Pablo Loza-Alvarez

ICFO Institute of Photonic Sciences, Barcino, Catalonia, Spain

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Publications (115)181.18 Total impact

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    ABSTRACT: Carotenoids are isoprenoid compounds essential for plants to protect the photosynthetic apparatus against excess light. They also function as health-promoting natural pigments that provide colors to ripe fruit, promoting seed dispersal by animals. Work in Arabidopsis thaliana unveiled that transcription factors of the Phytochrome-Interacting Factor (PIF) family regulate carotenoid gene expression in response to environmental (i.e. light and temperature) signals, including those created when sunlight reflects from or passes though nearby vegetation or canopy (referred to as shade). Here we show that PIFs use a virtually identical mechanism to modulate carotenoid biosynthesis during fruit ripening in tomato (Solanum lycopersicum). Instead of integrating environmental information, however, PIF-mediated signaling pathways appear to fulfill a completely new function in the fruit. As tomatoes ripe, they turn from green to red due to chlorophyll breakdown and carotenoid accumulation. When sunlight passes through the flesh of green fruit, a self-shading effect within the tissue maintains high levels of PIFs that directly repress the master gene of the fruit carotenoid pathway, hence preventing undue production of carotenoids. This effect attenuates as chlorophyll degrades, causing the degradation of PIF proteins and boosting carotenoid biosynthesis as ripening progresses. Together, shade signaling components might have been coopted in tomato fruit to inform on the actual stage of ripening (based on the pigment profile of the fruit at every moment) and thus finely coordinate fruit color change. We also show how this mechanism can be manipulated for tailoring carotenoid-enriched fruits. This article is protected by copyright. All rights reserved.
    No preview · Article · Dec 2015 · The Plant Journal
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    ABSTRACT: We perform rapid spontaneous Raman 2D imaging in light-sheet microscopy using continuous wave lasers and interferometric tunable filters. By angularly tuning the filter, the cut-on/off edge transitions are scanned along the excited Stokes wavelengths. This allows obtaining cumulative intensity profiles of the scanned vibrational bands, which are recorded on image stacks; resembling a spectral version of the knife-edge technique to measure intensity profiles. A further differentiation of the stack retrieves the Raman spectra at each pixel of the image which inherits the 3D resolution of the host light sheet system. We demonstrate this technique using solvent solutions and composites of polystyrene beads and lipid droplets immersed in agar and by imaging the C–H (2800-3100cm−1) region in a C. elegans worm. The image acquisition time results in 4 orders of magnitude faster than confocal point scanning Raman systems, allowing the possibility of performing fast spontaneous Raman·3D-imaging on biological samples.
    No preview · Article · Sep 2015 · Biomedical Optics Express
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    Marco Lombardo · David Merino · Pablo Loza-Alvarez · Giuseppe Lombardo
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    ABSTRACT: Diseases that affect the cornea can lead to severe vision loss and have tremendous social impact. These diseases are associated to deviations from normal structural order and orientation of collagen fibril bundles. Unfortunately, resolving non-invasively the corneal collagen structure is not possible to date. In this work, polarization sensitive second harmonic generation (pSHG) microscopy is used to obtain information with molecular specificity on microstructure of human corneas. This information is used to develop a set of label-free imaging biomarkers that were generated by means of a novel methodology based on mathematical tensorial calculus. The method is proven to be highly sensitive and robust. The use of these biomarkers permits accurate characterization of the anisotropic, depth-dependent, structural organization of corneal collagen fibril bundles without any a priori information. The method can be valuable to improve understanding of microstructural pathophysiological changes of the human cornea close to in vivo conditions.
    Full-text · Article · Aug 2015 · Biomedical Optics Express
  • Omar E Olarte · Jordi Andilla · David Artigas · Pablo Loza-Alvarez
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    ABSTRACT: Current microscopy demands the visualization of large three-dimensional samples with increased sensitivity, higher resolution, and faster speed. Several imaging techniques based on widefield, point-scanning, and light-sheet strategies have been designed to tackle some of these demands. Although successful, all these require the illuminated volumes to be tightly coupled with the detection optics to accomplish efficient optical sectioning. Here, we break this paradigm and produce optical sections from out-of-focus planes. This is done by extending the depth of field of the detection optics in a light-sheet microscope using wavefront-coding techniques. This passive technique allows accommodation of the light sheet at any place within the extended axial range. We show that this enables quick scanning of the light sheet across a volumetric sample. As a consequence, imaging speeds faster than twice the volumetric video rate (>70 volumes/s) can be achieved without needing to move the sample. These capabilities are demonstrated for volumetric imaging of fast dynamics in vivo as well as for fast, three-dimensional particle tracking.
    No preview · Article · Aug 2015 · Optica
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    Marco Lombardo · David Merino · Pablo Loza-Alvarez · Giuseppe Lombardo

    Full-text · Dataset · Jul 2015
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    ABSTRACT: We investigate the feasibility of both one photon and two photon fluorescence excitation using whispering gallery mode microresonators. We report the linear and non linear fluorescence real-time detection of labeled IgG covalently bonded to the surface of a silica whispering gallery mode resonator (WGMR). The immunoreagents have been immobilized onto the surface of the WGMR sensor after being activated with an epoxy silane and an orienting layer. The developed immunosensor presents great potential as a robust sensing device for fast and early detection of immunoreactions. We also investigate the potential of microbubbles as nonlinear enhancement platform. The dyes used in these studies are dylight800, tetramethyl rhodamine isothiocyanate, rhodamine 6G and fluorescein. All measurements were performed in a modified confocal microscope.
    No preview · Article · Jul 2015 · Journal of Luminescence
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    ABSTRACT: We report the non linear fluorescence real-time detection of labeled IgG covalently bonded to the surface of a microspherical whispering gallery mode resonator (WGMR). The immunoreagents have been immobilized onto the surface of the WGMR sensor after being activated with an epoxy silane and an orienting layer. The developed immunosensor presents great potential as a robust sensing device for fast and early detection of immunoreactions. We also tested the potential of microbubbles as nonlinear enhancement platform. The dyes used in these studies are tetramethyl rhodamine isothiocyanate and Rhodamine 6G. All measurements were performed in a modified confocal microscope.
    No preview · Article · Mar 2015 · Proceedings of SPIE - The International Society for Optical Engineering
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    Sotiris Psilodimitrakopoulos · Pablo Loza-Alvarez · David Artigas
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    ABSTRACT: Fast imaging of molecular changes under high-resolution and label-free conditions are essential for understanding in-vivo processes, however, current techniques are not able to monitor such changes in real time. Polarization sensitive second harmonic generation (PSHG) imaging is a minimally invasive optical microscopy technique capable of quantifying molecular conformational changes occurring below the diffraction limit. Up to now, such information is generally retrieved by exciting the sample with different linear polarizations. This procedure requires the sample to remain static during measurements (from a few second to minutes), preventing the use of PSHG microscopy from studying moving samples or molecular dynamics in living organisms. Here we demonstrate an imaging method that is one order of magnitude faster than conventional PSHG. Based on circular polarization excitation and instantaneous polarimetry analysis of the second harmonic signal generated in the tissue, the method is able to instantaneously obtain molecular information within a pixel dwell time. As a consequence, a single scan is only required to retrieve all the information. This allowed us to perform PSHG imaging in moving C. elegans, monitoring myosin’s dynamics during the muscular contraction and relaxation. Since the method provides images of the molecular state, an unprecedented global understanding of the muscles dynamics is possible by correlating changes in different regions of the sample.
    Full-text · Article · Dec 2014 · Biomedical Optics Express
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    ABSTRACT: We developed a low-cost, low-noise, tunable, high-peak-power, ultrafast laser system based on a SESAM-modelocked, solid-state Yb tungstate laser plus spectral broadening via a microstructured fiber followed by pulse compression. The spectral selection, tuning, and pulse compression are performed with a simple prism compressor. The output pulses are tunable from 800 to 1250 nm, with the pulse duration down to 25 fs, and average output power up to 150 mW, at 80 MHz pulse repetition rate. We introduce the figure of merit (FOM) for the two-photon and multi-photon imaging (or other nonlinear processes), which is a useful guideline in discussions and for designing the lasers for an improved microscopy signal. Using a 40 MHz pulse repetition rate laser system, with twice lower FOM, we obtained high signal-to-noise ratio two-photon fluorescence images with or without averaging, of mouse intestine section and zebra fish embryo. The obtained images demonstrate that the developed system is capable of nonlinear (TPE, SHG) imaging in a multimodal operation. The system could be potentially used in a variety of other techniques including, THG, CARS and applications such as nanosurgery.
    No preview · Article · Jun 2014 · Optics Express
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    ABSTRACT: Acquisition of images deep inside large samples is one of the most demanded improvements that current biology applications ask for. Absorption, scattering and optical aberrations are the main difficulties encountered in these types of samples. Adaptive optics has been imported form astronomy to deal with the optical aberrations induced by the sample. Nonlinear microscopy and SPIM have been proposed as interesting options to image deep into a sample. Particularly, light-sheet microscopy, due to its low photo bleaching properties, opens new opportunities to obtain information for example in long time lapses for large 3D imaging. In this work, we perform an overview of the application of adaptive optics to the fluorescence microscopy in linear and non-linear modalities. Then we will focus in the light-sheet microscopy architecture of two orthogonal optical paths which implies new requirements in terms of optical correction. We will see the different issues that appear in light-sheet microscopy particularly when imaging large and non-flat samples. Finally, we will study the problem of the isoplanetic patches.
    No preview · Article · Feb 2014 · Proceedings of SPIE - The International Society for Optical Engineering
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    ABSTRACT: This chapter contains sections titled: Compact Laser Systems for Nonlinear Imaging Applications QD Devices and Their Application in Optical Coherence Tomography Infrared QD Laser Application in Cancer Photodynamic Therapy: Killing Tumor Cells without Photosensitizers Acknowledgments References
    No preview · Chapter · Jan 2014
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    ABSTRACT: Bone metastasis is the most common distant relapse in breast cancer. The identification of key proteins involved in the osteotropic phenotype would represent a major step toward the development of new prognostic markers and therapeutic improvements. The aim of this study was to characterize functional phenotypes that favor bone metastasis in human breast cancer. We used the human breast cancer cell line MDAMB231 and its osteotropic BO2 subclone to identify crucial proteins in bone metastatic growth. We identified 31 proteins, 15 underexpressed and 16 overexpressed, in BO2 cells compared to parental cells. We employed a network modeling approach in which these 31 candidate proteins were prioritized with respect to their potential in metastasis formation, based on the topology of the protein protein interaction network and differential expression. The protein protein interaction network provided a framework to study the functional relationships between biological molecules by attributing functions to genes whose functions had not been characterized. The combination of expression profiles and protein interactions revealed an endoplasmic reticulum thiol oxidoreductase, ERp57, functioning as a hub which retained 4 downregulated nodes involved in antigen presentation associated with the human major histocompatibility complex class I molecules, including HLA A, HLA B, HLA E and HLA F. Further analysis of the interaction network revealed an inverse correlation between ERp57 and vimentin, which influences cytoskeleton reorganization. Moreover, knockdown of ERp57 in BO2 cells confirmed its bone organspecific prometastatic role. Altogether, ERp57 appears as a multifunctional chaperone that can regulate diverse biological processes to maintain the homeostasis of breast cancer cells and promote the development of bone metastasis.
    Full-text · Article · Apr 2013 · Molecular & Cellular Proteomics
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    ABSTRACT: In this work highly localized femtosecond laser ablation is used to dissect single axons within a living Caenorhabditis elegans (C. elegans). We present a multimodal imaging methodology for the assessment of the collateral damage induced by the laser. This relies on the observation of the tissues surrounding the targeted region using a combination of different high resolution microscopy modalities. We present the use of Second Harmonic Generation (SHG) and Polarization Sensitive SHG (PSHG) to determine damage in the neighbor muscle cells. All the above is done using a single instrument: multimodal microscopy setup that allows simultaneous imaging in the linear and non-linear regimes and femtosecond-laser ablation.
    Full-text · Article · Mar 2013 · PLoS ONE
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    ABSTRACT: Neuronal death can be preceded by progressive dysfunction of axons. Several pathological conditions such as ischemia can disrupt the neuronal cytoskeleton. Microtubules are basic structural components of the neuronal cytoskeleton that regulate axonal transport and neuronal function. Up-to-date, high-resolution observation of microtubules in living neuronal cells is usually accomplished using fluorescent-based microscopy techniques. However, this needs exogenous fluorescence markers to produce the required contrast. This is an invasive procedure that may interfere with the microtubule dynamics. In this work, we show, for the first time to our knowledge, that by using the endogenous (label-free) contrast provided by second harmonic generation (SHG) microscopy, it is possible to identify early molecular changes occurring in the microtubules of living neurons under ischemic conditions. This is done by measuring the intensity modulation of the SHG signal as a function of the angular rotation of the incident linearly polarized excitation light (technique referred to as PSHG). Our experiments were performed in microtubules from healthy control cultured cortical neurons and were compared to those upon application of several periods of oxygen and glucose deprivation (up to 120 min) causing ischemia. After 120-min oxygen and glucose deprivation, a change in the SHG response to the polarization was measured. Then, by using a three-dimensional PSHG biophysical model, we correlated this finding with the structural changes occurring in the microtubules under oxygen and glucose deprivation. To our knowledge, this is the first study performed in living neuronal cells that is based on direct imaging of axons and that provides the means of identifying the early symptoms of ischemia. Live observation of this process might bring new insights into understanding the dynamics and the mechanisms underlying neuronal degeneration or mechanisms of protection or regeneration.
    No preview · Article · Mar 2013 · Biophysical Journal
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    ABSTRACT: An immunosensor approach based on non-linear two-photon fluorescence spectroscopy (TPF) coupled to a resonant double grating waveguide structure (DGWS) has been used for the detection of methylboldenone (MB), an androgenic anabolic steroid used illegally as growth promoter. This synthetic steroid is detected by the biosensor down to 0.1 μg l−1, two orders of magnitude lower than the minimum required performance limit (MRPL) required by the World Anti-Doping Agency (WADA). Nevertheless, we have focused this work on the synthesis and characterization of small fluorescent-labeled conjugates, as well as on the modification of the waveguide surfaces. Detection relies on a direct competitive format, using a boldenone–rhodamine conjugate as fluorescent competitor and a specific anti-MB antibody. The immunoreagents are immobilized onto a resonant Ta2O5 sensing chip after being activated with phosphonohexanoic acid spacers, a novel methodology for covalently surface immobilization of biomolecules, which shows better stability and homogeneity than classical silane chemistries. The developed immunosensor presents great potential as a robust device for its implementation on the detection of small illegal and toxic contaminants.
    Full-text · Article · Nov 2012 · Sensors and Actuators B Chemical
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    ABSTRACT: Based on its polarization dependency, second harmonic generation (PSHG) microscopy has been proven capable to structurally characterize molecular architectures in different biological samples. By exploiting this polarization dependency of the SHG signal in every pixel of the image, average quantitative structural information can be retrieved in the form of PSHG image histograms. In the present study we experimentally show how the PSHG image histograms can be affected by the organization of the SHG active molecules. Our experimental scenario grounds on two inherent properties of starch granules. Firstly, we take advantage of the radial organization of amylopectin molecules (the SHG source in starch) to attribute shifts of the image histograms to the existence of tilted off the plane molecules. Secondly, we use the property of starch to organize upon hydration to demonstrate that the degree of structural order at the molecular level affects the width of the PSHG image histograms. The shorter the width is the more organized the molecules in the sample are, resulting in a reliable method to measure order. The implication of this finding is crucial to the interpretation of PSHG images used for example in tissue diagnostics.
    Full-text · Article · Oct 2012 · Biomedical Optics Express
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    ABSTRACT: The C-type lectin DC-SIGN expressed on dendritic cells (DCs) facilitates capture and internalization of a plethora of different pathogens. Although it is known that DC-SIGN organizes in nanoclusters at the surface of DCs, the molecular mechanisms responsible for this well-defined nanopatterning and role in viral binding remain enigmatic. By combining biochemical and advanced biophysical techniques, including optical superresolution and single particle tracking, we demonstrate that DC-SIGN intrinsic nanoclustering strictly depends on its molecular structure. DC-SIGN nanoclusters exhibited free, Brownian diffusion on the cell membrane. Truncation of the extracellular neck region, known to abrogate tetramerization, significantly reduced nanoclustering and concomitantly increased lateral diffusion. Importantly, DC-SIGN nanocluster dissolution exclusively compromised binding to nanoscale size pathogens. Monte Carlo simulations revealed that heterogeneity on nanocluster density and spatial distribution confers broader binding capabilities to DC-SIGN. As such, our results underscore a direct relationship between spatial nanopatterning, driven by intermolecular interactions between the neck regions, and receptor diffusion to provide DC-SIGN with the exquisite ability to dock pathogens at the virus length-scale. Insight on how virus receptors are organized prior to virus binding and how they assemble into functional platforms for virus docking is helpful to develop novel strategies to prevent virus entry and infection.
    Full-text · Article · Sep 2012 · Journal of Biological Chemistry
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    ABSTRACT: Two photon fluorescent spectroscopy (TPF) Double grating waveguide structure (DGWS) Alkyl phosphates and phosphonates SAM Tantalum oxide (Ta2O5) a b s t r a c t An immunosensor approach based on non-linear two-photon fluorescence spectroscopy (TPF) coupled to a resonant double grating waveguide structure (DGWS) has been used for the detection of methyl-boldenone (MB), an androgenic anabolic steroid used illegally as growth promoter. This synthetic steroid is detected by the biosensor down to 0.1 g l −1 , two orders of magnitude lower than the minimum required performance limit (MRPL) required by the World Anti-Doping Agency (WADA). Nevertheless, we have focused this work on the synthesis and characterization of small fluorescent-labeled conju-gates, as well as on the modification of the waveguide surfaces. Detection relies on a direct competitive format, using a boldenone–rhodamine conjugate as fluorescent competitor and a specific anti-MB anti-body. The immunoreagents are immobilized onto a resonant Ta 2 O 5 sensing chip after being activated with phosphonohexanoic acid spacers, a novel methodology for covalently surface immobilization of biomolecules, which shows better stability and homogeneity than classical silane chemistries. The devel-oped immunosensor presents great potential as a robust device for its implementation on the detection of small illegal and toxic contaminants.
    Full-text · Dataset · Aug 2012
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    ABSTRACT: We present the implementation of a combined digital scanned light-sheet microscope (DSLM) able to work in the linear and nonlinear regimes under either Gaussian or Bessel beam excitation schemes. A complete characterization of the setup is performed and a comparison of the performance of each DSLM imaging modality is presented using in vivo Caenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view. (C) 2012 Optical Society of America
    Full-text · Article · Jul 2012 · Biomedical Optics Express
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    ABSTRACT: In this paper, we present the generation of high peak-power picosecond optical pulses in the 1.26 μm spectral band from a repetition-rate-tunable quantum-dot external-cavity passively mode-locked laser (QD-ECMLL), amplified by a tapered quantum-dot semiconductor optical amplifier (QD-SOA). The laser emission wavelength was controlled through a chirped volume Bragg grating which was used as an external cavity output coupler. An average power of 208.2 mW, pulse energy of 321 pJ, and peak power of 30.3 W were achieved. Preliminary nonlinear imaging investigations indicate that this system is promising as a high peak-power pulsed light source for nonlinear bio-imaging applications across the 1.0 μm - 1.3 μm spectral range.
    Full-text · Article · Jun 2012 · Optics Express

Publication Stats

1k Citations
181.18 Total Impact Points

Institutions

  • 2003-2015
    • ICFO Institute of Photonic Sciences
      Barcino, Catalonia, Spain
  • 2002-2008
    • Polytechnic University of Catalonia
      • • Department of Signal Theory and Communications (TSC)
      • • Department of Optics and Optometry (OO)
      Barcino, Catalonia, Spain
  • 1997-2001
    • University of St Andrews
      • School of Physics and Astronomy
      Saint Andrews, Scotland, United Kingdom