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by Keyword: Resolution


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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.

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.

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


Fernandez, L., Yan, J., Fonollosa, J., Burgués, J., Gutierrez, A., Marco, S., (2018). A practical method to estimate the resolving power of a chemical sensor array: Application to feature selection Frontiers in Chemistry 6, Article 209

A methodology to calculate analytical figures of merit is not well established for detection systems that are based on sensor arrays with low sensor selectivity. In this work, we present a practical approach to estimate the Resolving Power of a sensory system, considering non-linear sensors and heteroscedastic sensor noise. We use the definition introduced by Shannon in the field of communication theory to quantify the number of symbols in a noisy environment, and its version adapted by Gardner and Barlett for chemical sensor systems. Our method combines dimensionality reduction and the use of algorithms to compute the convex hull of the empirical data to estimate the data volume in the sensor response space. We validate our methodology with synthetic data and with actual data captured with temperature-modulated MOX gas sensors. Unlike other methodologies, our method does not require the intrinsic dimensionality of the sensor response to be smaller than the dimensionality of the input space. Moreover, our method circumvents the problem to obtain the sensitivity matrix, which usually is not known. Hence, our method is able to successfully compute the Resolving Power of actual chemical sensor arrays. We provide a relevant figure of merit, and a methodology to calculate it, that was missing in the literature to benchmark broad-response gas sensor arrays.

Keywords: Gas sensor array, MOX sensors, Resolving Power, Sensor resolution, Dimensionality reduction, Machine olfaction


Feiner-Gracia, Natalia, Beck, Michaela, Pujals, Sílvia, Tosi, Sébastien, Mandal, Tamoghna, Buske, Christian, Linden, Mika, Albertazzi, Lorenzo, (2017). Super-resolution microscopy unveils dynamic heterogeneities in nanoparticle protein corona Small 13, (41), 1701631

The adsorption of serum proteins, leading to the formation of a biomolecular corona, is a key determinant of the biological identity of nanoparticles in vivo. Therefore, gaining knowledge on the formation, composition, and temporal evolution of the corona is of utmost importance for the development of nanoparticle-based therapies. Here, it is shown that the use of super-resolution optical microscopy enables the imaging of the protein corona on mesoporous silica nanoparticles with single protein sensitivity. Particle-by-particle quantification reveals a significant heterogeneity in protein absorption under native conditions. Moreover, the diversity of the corona evolves over time depending on the surface chemistry and degradability of the particles. This paper investigates the consequences of protein adsorption for specific cell targeting by antibody-functionalized nanoparticles providing a detailed understanding of corona-activity relations. The methodology is widely applicable to a variety of nanostructures and complements the existing ensemble approaches for protein corona study.

Keywords: Heterogeneity, Mesoporous silica nanoparticles, Protein corona, Super-resolution imaging, Targeting


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

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

Keywords: Compressed sensing, High resolution, Traction force microscopy


Oller-Moreno, S., Singla-Buxarrais, G., Jiménez-Soto, J. M., Pardo, Antonio, Garrido-Delgado, R., Arce, L., Marco, Santiago, (2015). Sliding window multi-curve resolution: Application to gas chromatography - Ion Mobility Spectrometry Sensors and Actuators B: Chemical 15th International Meeting on Chemical Sensors , Elsevier (Buenos Aires, Argentina) 217, 13-21

Abstract Blind Source Separation (BSS) techniques aim to extract a set of source signals from a measured mixture in an unsupervised manner. In the chemical instrumentation domain source signals typically refer to time-varying analyte concentrations, while the measured mixture is the set of observed spectra. Several techniques exist to perform BSS on Ion Mobility Spectrometry, being Simple-to-use interactive self-modeling mixture analysis (SIMPLISMA) and Multivariate Curve Resolution (MCR) the most commonly used. The addition of a multi-capillary gas chromatography column using the ion mobility spectrometer as detector has been proposed in the past to increase chemical resolution. Short chromatography times lead to high levels of co-elution, and ion mobility spectra are key to resolve them. For the first time, BSS techniques are used to deconvolve samples of the gas chromatography - ion mobility spectrometry tandem. We propose a method to extract spectra and concentration profiles based on the application of MCR in a sliding window. Our results provide clear concentration profiles and pure spectra, resolving peaks that were not detected by the conventional use of MCR. The proposed technique could also be applied to other hyphenated instruments with similar strong co-elutions.

Keywords: Blind Source Separation, Multivariate Curve Resolution, Ion Mobility Spectrometry, Gas Chromatography, Hyphenated instrumentation, SIMPLISMA, co-elution


Pomareda, Víctor, Guamán, Ana V., Mohammadnejad, Masoumeh, Calvo, Daniel, Pardo, Antonio, Marco, Santiago, (2012). Multivariate curve resolution of nonlinear ion mobility spectra followed by multivariate nonlinear calibration for quantitative prediction Chemometrics and Intelligent Laboratory Systems , 118, 219-229

In this work, a new methodology to analyze spectra time-series obtained from ion mobility spectrometry (IMS) has been investigated. The proposed method combines the advantages of multivariate curve resolution-alternating least squares (MCR-ALS) for an optimal physical and chemical interpretation of the system (qualitative information) and a multivariate calibration technique such as polynomial partial least squares (poly-PLS) for an improved quantification (quantitative information) of new samples. Ten different concentrations of 2-butanone and ethanol were generated using a volatile generator based on permeation tubes. The different concentrations were measured with IMS. These data present a non-linear behaviour as substance concentration increases. Although MCR-ALS is based on a bilinear decomposition, non-linear behaviour can be modelled adding new components to the model. After spectral pre-processing, MCR-ALS was applied aiming to get information about the ionic species that appear in the drift tube and their evolution with the analyte concentration. By resolving the IMS data matrix, concentration profiles and pure spectra of the different ionic species have been obtained for both analytes. Finally, poly-PLS was used in order to build a calibration model using concentration profiles obtained from MCR-ALS for ethanol and 2-butanone. The results, with more than 99% of explained variance for both substances, show the feasibility of using MCR-ALS to resolve IMS datasets. Furthermore, similar or better prediction accuracy is achieved when concentration profiles from MCR-ALS are used to build a calibration model (using poly-PLS) compared to other standard univariate and multivariate calibration methodologies.

Keywords: Ion Mobility Spectrometry, Multivariate Curve Resolution, Gas phase ion chemistry, Multivariate calibration


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

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

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


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

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

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


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

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

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


Montoliu, I., Tauler, R., Padilla, M., Pardo, A., Marco, S., (2010). Multivariate curve resolution applied to temperature modulated metal oxide gas sensors Sensors and Actuators B: Chemical 145, (1), 464-473

Metal oxide (MOX) gas sensors have been widely used for years. Temperature modulation of gas sensors is as an alternative to increase their sensitivity and selectivity to different gas species. In order to enhance the extraction of useful information from this kind of signals, data processing techniques are needed. In this work, the use of self-modelling curve resolution techniques, in particular multivariate curve resolution-alternating least squares (MCR-ALS), is presented for the analysis of these signals. First, the performance of MCR in a synthetic dataset generated from temperature-modulated gas sensor response models has been evaluated, showing good results both in the resolution of gas mixtures and in the determination of concentration/sensitivity profiles. Secondly, experimental confirmation of previously obtained conclusions is attempted using temperature-modulated MOX sensors together with MCR-ALS for the analysis of carbon monoxide (CO) and methane (CH4) gas mixtures in dry air. Results allow confirming the possibility of using the proposed approach as a quantitative technique for gas mixtures analysis, and also reveal some limitations.

Keywords: Temperature modulation, Multivariate curve resolution, MCR-ALS, Metal oxide sensors


Pomareda, V., Calvo, D., Pardo, A., Marco, S., (2010). Hard modeling multivariate curve resolution using LASSO: Application to ion mobility spectra Chemometrics and Intelligent Laboratory Systems , 104, (2), 318-332

Multivariate Curve Resolution (MCR) aims to blindly recover the concentration profile and the source spectra without any prior supervised calibration step. It is well known that imposing additional constraints like positiveness, closure and others may improve the quality of the solution. When a physico-chemical model of the process is known, this can be also introduced constraining even more the solution. In this paper, we apply MCR to Ion Mobility Spectra. Since instrumental models suggest that peaks are of Gaussian shape with a width depending on the instrument resolution, we introduce that each source is characterized by a linear superposition of Gaussian peaks of fixed spread. We also prove that this model is able to fit wider peaks departing from pure Gaussian shape. Instead of introducing a non-linear Gaussian peak fitting, we use a very dense model and rely on a least square solver with L1-norm regularization to obtain a sparse solution. This is accomplished via Least Absolute Shrinkage and Selection Operator (LASSO). Results provide nicely resolved concentration profiles and spectra improving the results of the basic MCR solution.

Keywords: Blind source separation, Ion mobility spectrometry, Multivariate curve resolution, Sparse solution, Non negative matrix factorization


Correa, R., Laciar, E., Arini, P., Jané, R., (2010). Analysis of QRS loop in the Vectorcardiogram of patients with Chagas' disease Engineering in Medicine and Biology Society (EMBC) 32nd Annual International Conference of the IEEE , IEEE (Buenos Aires, Argentina) , 2561-2564

In the present work, we have studied the QRS loop in the Vectorcardiogram (VCG) of 95 chronic chagasic patients classified in different groups (I, II and III) according to their degree of myocardial damage. For comparison, the VCGs of 11 healthy subjects used as control group (Group O) were also examined. The QRS loop was obtained for each patient from the XYZ orthogonal leads of their High-Resolution Electrocardiogram (HRECG) records. In order to analyze the variations of QRS loop in each detected beat, it has been proposed in this study the following vectorcardiographic parameters a) Maximum magnitude of the cardiac depolarization vector, b) Volume, c) Area of QRS loop, d) Ratio between the Area and Perimeter, e) Ratio between the major and minor axes of the QRS loop and f) QRS loop Energy. It has been found that one or more indexes exhibited statistical differences (p<0.05) between groups 0-II, O-III, I-II, I-III and II-III. We concluded that the proposed method could be use as complementary diagnosis technique to evaluate the degree of myocardial damage in chronic chagasic patients.

Keywords: Practical, Experimental/ bioelectric phenomena, Diseases, Electrocardiography, Medical signal, Processing/ QRS loop, Vectorcardiogram, Cardiac depolarization vector, Myocardial damage, Chagas disease, Complementary diagnosis technique, High-resolution electrocardiogram


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

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

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


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

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

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


Montoliu, I., Pomareda, V., Kalms, A., Pardo, A., Gobel, J., Kessler, M., Muller, G., Marco, S., (2009). Resolution of ion mobility spectra for the detection of hazardous substances in real sampling conditions Olfaction and Electronic Nose: Proceedings of the 13th International Symposium on Olfaction and Electronic Nose 13th International Symposium on Olfaction and the Electronic Nose (ed. Pardo, M., Sberveglieri, G.), Amer Inst Physics (Brescia, Italy) 1137, 576-578

This work presents the possibilities offered by a blind source separation method such Multivariate Curve Resolution- Alternating Least Squares (MCR-ALS) in the analysis of Ion Mobility Spectra (IMS). Two security applications are analyzed in this context: the detection of TNT both in synthetic and real samples. Results obtained show the possibilities offered by the direct analysis of the drift time spectra when an appropriate resolution method is used.

Keywords: Ion Mobility Spectrometry, Multivariate Curve Resolution, Security, LIMS, MCR-ALS


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

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

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