David Artigas

Polytechnic University of Catalonia, Barcino, Catalonia, Spain

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Publications (99)177.76 Total impact

  • [Show abstract] [Hide abstract]
    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.
    Biomedical Optics Express 12/2014; 5(12). · 3.18 Impact Factor
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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.
    Optics Express 06/2014; 22(13):16456-16461. · 3.55 Impact Factor
  • Osamu Takayama, David Artigas, Lluis Torner
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    ABSTRACT: Guiding light at the nanoscale is usually accomplished using surface plasmons. However, plasmons propagating at the surface of a metal sustain propagation losses. A different type of surface excitation is the Dyakonov surface wave. These waves, which exist in lossless media, were predicted more than two decades ago but observed only recently. Dyakonov surface waves exist when at least one of the two media forming the surface exhibits a suitable anisotropy of refractive indexes. Although propagating only within a narrow range of directions, these waves can be used to create modes supported by ultrathin films that confine light efficiently within film thicknesses well below the cutoff thickness required in standard waveguides. Here, we show that 10 nm and 20 nm dielectric nanosheets of aluminium oxide clad between an anisotropic crystal (lithium triborate) and different liquids support Dyakonov-like modes. The direction of light propagation can be controlled by modulating the refractive index of the cladding. The possibility of guiding light in nanometre-thick films with no losses and high directionality makes Dyakonov wave modes attractive for planar photonic devices in schemes similar to those currently employing long-range plasmons.
    Nature Nanotechnology 05/2014; · 31.17 Impact Factor
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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.
    Biophysical Journal 03/2013; 104(5):968-75. · 3.67 Impact Factor
  • Osamu Takayama, David Artigas, Lluis Torner
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    ABSTRACT: We show that engineered photonic metamaterials composed of alternating layers of suitable dielectrics and metals can support different kinds of surface waves (SWs) under robust and readily achievable experimental conditions. The supported SWs include Dyakonov SWs, hybrid plasmons, and Dyakonov plasmons. In particular, in contrast to conventional physical settings, we show that the high form birefringence exhibited by the metamaterials allows Dyakonov SWs, or dyakonons, to exist within large angular existence domains and levels of localization similar to plasmons, thus making dyakonons available for practical applications.
    Optics Letters 10/2012; 37(20):4311-3. · 3.39 Impact Factor
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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.
    Biomedical Optics Express 10/2012; 3(10):2681-93. · 3.18 Impact Factor
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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 vivoCaenorhabditis elegans samples. We found that the use of Bessel beam nonlinear excitation results in better image contrast over a wider field of view.
    Biomedical Optics Express 07/2012; 3(7):1492-505. · 3.18 Impact Factor
  • Osamu Takayama, David Artigas, Lluis Torner
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    ABSTRACT: We study the coupling of plasmons and Dyakonov surface waves propagating at the interfaces between isotropic-birefringent-metal layered structures. Efficient coupling is shown to occur with a proper choice of the crystal birefringence, the refractive index of the isotropic medium, and the light propagation direction relative to the crystal optical axis. In the case of low-loss metals, coupling efficiencies as high as 90% are predicted to be possible.
    Optics Letters 06/2012; 37(11):1983-5. · 3.39 Impact Factor
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    ABSTRACT: The performance of imaging devices such as linear and nonlinear microscopes (NLM) can be limited by the optical properties of the imaged sample. Such an important aspect has already been described using theoretical models due to the difficulties of implementing a direct wavefront sensing scheme. However, these only stand for simple interfaces and cannot be generalized to biological samples given its structural complexity. This has leaded to the development of sensor-less adaptive optics (AO) implementations. In this approach, aberrations are iteratively corrected trough an image related parameter (aberrations are not measured), being prone of causing sample damage. In this work, we perform a practical implementation of a Shack-Hartman wavefront sensor to compensate for sample induced aberrations, demonstrating its applicability in linear and NLM. We perform an extensive analysis of wavefront distortion effects through different depths employing phantom samples. Aberration effects originated by the refractive index mismatch and depth are quantified using the linear and nonlinear guide-star concept. More over we analyze offaxis aberrations in NLM, an important aspect that is commonly overlooked. In this case spherical aberration behaves similarly to the wavefront error compared with the on-axis case. Finally we give examples of aberration compensation using epi-fluorescence and nonlinear microscopy.
    Proc SPIE 02/2012;
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    ABSTRACT: We demonstrate that sample induced aberrations can be measured in a nonlinear microscope. This uses the fact that two-photon excited fluorescence naturally produces a localized point source inside the sample: the nonlinear guide-star (NL-GS). The wavefront emitted from the NL-GS can then be recorded using a Shack-Hartmann sensor. Compensation of the recorded sample aberrations is performed by the deformable mirror in a single-step. This technique is applied to fixed and in vivo biological samples, showing, in some cases, more than one order of magnitude improvement in the total collected signal intensity.
    Biomedical Optics Express 11/2011; 2(11):3135-49. · 3.18 Impact Factor
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    ABSTRACT: Long term in vivo observations at large penetration depths and minimum sample disturbance are some of the key factors that have enabled the study of different cellular and tissue mechanisms. The continuous optimization of these aspects is the main driving force for the development of advanced microscopy techniques such as those based on nonlinear effects. Its wide implementation for general biomedical applications is however, limited as the currently used nonlinear microscopes are based on bulky, maintenance-intensive and expensive excitation sources such as Ti:sapphire ultrafast lasers. We present the suitability of a portable (140x240x70 mm) ultrafast semiconductor disk laser (SDL) source, to be used in nonlinear microscopy. The SDL is modelocked by a quantum-dot semiconductor saturable absorber mirror (SESAM). This enables the source to deliver an average output power of 287 mW with 1.5 ps pulses at 500 MHz, corresponding to a peak power of 0.4 kW. The laser center wavelength (965 nm) virtually matches the two-photon absorption cross-section of the widely used Green Fluorescent Protein (GFP). This property greatly relaxes the required peak powers, thus maximizing sample viability. This is demonstrated by presenting two-photon excited fluorescence images of GFP labeled neurons and second-harmonic generation images of pharyngeal muscles in living C. elegans nematodes. Our results also demonstrate that this compact laser is well suited for efficiently exciting different biological dyes. Importantly this non expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices, facilitating its widespread adoption in biomedical applications.
    Proc SPIE 06/2011;
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    ABSTRACT: The role of Dyakonov surface waves in the transmission through structures composed of birefringent media is theoretically studied, showing that highly directional and efficient transmission above the critical angle takes place.
    05/2011;
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    ABSTRACT: In minimally destructive SHG biomedical imaging (high resolution optical slicing) is greatly desirable to extract the maximum of information from the light matter interaction. Here we develop a 3-D biophysical model and a methodology, which extracts molecular information below the experimental resolution limit. Firstly, it provides the pitch angle (SHG effective orientation) of the SHG source helix of the sample. This information is used to characterize and categorize the SHG sources among them. And secondly, it provides the degree of organization of the SHG source molecules. This can be used as a quantitative imaging biomarker able to characterize the degree of organization (homeostasis) of the sample. Here we applied the model in dried and hydrated wheat starch granules. Our results show that the SHG source molecule in starch is amylopectin. We also conclude that under hydration, the amylopectin molecules are further organized but they do not change structure. This organization is reflected to the width of the pitch angles pixels' histograms' distributions. The shorter the width is, the more organized the amylopectin molecules in starch are.
    Proc SPIE 05/2011;
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    ABSTRACT: The recent linkage between adaptive optics, a technique borrowed from astronomy and various imaging devices, has enabled to push forward their imaging capabilities by improving its contrast and resolution. A specific case is nonlinear microscopy (NLM) that, although it brings several inherent advantages (compared to linear fluorescence techniques) due to its nonlinear dependence on the excitation beam, its enhanced capabilities can be limited by the sample inhomogeneous structure. In this work, we demonstrate how these imaging capabilities can be enhanced by, employing adaptive optics in a two step correction process. Firstly, a closed-loop methodology aided by Shack-Hartman Wavefront sensing scheme is implemented for compensating the aberrations produced by the laser and the optical elements before the high numerical aperture microscope objective, resulting in a one-time calibration process. Then the residual aberrations are produced by the microscope objective and the sample. These are measured in a similar way as it is done in astronomy (employing a laser guide-star), using the two-photon excited fluorescence. The properties of this incoherent emission produced inside a test sample are compared to a genetically modified Caenorhabditis. elegans nematode expressing GFP showing that the emission of this protein (at 810nm) can be sensed efficiently with our WFS by modifying the exposure time. Therefore the recorded wavefront will capture the sample aberrations which are used to shape a deformable mirror in an open-loop configuration. This correction principle is demonstrated in a test sample by correcting aberrations in a "single-shot" resulting in a reduced sample exposure.
    Proc SPIE 05/2011;
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    ABSTRACT: The use of the nonlinear guide-star concept is proposed. This principle is used to directly measure sample aberrations employing a wave front sensor and correcting them in a single step by shaping a deformable mirror.
    Novel Techniques in Microscopy; 04/2011
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    ABSTRACT: The role of Dyakonov surface waves in the transmission through structures composed of birefringent media is theoretically explored. In the case of structures using prisms, unexpected high transmission above the critical angle due to resonant excitation of Dyakonov surface waves is predicted. This transmission is produced only when TE polarized incident wave reaches the interface supporting the surface waves within a narrow interval of angles, for both the angle of incidence and the angle with respect to the optic axis of the birefringent media. As a result, over 90% transmission for a single and isolated peak confined in the two transversal directions, with hybrid TE and TM polarization, can be obtained.
    Optics Express 03/2011; 19(7):6339-47. · 3.55 Impact Factor
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    ABSTRACT: Polarization sensitive second harmonic generation (PSHG) provides additional information in intensity only SHG imaging. In particular, it offers the SHG effective orientation of the implicated SHG active structures. Assuming that those structures possess cylindrical symmetry, the supplementary contrast is based on the ratio of two non-vanishing, independent elements of the χ2 tensor. This ratio is experimentally extracted by fitting pixel by pixel a theoretical model to the PSHG images and by finding the maximum frequency value or the mean of the consequent pixels' histogram. In the present study we show that the above χ2 elements' ratio critically depends on the tilted-off the plane SHG active structures. We performed PSHG in different z-planes of a starch granule (presenting radially oriented amylopectin molecules, the SHG source in starch) and we found different pick values of the χ2 elements' ratio histogram for each plane. By assuming a fixed value for the χ2 elements' ratio, we present here a generalized three dimensional (3D) model that determines the 3D orientation of the SHG active structures.
    Proc SPIE 02/2011;
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    ABSTRACT: Ultrashort pulsed laser systems (such as Ti:sapphire) have been used in nonlinear microscopy during the last years. However, its implementation is not straight forward as they are maintenance-intensive, bulky and expensive. These limitations have prevented their wide-spread use for nonlinear imaging, especially in "real-life" biomedical applications. In this work we present the suitability of a compact ultrafast semiconductor disk laser source, with a footprint of 140x240x70 mm, to be used for nonlinear microscopy. The modelocking mechanism of the laser is based on a quantumdot semiconductor saturable absorber mirror (SESAM). The laser delivers an average output power of 287 mW with 1.5 ps pulses at 500 MHz, corresponding to a peak power of 0.4 kW. Its center wavelength is 965 nm which is ideally suited for two-photon excitation of the widely used Green Fluorescent Protein (GFP) marker as it virtually matches its twophoton action cross section. We reveal that it is possible to obtain two photon excited fluorescence images of GFP labeled neurons and secondharmonic generation images of pharynx and body wall muscles in living C. elegans nematodes. Our results demonstrate that this compact laser is well suited for long-term time-lapse imaging of living samples as very low powers provide a bright signal. Importantly this non expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices, facilitating its wide-spread adoption in "real-life" applications.
    Proc SPIE 02/2011;
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    ABSTRACT: In-vivo microscopic long term time-lapse studies require controlled imaging conditions to preserve sample viability. Therefore it is crucial to meet specific exposure conditions as these may limit the applicability of established techniques. In this work we demonstrate the use of third harmonic generation (THG) microscopy for long term time-lapse three-dimensional studies (4D) in living Caenorhabditis elegans embryos employing a 1550 nm femtosecond fiber laser. We take advantage of the fact that THG only requires the existence of interfaces to generate signal or a change in the refractive index or in the chi3 nonlinear coefficient, therefore no markers are required. In addition, by using this wavelength the emitted THG signal is generated at visible wavelengths (516 nm) enabling the use of standard collection optics and detectors operating near their maximum efficiency. This enables the reduction of the incident light intensity at the sample plane allowing to image the sample for several hours. THG signal is obtained through all embryo development stages, providing different tissue/structure information. By means of control samples, we demonstrate that the expected water absorption at this wavelength does not severely compromise sample viability. Certainly, this technique reduces the complexity of sample preparation (i.e. genetic modification) required by established linear and nonlinear fluorescence based techniques. We demonstrate the non-invasiveness, reduced specimen interference, and strong potential of this particular wavelength to be used to perform long-term 4D recordings.
    Proc SPIE 02/2011;
  • American Journal of Obstetrics and Gynecology - AMER J OBSTET GYNECOL. 01/2011; 204(1).

Publication Stats

526 Citations
177.76 Total Impact Points

Institutions

  • 1994–2014
    • Polytechnic University of Catalonia
      • • Department of Signal Theory and Communications (TSC)
      • • ICFO - Institute of Photonic Sciences
      Barcino, Catalonia, Spain
  • 2004–2013
    • ICFO Institute of Photonic Sciences
      Barcino, Catalonia, Spain
  • 2005
    • Institut Marqués, Spain, Barcelona
      Barcino, Catalonia, Spain