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Publications

by Keyword: microscopy

Moussa, Dina G., Sharma, Ashok K., Mansour, Tamer A, Witthuhn, Bruce, Perdigão, Jorge, Rudney, Joel D., Aparicio, Conrado, Gomez, Andres, (2022). Functional signatures of ex-vivo dental caries onset Journal Of Oral Microbiology 14, 2123624

Lagunas, Anna, Belloir, Christine, Briand, Loïc, Gorostiza, Pau, Samitier, Josep, (2022). Determination of the nanoscale electrical properties of olfactory receptor hOR1A1 and their dependence on ligand binding: Towards the development of capacitance-operated odorant biosensors Biosensors & Bioelectronics 218, 114755

Martens KJA, Gobes M, Archontakis E, Brillas RR, Zijlstra N, Albertazzi L, Hohlbein J, (2022). Enabling Spectrally Resolved Single-Molecule Localization Microscopy at High Emitter Densities Nano Letters 22, 8618-8625

Single-molecule localization microscopy (SMLM) is a powerful super-resolution technique for elucidating structure and dynamics in the life- and material sciences. Simultaneously acquiring spectral information (spectrally resolved SMLM, sSMLM) has been hampered by several challenges: an increased complexity of the optical detection pathway, lower accessible emitter densities, and compromised spatio-spectral resolution. Here we present a single-component, low-cost implementation of sSMLM that addresses these challenges. Using a low-dispersion transmission grating positioned close to the image plane, the +1stdiffraction order is minimally elongated and is analyzed using existing single-molecule localization algorithms. The distance between the 0th and 1st order provides accurate information on the spectral properties of individual emitters. This method enables a 5-fold higher emitter density while discriminating between fluorophores whose peak emissions are less than 15 nm apart. Our approach can find widespread use in single-molecule applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

JTD Keywords: Multicolor imaging, Point accumulation for imaging in nanoscale topography (paint), Single-molecule förster resonance energy transfer (smfret), Single-molecule spectroscopy, Stochastic optical reconstruction microscopy (storm)


Altay G, Abad-Lázaro A, Gualda EJ, Folch J, Insa C, Tosi S, Hernando-Momblona X, Batlle E, Loza-Álvarez P, Fernández-Majada V, Martinez E, (2022). Modeling Biochemical Gradients In Vitro to Control Cell Compartmentalization in a Microengineered 3D Model of the Intestinal Epithelium Advanced Healthcare Materials , 2201172

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.

JTD Keywords: biomolecular gradients, colon, crypt, engineering organoids, hydrogels, identification, in silico modeling, intestinal stem cell niches, light sheet fluorescence microscopy, niche, permeability, photolithography, regeneration, villus, wnt, 3d architectures, Biomolecular gradients, Engineering organoids, In silico modeling, Intestinal stem cell niches, Light sheet fluorescence microscopy, Photolithography, Stem-cell


Archontakis, E, Woythe, L, van Hoof, B, Albertazzi, L, (2022). Mapping the relationship between total and functional antibodies conjugated to nanoparticles with spectrally-resolved direct stochastic optical reconstruction microscopy (SR-dSTORM) Nanoscale Advances 4, 4402-4409

Antibody-functionalized nanoparticles (NPs) have shown numerous benefits in drug delivery and biosensing, improving the specificity of cell targeting and analyte detection, respectively. However, one of the main challenges is the lack of control over antibody orientation on the NP surface. Popular and easy conjugation strategies, such as carbodiimide-based conjugations, lead to a random orientation of antibodies on the NPs, compromising ligand functionality and contributing to undesired biological effects and reduced target recognition. While new methods for more controlled NP functionalization have been proposed, there is a lack of techniques that can elucidate the orientation of the antibodies at the single-particle level to determine the conjugation outcome and, therefore, the NPs' potential in selective targeting. Here, spectrally-resolved direct stochastic optical reconstruction microscopy (SR-dSTORM), an optical super-resolution technique, is introduced to quantify the relationship between total and functional NP conjugated cetuximab antibodies at the single-particle level. An evident single-particle heterogeneity in total and functional cetuximab is observed, leading to particles with different functional : total ratios. Additionally, the results indicate that the functional : total ratio of cetuximab highly depends on the conjugated cetuximab concentration. Overall, SR-dSTORM represents a direct approach for the NP structure-functionality relationship quantification, providing a platform to improve antibody-conjugated NPs characterization and facilitating their rational design.

JTD Keywords: Heterogeneity, Superresolution microscopy


Wang, Yuyang, Soto Rodriguez, Paul ED., Woythe, Laura, Sánchez, Samuel, Samitier, Josep, Zijlstra, Peter, Albertazzi, Lorenzo, (2022). Multicolor Super-Resolution Microscopy of Protein Corona on Single Nanoparticles Acs Applied Materials & Interfaces 14, 37345-37355

Riera, Roger, Tauler, Jana, Feiner Gracia, Natàlia, Borrós, Salvador, Fornaguera, Cristina, Albertazzi, Lorenzo, (2022). Complex pBAE Nanoparticle Cell Trafficking: Tracking Both Position and Composition Using Super Resolution Microscopy Chemmedchem 17, e202100633

Nanomedicine emerged some decades ago with the hope to be the solution for most unmet medical needs. However, tracking materials at nanoscale is challenging to their reduced size, below the resolution limit of most conventional techniques. In this context, we propose the use of direct stochastic optical reconstruction microscopy (dSTORM) to study time stability and cell trafficking after transfection of oligopeptide end-modified poly(?-aminoester) (OM-pBAE) nanoparticles. We selected different combinations of cationic end oligopeptides (arginine - R; histidine - H; and lysine - K) among polymer libraries, since the oligopeptide combination demonstrated to be useful for different applications, such as vaccination and gene silencing. We demonstrate that their time evolution as well as their cell uptake and trafficking are dependent on the oligopeptide. This study opens the pave to broad mechanistic studies at nanoscale that could enable a rational selection of specific pBAE nanoparticles composition after determining their stability and cell trafficking.© 2022 The Authors. ChemMedChem published by Wiley-VCH GmbH.

JTD Keywords: cancer nanomedicine, cell trafficking, delivery, direct stochastic optical reconstruction microscopy (dstorm), nanoparticle stability, poly(beta-aminoester) nanoparticles, Direct stochastic optical reconstruction microscopy (dstorm), Poly(?-aminoester) nanoparticles, Poly(beta-amino ester)s


Clark, AG, Maitra, A, Jacques, C, Bergert, M, Perez-Gonzalez, C, Simon, A, Lederer, L, Diz-Munoz, A, Trepat, X, Voituriez, R, Vignjevic, DM, (2022). Self-generated gradients steer collective migration on viscoelastic collagen networks Nature Materials 21, 1200-1210

Growing evidence suggests that the physical properties of the cellular microenvironment influence cell migration. However, it is not currently understood how active physical remodelling by cells affects migration dynamics. Here we report that cell clusters seeded on deformable collagen-I networks display persistent collective migration despite not showing any apparent intrinsic polarity. Clusters generate transient gradients in collagen density and alignment due to viscoelastic relaxation of the collagen networks. Combining theory and experiments, we show that crosslinking collagen networks or reducing cell cluster size results in reduced network deformation, shorter viscoelastic relaxation time and smaller gradients, leading to lower migration persistence. Traction force and Brillouin microscopy reveal asymmetries in force distributions and collagen stiffness during migration, providing evidence of mechanical cross-talk between cells and their substrate during migration. This physical model provides a mechanism for self-generated directional migration on viscoelastic substrates in the absence of internal biochemical polarity cues.; Cell clusters mechanically reorganize viscoelastic collagen networks, resulting in transient gradients in collagen density, alignment and stiffness that promote spontaneous persistent migration.

JTD Keywords: Cell-migration, Design, Invasion, Limits, Mechanics, Microscopy, Morphogenesis, Motility, Rear, Rigidity


Lopez-Mengual, A, Segura-Feliu, M, Sunyer, R, Sanz-Fraile, H, Otero, J, Mesquida-Veny, F, Gil, V, Hervera, A, Ferrer, I, Soriano, J, Trepat, X, Farre, R, Navajas, D, del Rio, JA, (2022). Involvement of Mechanical Cues in the Migration of Cajal-Retzius Cells in the Marginal Zone During Neocortical Development Frontiers In Cell And Developmental Biology 10,

Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone.

JTD Keywords: Atomic force microscopy, Cajal-retzius cells, Central-nervous-system, Cortical development, Cortical hem, Developing cerebral-cortex, Expression, Growth, Marginal zone, Mechanical cues, Mouse, Neuronal migration, Nogo receptor, Olfactory ensheathing cells, Tangential migration, Traction force microscopy


Sans J, Arnau M, Sanz V, Turon P, Alemán C, (2022). Hydroxyapatite-based biphasic catalysts with plasticity properties and its potential in carbon dioxide fixation Chemical Engineering Journal 433, 133512

The design of catalysts with controlled selectivity at will, also known as catalytic plasticity, is a very attractive approach for the recycling of carbon dioxide (CO2). In this work, we study how catalytically active hydroxyapatite (HAp) and brushite (Bru) interact synergistically, allowing the production of formic acid or acetic acid depending on the HAp/Bru ratio in the catalyst. Raman, wide angle X-ray scattering, X-ray photoelectron spectroscopy, scanning electron microscopy and electrochemical impedance spectroscopy studies, combined with an exhaustive revision of the crystalline structure of the catalyst at the atomic level, allowed to discern how the Bru phase can be generated and stabilized at high temperatures. Results clearly indicate that the presence of OH– groups to maintain the crystalline structural integrity in conjunction with Ca2+ ions less bonded to the lattice fixate carbon into C1, C2 and C3 molecules from CO2 and allow the evolution from formic to acetic acid and acetone. In this way, the plasticity of the HAp-Bru system is demonstrated, representing a promising green alternative to the conventional metal-based electrocatalysts used for CO2 fixation. Thus, the fact that no electric voltage is necessary for the CO2 reduction has a very favorable impact in the final energetic net balance of the carbon fixation reaction. © 2021

JTD Keywords:

ethanol production & nbsp, brushite, co2 reduction, conversion, electrocatalytic reduction, electrode, formate, heterogeneous catalysis & nbsp, hydrogen evolution, insights, monetite, polarized hydroxyapatite,

, Acetic acid, Acetone, Biphasic catalyst, Brushite, Calcium phosphate, Carbon dioxide, Carbon dioxide fixation, Catalysis, Catalyst selectivity, Co 2 reduction, Co2 reduction, Electrocatalysts, Electrochemical impedance spectroscopy, Electrochemical reduction, Electrochemical-impedance spectroscopies, Ethanol production, Formic acid, Heterogeneous catalysis, Hydroxyapatite, Ph, Polarized hydroxyapatite, Property, Reduction, Scanning electron microscopy, Temperature programmed desorption, Wide angle x-ray scattering, X ray photoelectron spectroscopy, X ray scattering, ]+ catalyst


Valles, Morgane, Pujals, Sílvia, Albertazzi, Lorenzo, Sánchez, Samuel, (2022). Enzyme Purification Improves the Enzyme Loading, Self-Propulsion, and Endurance Performance of Micromotors Acs Nano 16, 5615-5626

Woythe L, Madhikar P, Feiner-Gracia N, Storm C, Albertazzi L, (2022). A Single-Molecule View at Nanoparticle Targeting Selectivity: Correlating Ligand Functionality and Cell Receptor Density Acs Nano 16, 3785-3796

Antibody-functionalized nanoparticles (NPs) are commonly used to increase the targeting selectivity toward cells of interest. At a molecular level, the number of functional antibodies on the NP surface and the density of receptors on the target cell determine the targeting interaction. To rationally develop selective NPs, the single-molecule quantitation of both parameters is highly desirable. However, techniques able to count molecules with a nanometric resolution are scarce. Here, we developed a labeling approach to quantify the number of functional cetuximabs conjugated to NPs and the expression of epidermal growth factor receptors (EGFRs) in breast cancer cells using direct stochastic optical reconstruction microscopy (dSTORM). The single-molecule resolution of dSTORM allows quantifying molecules at the nanoscale, giving a detailed insight into the distributions of individual NP ligands and cell receptors. Additionally, we predicted the fraction of accessible antibody-conjugated NPs using a geometrical model, showing that the total number exceeds the accessible number of antibodies. Finally, we correlated the NP functionality, cell receptor density, and NP uptake to identify the highest cell uptake selectivity regimes. We conclude that single-molecule functionality mapping using dSTORM provides a molecular understanding of NP targeting, aiding the rational design of selective nanomedicines.

JTD Keywords: active targeting, active targeting dstorm, antibodies, dstorm, heterogeneity, multivalency, nanomedicine, nanoparticle functionality, size, super-resolution microscopy, surface, Active targeting, Antibodies, Cell membranes, Cell receptors, Cytology, Direct stochastic optical reconstruction microscopy, Dstorm, Heterogeneity, Ligands, Medical nanotechnology, Molecules, Nanomedicine, Nanoparticle functionality, Nanoparticle targeting, Nanoparticles, Optical reconstruction, Single molecule, Stochastic systems, Stochastics, Super-resolution microscopy, Superresolution microscopy


Bonilla-Pons SÀ, Nakagawa S, Bahima EG, Fernández-Blanco Á, Pesaresi M, D'Antin JC, Sebastian-Perez R, Greco D, Domínguez-Sala E, Gómez-Riera R, Compte RIB, Dierssen M, Montserrat Pulido, N, Cosma MP, (2022). Müller glia fused with adult stem cells undergo neural differentiation in human retinal models Ebiomedicine 77, 103914

Visual impairments are a critical medical hurdle to be addressed in modern society. Müller glia (MG) have regenerative potential in the retina in lower vertebrates, but not in mammals. However, in mice, in vivo cell fusion between MG and adult stem cells forms hybrids that can partially regenerate ablated neurons.We used organotypic cultures of human retina and preparations of dissociated cells to test the hypothesis that cell fusion between human MG and adult stem cells can induce neuronal regeneration in human systems. Moreover, we established a microinjection system for transplanting human retinal organoids to demonstrate hybrid differentiation.We first found that cell fusion occurs between MG and adult stem cells, in organotypic cultures of human retina as well as in cell cultures. Next, we showed that the resulting hybrids can differentiate and acquire a proto-neural electrophysiology profile when the Wnt/beta-catenin pathway is activated in the adult stem cells prior fusion. Finally, we demonstrated the engraftment and differentiation of these hybrids into human retinal organoids.We show fusion between human MG and adult stem cells, and demonstrate that the resulting hybrid cells can differentiate towards neural fate in human model systems. Our results suggest that cell fusion-mediated therapy is a potential regenerative approach for treating human retinal dystrophies.This work was supported by La Caixa Health (HR17-00231), Velux Stiftung (976a) and the Ministerio de Ciencia e Innovación, (BFU2017-86760-P) (AEI/FEDER, UE), AGAUR (2017 SGR 689, 2017 SGR 926).Published by Elsevier B.V.

JTD Keywords: cell fusion, expression, fusion, ganglion-cells, in-vitro, mouse, müller glia, neural differentiation, organoids, regeneration, retina regeneration, stem cells, stromal cells, transplantation, 4',6 diamidino 2 phenylindole, 5' nucleotidase, Agarose, Alcohol, Arpe-19 cell line, Article, Beta catenin, Beta tubulin, Bone-marrow-cells, Bromophenol blue, Buffer, Calcium cell level, Calcium phosphate, Calretinin, Canonical wnt signaling, Cd34 antigen, Cell culture, Cell fusion, Cell viability, Coculture, Complementary dna, Confocal microscopy, Cornea transplantation, Cryopreservation, Cryoprotection, Crystal structure, Current clamp technique, Dimethyl sulfoxide, Dodecyl sulfate sodium, Edetic acid, Electrophysiology, Endoglin, Fetal bovine serum, Fibroblast growth factor 2, Flow cytometry, Fluorescence activated cell sorting, Fluorescence intensity, Glyceraldehyde 3 phosphate dehydrogenase, Glycerol, Glycine, Hoe 33342, Immunofluorescence, Immunohistochemistry, Incubation time, Interleukin 1beta, Lentivirus vector, Matrigel, Mercaptoethanol, Microinjection, Mueller cell, Müller glia, N methyl dextro aspartic acid, Nerve cell differentiation, Neural differentiation, Nitrogen, Nonhuman, Organoids, Paraffin, Paraffin embedding, Paraformaldehyde, Patch clamp technique, Penicillin derivative, Phenolsulfonphthalein, Phenotype, Phosphate buffered saline, Phosphoprotein phosphatase inhibitor, Polyacrylamide gel electrophoresis, Potassium chloride, Povidone iodine, Promoter region, Proteinase inhibitor, Real time polymerase chain reaction, Receptor type tyrosine protein phosphatase c, Restriction endonuclease, Retina, Retina dystrophy, Retina regeneration, Retinol, Rhodopsin, Rna extraction, Stem cell, Stem cells, Subcutaneous fat, Tunel assay, Visual impairment, Western blotting


Dhiman, Shikha, Andrian, Teodora, Gonzalez, Beatriz Santiago, Tholen, Marrit ME., Wang, Yuyang, Albertazzi, Lorenzo, (2022). Can super-resolution microscopy become a standard characterization technique for materials chemistry? Chemical Science 13, 2152-2166

The characterization of newly synthesized materials is a cornerstone of all chemistry and nanotechnology laboratories. For this purpose, a wide array of analytical techniques have been standardized and are used routinely by laboratories across the globe. With these methods we can understand the structure, dynamics and function of novel molecular architectures and their relations with the desired performance, guiding the development of the next generation of materials. Moreover, one of the challenges in materials chemistry is the lack of reproducibility due to improper publishing of the sample preparation protocol. In this context, the recent adoption of the reporting standard MIRIBEL (Minimum Information Reporting in Bio–Nano Experimental Literature) for material characterization and details of experimental protocols aims to provide complete, reproducible and reliable sample preparation for the scientific community. Thus, MIRIBEL should be immediately adopted in publications by scientific journals to overcome this challenge. Besides current standard spectroscopy and microscopy techniques, there is a constant development of novel technologies that aim to help chemists unveil the structure of complex materials. Among them super-resolution microscopy (SRM), an optical technique that bypasses the diffraction limit of light, has facilitated the study of synthetic materials with multicolor ability and minimal invasiveness at nanometric resolution. Although still in its infancy, the potential of SRM to unveil the structure, dynamics and function of complex synthetic architectures has been highlighted in pioneering reports during the last few years. Currently, SRM is a sophisticated technique with many challenges in sample preparation, data analysis, environmental control and automation, and moreover the instrumentation is still expensive. Therefore, SRM is currently limited to expert users and is not implemented in characterization routines. This perspective discusses the potential of SRM to transition from a niche technique to a standard routine method for material characterization. We propose a roadmap for the necessary developments required for this purpose based on a collaborative effort from scientists and engineers across disciplines.

JTD Keywords: blinking, fluorophore, intramolecular spirocyclization, localization, nanoparticles, resolution limit, reveals, single-molecule fluorescence, stimulated-emission, Characterization techniques, Diffraction, Distributed computer systems, Environmental management, Information reporting, Material chemistry, Materials characterization, Minimum information, Optical reconstruction microscopy, Optical resolving power, Sample preparation, Structure dynamics, Structure functions, Super-resolution microscopy, Synthesized materials


Bar L, Perissinotto F, Redondo-Morata L, Giannotti MI, Goole J, Losada-Pérez P, (2022). Interactions of hydrophilic quantum dots with defect-free and defect containing supported lipid membranes Colloids And Surfaces B-Biointerfaces 210, 112239

Quantum dots (QDs) are semiconductor nanoparticles with unique optical and electronic properties, whose interest as potential nano-theranostic platforms for imaging and sensing is increasing. The design and use of QDs requires the understanding of cell-nanoparticle interactions at a microscopic and nanoscale level. Model systems such as supported lipid bilayers (SLBs) are useful, less complex platforms mimicking physico-chemical properties of cell membranes. In this work, we investigated the effect of topographical homogeneity of SLBs bearing different surface charge in the adsorption of hydrophilic QDs. Using quartz-crystal microbalance, a label-free surface sensitive technique, we show significant differences in the interactions of QDs onto homogeneous and inhomogeneous SLBs formed following different strategies. Within short time scales, QDs adsorb onto topographically homogeneous, defect-free SLBs is driven by electrostatic interactions, leading to no layer disruption. After prolonged QD exposure, the nanomechanical stability of the SLB decreases suggesting nanoparticle insertion. In the case of inhomogeneous, defect containing layers, QDs target preferentially membrane defects, driven by a subtle interplay of electrostatic and entropic effects, inducing local vesicle rupture and QD insertion at membrane edges. © 2021

JTD Keywords: adsorption, atomic force microscopy, bilayer formation, gold nanoparticles, hydrophilic quantum dots, lipid membrane defects, model, nanomechanics, quartz crystal microbalance with dissipation, size, supported lipid bilayers, surfaces, Atomic force microscopy, Atomic-force-microscopy, Cytology, Defect-free, Electronic properties, Electrostatics, Hydrophilic quantum dot, Hydrophilic quantum dots, Hydrophilicity, Hydrophilics, Lipid bilayers, Lipid membrane defect, Lipid membrane defects, Lipid membranes, Lipids, Nanocrystals, Nanomechanics, Optical and electronic properties, Quartz, Quartz crystal microbalance with dissipation, Quartz crystal microbalances, Quartz-crystal microbalance, Semiconductor nanoparticles, Semiconductor quantum dots, Supported lipid bilayers


López Ortiz, Manuel, Zamora, Ricardo A., Giannotti, Marina Inés, Hu, Chen, Croce, Roberta, Gorostiza, Pau, (2022). Distance and Potential Dependence of Charge Transport Through the Reaction Center of Individual Photosynthetic Complexes Small 18, 2104366

Charge separation and transport through the reaction center of photosystem I (PSI) is an essential part of the photosynthetic electron transport chain. A strategy is developed to immobilize and orient PSI complexes on gold electrodes allowing to probe the complex's electron acceptor side, the chlorophyll special pair P700. Electrochemical scanning tunneling microscopy (ECSTM) imaging and current-distance spectroscopy of single protein complex shows lateral size in agreement with its known dimensions, and a PSI apparent height that depends on the probe potential revealing a gating effect in protein conductance. In current-distance spectroscopy, it is observed that the distance-decay constant of the current between PSI and the ECSTM probe depends on the sample and probe electrode potentials. The longest charge exchange distance (lowest distance-decay constant ?) is observed at sample potential 0 mV/SSC (SSC: reference electrode silver/silver chloride) and probe potential 400 mV/SSC. These potentials correspond to hole injection into an electronic state that is available in the absence of illumination. It is proposed that a pair of tryptophan residues located at the interface between P700 and the solution and known to support the hydrophobic recognition of the PSI redox partner plastocyanin, may have an additional role as hole exchange mediator in charge transport through PSI.© 2021 Wiley-VCH GmbH.

JTD Keywords: azurin, current distance decay spectroscopy, cytochrome c(6), electrochemical scanning tunneling microscopy (ecstm), electrochemistry, photosystem i, photosystem-i, plastocyanin, protein electron transfer, recognition, single metalloprotein, single molecules, structural basis, tunneling spectroscopy, 'current, Amino acids, Charge transfer, Chlorine compounds, Current distance decay spectroscopy, Decay spectroscopies, Distance decay, Electrochemical scanning tunneling microscopy, Electrochemical scanning tunneling microscopy (ecstm), Electrodes, Electron transfer, Electron transport properties, Gold compounds, Photosystem i, Photosystems, Protein electron transfer, Protein electron-transfer, Proteins, Scanning tunneling microscopy, Silver halides, Single molecule, Single molecules


Yazıcı N, Opar E, Kodal M, Tanören B, Sezen M, Özkoç G, (2022). A novel practical approach for monitoring the crosslink density of an ethylene propylene diene monomer compound: Complementary scanning acoustic microscopy and FIB-SEM-EDS analyses Polymers & Polymer Composites 30,

Tuning of the crosslink density (CLD) in the rubber compounds is very crucial for optimizing the physical and mechanical properties of the ultimate rubber products. Conventionally, CLD can be measured via rheological methods such as moving die rheometer (MDR), via mechanical tests such as temperature scanning stress relaxation analysis (TSSR), or via direct swelling experiments using Flory–Rehner approach. In the current study, two novel techniques, focused ion beam - scanning electron microscopy (FIB-SEM) processing, with simultaneous energy dispersive X-ray spectrometry (EDS) mapping analysis and scanning acoustic microscopy (SAM) were combined and correlated to conventional methods on a model recipe of ethylene propylene diene monomer (EPDM) compound having different sulphur contents. Depending on the applied technique, the increase in the crosslink density with sulphur content was found to be 1.7 fold for the Flory–Rehner approach and 1.2 fold for both TSSR and MDR. It is directly monitored from the FIB-SEM-EDS analysis that the sulphur distribution and agglomeration behavior increased in line with ZnO content, which is an indirect indication of the rise in crosslink density. The impedance maps of the crosslinked samples obtained through SAM analysis revealed that the impedance of the samples increased with the increasing sulphur content, which can be attributed to higher level of crosslink density. A quantified correlation was obtained between SAM images and the crosslink density of the samples. It was shown that SAM is a promising tool for practical and non-destructive analysis for determining the formation of crosslink density of the rubbers. © The Author(s) 2022.

JTD Keywords: Cross-link densities, Crosslink density, Crosslinking, Density (specific gravity), Ethylene, Ethylene propylene diene monomer, Flory-rehner, Focused ion beam - scanning electron microscopy, Focused ion beam-scanning electron microscopies, Ii-vi semiconductors, Monomers, Moving die rheometers, Physical and mechanical properties, Propylene, Relaxation analysis, Rubber, Scanning acoustic microscopy, Scanning electron microscopy, Stress relaxation, Sulfur contents, Temperature scanning stress relaxations, Zinc oxide


Arista-Romero M, Delcanale P, Pujals S, Albertazzi L, (2022). Nanoscale Mapping of Recombinant Viral Proteins: From Cells to Virus-Like Particles Acs Photonics 9, 101-109

Influenza recombinant proteins and virus-like particles (VLPs) play an important role in vaccine development (e.g., CadiFluS). However, their production from mammalian cells suffers from low yields and lack of control of the final VLPs. To improve these issues, characterization techniques able to visualize and quantify the different steps of the process are needed. Fluorescence microscopy represents a powerful tool able to image multiple protein targets; however, its limited resolution hinders the study of viral constructs. Here, we propose the use of super-resolution microscopy and in particular of DNA-point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy as a characterization method for recombinant viral proteins on both cells and VLPs. We were able to quantify the amount of the three main influenza proteins (hemagglutinin (HA), neuraminidase (NA), and ion channel matrix protein 2 (M2)) per cell and per VLP with nanometer resolution and single-molecule sensitivity, proving that DNA-PAINT is a powerful technique to characterize recombinant viral constructs.

JTD Keywords: dna-paint, hemagglutinin, influenza, neuraminidase, paint, recombinant proteins, single-molecule localization microscopy, single-particle analysis, virus-like particles, Dna-paint, Hemagglutinin, Influenza, Neuraminidase, Paint, Recombinant proteins, Single particle analysis, Single-molecule localization microscopy, Single-particle analysis, Super-resolution microscopy, Superresolution microscopy, Virus-like particles


Jain, A, Calo, A, Barcelo, D, Kumar, M, (2022). Supramolecular systems chemistry through advanced analytical techniques Analytical And Bioanalytical Chemistry 414, 5105-5119

Supramolecular chemistry is the quintessential backbone of all biological processes. It encompasses a wide range from the metabolic network to the self-assembled cytoskeletal network. Combining the chemical diversity with the plethora of functional depth that biological systems possess is a daunting task for synthetic chemists to emulate. The only route for approaching such a challenge lies in understanding the complex and dynamic systems through advanced analytical techniques. The supramolecular complexity that can be successfully generated and analyzed is directly dependent on the analytical treatment of the system parameters. In this review, we illustrate advanced analytical techniques that have been used to investigate various supramolecular systems including complex mixtures, dynamic self-assembly, and functional nanomaterials. The underlying theme of such an overview is not only the exceeding detail with which traditional experiments can be probed but also the fact that complex experiments can now be attempted owing to the analytical techniques that can resolve an ensemble in astounding detail. Furthermore, the review critically analyzes the current state of the art analytical techniques and suggests the direction of future development. Finally, we envision that integrating multiple analytical methods into a common platform will open completely new possibilities for developing functional chemical systems.

JTD Keywords: analytical techniques, dynamic self-assembly, high-speed afm, liquid cell tem, Analytical technique, Analytical techniques, Biological process, Chemical analysis, Chemical diversity, Complex networks, Cytoskeletal network, Dynamic self-assembly, High-speed afm, Hydrogels, In-situ, Liquid cell tem, Metabolic network, Microscopy, Nanoscale, Proteins, Self assembly, Supramolecular chemistry, Supramolecular systems, System chemistry, Systems chemistry


Andrian, T, Pujals, S, Albertazzi, L, (2021). Quantifying the effect of PEG architecture on nanoparticle ligand availability using DNA-PAINT Nanoscale Advances 3, 6876-6881

The importance of PEG architecture on nanoparticle (NP) functionality is known but still difficult to investigate, especially at a single particle level. Here, we apply DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT), a super-resolution microscopy (SRM) technique, to study the surface functionality in poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) NPs with different PEG structures. We demonstrated how the length of the PEG spacer can influence the accessibility of surface chemical functionality, highlighting the importance of SRM techniques to support the rational design of functionalized NPs.

JTD Keywords: chain-length, density, plga, surface, systems, Chain-length, Density, Dna, Microscopy technique, Nanoparticles, Nanoscale topography, Paint, Peg spacers, Plga, Poly lactide-co-glycolide, Poly-lactide-co-glycolide, Polyethylene glycols, Polylactide-co-glycolide, Single-particle, Super-resolution microscopy, Superresolution microscopy, Surface, Surface chemicals, Surface functionalities, Systems


Júnior C, Narciso M, Marhuenda E, Almendros I, Farré R, Navajas D, Otero J, Gavara N, (2021). Baseline stiffness modulates the non-linear response to stretch of the extracellular matrix in pulmonary fibrosis International Journal Of Molecular Sciences 22, 12928

Pulmonary fibrosis (PF) is a progressive disease that disrupts the mechanical homeostasis of the lung extracellular matrix (ECM). These effects are particularly relevant in the lung context, given the dynamic nature of cyclic stretch that the ECM is continuously subjected to during breathing. This work uses an in vivo model of pulmonary fibrosis to characterize the macro-and micromechanical properties of lung ECM subjected to stretch. To that aim, we have compared the micromechanical properties of fibrotic ECM in baseline and under stretch conditions, using a novel combination of Atomic Force Microscopy (AFM) and a stretchable membrane-based chip. At the macroscale, fibrotic ECM displayed strain-hardening, with a stiffness one order of magnitude higher than its healthy counterpart. Conversely, at the microscale, we found a switch in the stretch-induced mechanical behaviour of the lung ECM from strain-hardening at physiological ECM stiffnesses to strain-softening at fibrotic ECM stiffnesses. Similarly, we observed solidification of healthy ECM versus fluidization of fibrotic ECM in response to stretch. Our results suggest that the mechanical behaviour of fibrotic ECM under stretch involves a potential built-in mechanotransduction mechanism that may slow down the progression of PF by steering resident fibroblasts away from a pro-fibrotic profile. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: atomic force microscopy, extracellular matrix, fibrosis, mechanics, mechanosensing, strain, system, viscoelasticity, Atomic force microscopy, Extracellular matrix, Fibrosis, Lung fibrosis, Mechanosensing


Nashimoto Y, Abe M, Fujii R, Taira N, Ida H, Takahashi Y, Ino K, Ramon-Azcon J, Shiku H, (2021). Topography and Permeability Analyses of Vasculature-on-a-Chip Using Scanning Probe Microscopies Advanced Healthcare Materials 10, 2101186

Microphysiological systems (MPS) or organs-on-chips (OoC) can emulate the physiological functions of organs in vitro and are effective tools for determining human drug responses in preclinical studies. However, the analysis of MPS has relied heavily on optical tools, resulting in difficulties in real-time and high spatial resolution imaging of the target cell functions. In this study, the role of scanning probe microscopy (SPM) as an analytical tool for MPS is evaluated. An access hole is made in a typical MPS system with stacked microchannels to insert SPM probes into the system. For the first study, a simple vascular model composed of only endothelial cells is prepared for SPM analysis. Changes in permeability and local chemical flux are quantitatively evaluated during the construction of the vascular system. The morphological changes in the endothelial cells after flow stimulation are imaged at the single-cell level for topographical analysis. Finally, the possibility of adapting the permeability and topographical analysis using SPM for the intestinal vascular system is further evaluated. It is believed that this study will pave the way for an in situ permeability assay and structural analysis of MPS using SPM.

JTD Keywords: cell, electrochemical microscopy, membrane-permeability, microphysiological systems, organs-chips, platform, scanning electrochemical microscopy, scanning ion conductance microscopy, scanning probe microscopy, shear-stress, surface-topography, Ion conductance microscopy, Microphysiological systems, Organs-chips, Scanning electrochemical microscopy, Scanning ion conductance microscopy, Scanning probe microscopy


Avalos-Padilla Y, Georgiev VN, Dimova R, (2021). ESCRT-III induces phase separation in model membranes prior to budding and causes invagination of the liquid-ordered phase Biochimica et Biophysica Acta - Biomembranes 1863, 183689

Membrane fission triggered by the endosomal sorting complex required for transport (ESCRT) is an important process observed in several pathogenic and non-pathogenic cellular events. From a synthetic-biology viewpoint, ESCRT proteins represent an interesting machinery for the construction of cell mimetic sub-compartments produced by fission. Since their discovery, the studies on ESCRT-III-mediated action, have mainly focused on protein dynamics, ignoring the role of lipid organization and membrane phase state. Recently, it has been suggested that membrane buds formed by the action of ESCRT-III are generated from transient microdomains in endosomal membranes. However, the interplay between membrane domain formation and ESCRT remodeling pathways has not been investigated. Here, giant unilamellar vesicles made of ternary lipid mixtures, either homogeneous in phase or exhibiting liquid-ordered/liquid-disordered phase coexistence, were employed as a model membrane system. These vesicles were incubated with purified recombinant ESCRT-III proteins from the parasite Entamoeba histolytica. In homogeneous membranes, we observe that EhVps32 can trigger domain formation while EhVps20 preferentially co-localizes in the liquid disordered phase. The addition of EhVps24 appears to induce the formation of intraluminal vesicles produced from the liquid-ordered phase. In phase separated membranes, the intraluminal vesicles are also generated from the liquid-ordered phase and presumably emerge from the phase boundary region. Our findings reinforce the hypothesis that ESCRT-mediated remodeling depends on the membrane phase state. Furthermore, the obtained results point to a potential synthetic biology approach for establishing eukaryotic mimics of artificial cells with microcompartments of specific membrane composition, which can also differ from that of the mother vesicle.

JTD Keywords: cell-membranes, coexistence, complex, escrt-iii, fission, guvs, lipid domains, lipid rafts, membrane fission, microcompartments, microscopy, phase separation, plasma-membrane, protein microarrays, structural basis, ternary mixtures, Escrt-iii, Giant unilamellar vesicles, Guvs, Lipid domains, Membrane fission, Microcompartments, Phase separation, Ternary mixtures


Calò A, Eleta-Lopez A, Ondarçuhu T, Verdaguer A, Bittner AM, (2021). Nanoscale wetting of single viruses Molecules 26, 5184

The epidemic spread of many viral infections is mediated by the environmental conditions and influenced by the ambient humidity. Single virus particles have been mainly visualized by atomic force microscopy (AFM) in liquid conditions, where the effect of the relative humidity on virus topography and surface cannot be systematically assessed. In this work, we employed multi-frequency AFM, simultaneously with standard topography imaging, to study the nanoscale wetting of individual Tobacco Mosaic virions (TMV) from ambient relative humidity to water condensation (RH > 100%). We recorded amplitude and phase vs. distance curves (APD curves) on top of single virions at various RH and converted them into force vs. distance curves. The high sensitivity of multifrequency AFM to visualize condensed water and sub-micrometer droplets, filling gaps between individual TMV particles at RH > 100%, is demonstrated. Dynamic force spectroscopy allows detecting a thin water layer of thickness ⁓1 nm, adsorbed on the outer surface of single TMV particles at RH < 60%.

JTD Keywords: amplitude-modulation am-afm, atomic-force microscopy, capillary, force reconstruction, multifrequency afm, nanoscale wetting, persistence, reconstruction, relative-humidity, surfaces, tobacco mosaic virus (tmv), tobamovirus, transmission, water, Amplitude-modulation am-afm, Force reconstruction, Multifrequency afm, Nanoscale wetting, Tobacco mosaic virus (tmv), Tobacco mosaic virus (tmv), nanoscale wetting, Tobacco-mosaic-virus


Balakrishnan H, Fabregas R, Millan-Solsona R, Fumagalli L, Gomila G, (2021). Spatial Resolution and Capacitive Coupling in the Characterization of Nanowire Nanocomposites by Scanning Dielectric Microscopy Microscopy And Microanalysis 27, 1026-1034

Nanowire-based nanocomposite materials are being developed as transparent and flexible electrodes or as stretchable conductors and dielectrics for biosensing. Here, we theoretically investigate the use of scanning dielectric microscopy (SDM) to characterize these materials in a nondestructive way, with a special focus on the achievable spatial resolution and the possibility of detection of the capacitive coupling between nearby nanowires. Numerical calculations with models involving single and multiple buried nanowires have been performed. We demonstrate that the capacitance gradient spread function of a single buried nanowire consists of a modified Lorenzianan with a cubic decay. We show that the achievable spatial resolution can be determined with good accuracy with the help of this spread function. It is shown that, in general, the spatial resolution worsens when any system parameter decreases the maximum of the nanowire spread function or increases its width, or both. Finally, we show that SDM measurements are also sensitive to the capacitive coupling between nearby nanowires. This latter result is of utmost relevance since the macroscopic electric properties of nanowire nanocomposites largely depend on the electric interaction between nearby nanowires. The present results show that SDM can be a valuable nondestructive subsurface characterization technique for nanowire nanocomposite materials.

JTD Keywords: depth, electrodes, nanocomposites, nanowires, sdm, spatial resolution, subsurface, tomography, Capacitive coupling, Force microscopy, Nanocomposites, Nanowires, Sdm, Spatial resolution, Subsurface


Andreu, I, Falcones, B, Hurst, S, Chahare, N, Quiroga, X, Le Roux, AL, Kechagia, Z, Beedle, AEM, Elosegui-Artola, A, Trepat, X, Farre, R, Betz, T, Almendros, I, Roca-Cusachs, P, (2021). The force loading rate drives cell mechanosensing through both reinforcement and cytoskeletal softening Nature Communications 12, 4229

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat


Checa M, Millan-Solsona R, Mares AG, Pujals S, Gomila G, (2021). Fast Label-Free Nanoscale Composition Mapping of Eukaryotic Cells Via Scanning Dielectric Force Volume Microscopy and Machine Learning Small Methods 5, 2100279

Mapping the biochemical composition of eukaryotic cells without the use of exogenous labels is a long-sought objective in cell biology. Recently, it has been shown that composition maps on dry single bacterial cells with nanoscale spatial resolution can be inferred from quantitative nanoscale dielectric constant maps obtained with the scanning dielectric microscope. Here, it is shown that this approach can also be applied to the much more challenging case of fixed and dry eukaryotic cells, which are highly heterogeneous and show micrometric topographic variations. More importantly, it is demonstrated that the main bottleneck of the technique (the long computation times required to extract the nanoscale dielectric constant maps) can be shortcut by using supervised neural networks, decreasing them from weeks to seconds in a wokstation computer. This easy-to-use data-driven approach opens the door for in situ and on-the-fly label free nanoscale composition mapping of eukaryotic cells with scanning dielectric microscopy. © 2021 The Authors. Small Methods published by Wiley-VCH GmbH

JTD Keywords: label-free mapping, machine learning, nanoscale, scanning dielectric microscopy, Biochemical composition, Cells, Constant, Cytology, Data-driven approach, Dielectric forces, Dielectric materials, Eukaryotic cells, Label-free mapping, Machine learning, Mapping, Nanoscale, Nanoscale composition, Nanoscale spatial resolution, Nanotechnology, Scanning, Scanning dielectric microscopy, Supervised neural networks


Boschker, HTS, Cook, PLM, Polerecky, L, Eachambadi, RT, Lozano, H, Hidalgo-Martinez, S, Khalenkow, D, Spampinato, V, Claes, N, Kundu, P, Wang, D, Bals, S, Sand, KK, Cavezza, F, Hauffman, T, Bjerg, JT, Skirtach, AG, Kochan, K, McKee, M, Wood, B, Bedolla, D, Gianoncelli, A, Geerlings, NMJ, Van Gerven, N, Remaut, H, Geelhoed, JS, Millan-Solsona, R, Fumagalli, L, Nielsen, LP, Franquet, A, Manca, JV, Gomila, G, Meysman, FJR, (2021). Efficient long-range conduction in cable bacteria through nickel protein wires Nature Communications 12, 3996

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures. Filamentous cable bacteria conduct electrical currents over centimeter distances through fibers embedded in their cell envelope. Here, Boschker et al. show that the fibers consist of a conductive core containing nickel proteins that is surrounded by an insulating protein shell.

JTD Keywords: Bacteria (microorganisms), Bacterial protein, Bacterial proteins, Bacterium, Chemistry, Deltaproteobacteria, Electric conductivity, Electricity, Electron, Electron transport, Metabolism, Microscopy, Nanowires, Nickel, Physiology, Protein, Resonance raman, Spectroscopy, Transport electrons


Andrian T, Delcanale P, Pujals S, Albertazzi L, (2021). Correlating Super-Resolution Microscopy and Transmission Electron Microscopy Reveals Multiparametric Heterogeneity in Nanoparticles Nano Letters 21, 5360-5368

The functionalization of nanoparticles with functional moieties is a key strategy to achieve cell targeting in nanomedicine. The interplay between size and ligand number is crucial for the formulation performance and needs to be properly characterized to understand nanoparticle structure-activity relations. However, there is a lack of methods able to measure both size and ligand number at the same time and at the single particle level. Here, we address this issue by introducing a correlative light and electron microscopy (CLEM) method combining super-resolution microscopy (SRM) and transmission electron microscopy (TEM) imaging. We apply our super-resCLEM method to characterize the relationship between size and ligand number and density in PLGA-PEG nanoparticles. We highlight how heterogeneity found in size can impact ligand distribution and how a significant part of the nanoparticle population goes completely undetected in the single-technique analysis. Super-resCLEM holds great promise for the multiparametric analysis of other parameters and nanomaterials.

JTD Keywords: cellular uptake, correlative light and electron microscopy (clem), density, electron microscopy (em), functionalization, heterogeneity, nanomedicine, nanoparticles, pegylation, plga, progress, quantification, size, Correlative light and electron microscopy (clem), Electron microscopy (em), Heterogeneity, Nanomedicine, Nanoparticles, Physicochemical characterization, Super-resolution microscopy (srm)


Wang Y, Friedrich H, Voets IK, Zijlstra P, Albertazzi L, (2021). Correlative imaging for polymer science Journal Of Polymer Science 59, 1232-1240

The characterization of polymeric materials is key towards the understanding of structure–activity relations and therefore for the rational design of novel and improved materials for a myriad of applications. Many microscopy techniques are currently used, with electron microscopy, fluorescence microscopy, and atomic force microscopy being the most relevant. In this perspective paper, we discuss the use of correlative imaging, that is, the combination of multiple imaging methodologies on the same sample, in the field of polymeric materials. This innovative approach is emerging as a powerful tool to unveil the structure and functional properties of biological and synthetic structures. Here we discuss the possibilities of correlative imaging and highlight their potential to answer open questions in polymer science.

JTD Keywords: correlative imaging, electron microscopy, material characterization, resolution microscopy, super‐, Atomic force microscopy, Correlative imaging, Electron microscopy, Material characterization, Super-resolution microscopy


Balakrishnan H, Millan-Solsona R, Checa M, Fabregas R, Fumagalli L, Gomila G, (2021). Depth mapping of metallic nanowire polymer nanocomposites by scanning dielectric microscopy Nanoscale 13, 10116-10126

Polymer nanocomposite materials based on metallic nanowires are widely investigated as transparent and flexible electrodes or as stretchable conductors and dielectrics for biosensing. Here we show that Scanning Dielectric Microscopy (SDM) can map the depth distribution of metallic nanowires within the nanocomposites in a non-destructive way. This is achieved by a quantitative analysis of sub-surface electrostatic force microscopy measurements with finite-element numerical calculations. As an application we determined the three-dimensional spatial distribution of ?50 nm diameter silver nanowires in ?100 nm-250 nm thick gelatin films. The characterization is done both under dry ambient conditions, where gelatin shows a relatively low dielectric constant, ?r ? 5, and under humid ambient conditions, where its dielectric constant increases up to ?r ? 14. The present results show that SDM can be a valuable non-destructive subsurface characterization technique for nanowire-based nanocomposite materials, which can contribute to the optimization of these materials for applications in fields such as wearable electronics, solar cell technologies or printable electronics. © The Royal Society of Chemistry.

JTD Keywords: composite, constant, electrodes, mode, nanostructures, objects, progress, subsurface, tomography, Composite materials, Dielectric materials, Electric force microscopy, Electrostatic force, Force microscopy, Low dielectric constants, Nanocomposites, Numerical calculation, Polymer nanocomposite, Printable electronics, Scanning dielectric microscopy, Silver nanowires, Solar cell technology, Stretchable conductors, Subsurface characterizations, Transparent electrodes, Wearable technology


Di Muzio M, Millan-Solsona R, Dols-Perez A, Borrell JH, Fumagalli L, Gomila G, (2021). Dielectric properties and lamellarity of single liposomes measured by in-liquid scanning dielectric microscopy Journal Of Nanobiotechnology 19, 167

Liposomes are widely used as drug delivery carriers and as cell model systems. Here, we measure the dielectric properties of individual liposomes adsorbed on a metal electrode by in-liquid scanning dielectric microscopy in force detection mode. From the measurements the lamellarity of the liposomes, the separation between the lamellae and the specific capacitance of the lipid bilayer can be obtained. As application we considered the case of non-extruded DOPC liposomes with radii in the range ~ 100–800 nm. Uni-, bi- and tri-lamellar liposomes have been identified, with the largest population corresponding to bi-lamellar liposomes. The interlamellar separation in the bi-lamellar liposomes is found to be below ~ 10 nm in most instances. The specific capacitance of the DOPC lipid bilayer is found to be ~ 0.75 µF/cm2 in excellent agreement with the value determined on solid supported planar lipid bilayers. The lamellarity of the DOPC liposomes shows the usual correlation with the liposome's size. No correlation is found, instead, with the shape of the adsorbed liposomes. The proposed approach offers a powerful label-free and non-invasive method to determine the lamellarity and dielectric properties of single liposomes. [Figure not available: see fulltext.].

JTD Keywords: constant, force, lamellarity, liposomes, membrane capacitance, model, nanoscale, scanning dielectric microscopy, Lamellarity, Liposomes, Membrane capacitance, Nanoscale, Polarization properties, Scanning dielectric microscopy


Checa M, Millan‐solsona R, Mares AG, Pujals S, Gomila G, (2021). Dielectric imaging of fixed hela cells by in‐liquid scanning dielectric force volume microscopy Nanomaterials 11, 1402

Mapping the dielectric properties of cells with nanoscale spatial resolution can be an im-portant tool in nanomedicine and nanotoxicity analysis, which can complement structural and mechanical nanoscale measurements. Recently we have shown that dielectric constant maps can be obtained on dried fixed cells in air environment by means of scanning dielectric force volume mi-croscopy. Here, we demonstrate that such measurements can also be performed in the much more challenging case of fixed cells in liquid environment. Performing the measurements in liquid media contributes to preserve better the structure of the fixed cells, while also enabling accessing the local dielectric properties under fully hydrated conditions. The results shown in this work pave the way to address the nanoscale dielectric imaging of living cells, for which still further developments are required, as discussed here.

JTD Keywords: atomic force microscopy (afm), capacitance, constant, dielectric properties, electrostatic force microscopy (efm), functional microscopy, nanoscale, scanning dielectric microscopy (sdm), Atomic force microscopy (afm), Dielectric properties, Dielectrophoretic separation, Electrostatic force microscopy (efm), Functional micros-copy, Scanning dielectric microscopy (sdm), Scanning probe microscopy (spm)


Andrian T, Bakkum T, van Elsland DM, Bos E, Koster AJ, Albertazzi L, van Kasteren SI, Pujals S, (2021). Super-resolution correlative light-electron microscopy using a click-chemistry approach for studying intracellular trafficking Methods In Cell Biology 162, 303-331

© 2020 Elsevier Inc. Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples.

JTD Keywords: cells, click-chemistry, complex, correlative light and electron microscopy, cycloaddition, ligation, localization, proteins, resolution limit, single molecule localization microscopy, stochastic optical reconstruction microscopy (storm), storm, super-resolution microscopy, tokuyasu cryo-sectioning, tool, Click-chemistry, Correlative light and electron microscopy, Fluorescent-probes, Single molecule localization microscopy, Stochastic optical reconstruction microscopy (storm), Super-resolution microscopy, Tokuyasu cryo-sectioning, Transmission electron microscopy


Woythe L, Tito NB, Albertazzi L, (2021). A quantitative view on multivalent nanomedicine targeting Advanced Drug Delivery Reviews 169, 1-21

© 2020 The Authors Although the concept of selective delivery has been postulated over 100 years ago, no targeted nanomedicine has been clinically approved so far. Nanoparticles modified with targeting ligands to promote the selective delivery of therapeutics towards a specific cell population have been extensively reported. However, the rational design of selective particles is still challenging. One of the main reasons for this is the lack of quantitative theoretical and experimental understanding of the interactions involved in cell targeting. In this review, we discuss new theoretical models and experimental methods that provide a quantitative view of targeting. We show the new advancements in multivalency theory enabling the rational design of super-selective nanoparticles. Furthermore, we present the innovative approaches to obtain key targeting parameters at the single-cell and single molecule level and their role in the design of targeting nanoparticles. We believe that the combination of new theoretical multivalent design and experimental methods to quantify receptors and ligands aids in the rational design and clinical translation of targeted nanomedicines.

JTD Keywords: binding-kinetics, biological identity, biomolecular corona, blood-brain-barrier, drug-delivery, gold nanoparticles, multivalency, nanotechnology, protein corona, quantitative characterization, rational design, super-selectivity, superresolution microscopy, tumor heterogeneity, Ligand-receptor interactions, Multivalency, Nanotechnology, Quantitative characterization, Rational design, Super-selectivity


Kyndiah A, Checa M, Leonardi F, Millan-Solsona R, Di Muzio M, Tanwar S, Fumagalli L, Mas-Torrent M, Gomila G, (2021). Nanoscale Mapping of the Conductivity and Interfacial Capacitance of an Electrolyte-Gated Organic Field-Effect Transistor under Operation Advanced Functional Materials 31, 2008032

© 2020 Wiley-VCH GmbH Probing nanoscale electrical properties of organic semiconducting materials at the interface with an electrolyte solution under externally applied voltages is key in the field of organic bioelectronics. It is demonstrated that the conductivity and interfacial capacitance of the active channel of an electrolyte-gated organic field-effect transistor (EGOFET) under operation can be probed at the nanoscale using scanning dielectric microscopy in force detection mode in liquid environment. Local electrostatic force versus gate voltage transfer characteristics are obtained on the device and correlated with the global current–voltage transfer characteristics of the EGOFET. Nanoscale maps of the conductivity of the semiconducting channel show the dependence of the channel conductivity on the gate voltage and its variation along the channel due to the space charge limited conduction. The maps reveal very small electrical heterogeneities, which correspond to local interfacial capacitance variations due to an ultrathin non-uniform insulating layer resulting from a phase separation in the organic semiconducting blend. Present results offer insights into the transduction mechanism at the organic semiconductor/electrolyte interfaces at scales down to ≈100 nm, which can bring substantial optimization of organic electronic devices for bioelectronic applications such as electrical recording on excitable cells or label-free biosensing.

JTD Keywords: Atomic force microscopy, Bioelectronic devices, Electrolyte gated organic field effect transistors, In-liquid scanning dielectric microscopy, Organic semiconducting blend


Tian, X., De Pace, C., Ruiz-Perez, L., Chen, B., Su, R., Zhang, M., Zhang, R., Zhang, Q., Wang, Q., Zhou, H., Wu, J., Zhang, Z., Tian, Y., Battaglia, G., (2020). A Cyclometalated iridium (III) complex as a microtubule probe for correlative super-resolution fluorescence and electron microscopy Advanced Materials 32, (39), 2003901

The visualization of microtubules by combining optical and electron microscopy techniques provides valuable information to understand correlated intracellular activities. However, the lack of appropriate probes to bridge both microscopic resolutions restricts the areas and structures that can be comprehended within such highly assembled structures. Here, a versatile cyclometalated iridium (III) complex is designed that achieves synchronous fluorescence–electron microscopy correlation. The selective insertion of the probe into a microtubule triggers remarkable fluorescence enhancement and promising electron contrast. The long-life, highly photostable probe allows live-cell super-resolution imaging of tubulin localization and motion with a resolution of ≈30 nm. Furthermore, correlative light–electron microscopy and energy-filtered transmission electron microscopy reveal the well-associated optical and electron signal at a high specificity, with an interspace of ≈41 Å of microtubule monomer in cells.

JTD Keywords: Correlation light–electron microscopy, Microtubules, Organometallic probes, Super-resolution microscopy


Delcanale, P., Albertazzi, L., (2020). DNA-PAINT super-resolution imaging data of surface exposed active sites on particles Data in Brief 30, 105468

Surface functionalization with targeting ligands confers to nanomaterials the ability of selectively recognize a biological target. Therefore, a quantitative characterization of surface functional molecules is critical for the rational development of nanomaterials-based applications, especially in nanomedicine research. Single-molecule localization microscopy can provide visualization of surface molecules at the level of individual particles, preserving the integrity of the material and overcoming the limitations of analytical methods based on ensemble averaging. Here we provide single-molecule localization data obtained on streptavidin-coated polystyrene particles, which can be exploited as a model system for surface-functionalized materials. After loading of the active sites of streptavidin molecules with a biotin-conjugated probe, they were imaged with a DNA-PAINT imaging approach, which can provide single-molecule imaging at subdiffraction resolution and molecule counting. Both raw records and analysed data, consisting in a list of space-time single-molecule coordinates, are shared. Additionally, Matlab functions are provided that analyse the single-molecule coordinates in order to quantify features of individual particles. These data might constitute a valuable reference for applications of similar quantitative imaging methodologies to other types of functionalized nanomaterials.

JTD Keywords: DNA-PAINT, Functional materials, Nanoparticles, Single-molecule localization microscopy, Super-resolution microscopy


Altay, Gizem, Tosi, Sébastien, García-Díaz, María, Martínez, Elena, (2020). Imaging the cell morphological response to 3D topography and curvature in engineered intestinal tissues Frontiers in Bioengineering and Biotechnology 8, 294

While conventional cell culture methodologies have relied on flat, two-dimensional cell monolayers, three-dimensional engineered tissues are becoming increasingly popular. Often, engineered tissues can mimic the complex architecture of native tissues, leading to advancements in reproducing physiological functional properties. In particular, engineered intestinal tissues often use hydrogels to mimic villi structures. These finger-like protrusions of a few hundred microns in height have a well-defined topography and curvature. Here, we examined the cell morphological response to these villus-like microstructures at single-cell resolution using a novel embedding method that allows for the histological processing of these delicate hydrogel structures. We demonstrated that by using photopolymerisable poly(ethylene) glycol as an embedding medium, the villus-like microstructures were successfully preserved after sectioning with vibratome or cryotome. Moreover, high-resolution imaging of these sections revealed that cell morphology, nuclei orientation, and the expression of epithelial polarization markers were spatially encoded along the vertical axis of the villus-like microstructures and that this cell morphological response was dramatically affected by the substrate curvature. These findings, which are in good agreement with the data reported for in vivo experiments on the native tissue, are likely to be the origin of more physiologically relevant barrier properties of engineered intestinal tissues when compared with standard monolayer cultures. By showcasing this example, we anticipate that the novel histological embedding procedure will have a positive impact on the study of epithelial cell behavior on three-dimensional substrates in both physiological and pathological situations.

JTD Keywords: Hydrogel scaffold, Confocal microscopy, Substrate curvature, Cell morphology, Cell orientation, Histological section, Small intestine, Villus


Mencattini, A., Di Giuseppe, D., D'Orazio, M., Rizzuto, V., Manu Pereira, M. M., Colomba Comes, M., Lopez-Martinez, M. J., Samitier, J., Martinelli, E., (2020). A microfluidic device for shape measurement in red blood cells (RBCs) IEEE International Workshop on Medical Measurement and Applications (MEMEA) , IEEE (Bari, Italy) , 1-5

Modern optical sensors coupled with time-lapse microscopy devices and dedicated software tools allow the miniaturization of laboratories for biological experiments leading to the Organ-On-Chip (OoC) framework. OoCs allow performing massive measurements on a large number of cells under the assumption of reproducibility conditions, permitting to investigate the cell dynamics in terms of motility and shape changes over time. In this work, we present the OoC platform used in a preliminary study of the Rare Haemolytic Anaemia (RHA) disease, a group of rare diseases characterized by haemolysis, which is the premature loss of red blood cells (RBCs). Preliminary results demonstrate the effectiveness of shape measurement for the diagnosis of RHA.

JTD Keywords: Anaemia diagnosis, Cell tracking, Plasticity measurement, Time-lapse microscopy


Otero, J., Navajas, D., Alcaraz, J., (2020). Characterization of the elastic properties of extracellular matrix models by atomic force microscopy Methods in Cell Biology (ed. Caballero, David, Kundu, Subhas C., Reis, Rui L.), Academic Press (Cambridge, USA) 156, 59-83

Tissue elasticity is a critical regulator of cell behavior in normal and diseased conditions like fibrosis and cancer. Since the extracellular matrix (ECM) is a major regulator of tissue elasticity and function, several ECM-based models have emerged in the last decades, including in vitro endogenous ECM, decellularized tissue ECM and ECM hydrogels. The development of such models has urged the need to quantify their elastic properties particularly at the nanometer scale, which is the relevant length scale for cell-ECM interactions. For this purpose, the versatility of atomic force microscopy (AFM) to quantify the nanomechanical properties of soft biomaterials like ECM models has emerged as a very suitable technique. In this chapter we provide a detailed protocol on how to assess the Young's elastic modulus of ECM models by AFM, discuss some of the critical issues, and provide troubleshooting guidelines as well as illustrative examples of AFM measurements, particularly in the context of cancer.

JTD Keywords: 3D ECM hydrogels, Atomic force microscopy, Decellularized tissue, Elastic modulus, Endogenous ECM, Extracellular matrix


Redondo-Morata, Lorena, Losada-Pérez, Patricia, Giannotti, Marina Inés, (2020). Lipid bilayers: Phase behavior and nanomechanics Current Topics in Membranes (ed. Levitan, Irena, Trache, Andreea), Academic Press (Berlin, Germany) 86, 1-55

Lipid membranes are involved in many physiological processes like recognition, signaling, fusion or remodeling of the cell membrane or some of its internal compartments. Within the cell, they are the ultimate barrier, while maintaining the fluidity or flexibility required for a myriad of processes, including membrane protein assembly. The physical properties of in vitro model membranes as model cell membranes have been extensively studied with a variety of techniques, from classical thermodynamics to advanced modern microscopies. Here we review the nanomechanics of solid-supported lipid membranes with a focus in their phase behavior. Relevant information obtained by quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM) as complementary techniques in the nano/mesoscale interface is presented. Membrane morphological and mechanical characterization will be discussed in the framework of its phase behavior, phase transitions and coexistence, in simple and complex models, and upon the presence of cholesterol.

JTD Keywords: Lipid phase behavior, Phase transition, Phase coexistence, Nanomechanics, Thermodynamics, Atomic force microscopy (AFM), Quartz crystal microbalance with dissipation monitoring (QCM-D)


Baranov, M. V., Olea, R. A., van den Bogaart, G., (2019). Chasing uptake: Super-resolution microscopy in endocytosis and phagocytosis Trends in Cell Biology 29, (9), 727-739

Since their invention about two decades ago, super-resolution microscopes have become a method of choice in cell biology. Owing to a spatial resolution below 50 nm, smaller than the size of most organelles, and an order of magnitude better than the diffraction limit of conventional light microscopes, superresolution microscopy is a powerful technique for resolving intracellular trafficking. In this review we discuss discoveries in endocytosis and phagocytosis that have been made possible by super-resolution microscopy – from uptake at the plasma membrane, endocytic coat formation, and cytoskeletal rearrangements to endosomal maturation. The detailed visualization of the diverse molecular assemblies that mediate endocytic uptake will provide a better understanding of how cells ingest extracellular material.

JTD Keywords: Endocytosis, Endosomes, Organelles, Super-resolution microscopy, Trafficking


Feiner-Gracia, N., Olea, R. A., Fitzner, R., El Boujnouni, N., Van Asbeck, A. H., Brock, R., Albertazzi, L., (2019). Super-resolution imaging of structure, molecular composition, and stability of single oligonucleotide polyplexes Nano Letters 19, (5), 2784-2792

The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.

JTD Keywords: dSTORM, Gene delivery, Polyplexes, Stability, Super-resolution microscopy


Pollastri, S., Jorba, I., Hawkins, T. J., Llusià , J., Michelozzi, M., Navajas, D., Peñuelas, J., Hussey, P. J., Knight, M. R., Loreto, F., (2019). Leaves of isoprene-emitting tobacco plants maintain PSII stability at high temperatures New Phytologist 223, (3), 1307-1318

At high temperatures, isoprene-emitting plants display a higher photosynthetic rate and a lower nonphotochemical quenching (NPQ) compared with nonemitting plants. The mechanism of this phenomenon, which may be very important under current climate warming, is still elusive. NPQ was dissected into its components, and chlorophyll fluorescence lifetime imaging microscopy (FLIM) was used to analyse the dynamics of excited chlorophyll relaxation in isoprene-emitting and nonemitting plants. Thylakoid membrane stiffness was also measured using atomic force microscope (AFM) to identify a possible mode of action of isoprene in improving photochemical efficiency and photosynthetic stability. We show that, when compared with nonemitters, isoprene-emitting tobacco plants exposed at high temperatures display a reduced increase of the NPQ energy-dependent component (qE) and stable (1) chlorophyll fluorescence lifetime; (2) amplitude of the fluorescence decay components; and (3) thylakoid membrane stiffness. Our study shows for the first time that isoprene maintains PSII stability at high temperatures by preventing the modifications of the surrounding environment, namely providing a more steady and homogeneous distribution of the light-absorbing centres and a stable thylakoid membrane stiffness. Isoprene photoprotects leaves with a mechanism alternative to NPQ, enabling plants to maintain a high photosynthetic rate at rising temperatures.

JTD Keywords: (High) temperature, Atomic force microscopy (AFM), Chlorophyll fluorescence (quenching and lifetime), Fluorescence lifetime imaging microscopy (FLIM), Isoprene, Nonphotochemical quenching (NPQ), Photosynthesis


Garcia-Puig, A., Mosquera, J. L., Jiménez-Delgado, S., García-Pastor, C., Jorba, I., Navajas, D., Canals, F., Raya, A., (2019). Proteomics analysis of extracellular matrix remodeling during zebrafish heart regeneration Molecular & cellular proteomics 18, (9), 1745-1755

Adult zebrafish, in contrast to mammals, are able to regenerate their hearts in response to injury or experimental amputation. Our understanding of the cellular and molecular bases that underlie this process, although fragmentary, has increased significantly over the last years. However, the role of the extracellular matrix (ECM) during zebrafish heart regeneration has been comparatively rarely explored. Here, we set out to characterize the ECM protein composition in adult zebrafish hearts, and whether it changed during the regenerative response. For this purpose, we first established a decellularization protocol of adult zebrafish ventricles that significantly enriched the yield of ECM proteins. We then performed proteomic analyses of decellularized control hearts and at different times of regeneration. Our results show a dynamic change in ECM protein composition, most evident at the earliest (7 days post-amputation) time-point analyzed. Regeneration associated with sharp increases in specific ECM proteins, and with an overall decrease in collagens and cytoskeletal proteins. We finally tested by atomic force microscopy that the changes in ECM composition translated to decreased ECM stiffness. Our cumulative results identify changes in the protein composition and mechanical properties of the zebrafish heart ECM during regeneration.

JTD Keywords: Animal models, Atomic force microscopy, Cardiovascular disease, Cardiovascular function or biology, Developmental biology, Extracellular matrix, Heart regeneration, Proteomic analysis


de Goede, M., Dijkstra, M., Obregón, R., Ramón-Azcón, J., Martínez, Elena, Padilla, L., Mitjans, F., Garcia-Blanco, S. M., (2019). Al2O3 microring resonators for the detection of a cancer biomarker in undiluted urine Optics Express 27, (13), 18508-18521

Concentrations down to 3 nM of the rhS100A4 protein, associated with human tumor development, have been detected in undiluted urine using an integrated sensor based on microring resonators in the emerging Al2O3 photonic platform. The fabricated microrings were designed for operation in the C-band (λ = 1565 nm) and exhibited a high-quality factor in air of 3.2 × 105. The bulk refractive index sensitivity of the devices was ~100 nm/RIU (for TM polarization) with a limit of detection of ~10−6 RIU. A surface functionalization protocol was developed to allow for the selective binding of the monoclonal antibodies designed to capture the target biomarker to the surface of the Al2O3 microrings. The detection of rhS100A4 proteins at clinically relevant concentrations in urine is a big milestone towards the use of biosensors for the screening and early diagnosis of different cancers. Biosensors based on this microring technology can lead to portable, multiplexed and easy-to-use point of care devices.

JTD Keywords: Distributed feedback lasers, Effective refractive index, Laser coupling, Polarization maintaining fibers, Refractive index, Scanning electron microscopy


Aguiar, L., Biosca, A., Lantero, E., Gut, J., Vale, N., Rosenthal, P. J., Nogueira, F., Andreu, D., Fernàndez-Busquets, X., Gomes, P., (2019). Coupling the antimalarial cell penetrating peptide TP10 to classical antimalarial drugs primaquine and chloroquine produces strongly hemolytic conjugates Molecules 24, (24), 4559

Recently, we disclosed primaquine cell penetrating peptide conjugates that were more potent than parent primaquine against liver stage Plasmodium parasites and non-toxic to hepatocytes. The same strategy was now applied to the blood-stage antimalarial chloroquine, using a wide set of peptides, including TP10, a cell penetrating peptide with intrinsic antiplasmodial activity. Chloroquine-TP10 conjugates displaying higher antiplasmodial activity than the parent TP10 peptide were identified, at the cost of an increased hemolytic activity, which was further confirmed for their primaquine analogues. Fluorescence microscopy and flow cytometry suggest that these drug-peptide conjugates strongly bind, and likely destroy, erythrocyte membranes. Taken together, the results herein reported put forward that coupling antimalarial aminoquinolines to cell penetrating peptides delivers hemolytic conjugates. Hence, despite their widely reported advantages as carriers for many different types of cargo, from small drugs to biomacromolecules, cell penetrating peptides seem unsuitable for safe intracellular delivery of antimalarial aminoquinolines due to hemolysis issues. This highlights the relevance of paying attention to hemolytic effects of cell penetrating peptide-drug conjugates.

JTD Keywords: Antimalarial, Cell penetrating peptide, Chloroquine, Erythrocyte fluorescence, Flow cytometry, Hemolysis, Microscopy, Plasmodium, Primaquine, Red blood cell


Pellequer, J. L., Parot, P., Navajas, D., Kumar, S., Svetli, Scheuring, S., Hu, J., Li, B., Engler, A., Sousa, S., Lekka, M., Szymo, Schillers, H., Odorico, M., Lafont, F., Janel, S., Rico, F., (2019). Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed Journal of Molecular Recognition 32, (3), e2773

AFMBioMed is the founding name under which international conferences and summer schools are organized around the application of atomic force microscopy in life sciences and nanomedicine. From its inception at the Atomic Energy Commission in Marcoule near 2004 to its creation in 2007 and to its 10th anniversary conference in Krakow, a brief narrative history of its birth and rise will demonstrate how and what such an organization brings to laboratories and the AFM community. With the current planning of the next AFMBioMed conference in Münster in 2019, it will be 15 years of commitment to these events.

JTD Keywords: Atomic Force Microscopy, Single molecules, Biomechanics, Force spectroscopy, High-speed AFM, Imaging, Nanoindentation, Nanomedicine, Nanotoxicology


Gumí-Audenis, B., Giannotti, M. I., (2019). Structural and mechanical characterization of supported model membranes by AFM Biomimetic Lipid Membranes: Fundamentals, Applications, and Commercialization (ed. Kök, Fatma N., Arslan Yildiz, Ahu, Inci, Fatih), Springer International Publishing (Cham, Germany) , 1-27

Several cellular processes, including adhesion, signaling and transcription, endocytosis, and membrane resealing, among others, involve conformational changes such as bending, vesiculation, and tubulation. These mechanisms generally involve membrane separation from the cytoskeleton as well as strong bending, for which the membrane chemical composition and physicochemical properties, often highly localized and dynamic, are key players. The mechanical role of the lipid membrane in force triggered (or sensing) mechanisms in cells is important, and understanding the lipid bilayers’ physical and mechanical properties is essential to comprehend their contribution to the overall membrane. Atomic force microscopy (AFM)-based experimental approaches have been to date very valuable to deepen into these aspects. As a stand-alone, high-resolution imaging technique and force transducer with the possibility to operate in aqueous environment, it defies most other surface instrumentation in ease of use, sensitivity and versatility. In this chapter, we introduce the different AFM-based methods to assess topological and nanomechanical information on model membranes, specifically to supported lipid bilayers (SLBs), including several examples ranging from pure phospholipid homogeneous bilayers to multicomponent and phase-separated SLBs, increasing the bilayer complexity, in the direction of mimicking biological membranes.

JTD Keywords: Atomic force microscopy, Force spectroscopy, Model membranes, Nanomechanics, Supported lipid bilayers


Samitier, Josep, Correia, A., (2019). Biomimetic Nanotechnology for Biomedical Applications (NanoBio&Med 2018) Biomimetics MDPI

Emerging nanobiotechnologies can offer solutions to the current and future challenges in medicine. By covering topics from regenerative medicine, tissue engineering, drug delivery, bionanofabrication, and molecular biorecognition, this Special Issue aims to provide an update on the trends in nanomedicine and drug delivery using biomimetic approaches, and the development of novel biologically inspired devices for the safe and effective diagnosis, prevention, and treatment of disease.

JTD Keywords: Bioinspired nanotechnologies, Bionanofabrication, Bio-nano measurement and microscopy, Nanomaterials for biological and medical applications, Nanoassemblies, Nanostructured surfaces, Drug delivery, Nanobioelectronics, Integrated systems/nanobiosensors, Nanotoxicology, Graphene-based applications


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 β1 integrin/FAK(Y397) signaling and the actomyosin cytoskeleton in the mechanoresponses of both parenchymal and stromal cells. Moreover AFM has unveiled that the micromechanics of the ECM obtained by tissue decellularization is unique for each anatomical compartment, which may support both its specific function and cell differentiation. AFM has also enabled identifying critical mechanoregulatory proteins involved in branching morphogenesis (MMP14) and acinar differentiation (α3β1 integrin), and has clarified the role of altered tissue mechanics and architecture in a variety of pathologic conditions. Critical technical issues of AFM mechanical measurements like tip geometry effects are also discussed.

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


Dols-Perez, Aurora, Fumagalli, Laura, Gomila, Gabriel, (2018). Interdigitation in spin-coated lipid layers in air Colloids and Surfaces B: Biointerfaces 172, 400-406

In this study, we show that dry saturated phospholipid layers prepared by the spin-coating technique could present thinner regions associated to interdigitated phases under some conditions. The morphological characteristics of lipid layers of saturated phosphocholines, such as dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine (DSPC), have been measured by Atomic Force Microscopy and revealed that the presence of interdigitated regions is not induced by the same parameters that induce them in hydrated samples. To achieve these results the effect of the lipid hidrocabonated chain length, the presence of alcohol in the coating solution, the spinning velocity and the presence of cholesterol were tested. We showed that DPPC and DSPC bilayers, on the one side, can show structures with similar height than interdigitated regions observed in hydrated samples, while, on the other side, DLPC and DMPC tend to show no evidence of interdigitation. Results indicate that the presence of interdigitated areas is due to the presence of lateral tensions and, hence, that they can be eliminated by releasing these tensions by, for instance, the addition of cholesterol. These results demonstrate that interdigitation in lipid layers is a rather general phenomena and can be observed in lipid bilayers in dry conditions.

JTD Keywords: Spin-coating, Lipid layers, Atomic Force Microscopy, Interdigitation


Crespo-Villanueva, Adrián, Gumí-Audenis, Berta, Sanz, Fausto, Artzner, Franck, Mériadec, Cristelle, Rousseau, Florence, Lopez, Christelle, Giannotti, M. I., Guyomarc'h, Fanny, (2018). Casein interaction with lipid membranes: Are the phase state or charge density of the phospholipids affecting protein adsorption? Biochimica et Biophysica Acta (BBA) - Biomembranes 1860, (12), 2588-2598

Casein micelles are ~200 nm electronegative particles that constitute 80 wt% of the milk proteins. During synthesis in the lactating mammary cells, caseins are thought to interact in the form of ~20 nm assemblies, directly with the biological membranes of the endoplasmic reticulum and/or the Golgi apparatus. However, conditions that drive this interaction are not yet known. Atomic force microscopy imaging and force spectroscopy were used to directly observe the adsorption of casein particles on supported phospholipid bilayers with controlled compositions to vary their phase state and surface charge density, as verified by X-ray diffraction and zetametry. At pH 6.7, the casein particles adsorbed onto bilayer phases with zwitterionic and liquid-disordered phospholipid molecules, but not on phases with anionic or ordered phospholipids. Furthermore, the presence of adsorbed caseins altered the stability of the yet exposed bilayer. Considering their respective compositions and symmetry/asymmetry, these results cast light on the possible interactions of casein assemblies with the organelles’ membranes of the lactating mammary cells.

JTD Keywords: Casein proteins, Phospholipid membrane, Supported lipid bilayer, Atomic force microscopy


Farré, N., Otero, J., Falcones, B., Torres, M., Jorba, I., Gozal, D., Almendros, I., Farré, R., Navajas, D., (2018). Intermittent hypoxia mimicking sleep apnea increases passive stiffness of myocardial extracellular matrix. A multiscale study Frontiers in Physiology 9, Article 1143

Background: Tissue hypoxia-reoxygenation characterizes obstructive sleep apnea (OSA), a very prevalent respiratory disease associated with increased cardiovascular morbidity and mortality. Experimental studies indicate that intermittent hypoxia (IH) mimicking OSA induces oxidative stress and inflammation in heart tissue at the cell and molecular levels. However, it remains unclear whether IH modifies the passive stiffness of the cardiac tissue extracellular matrix (ECM). Aim: To investigate multiscale changes of stiffness induced by chronic IH in the ECM of left ventricular (LV) myocardium in a murine model of OSA. Methods: Two-month and 18-month old mice (N = 10 each) were subjected to IH (20% O2 40 s–6% O2 20 s) for 6 weeks (6 h/day). Corresponding control groups for each age were kept under normoxia. Fresh LV myocardial strips (~7 mm × 1 mm × 1 mm) were prepared, and their ECM was obtained by decellularization. Myocardium ECM macroscale mechanics were measured by performing uniaxial stress–strain tensile tests. Strip macroscale stiffness was assessed as the stress value (σ) measured at 0.2 strain and Young’s modulus (EM) computed at 0.2 strain by fitting Fung’s constitutive model to the stress–strain relationship. ECM stiffness was characterized at the microscale as the Young’s modulus (Em) measured in decellularized tissue slices (~12 μm tick) by atomic force microscopy. Results: Intermittent hypoxia induced a ~1.5-fold increase in σ (p < 0.001) and a ~2.5-fold increase in EM (p < 0.001) of young mice as compared with normoxic controls. In contrast, no significant differences emerged in Em among IH-exposed and normoxic mice. Moreover, the mechanical effects of IH on myocardial ECM were similar in young and aged mice. Conclusion: The marked IH-induced increases in macroscale stiffness of LV myocardium ECM suggests that the ECM plays a role in the cardiac dysfunction induced by OSA. Furthermore, absence of any significant effects of IH on the microscale ECM stiffness suggests that the significant increases in macroscale stiffening are primarily mediated by 3D structural ECM remodeling.

JTD Keywords: Atomic force microscopy, Heart mechanics, Myocardial stiffness, Obstructive sleep apnea, Tensile test, Ventricular strain


Menal, M. J., Jorba, I., Torres, M., Montserrat, J. M., Gozal, D., Colell, A., Piñol-Ripoll, G., Navajas, D., Almendros, I., Farré, R., (2018). Alzheimer's disease mutant mice exhibit reduced brain tissue stiffness compared to wild-type mice in both normoxia and following intermittent hypoxia mimicking sleep apnea Frontiers in Neurology 9, Article 1

Background: Evidence from patients and animal models suggests that obstructive sleep apnea (OSA) may increase the risk of Alzheimer’s disease (AD) and that AD is associated with reduced brain tissue stiffness. Aim: To investigate whether intermittent hypoxia (IH) alters brain cortex tissue stiffness in AD mutant mice exposed to IH mimicking OSA. Methods: Six-eight month old (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) AD mutant mice and wild-type (WT) littermates were subjected to IH (21% O2 40 s to 5% O2 20 s; 6 h/day) or normoxia for 8 weeks. After euthanasia, the stiffness (E) of 200-μm brain cortex slices was measured by atomic force microscopy. Results: Two-way ANOVA indicated significant cortical softening and weight increase in AD mice compared to WT littermates, but no significant effects of IH on cortical stiffness and weight were detected. In addition, reduced myelin was apparent in AD (vs. WT), but no significant differences emerged in the cortex extracellular matrix components laminin and glycosaminoglycans when comparing baseline AD and WT mice. Conclusion: AD mutant mice exhibit reduced brain tissue stiffness following both normoxia and IH mimicking sleep apnea, and such differences are commensurate with increased edema and demyelination in AD.

JTD Keywords: Animal model, Atomic force microscopy, Brain mechanics, Cortex stiffness, Neurodegenerative disease


Casanellas, Ignasi, Lagunas, Anna, Tsintzou, Iro, Vida, Yolanda, Collado, Daniel, Pérez-Inestrosa, Ezequiel, Rodríguez-Pereira, Cristina, Magalhaes, Joana, Gorostiza, Pau, Andrades, José A., Becerra, José, Samitier, Josep, (2018). Dendrimer-based uneven nanopatterns to locally control surface adhesiveness: A method to direct chondrogenic differentiation Journal of Visualized Experiments Bioengineering, (131), e56347

Cellular adhesion and differentiation is conditioned by the nanoscale disposition of the extracellular matrix (ECM) components, with local concentrations having a major effect. Here we present a method to obtain large-scale uneven nanopatterns of arginine-glycine-aspartic acid (RGD)-functionalized dendrimers that permit the nanoscale control of local RGD surface density. Nanopatterns are formed by surface adsorption of dendrimers from solutions at different initial concentrations and are characterized by water contact angle (CA), X-ray photoelectron spectroscopy (XPS), and scanning probe microscopy techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). The local surface density of RGD is measured using AFM images by means of probability contour maps of minimum interparticle distances and then correlated with cell adhesion response and differentiation. The nanopatterning method presented here is a simple procedure that can be scaled up in a straightforward manner to large surface areas. It is thus fully compatible with cell culture protocols and can be applied to other ligands that exert concentration-dependent effects on cells.

JTD Keywords: Bioengineering, Dendrimer, Nanopattern, Arginine-Glycine-Aspartic Acid (RGD), Atomic Force Microscopy (AFM), Cell Adhesion, Mesenchymal Stem Cells (Mscs), Chondrogenesis


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.

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


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.

JTD 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.

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


Marsal, Maria, Jorba, Ignasi, Rebollo, Elena, Luque, Tomas, Navajas, Daniel, Martín-Blanco, Enrique, (2017). AFM and microrheology in the zebrafish embryo yolk cell Journal of Visualized Experiments Developmental Biology, (129), e56224

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

JTD Keywords: Developmental Biology, Zebrafish, Yolk, Atomic Force Microscopy, Cortical Tension, Microrheology, Nanoparticle tracking


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.

JTD 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.

JTD 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.

JTD 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, M. I., (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

JTD 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.

JTD 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.

JTD 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.

JTD 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.

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


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.

JTD 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.

JTD 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.

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


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.

JTD 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.

JTD 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.

JTD 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.

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


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.

JTD 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.

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


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.

JTD 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.

JTD 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.

JTD 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.

JTD 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(ε-caprolactone) fibrous scaffold modified with calcium phosphate for bone tissue engineering 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.

JTD 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


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.

JTD 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.

JTD 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.

JTD 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.

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


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.

JTD 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.

JTD 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.

JTD 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 β complex formation 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 β peptide (Aβ). However, few clues about the formation of the magnetite-Aβ complex have been reported. We have investigated the interaction between these important players in the AD with superconducting quantum interference, scanning electron microscope, surface plasmon resonance, and magnetic force microscopy. The results support the notion that the magnetite-Aβ complex is created before the synthesis of the magnetic nanoparticles, bringing a highly stable interaction of this couple.

JTD 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.

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


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.

JTD 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.

JTD 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.

JTD 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.

JTD 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.

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


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.

JTD 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 (β) strongly supports a two-step tunneling ET mechanism. Statistical analysis of decay constant measurements reveals differences between the reduced and oxidized states that may be relevant to the control of ET rates in enzymes and biological electron transport chains.

JTD 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.

JTD 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.

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


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.

JTD 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.

JTD 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.

JTD 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).

JTD 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.

JTD 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.

JTD 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.

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


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.

JTD 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.

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


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.

JTD 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.

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


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.

JTD 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.

JTD 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.

JTD 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.

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


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.

JTD 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.

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


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.

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


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.

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


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.

JTD 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.

JTD 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


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.

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