Suchergebnisse

In this study, the second harmonic generation (SHG) response to polarization and subsequent data analysis is used to discriminate, in the same image, different SHG source architectures with pixel resolution. This is demonstrated in a mammalian tissue containing both skeletal muscle and fibrilar collagen. The SHG intensity variation with the input polarization (PSHG) is fitted pixel by pixel in the image using an algorithm based on a generalized biophysical model. The analysis provides the effective orientation, θe, of the different SHG active structures (harmonophores) at every pixel. This results in a new image in which collagen and muscle are clearly differentiated. In order to quantify the SHG response, the distribution of θe for every harmonophore is obtained. We found that for collagen, the distribution was centered at θe = 42.7° with a full width at half maximum of ∆θ = 5.9° while for muscle θe = 65.3°, with ∆θ = 7.7°. By comparing these distributions, a quantitative measurement of the discrimination procedure is provided. ; This work is supported by the Generalitat de Catalunya and by the Spanish government grant TEC2006-12654 SICO. Authors also acknowledge The Centre for Innovacio i Desenvolupament Empresarial-CIDEM (RDITSCON07-1-0006), Grupo Ferrer and the European Regional Development Fund. This research has been partially supported by Fundació Cellex Barcelona. ; Peer reviewed

We outline criteria for fast and accurate acquisition of collinear FROG (CFROG) trace and how it can be transformed into the more traditional noncollinear FROG trace. The CFROG has an intrinsically simple geometry that provides greater versatility as well as the ability for built-in delay calibration and enhanced error-checking. The procedure, based on data processing, allows conventional SHG-FROG retrieval algorithms to be used. This technique is tested numerically and experimentally giving excellent results. This work represents an attractive alternative to the traditional, more complex non-collinear FROG technique while, at the same time, extending its use to experiments where collinear geometry is imposed. ; This work was supported by the Generalitat of Catalunya, the Spanish Government under grant TIC2003-07485 and by the European Regional Development Fund. P. Loza-Alvarez acknowledges funding from the Spanish Government through the Ramón y Cajal program. ; Peer Reviewed ; Postprint (published version)

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. ; This work is supported by the Spanish government through the Ministry of Economy and Competitiveness, grants No. TEC2009-09698 and No. FIS2009-09928; the Ministerio de Sanidad, grant No. FIS PI081932; the Laserlab-Europe Cont. grant No. JRA4:Optobio 212025; and the Photonics for Life Networks of Excellence. This research has also been partially supported by Fundació Cellex Barcelona and partially conducted at the Institute of Photonic Sciences' Super-Resolution Light Nanoscopy Facility. ; Peer reviewed

In this paper we provide, for the first time to our knowledge, the effective orientation of the SHG source in cultured cortical neuronal processes in vitro. This is done by the use of the polarization sensitive second harmonic generation (PSHG) imaging microscopy technique. By performing a pixel-level resolution analysis we found that the SHG dipole source has a distribution of angles centered at θe =33.96°, with a bandwidth of ∆θe = 12.85°. This orientation can be related with the molecular geometry of the tubulin heterodimmer contained in microtubules. ; This work is supported by the Generalitat de Catalunya and by the Spanish government grant TEC2006-12654. Authors also acknowledge The Centre for Innovacio i Desenvolupament Empresarial - CIDEM (RDITSCON07-1-0006), Grupo Ferrer and the European Regional Development Fund. This research has been partially supported by Fundació Cellex Barcelona. ; Peer reviewed

In this study, polarization second harmonic generation (SHG) imaging is used and data analysis is developed to gain contrast and to discriminate with pixel resolution, in the same image, SHG source architectures. We use mammalian tissue in which both skeletal muscle and fibrilar collagen can be found. The images are fitted point by point using an algorithm based on a biophysical model, where the coefficient of determination is utilized as a filtering mechanism. For the whole image we retrieve for every pixel, the effective orientation, θe, of the SHG active structures. As a result a new image is formed which its contrast depends on the values of θe. Collagen presented in the forward direction for a predefined region of interest (ROI), peak distribution of angles θe centered in the region of ∼45°, while muscle in the region of ∼65°. Consequently, collagen and muscle are represented in different colors in the same image. Thus, here we show that it is possible to gain contrast and to discriminate between collagen and muscle without the use of any exogenous labeling or any co-localization with fluorescence imaging. © 2009 SPIE-OSA. ; This work is supported by the Generalitat de Catalunya and by the Spanish government grant TEC2006-12654 SICO. Authors also acknowledge The Centre Innovacio i Desenvolupament Empresarial-CIDEM (RDITSCON07-1-0006), Grupo Ferrer and the European Regional Development Fund. This research has been partially supported by Fundació Cellex Barcelona. ; Peer Reviewed

Bacillus firmus I-1582 is approved in Europe for the management of Meloidogyne on vegetable crops. However, little information about its modes of action and temperature requirements is available, despite the effect of these parameters in its efficacy. The cardinal temperatures for bacterial growth and biofilm formation were determined. The bacteria was transformed with GFP to study its effect on nematode eggs and root colonization of tomato (Solanum lycopersicum) and cucumber (Cucumis sativus) by laser-scanning confocal microscopy. Induction of plant resistance was determined in split-root experiments and the dynamic regulation of genes related to jasmonic acid (JA) and salicylic acid (SA) by RT-qPCR at three different times after nematode inoculation. The bacteria was able to grow and form biofilms between 15 and 45 C; it degraded eggshells and colonized eggs; it colonized tomato roots more extensively than cucumber roots; it induced systemic resistance in tomato, but not in cucumber; SA and JA related genes were primed at different times after nematode inoculation in tomato, but only the SA-related gene was up-regulated at 7 days after nematode inoculation in cucumber. In conclusion, B. firmus I-1582 is active at a wide range of temperatures; its optimal growth temperature is 35 C; it is able to degrade Meloidogyne eggs, and to colonize plant roots, inducing systemic resistance in a plant dependent species manner. ; Thanks are given to the Spanish Ministry of Economy and Competitiveness (MINECO) for funding the AGL2013-49040- C2-1-R project, to the Ministry of Science and Innovation from the Spanish Government for funding the AGL2017-89785-R, and to the European Regional Development Fund (FEDER). MC,JA, and PL-A acknowledges financial support from the Spanish Ministry of Economy and Competitiveness through the "Severo Ochoa" program for Centers of Excellence in R&D (SEV-2015-0522), from Fundació Privada Cellex, Fundación Mig-Puig and from Generalitat de Catalunya through the CERCA program and Laser lab Europe through the research and innovation programme under grant agreement No. 654148 ; Postprint (published version)

The cerebellum harbors a specialized area on the roof of the fourth ventricle that is composed of glial cells and neurons that interface with the cerebrospinal fluid. This region includes the so-called ventromedial cord (VMC), which is composed of cells that are glial fibrillary acidic protein (GFAP)-positive and nestin-positive and distributes along the midline in association with blood vessels. We hypothesized that these cells should compare to GFAP and nestin-positive cells that are known to exist in other areas of the brain, which undergo proliferation and differentiation under hypoxic conditions ; . This work wassupported by Grant A1-S-7659 from CONACyT to AMT.MBG and GBGG were supported by CONACyT(Fellowships 330119 and 277694), MBG is a doctoralstudent from the Programa de Doctorado en CienciasBiome ́dicas, Universidad Nacional Auto ́noma de Me ́xico(UNAM), MBG was supported by a PAEP travel grant.RVD was supported by a postdoctoral fellowship fromUNAM-DGAPA. We thank to K. Engberg and K.Deisseroth from Stanford University. EJG and PLAacknowledge financial support from the Spanish Ministryof Economy and Competitiveness (AEI/FEDER),through founded programs BIO2014-59614-JIN, RYC-2015-17935 and FIS2016-80455-R (AEI/FEDER),European Union grant EU-H2020 713140; the ''SeveroOchoa" Programme for Centres of Excellence in R&D(SEV-2015-0522), Fundacio ́Privada Cellex, Fundacio ́nMig-Puig and the CERCA program and Laserlab-Europe(EU-H2020 654148 ; Postprint (published version)