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1. Detection of capillary abnormalities in early diabetic retinopathy using scanning laser ophthalmoscopy and optical coherence tomography combined with adaptive optics

Author

Torm, Marie Elise Wistrup, Pircher, Michael, Bonnin, Sophie, Johannesen, Jesper, Klefter, Oliver Niels, Schmidt, Mathias Falck, Frederiksen, Jette Lautrup, Lefaudeux, Nicolas, Andilla, Jordi, Valdes, Claudia, Loza-Alvarez, Pablo, Brea, Luisa Sanchez, De Jesus, Danilo Andrade, Grieve, Kate, Paques, Michel, Larsen, Michael, and Gocho, Kiyoko

Published
2024
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2. TANGO1 inhibitors reduce collagen secretion and limit tissue scarring

Author

Raote, Ishier, Rosendahl, Ann-Helen, Häkkinen, Hanna-Maria, Vibe, Carina, Küçükaylak, Ismail, Sawant, Mugdha, Keufgens, Lena, Frommelt, Pia, Halwas, Kai, Broadbent, Katrina, Cunquero, Marina, Castro, Gustavo, Villemeur, Marie, Nüchel, Julian, Bornikoel, Anna, Dam, Binita, Zirmire, Ravindra K., Kiran, Ravi, Carolis, Carlo, Andilla, Jordi, Loza-Alvarez, Pablo, Ruprecht, Verena, Jamora, Colin, Campelo, Felix, Krüger, Marcus, Hammerschmidt, Matthias, Eckes, Beate, Neundorf, Ines, Krieg, Thomas, and Malhotra, Vivek

Published
2024
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3. Multi-modal and multi-scale clinical retinal imaging system with pupil and retinal tracking

Author

Shirazi, Muhammad Faizan, Andilla, Jordi, Lefaudeux, Nicolas, Valdes, Claudia, Schwarzhans, Florian, Durand, Marine, Ntatsis, Konstantinos, De Jesus, Danilo Andrade, Sanchez Brea, Luisa, Gocho, Kiyoko, Gautier, Josselin, Eckmann-Hansen, Christina, Torm, Marie Elise Wistrup, Amini, Abdullah, Klein, Stefan, Van Walsum, Theo, Grieve, Kate, Paques, Michel, Larsen, Michael, Loza-Alvarez, Pablo, Levecq, Xavier, Chateau, Nicolas, and Pircher, Michael

Published
2022
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14. Adaptive optics scanning laser ophthalmoscope imaging: technology update

Author

Merino D and Loza-Alvarez P

Subjects
High Resolution Retinal Imaging, AOSLO, Ophthalmology, RE1-994
Abstract

David Merino, Pablo Loza-Alvarez The Institute of Photonic Sciences (ICFO), The Barcelona Institute of Science and Technology, Castelldefels, Barcelona, Spain Abstract: Adaptive optics (AO) retinal imaging has become very popular in the past few years, especially within the ophthalmic research community. Several different retinal techniques, such as fundus imaging cameras or optical coherence tomography systems, have been coupled with AO in order to produce impressive images showing individual cell mosaics over different layers of the in vivo human retina. The combination of AO with scanning laser ophthalmoscopy has been extensively used to generate impressive images of the human retina with unprecedented resolution, showing individual photoreceptor cells, retinal pigment epithelium cells, as well as microscopic capillary vessels, or the nerve fiber layer. Over the past few years, the technique has evolved to develop several different applications not only in the clinic but also in different animal models, thanks to technological developments in the field. These developments have specific applications to different fields of investigation, which are not limited to the study of retinal diseases but also to the understanding of the retinal function and vision science. This review is an attempt to summarize these developments in an understandable and brief manner in order to guide the reader into the possibilities that AO scanning laser ophthalmoscopy offers, as well as its limitations, which should be taken into account when planning on using it. Keywords: high-resolution, in vivo retinal imaging, AOSLO

Published
2016

17. Deficiency of the ywhaz gene, involved in neurodevelopmental disorders, alters brain activity and behaviour in zebrafish

Author

Antón-Galindo E, Dalla Vecchia E, Orlandi JG, Castro G, Gualda EJ, Young AMJ, Guasch-Piqueras M, Arenas C, Herrera-Úbeda C, Garcia-Fernàndez J, Aguado F, Loza-Alvarez P, Cormand B, Norton WHJ, and Fernandez N

Abstract

Genetic variants in YWHAZ contribute to psychiatric disorders such as autism spectrum disorder and schizophrenia, and have been related to an impaired neurodevelopment in humans and mice. Here, we have used zebrafish to investigate the mechanisms by which YWHAZ contributes to neurodevelopmental disorders. We observed that ywhaz expression was pan-neuronal during developmental stages and restricted to Purkinje cells in the adult cerebellum, cells that are described to be reduced in number and size in autistic patients. We then performed whole-brain imaging in wild-type and ywhaz CRISPR/Cas9 knockout (KO) larvae and found altered neuronal activity and connectivity in the hindbrain. Adult ywhaz KO fish display decreased levels of monoamines in the hindbrain and freeze when exposed to novel stimuli, a phenotype that can be reversed with drugs that target monoamine neurotransmission. These findings suggest an important role for ywhaz in establishing neuronal connectivity during development and modulating both neurotransmission and behaviour in adults.

Published
2022

21. Comparability of Raman Spectroscopic Configurations: A Large Scale Cross-Laboratory Study

Author

Guo, S. Beleites, C. Neugebauer, U. Abalde-Cela, S. Afseth, N.K. Alsamad, F. Anand, S. Araujo-Andrade, C. Aškrabić, S. Avci, E. Baia, M. Baranska, M. Baria, E. Batista De Carvalho, L.A.E. De Bettignies, P. Bonifacio, A. Bonnier, F. Brauchle, E.M. Byrne, H.J. Chourpa, I. Cicchi, R. Cuisinier, F. Culha, M. Dahms, M. David, C. Duponchel, L. Duraipandian, S. El-Mashtoly, S.F. Ellis, D.I. Eppe, G. Falgayrac, G. Gamulin, O. Gardner, B. Gardner, P. Gerwert, K. Giamarellos-Bourboulis, E.J. Gizurarson, S. Gnyba, M. Goodacre, R. Grysan, P. Guntinas-Lichius, O. Helgadottir, H. Grošev, V.M. Kendall, C. Kiselev, R. Kölbach, M. Krafft, C. Krishnamoorthy, S. Kubryck, P. Lendl, B. Loza-Alvarez, P. Lyng, F.M. Machill, S. Malherbe, C. Marro, M. Marques, M.P.M. Matuszyk, E. Morasso, C.F. Moreau, M. Muhamadali, H. Mussi, V. Notingher, I. Pacia, M.Z. Pavone, F.S. Penel, G. Petersen, D. Piot, O. Rau, J.V. Richter, M. Rybarczyk, M.K. Salehi, H. Schenke-Layland, K. Schlücker, S. Schosserer, M. Schütze, K. Sergo, V. Sinjab, F. Smulko, J. Sockalingum, G.D. Stiebing, C. Stone, N. Untereiner, V. Vanna, R. Wieland, K. Popp, J. Bocklitz, T.

Abstract

The variable configuration of Raman spectroscopic platforms is one of the major obstacles in establishing Raman spectroscopy as a valuable physicochemical method within real-world scenarios such as clinical diagnostics. For such real world applications like diagnostic classification, the models should ideally be usable to predict data from different setups. Whether it is done by training a rugged model with data from many setups or by a primary-replica strategy where models are developed on a 'primary' setup and the test data are generated on 'replicate' setups, this is only possible if the Raman spectra from different setups are consistent, reproducible, and comparable. However, Raman spectra can be highly sensitive to the measurement conditions, and they change from setup to setup even if the same samples are measured. Although increasingly recognized as an issue, the dependence of the Raman spectra on the instrumental configuration is far from being fully understood and great effort is needed to address the resulting spectral variations and to correct for them. To make the severity of the situation clear, we present a round robin experiment investigating the comparability of 35 Raman spectroscopic devices with different configurations in 15 institutes within seven European countries from the COST (European Cooperation in Science and Technology) action Raman4clinics. The experiment was developed in a fashion that allows various instrumental configurations ranging from highly confocal setups to fibre-optic based systems with different excitation wavelengths. We illustrate the spectral variations caused by the instrumental configurations from the perspectives of peak shifts, intensity variations, peak widths, and noise levels. We conclude this contribution with recommendations that may help to improve the inter-laboratory studies. © 2020 American Chemical Society.

Published
2020

22. Development of two-photon polymerised scaffolds for optical interrogation and neurite guidance of human iPSC-derived cortical neuronal networks

Author

Crowe, J A, El-Tamer, A, Nagel, D, Koroleva, A V, Madrid-Wolff, J, Olarte, O E, Sokolovsky, S, Estevez-Priego, E, Ludl, A-A, Soriano, J, Loza-Alvarez, P, Chichkov, B N, Hill, E J, Parri, H R, Rafailov, E U, Crowe, J A, El-Tamer, A, Nagel, D, Koroleva, A V, Madrid-Wolff, J, Olarte, O E, Sokolovsky, S, Estevez-Priego, E, Ludl, A-A, Soriano, J, Loza-Alvarez, P, Chichkov, B N, Hill, E J, Parri, H R, and Rafailov, E U

Abstract

Recent progress in the field of human induced pluripotent stem cells (iPSCs) has led to the efficient production of human neuronal cell models for in vitro study. This has the potential to enable the understanding of live human cellular and network function which is otherwise not possible. However, a major challenge is the generation of reproducible neural networks together with the ability to interrogate and record at the single cell level. A promising aid is the use of biomaterial scaffolds that would enable the development and guidance of neuronal networks in physiologically relevant architectures and dimensionality. The optimal scaffold material would need to be precisely fabricated with submicron resolution, be optically transparent, and biocompatible. Two-photon polymerisation (2PP) enables precise microfabrication of three-dimensional structures. In this study, we report the identification of two biomaterials that support the growth and differentiation of human iPSC-derived neural progenitors into functional neuronal networks. Furthermore, these materials can be patterned to induce alignment of neuronal processes and enable the optical interrogation of individual cells. 2PP scaffolds with tailored topographies therefore provide an effective method of producing defined in vitro human neural networks for application in influencing neurite guidance and complex network activity.

Published
2020

23. Post-translational regulation of retinal IMPDH1 in vivo to adjust GTP synthesis to illumination conditions

Author

Universitat Rovira i Virgili, Plana-Bonamaisó A; López-Begines S; Fernández-Justel D; Junza A; Soler-Tapia A; Andilla J; Loza-Alvarez P; Rosa JL; Miralles E; Casals I; Yanes O; Villa Pdl; Buey RM; Méndez A, Universitat Rovira i Virgili, and Plana-Bonamaisó A; López-Begines S; Fernández-Justel D; Junza A; Soler-Tapia A; Andilla J; Loza-Alvarez P; Rosa JL; Miralles E; Casals I; Yanes O; Villa Pdl; Buey RM; Méndez A

Abstract

© Plana-Bonamaisó et al. We report the in vivo regulation of Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) in the retina. IMPDH1 catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness. We unveil a light-dependent phosphorylation of retinal IMPDH1 at Thr159/Ser160 in the Bateman domain that desensitizes the enzyme to allosteric inhibition by GDP/GTP. When exposed to bright light, living mice increase the rate of GTP and ATP synthesis in their retinas; concomitant with IMPDH1 aggregate formation at the outer segment layer. Inhibiting IMPDH activity in living mice delays rod mass recovery. We unveil a novel mechanism of regulation of IMPDH1 in vivo, important for understanding GTP homeostasis in the retina and the pathogenesis of adRP10 IMPDH1 mutations.

Published
2020

24. Nanoscale structure of amyloid-beta plaques in Alzheimer's disease

Author

Querol-Vilaseca, M, Colom-Cadena, M, Pegueroles, J, Nunez-Llaves, R, Luque-Cabecerans, J, Munoz-Llahuna, L, Andilla, J, Belbin, O, Spires-Jones, TL, Gelpi, E, Clarimon, J, Loza-Alvarez, P, Fortea, J, and Lleo, A

Abstract

Soluble amyloid-beta (A beta) is considered to be a critical component in the pathogenesis of Alzheimer's disease (AD). Evidence suggests that these non-fibrillar A beta assemblies are implicated in synaptic dysfunction, neurodegeneration and cell death. However, characterization of these species comes mainly from studies in cellular or animal models, and there is little data in intact human samples due to the lack of adequate optical microscopic resolution to study these small structures. Here, to achieve super-resolution in all three dimensions, we applied Array Tomography (AT) and Stimulated Emission Depletion microscopy (STED), to characterize in postmortem human brain tissue non-fibrillar A beta structures in amyloid plaques of cases with autosomal dominant and sporadic AD. Ultrathin sections scanned with super-resolution STED microscopy allowed the detection of small A beta structures of the order of 100 nm. We reconstructed a whole human amyloid plaque and established that plaques are formed by a dense core of higher order A beta species (similar to 0.022 mu m(3)) and a peripheral halo of smaller A beta structures (similar to 0.003 mu m(3)). This work highlights the potential of AT-STED for human neuropathological studies.

Published
2019

26. Analysis of intracellular protein dynamics in living zebrafish embryos using light-sheet fluorescence single-molecule microscopy

Author

Bernardello, Matteo, Gora, Radoslaw J., Van Hage, Patrick, Castro-Olvera, Gustavo, Gualda, Emilio J., Schaaf, Marcel J. M., and Loza-Alvarez, Pablo

Abstract

Single-molecule microscopy techniques have emerged as useful tools to image individual molecules and analyze their dynamics inside cells, but their application has mostly been restricted to cell cultures. Here, a light-sheet fluorescence microscopy setup is presented for imaging individual proteins inside living zebrafish embryos. The optical configuration makes this design accessible to many laboratories and a dedicated sample-mounting system ensures sample viability and mounting flexibility. Using this setup, we have analyzed the dynamics of individual glucocorticoid receptors, which demonstrates that this approach creates multiple possibilities for the analysis of intracellular protein dynamics in intact living organisms.

Published
2021

27. Light-sheet fluorescence microscopy for the in vivo study of microtubule dynamics in the zebrafish embryo

Author

Bernardello, Matteo, Marsal, Maria, Gualda, Emilio J., and Loza-Alvarez, Pablo

Abstract

During its first hours of development, the zebrafish embryo presents a large microtubule array in the yolk region, essential for its development. Despite of its size and dynamic behavior, this network has been studied only in limited field of views or in fixed samples. We designed and implemented different strategies in Light Sheet Fluorescence microscopy for imaging the entire yolk microtubule (MT) network in vivo. These have allowed us to develop a novel image analysis from which we clearly observe a cyclical re-arrangement of the entire MT network in synchrony with blastoderm mitotic waves. These dynamics also affect a previously unreported microtubule array deep within the yolk, here described. These findings provide a new vision of the zebrafish yolk microtubules arrangement, and offers novel insights in the interaction between mitotic events and microtubules reorganization.

Published
2021

28. Transcriptome analysis in tissue sectors with contrasting crocins accumulation provides novel insights into apocarotenoid biosynthesis and regulation during chromoplast biogenesis

Author

Universitat Politècnica de Catalunya. Institut de Ciències Fotòniques, Ahrazem, O., Argandoña, J., Fiore, A., Aguado, C., Luján, R., Rubio-Moraga, A., Marro, M., Araujo-Andrade, C., Loza-Alvarez, P., Diretto, G., Gómez-Gómez, L., Universitat Politècnica de Catalunya. Institut de Ciències Fotòniques, Ahrazem, O., Argandoña, J., Fiore, A., Aguado, C., Luján, R., Rubio-Moraga, A., Marro, M., Araujo-Andrade, C., Loza-Alvarez, P., Diretto, G., and Gómez-Gómez, L.

Abstract

Crocins, the red soluble apocarotenoids of saffron, accumulate in the flowers of Crocus species in a developmental and tissue-specific manner. In Crocus sieberi, crocins accumulate in stigmas but also in a distinct yellow tepal sector, which we demonstrate contains chromoplast converted from amyloplasts. Secondary metabolites were analysed by LC-DAD-HRMS, revealing the progressive accumulation of crocetin and crocins in the yellow sector, which were also localized in situ by Raman microspectroscopy. To understand the underlying mechanisms of crocin biosynthesis, we sequenced the C. sieberi tepal transcriptome of two differentially pigmented sectors (yellow and white) at two developmental stages (6 and 8) by Illumina sequencing. A total of 154 million high-quality reads were generated and assembled into 248,099 transcripts. Differentially expressed gene analysis resulted in the identification of several potential candidate genes involved in crocin metabolism and regulation. The results provide a first profile of the molecular events related to the dynamics of crocetin and crocin accumulation during tepal development, and present new information concerning apocarotenoid biosynthesis regulators and their accumulation in Crocus. Further, reveals genes that were previously unknown to affect crocin formation, which could be used to improve crocin accumulation in Crocus plants and the commercial quality of saffron spice., Peer Reviewed, Postprint (published version)

Published
2018

29. Synaptic phosphorylated alpha-synuclein in dementia with Lewy bodies

Author

Colom-Cadena, M, Pegueroles, J, Herrmann, AG, Henstridge, CM, Munoz, L, Querol-Vilaseca, M, Martin-Paniello, C, Luque-Cabecerans, J, Clarimon, J, Belbin, O, Nunez-Llaves, R, Blesa, R, Smith, C, McKenzie, CA, Frosch, MP, Roe, A, Fortea, J, Andilla, J, Loza-Alvarez, P, Gelpi, E, Hyman, BT, Spires-Jones, TL, and Lleo, A

Subjects
p-alpha-synuclein, nervous system, mental disorders, synapses, dementia with Lewy bodies, array tomography, human tissue, nervous system diseases
Abstract

Synaptic loss occurs early in dementia with Lewy bodies (DLB), but its relationship to alpha-synuclein pathology remains unclear. Using array tomography microscopy, Colom-Cadena et al. reveal small phosphorylated alpha-synuclein aggregates at synaptic terminals of DLB cases, supporting a direct association between alpha-synuclein accumulation and synaptic dysfunction.Dementia with Lewy bodies is characterized by the accumulation of Lewy bodies and Lewy neurites in the CNS, both of which are composed mainly of aggregated alpha-synuclein phosphorylated at Ser129. Although phosphorylated alpha-synuclein is believed to exert toxic effects at the synapse in dementia with Lewy bodies and other alpha-synucleinopathies, direct evidence for the precise synaptic localization has been difficult to achieve due to the lack of adequate optical microscopic resolution to study human synapses. In the present study we applied array tomography, a microscopy technique that combines ultrathin sectioning of tissue with immunofluorescence allowing precise identification of small structures, to quantitatively investigate the synaptic phosphorylated alpha-synuclein pathology in dementia with Lewy bodies. We performed array tomography on human brain samples from five patients with dementia with Lewy bodies, five patients with Alzheimer's disease and five healthy control subjects to analyse the presence of phosphorylated alpha-synuclein immunoreactivity at the synapse and their relationship with synapse size. Main analyses were performed in blocks from cingulate cortex and confirmed in blocks from the striatum of cases with dementia with Lewy bodies. A total of 1 318 700 single pre- or postsynaptic terminals were analysed. We found that phosphorylated alpha-synuclein is present exclusively in dementia with Lewy bodies cases, where it can be identified in the form of Lewy bodies, Lewy neurites and small aggregates (< 0.16 A mu m(3)). Between 19% and 25% of phosphorylated alpha-synuclein deposits were found in presynaptic terminals mainly in the form of small aggregates. Synaptic terminals that co-localized with small aggregates of phosphorylated alpha-synuclein were significantly larger than those that did not. Finally, a gradient of phosphorylated alpha-synuclein aggregation in synapses (pre > pre + post > postsynaptic) was observed. These results indicate that phosphorylated alpha-synuclein is found at the presynaptic terminals of dementia with Lewy bodies cases mainly in the form of small phosphorylated alpha-synuclein aggregates that are associated with changes in synaptic morphology. Overall, our data support the notion that pathological phosphorylated alpha-synuclein may disrupt the structure and function of the synapse in dementia with Lewy bodies.

Published
2017

30. High peak-power picosecond pulse generation at 1.26 μm using a quantum-dot-based externalcavity mode-locked laser and tapered optical amplifier

Author

Ding, Y. Aviles-Espinosa, R. Cataluna, M.A. Nikitichev, D. Ruiz, M. Tran, M. Robert, Y. Kapsalis, A. Simos, H. Mesaritakis, C. Xu, T. Bardella, P. Rossetti, M. Krestnikov, I. Livshits, D. Montrosset, I. Syvridis, D. Krakowski, M. Loza-Alvarez, P. Rafailov, E.

Subjects
Physics::Optics
Abstract

In this paper, we present the generation of high peak-power picosecond optical pulses in the 1.26 μm spectral band from a repetitionrate- tunable quantum-dot external-cavity passively mode-locked laser (QDECMLL), 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 peakpower pulsed light source for nonlinear bio-imaging applications across the 1.0 μm - 1.3 μm spectral range. © 2012 Optical Society of America.

Published
2012

31. The Neck Region of the C-type Lectin DC-SIGN Regulates Its Surface Spatiotemporal Organization and Virus-binding Capacity on Antigen-presenting Cells

Author

Manzo, C., Torreno-Pina, J.A., Joosten, B., Reinieren-Beeren, I., Gualda, E.J., Loza-Alvarez, P., Figdor, C.G., Garcia-Parajo, M.F., Cambi, A., Manzo, C., Torreno-Pina, J.A., Joosten, B., Reinieren-Beeren, I., Gualda, E.J., Loza-Alvarez, P., Figdor, C.G., Garcia-Parajo, M.F., and Cambi, A.

Abstract

Contains fulltext : 109735.pdf (Publisher’s version ) (Open Access), 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 into 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.

Published
2012

32. High peak-power picosecond pulse generation at 126 µm using a quantum-dot-based external-cavity mode-locked laser and tapered optical amplifier

Author

Ding, Y., primary, Aviles-Espinosa, R., additional, Cataluna, M. A., additional, Nikitichev, D., additional, Ruiz, M., additional, Tran, M., additional, Robert, Y., additional, Kapsalis, A., additional, Simos, H., additional, Mesaritakis, C., additional, Xu, T., additional, Bardella, P., additional, Rossetti, M., additional, Krestnikov, I., additional, Livshits, D., additional, Montrosset, Ivo, additional, Syvridis, D., additional, Krakowski, M., additional, Loza-Alvarez, P., additional, and Rafailov, E., additional

Published
2012
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33. Poster session 3

Author

Nanka, O., primary, Krejci, E., additional, Pesevski, Z., additional, Sedmera, D., additional, Smart, N., additional, Rossdeutsch, A., additional, Dube, K. N., additional, Riegler, J., additional, Price, A. N., additional, Taylor, A., additional, Muthurangu, V., additional, Turner, M., additional, Lythgoe, M. F., additional, Riley, P. R., additional, Kryvorot, S., additional, Vladimirskaya, T., additional, Shved, I., additional, Schwarzl, M., additional, Seiler, S., additional, Huber, S., additional, Steendijk, P., additional, Maechler, H., additional, Truschnig-Wilders, M., additional, Pieske, B., additional, Post, H., additional, Caprio, C., additional, Baldini, A., additional, Chiavacci, E., additional, Dolfi, L., additional, Verduci, L., additional, Meghini, F., additional, Cremisi, F., additional, Pitto, L., additional, Kuan, T.-C., additional, Chen, M.-C., additional, Yang, T.-H., additional, Wu, W.-T., additional, Lin, C. S., additional, Rai, H., additional, Kumar, S., additional, Sharma, A. K., additional, Mastana, S., additional, Kapoor, A., additional, Pandey, C. M., additional, Agrawal, S., additional, Sinha, N., additional, Orlowska-Baranowska, E. H., additional, Placha, G., additional, Gora, J., additional, Baranowski, R., additional, Abramczuk, E., additional, Hryniewiecki, T., additional, Gaciong, Z., additional, Verschuren, J. J. W., additional, Wessels, J. A. M., additional, Trompet, S., additional, Stott, D. J., additional, Sattar, N., additional, Buckley, B., additional, Guchelaar, H. J., additional, Jukema, J. W., additional, Gharanei, M., additional, Hussain, A., additional, Mee, C. J., additional, Maddock, H. L., additional, Wijnen, W. J., additional, Van Den Oever, S., additional, Van Der Made, I., additional, Hiller, M., additional, Tijsen, A. J., additional, Pinto, Y. M., additional, Creemers, E. E., additional, Nikulina, S. U. Y., additional, Chernova, A., additional, Petry, A., additional, Rzymski, T., additional, Kracun, D., additional, Riess, F., additional, Pike, L., additional, Harris, A. L., additional, Gorlach, A., additional, Katare, R., additional, Oikawa, A., additional, Riu, F., additional, Beltrami, A. P., additional, Cesseli, D., additional, Emanueli, C., additional, Madeddu, P., additional, Zaglia, T., additional, Milan, G., additional, Franzoso, M., additional, Pesce, P., additional, Sarais, C., additional, Sandri, M., additional, Mongillo, M., additional, Butler, T. J., additional, Seymour, A.-M. L., additional, Ashford, D., additional, Jaffre, F., additional, Bussen, M., additional, Flohrschutz, I., additional, Martin, G. R., additional, Engelhardt, S., additional, Kararigas, G., additional, Nguyen, B. T., additional, Jarry, H., additional, Regitz-Zagrosek, V., additional, Van Bilsen, M., additional, Daniels, A., additional, Munts, C., additional, Janssen, B. J. A., additional, Van Der Vusse, G. J., additional, Van Nieuwenhoven, F. A., additional, Montalvo, C., additional, Villar, A. V., additional, Merino, D., additional, Garcia, R., additional, Llano, M., additional, Ares, M., additional, Hurle, M. A., additional, Nistal, J. F., additional, Dembinska-Kiec, A., additional, Beata Kiec-Wilk, B. K. W., additional, Anna Polus, A. P., additional, Urszula Czech, U. C., additional, Tatiana Konovaleva, T. K., additional, Gerd Schmitz, G. S., additional, Bertrand, L., additional, Balteau, M., additional, Timmermans, A., additional, Viollet, B., additional, Sakamoto, K., additional, Feron, O., additional, Horman, S., additional, Vanoverschelde, J. L., additional, Beauloye, C., additional, De Meester, C., additional, Martinez, E., additional, Martin, R., additional, Miana, M., additional, Jurado, R., additional, Gomez-Hurtado, N., additional, Bartolome, M. V., additional, San Roman, J. A., additional, Lahera, V., additional, Nieto, M. L., additional, Cachofeiro, V., additional, Rochais, F., additional, Sturny, R., additional, Mesbah, K., additional, Miquerol, L., additional, Kelly, R. G., additional, Messaoudi, S., additional, Gravez, B., additional, Tarjus, A., additional, Pelloux, V., additional, Samuel, J. L., additional, Delcayre, C., additional, Launay, J. M., additional, Clement, K., additional, Farman, N., additional, Jaisser, F., additional, Hadyanto, L., additional, Castellani, C., additional, Vescovo, G., additional, Ravara, B., additional, Tavano, R., additional, Pozzobon, M., additional, De Coppi, P., additional, Papini, E., additional, Vettor, R., additional, Thiene, G., additional, Angelini, A., additional, Meloni, M., additional, Caporali, A., additional, Cesselli, D., additional, Fortunato, O., additional, Avolio, E., additional, Schindler, R., additional, Simrick, S., additional, Brand, T., additional, Smart, N. S., additional, Herman, A., additional, Roura Ferrer, S., additional, Rodriguez Bago, J., additional, Soler-Botija, C., additional, Pujal, J. M., additional, Galvez-Monton, C., additional, Prat-Vidal, C., additional, Llucia-Valldeperas, A., additional, Blanco, J., additional, Bayes-Genis, A., additional, Foldes, G., additional, Maxime, M., additional, Ali, N. N., additional, Schneider, M. D., additional, Harding, S. E., additional, Reni, C., additional, Mangialardi, G., additional, De Pauw, A., additional, Sekkali, B., additional, Friart, A., additional, Ding, H., additional, Graffeuil, A., additional, Catalucci, D., additional, Balligand, J. 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2012
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34. Poster session 2

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Perez-Pomares, J. M., primary, Ruiz-Villalba, A., additional, Ziogas, A., additional, Segovia, J. C., additional, Ehrbar, M., additional, Munoz-Chapuli, R., additional, De La Rosa, A., additional, Dominguez, J. N., additional, Hove-Madsen, L., additional, Sankova, B., additional, Sedmera, D., additional, Franco, D., additional, Aranega Jimenez, A., additional, Babaeva, G., additional, Chizh, N., additional, Galchenko, S., additional, Sandomirsky, B., additional, Schwarzl, M., additional, Seiler, S., additional, Steendijk, P., additional, Huber, S., additional, Maechler, H., additional, Truschnig-Wilders, M., additional, Pieske, B., additional, Post, H., additional, Simrick, S., additional, Kreutzer, R., additional, Rao, C., additional, Terracciano, C. M., additional, Kirchhof, P., additional, Fabritz, L., additional, Brand, T., additional, Theveniau-Ruissy, M., additional, Parisot, P., additional, Francou, A., additional, Saint-Michel, E., additional, Mesbah, K., additional, Kelly, R. G., additional, Wu, H.-T., additional, Sie, S.-S., additional, Chen, C.-Y., additional, Kuan, T.-C., additional, Lin, C. S., additional, Ismailoglu, Z., additional, Guven, M., additional, Yakici, A., additional, Ata, Y., additional, Ozcan, S., additional, Yildirim, E., additional, Ongen, Z., additional, Miroshnikova, V., additional, Demina, E., additional, Rodygina, T., additional, Kurjanov, P., additional, Denisenko, A., additional, Schwarzman, A., additional, Rubanenko, A., additional, Shchukin, Y., additional, Germanov, A., additional, Goldbergova, M., additional, Parenica, J., additional, Lipkova, J., additional, Pavek, N., additional, Kala, P., additional, Poloczek, M., additional, Vasku, A., additional, Parenicova, I., additional, Spinar, J., additional, Gambacciani, C., additional, Chiavacci, E., additional, Evangelista, M., additional, Vesentini, N., additional, Kusmic, C., additional, Pitto, L., additional, Chernova, A., additional, Nikulina, S. U. Y., additional, Arvanitis, D. A., additional, Mourouzis, I., additional, Pantos, C., additional, Kranias, E. G., additional, Cokkinos, D. V., additional, Sanoudou, D., additional, Vladimirskaya, T. E., additional, Shved, I. A., additional, Kryvorot, S. G., additional, Schirmer, I. M., additional, Appukuttan, A., additional, Pott, L., additional, Jaquet, K., additional, Ladilov, Y., additional, Archer, C. R., additional, Bootman, M. D., additional, Roderick, H. L., additional, Fusco, A., additional, Sorriento, D., additional, Santulli, G., additional, Trimarco, B., additional, Iaccarino, G., additional, Hagenmueller, M., additional, Riffel, J., additional, Bernhold, E., additional, Katus, H. A., additional, Hardt, S. 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Published
2012
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35. Sub-diffraction discrimination with polarization-resolved two-photon excited fluorescence microscopy

Author

Artigas, David, Merino, David, Polzer, Christoph, and Loza-Alvarez, Pablo

Abstract

Imaging molecular structures separated by distances of a few nanometers still represents a complex challenge. Moreover, it is normally restricted to observations on thin (few micrometers) samples. In this work, we rotate the polarization of the excitation beam of two-photon excited fluorescence (TPEF) images to show that fluorescent structures at the molecular scale can be discriminated in a living organism. The polarization rotation generates a modulation of the signal intensity in each pixel of the TPEF images that carry information related to the fluorophore orientation. We analyze the signal modulation in every pixel of the polarization-resolved (PR) TPEF images through a Fourier analysis and generate images for the different Fourier components. Doing that, we show that two fluorophores oriented in different directions can be distinguished. Although by imaging the Fourier components the resolution of the optical system restricts the exact localization of two close molecules, discrimination is still possible even when the molecules are located at sub-diffraction distances. We propose a model that predicts this behavior, and demonstrate it experimentally in the neurons of a living Caenorhabditis elegans nematode, where we distinguish the walls of an axon with a diameter below the objective resolution. Since the technique is based in TPEF, the method can be extended to deep tissue imaging and has potential applications in single molecule detection, biological sensors, or super-resolution imaging techniques.

Published
2017

36. STED imaging performance estimation by means of Fourier transform analysis

Author

Merino, David, Mallabiabarrena, Arrate, Andilla, Jordi, Artigas, David, Zimmermann, Timo, and Loza-Alvarez, Pablo

Abstract

Due to relatively high powers used in STED, biological samples may be affected by the illumination in the process of image acquisition. Similarly, the performance of the system may be limited by the sample itself. Optimization of the STED parameters taking into account the sample itself is therefore a complex task as there is no clear methodology that can determine the image improvement in an objective and quantitative manner. In this work, a method based on Fourier transform formalism is presented to analyze the performance of a STED system. The spatial frequency distribution of pairs of confocal and STED images are compared to obtain an objective parameter, the Azimuth Averaged Spectral Content Spread (AASCS), that is related to the performance of the system in which the sample is also considered. The method has been first tested on samples of beads, and then applied to cell samples labeled with multiple fluorescent dyes. The results show that a single parameter, the AASCS, can be used to determine the optimal settings for STED image acquisition in an objective way, only by using the information provided by the images from the sample themselves. The AASCS also helps minimize the depletion power, for better preservation of the samples.

Published
2017

42. Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters

Author

Rocha-Mendoza, Israel, Licea-Rodriguez, Jacob, Marro, Mónica, Olarte, Omar E., Plata-Sanchez, Marcos, and Loza-Alvarez, Pablo

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.

Published
2015

43. Decoupled illumination detection in light sheet microscopy for fast volumetric imaging

Author

Olarte, Omar E., Andilla, Jordi, Artigas, David, and Loza-Alvarez, Pablo

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.

Published
2015

44. Translational label-free nonlinear imaging biomarkers to classify the human corneal microstructure

Author

Lombardo, Marco, Merino, David, Loza-Alvarez, Pablo, and Lombardo, Giuseppe

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.

Published
2015

45. Fast monitoring of in-vivo conformational changes in myosin using single scan polarization-SHG microscopy

Author

Psilodimitrakopoulos, Sotiris, Loza-Alvarez, Pablo, and Artigas, David

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.

Published
2014

46. A Transcriptome-proteome Integrated Network Identifies Endoplasmic Reticulum thiol oxidoreductase (ERp57) as a Hub that Mediates Bone Metastasis*

Author

Santana-Codina, Naiara, Carretero, Rafael, Sanz-Pamplona, Rebeca, Cabrera, Teresa, Guney, Emre, Oliva, Baldo, Clezardin, Philippe, Olarte, Omar E., Loza-Alvarez, Pablo, Méndez-Lucas, Andrés, Perales, Jose Carlos, and Sierra, Angels

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 MDA-MB-231 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 with 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 that retained four down-regulated 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 organ-specific 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.

Published
2013
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47. Quantitative Imaging of Microtubule Alteration as an Early Marker of Axonal Degeneration after Ischemia in Neurons

Author

Psilodimitrakopoulos, Sotiris, Petegnief, Valerie, de Vera, Nuria, Hernandez, Oscar, Artigas, David, Planas, Anna M., and Loza-Alvarez, Pablo

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.

Published
2013
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48. Image formation by linear and nonlinear digital scanned light-sheet fluorescence microscopy with Gaussian and Bessel beam profiles

Author

Olarte, Omar E., Licea-Rodriguez, Jacob, Palero, Jonathan A., Gualda, Emilio J., Artigas, David, Mayer, Jürgen, Swoger, Jim, Sharpe, James, Rocha-Mendoza, Israel, Rangel-Rojo, Raul, and Loza-Alvarez, Pablo

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.

Published
2012

49. Measurement and correction of in vivo sample aberrations employing a nonlinear guide-star in two-photon excited fluorescence microscopy

Author

Aviles-Espinosa, Rodrigo, Andilla, Jordi, Porcar-Guezenec, Rafael, Olarte, Omar E., Nieto, Marta, Levecq, Xavier, Artigas, David, and Loza-Alvarez, Pablo

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.

Published
2011

50. Compact ultrafast semiconductor disk laser: targeting GFP based nonlinear applications in living organisms

Author

Aviles-Espinosa, Rodrigo, Filippidis, George, Hamilton, Craig, Malcolm, Graeme, Weingarten, Kurt J., Südmeyer, Thomas, Barbarin, Yohan, Keller, Ursula, Santos, Susana I.C.O, Artigas, David, and Loza-Alvarez, Pablo

Abstract

We present a portable ultrafast Semiconductor Disk Laser (SDL) (or vertical extended cavity surface emitting laser—VECSELs), to be used for nonlinear microscopy. The SDL is modelocked using a quantum-dot semiconductor saturable absorber mirror (SESAM), delivering an average output power of 287 mW, with 1.5 ps pulses at 500 MHz and a central wavelength of 965 nm. Specifically, despite the fact of having long pulses and high repetition rates, we demonstrate the potential of this laser for Two-Photon Excited Fluorescence (TPEF) imaging of in vivo Caenorhabditis elegans (C. elegans) expressing Green Fluorescent Protein (GFP) in a set of neuronal processes and cell bodies. Efficient TPEF imaging is achieved due to the fact that this wavelength matches the peak of the two-photon action cross section of this widely used fluorescent marker. The SDL extended versatility is shown by presenting Second Harmonic Generation images of pharynx, uterus, body wall muscles and its potential to be used to excite other different commercial dyes. Importantly this non-expensive, turn-key, compact laser system could be used as a platform to develop portable nonlinear bio-imaging devices.

Published
2011

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