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Alcaraz, J., Otero, J., Jorba, I., Navajas, D., (2018). Bidirectional mechanobiology between cells and their local extracellular matrix probed by atomic force microscopy Seminars in Cell and Developmental Biology 73, 71-81

There is growing recognition that the mechanical interactions between cells and their local extracellular matrix (ECM) are central regulators of tissue development, homeostasis, repair and disease progression. The unique ability of atomic force microscopy (AFM) to probe quantitatively mechanical properties and forces at the nanometer or micrometer scales in all kinds of biological samples has been instrumental in the recent advances in cell and tissue mechanics. In this review we illustrate how AFM has provided important insights on our current understanding of the mechanobiology of cells, ECM and cell-ECM bidirectional interactions, particularly in the context of soft acinar tissues like the mammary gland or pulmonary tissue. AFM measurements have revealed that intrinsic cell micromechanics is cell-type specific, and have underscored the prominent role of

Keywords: Atomic force microscopy, Beta1 integrin, Elastic modulus, Extracellular matrix, Morphogenesis, Tissue decellularization

Elosegui-Artola, A., Andreu, I., Beedle, A. E. M., Lezamiz, A., Uroz, M., Kosmalska, A. J., Oria, R., Kechagia, J. Z., Rico-Lastres, P., Le Roux, A. L., Shanahan, C. M., Trepat, X., Navajas, D., Garcia-Manyes, S., Roca-Cusachs, P., (2017). Force triggers YAP nuclear entry by regulating transport across nuclear pores Cell 171, (6), 1397-1410

YAP is a mechanosensitive transcriptional activator with a critical role in cancer, regeneration, and organ size control. Here, we show that force applied to the nucleus directly drives YAP nuclear translocation by decreasing the mechanical restriction of nuclear pores to molecular transport. Exposure to a stiff environment leads cells to establish a mechanical connection between the nucleus and the cytoskeleton, allowing forces exerted through focal adhesions to reach the nucleus. Force transmission then leads to nuclear flattening, which stretches nuclear pores, reduces their mechanical resistance to molecular transport, and increases YAP nuclear import. The restriction to transport is further regulated by the mechanical stability of the transported protein, which determines both active nuclear transport of YAP and passive transport of small proteins. Our results unveil a mechanosensing mechanism mediated directly by nuclear pores, demonstrated for YAP but with potential general applicability in transcriptional regulation. Force-dependent changes in nuclear pores control protein access to the nucleus.

Keywords: Atomic force microscopy, Hippo pathway, Mechanosensing, Mechanotransduction, Molecular mechanical stability, Nuclear mechanics, Nuclear pores, Nuclear transport, Rigidity sensing, Transcription regulation

Wang, Y., van Merwyk, L., Tönsing, K., Walhorn, V., Anselmetti, D., Fernàndez-Busquets, X., (2017). Biophysical characterization of the association of histones with single-stranded DNA Biochimica et Biophysica Acta (BBA) - General Subjects 1861, (11), 2739-2749

Background: Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. Methods: Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. Results: Histones have a high affinity for ssDNA in 0.15 M NaCl ionic strength, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Although histone-ssDNA complexes show a high tendency to aggregate, nucleosome-like structures are formed at physiological salt concentrations. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (−) strand. Conclusions: At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. General significance: In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin.

Keywords: Electrophoresis, Force spectroscopy, Histones, Magnetic tweezers, Nucleosome, Single-stranded DNA

Diez-Escudero, A., Espanol, M., Montufar, E. B., Di Pompo, G., Ciapetti, G., Baldini, N., Ginebra, M. P., (2017). Focus ion beam/scanning electron microscopy characterization of osteoclastic resorption of calcium phosphate substrates Tissue Engineering Part C: Methods 23, (2), 118-124

This article presents the application of dual focused ion beam/scanning electron microscopy (FIB-SEM) imaging for preclinical testing of calcium phosphates with osteoclast precursor cells and how this high-resolution imaging technique is able to reveal microstructural changes at a level of detail previously not possible. Calcium phosphate substrates, having similar compositions but different microstructures, were produced using low-and high-Temperature processes (biomimetic calcium-deficient hydroxyapatite [CDHA] and stoichiometric sintered hydroxyapatite, respectively). Human osteoclast precursor cells were cultured for 21 days before evaluating their resorptive potential on varying microstructural features. Alternative to classical morphological evaluation of osteoclasts (OC), FIB-SEM was used to observe the subjacent microstructure by transversally sectioning cells and observing both the cells and the substrates. Resorption pits, indicating OC activity, were visible on the smoother surface of high-Temperature sintered hydroxyapatite. FIB-SEM analysis revealed signs of acidic degradation on the grain surface under the cells, as well as intergranular dissolution. No resorption pits were evident on the surface of the rough CDHA substrates. However, whereas no degradation was detected by FIB sections in the material underlying some of the cells, early stages of OC-mediated acidic degradation were observed under cells with more spread morphology. Collectively, these results highlight the potential of FIB to evaluate the resorptive activity of OC, even in rough, irregular, or coarse surfaces where degradation pits are otherwise difficult to visualize.

Keywords: Bone Regeneration, Calcium Phosphate, Focus Ion Beam, Osteoclast, Resorption, Scanning Electron Microscopy

Jorba, I., Menal, M. J., Torres, M., Gozal, D., Piñol-Ripoll, G., Colell, A., Montserrat, J. M., Navajas, D., Farré, R., Almendros, I., (2017). Ageing and chronic intermittent hypoxia mimicking sleep apnea do not modify local brain tissue stiffness in healthy mice Journal of the Mechanical Behavior of Biomedical Materials 71, 106-113

Recent evidence suggests that obstructive sleep apnea (OSA) may increase the risk of Alzheimer´s disease (AD), with the latter promoting alterations in brain tissue stiffness, a feature of ageing. Here, we assessed the effects of age and intermittent hypoxia (IH) on brain tissue stiffness in a mouse model of OSA. Two-month-old and 18-month-old mice (N=10 each) were subjected to IH (20% O2 40 s – 6% O2 20 s) for 8 weeks (6 h/day). Corresponding control groups for each age were kept under normoxic conditions in room air (RA). After sacrifice, the brain was excised and 200-micron coronal slices were cut with a vibratome. Local stiffness of the cortex and hippocampus were assessed in brain slices placed in an Atomic Force Microscope. For both brain regions, the Young's modulus (E) in each animal was computed as the average values from 9 force-indentation curves. Cortex E mean (±SE) values were 442±122 Pa (RA) and 455±120 (IH) for young mice and 433±44 (RA) and 405±101 (IH) for old mice. Hippocampal E values were 376±62 (RA) and 474±94 (IH) for young mice and 486±93 (RA) and 521±210 (IH) for old mice. For both cortex and hippocampus, 2-way ANOVA indicated no statistically significant effects of age or challenge (IH vs. RA) on E values. Thus, neither chronic IH mimicking OSA nor ageing up to late middle age appear to modify local brain tissue stiffness in otherwise healthy mice.

Keywords: Atomic Force Microscopy, Brain mechanics, Cortex stiffness, Hippocampus stiffness, Obstructive sleep apnea, Young's modulus

Giménez, A., Uriarte, J. J., Vieyra, J., Navajas, D., Alcaraz, J., (2017). Elastic properties of hydrogels and decellularized tissue sections used in mechanobiology studies probed by atomic force microscopy Microscopy Research and Technique 80, (1), 85-96

The increasing recognition that tissue elasticity is an important regulator of cell behavior in normal and pathologic conditions such as fibrosis and cancer has driven the development of cell culture substrata with tunable elasticity. Such development has urged the need to quantify the elastic properties of these cell culture substrata particularly at the nanometer scale, since this is the relevant length scale involved in cell-extracellular matrix (ECM) mechanical interactions. To address this need, we have exploited the versatility of atomic force microscopy to quantify the elastic properties of a variety of cell culture substrata used in mechanobiology studies, including floating collagen gels, ECM-coated polyacrylamide gels, and decellularized tissue sections. In this review we summarize major findings in this field from our group within the context of the state-of-the-art in the field, and provide a critical discussion on the applicability and complementarity of currently available cell culture assays with tunable elasticity. In addition, we briefly describe how the limitations of these assays provide opportunities for future research, which is expected to continue expanding our understanding of the mechanobiological aspects that support both normal and diseased conditions.

Keywords: 3D culture, Atomic force microscopy, Elastic modulus, Extracellular matrix, Polyacrylamide

Climent, A. M., Hernandez-Romero, I., Guillem, M. S., Montserrat, N., Fernandez, M. E., Atienza, F., Fernandez-Aviles, F., (2017). High resolution microscopic optical mapping of anatomical and functional reentries in human cardiac cell cultures IEEE Conference Publications Computing in Cardiology Conference (CinC), 2016 , IEEE (Vancouver, Canada) 43, 233-236

Anatomical and/or functional reentries have been proposed as one of the main mechanism of perpetuation of cardiac fibrillation processes. However, technical limitations have difficult the characterization of those reentries and are hampering the development of effective anti-arrhythmic treatments. The goal of this study is to present a novel technology to map with high resolution the center of fibrillation drivers in order to characterize the mechanisms of reentry. Cell cultures of human cardiac-like cells differentiated from pluripotent stem cells were analyzed with a novel microscopic optical mapping system. The pharmacological response to verapamil administration of each type of reentry was analyzed. In all analyzed cell cultures, a reentry was identified as the mechanism of maintenance of the arrhythmia. Interestingly, the administration of verapamil produced opposite effects on activation rate depending on the mechanisms of reentry (i.e. anatomical or functional). Microscopic optical mapping of reentries allows the identification of perpetuation mechanisms which has been demonstrated to be linked with different pharmacological response.

Keywords: Stem cells, Rotors, Microscopy, Optical filters, Calcium, Optical microscopy, Biomedical optical imaging

Bosch, M., Castro, J., Sur, M., Hayashi, Y., (2017). Photomarking relocalization technique for correlated two-photon and electron microcopy imaging of single stimulated synapses Synapse Development - Methods and Protocols (Methods in Molecular Biology) (ed. Poulopoulos , A.), Humana Press (New York, USA) 1538, 185-214

Synapses learn and remember by persistent modifications of their internal structures and composition but, due to their small size, it is difficult to observe these changes at the ultrastructural level in real time. Two-photon fluorescence microscopy (2PM) allows time-course live imaging of individual synapses but lacks ultrastructural resolution. Electron microscopy (EM) allows the ultrastructural imaging of subcellular components but cannot detect fluorescence and lacks temporal resolution. Here, we describe a combination of procedures designed to achieve the correlated imaging of the same individual synapse under both 2PM and EM. This technique permits the selective stimulation and live imaging of a single dendritic spine and the subsequent localization of the same spine in EM ultrathin serial sections. Landmarks created through a photomarking method based on the 2-photon-induced precipitation of an electrodense compound are used to unequivocally localize the stimulated synapse. This technique was developed to image, for the first time, the ultrastructure of the postsynaptic density in which long-term potentiation was selectively induced just seconds or minutes before, but it can be applied for the study of any biological process that requires the precise relocalization of micron-wide structures for their correlated imaging with 2PM and EM.

Keywords: Correlated imaging, DAB, Dendritic spine, Photobranding, Photoetching, Photomarking, Postsynaptic density, Serial-section transmission electron microscopy, Synapse, Time-lapse live two-photon fluorescence microscopy

Gumí-Audenis, Berta, Costa, Luca, Carlá, Francesco, Comin, Fabio, Sanz, Fausto, Giannotti, Marina, (2016). Structure and nanomechanics of model membranes by atomic force microscopy and spectroscopy: Insights into the role of cholesterol and sphingolipids Membranes 6, (4), 58

Biological membranes mediate several biological processes that are directly associated with their physical properties but sometimes difficult to evaluate. Supported lipid bilayers (SLBs) are model systems widely used to characterize the structure of biological membranes. Cholesterol (Chol) plays an essential role in the modulation of membrane physical properties. It directly influences the order and mechanical stability of the lipid bilayers, and it is known to laterally segregate in rafts in the outer leaflet of the membrane together with sphingolipids (SLs). Atomic force microscope (AFM) is a powerful tool as it is capable to sense and apply forces with high accuracy, with distance and force resolution at the nanoscale, and in a controlled environment. AFM-based force spectroscopy (AFM-FS) has become a crucial technique to study the nanomechanical stability of SLBs by controlling the liquid media and the temperature variations. In this contribution, we review recent AFM and AFM-FS studies on the effect of Chol on the morphology and mechanical properties of model SLBs, including complex bilayers containing SLs. We also introduce a promising combination of AFM and X-ray (XR) techniques that allows for in situ characterization of dynamic processes, providing structural, morphological, and nanomechanical information

Keywords: Atomic force microscopy, Force spectroscopy, Lipid membranes, Supported lipid bilayers, Nanomechanics, Cholesterol, Sphingolipids, Membrane structure, XR-AFM combination

Aragonès, Albert C., Haworth, Naomi L., Darwish, Nadim, Ciampi, Simone, Bloomfield, Nathaniel J., Wallace, Gordon G., Diez-Perez, Ismael, Coote, Michelle L., (2016). Electrostatic catalysis of a Diels–Alder reaction Nature 531, (7592), 88-91

It is often thought that the ability to control reaction rates with an applied electrical potential gradient is unique to redox systems. However, recent theoretical studies suggest that oriented electric fields could affect the outcomes of a range of chemical reactions, regardless of whether a redox system is involved1, 2, 3, 4. This possibility arises because many formally covalent species can be stabilized via minor charge-separated resonance contributors. When an applied electric field is aligned in such a way as to electrostatically stabilize one of these minor forms, the degree of resonance increases, resulting in the overall stabilization of the molecule or transition state. This means that it should be possible to manipulate the kinetics and thermodynamics of non-redox processes using an external electric field, as long as the orientation of the approaching reactants with respect to the field stimulus can be controlled. Here, we provide experimental evidence that the formation of carbon–carbon bonds is accelerated by an electric field. We have designed a surface model system to probe the Diels–Alder reaction, and coupled it with a scanning tunnelling microscopy break-junction approach5, 6, 7. This technique, performed at the single-molecule level, is perfectly suited to deliver an electric-field stimulus across approaching reactants. We find a fivefold increase in the frequency of formation of single-molecule junctions, resulting from the reaction that occurs when the electric field is present and aligned so as to favour electron flow from the dienophile to the diene. Our results are qualitatively consistent with those predicted by quantum-chemical calculations in a theoretical model of this system, and herald a new approach to chemical catalysis.

Keywords: Electrocatalysis, Scanning probe microscopy

Tekeli, I., Aujard, I., Trepat, X., Jullien, L., Raya, A., Zalvidea, D., (2016). Long-term in vivo single-cell lineage tracing of deep structures using three-photon activation Light: Science and Applications 5, (6), e16084

Genetic labeling techniques allow for noninvasive lineage tracing of cells in vivo. Two-photon inducible activators provide spatial resolution for superficial cells, but labeling cells located deep within tissues is precluded by scattering of the far-red illumination required for two-photon photolysis. Three-photon illumination has been shown to overcome the limitations of two-photon microscopy for in vivo imaging of deep structures, but whether it can be used for photoactivation remains to be tested. Here we show, both theoretically and experimentally, that three-photon illumination overcomes scattering problems by combining longer wavelength excitation with high uncaging three-photon cross-section molecules. We prospectively labeled heart muscle cells in zebrafish embryos and found permanent labeling in their progeny in adult animals with negligible tissue damage. This technique allows for a noninvasive genetic manipulation in vivo with spatial, temporal and cell-type specificity, and may have wide applicability in experimental biology.

Keywords: Multi-photon microscopy, Photoactivation, Three-photon microscopy, Zebrafish

Beun, L. H., Albertazzi, L., Van Der Zwaag, D., De Vries, R., Cohen Stuart, M. A., (2016). Unidirectional living growth of self-assembled protein nanofibrils revealed by super-resolution microscopy ACS Nano 10, (5), 4973-4980

Protein-based nanofibrils are emerging as a promising class of materials that provide unique properties for applications such as biomedical and food engineering. Here, we use atomic force microscopy and stochastic optical reconstruction microscopy imaging to elucidate the growth dynamics, exchange kinetics, and polymerization mechanism for fibrils composed of a de novo designed recombinant triblock protein polymer. This macromolecule features a silk-inspired self-assembling central block composed of GAGAGAGH repeats, which are known to fold into a β roll with turns at each histidine and, once folded, to stack, forming a long, ribbon-like structure. We find several properties that allow the growth of patterned protein nanofibrils: the self-assembly takes place on only one side of the growing fibrils by the essentially irreversible addition of protein polymer subunits, and these fibril ends remain reactive indefinitely in the absence of monomer ("living ends"). Exploiting these characteristics, we can grow stable diblock protein nanofibrils by the sequential addition of differently labeled proteins. We establish control over the block length ratio by simply varying monomer feed conditions. Our results demonstrate the use of engineered protein polymers in creating precisely patterned protein nanofibrils and open perspectives for the hierarchical self-assembly of functional biomaterials.

Keywords: Nanofibrils, Protein polymers, Self-assembly, STORM microscopy

Przybyla, L., Lakins, J. N., Sunyer, R., Trepat, X., Weaver, V. M., (2016). Monitoring developmental force distributions in reconstituted embryonic epithelia Methods 94, 101-113

The way cells are organized within a tissue dictates how they sense and respond to extracellular signals, as cues are received and interpreted based on expression and organization of receptors, downstream signaling proteins, and transcription factors. Part of this microenvironmental context is the result of forces acting on the cell, including forces from other cells or from the cellular substrate or basement membrane. However, measuring forces exerted on and by cells is difficult, particularly in an in vivo context, and interpreting how forces affect downstream cellular processes poses an even greater challenge. Here, we present a simple method for monitoring and analyzing forces generated from cell collectives. We demonstrate the ability to generate traction force data from human embryonic stem cells grown in large organized epithelial sheets to determine the magnitude and organization of cell-ECM and cell-cell forces within a self-renewing colony. We show that this method can be used to measure forces in a dynamic hESC system and demonstrate the ability to map intracolony protein localization to force organization.

Keywords: Epiblast, Human embryonic stem cells, Mechanotransduction, Monolayer stress microscopy, Self-organization, Traction force

Botaya, L., Coromina, X., Samitier, J., Puig-Vidal, M., Otero, J., (2016). Visualized multiprobe electrical impedance measurements with STM tips using shear force feedback control Sensors 16, (6), 757

Here we devise a multiprobe electrical measurement system based on quartz tuning forks (QTFs) and metallic tips capable of having full 3D control over the position of the probes. The system is based on the use of bent tungsten tips that are placed in mechanical contact (glue-free solution) with a QTF sensor. Shear forces acting in the probe are measured to control the tip-sample distance in the Z direction. Moreover, the tilting of the tip allows the visualization of the experiment under the optical microscope, allowing the coordination of the probes in X and Y directions. Meanwhile, the metallic tips are connected to a current-voltage amplifier circuit to measure the currents and thus the impedance of the studied samples. We discuss here the different aspects that must be addressed when conducting these multiprobe experiments, such as the amplitude of oscillation, shear force distance control, and wire tilting. Different results obtained in the measurement of calibration samples and microparticles are presented. They demonstrate the feasibility of the system to measure the impedance of the samples with a full 3D control on the position of the nanotips.

Keywords: Impedance measurement, Multiprobe SPM, Quartz tuning forks, Scanning probe microscopy, Scanning tunneling microscope (STM) tip

Paéz Aviles, C. , Juanola-Feliu, E., Tahirbegi, I.B. , Mir, M., Gonzalez-Piñero, M., Samitier, J., (2015). Innovation and technology transfer of medical devices fosterd by cross disciplinary communities of practitioners International Journal of Innovation Management 19, (6), 1540012

Commercialisation of emerging technological innovations such as medical devices can be a time-consuming and lengthy process resulting in a market entrance failure. To tackle this general problem, major challenges are being analysed, principally focusing on the role of Communities of Practitioners (CoPs) in the process of effective transfer of high-value emerging technologies from academia to market. Taking a case study approach, this document describes the role of a cross-disciplinary CoP in the technology transfer process within a convergence scenario. The case presented is a sensor array for ischemia detection developed by different practitioners from diverse organisations: university, research institution, hospital, and a scientific park. The analysis also involves the innovation ecosystem where all stakeholders are taken into account. This study contributes to a better understanding of the managerial implications of CoP fostering technology transfer and innovation, principally focused on the current need for new biomedical technologies and tools.

Keywords: CoP, Medical devices, Innovation, Technology transfer, Ischemia

Brask, J. B., Singla-Buxarrais, G., Uroz, M., Vincent, R., Trepat, X., (2015). Compressed sensing traction force microscopy Acta Biomaterialia 26, 286-294

Adherent cells exert traction forces on their substrate, and these forces play important roles in biological functions such as mechanosensing, cell differentiation and cancer invasion. The method of choice to assess these active forces is traction force microscopy (TFM). Despite recent advances, TFM remains highly sensitive to measurement noise and exhibits limited spatial resolution. To improve the resolution and noise robustness of TFM, here we adapt techniques from compressed sensing (CS) to the reconstruction of the traction field from the substrate displacement field. CS enables the recovery of sparse signals at higher resolution from lower resolution data. Focal adhesions (FAs) of adherent cells are spatially sparse implying that traction fields are also sparse. Here we show, by simulation and by experiment, that the CS approach enables circumventing the Nyquist-Shannon sampling theorem to faithfully reconstruct the traction field at a higher resolution than that of the displacement field. This allows reaching state-of-the-art resolution using only a medium magnification objective. We also find that CS improves reconstruction quality in the presence of noise. Statement of Significance A great scientific advance of the past decade is the recognition that physical forces determine an increasing list of biological processes. Traction force microscopy which measures the forces that cells exert on their surroundings has seen significant recent improvements, however the technique remains sensitive to measurement noise and severely limited in spatial resolution. We exploit the fact that the force fields are sparse to boost the spatial resolution and noise robustness by applying ideas from compressed sensing. The novel method allows high resolution on a larger field of view. This may in turn allow better understanding of the cell forces at the multicellular level, which are known to be important in wound healing and cancer invasion.

Keywords: Compressed sensing, High resolution, Traction force microscopy

Reginensi, Diego, Carulla, Patricia, Nocentini, Sara, Seira, Oscar, Serra-Picamal, Xavier, Torres-Espín, Abel, Matamoros-Angles, Andreu, Gavín, Rosalina, Moreno-Flores, María Teresa, Wandosell, Francisco, Samitier, Josep, Trepat, Xavier, Navarro, Xavier, del Río, José Antonio, (2015). Increased migration of olfactory ensheathing cells secreting the Nogo receptor ectodomain over inhibitory substrates and lesioned spinal cord Cellular and Molecular Life Sciences 72, (14), 2719-2737

Olfactory ensheathing cell (OEC) transplantation emerged some years ago as a promising therapeutic strategy to repair injured spinal cord. However, inhibitory molecules are present for long periods of time in lesioned spinal cord, inhibiting both OEC migration and axonal regrowth. Two families of these molecules, chondroitin sulphate proteoglycans (CSPG) and myelin-derived inhibitors (MAIs), are able to trigger inhibitory responses in lesioned axons. Mounting evidence suggests that OEC migration is inhibited by myelin. Here we demonstrate that OEC migration is largely inhibited by CSPGs and that inhibition can be overcome by the bacterial enzyme Chondroitinase ABC. In parallel, we have generated a stable OEC cell line overexpressing the Nogo receptor (NgR) ectodomain to reduce MAI-associated inhibition in vitro and in vivo. Results indicate that engineered cells migrate longer distances than unmodified OECs over myelin or oligodendrocyte-myelin glycoprotein (OMgp)-coated substrates. In addition, they also show improved migration in lesioned spinal cord. Our results provide new insights toward the improvement of the mechanisms of action and optimization of OEC-based cell therapy for spinal cord lesion.

Keywords: Olfactory ensheathing cells, Traction force microscopy, Chondroitin sulphate proteoglycans, Cell migration, Nogo receptor ectodomain

Abadías, Clara, Serés, Carme, Torrent-Burgués, J., (2015). AFM in peak force mode applied to worn siloxane-hydrogel contact lenses Colloids and Surfaces B: Biointerfaces 128, 61-66

The objective of this work is to apply Atomic Force Microscopy in Peak Force mode to obtain topographic characteristics (mean roughness, root-mean-square roughness, skewness and kurtosis) and mechanical characteristics (adhesion, elastic modulus) of Siloxane-Hydrogel Soft Contact Lenses (CLs) of two different materials, Lotrafilcon B of Air Optix (AO) and Asmofilcon A of PremiO (P), after use (worn CLs). Thus, the results obtained with both materials will be compared, as well as the changes produced by the wear at a nanoscopic level. The results show significant changes in the topographic and mechanical characteristics of the CLs, at a nanoscopic level, due to wear. The AO CL show values of the topographic parameters lower than those of the P CL after wear, which correlates with a better comfort qualification given to the former by the wearers. A significant correlation has also been obtained between the adhesion values found after the use of the CLs with tear quality tests, both break-up-time and Schirmer.

Keywords: Adhesion, Atomic force microscopy-peak force mode, Surface topography, Worn siloxane-hydrogel contact lenses, Young modulus

Barreiros dos Santos, M., Azevedo, S., Agusil, J. P., Prieto-Simón, B., Sporer, C., Torrents, E., Juárez, A., Teixeira, V., Samitier, J., (2015). Label-free ITO-based immunosensor for the detection of very low concentrations of pathogenic bacteria Bioelectrochemistry 101, 146-152

Abstract Here we describe the fabrication of a highly sensitive and label-free ITO-based impedimetric immunosensor for the detection of pathogenic bacteria Escherichia coli O157:H7. Anti-E. coli antibodies were immobilized onto ITO electrodes using a simple, robust and direct methodology. First, the covalent attachment of epoxysilane on the ITO surface was demonstrated by Atomic Force Microscopy and cyclic voltammetry. The immobilization of antibody on the epoxysilane layer was quantified by Optical Waveguide Lightmode Spectroscopy, obtaining a mass variation of 12 ng cm− 2 (0.08 pmol cm− 2). Microcontact printing and fluorescence microscopy were used to demonstrate the specific binding of E. coli O157:H7 to the antibody-patterned surface. We achieved a ratio of 1:500 Salmonella typhimurium/E. coli O157:H7, thus confirming the selectivity of the antibodies and efficiency of the functionalization procedure. Finally, the detection capacity of the ITO-based immunosensor was evaluated by Electrochemical Impedance Spectroscopy. A very low limit of detection was obtained (1 CFU mL− 1) over a large linear working range (10–106 CFU mL− 1). The specificity of the impedimetric immunosensor was also examined. Less than 20% of non-specific bacteria (S. typhimurium and E. coli K12) was observed. Our results reveal the applicability of ITO for the development of highly sensitive and selective impedimetric immunosensors.

Keywords: E. coli O157:H7, Electrochemical Impedance Spectroscopy, Immunosensor, Indium tin oxide, Label-free detection

Van Der Hofstadt, M., Hüttener, M., Juárez, A., Gomila, G., (2015). Nanoscale imaging of the growth and division of bacterial cells on planar substrates with the atomic force microscope Ultramicroscopy 154, 29-36

Abstract With the use of the atomic force microscope (AFM), the Nanomicrobiology field has advanced drastically. Due to the complexity of imaging living bacterial processes in their natural growing environments, improvements have come to a standstill. Here we show the in situ nanoscale imaging of the growth and division of single bacterial cells on planar substrates with the atomic force microscope. To achieve this, we minimized the lateral shear forces responsible for the detachment of weakly adsorbed bacteria on planar substrates with the use of the so called dynamic jumping mode with very soft cantilever probes. With this approach, gentle imaging conditions can be maintained for long periods of time, enabling the continuous imaging of the bacterial cell growth and division, even on planar substrates. Present results offer the possibility to observe living processes of untrapped bacteria weakly attached to planar substrates.

Keywords: Atomic Force Microscope (AFM), Living cell imaging, Bacteria division, Gelatine immobilization, Dynamic jumping mode

Gumí-Audenis, B., Carlà, F., Vitorino, M. V., Panzarella, A., Porcar, L., Boilot, M., Guerber, S., Bernard, P., Rodrigues, M. S., Sanz, F., Giannotti, M. I., Costa, L., (2015). Custom AFM for X-ray beamlines: in situ biological investigations under physiological conditions Journal of Synchrotron Radiation 22, 1364-1371

A fast atomic force microscope (AFM) has been developed that can be installed as a sample holder for grazing-incidence X-ray experiments at solid/gas or solid/liquid interfaces. It allows a wide range of possible investigations, including soft and biological samples under physiological conditions (hydrated specimens). The structural information obtained using the X-rays is combined with the data gathered with the AFM (morphology and mechanical properties), providing a unique characterization of the specimen and its dynamics in situ during an experiment. In this work, lipid monolayers and bilayers in air or liquid environment have been investigated by means of AFM, both with imaging and force spectroscopy, and X-ray reflectivity. In addition, this combination allows the radiation damage induced by the beam on the sample to be studied, as has been observed on DOPC and DPPC supported lipid bilayers under physiological conditions.

Keywords: In situ atomic force microscopy, Grazing-incidence scattering and reflectivity, Radiation damage, Model lipid membranes

Serra-Picamal, Xavier, Conte, Vito, Sunyer, Raimon, Muñoz, José J., Trepat, Xavier, (2015). Mapping forces and kinematics during collective cell migration Methods in Cell Biology - Biophysical Methods in Cell Biology (ed. Wilson, L., Tran, P.), Academic Press (Santa Barbara, USA) 125, 309-330

Abstract Fundamental biological processes including morphogenesis and tissue repair require cells to migrate collectively. In these processes, epithelial or endothelial cells move in a cooperative manner coupled by intercellular junctions. Ultimately, the movement of these multicellular systems occurs through the generation of cellular forces, exerted either on the substrate via focal adhesions (cell–substrate forces) or on neighboring cells through cell–cell junctions (cell–cell forces). Quantitative measurements of multicellular forces and kinematics with cellular or subcellular resolution have become possible only in recent years. In this chapter, we describe some of these techniques, which include particle image velocimetry to map cell velocities, traction force microscopy to map forces exerted by cells on the substrate, and monolayer stress microscopy to map forces within and between cells. We also describe experimental protocols to perform these measurements. The combination of these techniques with high-resolution imaging tools and molecular perturbations will lead to a better understanding of the mechanisms underlying collective cell migration in health and disease.

Keywords: Collective cell migration, Monolayer stress microscopy, Traction force microscopy

Cuervo, A., Dans, P. D., Carrascosa, J. L., Orozco, M., Gomila, G., Fumagalli, L., (2014). Direct measurement of the dielectric polarization properties of DNA Proceedings of the National Academy of Sciences of the United States of America 111, (35), E3624-E3630

The electric polarizability of DNA, represented by the dielectric constant, is a key intrinsic property that modulates DNA interaction with effector proteins. Surprisingly, it has so far remained unknown owing to the lack of experimental tools able to access it. Here, we experimentally resolved it by detecting the ultraweak polarization forces of DNA inside single T7 bacteriophages particles using electrostatic force microscopy. In contrast to the common assumption of low-polarizable behavior like proteins (εr ∼ 2-4), we found that the DNA dielectric constant is ∼8, considerably higher than the value of ∼3 found for capsid proteins. State-of-the-art molecular dynamic simulations confirm the experimental findings, which result in sensibly decreased DNA interaction free energy than normally predicted by Poisson-Boltzmann methods. Our findings reveal a property at the basis of DNA structure and functions that is needed for realistic theoretical descriptions, and illustrate the synergetic power of scanning probe microscopy and theoretical computation techniques.

Keywords: Atomic force microscopy, Atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, capsid protein, DNA, double stranded DNA, amino acid composition, article, atomic force microscopy, bacteriophage, bacteriophage T7, dielectric constant, dipole, DNA binding, DNA packaging, DNA structure, electron microscopy, ligand binding, nonhuman, polarization, priority journal, protein analysis, protein DNA interaction, scanning probe microscopy, static electricity, virion, virus capsid, virus particle, atomic force microscopy, atomistic simulations, DNA packaging, DNA-ligand binding, Poisson-Boltzmann equation, Bacteriophage T7, Capsid, Cations, Dielectric Spectroscopy, DNA, DNA, Viral, DNA-Binding Proteins, Electrochemical Techniques, Ligands, Microscopy, Atomic Force, Models, Chemical, Nuclear Proteins

Lagunas, A., Garcia, A., Artés, J. M., Vida, Y., Collado, D., Pérez-Inestrosa, E., Gorostiza, P., Claros, S., Andrades, J. A., Samitier, J., (2014). Large-scale dendrimer-based uneven nanopatterns for the study of local arginine-glycine-aspartic acid (RGD) density effects on cell adhesion Nano Research 7, (3), 399-409

Cell adhesion processes are governed by the nanoscale arrangement of the extracellular matrix (ECM), being more affected by local rather than global concentrations of cell adhesive ligands. In many cell-based studies, grafting of dendrimers on surfaces has shown the benefits of the local increase in concentration provided by the dendritic configuration, although the lack of any reported surface characterization has limited any direct correlation between dendrimer disposition and cell response. In order to establish a proper correlation, some control over dendrimer surface deposition is desirable. Here, dendrimer nanopatterning has been employed to address arginine-glycine-aspartic acid (RGD) density effects on cell adhesion. Nanopatterned surfaces were fully characterized by atomic force microscopy (AFM), scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS), showing that tunable distributions of cell adhesive ligands on the surface are obtained as a function of the initial dendrimer bulk concentration. Cell experiments showed a clear correlation with dendrimer surface layout: Substrates presenting regions of high local ligand density resulted in a higher percentage of adhered cells and a higher degree of maturation of focal adhesions (FAs). Therefore, dendrimer nanopatterning is presented as a suitable and controlled approach to address the effect of local ligand density on cell response. Moreover, due to the easy modification of dendrimer peripheral groups, dendrimer nanopatterning can be further extended to other ECM ligands having density effects on cells.

Keywords: Arginine-glycine-aspartic acid, Atomic force microscopy, Cell adhesion, Dendrimer, Focal adhesions, Scanning tunneling microscopy

Tahirbegi, I. B., Mir, M., Schostek, S., Schurr, M., Samitier, J., (2014). In vivo ischemia monitoring array for endoscopic surgery Biosensors and Bioelectronics 61, 124-130

An array with all-solid-state, potentiometric, miniaturized sensors for pH and potassium was developed to be introduced into the stomach or other sectors of the digestive tract by means of flexible endoscopy. These sensors perform continuous and simultaneous measurement of extracellular pH and potassium. This detection seeks to sense ischemia in the gastric mucosa inside the stomach, an event indicative of local microvascular perfusion and tissue oxygenation status. Our array is proposed as a medical tool to identify the occurrence of the ischemia after gastrointestinal or gastroesophageal anastomosis. The stability and feasibility of the miniaturized working and reference electrodes integrated in the array were studied under in vitro conditions, and the behavior of the potassium and pH ion-selective membranes were optimized to work under acidic gastric conditions with high concentrations of HCl. The array was tested in vivo in pigs to measure the ischemia produced by clamping the blood flow into the stomach. Our results indicate that ischemic and reperfusion states can be sensed in vivo and that information on tissue damage can be collected by this sensor array. The device described here provides a miniaturized, inexpensive, and mass producible sensor array for detecting local ischemia caused by unfavorable anastomotic perfusion and will thus contribute to preventing anastomotic leakage and failure caused by tissue necrosis.

Keywords: Endoscopy, Surgery, Tissue, Gastric anastomosis, Gastric conditions, Ion selective sensors, Ischemia, pH detection, Reference electrodes, Simultaneous measurement, Tissue oxygenation, Sensors

Marques, J., Moles, E., Urbán, P., Prohens, R., Busquets, M. A., Sevrin, C., Grandfils, C., Fernàndez-Busquets, X., (2014). Application of heparin as a dual agent with antimalarial and liposome targeting activities toward Plasmodium-infected red blood cells Nanomedicine: Nanotechnology, Biology, and Medicine 10, (8), 1719-1728

Heparin had been demonstrated to have antimalarial activity and specific binding affinity for Plasmodium-infected red blood cells (pRBCs) vs. non-infected erythrocytes. Here we have explored if both properties could be joined into a drug delivery strategy where heparin would have a dual role as antimalarial and as a targeting element of drug-loaded nanoparticles. Confocal fluorescence and transmission electron microscopy data show that after 30. min of being added to living pRBCs fluorescein-labeled heparin colocalizes with the intracellular parasites. Heparin electrostatically adsorbed onto positively charged liposomes containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane and loaded with the antimalarial drug primaquine was capable of increasing three-fold the activity of encapsulated drug in Plasmodium falciparum cultures. At concentrations below those inducing anticoagulation of mouse blood in vivo, parasiticidal activity was found to be the additive result of the separate activities of free heparin as antimalarial and of liposome-bound heparin as targeting element for encapsulated primaquine. From the Clinical Editor: Malaria remains an enormous global public health concern. In this study, a novel functionalized heparin formulation used as drug delivery agent for primaquine was demonstrated to result in threefold increased drug activity in cell cultures, and in a murine model it was able to provide these benefits in concentrations below what would be required for anticoagulation. Further studies are needed determine if this approach is applicable in the human disease as well.

Keywords: Heparin, Liposomes, Malaria, Plasmodium, Targeted drug delivery, Heparin, Malaria, Plasmodium, Red blood cell, Targeted drug delivery, Liposomes, 1,2 dioleoyl 3 trimethylammoniopropane, fluorescein, heparin, liposome, nanoparticle, primaquine, adsorption, animal experiment, anticoagulation, antimalarial activity, Article, binding affinity, confocal microscopy, controlled study, drug targeting, encapsulation, erythrocyte, female, fluorescence microscopy, human, human cell, in vivo study, liposomal delivery, mouse, nonhuman, Plasmodium falciparum, transmission electron microscopy

Andreu, I., Luque, T., Sancho, A., Pelacho, B., Iglesias-García, O., Melo, E., Farré, R., Prósper, F., Elizalde, M. R., Navajas, D., (2014). Heterogeneous micromechanical properties of the extracellular matrix in healthy and infarcted hearts Acta Biomaterialia 10, (7), 3235-3242

Infarcted hearts are macroscopically stiffer than healthy organs. Nevertheless, although cell behavior is mediated by the physical features of the cell niche, the intrinsic micromechanical properties of healthy and infarcted heart extracellular matrix (ECM) remain poorly characterized. Using atomic force microscopy, we studied ECM micromechanics of different histological regions of the left ventricle wall of healthy and infarcted mice. Hearts excised from healthy (n = 8) and infarcted mice (n = 8) were decellularized with sodium dodecyl sulfate and cut into 12 μm thick slices. Healthy ventricular ECM revealed marked mechanical heterogeneity across histological regions of the ventricular wall with the effective Young's modulus ranging from 30.2 ± 2.8 to 74.5 ± 8.7 kPa in collagen- and elastin-rich regions of the myocardium, respectively. Infarcted ECM showed a predominant collagen composition and was 3-fold stiffer than collagen-rich regions of the healthy myocardium. ECM of both healthy and infarcted hearts exhibited a solid-like viscoelastic behavior that conforms to two power-law rheology. Knowledge of intrinsic micromechanical properties of the ECM at the length scale at which cells sense their environment will provide further insight into the cell-scaffold interplay in healthy and infarcted hearts.

Keywords: Atomic force microscopy, Extracellular matrix, Heart scaffold, Nanoindentation, Viscoelasticity

Dols-Perez, A., Fumagalli, L., Gomila, G., (2014). Structural and nanomechanical effects of cholesterol in binary and ternary spin-coated single lipid bilayers in dry conditions Colloids and Surfaces B: Biointerfaces 116, 295-302

We investigate the effects of Cholesterol (Chol) in the structural and nanomechanical properties of binary and ternary spin-coated single lipid bilayers made of Dioleoylphosphatidylcholine (DOPC) and Sphingomyelin (SM) in dry conditions. We show that for the DOPC/Chol bilayers, Chol induces an initial increase of the bilayer thickness, followed by decrease for concentrations above 30% Chol. The mechanical properties, instead, appear practically insensitive to the Chol content. For the SM/Chol bilayers we have observed both the thinning of the bilayer and the decrease of the force necessary to break it for Chol content above 40. mol%. In both binary mixtures phase separation is not observed. For ternary single bilayers of DOPC/SM/Chol, Chol induces phase segregation and the formation of domains resembling lipid rafts. The domains show a thickness and mechanical response clearly distinct from the surrounding phase and dependent on the relative Chol content. Based on the results obtained for the binary mixtures, DOPC- and SM-enriched domains can be identified. We highlight that many of the effects of Chol reported here for the dry multicomponent single lipid bilayers resemble closely those observed in hydrated bilayers, thus offering an additional insight into their properties.

Keywords: AFM, Air-stable lipid layer, Force spectroscopy, Lipid raft, Spin-coating

Gramse, G., Kasper, M., Fumagalli, L., Gomila, G., Hinterdorfer, P., Kienberger, F., (2014). Calibrated complex impedance and permittivity measurements with scanning microwave microscopy Nanotechnology 25, (14), 145703 (8)

We present a procedure for calibrated complex impedance measurements and dielectric quantification with scanning microwave microscopy. The calibration procedure works in situ directly on the substrate with the specimen of interest and does not require any specific calibration sample. In the workflow tip-sample approach curves are used to extract calibrated complex impedance values and to convert measured S11 reflection signals into sample capacitance and resistance images. The dielectric constant of thin dielectric SiO2 films were determined from the capacitance images and approach curves using appropriate electrical tip-sample models and the εr value extracted at f = 19.81 GHz is in good agreement with the nominal value of εr ∼ 4. The capacitive and resistive material properties of a doped Si semiconductor sample were studied at different doping densities and tip-sample bias voltages. Following a simple serial model the capacitance-voltage spectroscopy curves are clearly related to the semiconductor depletion zone while the resistivity is rising with falling dopant density from 20 Ω to 20 kΩ. The proposed procedure of calibrated complex impedance measurements is simple and fast and the accuracy of the results is not affected by varying stray capacitances. It works for nanoscale samples on either fully dielectric or highly conductive substrates at frequencies between 1 and 20 GHz.

Keywords: Complex impedance, Dielectric constant, Nanotechnology: calibration, Resistivity, Scanning microwave microscopy

Gomila, G., Gramse, G., Fumagalli, L., (2014). Finite-size effects and analytical modeling of electrostatic force microscopy applied to dielectric films Nanotechnology 25, (25), 255702 (11)

A numerical analysis of the polarization force between a sharp conducting probe and a dielectric film of finite lateral dimensions on a metallic substrate is presented with the double objective of (i) determining the conditions under which the film can be approximated by a laterally infinite film and (ii) proposing an analytical model valid in this limit. We show that, for a given dielectric film, the critical diameter above which the film can be modeled as laterally infinite depends not only on the probe geometry, as expected, but mainly on the film thickness. In particular, for films with intermediate to large thicknesses (>100 nm), the critical diameter is nearly independent from the probe geometry and essentially depends on the film thickness and dielectric constant following a relatively simple phenomenological expression. For films that can be considered as laterally infinite, we propose a generalized analytical model valid in the thin-ultrathin limit (<20-50 nm) that reproduces the numerical calculations and the experimental data. Present results provide a general framework under which accurate quantification of electrostatic force microscopy measurements on dielectric films on metallic substrates can be achieved.

Keywords: Dielectric constant, Dielectric films, Electrostatic force microscopy, Quantification, Analytical models, Electric force microscopy, Electrostatic force, Film thickness, Permittivity, Probes, Substrates, Ultrathin films, Accurate quantifications, Electrostatic force microscopy, Finite size effect, Lateral dimension, Metallic substrate, Numerical calculation, Polarization forces, Quantification, Dielectric films

Melo, E., Cárdenes, N., Garreta, E., Luque, T., Rojas, M., Navajas, D., Farré, R., (2014). Inhomogeneity of local stiffness in the extracellular matrix scaffold of fibrotic mouse lungs Journal of the Mechanical Behavior of Biomedical Materials 37, 186-195

Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1. kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7. kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6. kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8. kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs.

Keywords: Ageing, Atomic force microscopy, Decellularization, Lung fibrosis, Tissue engineering, Atomic force microscopy, Biological organs, Peptides, Sodium dodecyl sulfate, Sodium sulfate, Tissue engineering, Ageing, Decellularization, Extracellular matrices, Healthy controls, Inhomogeneities, Lung fibrosis, Micro-mechanical, Statistically significant difference, Mammals, bleomycin, adventitia, animal experiment, animal model, article, atomic force microscopy, bleomycin-induced pulmonary fibrosis, cell fate, controlled study, extracellular matrix, female, intima, lung alveolus, lung fibrosis, lung mechanics, mechanical probe, microenvironment, mouse, nonhuman, pleura, priority journal, rigidity, tissue engineering

Uriarte, J. J., Nonaka, P. N., Campillo, N., Palma, R. K., Melo, E., de Oliveira, L. V. F., Navajas, D., Farré, R., (2014). Mechanical properties of acellular mouse lungs after sterilization by gamma irradiation Journal of the Mechanical Behavior of Biomedical Materials 40, 168-177

Lung bioengineering using decellularized organ scaffolds is a potential alternative for lung transplantation. Clinical application will require donor scaffold sterilization. As gamma-irradiation is a conventional method for sterilizing tissue preparations for clinical application, the aim of this study was to evaluate the effects of lung scaffold sterilization by gamma irradiation on the mechanical properties of the acellular lung when subjected to the artificial ventilation maneuvers typical within bioreactors. Twenty-six mouse lungs were decellularized by a sodium dodecyl sulfate detergent protocol. Eight lungs were used as controls and 18 of them were submitted to a 31kGy gamma irradiation sterilization process (9 kept frozen in dry ice and 9 at room temperature). Mechanical properties of acellular lungs were measured before and after irradiation. Lung resistance (RL) and elastance (EL) were computed by linear regression fitting of recorded signals during mechanical ventilation (tracheal pressure, flow and volume). Static (Est) and dynamic (Edyn) elastances were obtained by the end-inspiratory occlusion method. After irradiation lungs presented higher values of resistance and elastance than before irradiation: RL increased by 41.1% (room temperature irradiation) and 32.8% (frozen irradiation) and EL increased by 41.8% (room temperature irradiation) and 31.8% (frozen irradiation). Similar increases were induced by irradiation in Est and Edyn. Scanning electron microscopy showed slight structural changes after irradiation, particularly those kept frozen. Sterilization by gamma irradiation at a conventional dose to ensure sterilization modifies acellular lung mechanics, with potential implications for lung bioengineering.

Keywords: Gamma irradiation, Lung bioengineering, Lung decellularization, Organ scaffold, Pulmonary mechanics, Decellularization, Gamma irradiation, Mouse lung, Pulmonary mechanics, dodecyl sulfate sodium, animal tissue, Article, artificial ventilation, bioengineering, bioreactor, compliance (physical), controlled study, freezing, gamma irradiation, lung, lung mechanics, lung resistance, male, mouse, nonhuman, room temperature, scanning electron microscopy, tissue scaffold, trachea pressure

Rajzer, I., Menaszek, E., Kwiatkowski, R., Planell, J. A., Castaño, O., (2014). Electrospun gelatin/poly( Materials Science and Engineering: C 44, 183-190

In this study gelatin (Gel) modified with calcium phosphate nanoparticles (SG5) and polycaprolactone (PCL) were used to prepare a 3D bi-layer scaffold by collecting electrospun PCL and gelatin/SG5 fibers separately in the same collector. The objective of this study was to combine the desired properties of PCL and Gel/SG5 in the same scaffold in order to enhance mineralization, thus improving the ability of the scaffold to bond to the bone tissue. The scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and the wide angle X-ray diffraction (WAXD) measurements confirmed that SG5 nanoparticles were successfully incorporated into the fibrous gelatin matrix. The composite Gel/SG5/PCL scaffold exhibited more enhanced mechanical properties than individual Gel and Gel/SG5 scaffolds. The presence of SG5 nanoparticles accelerated the nucleation and growth of apatite crystals on the surface of the composite Gel/SG5/PCL scaffold in simulated body fluid (SBF). The osteoblast response in vitro to developed electrospun scaffolds (PCL and Gel/SG5/PCL) was investigated by using normal human primary NHOst cell lines. NHOst cell culture studies showed that higher alkaline phosphatase (ALP) activity and better mineralization were obtained in the case of composite materials than in pure PCL scaffolds. The mechanically strong PCL scaffold served as a skeleton, while the Gel/SG5 fibers facilitated cell spreading and mineralization of the scaffold.

Keywords: Bilayer fibrous scaffold, Ceramic nanoparticles, Electrospinning, Gelatin, Polycaprolactone, Biomechanics, Bone, Calcium phosphate, Cell culture, Electrospinning, Fourier transform infrared spectroscopy, Mechanical properties, Mineralogy, Nanoparticles, Phosphatases, Polycaprolactone, Scanning electron microscopy, X ray diffraction, Polycaprolactone, Alkaline phosphatase activity, Bone tissue engineering, Calcium phosphate nanoparticles, Ceramic nanoparticles, Fibrous scaffolds, Gelatin, Simulated body fluids, Wide-angle x-ray diffraction, Electrospuns, Scaffolds (biology), Electrospinning

Mir, M., Lugo, R., Tahirbegi, I. B., Samitier, J., (2014). Miniaturizable ion-selective arrays based on highly stable polymer membranes for biomedical applications Sensors 14, (7), 11844-11854

Poly(vinylchloride) (PVC) is the most common polymer matrix used in the fabrication of ion-selective electrodes (ISEs). However, the surfaces of PVC-based sensors have been reported to show membrane instability. In an attempt to overcome this limitation, here we developed two alternative methods for the preparation of highly stable and robust ion-selective sensors. These platforms are based on the selective electropolymerization of poly(3,4-ethylenedioxythiophene) (PEDOT), where the sulfur atoms contained in the polymer covalently interact with the gold electrode, also permitting controlled selective attachment on a miniaturized electrode in an array format. This platform sensor was improved with the crosslinking of the membrane compounds with poly(ethyleneglycol) diglycidyl ether (PEG), thus also increasing the biocompatibility of the sensor. The resulting ISE membranes showed faster signal stabilization of the sensor response compared with that of the PVC matrix and also better reproducibility and stability, thus making these platforms highly suitable candidates for the manufacture of robust implantable sensors.

Keywords: Biomedicine, Electrochemistry, Endoscope, Implantable device, Ion-selective electrode (ISE) sensor, Ischemia, pH detection, Biocompatibility, Chemical sensors, Electrochemistry, Electrodes, Electropolymerization, Endoscopy, Functional polymers, Implants (surgical), Ion selective electrodes, Medical applications, Polyvinyl chlorides, Stabilization, Biomedical applications, Biomedicine, Implantable devices, Ion selective sensors, Ischemia, Membrane instability, pH detection, Poly(3 ,4 ethylenedioxythiophene) (PEDOT), Ion selective membranes

Tahirbegi, I. B., Alvira, M., Mir, M., Samitier, J., (2014). Simple and fast method for fabrication of endoscopic implantable sensor arrays Sensors 14, (7), 11416-11426

Here we have developed a simple method for the fabrication of disposable implantable all-solid-state ion-selective electrodes (ISE) in an array format without using complex fabrication equipment or clean room facilities. The electrodes were designed in a needle shape instead of planar electrodes for a full contact with the tissue. The needle-shape platform comprises 12 metallic pins which were functionalized with conductive inks and ISE membranes. The modified microelectrodes were characterized with cyclic voltammetry, scanning electron microscope (SEM), and optical interferometry. The surface area and roughness factor of each microelectrode were determined and reproducible values were obtained for all the microelectrodes on the array. In this work, the microelectrodes were modified with membranes for the detection of pH and nitrate ions to prove the reliability of the fabricated sensor array platform adapted to an endoscope.

Keywords: Chemical sensors, Cyclic voltammetry, Electrochemistry, Endoscopy, Fabrication, Implants (surgical), Microelectrodes, Needles, Nitrates, Scanning electron microscopy, Biomedicine, Fabricated sensors, Fabrication equipment, Implantable devices, Implantable sensors, Optical interferometry, Planar electrode, Roughness factor, Ion selective electrodes

Torrent-Burgués, J., Cea, P., Giner, I., Guaus, E., (2014). Characterization of Langmuir and Langmuir-Blodgett films of an octasubstituted zinc phthalocyanine Thin Solid Films 556, 485-494

In this work we report the fabrication of Langmuir and Langmuir-Blodgett (LB) films of a substituted ZnPc (octakis(oxyoctyl)phthalocyanine of zinc), and their characterization by means of several techniques. These characterization techniques include surface pressure (π-A) and surface potential (ΔV-A) isotherms as well as UV-vis Reflection spectroscopy and Brewster Angle Microscopy (BAM) for the films at the air-water interface together with UV-vis absorption and IR spectroscopies and Atomic Force Microscopy (AFM) for the LB films. The π-A and ΔV-A isotherms and BAM images indicate a phase transition at a surface pressure of ca. 9 mN/m and a multilayer formation at surface pressures around 19-20 mN/m; at a surface pressure around 27 mN/m a disordered collapse of the film occurs. In addition, AFM images of LB films at π = 10 mN/m and π = 20 mN/m show a monomolecular and a multilayered film, respectively. The comparison of the UV-vis spectrum of ZnPc in solution, the reflection spectra of the Langmuir films and UV-vis spectra of LB films reveals a significant reduction in the Q band intensity for the films, indicative of an organization of ZnPc in the Langmuir and LB films versus the random distribution in solution. The UV-vis Reflection spectra are also consistent with multilayer formation at surface pressures around 19-20 mN/m. The relative intensities of the IR spectrum bands change from the KBr pellet to the LB film which is also attributable to orientation effects in the film. Cyclic voltammetric experiments of LB films incorporating the ZnPc derivative show peaks that can be correlated with redox processes occurring in the phthalocyanine ring. A small but significant influence of the surface pressure and the number of deposited layers in the electrochemical behaviour is observed. The electrochemical response of cast films exhibits some differences with respect to that of LB films which have been attributed to their different molecular organizations.

Keywords: Atomic Force Microscopy, Electrochemistry, Langmuir-Blodgett, Multilayers, Optical spectroscopy techniques, Zinc phthalocyanine, Atomic force microscopy, Electrochemistry, Interfaces (materials), Isotherms, Multilayers, Nitrogen compounds, Optical multilayers, Organic polymers, Zinc compounds, Brewster angle microscopy, Characterization techniques, Electrochemical behaviour, Langmuir and langmuir-blodgett films, Langmuir-blodgett, Optical spectroscopy techniques, UV-Vis Reflection Spectroscopy, Zinc phthalocyanines, Langmuir Blodgett films

Redondo-Morata, L., Giannotti, M. I., Sanz, F., (2014). Structural impact of cations on lipid bilayer models: Nanomechanical properties by AFM-force spectroscopy Molecular Membrane Biology 31, (1), 17-28

Atomic Force Microscopy (AFM) has become an invaluable tool for studying the micro-and nanoworlds. As a stand-alone, high-resolution imaging technique and force transducer, it defies most other surface instrumentation in ease of use, sensitivity and versatility. The main strength of AFM relies on the possibility to operate in an aqueous environment on a wide variety of biological samples, from single molecules-DNA or proteins-to macromolecular assemblies like biological membranes. Understanding the effect of mechanical stress on membranes is of primary importance in biophysics, since cells are known to perform their function under a complex combination of forces. In the later years, AFM-based Force-Spectroscopy (AFM-FS) has provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Lipid membranes are electrostatically charged entities that physiologically coexist with electrolyte solutions. Thus, specific interactions with ions are a matter of considerable interest. The distribution of ions in the solution and their interaction with the membranes are factors that substantially modify the structure and dynamics of the cell membranes. Furthermore, signaling processes are modified by the membrane capability of retaining ions. Supported Lipid Bilayers (SLBs) are a versatile tool to investigate phospholipid membranes mimicking biological surfaces. In the present contribution, we review selected experiments on the mechanical stability of SLBs as models of lipid membranes by means of AFM-FS, with special focus on the effect of cations and ionic strength in the overall nanomechanical stability.

Keywords: Atomic force microscopy, Cations, Force spectroscopy, Lipid bilayer, Mechanical stability

Birhane, Y., Otero, J., Pérez-Murano, F., Fumagalli, L., Gomila, G., Bausells, J., (2014). Batch fabrication of insulated conductive scanning probe microscopy probes with reduced capacitive coupling Microelectronic Engineering 119, 44-47

We report a novel fabrication process for the batch fabrication of insulated conductive scanning probe microscopy (SPM) probes for electrical and topographic characterization of soft samples in liquid media at the nanoscale. The whole SPM probe structure is insulated with a dielectric material except at the very tip end and at the contact pad area to minimize the leakage current in liquid. Additionally, the geometry of the conducting layer in the probe cantilever and substrate is engineered to reduce the parasitic capacitance coupling with the sample. The electrical characterization of the probes has shown that parasitic capacitances are significantly reduced as compared to fully metallized cantilevers.

Keywords: Conductive scanning probe microscopy (C-SPM), EFM, SECM, SECM-AFM, SIM

Correa, L.S., Giraldo, B., Correa, R., Arini, P.D., Laciar, E., (2014). Estudio de la pausa espiratoria en pacientes con enfermedades obstructivas en proceso de desconexión de la ventilación mecánica IFMBE Proceedings VI Latin American Congress on Biomedical Engineering (CLAIB 2014) , Springer (Paraná, Argentina) 49, 705-708

In this work, the flow signal Expiratory Pause (EP) temporal analysis is used in 18 patients with obstructive lung diseases going through spontaneous breathing trial at weaning process. The main objective was to identify the patients who were successfully disconnected (success group: 9 patients), and those who were not (failure and reintubated group: 9 patients). A variable selection stage was done by mean group comparison and step wise variable inclusion, leading to a 3 parameters set: EP time median; cycle time mean; and median absolute deviation of the EP maxima local number. Next, this set was used in a classifier based on linear discriminant analysis, which results in 17 patients (94.4%) correctly classified, with 88.9% of specificity (Sp) and 100% of sensitivity (Se). Finally, applying the leave-one-out cross validation method, results were 88.9% of correctly classified patients (Sp=77.8% and Se=100%). In conclusion, the proposed parameters showed a good performance and could be used to help therapists to wean patients with obstructive diseases.

Keywords: Chronic Obstructive Pulmonary Disease (COPD), Weaning, Mechanical ventilation, Expiratory pause

Barreto, S., Clausen, C. H., Perrault, C. M., Fletcher, D. A., Lacroix, D., (2013). A multi-structural single cell model of force-induced interactions of cytoskeletal components Biomaterials 34, (26), 6119-6126

Several computational models based on experimental techniques and theories have been proposed to describe cytoskeleton (CSK) mechanics. Tensegrity is a prominent model for force generation, but it cannot predict mechanics of individual CSK components, nor explain the discrepancies from the different single cell stimulating techniques studies combined with cytoskeleton-disruptors. A new numerical concept that defines a multi-structural 3D finite element (FE) model of a single-adherent cell is proposed to investigate the biophysical and biochemical differences of the mechanical role of each cytoskeleton component under loading. The model includes prestressed actin bundles and microtubule within cytoplasm and nucleus surrounded by the actin cortex. We performed numerical simulations of atomic force microscopy (AFM) experiments by subjecting the cell model to compressive loads. The numerical role of the CSK components was corroborated with AFM force measurements on U2OS-osteosarcoma cells and NIH-3T3 fibroblasts exposed to different cytoskeleton-disrupting drugs. Computational simulation showed that actin cortex and microtubules are the major components targeted in resisting compression. This is a new numerical tool that explains the specific role of the cortex and overcomes the difficulty of isolating this component from other networks invitro. This illustrates that a combination ofcytoskeletal structures with their own properties is necessary for a complete description of cellular mechanics.

Keywords: Actin bundles, Actin cortex, AFM (atomic force microscopy), Cytoskeleton, Finite element modeling, Microtubules

Dols-Perez, A., Sisquella, X., Fumagalli, L., Gomila, G., (2013). Optical visualization of ultrathin mica flakes on semitransparent gold substrates Nanoscale Research Letters 8, (1), 1-5

We show that optical visualization of ultrathin mica flakes on metallic substrates is viable using semitransparent gold as substrates. This enables to easily localize mica flakes and rapidly estimate their thickness directly on gold substrates by conventional optical reflection microscopy. We experimentally demonstrate it by comparing optical images with atomic force microscopy images of mica flakes on semitransparent gold. Present results open the possibility for simple and rapid characterization of thin mica flakes as well as other thin sheets directly on metallic substrates.

Keywords: Atomic force, Conductive AFM, Gold substrates, Metallic substrate, Optical image, Optical reflection, Optical visualization, Ultrathin layers, Atomic force microscopy, Geometrical optics, Gold, Mica, Optical microscopy, Substrates

Esteban, O., Christ, D., Stock, D., (2013). Purification of molecular machines and nanomotors using phage-derived monoclonal antibody fragments Protein Nanotechnology - Methods in Molecular Biology (ed. Gerrard, J. A.), Humana Press (New York, USA) 996, 203-217

Molecular machines and nanomotors are sophisticated biological assemblies that convert potential energy stored either in transmembrane ion gradients or in ATP into kinetic energy. Studying these highly dynamic biological devices by X-ray crystallography is challenging, as they are difficult to produce, purify, and crystallize. Phage display technology allows us to put a handle on these molecules in the form of highly specific antibody fragments that can also stabilize conformations and allow versatile labelling for electron microscopy, immunohistochemistry, and biophysics experiments. Here, we describe a widely applicable protocol for selecting high-affinity monoclonal antibody fragments against a complex molecular machine, the A-type ATPase from T. thermophilus that allows fast and simple purification of this transmembrane rotary motor from its wild-type source. The approach can be readily extended to other integral membrane proteins and protein complexes as well as to soluble molecular machines and nanomotors.

Keywords: ATP synthase, Crystallization, Domain antibodies, Electron microscopy, Labelling, Membrane proteins, Monoclonal antibody fragments, Phage display, Protein purification, X-ray crystallography

Mir, Mònica , Tahirbegi, Islam Bogachan , Valle-Delgado, Juan José , Fernàndez-Busquets, X., Samitier, Josep , (2012). In vitro study of magnetite-amyloid Nanomedicine: Nanotechnology, Biology and Medicine 8, (6), 974-980

Biogenic magnetite (Fe3O4) has been identified in human brain tissue. However, abnormal concentration of magnetite nanoparticles in the brain has been observed in different neurodegenerative pathologies. In the case of Alzheimer's disease (AD), these magnetic nanoparticles have been identified attached to the characteristic brain plaques, which are mainly formed by fibrils of amyloid

Keywords: Alzheimer's disease, Biogenic magnetite, Amyloid β peptide (Aβ), Superconducting quantum interference, Scanning electron microscope, Surface plasmon resonance, Magnetic force microscopy

Nocentini, S., Reginensi, D., Garcia, S., Carulla, P., Moreno-Flores, Wandosell, F., Trepat, X., Bribian, A., Del Rí, (2012). Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single-cell tracking and traction force microscopy Cellular and Molecular Life Sciences 69, (10), 1689-1703

Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but instead a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion, and crossover during cell–cell and cell–matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo receptor complex and that their migration is blocked by myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over myelin. Our data relate the decrease of traction force of OEC with lower migratory capacity over myelin, which correlates with changes in the F-actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo receptor inhibitor NEP1-40.

Keywords: Ensheathing glia, Traction force, microscopy, Migration, Myelin-associated inhibitors

Caballero, D., Martinez, E., Bausells, J., Errachid, A., Samitier, J., (2012). Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface Analytica Chimica Acta 720, 43-48

In this work we report the fabrication and characterization of a label-free impedimetric immunosensor based on a silicon nitride (Si 3N 4) surface for the specific detection of human serum albumin (HSA) proteins. Silicon nitride provides several advantages compared with other materials commonly used, such as gold, and in particular in solid-state physics for electronic-based biosensors. However, few Si 3N 4-based biosensors have been developed; the lack of an efficient and direct protocol for the integration of biological elements with silicon-based substrates is still one of its the main drawbacks. Here, we use a direct functionalization method for the direct covalent binding of monoclonal anti-HSA antibodies on an aldehyde-functionalized Si-p/SiO 2/Si 3N 4 structure. This methodology, in contrast with most of the protocols reported in literature, requires less chemical reagents, it is less time-consuming and it does not need any chemical activation. The detection capability of the immunosensor was tested by performing non-faradaic electrochemical impedance spectroscopy (EIS) measurements for the specific detection of HSA proteins. Protein concentrations within the linear range of 10 -13-10 -7M were detected, showing a sensitivity of 0.128ΩμM -1 and a limit of detection of 10 -14M. The specificity of the sensor was also addressed by studying the interferences with a similar protein, bovine serum albumin. The results obtained show that the antibodies were efficiently immobilized and the proteins detected specifically, thus, establishing the basis and the potential applicability of the developed silicon nitride-based immunosensor for the detection of proteins in real and more complex samples.

Keywords: Aldehyde, Electrochemical impedance spectroscopy, Human serum albumin, Immunosensor, Silicon nitride, Bovine serum albumins, Chemical reagents, Complex samples, Covalent binding, Detection capability, Electrochemical impedance, Electrochemical impedance spectroscopy measurements, Functionalizations, Human serum albumins, Impedimetric immunosensors, Label free, Limit of detection, Linear range, Protein concentrations, Silicon-based, Specific detection, Aldehydes

Redondo-Morata, L., Giannotti, M. I., Sanz, F., (2012). AFM-based force-clamp monitors lipid bilayer failure kinetics Langmuir 28, (15), 6403-6410

The lipid bilayer rupture phenomenon is here explored by means of atomic force microscopy (AFM)-based force clamp, for the first time to our knowledge, to evaluate how lipid membranes respond when compressed under an external constant force, in the range of nanonewtons. Using this method, we were able to directly quantify the kinetics of the membrane rupture event and the associated energy barriers, for both single supported bilayers and multibilayers, in contradistinction to the classic studies performed at constant velocity. Moreover, the affected area of the membrane during the rupture process was calculated using an elastic deformation model. The elucidated information not only contributes to a better understanding of such relevant process, but also proves the suitability of AFM-based force clamp to study model structures as lipid bilayers. These findings on the kinetics of lipid bilayers rupture could be extended and applied to the study of other molecular thin films. Furthermore, systems of higher complexity such as models mimicking cell membranes could be studied by means of AFM-based force-clamp technique.

Keywords: Chain-Length, Spectroscopy, Nanomechanics, Microscopy, Elasticity, Stability, Membranes, Reveals, Fusion, Ions

Valle-Delgado, J. J., Liepina, I., Lapidus, D., Sabaté, R., Ventura, S., Samitier, J., Fernàndez-Busquets, X., (2012). Self-assembly of human amylin-derived peptides studied by atomic force microscopy and single molecule force spectroscopy Soft Matter 8, (4), 1234-1242

The self-assembly of peptides and proteins into amyloid fibrils of nanometric thickness and up to several micrometres in length, a phenomenon widely observed in biological systems, has recently aroused a growing interest in nanotechnology and nanomedicine. Here we have applied atomic force microscopy and single molecule force spectroscopy to study the amyloidogenesis of a peptide derived from human amylin and of its reverse sequence. The spontaneous formation of protofibrils and their orientation along well-defined directions on graphite and DMSO-coated graphite substrates make the studied peptides interesting candidates for nanotechnological applications. The measured binding forces between peptides correlate with the number of hydrogen bonds between individual peptides inside the fibril structure according to molecular dynamics simulations.

Keywords: Amyloid fibril, Amyloidogenesis, Binding forces, Fibril structure, Graphite substrate, Molecular dynamics simulations, Nanometrics, Protofibrils, Single molecule force spectroscopy, Spontaneous formation, Atomic force microscopy, Atomic spectroscopy, Graphite, Hydrogen bonds, Medical nanotechnology, Molecular dynamics, Molecular physics, Self assembly, Thickness measurement, Peptides

Redondo-Morata, Lorena, Oncins, Gerard, Sanz, Fausto, (2012). Force spectroscopy reveals the effect of different ions in the nanomechanical behavior of phospholipid model membranes: The case of potassium cation Biophysical Journal 102, (1), 66-74

How do metal cations affect the stability and structure of phospholipid bilayers? What role does ion binding play in the insertion of proteins and the overall mechanical stability of biological membranes? Investigators have used different theoretical and microscopic approaches to study the mechanical properties of lipid bilayers. Although they are crucial for such studies, molecular-dynamics simulations cannot yet span the complexity of biological membranes. In addition, there are still some experimental difficulties when it comes to testing the ion binding to lipid bilayers in an accurate way. Hence, there is a need to establish a new approach from the perspective of the nanometric scale, where most of the specific molecular phenomena take place. Atomic force microscopy has become an essential tool for examining the structure and behavior of lipid bilayers. In this work, we used force spectroscopy to quantitatively characterize nanomechanical resistance as a function of the electrolyte composition by means of a reliable molecular fingerprint that reveals itself as a repetitive jump in the approaching force curve. By systematically probing a set of bilayers of different composition immersed in electrolytes composed of a variety of monovalent and divalent metal cations, we were able to obtain a wealth of information showing that each ion makes an independent and important contribution to the gross mechanical resistance and its plastic properties. This work addresses the need to assess the effects of different ions on the structure of phospholipid membranes, and opens new avenues for characterizing the (nano)mechanical stability of membranes.

Keywords: Molecular-dynamics simulation, Liquid expanded monolayers, Lipid-bilayers, Hofmeister series, Monovalent salt, Phosphatidylcholine, Microscopy, Binding, Surfaces, NaCl

Serra, T., Navarro, M., Planell, J. A., (2012). Fabrication and characterization of biodegradable composite scaffolds for tissue engineering Innovative Developments in Virtual and Physical Prototyping 5th International Conference on Advanced Research and Rapid Prototyping (ed. Margarida, T., Ferreira, D.), Taylor & Francis (Leiria, Portugal) VR@P, 67-72

In this study, polylactic acid (PLA) and polyethylene glycol (PEG) were combined with soluble CaP glass particles and processed by rapid prototyping to obtain fully biodegradable structures for Tissue Engineering applications. The obtained 3D biodegradable structures were characterized in terms of their architecture and mechanical properties. The scaffold morphology, internal micro-architecture and mechanical properties were evaluated using Scanning Electron Microscopy (SEM), micro-computed tomography (micro-CT) and mechanical testing, respectively. Well defined structures with pore size of 350-400μm (in the axial view), struts width of approximately 70-80μm, and a porosity ranging between 60-65% were obtained. The combination RP and PLA/PEG/CaP glass turned into promising fully degradable, mechanically stable, bioactive and biocompatible composite scaffolds for TE.

Keywords: Axial view, Biodegradable composites, Composite scaffolds, Glass particles, Mechanically stable, Micro architectures, Micro computed tomography (micro-CT), Poly lactic acid, Scaffold morphology, Tissue engineering applications, Well-defined structures, Bioactive glass, Mechanical properties, Mechanical testing, Polyethylene glycols, Polymer blends, Rapid prototyping, Scaffolds (biology), Scanning electron microscopy, Computerized tomography

Garcia-Parajo, M. F., (2012). The role of nanophotonics in regenerative medicine Nanotechnology in Regenerative Medicine - Methods and Protocols (Methods in Molecular Biology) (ed. Navarro, M., Planell, J. A.), Springer (New York, USA) 811, 267-284

Cells respond to biochemical and mechanical stimuli through a series of steps that begin at the molecular, nanometre level, and translate finally in global cell response. Defects in biochemical- and/or mechanical-sensing, transduction or cellular response are the cause of multiple diseases, including cancer and immune disorders among others. Within the booming field of regenerative medicine, there is an increasing need for developing and applying nanotechnology tools to bring understanding on the cellular machinery and molecular interactions at the nanoscale. Nanotechnology, nanophotonics and in particular, high-resolution-based fluorescence approaches are already delivering crucial information on the way that cells respond to their environment and how they organize their receptors to perform specialized functions. This chapter focuses on emerging super-resolution optical techniques, summarizing their principles, technical implementation, and reviewing some of the achievements reached so far.

Keywords: Cell membrane organization, Nanophotonics, Near-field optical microscopy, Super-resolution optical microscopy

Redondo, L., Giannotti, M. I., Sanz, F., (2012). Stability of lipid bilayers as model membranes: Atomic force microscopy and spectroscopy approach Atomic force microscopy in liquid (ed. Baró, A. M., Reifenberger, R. G.), Wiley-VCH Verlag GmbH & Co.KGaA (Weinheim, Germany) Part I: General Atomic Force Microscopy, 259-284

van Zanten, T. S., Garcia-Parajo, M. F., (2012). Super-resolution near-field optical microscopy Comprehensive Biophysics (ed. Egelman, E. H.), Elsevier (Desdren, Germany) Volume 2: Biophysical Techniques for Characterization of Cells, 144-164

Near-field optical microscopy is a technique not limited by the laws of diffraction that enables simultaneous high-resolution fluorescence and topographic measurements at the nanometer scale. This chapter highlights the intrinsic advantages of near-field optics in the study of cellular structures. The first part of the chapter lays the foundations of the near-field concept and technical implementation of near-field scanning optical microscopy (NSOM), whereas the second part of the chapter focuses on applications of NSOM to the study of model membranes and cellular structures on the plasma membrane. The last part of the chapter discusses further directions of near-field optics, including optical antennas and fluorescence correlation spectroscopy approaches in the near-field regime.

Keywords: Biological membranes, Cell membrane nanoscale compartmentalization, Cellular nanodomains, Fluorescence correlation spectroscopy in reduced volumes, Immunoreceptor imaging, Lipid rafts, Near-field scanning optical microscopy, Optical nano-antennas, Shear force imaging, Single molecule detection, Super-resolution microscopy

Artés, Juan M., Díez-Pérez, Ismael, Sanz, Fausto, Gorostiza, Pau, (2011). Direct measurement of electron transfer distance decay constants of single redox proteins by electrochemical tunneling spectroscopy ACS Nano 5, (3), 2060-2066

We present a method to measure directly and at the single-molecule level the distance decay constant that characterizes the rate of electron transfer (ET) in redox proteins. Using an electrochemical tunneling microscope under bipotentiostatic control, we obtained current-distance spectroscopic recordings of individual redox proteins confined within a nanometric tunneling gap at a well-defined molecular orientation. The tunneling current decays exponentially, and the corresponding decay constant (

Keywords: Long-range electron transfer (LRET), Distance decay constant, Single-molecule electrochemistry, Redox enzyme, Metalloprotein, Blue copper protein, Azurin, Electrochemical scanning tunneling microscopy and spectroscopy, Nanoelectrodes, Debye length, Electrochemical charge screening

Melchels, Ferry P. W., Tonnarelli, Beatrice, Olivares, Andy L., Martin, Ivan, Lacroix, Damien, Feijen, Jan, Wendt, David J., Grijpma, Dirk W., (2011). The influence of the scaffold design on the distribution of adhering cells after perfusion cell seeding Biomaterials 32, (11), 2878-2884

In natural tissues, the extracellular matrix composition, cell density and physiological properties are often non-homogeneous. Here we describe a model system, in which the distribution of cells throughout tissue engineering scaffolds after perfusion seeding can be influenced by the pore architecture of the scaffold. Two scaffold types, both with gyroid pore architectures, were designed and built by stereolithography: one with isotropic pore size (412 ± 13 [mu]m) and porosity (62 ± 1%), and another with a gradient in pore size (250-500 [mu]m) and porosity (35%-85%). Computational fluid flow modelling showed a uniform distribution of flow velocities and wall shear rates (15-24 s-1) for the isotropic architecture, and a gradient in the distribution of flow velocities and wall shear rates (12-38 s-1) for the other architecture. The distribution of cells throughout perfusion-seeded scaffolds was visualised by confocal microscopy. The highest densities of cells correlated with regions of the scaffolds where the pores were larger, and the fluid velocities and wall shear rates were the highest. Under the applied perfusion conditions, cell deposition is mainly determined by local wall shear stress, which, in turn, is strongly influenced by the architecture of the pore network of the scaffold.

Keywords: Scaffolds, Microstructure, Cell adhesion, Confocal microscopy, Image analysis, Computational fluid dynamics

Miranda Coelho, Nuno, Gonzalez-Garcia, Cristina, Salmeron-Sanchez, Manuel, Altankov, George, (2011). Arrangement of type IV collagen and laminin on substrates with controlled density of -OH groups Tissue Engineering Part A 17, (17-18), 2245-2257

Collagen IV (Col IV) and laminin (Lam) are the main structural components of the basement membrane where they form two overlapping polymeric networks. We studied the adsorption pattern of these proteins on five model surfaces with tailored density of -OH groups obtained by copolymerization of different ratios ethyl acrylate (EA) and hydroxyl EA (HEA): X(OH) = 0, X(OH) = 0.3, X(OH) = 0.5, X(OH) = 0.7, and X(OH) = 1 (where X refers the ratio of HEA). Atomic force microscopy revealed substratum-specific adsorption patterns of Col IV and Lam, ranging from single molecules deposition on more hydrophilic substrata to the formation of complex networks on hydrophobic ones. Human umbilical endothelial cells were used to study the biological performance of adsorbed proteins, following the overall cell morphology, the quantities for cell adhesion and spreading, and the development of focal adhesion complexes and actin cytoskeleton. Surprisingly, two optima in the cellular interaction were observed-one on the most hydrophilic X(OH) = 1 and other on the relatively hydrophobic X(OH) = 0.3 substrate-valid for both Col IV and Lam. When the proteins were adsorbed consecutively, a hydrophobic shift to X(OH) = 0 substratum was obtained. Collectively, these data suggest that varying with the density of -OH groups one can tailor the conformation and the functional activity of adsorbed basement membrane proteins.

Keywords: Atomic-force microscopy, Fibronectin adsorption, Basement-membranes, Polymer surfaces, Cell-adhesion, Biomaterials, Wettability, Fibrinogen

Morgenstern, R., Morgenstern, C., Jané, R., Lee, S. H., (2011). Usefulness of an expandable interbody spacer for the treatment of foraminal stenosis in extremely collapsed disks preliminary clinical experience with endoscopic posterolateral transforaminal approach Journal of Spinal Disorders & Techniques 24, (8), 485-491

Study Design: Clinical series of patients with degenerative disk disease undergoing an endoscopic posterolateral transforaminal procedure that used a reaming foraminoplasty technique to enlarge the foramen coupled with insertion of the B-Twin expandable spacer. Objectives: This retrospective analysis of 107 consecutive patients sought to assess the outcome of this surgical procedure. Summary of Background Data: Reamed endoscopic foraminoplasty under direct endoscopic vision has been shown to be suitable for extremely collapsed disks (> 50% total disk height) despite the difficult access, especially at L5-S1. The authors tried to investigate the efficacy of an expandable spacer being inserted by the endoscopic transforaminal approach to solve foraminal stenosis without bone fusion techniques. Methods: The procedure consists of bone reaming under direct endoscopic control to wide the foramen followed by insertion of the B-Twin expandable device as a disk spacer to restore partially or to maintain the height of the collapsed disk. Outcome measures included visual analog scale (VAS) for pain, the Oswestry Disability Index (ODI) for functional disability, and radioimaging studies. Results: Mean follow-up was 27.2 months. Clinical outcome was considered excellent in 64 patients, good in 25, fair in 10, and poor in 8. Results were similar in single and double B-Twin spacer insertions. Postoperative mean values for VAS and ODI scores improved significantly as compared with preoperative data. Mean VAS and ODI scores were significantly higher in patients with fair or poor results than in those with excellent or good outcome. In 2 cases, clear signs of end plate bone resorption in the control computed tomographic scans at 6 months and 12 months leading to a substantial loss of disk height were documented. Conclusions: This preliminary study has shown the efficacy of an endoscopic surgical technique for the treatment of foraminal stenosis in extremely collapsed disks.

Keywords: Foraminal stenosis, B-twin expandable spacer, Endoscopic foraminoplasty, Minimally invasive surgery, Surgical technique, Spinal spacer, Lumbar, Diskectomy, Fusion, Discectomy

Moore, S. W., Roca-Cusachs, P., Sheetz, M. P., (2010). Stretchy proteins on stretchy substrates: The important elements of integrin-mediated rigidity sensing Developmental Cell 19, (2), 194-206

Matrix and tissue rigidity guides many cellular processes, including the differentiation of stem cells and the migration of cells in health and disease. Cells actively and transiently test rigidity using mechanisms limited by inherent physical parameters that include the strength of extracellular attachments, the pulling capacity on these attachments, and the sensitivity of the mechanotransduction system. Here, we focus on rigidity sensing mediated through the integrin family of extracellular matrix receptors and linked proteins and discuss the evidence supporting these proteins as mechanosensors.

Keywords: Focal adhesion kinase, Atomic Force Microscopy, Smooth-muscle cells, Traction forces, Living cells, Mechanical force, Locomoting cells

van Zanten, T. S., Gomez, J., Manzo, C., Cambi, A., Buceta, J., Reigada, R., Garcia-Parajo, M. F., (2010). Direct mapping of nanoscale compositional connectivity on intact cell membranes Proceedings of the National Academy of Sciences of the United States of America 107, (35), 15437-15442

Lateral segregation of cell membranes is accepted as a primary mechanism for cells to regulate a diversity of cellular functions. In this context, lipid rafts have been conceptualized as organizing principle of biological membranes where underlying cholesterol-mediated selective connectivity must exist even at the resting state. However, such a level of nanoscale compositional connectivity has been challenging to prove. Here we used single-molecule near-field scanning optical microscopy to visualize the nanolandscape of raft ganglioside GM1 after tightening by its ligand cholera toxin (CTxB) on intact cell membranes. We show that CTxB tightening of GM1 is sufficient to initiate a minimal raft coalescence unit, resulting in the formation of cholesterol-dependent GM1 nanodomains <120 nm in size. This particular arrangement appeared independent of cell type and GM1 expression level on the membrane. Simultaneous dual color high-resolution images revealed that GPI anchored and certain transmembrane proteins were recruited to regions proximal (<150 nm) to CTxB-GM1 nanodomains without physical intermixing. Together with in silico experiments, our high-resolution data conclusively demonstrate the existence of raft-based interconnectivity at the nanoscale. Such a linked state on resting cell membranes constitutes thus an obligatory step toward the hierarchical evolution of large-scale raft coalescence upon cell activation.

Keywords: Cholera toxin, Membrane heterogeneity, Near-field scanning optical microscopy, Raft ganglioside GM1, Single-molecule detection

Garcia-Manyes, S., Redondo-Morata, L., Oncins, G., Sanz, F., (2010). Nanomechanics of lipid bilayers: Heads or tails? Journal of the American Chemical Society American Chemical Society 132, (37), 12874-12886

Understanding the effect of mechanical stress on membranes is of primary importance in biophysics. Here we use force spectroscopy AFM to quantitatively characterize the nanomechanical stability of supported lipid bilayers as a function of their chemical composition. The onset of plastic deformation reveals itself as a repetitive jump in the approaching force curve, which represents a molecular fingerprint for the bilayer mechanical stability. By systematically probing a set of chemically distinct supported lipid bilayers (SLBs), we first show that both the headgroup and tail have a decisive effect on their mechanical properties. While the mechanical stability of the probed SLBs linearly increases by 3.3 nN upon the introduction of each additional -CH2- in the chain, it exhibits a significant dependence on the phospholipid headgroup, ranging from 3 nN for DPPA to 66 nN for DPPG. Furthermore, we also quantify the reduction of the membrane mechanical stability as a function of the number of unsaturations and molecular branching in the chemical structure of the apolar tails. Finally, we demonstrate that, upon introduction of cholesterol and ergosterol, contrary to previous belief the mechanical stability of membranes not only increases linearly in the liquid phase (DLPC) but also for phospholipids present in the gel phase (DPPC). Our results are discussed in the framework of the continuum nucleation model. This work highlights the compelling effect of subtle variations in the chemical structure of phospholipid molecules on the membrane response when exposed to mechanical forces, a mechanism of common occurrence in nature.

Keywords: Atomic-force microscopy, Molecular-dynamics simulation, Aqueous-electrolyte solutions, Supported planar membranes, Phospholipid-bilayers, Biological-membranes, Physical-properties, Fluid membranes, Model membranes, Chain-length

Comelles, J., Estevez, M., Martinez, E., Samitier, J., (2010). The role of surface energy of technical polymers in serum protein adsorption and MG-63 cells adhesion Nanomedicine: Nanotechnology Biology and Medicine 6, (1), 44-51

Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. However, novel applications in the biosensor field require materials to be compatible with cell growth and at the same time be suitable for technological processing. Technological polymers are key materials in the fabrication of disposable parts and other sensing elements. As such, it is essential to characterize the surface properties of technological polymers, especially after processing and sterilization. It is also important to understand how technological polymers affect cell behavior when in contact with polymer materials. Therefore, the aim of this research was to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly (methyl methacrylate), polystyrene, and poly(dimethylsiloxane). Glass was used as the control material. From the Clinical Editor: Polymeric materials are widely used as supports for cell culturing in medical implants and as scaffolds for tissue regeneration. The aim of this research is to study how surface energy and surface roughness affect the biocompatibility of three polymeric materials widely used in research and industry: poly(methylmethacrylate) (PMMA), polystyrene (PS), and poly(dimethylsiloxane) (PDMS).

Keywords: Thin-films, Poly(methyl methacrylate), Osteoblast adhesion, Electron-microscopy, Fibronectin, Polystyrene, Oly(dimethylsiloxane), Biocompatibility, Hydroxyapatite, Behavior

Garcia-Manyes, S., Sanz, F., (2010). Nanomechanics of lipid bilayers by force spectroscopy with AFM: A perspective Biochimica et Biophysica Acta - Biomembranes 1798, (4), 741-749

Lipid bilayers determine the architecture of cell membranes and regulate a myriad of distinct processes that are highly dependent on the lateral organization of the phospholipid molecules that compose the membrane. Indeed, the mechanochemical properties of the membrane are strongly correlated with the function of several membrane proteins, which demand a very specific, highly localized physicochemical environment to perform their function. Several mesoscopic techniques have been used in the past to investigate the mechanical properties of lipid membranes. However, they were restricted to the study of the ensemble properties of giant bilayers. Force spectroscopy with AFM has emerged as a powerful technique able to provide valuable insights into the nanomechanical properties of supported lipid membranes at the nanometer/nanonewton scale in a wide variety of systems. In particular, these measurements have allowed direct measurement of the molecular interactions arising between neighboring phospholipid molecules and between the lipid molecules and the surrounding solvent environment. The goal of this review is to illustrate how these novel experiments have provided a new vista on membrane mechanics in a confined area within the nanometer realm, where most of the specific molecular interactions take place. Here we report in detail the main discoveries achieved by force spectroscopy with AFM on supported lipid bilayers, and we also discuss on the exciting future perspectives offered by this growing research field.

Keywords: Force spectroscopy, Atomic force microscopy, Lipid bilayer, Nanomechanics

van Zanten, T. S., Cambi, A., Garcia-Parajo, M. F., (2010). A nanometer scale optical view on the compartmentalization of cell membranes Biochimica et Biophysica Acta - Biomembranes 1798, (4), 777-787

For many years, it was believed that the laws of diffraction set a fundamental limit to the spatial resolution of conventional light microscopy. Major developments, especially in the past few years, have demonstrated that the diffraction barrier can be overcome both in the near- and far-field regime. Together with dynamic measurements, a wealth of new information is now emerging regarding the compartmentalization of cell membranes. In this review we focus on optical methods designed to explore the nanoscale architecture of the cell membrane, with a focal point on near-field optical microscopy (NSOM) as the first developed technique to provide truly optical super-resolution beyond the diffraction limit of light. Several examples illustrate the unique capabilities offered by NSOM and highlight its usefulness on cell membrane studies, complementing the palette of biophysical techniques available nowadays.

Keywords: Membrane nanodomain, Lipid raft, Single molecule detection, Near-field scanning optical microscopy, Super-resolution optical microscopy

Harder, A., Walhorn, V., Dierks, T., Fernàndez-Busquets, X., Anselmetti, D., (2010). Single-molecule force spectroscopy of cartilage aggrecan self-adhesion Biophysical Journal 99, (10), 3498-3504

We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca2+-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of I-p = 0.31 +/- 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of tau = 7.9 +/- 4.9 s and a reaction bond length of x(beta) = 0.31 +/- 0.08 nm. Whereas the x(beta)-value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans, the rather short GAG interaction lifetime reflects high intermolecular dynamics within aggrecan complexes, which may be relevant for the viscoelastic properties of cartilage tissue.

Keywords: Bovine nasal cartilage, Articular-cartilage, Sinorhizobium-meliloti, Proteoglycan, Microscopy, DNA, Macromolecules, Binding, Protein, Glycosaminoglycans

Toromanov, Georgi, González-García, Cristina, Altankov, George, Salmerón-Sánchez, Manuel, (2010). Vitronectin activity on polymer substrates with controlled -OH density Polymer 51, (11), 2329-2336

Vitronectin (VN) adsorption on a family of model substrates consisting of copolymers of ethyl acrylate and hydroxyl ethylacrylate in different ratios (to obtain a controlled surface density of -OH groups) was investigated by Atomic Force Microscopy (AFM). It is shown that the fraction of the substrate covered by the protein depends strongly on the amount of hydroxyl groups in the sample and it monotonically decreases as the -OH density increases. Isolated globular-like VN molecules are observed on the surfaces with the higher OH density. As the fraction of hydroxyl groups decreases, aggregates of 3-5 VN molecules are observed on the sample. Overall cell morphology, focal adhesion formation and actin cytoskeleton development are investigated to assess the biological activity of the adsorbed VN on the different surfaces. Dermal fibroblast cells show excellent material interaction on the more hydrophobic samples (OH contents lower than 0.5), which reveals enhanced VN activity on this family of substrates as compared with other extracellular matrix proteins (e.g., fibronectin and fibrinogen).

Keywords: Copolymers, Vitronectin, AFM, Self-assembled monolayers, Cell-adhesion, Thermal transitions, Protein adsorption, Surfaces, Fibronectin, Biomaterials, Attachment, Fibrinogen

Fumagalli, L., Gramse, G., Esteban-Ferrer, D., Edwards, M. A., Gomila, G., (2010). Quantifying the dielectric constant of thick insulators using electrostatic force microscopy Applied Physics Letters 96, (18), 183107

Quantitative measurement of the low-frequency dielectric constants of thick insulators at the nanoscale is demonstrated utilizing ac electrostatic force microscopy combined with finite-element calculations based on a truncated cone with hemispherical apex probe geometry. The method is validated on muscovite mica, borosilicate glass, poly(ethylene naphthalate), and poly(methyl methacrylate). The dielectric constants obtained are essentially given by a nanometric volume located at the dielectric-air interface below the tip, independently of the substrate thickness, provided this is on the hundred micrometer-length scale, or larger.

Keywords: Borosilicate glasses, Finite element analysis, Insulating thin films, Mica, Nanostructured materials, Permittivity, Polymers, Scanning probe microscopy

Toset, J., Gomila, G., (2010). Three-dimensional manipulation of gold nanoparticles with electro-enhanced capillary forces Applied Physics Letters 96, (4), 043117

We demonstrate the possibility to manipulate 25 nm radius gold nanoparticles in the three spatial dimensions with an atomic force microscope with the use of electroenhanced capillary forces. We show that an enhanced water-bridge can be electrostatically induced between a conducting probe and a metallic nanoparticle by the application of a voltage pulse, which is able to exert a pulling capillary force on the nanoparticle strong enough to detach it from the substrate. The nanoparticle can then be moved, attached to the probe, and placed back to the desired location on the substrate simply by contacting it.

Keywords: Atomic force microscopy, Capillarity, Gold, Nanoparticles, Nanotechnology

Almendros, I., Montserrat, J. M., Torres, M., Gonzalez, C., Navajas, D., Farre, R., (2010). Changes in oxygen partial pressure of brain tissue in an animal model of obstructive apnea Respiratory Research 11, (3), 1-6

Cognitive impairment is one of the main consequences of obstructive sleep apnea (OSA) and is usually attributed in part to the oxidative stress caused by intermittent hypoxia in cerebral tissues. The presence of oxygen-reactive species in the brain tissue should be produced by the deoxygenation-reoxygenation cycles which occur at tissue level during recurrent apneic events. However, how changes in arterial blood oxygen saturation (SpO(2)) during repetitive apneas translate into oxygen partial pressure (PtO2) in brain tissue has not been studied. The objective of this study was to assess whether brain tissue is partially protected from intermittently occurring interruption of O-2 supply during recurrent swings in arterial SpO(2) in an animal model of OSA. Methods: Twenty-four male Sprague-Dawley rats (300-350 g) were used. Sixteen rats were anesthetized and noninvasively subjected to recurrent obstructive apneas: 60 apneas/h, 15 s each, for 1 h. A control group of 8 rats was instrumented but not subjected to obstructive apneas. PtO2 in the cerebral cortex was measured using a fast-response oxygen microelectrode. SpO(2) was measured by pulse oximetry. The time dependence of arterial SpO(2) and brain tissue PtO2 was carried out by Friedman repeated measures ANOVA. Results: Arterial SpO(2) showed a stable periodic pattern (no significant changes in maximum [95.5 +/- 0.5%; m +/- SE] and minimum values [83.9 +/- 1.3%]). By contrast, brain tissue PtO2 exhibited a different pattern from that of arterial SpO(2). The minimum cerebral cortex PtO2 computed during the first apnea (29.6 +/- 2.4 mmHg) was significantly lower than baseline PtO2 (39.7 +/- 2.9 mmHg; p = 0.011). In contrast to SpO(2), the minimum and maximum values of PtO2 gradually increased (p < 0.001) over the course of the 60 min studied. After 60 min, the maximum (51.9 +/- 3.9 mmHg) and minimum (43.7 +/- 3.8 mmHg) values of PtO2 were significantly greater relative to baseline and the first apnea dip, respectively. Conclusions: These data suggest that the cerebral cortex is partially protected from intermittently occurring interruption of O-2 supply induced by obstructive apneas mimicking OSA.

Keywords: Near-infrared spectroscopy, Sleep-apnea, Iintermittent hypoxia, Cerebral oxygenation, Oxidative stress, Blood-flow, Rat, Apoptosis, Inflammation, Hypercapnia

Hofer, M., Adamsmaier, S., van Zanten, T. S., Chtcheglova, L. A., Manzo, C., Duman, M., Mayer, B., Ebner, A., Moertelmaier, M., Kada, G., Garcia-Parajo, M. F., Hinterdorfer, P., Kienberger, F., (2010). Molecular recognition imaging using tuning fork-based transverse dynamic force microscopy Ultramicroscopy 110, (6), 605-611

We demonstrate simultaneous transverse dynamic force microscopy and molecular recognition imaging using tuning forks as piezoelectric sensors. Tapered aluminum-coated glass fibers were chemically functionalized with biotin and anti-lysozyme molecules and attached to one of the prongs of a 32 kHz tuning fork. The lateral oscillation amplitude of the tuning fork was used as feedback signal for topographical imaging of avidin aggregates and lysozyme molecules on mica substrate. The phase difference between the excitation and detection signals of the tuning fork provided molecular recognition between avidin/biotin or lysozyme/anti-lysozyme. Aggregates of avidin and lysozyme molecules appeared as features with heights of 1-4 nm in the topographic images, consistent with single molecule atomic force microscopy imaging. Recognition events between avidin/biotin or lysozyme/anti-lysozyme were detected in the phase image at high signal-to-noise ratio with phase shifts of 1-2 degrees. Because tapered glass fibers and shear-force microscopy based on tuning forks are commonly used for near-field scanning optical microscopy (NSOM), these results open the door to the exciting possibility of combining optical, topographic and biochemical recognition at the nanometer scale in a single measurement and in liquid conditions.

Keywords: Tuning fork, Atomic force microscopy, Shear-force microscopy, Molecular recognition, Avidin-biotin

de Oliveira, I. A. M., Vocanson, F., Uttaro, J. P., Asfari, Z., Mills, C. A., Samitier, J., Errachid, A., (2010). Characterization of a self-assembled monolayer based on a calix[4]crown-5 derivate: fabrication of a chemical sensor sensitive to calcium Journal of Nanoscience and Nanotechnology 10, (1), 413-420

The synthesis and self-assembled monolayer (SAM) formation of a calix[4]crown-5 derivative are reported. Several techniques, including electrochemistry, atomic force microscopy (AFM), Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and contact angle measurements have been applied to characterise the monolayer film designed for chemical sensor applications. The recognition properties of this SAM for metal cations has been investigated using impedance spectroscopy (IS) showing an electrochemical response proportional to calcium ion concentration in the range from 10(-7) M to 10(-2) M. This response is related to microscopic changes at the gold surface induced by selective binding by the immobilised calixarene.

Keywords: Calixarenes, Self assembled monolayer, Micro-contact printing, Atomic force microscopy, Impedance spectroscopy

Caballero, D., Villanueva, G., Plaza, J. A., Mills, C. A., Samitier, J., Errachid, A., (2010). Sharp high-aspect-ratio AFM tips fabricated by a combination of deep reactive ion etching and focused ion beam techniques Journal of Nanoscience and Nanotechnology 10, (1), 497-501

The shape and dimensions of an atomic force microscope tip are crucial factors to obtain high resolution images at the nanoscale. When measuring samples with narrow trenches, inclined sidewalls near 90 or nanoscaled structures, standard silicon atomic force microscopy (AFM) tips do not provide satisfactory results. We have combined deep reactive ion etching (DRIE) and focused ion beam (FIB) lithography techniques in order to produce probes with sharp rocket-shaped silicon AFM tips for high resolution imaging. The cantilevers were shaped and the bulk micromachining was performed using the same DRIE equipment. To improve the tip aspect ratio we used FIB nanolithography technique. The tips were tested on narrow silicon trenches and over biological samples showing a better resolution when compared with standard AFM tips, which enables nanocharacterization and nanometrology of high-aspect-ratio structures and nanoscaled biological elements to be completed, and provides an alternative to commercial high aspect ratio AFM tips.

Keywords: Atomic-Force Microscope, Carbon nanotube tips, Probes, Roughness, Cells, Microfabrication, Calibration, Surfaces

Trepat, X., Wasserman, M. R., Angelini, T. E., Millet, E., Weitz, D. A., Butler, J. P., Fredberg, J. J., (2009). Physical forces during collective cell migration Nature Physics 5, (6), 426-430

Fundamental biological processes including morphogenesis, tissue repair and tumour metastasis require collective cell motions(1-3), and to drive these motions cells exert traction forces on their surroundings(4). Current understanding emphasizes that these traction forces arise mainly in 'leader cells' at the front edge of the advancing cell sheet(5-9). Our data are contrary to that assumption and show for the first time by direct measurement that traction forces driving collective cell migration arise predominately many cell rows behind the leading front edge and extend across enormous distances. Traction fluctuations are anomalous, moreover, exhibiting broad non-Gaussian distributions characterized by exponential tails(10-12). Taken together, these unexpected findings demonstrate that although the leader cell may have a pivotal role in local cell guidance, physical forces that it generates are but a small part of a global tug-of-war involving cells well back from the leading edge.

Keywords: Focal adhesions, Granular matter, Bead packs, Morphogenesis, Sheets, Actin, Fluctuations, Fibroblasts, Microscopy, Diversity

Fumagalli, L., Ferrari, G., Sampietro, M., Gomila, G., (2009). Quantitative nanoscale dielectric microscopy of single-layer supported biomembranes Nano Letters 9, (4), 1604-1608

We present the experimental demonstration of low-frequency dielectric constant imaging of single-layer supported biomembranes at the nanoscale. The dielectric constant image has been quantitatively reconstructed by combining the thickness and local capacitance obtained using a scanning force microscope equipped with a sub-attofarad low-frequency capacitance detector. This work opens new possibilities for studying bioelectric phenomena and the dielectric properties of biological membranes at the nanoscale.

Keywords: Atomic-force microscopy, Nnear-field microscopy, Purple membrane, Scanning capacitance, Biological-systems, Fluid, Spectroscopy, Resolution, Proteins, Dynamics

van Zanten, T. S., Cambi, A., Koopman, M., Joosten, B., Figdor, Carl G., Garcia-Parajo, M. F., (2009). Hotspots of GPI-anchored proteins and integrin nanoclusters function as nucleation sites for cell adhesion Proceedings of the National Academy of Sciences of the United States of America 106, (44), 18557-18562

Recruitment of receptor proteins to lipid rafts has been proposed as an important mechanism to regulate their cellular function. In particular, rafts have been implicated in regulation of integrin-mediated cell adhesion, although the underlying mechanism remains elusive. We used single-molecule near-field optical microscopy (NSOM) with localization accuracy of approximately 3 nm, to capture the spatio-functional relationship between the integrin LFA-1 and raft components (GPI-APs) on immune cells. Dual color nanoscale imaging revealed the existence of a nanodomain GPI-AP subpopulation that further concentrated in regions smaller than 250 nm, suggesting a hierarchical prearrangement of GPI-APs on resting monocytes. We previously demonstrated that in quiescent monocytes, LFA-1 preorganizes in nanoclusters. We now show that integrin nanoclusters are spatially different but reside proximal to GPI-AP nanodomains, forming hotspots on the cell surface. Ligand-mediated integrin activation resulted in an interconversion from monomers to nanodomains of GPI-APs and the generation of nascent adhesion sites where integrin and GPI-APs colocalized at the nanoscale. Cholesterol depletion significantly affected the reciprocal distribution pattern of LFA-1 and GPI-APs in the resting state, and LFA-1 adhesion to its ligand. As such, our data demonstrate the existence of nanoplatforms as essential intermediates in nascent cell adhesion. Since raft association with a variety of membrane proteins other than LFA-1 has been documented, we propose that hotspots regions enriched with raft components and functional receptors may constitute a prototype of nanoscale inter-receptor assembly and correspond to a generic mechanism to offer cells with privileged areas for rapid cellular function and responses to the outside world.

Keywords: Integrin LFA-1, Membrane nanocompartments, Near-field scanning optical microscopy (NSOM), Single molecule detection

Pla, D., Sischka, A., Albericio, F., Alvarez, M., Fernàndez-Busquets, X., Anselmetti, D., (2009). Optical-tweezers study of topoisomerase inhibition Small 5, (11), 1269-1272

Optical tweezers force-stretching of highly nicked dsDNA, as indicated by the large hysteresis area (black and red curves). Topoisomerase activity is evidenced by a higher level plateau and a complete vanishing of the overstretching hysteresis (green curve), indicating total repair of the DNA nicks. The arrow indicates a drop in the stretching curve resulting from topoisomerase cleavage during the cycle.

Keywords: Atomic force microscopy, DNA, Lamellarin D, Optical tweezers, Topoisomerase

Caballero-Briones, F., Artes, J. M., Diez-Perez, I., Gorostiza, P., Sanz, F., (2009). Direct observation of the valence band edge by in situ ECSTM-ECTS in p-type Cu2O layers prepared by copper anodization Journal of Physical Chemistry C 113, (3), 1028-1036

Polycrystalline Cu2O layers have been selectively grown by electrochemical anodization of polycrystalline Cu electrodes in an alkaline medium (pH 12.85). Uniform layers with thicknesses around 100 nm have been obtained. Using electrochemical impedance spectroscopy, it was concluded that the Cu2O films behave as a p-type semiconductor. The Mott-Schottky plot gives a value for the flat band potential of U-FB = -255 mV vs silver/silver chloride electrode (SSC), an estimated carrier density N-A = 6.1 x 10(17) cm(-3), and the space charge layer width was calculated to be W-SCL = 9 nm at a band bending of 120 mV. The electronic structure of the Cu vertical bar Cu2O vertical bar electrolyte interface was for the first time probed by in situ electrochemical tunneling spectroscopy. The use of in situ electrochemical scanning tunneling microscopy allows us to directly observed the valence band edge and determine its position against the absolute energy scale to be E-VB = -4.9 eV. Finally, we constructed a quantitative electronic diagram of the Cu vertical bar Cu2O vertical bar electrolyte interface, where the positions of the valence and conduction band edges are depicted, as well as the edge of the previously reported electronic subband.

Keywords: 0.1 m NaOH, Electrochemical tunneling spectroscopy, Cuprous-oxide films, Anodic-oxidation, Electronic-structure, Alkaline-solution, Aqueous-solution, Initial-stages, Passive film, Thin-films

Gramse, G., Casuso, I., Toset, J., Fumagalli, L., Gomila, G., (2009). Quantitative dielectric constant measurement of thin films by DC electrostatic force microscopy Nanotechnology 20, (39), 395702

A simple method to measure the static dielectric constant of thin films with nanometric spatial resolution is presented. The dielectric constant is extracted from DC electrostatic force measurements with the use of an accurate analytical model. The method is validated here on thin silicon dioxide films (8 nm thick, dielectric constant approximately 4) and purple membrane monolayers (6 nm thick, dielectric constant approximately 2), providing results in excellent agreement with those recently obtained by nanoscale capacitance microscopy using a current-sensing approach. The main advantage of the force detection approach resides in its simplicity and direct application on any commercial atomic force microscope with no need of additional sophisticated electronics, thus being easily available to researchers in materials science, biophysics and semiconductor technology.

Keywords: Roscopy, Membrane, Tip, Polarizability, Polarization, Resolution, Nanotubes, Charge

Rico, P., Rodriguez Hernandez, J. C., Moratal, D., Altankov, G., Monleon Pradas, M., Salmeron-Sanchez, M., (2009). Substrate-induced assembly of fibronectin into networks. Influence of surface chemistry and effect on osteoblast adhesion Tissue Engineering Part A 15, (00), 1-11

The influence of surface chemistry -substrates with controlled surface density of -OH groups- on fibronectin conformation and distribution is directly observed by Atomic Force Microscopy (AFM). FN fibrillogenesis, which is known to be a process triggered by interaction with integrins, is shown in our case to be induced by the substrate (in absence of cells), which is able to enhance FN-FN interactions leading to the formation of a protein network on the material surface. This phenomenon depends both on surface chemistry and protein concentration. The level of the FN fibrillogenesis was quantified by calculating the fractal dimension of the adsorbed protein from image analysis of the AFM results. The total amount of adsorbed FN is obtained by making use of a methodology which employs western-blotting combined with image analysis of the corresponding protein bands, with the lowest sensitivity threshold equal to 15 ng of adsorbed protein. Furthermore, FN adsorption is correlated to human osteoblast adhesion through morphology and actin cytoskeleton formation. Actin polymerization is in need of the formation of the protein network on the substrate's surface. Cell morphology is more rounded (as quantified by calculating the circularity of the cells by image analysis) the lower the degree of FN fibrillogenesis on the substrate.

Keywords: Cell-adhesion, Conformational-changes, Electron-microscopy, Protein adsorption, Fractal dimension, Integrin binding, Biocompatibility, Monolayers, Matrix, Fibrillogenesis

Puig, F., Gavara, N., Sunyer, R., Carreras, A., Farre, R., Navajas, D., (2009). Stiffening and contraction induced by dexamethasone in alveolar epithelial cells Experimental Mechanics 49, (1), 47-55

The structural integrity of the alveolar monolayer, which is compromised during lung inflammation, is determined by the balance between cell-cell and cell-matrix tethering forces and the centripetal forces owing to cell viscoelasticity and contraction. Dexamethasone is an anti-inflammatory glucocorticoid with protective effects in lung injury. To determine the effects of Dexamethasone on the stiffness and contractility of alveolar epithelial cells. Cell stiffness (G') and average traction exerted by the cell (T) were measured by magnetic twisting cytometry and by traction microscopy, respectively. A549 cells were treated 24 h with Dexamethasone (1 mu M) or vehicle (control). G' and T were measured before and 5 min after challenge with the inflammatory mediator Thrombin (0.5 U/ml). Changes induced by Dexamethasone in actin cytoskeleton polymerization were assessed by the fluorescent ratio between F-actin and G-actin obtained by staining cells with phalloidin and DNase I. Dexamethasone significantly increased G' and T by 56% (n = 11; p < 0.01) and by 80% (n = 17; p < 0.05), respectively. Dexamethasone also increased F/G-actin ratio from 2.68 +/- 0.07 to 2.96 +/- 0.09 (n = 10; p < 0.05). The relative increase in stiffness and contraction induced by Thrombin in control cells was significantly (p < 0.05) reduced by Dexamethasone treatment: from 190 to 98% in G' and from 318 to 105% in T. The cytoskeleton remodelling and the increase in cell stiffness and contraction induced by Dexamethasone could account for its protective effect in the alveolar epithelium when subjected to inflammatory challenge.

Keywords: Cell mechanics, Cytoskeleton, Magnetic twisting cytometry, Traction microscopy, Respiratory diseases

Barreiros dos Santos, M., Sporer, C., Sanvicens, N., Pascual, N., Errachid, A., Martinez, E., Marco, M. P., Teixeira, V., Samiter, J., (2009). Detection of pathogenic Bacteria by Electrochemical Impedance Spectroscopy: Influence of the immobilization strategies on the sensor performance Procedia Chemistry 23rd Eurosensors Conference (ed. Brugger, J., Briand, D.), Elsevier Science, BV (Lausanne, Switzerland) 1, 1291-1294

Electrochemical impedance spectroscopy (EIS) is applied to detect pathogenic E. coli O157:H7 bacteria via a label free immunoassay-based detection method. Polyclonal anti-E.coli antibodies (PAb) are immobilized onto gold electrodes following two different strategies, via chemical bond formation between antibody amino groups and a carboxylic acid containing self-assembled molecular monolayer (SAM) and alternatively by linking a biotinylated anti-E. coli to Neutravidin on a mixed-SAM. Impedance spectra for sensors of both designs for increasing concentrations of E. coli are recorded in phosphate buffered saline (PBS). The Nyquist plots can be modeled with a Randle equivalent circuit, identifying the charge transfer resistance RCT as the relevant concentration dependent parameter. Sensors fabricated from both designs are able to detect very low concentration of E. coli with limits of detection as low as 10-100 cfu/ml. The influence of the different immobilization protocols on the sensor performance is evaluated in terms of sensitivity, dynamic range and resistance against nonspecific absorption.

Keywords: Bacteria detection, Biosensors, E-coli, Impedance spectroscopy

Guaus, E., Torrent-Burgues, J., Zine, N., Errachid, A., (2009). Glassy carbon electrode modified with a langmuir-blodgett film of a thiomacrocyclic ionophore for Cu(II) recognition Sensor Letters 6th Maghreb-Europe Meeting on Materials and Their Applications for Devices and Physical, Chemical and Biological Sensors , AMER SCIENTIFIC PUBLISHERS (Rabat, Morocco) 7, (5), 1006-1011

Nanometric films of a thiomacrocyclic ionophore, 4-phenyl-4-sulfide-11(1- oxodecyl)-1,7-dithia-11-aza-4-phosphacyclotetradecane (ThM), have been deposited on the surface of a Glassy Carbon Electrode (GCE) by the Langmuir-Blodgett (LB) technique. The films have been characterised by using AFM. The influence of these modified electrodes (GCE-ThM) on the reduction of Cu(II) ions has been investigated by using Cyclic Voltammetry (CV) and Electrochemical Impedance Spectroscopy (EIS), and its sensor response has been checked. The CV and EIS responses of LB films on GCE indicate that these ThM films are sensitive to Cu(II) ions. The analysis by EIS of the interference of some other cations, as Mg(II) and Co(II), shows that LB films of ThM can be used for specific Cu(II) sensing applications.

Keywords: Cu(II) sensor, Cyclic voltammetry, Electrochemical impedance spectroscopy, Langmuir-blodgett films

de Bakker, Barbel I., Bodnar, Andrea, van Dijk, Erik M. H. P., Vamosi, Gyorgy, Damjanovich, Sandor, Waldmann, Thomas A., van Hulst, Niek F., Jenei, Attila, Garcia-Parajo, M. F., (2008). Nanometer-scale organization of the alpha subunits of the receptors for IL2 and IL15 in human T lymphoma cells Journal of Cell Science 121, (5), 627-633

Interleukin 2 and interleukin 15 (IL2 and IL15, respectively) provide quite distinct contributions to T-cell-mediated immunity, despite having similar receptor composition and signaling machinery. As most of the proposed mechanisms underlying this apparent paradox attribute key significance to the individual {alpha}-chains of IL2 and IL15 receptors, we investigated the spatial organization of the receptors IL2R{alpha} and IL15R{alpha} at the nanometer scale expressed on a human CD4+ leukemia T cell line using single-molecule-sensitive near-field scanning optical microscopy (NSOM). In agreement with previous findings, we here confirm clustering of IL2R{alpha} and IL15R{alpha} at the submicron scale. In addition to clustering, our single-molecule data reveal that a non-negligible percentage of the receptors are organized as monomers. Only a minor fraction of IL2R{alpha} molecules reside outside the clustered domains, whereas [~]30% of IL15R{alpha} molecules organize as monomers or small clusters, excluded from the main domain regions. Interestingly, we also found that the packing densities per unit area of both IL2R{alpha} and IL15R{alpha} domains remained constant, suggesting a `building block' type of assembly involving repeated structures and composition. Finally, dual-color NSOM demonstrated co-clustering of the two {alpha}-chains. Our results should aid understanding the action of the IL2R-IL15R system in T cell function and also might contribute to the more rationale design of IL2R- or IL15R-targeted immunotherapy agents for treating human leukemia.

Keywords: Near-field scanning optical microscopy (NSOM), Interleukin receptors IL2R, IL15R, Single-molecule detection, Nanometer-scale membrane organization

Caballero-Briones, F., Palacios-Padros, A., Pena, J. L., Sanz, F., (2008). Phase tailored, potentiodynamically grown P-Cu2-xTe/Cu layers Electrochemistry Communications 10, (11), 1684-1687

In this work we successfully prepared p-type semiconducting Cu2-xTe layers on Cu substrates by applying a potential multistep signal. Spontaneously deposited tellurium layers were reduced in a single cathodic sweep. The X-ray diffraction characterization showed the presence of single-phased, crystalline Cu2-xTe in the weissite form. A further anodization step allows crystallization of several phases such as CU1.75Te, Cu0.664Te0.336 and CU7Te4. This type of sample was found to be photoactive. The prepared films are p-type and have carrier concentrations in the order of 10(21) CM-3, suitable for CdTe-CU2-xTe contacts.

Keywords: Copper telluride, Electrochemical signal, XRD, Morphology, EIS, Photocurrent, Telluride thin-films, Solar cells, Deposition, Cu

Navarro, M., Benetti, E. M., Zapotoczny, S., Planell, J. A., Vancso, G. J., (2008). Buried, covalently attached RGD peptide motifs in poly(methacrylic acid) brush layers: The effect of brush structure on cell adhesion Langmuir 24, (19), 10996-11002

Iniferter-mediated surface-initiated photopolymerization was used to graft poly(methacrylic acid) (PMAA) brush layers obtained from surface-attached iniferters in self-assembled monolayers to a gold surface. The tethered chains were subsequently functionalized with the cell-adhesive arginine-glycine-aspartic acid (RGD) motif. The modified brushes were extended by reinitiating the polymerization to obtain an additional layer of PMAA, thereby burying the peptide-functionalized segments inside the brush structure. Contact angle measurements and Fourier transform infrared (FTIR) spectroscopy were employed to characterize the wettability and the chemical properties of these platforms. Time of flight secondary ion mass spectroscopy (TOF-SIMS) measurements were performed to monitor the chemical composition of the polymer layer as a function of the distance to the gold surface and obtain information concerning the depth of the RGD motifs inside the brush structure. The brush thickness was evaluated as a function of the polymerization (i.e.. UV-irradiation) time with atomic force microscopy (AFM) and ellipsometry. Cell adhesion tests employing human osteoblasts were performed on substrates with the RGD peptides exposed at the surface as well as covered by a PMAA top brush layer. Immunofluorescence studies demonstrated a variation of the cell morphology as a function of the position of the peptide units along the grafted chains.

Keywords: Ion mass-spectrometry, Transfer radical polymerization, Asymmetric diblock copolymers, Arg-gly-asp, Swelling behaviour, Endothelial-cells, Thin-films, fibronectin, Surfaces, SIMS

Oncins, G., Torrent-Burgues, J., Sanz, F., (2008). Nanomechanical properties of arachidic acid Langmuir-Blodgett films Journal of Physical Chemistry C 112, (6), 1967-1974

The nanomechanical properties of Langmuir-Blodgett monolayers of arachidic acid extracted at surface pressures of 1, 15, and 35 mN/m and deposited on mica were investigated by atomic force microscopy, force spectroscopy, and lateral force microscopy. It was experimentally demonstrated that the arachidic acid molecular orientation depends on the extraction pressure. According to this, tilting angles of 50, 34, and 22 degrees with respect to the surface perpendicular were detected and identified as conformations that maximize van der Waals interactions between the arachidic acid alkyl chains. The vertical force needed to puncture the monolayers with the AFM tip strongly depends on the molecular tilting angles attained at different monolayer extraction surface pressures, obtaining values that range from 13.07 +/- 3.24 nN for 50 degrees to 22.94 +/- 5.49 nN for 22 degrees tilting angles. The different molecular interactions involved in the monolayer cohesion are discussed and quantitatively related to the experimental monolayer breakthrough forces. The friction measurements performed from low vertical forces up to monolayer disruption reveal the existence of three well-defined regimes: first, a low friction response due to the elastic deformation of the monolayer, which is followed by a sharp increase in the friction force due to the onset of a sudden plastic deformation. The last regime corresponds to the monolayer rupture and the contact between tip and substrate. The friction coefficient of the substrate is seen to depend on the monolayer extraction pressure, a fact that is discussed in terms of the relationship between the sample compactness and its rupture mechanism.

Keywords: AFM, SAM, Reflection-absortion spectroscopy, Lipid-bilayers, Frictional-properies, Molecular-structure, Thermal behavior, Nanometer-scale, Chain-length, LB films

Manara, S., Paolucci, F., Palazzo, B., Marcaccio, M., Foresti, E., Tosi, G., Sabbatini, S., Sabatino, P., Altankov, G., Roveri, N., (2008). Electrochemically-assisted deposition of biomimetic hydroxyapatite-collagen coatings on titanium plate Inorganica Chimica Acta 361, (6), 1634-1645

A biomimetic bone-like composite, made of self-assembled collagen fibrils and carbonate hydroxyapatite nanocrystals, has been performed by an electrochemically-assisted deposition on titanium plate. The electrolytic processes have been carried out using a single type I collagen molecules suspension in a diluted Ca(NO3)(2) and NH4H2PO4 solution at room temperature and applying a constant current for different periods of time. Using the same electrochemical conditions, carbonate hydroxyapatite nanocrystals or reconstituted collagen. brils coatings were obtained. The reconstituted collagen. brils, hydroxyapatite nanocrystals and collagen fibrils/apatite nanocrystals coatings have been characterized chemically, structurally and morphologically, as well as for their ability to bind fibronectin (FN). Fourier Transform Infrared microscopy has been used to map the topographic distribution of the coating components at different times of electrochemical deposition, allowing to single out the individual deposition steps. Moreover, roughness of Ti plate has been found to affect appreciably the nucleation region of the inorganic nanocrystals. Laser scanning confocal microscopy has been used to characterize the FN adsorption pattern on a synthetic biomimetic apatitic phase, which exhibits a higher affinity when it is inter-grown with the collagen fibrils. The results offer auspicious applications in the preparation of medical devices such as biomimetic bone-like composite-coated metallic implants.

Keywords: Hydroxyapatite-collagen coating, Electrochemically-assisted deposition, Micro-imaging FTIR spectroscopy, Laser scanning confocal microscopy, Biomimetic crystal growth, Fibronectin binding

Cho, S., Castellarnau, M., Samitier, J., Thielecke, H., (2008). Dependence of impedance of embedded single cells on cellular behaviour Sensors 8, (2), 1198-1211

Non-invasive single cell analyses are increasingly required for the medical diagnostics of test substances or the development of drugs and therapies on the single cell level. For the non-invasive characterisation of cells, impedance spectroscopy which provides the frequency dependent electrical properties has been used. Recently, microfludic systems have been investigated to manipulate the single cells and to characterise the electrical properties of embedded cells. In this article, the impedance of partially embedded single cells dependent on the cellular behaviour was investigated by using the microcapillary. An analytical equation was derived to relate the impedance of embedded cells with respect to the morphological and physiological change of extracellular interface. The capillary system with impedance measurement showed a feasibility to monitor the impedance change of embedded single cells caused by morphological and physiological change of cell during the addition of DMSO. By fitting the derived equation to the measured impedance of cell embedded at different negative pressure levels, it was able to extrapolate the equivalent gap and gap conductivity between the cell and capillary wall representing the cellular behaviour.

Keywords: Frequency-domain, Spectroscopy, Erythrocytes, Biosensor, Membrane, System

Martinez, E., Engel, E., Lopez-Iglesias, C., Mills, C. A., Planell, J. A., Samitier, J., (2008). Focused ion beam/scanning electron microscopy characterization of cell behavior on polymer micro-/nanopatterned substrates: A study of cell-substrate interactions Micron 39, (2), 111-116

Topographic micro and nanostructures can play an interesting role in cell behaviour when cells are cultured on these kinds of patterned substrates. It is especially relevant to investigate the influence of the nanometric dimensions topographic features on cell morphology, proliferation, migration and differentiation. To this end, some of the most recent fabrication technologies, developed for the microelectronics industry, can be used to produce well-defined micro and nanopatterns on biocompatible polymer substrates. In this work, osteoblast-like cells are grown on poly(methyl methacrylate) substrates patterned by nanoimprint lithography techniques. Examination of the cell-substrate interface can reveal important details about the cell morphology and the distribution of the focal contacts on the substrate surface. For this purpose, a combination of focused ion beam milling and scanning electron microscopy techniques has been used to image the cell-substrate interface. This technique, if applied to samples prepared by freeze-drying methods, allows high-resolution imaging of cross-sections through the cell and the substrate, where the interactions between the nanopatterned substrate, the cell and the extracellular matrix, which are normally hidden by the bulk of the cell, can be studied.

Keywords: Electron microscopy, Interface, Nanotopography, Osteoblast, Adhesion molecule, Cell morphology

Charles-Harris, M., Koch, M. A., Navarro, M., Lacroix, D., Engel, E., Planell, J. A., (2008). A PLA/calcium phosphate degradable composite material for bone tissue engineering: an in vitro study Journal of Materials Science-Materials in Medicine 19, (4), 1503-1513

Biodegradable polymers reinforced with an inorganic phase such as calcium phosphate glasses may be a promising approach to fulfil the challenging requirements presented by 3D porous scaffolds for tissue engineering. Scaffolds' success depends mainly on their biological behaviour. This work is aimed to the in vitro study of polylactic acid (PLA)/CaP glass 3D porous constructs for bone regeneration. The scaffolds were elaborated using two different techniques, namely solvent-casting and phase-separation. The effect of scaffolds' micro and macrostructure on the biological response of these scaffolds was assayed. Cell proliferation, differentiation and morphology within the scaffolds were studied. Furthermore, polymer/glass scaffolds were seeded under dynamic conditions in a custom-made perfusion bioreactor. Results indicate that the final architecture of the solvent-cast or phase separated scaffolds have a significant effect on cells' behaviour. Solvent-cast scaffolds seem to be the best candidates for bone tissue engineering. Besides, dynamic seeding yielded a higher seeding efficiency in comparison with the static method.

Keywords: Biocompatible Materials/ chemistry, Bone and Bones/ metabolism, Calcium Phosphates/ chemistry, Cell Differentiation, Cell Proliferation, Humans, Lactic Acid/ chemistry, Microscopy, Confocal, Microscopy, Electron, Scanning, Osteoblasts/metabolism, Permeability, Polymers/ chemistry, Porosity, Solvents/chemistry, Tissue Engineering/ methods

Torrent-Burgues, J., Oncins, G., Sanz, F., (2008). Study of mixed Langmuir and Langmuir-Blodgett films of dissimilar components by AFM and force spectroscopy Colloids and Surfaces a-Physicochemical and Engineering Aspects 12th International Conference on Organized Molecular Films , Elsevier Science (Krakow, Poland) 321, (1-3), 70-75

In this study the structure of mixed Langmuir-Blodgett (LB) monolayers has been investigated using atomic force microscopy, lateral force microscopy and force spectroscopy, as well as the characteristics of the Langmuir monolayers by surface pressure-area isotherms and Brewster angle microscopy. Mixed films were of dissimilar compounds, a fatty acid such as arachidic acid and a macrocyclic compound. The mixture forms separated phases, but some degree of partial miscibility occurs, with domains at the micro-scale that have different nanomechanical and nanotribological properties. LB films transferred at the same surface pressure show different characteristics depending on the composition. The higher domains correspond to arachidic acid and some of these domains show the presence of two phases, which have been identified as phases with discrete molecular tilting angles.

Keywords: Mixed monolayers, Pressure-area isotherm, Langmuir-Blodgett, AFM, Force spectroscopy

De Bakker, B. I., De Lange, F., Cambi, A., Korterik, J. P., Van Dijk, E. M. H. P., Van Hulst, N. F., Figdor, C. G., Garcia-Parajo, M. F., (2007). Nanoscale organization of the pathogen receptor DC-SIGN mapped by single-molecule high-resolution fluorescence microscopy ChemPhysChem 8, (10), 1473-1480

DC-SIGN, a C-type lectin exclusively expressed on dendritic cells (DCs), plays an important role in pathogen recognition by binding with high affinity to a large variety of microorganisms. Recent experimental evidence points to a direct relation between the function of DC-SIGN as a viral receptor and its spatial arrangement on the plasma membrane. We have investigated the nanoscale organization of fluorescently labeled DC-SIGN on intact isolated DCs by means of near-field scanning optical microscopy (NSOM) combined with single-molecule detection. Fluorescence spots of different intensity and size have been directly visualized by optical means with a spatial resolution of less than 100 nm. Intensity- and size-distribution histograms of the DC-SIGN fluorescent spots confirm that approximately 80% of the receptors are organized in nanosized domains randomly distributed on the cell membrane. Intensity-size correlation analysis revealed remarkable heterogeneity in the molecular packing density of the domains. Furthermore, we have mapped the intermolecular organization within a dense cluster by means of sequential NSOM imaging combined with discrete single-molecule photobleaching. In this way we have determined the spatial coordinates of 13 different individual dyes, with a localization accuracy of 6 nm. Our experimental observations are all consistent with an arrangement of DC-SIGN designed to maximize its chances of binding to a wide range of microorganisms. Our data also illustrate the potential of NSOM as an ultrasensitive, high-resolution technique to probe nanometer-scale organization of molecules on the cell membrane.

Keywords: High-resolution optical microscopy, Lectins, Membranes, Receptors, Single-molecule studies

Díez-Pérez, Ismael, Guell, Aleix Garcia, Sanz, Fausto, Gorostiza, Pau, (2006). Conductance maps by electrochemical tunneling spectroscopy to fingerprint the electrode electronic structure Analytical Chemistry 78, (20), 7325-7329

We describe a methodology to perform reliable tunneling spectroscopy in electrochemical media. Sequential in situ tunneling spectra are recorded while the electrochemical potential of the electrode is scanned. Spectroscopic data are presented as conductance maps or conductograms that show the in situ electronic structure of an electrode surface while it undergoes an electrochemical reaction. The conductance map or conductogram represents the redox fingerprint of an electrode/liquid interface in a specific medium and can serve to predict its electrochemical behavior in a quantitative energy scale. The methodology is validated studying the reversible oxidation and passivity of an iron electrode in borate buffer, and we describe the main quantitative information that can be extracted concerning the semiconducting properties of the Fe passive film. This methodology is useful to study heterogeneous catalysis, electrochemical sensing and bioelectronic systems.

Keywords: Passive film, Oxide-film, Stainless-steel, Iron, Microscope, Surfaces, STM, Probes

Díez-Pérez, Ismael, Sanz, Fausto, Gorostiza, Pau, (2006). Electronic barriers in the iron oxide film govern its passivity and redox behavior: Effect of electrode potential and solution pH Electrochemistry Communications 8, (10), 1595-1602

We have measured in situ the electronic conductance spectra of the passive film formed on an Fe electrode immersed in a borate buffer solution using electrochemical tunneling spectroscopy (ECTS) and electrochemical impedance spectroscopy (EIS) techniques, and we have followed their changes as the electrode is electrochemically oxidized and reduced. We demonstrate that pre-passive Fe(II) oxide and the passive Fe(II)/Fe(III) film, behave as p- and n-type semiconductors, respectively and that their reversible inter-conversion is mediated by the availability of free charge carriers on the electrode surface. ECTS spectra have been also modeled to obtain the main electrochemical kinetic parameters of the electron transfer through both p-Fe(II) and n-Fe(III) oxides at different sample potentials and pHs values. We find that the electronic energy barrier in the oxide and its dependence with electrode potential and solution pH, determine the reactivity and passivity of iron.

Keywords: Electrochemical tunneling spectroscopy, Fe passivity Electronic energy barriers, pH effect on passivity

Díez-Pérez, Ismael, Vericat, Carolina, Gorostiza, Pau, Sanz, Fausto, (2006). The iron passive film breakdown in chloride media may be mediated by transient chloride-induced surface states located within the band gap Electrochemistry Communications 8, (4), 627-632

Despite its tremendous scientific and economic impact, the mechanism that triggers metal passive film breakdown in the presence of aggressive ions remains under discussion. We have studied the iron passive film in chloride media using X-ray photoelectron spectroscopy (XPS), electrochemical impedance spectroscopy and electrochemical tunneling spectroscopy (ECTS). Ex situ XPS reveal that the film consists exclusively of an Fe(III) oxide without chloride content. In situ ECTS has been used to build up conductance maps of the Fe electrode during its electrochemical oxidation in a borate buffer solution and its breakdown when the film is grown in the presence of chloride. This conductograms provide direct and in situ experimental evidence of chloride-induced surface states within the band gap of the oxide film (~3.3eV). These states enable new charge exchange pathways that allow hole capture at the surface of the n-type Fe(III) oxide. The blocking of VB processes that occurs in the iron passive film is no longer present in chloride media, and electrode corrosion can proceed through these new states. We propose a simple 3-step mechanism for the process, in which chloride anions form an oxidizing Fe(II) surface intermediate but do not participate directly in the reaction.

Keywords: Electrochemical tunneling spectroscopy, Electronic band structure, Fe passive film, Aqueous chloride corrosion, Semiconductor decomposition, Interface states