Brillouin microscopy: an emerging tool for mechanobiology.

The role and importance of mechanical properties of cells and tissues in cellular function, development and disease has widely been acknowledged, however standard techniques currently used to assess them exhibit intrinsic limitations. Recently, Brillouin microscopy, a type of optical elastography, has emerged as a non-destructive, label- and contact-free method that can probe the viscoelastic properties of biological samples with diffraction-limited resolution in 3D. This led to increased attention amongst the biological and medical research communities, but it also sparked debates about the interpretation and relevance of the measured physical quantities. Here, we review this emerging technology by describing the underlying biophysical principles and discussing the interpretation of Brillouin spectra arising from heterogeneous biological matter. We further elaborate on the technique's limitations, as well as its potential for gaining insights in biology, in order to guide interested researchers from various fields.

Mechano-Pharmacological Testing of L-Type Ca2+ Channel Modulators via Human Vascular Celldrum Model.

BACKGROUND/AIMS: This study aimed to establish a precise and well-defined working model, assessing pharmaceutical effects on vascular smooth muscle cell monolayer in-vitro. It describes various analysis techniques to determine the most suitable to measure the biomechanical impact of vasoactive agents by using CellDrum technology. METHODS: The so-called CellDrum technology was applied to analyse the biomechanical properties of confluent human aorta muscle cells (haSMC) in monolayer. The cell generated tensions deviations in the range of a few N/m² are evaluated by the CellDrum technology. This study focuses on the dilative and contractive effects of L-type Ca2+ channel agonists and antagonists, respectively. We analyzed the effects of Bay K8644, nifedipine and verapamil. Three different measurement modes were developed and applied to determine the most appropriate analysis technique for the study purpose. These three operation modes are called, particular time mode" (PTM), "long term mode" (LTM) and "real-time mode" (RTM). RESULTS: It was possible to quantify the biomechanical response of haSMCs to the addition of vasoactive agents using CellDrum technology. Due to the supplementation of 100nM Bay K8644, the tension increased approximately 10.6% from initial tension maximum, whereas, the treatment with nifedipine and verapamil caused a significant decrease in cellular tension: 10nM nifedipine decreased the biomechanical stress around 6,5% and 50nM verapamil by 2,8%, compared to the initial tension maximum. Additionally, all tested measurement modes provide similar results while focusing on different analysis parameters. CONCLUSION: The CellDrum technology allows highly sensitive biomechanical stress measurements of cultured haSMC monolayers. The mechanical stress responses evoked by the application of vasoactive calcium channel modulators were quantified functionally (N/m²). All tested operation modes resulted in equal findings, whereas each mode features operation-related data analysis.

In vivo quantification of spatially varying mechanical properties in developing tissues.

The mechanical properties of the cellular microenvironment and their spatiotemporal variations are thought to play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has yet been performed. Here we introduce a technique that employs biocompatible, magnetically responsive ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo. Using this technique, we show that vertebrate body elongation entails spatially varying tissue mechanics along the anteroposterior axis. Specifically, we find that the zebrafish tailbud is viscoelastic (elastic below a few seconds and fluid after just 1 min) and displays decreasing stiffness and increasing fluidity toward its posterior elongating region. This method opens new avenues to study mechanobiology in vivo, both in embryogenesis and in disease processes, including cancer.

Biological physics by high-speed atomic force microscopy.

While many fields have contributed to biological physics, nanotechnology offers a new scale of observation. High-speed atomic force microscopy (HS-AFM) provides nanometre structural information and dynamics with subsecond resolution of biological systems. Moreover, HS-AFM allows us to measure piconewton forces within microseconds giving access to unexplored, fast biophysical processes. Thus, HS-AFM provides a tool to nourish biological physics through the observation of emergent physical phenomena in biological systems. In this review, we present an overview of the contribution of HS-AFM, both in imaging and force spectroscopy modes, to the field of biological physics. We focus on examples in which HS-AFM observations on membrane remodelling, molecular motors or the unfolding of proteins have stimulated the development of novel theories or the emergence of new concepts. We finally provide expected applications and developments of HS-AFM that we believe will continue contributing to our understanding of nature, by serving to the dialogue between biology and physics. This article is part of a discussion meeting issue 'Dynamic in situ microscopy relating structure and function'.

Rational Design of Semiconductor Nanostructures for Functional Subcellular Interfaces.

One of the fundamental questions guiding research in the biological sciences is how cellular systems process complex physical and environmental cues and communicate with each other across multiple length scales. Importantly, aberrant signal processing in these systems can lead to diseases that can have devastating impacts on human lives. Biophysical studies in the past several decades have demonstrated that cells can respond to not only biochemical cues but also mechanical and electrical ones. Thus, the development of new materials that can both sense and modulate all of these pathways is necessary. Semiconducting nanostructures are an emerging class of discovery platforms and tools that can push the limits of our ability to modulate and sense biological behaviors for both fundamental research and clinical applications. These materials are of particular interest for interfacing with cellular systems due to their matched dimension with subcellular components (e.g., cytoskeletal filaments), and easily tunable properties in the electrical, optical and mechanical regimes. Rational design via traditional or new approaches, such as nanocasting and mesoscale chemical lithography, can allow us to control micro- and nanoscale features in nanowires to achieve new biointerfaces. Both processes endogenous to the target cell and properties of the material surface dictate the character of these interfaces. In this Account, we focus on (1) approaches for the rational design of semiconducting nanowires that exhibit unique structures for biointerfaces, (2) recent fundamental discoveries that yield robust biointerfaces at the subcellular level, (3) intracellular electrical and mechanical sensing, and (4) modulation of cellular behaviors through material topography and remote physical stimuli. In the first section, we discuss new approaches for the synthetic control of micro- and nanoscale features of these materials. In the second section, we focus on achieving biointerfaces with these rationally designed materials either intra- or extracellularly. We last delve into the use of these materials in sensing mechanical forces and electrical signals in various cellular systems as well as in instructing cellular behaviors. Future research in this area may shift the paradigm in fundamental biophysical research and biomedical applications through (1) the design and synthesis of new semiconductor-based materials and devices that interact specifically with targeted cells, (2) the clarification of many developmental, physiological, and anatomical aspects of cellular communications, (3) an understanding of how signaling between cells regulates synaptic development (e.g., information like this would offer new insight into how the nervous system works and provide new targets for the treatment of neurological diseases), (4) and the creation of new cellular materials that have the potential to open up completely new areas of application, such as in hybrid information processing systems.

Tank Circuit for Ultrafast Single-Particle Detection in Micropores.

We present an ultrafast single submicron particle detection method based on a half-bowtie coplanar waveguide. The method is capable of resolving the translocation of these particles at a bandwidth greater than 30 MHz. We compare experimentally the simultaneous use of our radio-frequency technique with conventional dc based resistive pulse recordings and find that our method has a throughput that is enhanced by 2 orders of magnitude. The technique incorporates a microfluidic circuit and has the potential to be employed for screening microparticles and biological cells at frequencies in excess of 1 GHz.

High-throughput platform for real-time monitoring of biological processes by multicolor single-molecule fluorescence.

Zero-mode waveguides provide a powerful technology for studying single-molecule real-time dynamics of biological systems at physiological ligand concentrations. We customized a commercial zero-mode waveguide-based DNA sequencer for use as a versatile instrument for single-molecule fluorescence detection and showed that the system provides long fluorophore lifetimes with good signal to noise and low spectral cross-talk. We then used a ribosomal translation assay to show real-time fluidic delivery during data acquisition, showing it is possible to follow the conformation and composition of thousands of single biomolecules simultaneously through four spectral channels. This instrument allows high-throughput multiplexed dynamics of single-molecule biological processes over long timescales. The instrumentation presented here has broad applications to single-molecule studies of biological systems and is easily accessible to the biophysical community.

A Wearable Magneto-Inertial System for Gait Analysis (H-Gait): Validation on Normal Weight and Overweight/Obese Young Healthy Adults.

Background: Wearable magneto-inertial sensors are being increasingly used to obtain human motion measurements out of the lab, although their performance in applications requiring high accuracy, such as gait analysis, are still a subject of debate. The aim of this work was to validate a gait analysis system (H-Gait) based on magneto-inertial sensors, both in normal weight (NW) and overweight/obese (OW) subjects. The validation is performed against a reference multichannel recording system (STEP32), providing direct measurements of gait timings (through foot-switches) and joint angles in the sagittal plane (through electrogoniometers). Methods: Twenty-two young male subjects were recruited for the study (12 NW, 10 OW). After positioning body-fixed sensors of both systems, each subject was asked to walk, at a self-selected speed, over a 14-m straight path for 12 trials. Gait signals were recorded, at the same time, with the two systems. Spatio-temporal parameters, ankle, knee, and hip joint kinematics were extracted analyzing an average of 89 ± 13 gait cycles from each lower limb. Intraclass correlation coefficient and Bland-Altmann plots were used to compare H-Gait and STEP32 measurements. Changes in gait parameters and joint kinematics of OW with respect NW were also evaluated. Results: The two systems were highly consistent for cadence, while a lower agreement was found for the other spatio-temporal parameters. Ankle and knee joint kinematics is overall comparable. Joint ROMs values were slightly lower for H-Gait with respect to STEP32 for the ankle (by 1.9° for NW, and 1.6° for OW) and for the knee (by 4.1° for NW, and 1.8° for OW). More evident differences were found for hip joint, with ROMs values higher for H-Gait (by 6.8° for NW, and 9.5° for OW). NW and OW showed significant differences considering STEP32 (p = 0.0004), but not H-Gait (p = 0.06). In particular, overweight/obese subjects showed a higher cadence (55.0 vs. 52.3 strides/min) and a lower hip ROM (23.0° vs. 27.3°) than normal weight subjects. Conclusions: The two systems can be considered interchangeable for what concerns joint kinematics, except for the hip, where discrepancies were evidenced. Differences between normal and overweight/obese subjects were statistically significant using STEP32. The same tendency was observed using H-Gait.

Plant pneumatics: stem air flow is related to embolism - new perspectives on methods in plant hydraulics.

Wood contains a large amount of air, even in functional xylem. Air embolisms in the xylem affect water transport and can determine plant growth and survival. Embolisms are usually estimated with laborious hydraulic methods, which can be prone to several artefacts. Here, we describe a new method for estimating embolisms that is based on air flow measurements of entire branches. To calculate the amount of air flowing out of the branch, a vacuum was applied to the cut bases of branches under different water potentials. We first investigated the source of air by determining whether it came from inside or outside the branch. Second, we compared embolism curves according to air flow or hydraulic measurements in 15 vessel- and tracheid-bearing species to test the hypothesis that the air flow is related to embolism. Air flow came almost exclusively from air inside the branch during the 2.5-min measurements and was strongly related to embolism. We propose a new embolism measurement method that is simple, effective, rapid and inexpensive, and that allows several measurements on the same branch, thus opening up new possibilities for studying plant hydraulics.

Characterization of a novel bioreactor system for 3D cellular mechanobiology studies.

In vitro engineering systems can be powerful tools for studying tissue development in response to biophysical stimuli as well as for evaluating the functionality of engineered tissue grafts. It has been challenging, however, to develop systems that adequately integrate the application of biomimetic mechanical strain to engineered tissue with the ability to assess functional outcomes in real time. The aim of this study was to design a bioreactor system capable of real-time conditioning (dynamic, uniaxial strain, and electrical stimulation) of centimeter-long 3D tissue engineered constructs simultaneously with the capacity to monitor local strains. The system addresses key limitations of uniform sample loading and real-time imaging capabilities. Our system features an electrospun fibrin scaffold, which exhibits physiologically relevant stiffness and uniaxial alignment that facilitates cell adhesion, alignment, and proliferation. We have demonstrated the capacity for directly incorporating human adipose-derived stromal/stem cells into the fibers during the electrospinning process and subsequent culture of the cell-seeded constructs in the bioreactor. The bioreactor facilitates accurate pre-straining of the 3D constructs as well as the application of dynamic and static uniaxial strains while monitoring bulk construct tensions. The incorporation of fluorescent nanoparticles throughout the scaffolds enables in situ monitoring of local strain fields using fluorescent digital image correlation techniques, since the bioreactor is imaging compatible, and allows the assessment of local sample stiffness and stresses when coupled with force sensor measurements. In addition, the system is capable of measuring the electromechanical coupling of skeletal muscle explants by applying an electrical stimulus and simultaneously measuring the force of contraction. The packaging of these technologies, biomaterials, and analytical methods into a single bioreactor system has produced a powerful tool that will enable improved engineering of functional 3D ligaments, tendons, and skeletal muscles. Biotechnol. Bioeng. 2016;113: 1825-1837. © 2016 Wiley Periodicals, Inc.

Cell motility regulation on a stepped micro pillar array device (SMPAD) with a discrete stiffness gradient.

Our tissues consist of individual cells that respond to the elasticity of their environment, which varies between and within tissues. To better understand mechanically driven cell migration, it is necessary to manipulate the stiffness gradient across a substrate. Here, we have demonstrated a new variant of the microfabricated polymeric pillar array platform that can decouple the stiffness gradient from the ECM protein area. This goal is achieved via a "stepped" micro pillar array device (SMPAD) in which the contact area with the cell was kept constant while the diameter of the pillar bodies was altered to attain the proper mechanical stiffness. Using double-step SU-8 mold fabrication, the diameter of the top of every pillar was kept uniform, whereas that of the bottom was changed, to achieve the desired substrate rigidity. Fibronectin was immobilized on the pillar tops, providing a focal adhesion site for cells. C2C12, HeLa and NIH3T3 cells were cultured on the SMPAD, and the motion of the cells was observed by time-lapse microscopy. Using this simple platform, which produces a purely physical stimulus, we observed that various types of cell behavior are affected by the mechanical stimulus of the environment. We also demonstrated directed cell migration guided by a discrete rigidity gradient by varying stiffness. Interestingly, cell velocity was highest at the highest stiffness. Our approach enables the regulation of the mechanical properties of the polymeric pillar array device and eliminates the effects of the size of the contact area. This technique is a unique tool for studying cellular motion and behavior relative to various stiffness gradients in the environment.

The investigation of the skin biophysical measurements focusing on daily activities, skin care habits, and gender differences.

BACKGROUND: Skin, as a protective barrier to exogenous substances, can be modulated by various internal and external factors that can affect its functional state. In order to prevent the early symptoms and signs of diseases of the skin, frequent skin health assessment should be performed. The aims of the study were to evaluate four skin properties of transepidermal water loss (TEWL), hydration, elasticity, and pigmentation using a non-invasive skin assessment tool, DermaLab Combo(®) , and also to determine possible factors that may influence skin condition. METHODS: DermaLab(®) Combo was used to measure TEWL, hydration, pigmentation, and elasticity on the forearm of volunteers by using different probes. In this study, four parameters were observed to reflect the health of the skin in 100 volunteers. RESULTS: There were significant differences (P < 0.05) between TEWL, hydration, pigmentation, and elasticity in different genders on the same anatomical site of the forearm. Female subjects have a higher average value of TEWL, hydration, and elasticity compared to male subjects. The differences may be due to an individual's daily activity and use of skin care products as well as environmental factors. The use of moisturiser and drinking lots of water may keep the skin hydrated and delay the process of skin ageing as shown by the better hydration and elasticity observed (P < 0.05). CONCLUSION: In this study, it can be concluded that DermaLab(®) Combo is a reliable skin analysis instrument that offers high precision, accuracy, and reproducibility for all the measuring parameters. It has also been found that daily activities and habits influence skin condition as reflected by the measurement of these biophysical skin parameters.

A soft, stretchable and conductive biointerface for cell mechanobiology.

In mechanobiology the study of cell response to mechanical stimuli is fundamental, and the involved processes (i.e., mechanotransduction) need to be investigated by interfacing (mechanically and electrically) with the cells in dynamic and non-invasive natural-like conditions. In this work, we present a novel soft, stretchable and conductive biointerface that allows both cell mechanical stimulation and dynamic impedance recording. The biointerface stretchability and conductivity, jointly to the biocompatibility and transparency needed to perform cell culture studies, were obtained by exploiting the formation of wrinkles on the surface of a 90 nm thick conductive layer of poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) on a pre-stretched 130 µm thick poly(dimethylsiloxane) (PDMS) substrate. Cell adhesion and proliferation of SH-SY5Y human neuroblastoma cells were evaluated, and cell differentiation on the corrugated surface was assessed. We demonstrate how the biointerface remains conductive when applying uniaxial strain up to 10%, and when cell culturing is performed. Finally, a reduction of about 30% of the relative impedance variation signal was measured, with respect to the control, as a result of the mechanical stimulation of cells.

Model building with wind and water: Friedrich Ahlborn's photo-optical flow analysis.

Around 1900, several experimenters investigated turbulences in wind tunnels or water basins by creating visualizations. One of them, the German zoologist Friedrich Ahlborn (1858-1937), was familiar with the works by his contemporaries but he struck a new path. He combined three different kinds of photographs taken at the same time and showed the same situation in his water trough-but each in a different way. With this first basic operation, Ahlborn heuristically opened up a previously non-existent space for experimentation, analysis, and recombination. He generated an astonishing diversity of information by adopting the tactics of 'inversions' in which he interpreted one part of the experimental setup, or its results, in different ways. Between the variants of the 'autographs' which he developed, he defined areas of intersection to be able to translate results from individual records into each other. To this end, Ahlborn created other sets of visual artifacts such as drawn diagrams, three-dimensional wire frame constructions, and clay reliefs. His working method can be described as a cascading array of successive modeling steps, as elaborated by Eric Winsberg (1999), or of inscriptions in Bruno Latour's words (Latour, 1986). By examining Ahlborn's procedures closely we propose conceptualizations for the experimenter's various operations.

Measurement of cell adhesion force by vertical forcible detachment using an arrowhead nanoneedle and atomic force microscopy.

The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered-integrin bindings link cells mechanically to the ECM. In order to quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin-substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.

Mass and density measurements of live and dead Gram-negative and Gram-positive bacterial populations.

Monitoring cell growth and measuring physical features of food-borne pathogenic bacteria are important for better understanding the conditions under which these organisms survive and proliferate. To address this challenge, buoyant masses of live and dead Escherichia coli O157:H7 and Listeria innocua were measured using Archimedes, a commercially available suspended microchannel resonator (SMR). Cell growth was monitored with Archimedes by observing increased cell concentration and buoyant mass values of live growing bacteria. These growth data were compared to optical density measurements obtained with a Bioscreen system. We observed buoyant mass measurements with Archimedes at cell concentrations between 10(5) and 10(8) cells/ml, while growth was not observed with optical density measurements until the concentration was 10(7) cells/ml. Buoyant mass measurements of live and dead cells with and without exposure to hydrogen peroxide stress were also compared; live cells generally had a larger buoyant mass than dead cells. Additionally, buoyant mass measurements were used to determine cell density and total mass for both live and dead cells. Dead E. coli cells were found to have a larger density and smaller total mass than live E. coli cells. In contrast, density was the same for both live and dead L. innocua cells, while the total mass was greater for live than for dead cells. These results contribute to the ongoing challenge to further develop existing technologies used to observe cell populations at low concentrations and to measure unique physical features of cells that may be useful for developing future diagnostics.

Theoretical and experimental analysis of pulse delay in bacteriorhodopsin films by a saturable absorber theory.

Time-delay of transmitted pulses with respect to the incident pulse in bacteriorhodopsin films has been studied without the use of a pump beam. Based on a modified saturable absorber model, analytical expressions of the transmitted pulse have been obtained. As a result, time delay, distortion and fractional delay have been analyzed for sinusoidal pulses with a low background. A good agreement between theory and experiences has been observed.

A microscale anisotropic biaxial cell stretching device for applications in mechanobiology.

A multi-layered polydimethylsiloxane microfluidic device with an integrated suspended membrane has been fabricated that allows dynamic and multi-axial mechanical deformation and simultaneous live-cell microscopy imaging. The transparent membrane's strain field can be controlled independently along two orthogonal directions. Human foreskin fibroblasts were immobilized on the membrane's surface and stretched along two orthogonal directions sequentially while performing live-cell imaging. Cyclic deformation of the cells induced a reversible reorientation perpendicular to the direction of the applied strain. Cells remained viable in the microdevice for several days. As opposed to existing microfluidic or macroscale stretching devices, this device can impose changing, anisotropic and time-varying strain fields in order to more closely mimic the complexities of strains occurring in vivo.