulation and dynamics of single molecule and organelles are reviewed.

Optical manipulation of single molecules in the living cell.

Optical tweezers are the only nano-tools capable of manipulating and performing force-measurements on individual molecules and organelles within the living cell without performing destructive penetration through the cell wall and without the need for inserting a non-endogenous probe. Here, we describe how optical tweezers are used to manipulate individual molecules and perform accurate force and distance measurements within the complex cytoplasm of the living cell. Optical tweezers can grab individual molecules or organelles, if their optical contrast to the medium is large enough, as is the case, e.g., for lipid granules or chromosomes. However, often the molecule of interest is specifically attached to a handle manipulated by the optical trap. The most commonly used handles, their insertion into the cytoplasm, and the relevant micro-rheology of the cell are discussed here and we also review recent and exciting results achieved through optical force manipulation of individual molecules in vivo.

The Interplay between Mechanoregulation and ROS in Heart Physiology, Disease, and Regeneration.

Cardiovascular diseases are currently the most common cause of death in developed countries. Due to lifestyle and environmental factors, this problem is only expected to increase in the future. Reactive oxygen species (ROS) are a key player in the onset of cardiovascular diseases but also have important functions in healthy cardiac tissue. Here, the interplay between ROS generation and cardiac mechanical forces is shown, and the state of the art and a perspective on future directions are discussed. To this end, an overview of what is currently known regarding ROS and mechanosignaling at a subcellular level is first given. There the role of ROS in mechanosignaling as well as the interplay between both factors in specific organelles is emphasized. The consequences at a larger scale across the population of heart cells are then discussed. Subsequently, the roles of ROS in embryogenesis, pathogenesis, and aging are further discussed, exemplifying some aspects of mechanoregulation. Finally, different models that are currently in use are discussed to study the topics above.

'Seeing' the electromagnetic spectrum: spotlight on the cryptochrome photocycle.

Cryptochromes are widely dispersed flavoprotein photoreceptors that regulate numerous developmental responses to light in plants, as well as to stress and entrainment of the circadian clock in animals and humans. All cryptochromes are closely related to an ancient family of light-absorbing flavoenzymes known as photolyases, which use light as an energy source for DNA repair but themselves have no light sensing role. Here we review the means by which plant cryptochromes acquired a light sensing function. This transition involved subtle changes within the flavin binding pocket which gave rise to a visual photocycle consisting of light-inducible and dark-reversible flavin redox state transitions. In this photocycle, light first triggers flavin reduction from an initial dark-adapted resting state (FADox). The reduced state is the biologically active or 'lit' state, correlating with biological activity. Subsequently, the photoreduced flavin reoxidises back to the dark adapted or 'resting' state. Because the rate of reoxidation determines the lifetime of the signaling state, it significantly modulates biological activity. As a consequence of this redox photocycle Crys respond to both the wavelength and the intensity of light, but are in addition regulated by factors such as temperature, oxygen concentration, and cellular metabolites that alter rates of flavin reoxidation even independently of light. Mechanistically, flavin reduction is correlated with conformational change in the protein, which is thought to mediate biological activity through interaction with biological signaling partners. In addition, a second, entirely independent signaling mechanism arises from the cryptochrome photocycle in the form of reactive oxygen species (ROS). These are synthesized during flavin reoxidation, are known mediators of biotic and abiotic stress responses, and have been linked to Cry biological activity in plants and animals. Additional special properties arising from the cryptochrome photocycle include responsivity to electromagnetic fields and their applications in optogenetics. Finally, innovations in methodology such as the use of Nitrogen Vacancy (NV) diamond centers to follow cryptochrome magnetic field sensitivity in vivo are discussed, as well as the potential for a whole new technology of 'magneto-genetics' for future applications in synthetic biology and medicine.

Quantitative determination of optical trapping strength and viscoelastic moduli inside living cells.

With the success of in vitro single-molecule force measurements obtained in recent years, the next step is to perform quantitative force measurements inside a living cell. Optical traps have proven excellent tools for manipulation, also in vivo, where they can be essentially non-invasive under correct wavelength and exposure conditions. It is a pre-requisite for in vivo quantitative force measurements that a precise and reliable force calibration of the tweezers is performed. There are well-established calibration protocols in purely viscous environments; however, as the cellular cytoplasm is viscoelastic, it would be incorrect to use a calibration procedure relying on a viscous environment. Here we demonstrate a method to perform a correct force calibration inside a living cell. This method (theoretically proposed in Fischer and Berg-Sørensen (2007 J. Opt. A: Pure Appl. Opt. 9 S239)) takes into account the viscoelastic properties of the cytoplasm and relies on a combination of active and passive recordings of the motion of the cytoplasmic object of interest. The calibration procedure allows us to extract absolute values for the viscoelastic moduli of the living cell cytoplasm as well as the force constant describing the optical trap, thus paving the way for quantitative force measurements inside the living cell. Here, we determine both the spring constant of the optical trap and the elastic contribution from the cytoplasm, influencing the motion of naturally occurring tracer particles. The viscoelastic moduli that we find are of the same order of magnitude as moduli found in other cell types by alternative methods.

Pulsed laser manipulation of an optically trapped bead: averaging thermal noise and measuring the pulsed force amplitude.

An experimental strategy for post-eliminating thermal noise on position measurements of optically trapped particles is presented. Using a nanosecond pulsed laser, synchronized to the detection system, to exert a periodic driving force on an optically trapped 10 µm polystyrene bead, the laser pulse-bead interaction is repeated hundreds of times. Traces with the bead position following the prompt displacement from equilibrium, induced by each laser pulse, are averaged and reveal the underlying deterministic motion of the bead, which is not visible in a single trace due to thermal noise. The motion of the bead is analyzed from the direct time-dependent position measurements and from the power spectrum. The results show that the bead is on average displaced 208 nm from the trap center and exposed to a force amplitude of 71 nanoNewton, more than five orders of magnitude larger than the trapping forces. Our experimental method may have implications for microrheology.

Creation of Boron Vacancies in Hexagonal Boron Nitride Exfoliated from Bulk Crystals for Quantum Sensing.

Boron vacancies (VB-) in hexagonal boron -nitride (hBN) have sparked great interest in recent years due to their optical and spin properties. Since hBN can be readily integrated into devices where it interfaces a huge variety of other 2D materials, boron vacancies may serve as a precise sensor which can be deployed at very close proximity to many important materials systems. Boron vacancy defects may be produced by a number of existing methods, the use of which may depend on the final application. Any method should reproducibly generate defects with controlled density and desired pattern. To date, however, detailed studies of such methods are missing. In this paper, we study various techniques for the preparation of hBN flakes from bulk crystals and relevant postprocessing treatments, namely, focused ion beam (FIB) implantation, for creation of VB-s as a function of flake thickness and defect concentrations. We find that flake thickness plays an important role when optimizing implantation parameters, while careful sample cleaning proved important to achieve consistent results.

Microscopic-scale magnetic recording of brain neuronal electrical activity using a diamond quantum sensor.

Quantum sensors using solid state qubits have demonstrated outstanding sensitivity, beyond that possible using classical devices. In particular, those based on colour centres in diamond have demonstrated high sensitivity to magnetic field through exploiting the field-dependent emission of fluorescence under coherent control using microwaves. Given the highly biocompatible nature of diamond, sensing from biological samples is a key interdisciplinary application. In particular, the microscopic-scale study of living systems can be possible through recording of temperature and biomagnetic field. In this work, we use such a quantum sensor to demonstrate such microscopic-scale recording of electrical activity from neurons in fragile living brain tissue. By recording weak magnetic field induced by ionic currents in mouse corpus callosum axons, we accurately recover signals from neuronal action potential propagation while demonstrating in situ pharmacology. Our sensor allows recording of the electrical activity in neural circuits, disruption of which can shed light on the mechanisms of disease emergence. Unlike existing techniques for recording activity, which can require potentially damaging direct interaction, our sensing is entirely passive and remote from the sample. Our results open a promising new avenue for the microscopic recording of neuronal signals, offering the eventual prospect of microscopic imaging of electrical activity in the living mammalian brain.

Analytic solutions and universal properties of sugar loading models in Münch phloem flow.

The transport of sugars in the phloem vascular system of plants is believed to be driven by osmotic pressure differences according to the Münch hypothesis. Thus, the translocation process is viewed as a passive reaction to the active sugar loading in the leaves and sugar unloading in roots and other places of growth or storage. The modelling of the loading and unloading mechanism is thus a key ingredient in the mathematical description of such flows, but the influence of particular choices of loading functions on the translocation characteristics is not well understood. Most of the work has relied on numerical solutions, which makes it difficult to draw general conclusions. Here, we present analytic solutions to the Münch-Horwitz flow equations when the loading and unloading rates are assumed to be linear functions of the concentration, thus allowing them to depend on the local osmotic pressure. We are able to solve the equations analytically for very small and very large Münch numbers (e.g., very small and very large viscosity) for the flow velocity and sugar concentration as a function of the geometric and material parameters of the system. We further show, somewhat surprisingly, that the constant loading case can be solved along the same lines and we speculate on possible universal properties of different loading and unloading functions applied in the literature.

In vivo anomalous diffusion and weak ergodicity breaking of lipid granules.

Combining extensive single particle tracking microscopy data of endogenous lipid granules in living fission yeast cells with analytical results we show evidence for anomalous diffusion and weak ergodicity breaking. Namely we demonstrate that at short times the granules perform subdiffusion according to the laws of continuous time random walk theory. The associated violation of ergodicity leads to a characteristic turnover between two scaling regimes of the time averaged mean squared displacement. At longer times the granule motion is consistent with fractional Brownian motion.

Bioinspired Microstructured Polymer Surfaces with Antireflective Properties.

Over the years, different approaches to obtaining antireflective surfaces have been explored, such as using index-matching, interference, or micro- and nanostructures. Structural super black colors are ubiquitous in nature, and biomimicry thus constitutes an interesting way to develop antireflective surfaces. Moth-eye nanostructures, for example, are well known and have been successfully replicated using micro- and nanofabrication. However, other animal species, such as birds of paradise and peacock spiders, have evolved to display larger structures with antireflective features. In peacock spiders, the antireflective properties of their super black patches arise from relatively simple microstructures with lens-like shapes organized in tightly packed hexagonal arrays, which makes them a good candidate for cheap mass replication techniques. In this paper, we present the fabrication and characterization of antireflective microarrays inspired by the peacock spider's super black structures encountered in nature. Firstly, different microarrays 3D models are generated from a surface equation. Secondly, the arrays are fabricated in a polyacrylate resin by super-resolution 3D printing using two-photon polymerization. Thirdly, the resulting structures are inspected using a scanning electron microscope. Finally, the reflectance and transmittance of the printed structures are characterized at normal incidence with a dedicated optical setup. The bioinspired microlens arrays display excellent antireflective properties, with a measured reflectance as low as 0.042 ± 0.004% for normal incidence, a wavelength of 550 nm, and a collection angle of 14.5°. These values were obtained using a tightly-packed array of slightly pyramidal lenses with a radius of 5 µm and a height of 10 µm.

Detection of biological signals from a live mammalian muscle using an early stage diamond quantum sensor.

The ability to perform noninvasive and non-contact measurements of electric signals produced by action potentials is essential in biomedicine. A key method to do this is to remotely sense signals by the magnetic field they induce. Existing methods for magnetic field sensing of mammalian tissue, used in techniques such as magnetoencephalography of the brain, require cryogenically cooled superconducting detectors. These have many disadvantages in terms of high cost, flexibility and limited portability as well as poor spatial and temporal resolution. In this work we demonstrate an alternative technique for detecting magnetic fields generated by the current from action potentials in living tissue using nitrogen vacancy centres in diamond. With 50 pT/[Formula: see text] sensitivity, we show the first measurements of magnetic sensing from mammalian tissue with a diamond sensor using mouse muscle optogenetically activated with blue light. We show these proof of principle measurements can be performed in an ordinary, unshielded lab environment and that the signal can be easily recovered by digital signal processing techniques. Although as yet uncompetitive with probe electrophysiology in terms of sensitivity, we demonstrate the feasibility of sensing action potentials via magnetic field in mammals using a diamond quantum sensor, as a step towards microscopic imaging of electrical activity in a biological sample using nitrogen vacancy centres in diamond.

Effect of energy metabolism on protein motility in the bacterial outer membrane.

We demonstrate the energy dependence of the motion of a porin, the lambda-receptor, in the outer membrane of living Escherichia coli by single molecule investigations. By poisoning the bacteria with arsenate and azide, the bacterial energy metabolism was stopped. The motility of individual lambda-receptors significantly and rapidly decreased upon energy depletion. We suggest two different causes for the ceased motility upon comprised energy metabolism: One possible cause is that the cell uses energy to actively wiggle its proteins, this energy being one order-of-magnitude larger than thermal energy. Another possible cause is an induced change in the connection between the lambda-receptor and the membrane structure, for instance by a stiffening of part of the membrane structure. Treatment of the cells with ampicillin, which directly targets the bacterial cell wall by inhibiting cross-linking of the peptidoglycan layer, had an effect similar to energy depletion and the motility of the lambda-receptor significantly decreased. Since the lambda-receptor is closely linked to the peptidoglycan layer, we propose that lambda-receptor motility is directly coupled to the constant and dynamic energy-consuming reconstruction of the peptidoglycan layer. The result of this motion could be to facilitate transport of maltose-dextrins through the porin.

Variety in intracellular diffusion during the cell cycle.

During the cell cycle, the organization of the cytoskeletal network undergoes dramatic changes. In order to reveal possible changes of the viscoelastic properties in the intracellular space during the cell cycle we investigated the diffusion of endogenous lipid granules within the fission yeast Schizosaccharomyces Pombe using optical tweezers. The cell cycle was divided into interphase and mitotic cell division, and the mitotic cell division was further subdivided in its stages. During all stages of the cell cycle, the granules predominantly underwent subdiffusive motion, characterized by an exponent alpha that is also linked to the viscoelastic moduli of the cytoplasm. The exponent alpha was significantly smaller during interphase than during any stage of the mitotic cell division, signifying that the cytoplasm was more elastic during interphase than during division. We found no significant differences in the subdiffusive exponents from granules measured in different stages of cell division. Also, our results for the exponent displayed no significant dependence on the position of the granule within the cell. The observation that the cytoplasm is more elastic during interphase than during mitotic cell division is consistent with the fact that elastic cytoskeletal elements such as microtubules are less abundantly present during cell division than during interphase.

Dermal Denticles of Three Slowly Swimming Shark Species: Microscopy and Flow Visualization.

Shark skin has for many years inspired engineers to produce biomimetic structures reducing surface drag or acting as an anti-fouling layer. Both effects are presumed to be consequences of the structure of shark skin that is composed of arrays of so-called dermal denticles. However, the understanding of the full functional role of the dermal denticles is still a topic of research. We report optical microscopy and scanning electron microscopy of dermal denticles from three slowly swimming shark species for which the functional role of the dermal denticles is suggested as one of defense (possibly understood as anti-fouling) and/or abrasion strength. The three species are Greenland shark (Somnosius microcephalus), small-spotted catshark (Scyliorhinus canicula) and spiny dogfish (Squalus acanthias). Samples were taken at over 30 different positions on the bodies of the sharks. In addition, we demonstrate that the flow pattern near natural shark skin can be measured by micro-PIV (particle image velocimetry). The microfluidic experiments are complemented by numerical flow simulations. Both visualize unsteady flow, small eddies, and recirculation bubbles behind the natural dermal denticles.

Quantifying Force and Viscoelasticity Inside Living Cells Using an Active-Passive Calibrated Optical Trap.

As described in the previous chapters, optical tweezers have become a tool of precision for in vitro single-molecule investigations, where the single molecule of interest most often is studied in purified form in an experimental assay with a well-controlled fluidic environment. A well-controlled fluidic environment implies that the physical properties of the liquid, most notably the viscosity, are known and the fluidic environment can, for calibrational purposes, be treated as a simple liquid.In vivo, however, optical tweezers have primarily been used as a tool of manipulation and not so often for precise quantitative force measurements, due to the unknown value of the spring constant of the optical trap formed within the cell's viscoelastic cytoplasm. Here, we describe a method for utilizing optical tweezers for quantitative in vivo force measurements. The experimental protocol and the protocol for data analysis rely on two types of experiments, passive observation of the thermal motion of a trapped object inside a living cell, followed by observations of the response of the trapped object when subject to controlled oscillations of the optical trap. One advantage of this calibration method is that the size and refractive properties of the trapped object and the viscoelastic properties of its environment need not be known. We explain the protocol and demonstrate its use with experiments of trapped granules inside live S. pombe cells.

Tuning biomimetic membrane barrier properties by hydrocarbon, cholesterol and polymeric additives.

The barrier properties of cellular membranes are increasingly attracting attention as a source of inspiration for designing biomimetic membranes. The broad range of potential technological applications makes the use of lipid and lately also polymeric materials a popular choice for constructing biomimetic membranes, where the barrier properties can be controlled by the composition of the membrane constituent elements. Here we investigate the membrane properties reported by the light-induced proton pumping activity of bacteriorhodopsin (bR) reconstituted in three vesicle systems of different membrane composition. Specifically we quantify how the resulting proton influx and efflux rates are influenced by the membrane composition using a variety of membrane modulators. We demonstrate that by adding hydrocarbons to vesicles with reconstituted bR formed from asolectin lipids the resulting transmembrane proton fluxes changes proportional to the carbon chain length when compared against control. We observe a similar proportionality in single-component 1,2-Dioleoyl-sn-glycero-3-phosphocholine model membranes when using cholesterol. Lastly we investigate the effects of adding the amphiphilic di-block co-polymer polybutadiene-polyethyleneoxide (PB12-PEO10) to phospholipid membranes formed from 1,2-Dioleoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-Dioleoyl-sn-glycero-3-phosphatidylserine. The proton pumping activity of bR (measured as a change in extra-vesicular pH) in mixed lipid/PB12-PEO10 lipid systems is up to six-fold higher compared to that observed for bR containing vesicles made from PB12-PEO10 alone. Interestingly, bR inserts with apparent opposite orientation in pure PB12-PEO10 vesicles as compared to pure lipid vesicles. Addition of equimolar amounts of lipids to PB12-PEO10 results in bR orientation similar to that observed for pure lipids. In conclusion our results show how the barrier properties of the membranes can be controlled by the composition of the membrane. In particular the use of mixed lipid-polymer systems may pave the way for constructing biomimetic membranes tailored for optimal properties in various applications including drug delivery systems, biosensors and energy conservation technology.

Quantitative approach to small-scale nonequilibrium systems.

In a nanoscale system out of thermodynamic equilibrium, it is important to account for thermal fluctuations. Typically, the thermal noise contributes fluctuations, e.g., of distances that are substantial in comparison to the size of the system and typical distances measured. If the thermal fluctuations are ignored, misinterpretation of measured quantities such as interaction forces, potentials, and constants may result. Here, we consider a particle moving in a time-dependent landscape, as, e.g., in an optical tweezers or atomic force nanoscopic measurement. Based on the Kramers equation [H. A. Kramers, Physica 7, 284 (1940)], we propose an approximate but quantitative way of dealing with such an out-of-equilibrium system. The limits of this approximate description of the escape process are determined through optical tweezers experiments and comparison to simulations. Also, this serves as a recipe for how to use the proposed method to obtain knowledge about the underlying energy landscape from a set of experimental measurements. Finally, we perform estimates of the error made if thermal fluctuations are ignored.