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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)


Pala, M, El Khannaji, H, Garay-Sarmiento, M, Ronda, JC, Cadiz, V, Galia, M, Percec, V, Rodriguez-Emmenegger, C, Lligadas, G, (2022). A green solvent-to-polymer upgrading approach to water-soluble LCST poly(N-substituted lactamide acrylate)s Green Chemistry 21, 8314-8323

We report a green solvent-to-polymer upgrading transformation of chemicals of the lactic acid portfolio into water-soluble lower critical solution temperature (LCST)-type acrylic polymers. Aqueous Cu(0)-mediated living radical polymerization (SET-LRP) was utilized for the rapid synthesis of N-substituted lactamide-type homo and random acrylic copolymers under mild conditions. A particularly unique aspect of this work is that the water-soluble monomers and the SET-LRP initiator used to produce the corresponding polymers were synthesized from biorenewable and non-toxic solvents, namely natural ethyl lactate and BASF's Agnique (R) AMD 3L (N,N-dimethyl lactamide, DML). The pre-disproportionation of Cu(I) Br in the presence of tris[2-(dimethylamino)ethyl]amine (Me6TREN) in water generated nascent Cu(0) and Cu(II) complexes that facilitated the fast polymerization of N-tetrahydrofurfuryl lactamide and N,N-dimethyl lactamide acrylate monomers (THFLA and DMLA, respectively) up to near-quantitative conversion with excellent control over molecular weight (5000 < M-n < 83 000) and dispersity (1.05 < D < 1.16). Interestingly, poly(THFLA) showed a degree of polymerization and concentration dependent LCST behavior, which can be fine-tuned (T-cp = 12-62 degrees C) through random copolymerization with the more hydrophilic DMLA monomer. Finally, covalent cross-linking of these polymers resulted in a new family of thermo-responsive hydrogels with excellent biocompatibility and tunable swelling and LCST transition. These illustrate the versatility of these neoteric green polymers in the preparation of smart and biocompatible soft materials.

JTD Keywords: Acid, Ethyl lactate, Living radical polymerization, Monomers, Pnipam, Reductive amination, Ruthenium nanoparticles, Set-lrp, Single, Thermoresponsive polymers


Zamora RA, López-Ortiz M, Sales-Mateo M, Hu C, Croce R, Maniyara RA, Pruneri V, Giannotti MI, Gorostiza P, (2022). Light- and Redox-Dependent Force Spectroscopy Reveals that the Interaction between Plastocyanin and Plant Photosystem I Is Favored when One Partner Is Ready for Electron Transfer Acs Nano 16, 15155-15164

Photosynthesis is a fundamental process that converts photons into chemical energy, driven by large protein complexes at the thylakoid membranes of plants, cyanobacteria, and algae. In plants, water-soluble plastocyanin (Pc) is responsible for shuttling electrons between cytochrome b6f complex and the photosystem I (PSI) complex in the photosynthetic electron transport chain (PETC). For an efficient turnover, a transient complex must form between PSI and Pc in the PETC, which implies a balance between specificity and binding strength. Here, we studied the binding frequency and the unbinding force between suitably oriented plant PSI and Pc under redox control using single molecule force spectroscopy (SMFS). The binding frequency (observation of binding-unbinding events) between PSI and Pc depends on their respective redox states. The interaction between PSI and Pc is independent of the redox state of PSI when Pc is reduced, and it is disfavored in the dark (reduced P700) when Pc is oxidized. The frequency of interaction between PSI and Pc is higher when at least one of the partners is in a redox state ready for electron transfer (ET), and the post-ET situation (PSIRed-PcOx) leads to lower binding. In addition, we show that the binding of ET-ready PcRed to PSI can be regulated externally by Mg2+ ions in solution.

JTD Keywords: architecture, binding-site, complexes, ferredoxin, force spectroscopy, induced structural-changes, interprotein electron transfer, light-dependent interaction, mg2+ concentration, photosystem i, plastocyanin, probe, recognition, reduction, Force spectroscopy, Interprotein electron transfer, Light-dependent interaction, Photosynthetic reaction-center, Photosystem i, Plastocyanin, Single molecule measurements


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


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


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


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


Macedo, MH, Barros, AS, Martinez, E, Barrias, CC, Sarmento, B, (2022). All layers matter: Innovative three-dimensional epithelium-stroma-endothelium intestinal model for reliable permeability outcomes Journal Of Controlled Release 341, 414-430

Drug development is an ever-growing field, increasingly requesting reliable in vitro tools to speed up early screening phases, reducing the need for animal experiments. In oral delivery, understanding the absorption pattern of a new drug in the small intestine is paramount. Classical two-dimensional (2D) in vitro models are generally too simplistic and do not accurately represent native tissues. The main goal of this work was to develop an advanced three-dimensional (3D) in vitro intestinal model to test absorption in a more reliable manner, by better mimicking the native environment. The 3D model is composed of a collagen-based stromal layer with embedded fibroblasts mimicking the intestinal lamina propria and providing support for the epithelium, composed of enterocytes and mucus-secreting cells. An endothelial layer, surrogating the absorptive capillary network, is also present. The cellular crosstalk between the different cells present in the model is unveiled, disclosing key players, namely those involved in the contraction of collagen by fibroblasts. The developed 3D model presents lower levels of P-glycoprotein (P-gp) and Multidrug Resistance Protein 2 (MRP2) efflux transporters, which are normally overexpressed in traditional Caco-2 models, and are paramount in the absorption of many compounds. This, allied with transepithelial electrical resistance (TEER) values closer to physiological ranges, leads to improved and more reliable permeability outcomes, which are observed when comparing our results with in vivo data.

JTD Keywords: 3d intestinal model, drug absorption, drug development, endothelium, hydrogel, 3d intestinal model, 3d modeling, 3d models, 3d-modeling, Alkaline-phosphatase, Animal experiments, Biopharmaceutics classification, Caco-2 cells, Cell culture, Collagen, Collagen gel, Drug absorption, Drug development, Endothelium, Fibroblasts, Glycoproteins, Hydrogel, In-vitro, Matrix metalloproteinases, Membrane-permeability, Paracellular transport, Permeability, Single-pass vs., Speed up


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


Riera, Roger, Hogervorst, Tim P., Doelman, Ward, Ni, Yan, Pujals, Silvia, Bolli, Evangelia, Codée, Jeroen DC., van Kasteren, Sander I., Albertazzi, Lorenzo, (2021). Single-molecule imaging of glycan–lectin interactions on cells with Glyco-PAINT Nature Chemical Biology 17, 1281-+

Most lectins bind carbohydrate ligands with relatively low affinity, making the identification of optimal ligands challenging. Here we introduce a point accumulation in nanoscale topography (PAINT) super-resolution microscopy method to capture weak glycan-lectin interactions at the single-molecule level in living cells (Glyco-PAINT). Glyco-PAINT exploits weak and reversible sugar binding to directly achieve single-molecule detection and quantification in cells and is used to establish the relative kon and koff rates of a synthesized library of carbohydrate-based probes, as well as the diffusion coefficient of the receptor-sugar complex. Uptake of ligands correlates with their binding affinity and residence time to establish structure-function relations for various synthetic glycans. We reveal how sugar multivalency and presentation geometry can be optimized for binding and internalization. Overall, Glyco-PAINT represents a powerful approach to study weak glycan-lectin interactions on the surface of living cells, one that can be potentially extended to a variety of lectin-sugar interactions.© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.

JTD Keywords: dc-sign, density, dimerization, endocytosis, lateral mobility, ligand-binding, mannose receptor, proteins, recognition, Animal, Animals, Cell membrane, Cell membrane permeability, Chemistry, Cho cell line, Cho cells, Cricetulus, Cysteine-rich domain, Kinetics, Lectin, Lectins, Ligand, Ligands, Molecular library, Multivariate analysis, Polysaccharide, Polysaccharides, Procedures, Protein binding, Single molecule imaging, Small molecule libraries, Structure activity relation, Structure-activity relationship


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


Dhillon P, Park J, Hurtado del Pozo C, Li L, Doke T, Huang S, Zhao J, Kang HM, Shrestra R, Balzer MS, Chatterjee S, Prado P, Han SY, Liu H, Sheng X, Dierickx P, Batmanov K, Romero JP, Prósper F, Li M, Pei L, Kim J, Montserrat N, Susztak K, (2021). The Nuclear Receptor ESRRA Protects from Kidney Disease by Coupling Metabolism and Differentiation Cell Metabolism 33,

© 2020 Elsevier Inc. Using single-cell RNA sequencing, Susztak and colleagues, show major changes in cell diversity in mouse models of kidney fibrosis. Proximal tubule (PT) cells are highly vulnerable to dysfunction in fibrosis and show altered differentiation. Nuclear receptors such as ESRRA maintain both PT cell metabolism and differentiation by directly regulating PT-cell-specific genes.

JTD Keywords: chronic kidney disease, esrra, fatty-acid oxidation, fibrosis, kidney, organoids, ppara, proximal tubule cells, single-cell atac sequencing, Chronic kidney disease, Esrra, Fatty-acid oxidation, Fibrosis, Kidney, Organoids, Ppara, Proximal tubule cells, Single-cell atac sequencing, Single-cell rna sequencing


Delcanale, P., Porciani, D., Pujals, S., Jurkevich, A., Chetrusca, A., Tawiah, K. D., Burke, D. H., Albertazzi, L., (2020). Aptamers with tunable affinity enable single-molecule tracking and localization of membrane receptors on living cancer cells Angewandte Chemie - International Edition 59, (42), 18546-18555

Tumor cell-surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single-molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single-molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live-cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single-molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.

JTD Keywords: Aptamers, Cell-surface receptors, Live-cell imaging, PAINT, Single-molecule tracking


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


Kechagia, Jenny Z., Ivaska, Johanna, Roca-Cusachs, Pere, (2019). Integrins as biomechanical sensors of the microenvironment Nature Reviews Molecular Cell Biology 20, (8), 457-473

Integrins, and integrin-mediated adhesions, have long been recognized to provide the main molecular link attaching cells to the extracellular matrix (ECM) and to serve as bidirectional hubs transmitting signals between cells and their environment. Recent evidence has shown that their combined biochemical and mechanical properties also allow integrins to sense, respond to and interact with ECM of differing properties with exquisite specificity. Here, we review this work first by providing an overview of how integrin function is regulated from both a biochemical and a mechanical perspective, affecting integrin cell-surface availability, binding properties, activation or clustering. Then, we address how this biomechanical regulation allows integrins to respond to different ECM physicochemical properties and signals, such as rigidity, composition and spatial distribution. Finally, we discuss the importance of this sensing for major cell functions by taking cell migration and cancer as examples.

JTD Keywords: Cell migration, Extracellular matrix, Integrins, Mechanotransduction, Single-molecule biophysics


de Goede, Michiel, Chang, Lantian, Mu, Jinfeng, Dijkstra, Meindert, Obregón, Raquel, Martínez, Elena, Padilla, Laura, Mitjans, Francesc, Garcia-Blanco, Sonia M., (2019). Al2O3:Yb3+ integrated microdisk laser label-free biosensor Optics Letters 44, (24), 5937-5940

Whispering gallery mode resonator lasers hold the promise of an ultralow intrinsic limit of detection. However, the widespread use of these devices for biosensing applications has been hindered by the complexity and lack of robustness of the proposed configurations. In this work, we demonstrate biosensing with an integrated microdisk laser. Al2O3doped with Yb3+ was utilized because of its low optical losses as well as its emission in the range 1020–1050 nm, outside the absorption band of water. Single-mode laser emission was obtained at a wavelength of 1024 nm with a linewidth of 250 kHz while the microdisk cavity was submerged in water. A limit of detection of 300 pM (3.6 ng/ml) of the protein rhS100A4 in urine was experimentally demonstrated, showing the potential of the proposed devices for biosensing.

JTD Keywords: Distributed feedback lasers, Fiber lasers, Laser modes, Microdisk lasers, Single mode lasers, Tunable lasers


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


Martinez-Hernandez, Uriel, Vouloutsi, Vasiliki, Mura, Anna, Mangan, Michael, Asada, Minoru, Prescott, T. J., Verschure, P., (2019). Biomimetic and Biohybrid Systems 8th International Conference, Living Machines 2019, Nara, Japan, July 9–12, 2019, Proceedings , Springer, Cham (Lausanne, Switzerland) 11556, 1-384

This book constitutes the proceedings of the 8th International Conference on Biomimetic and Biohybrid Systems, Living Machines 2019, held in Nara, Japan, in July 2019. The 26 full and 16 short papers presented in this volume were carefully reviewed and selected from 45 submissions. They deal with research on novel life-like technologies inspired by the scientific investigation of biological systems, biomimetics, and research that seeks to interface biological and artificial systems to create biohybrid systems.

JTD Keywords: Artificial intelligence, Biomimetics, Computer architecture, Human robot interaction, Human-Computer Interaction (HCI), Humanoid robot, Image processing, Learning algorithms, Mobile robots, Multipurpose robots, Neural networks, Quadruped robots, Reinforcement learning, Robot learning, Robotics, Robots, Sensor, Sensors, Swarm robotics, User interfaces


Aragonès, Albert C., Medina, Ernesto, Ferrer-Huerta, Miriam, Gimeno, Nuria, Teixidó, Meritxell, Palma, Julio L., Tao, Nongjian, Ugalde, Jesus M., Giralt, Ernest, Díez-Pérez, Ismael, Mujica, Vladimiro, (2017). Measuring the spin-polarization power of a single chiral molecule Small 13, (2), 1602519

The electronic spin filtering capability of a single chiral helical peptide is measured. A ferromagnetic electrode source is employed to inject spin-polarized electrons in an asymmetric single-molecule junction bridging an α-helical peptide sequence of known chirality. The conductance comparison between both isomers allows the direct determination of the polarization power of an individual chiral molecule.

JTD Keywords: Alpha-helical peptides, Chiral transport, Single-molecule wires, Spin-polarization power, Spin-polarized transmission


Santander-Nelli, M., Silva, C. P., Espinoza-Vergara, J., Silva, J. F., Olguín, C. F., Cortés-Arriagada, D., Zagal, J. H., Mendizabal, F., Díez-Pérez, I., Pavez, J., (2017). Tailoring electroactive surfaces by non-template molecular assembly. Towards electrooxidation of L-cysteine Electrochimica Acta 254, 201-213

We have prepared a nanoelectrode ensemble containing vertically aligned single walled carbon nanotubes (SWCNTs) using a non-template molecular self-assembling strategy. We used a bottom-up construction approach to assemble amino functionalized SWCNTs (af-SWCNTs) in a well-defined architecture. These af-SWCNTs were linked and vertically aligned to pre-formed self-assembled monolayers of 4-MBA. A Cobalt(II) tetracarboxyphthalocyanine (Co(COOH)4Pc) complex was covalently bonded to external portion of af-SWCNTs to complete the final nanoelectrode ensemble. X-ray photoelectron spectroscopy (XPS) and Atomic Force Microcopy (AFM) confirmed the effectiveness of the assembling steps on the gold surface starting from the Au/MBA SAMs. The system Au/4-MBA/af-SWCNTs shows an interface with large ordered array, which exhibits a high activity for the electrooxidation of L-cysteine (L-cys). Theoretical calculations suggest that the incorporation of the af-SWCNTs increased the activity of the assembly to electronic transfer and it was observed that the electrooxidation reaction is energetically favorable.

JTD Keywords: Bottom-up construction, DFT, Modified electrode, Molecular assembly, SAMs, Single walled carbon nanotube


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

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

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


Ramos, E., Pardo, W. A., Mir, M., Samitier, J., (2017). Dependence of carbon nanotubes dispersion kinetics on surfactants Nanotechnology 28, (13), 135702

Carbon nanotubes (CNTs) have been the subject of many studies due to their unique structure and desirable properties. However, the ability to solubilize and separate single CNTs from the bundles they form is still a challenge that needs to be overcome in order to extend their applications in the field of Nanotechnology. Covalent interactions are designed to modify CNTs surface and so prevent agglomeration. Though, this method alters the structures and intrinsic properties of CNTs. In the present work, noncovalent approaches to functionalize and solubilize CNTs are studied in detail. A dispersion kinetic study was performed to characterize the ability of different type of surfactants (non-ionic, anionic, cationic and biopolymer) to unzip CNT bundles. The dispersion kinetic study performed depicts the distinct CNTs bundles unzipping behavior of the different type of surfactants and the results elucidate specific wavelengths in relation with the degree of CNT clustering, which provides new tools for a deeper understanding and characterization of CNTs. Small angle x-ray scattering and transmission electron microscopy results are in agreement with UV-vis-NIR observations, revealing perfectly monodispersed CNTs for the biopolymer and cationic surfactant.

JTD Keywords: Dispersion, DNA, Single-walled carbon nanotubes (SWCNTs), Small angle x-ray scattering (SAXS), Sodium dodecyl sulfate (SDS), Surfactant, Triton X-100


Torras, Núria, Agusil, Juan Pablo, Vázquez, Patricia, Duch, Marta, Hernández-Pinto, Alberto M., Samitier, Josep, de la Rosa, Enrique J., Esteve, Jaume, Suárez, Teresa, Pérez-García, Lluïsa, Plaza, José A., (2016). Suspended planar-array chips for molecular multiplexing at the microscale Advanced Materials 28, (7), 1449–1454

A novel suspended planar-array chips technology is described, which effectively allows molecular multiplexing using a single suspended chip to analyze extraordinarily small volumes. The suspended chips are fabricated by combining silicon-based technology and polymer-pen lithography, obtaining increased molecular pattern flexibility, and improving miniaturization and parallel production. The chip miniaturization is so dramatic that it permits the intracellular analysis of living cells.

JTD Keywords: Chip-in-a-cell, Suspended-arrays, Planar-arrays, Silicon chips, Single-cell analysis


Aragonès, A. C., Aravena, D., Cerdá, J. I., Acís-Castillo, Z., Li, H., Real, J. A., Sanz, F., Hihath, J., Ruiz, E., Díez-Pérez, I., (2016). Large conductance switching in a single-molecule device through room temperature spin-dependent transport Nano Letters 16, (1), 218-226

Controlling the spin of electrons in nanoscale electronic devices is one of the most promising topics aiming at developing devices with rapid and high density information storage capabilities. The interface magnetism or spinterface resulting from the interaction between a magnetic molecule and a metal surface, or vice versa, has become a key ingredient in creating nanoscale molecular devices with novel functionalities. Here, we present a single-molecule wire that displays large (>10000%) conductance switching by controlling the spin-dependent transport under ambient conditions (room temperature in a liquid cell). The molecular wire is built by trapping individual spin crossover FeII complexes between one Au electrode and one ferromagnetic Ni electrode in an organic liquid medium. Large changes in the single-molecule conductance (>100-fold) are measured when the electrons flow from the Au electrode to either an α-up or a β-down spin-polarized Ni electrode. Our calculations show that the current flowing through such an interface appears to be strongly spin-polarized, thus resulting in the observed switching of the single-molecule wire conductance. The observation of such a high spin-dependent conductance switching in a single-molecule wire opens up a new door for the design and control of spin-polarized transport in nanoscale molecular devices at room temperature.

JTD Keywords: Density functional calculations, Magnetoresistance, Single-molecule junctions, Spin orbit coupling, Spin-crossover complexes, Spinterface, STM break-junction


Aragonès, Albert C., Darwish, Nadim, Im, JongOne, Lim, Boram, Choi, Jeongae, Koo, Sangho, Díez-Pérez, Ismael, (2015). Fine-tuning of single-molecule conductance by tweaking both electronic structure and conformation of side substituents Chemistry – A European Journal , 21, (21), 7716-7720

Herein, we describe a method to fine-tune the conductivity of single-molecule wires by employing a combination of chemical composition and geometrical modifications of multiple phenyl side groups as conductance modulators embedded along the main axis of the electronic pathway. We have measured the single-molecule conductivity of a novel series of phenyl-substituted carotenoid wires whose conductivity can be tuned with high precision over an order of magnitude range by modulating both the electron-donating character of the phenyl substituent and its dihedral angle. It is demonstrated that the electronic communication between the phenyl side groups and the molecular wire is maximized when the phenyl groups are twisted closer to the plane of the conjugated molecular wire. These findings can be refined to a general technique for precisely tuning the conductivity of molecular wires.

JTD Keywords: Carotenoids, Conductance, Self-assembly, Single-molecule studies, STM break junction


Ponce, I., Aragonès, A. C., Darwish, Nadrim, Pla-Vilanova, P., Oñate, R., Rezende, M. C., Zagal, J. H., Sanz, F., Pavez, J., Díez-Pérez, I., (2015). Building nanoscale molecular wires exploiting electrocatalytic interactions Electrochimica Acta 179, 611-167

Herein, we present a novel method to design nanoscale molecular wires by exploiting well-established electrocatalytic molecular platforms based on metallophthalocyanine blocks. Metallophthalocyanines exhibit high catalytic activity for a wide variety of electrochemical reactions of practical interests. To this aim, metallophthalocyanine molecules can be attached to an electrode surface via a conjugated mercaptopyridine axial ligand that provides (i) stable chemical binding to the metal surface through the thiol-anchoring group, and (ii) a good electrical communication between the metallophthalocyanine ring and the electrode surface. Our previous work demonstrates that long mercaptopyridinium blocks act as excellent linkers in such electrocatalytic platform, resulting in an optimal electrocatalytic activity of the metallophthalocyanine unit. Here we profit from this optimized electrocatalytic molecular platform to design new molecular wires that connect a metal nanoscale junction in a highly efficient and tunable way. To this aim, we use an STM break-junction approach to control the formation of a nanometric gap between two Au electrodes, both functionalized with mercaptopyridinium (bottom) and mercaptopyridine (top). When metallophthalocyanine is introduced into the functionalized metal nanojunction, stable molecular connections between the two electrodes are formed through axial coordination to the top and bottom pyridine moieties. We show that the highest conductance of the resulting nanoscale molecular wire corresponds to an Fe-phthalocyanine as compare to a Cu-phthalocyanine, which follows the electrocatalytic trend for such molecular systems. These results not only demonstrate a new strategy to design new families of highly conductive and tunable nanoscale molecular wires, but it also brings a new nanoscale electrical platform to help understanding some fundamental mechanistic aspects of molecular electrocatalysis.

JTD Keywords: Single-molecule wires, Metallophthalocyanine, Electrocatalytic molecular platform, Molecular Electronics, STM break-junction


Pla-Vilanova, P., Aragonès, A. C., Ciampi, S., Sanz, F., Darwish, N., Diez-Perez, I., (2015). The spontaneous formation of single-molecule junctions via terminal alkynes Nanotechnology 26, 381001

Herein, we report the spontaneous formation of single-molecule junctions via terminal alkyne contact groups. Self-assembled monolayers that form spontaneously from diluted solutions of 1, 4-diethynylbenzene (DEB) were used to build single-molecule contacts and assessed using the scanning tunneling microscopy-break junction technique (STM-BJ). The STM-BJ technique in both its dynamic and static approaches was used to characterize the lifetime (stability) and the conductivity of a single-DEB wire. It is demonstrated that single-molecule junctions form spontaneously with terminal alkynes and require no electrochemical control or chemical deprotonation. The alkyne anchoring group was compared against typical contact groups exploited in single-molecule studies, i.e. amine (benzenediamine) and thiol (benzendithiol) contact groups. The alkyne contact showed a conductance magnitude comparable to that observed with amine and thiol groups. The lifetime of the junctions formed from alkynes were only slightly less than that of thiols and greater than that observed for amines. These findings are important as (a) they extend the repertoire of chemical contacts used in single-molecule measurements to 1-alkynes, which are synthetically accessible and stable and (b) alkynes have a remarkable affinity toward silicon surfaces, hence opening the door for the study of single-molecule transport on a semiconducting electronic platform.

JTD Keywords: Ferrocene, Molecular electronics, Single-molecule electronics, Single-molecule junctions, Singlemolecule contacts, STM-break junction, Terminal alkyne


Hernansanz, A., Casals, A., Amat, J., (2015). A multi-robot cooperation strategy for dexterous task oriented teleoperation Robotics and Autonomous Systems , 68, 156-172

The use of multiple robots working cooperatively in a redundant way offers new possibilities in the execution of complex tasks in dynamic workspaces. The aim of this work is to increase the range of applicability of teleoperated systems by means of the automatic cooperation of multiple slave robots which, controlled by a human operator, act as if they were a unique robot: a Multi-Robot Cooperation Platform for Task-Oriented Teleoperation, MRCP. From the human operator commands, this robotic platform, the MRCP, dynamically selects the most suitable slave robot and manages, when necessary, a task transfer from one robot to another in order to achieve a smooth execution of teleoperated tasks. The result of the proposed methodology is an improved teleoperated system in terms of reachable workspace (volume, manoeuvrability and accessibility) and dexterity, thus widening its range of applicability. This approach allows human operators to focus their attention on the ongoing task more than on the teleoperated robots.

JTD Keywords: Multi-robot cooperation, Single-operator-multiple-robot, Task-oriented teleoperation


Darwish, Nadim., Aragonès, A. C., Darwish, T., Ciampi, S., Díez-Pérez, I., (2014). Multi-responsive photo- and chemo-electrical single-molecule switches Nano Letters 14, (12), 7064-7070

Incorporating molecular switches as the active components in nanoscale electrical devices represents a current challenge in molecular electronics. It demands key requirements that need to be simultaneously addressed including fast responses to external stimuli and stable attachment of the molecules to the electrodes while mimicking the operation of conventional electronic components. Here, we report a single-molecule switching device that responds electrically to optical and chemical stimuli. A light pointer or a chemical signal can rapidly and reversibly induce the isomerization of bifunctional spiropyran derivatives in the bulk reservoir and, consequently, switch the electrical conductivity of the single-molecule device between a low and a high level. The spiropyran derivatives employed are chemically functionalized such that they can respond in fast but practical time scales. The unique multistimuli response and the synthetic versatility to control the switching schemes of this single-molecule device suggest spiropyran derivatives as key candidates for molecular circuitry.

JTD Keywords: Molecular Electronics, Multi-Responsive Molecular Switches, Photo- and Chemo-Switches Spiropyran, Single-Molecule Conductance, STM Break-Junction, Electronic equipment, Isomerization, Molecular electronics, Photochromism, Electrical conductivity, Electronic component, Molecular switches, Single-molecule conductances, Single-molecule devices, Spiropyran derivatives, Spiropyrans, STM Break-Junction, Molecules


Artés, J. M., López-Martínez, M., Díez-Pérez, I., Sanz, F., Gorostiza, P., (2014). Nanoscale charge transfer in redox proteins and DNA: Towards biomolecular electronics Electrochimica Acta 140, 83-95

Understanding how charges move through and between biomolecules is a fundamental question that constitutes the basis for many biological processes. On the other hand, it has potential applications in the design of sensors based on biomolecules and single molecule devices. In this review we introduce the study of the electron transfer (ET) process in biomolecules, providing an overview of the fundamental theory behind it and the different experimental approaches. The ET in proteins is introduced by reviewing a complete electronic characterization of a redox protein (azurin) using electrochemical scanning tunnelling microscopy (ECSTM). The ET process in DNA is overviewed and results from different experimental approaches are discussed. Finally, future directions in the study of the ET process in biomolecules are introduced as well as examples of possible technological applications.

JTD Keywords: Bioelectrochemistry, Biomolecular electronics, Charge transfer, Nanobiodevice, Single-molecule junction


Zaffino, R. L., Mir, M., Samitier, J., (2014). Label-free detection of DNA hybridization and single point mutations in a nano-gap biosensor Nanotechnology 25, (10), 105501 (8)

We describe a conductance-based biosensor that exploits DNA-mediated long-range electron transport for the label-free and direct electrical detection of DNA hybridization. This biosensor platform comprises an array of vertical nano-gap biosensors made of gold and fabricated through standard photolithography combined with focused ion beam lithography. The nano-gap walls are covalently modified with short, anti-symmetric thiolated DNA probes, which are terminated by 19 bases complementary to both the ends of a target DNA strand. The nano-gaps are separated by a distance of 50nm, which was adjusted to fit the length of the DNA target plus the DNA probes. The hybridization of the target DNA closes the gap circuit in a switch on/off fashion, in such a way that it is readily detected by an increase in the current after nano-gap closure. The nano-biosensor shows high specificity in the discrimination of base-pair mismatching and does not require signal indicators or enhancing molecules. The design of the biosensor platform is applicable for multiplexed detection in a straightforward manner. The platform is well-suited to mass production, point-of-care diagnostics, and wide-scale DNA analysis applications.

JTD Keywords: Biosensor, DNA hybridization, Labelfree, Nanogap, Single nucleotide mutation


Fresco-Cala, B., Jimenez-Soto, J. M., Cardenas, S., Valcarcel, M., (2014). Single-walled carbon nanohorns immobilized on a microporous hollow polypropylene fiber as a sorbent for the extraction of volatile organic compounds from water samples Microchimica Acta , 181, (9-10), 1117-1124

We have evaluated the behavior of single-walled carbon nanohorns as a sorbent for headspace and direct immersion (micro)solid phase extraction using volatile organic compounds (VOCs) as model analytes. The conical carbon nanohorns were first oxidized in order to increase their solubility in water and organic solvents. A microporous hollow polypropylene fiber served as a mechanical support that provides a high surface area for nanoparticle retention. The extraction unit was directly placed in the liquid sample or the headspace of an aqueous standard or a water sample to extract and preconcentrate the VOCs. The variables affecting extraction have been optimized. The VOCs were then identified and quantified by GC/MS. We conclude that direct immersion of the fiber is the most adequate method for the extraction of VOCs from both liquid samples and headspace. Detection limits range from 3.5 to 4.3 ng L-1 (excepted for toluene with 25 ng L-1), and the precision (expressed as relative standard deviation) is between 3.9 and 9.6 %. The method was applied to the determination of toluene, ethylbenzene, various xylene isomers and styrene in bottled, river and tap waters, and the respective average recoveries of spiked samples are 95.6, 98.2 and 86.0 %.

JTD Keywords: (Micro)solid phase extraction, Nanotechnology, Oxidized single-walled carbon nanohorns, Volatiles compounds, Waters


Guo, S., Artés, J. M., Díez-Pérez, I., (2013). Electrochemically-gated single-molecule electrical devices Electrochimica Acta 63rd Annual Meeting of the International Society of Electrochemistry , Elsevier (Prague, Czech Republic) 110, 741-753

In the last decade, single-molecule electrical contacts have emerged as a new experimental platform that allows exploring charge transport phenomena in individual molecular blocks. This novel tool has evolved into an essential element within the Molecular Electronics field to understand charge transport processes in hybrid (bio)molecule/electrode interfaces at the nanoscale, and prospect the implementation of active molecular components into functional nanoscale optoelectronic devices. Within this area, three-terminal single-molecule devices have been sought, provided that they are highly desired to achieve full functionality in logic electronic circuits. Despite the latest experimental developments offer consistent methods to bridge a molecule between two electrodes (source and drain in a transistor notation), placing a third electrode (gate) close to the single-molecule electrical contact is still technically challenging. In this vein, electrochemically-gated single-molecule devices have emerged as an experimentally affordable alternative to overcome these technical limitations. In this review, the operating principle of an electrochemically-gated single-molecule device is presented together with the latest experimental methodologies to built them and characterize their charge transport characteristics. Then, an up-to-date comprehensive overview of the most prominent examples will be given, emphasizing on the relationship between the molecular structure and the final device electrical behaviour.

JTD Keywords: Electrochemical gate, Electrochemical switches, NDR, Single-molecule junctions, Unipolar/ambipolar FETs


Bakker, G. J., Eich, C., Torreno-Pina, J. A., Diez-Ahedo, R., Perez-Samper, G., Van Zanten, T. S., Figdor, C. G., Cambi, A., Garcia-Parajo, M. F., (2012). Lateral mobility of individual integrin nanoclusters orchestrates the onset for leukocyte adhesion Proceedings of the National Academy of Sciences of the United States of America 109, (13), 4869-4874

Integrins are cell membrane adhesion receptors involved in morphogenesis, immunity, tissue healing, and metastasis. A central, yet unresolved question regarding the function of integrins is how these receptors regulate both their conformation and dynamic nanoscale organization on the membrane to generate adhesion-competent microclusters upon ligand binding. Here we exploit the high spatial (nanometer) accuracy and temporal resolution of single-dye tracking to dissect the relationship between conformational state, lateral mobility, and microclustering of the integrin receptor lymphocyte function-associated antigen 1 (LFA-1) expressed on immune cells. We recently showed that in quiescent monocytes, LFA-1 preorganizes in nanoclusters proximal to nanoscale raft components. We now show that these nanoclusters are primarily mobile on the cell surface with a small (ca. 5%) subset of conformational- active LFA-1 nanoclusters preanchored to the cytoskeleton. Lateral mobility resulted crucial for the formation of microclusters upon ligand binding and for stable adhesion under shear flow. Activation of high-affinity LFA-1 by extracellular Ca 2+ resulted in an eightfold increase on the percentage of immobile nanoclusters and cytoskeleton anchorage. Although having the ability to bind to their ligands, these active nanoclusters failed to support firm adhesion in static and low shear-flow conditions because mobility and clustering capacity were highly compromised. Altogether, our work demonstrates an intricate coupling between conformation and lateral diffusion of LFA-1 and further underscores the crucial role of mobility for the onset of LFA-1 mediated leukocyte adhesion.

JTD Keywords: Cumulative probability distribution, Integrin lymphocyte function-associated antigen 1, Intercellular adhesion molecule, Single molecule detection


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


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


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


Kodippili, G. C., Spector, J., Kang, G. E., Liu, H., Wickrema, A., Ritchie, K., Low, P. S., (2010). Analysis of the kinetics of band 3 diffusion in human erythroblasts during assembly of the erythrocyte membrane skeleton British Journal of Haematology , 150, (5), 592-600

Summary During definitive erythropoiesis, erythroid precursors undergo differentiation through multiple nucleated states to an enucleated reticulocyte, which loses its residual RNA/organelles to become a mature erythrocyte. Over the course of these transformations, continuous changes in membrane proteins occur, including shifts in protein abundance, rates of expression, isoform prominence, states of phosphorylation, and stability. In an effort to understand when assembly of membrane proteins into an architecture characteristic of the mature erythrocyte occurs, we quantitated the lateral diffusion of the most abundant membrane protein, band 3 (AE1), during each stage of erythropoiesis using single particle tracking. Analysis of the lateral trajectories of individual band 3 molecules revealed a gradual reduction in mobility of the anion transporter as erythroblasts differentiated. Evidence for this progressive immobilization included a gradual decline in diffusion coefficients as determined at a video acquisition rate of 120 frames/s and a decrease in the percentage of compartment sizes >100 nm. Because complete acquisition of the properties of band 3 seen in mature erythrocytes is not observed until circulating erythrocytes are formed, we suggest that membrane maturation involves a gradual and cooperative assembly process that is not triggered by the synthesis of any single protein.

JTD Keywords: Band 3 diffusion, Erythrocyte, Progenitor cells, Single particle tracking, Streptavidin quantum dot


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


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


Diez-Ahedo, Ruth , Normanno, Davide , Esteban, Olga,, Bakker, Gert-Jan, Figdor, Carl, Cambi, Alessandra , Garcia-Parajo, M. F., (2009). Dynamic re-organization of individual adhesion nanoclusters in living cells by ligand-patterned surfaces Small 5, (11), 1258-1263

Ligand-patterned surfaces alter the spatio-temporal organization of specific receptors on the cell membrane. Chemically confined surfaces are fabricated using microcontact patterning. The dynamic re-organization of the integrin LFA-1 in living cells is monitored at the single-molecule level using total internal reflection fluorescence. The image on the left shows individual LFA-1 nanoclusters on a single cell being recruited to ligand-rich areas of the pattern.

JTD Keywords: Cell adhesion, Microcontact printing, Patterning, Single molecule studies


Sunyer, R., Trepat, X., Fredberg, J. J., Farre, R., Navajas, D., (2009). The temperature dependence of cell mechanics measured by atomic force microscopy Physical Biology 6, (2), 25009

The cytoskeleton is a complex polymer network that regulates the structural stability of living cells. Although the cytoskeleton plays a key role in many important cell functions, the mechanisms that regulate its mechanical behaviour are poorly understood. Potential mechanisms include the entropic elasticity of cytoskeletal filaments, glassy-like inelastic rearrangements of cross-linking proteins and the activity of contractile molecular motors that sets the tensional stress (prestress) borne by the cytoskeleton filaments. The contribution of these mechanisms can be assessed by studying how cell mechanics depends on temperature. The aim of this work was to elucidate the effect of temperature on cell mechanics using atomic force microscopy. We measured the complex shear modulus (G*) of human alveolar epithelial cells over a wide frequency range (0.1-25.6 Hz) at different temperatures (13-37 degrees C). In addition, we probed cell prestress by mapping the contractile forces that cells exert on the substrate by means of traction microscopy. To assess the role of actomyosin contraction in the temperature-induced changes in G* and cell prestress, we inhibited the Rho kinase pathway of the myosin light chain phosphorylation with Y-27632. Our results show that with increasing temperature, cells become stiffer and more solid-like. Cell prestress also increases with temperature. Inhibiting actomyosin contraction attenuated the temperature dependence of G* and prestress. We conclude that the dependence of cell mechanics with temperature is dominated by the contractile activity of molecular motors.

JTD Keywords: Membrane Stress Failure, Frog Skeletal-Muscle, Extracellular-Matrix, Glass-Transition, Energy Landscape, Actin-Filaments, Living Cell, Single, Traction, Cytoskeleton


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


Hernando, Jordi, Hoogenboom, Jacob, van Dijk, Erik, Garcia-Parajo, Maria, van Hulst, Niek F., (2008). Ultrafast single-molecule photonics: Excited state dynamics in coherently coupled complexes Journal of Luminescence 16th International Conference on Dynamical Processes in Excited States of Solids (ed. -----), Elsevier Science BV (Segovia, Spain) 128, (5-6), 1050-1052

We present a single-molecule study on femtosecond dynamics in multichromophoric systems, combining fs pump-probe, emission-spectra and fluorescence-lifetime analysis. The ultrafast fs approach gives direct information on the initial exciton dynamics after excitation. The lifetime data show superradiance, a direct measure for the extent of the coherent coupling and static disorder. The spectra finally reveal the role of exciton-phonon coupling. At the single-molecule level a wide range of exciton delocalization lengths and energy redistribution times is revealed.

JTD Keywords: Single-molecule detection, Pump-probe, Exciton delocalization, Superradiance, Exciton-phonon coupling


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