Development of novel multimodal light-sheet fluorescence microscopes for in-vivo imaging of vertebrate organisms

The observation of biological processes in their native environments is of critical importance for life science. While substantial information can be derived from the examination of in-vitro biological samples, in-vivo studies are necessary to reveal the complexity of the dynamics happening in real-time within a living organism. Between the possible biological model choices, vertebrates represent an important family due to the various characteristics they share with the human organism. The development of an embryo, the effect of a drug, the interaction between the immune system and pathogens, and the cellular machinery activities are all examples of highly-relevant applications requiring in-vivo observations on broadly used vertebrate models such as the zebrafish and the mouse. To perform such observations, appropriate devices have been devised. Fluorescence microscopy is one of the main approaches through which specific sample structures can be detected and registered in high-contrast images. Through micro-injections or transgenic lines, a living specimen can express fluorescence and can be imaged through such microscopes. Various fluorescence microscopy techniques have been developed, such as Widefield Microscopy (WM) and Laser Scanning Confocal Microscopy (LSCM). In WM the entire sample is visualized in a single 2D image, therefore losing the depth information, while LSCM can recover the 3D information of the sample but with inherent limitations, such as phototoxicity and limited imaging speed. In the last two decades, Light-Sheet Fluorescence Microscopy (LSFM) emerged as a technique providing fast and 3D imaging, while minimizing collateral damages to the specimen. However, due to the particular configuration of the microscope’s components, LSFM setups are normally optimized for a single application. Also, sample management is not trivial, as controlling the specimen positioning and keeping it alive for a long time within the microscope needs dedicated environmental conditioning. In this thesis, I aimed at advancing the imaging flexibility of LSFM, with particular attention to sample management. The conjugation of these aspects enabled novel observations and applications on living vertebrate samples. In Chapter 1, a brief review of the concepts employed within this thesis is presented, also pointing to the main challenges that the thesis aims to solve. In Chapter 2, a new design for multimodal LSFM is presented, which enables performing different experiments with the same instrument. Particularly, high-throughput studies would benefit from this imaging paradigm, conjugating the need for fast and reproducible mounting of multiple samples with the opportunity to image them in 3D. Additionally, from this design, a transportable setup has also been implemented. With these systems, I studied the dynamics of the yolk’s microtubule network of zebrafish embryos, describing novel features and underlining the importance of live imaging to have a whole view of the sample’s peculiarities. This is described in Chapter 3. Further applications on challenging live samples have been implemented, monitoring the macrophage recruitment in zebrafish and the development of mouse embryos. For these applications, described in Chapter 4, I devised specific mounting protocols for the samples, keeping them alive during the imaging sessions. In Chapter 5, an additional LSFM system is described, which allows for recording the sub-cellular machinery in a living vertebrate sample, while avoiding its damage thanks to the devised sample mounting. Through this, single-molecule microscopy (SMM) studies, normally performed on cultured cells, can be extended to the nuclei of living zebrafish embryos, which better recapitulate the native environment where biological processes take place. Finally, Chapter 6 recapitulates the conclusions, the impacts, future integrations, and experimental procedures that would be enabled by the work resumed in this thesis.La observación de los procesos biológicos en su entorno es de vital importancia para las ciencias de la vida. Si bien se puede derivar información sustancial desde muestras biológicas in-vitro, los estudios in-vivo son necesarios para revelar la complejidad de la dinámica que ocurre, en tiempo real, dentro de un organismo vivo. Entre las posibles elecciones de modelos biológicos, los vertebrados representan una familia importante debido a las diversas características que comparten con el organismo humano. El desarrollo de un embrión, la interacción entre el sistema inmunitario y los patógenos, el efecto de un fármaco y las actividades celulares son ejemplos de aplicaciones que requieren observaciones in-vivo en modelos de vertebrados, como el pez cebra y el ratón. La microscopía de fluorescencia es uno de los principales métodos mediante los cuales se pueden grabar imágenes, de alto contraste, de estructuras biológicas específicas. Utilizando microinyecciones o líneas transgénicas, es posible inducir una expresión de proteínas fluorescentes en la muestra y entonces puede ser observada a través de dichos microscopios. Existen varias técnicas de microscopía de fluorescencia, entre ellas las más utilizadas son la microscopía ¿widefield¿ (WM) y la microscopía ¿confocal¿ (LSCM). En WM, una sola imagen en 2D representa el volumen entero de la muestra, por lo cual la información de profundidad se pierde. Por otro lado, LSCM puede recuperar la información en 3D con algunas limitaciones como la fototoxicidad y una velocidad de generación de las imágenes limitada. En las últimas dos décadas, la microscopía de fluorescencia de hoja de luz (LSFM) surgió como técnica que ofrece imágenes de manera rápidas y en 3D, y que al mismo tiempo minimiza los daños colaterales de la muestra. Sin embargo, debido a la geometría de los componentes del microscopio, las configuraciones de LSFM normalmente se optimizan para una sola aplicación. Además, la gestión de las muestras no es trivial, ya que controlar su posición y mantenerlas vivas durante largos periodos de tiempo dentro del microscopio requiere una atención especifica. En esta tesis, me propuse mejorar la versatilidad que LSFM puede ofrecer, con especial atención a la gestión de muestras vivas. La conjugación de estos aspectos permitió nuevas observaciones y nuevas aplicaciones en vertebrados vivos. En el Capítulo 1, se presenta un breve resumen de los conceptos empleados dentro de esta tesis, señalando también los principales desafíos que la tesis pretende resolver. En el Capítulo 2, se presenta un nuevo diseño para un LSFM multimodal, que permite realizar diferentes experimentos con el mismo instrumento. Los estudios de High-Throughput se beneficiarían de este diseño, ya que conjuga la necesidad de un montaje rápido y reproducible de varias muestras con las ventajas de LSFM. Además, a partir de este diseño, también se ha desarrollado un otro microscopio LSFM transportable. Con estos sistemas, se estudió la dinámica de la red de microtúbulos en embriones de pez cebra, describiendo características nuevas y acentuando la importancia de los experimentos in-vivo para obtener una visión completa de la muestra. Esto se describe en el Capítulo 3. Para realizar otras aplicaciones, como la observación de la dinámica de macrófagos en el pez cebra y del desarrollo de embriones de ratón, descritas en el Capítulo 4, se establecieron protocolos de montaje específicos para las muestras, manteniéndolas vivas durante las sesiones experimentales. En el Capítulo 5, se describe otro sistema LSFM, que permite extender los estudios de microscopía de moléculas individuales (SMM), normalmente realizados en cultivos de células, a núcleos de embriones de pez cebra vivos, que recrean mejor el entorno natural de los procesos biológicos. Finalmente, el Capítulo 6 recapitula las conclusiones, los impactos, las integraciones futuras y los procedimientos experimentales que serían posibilitados por el trabajo resumido en esta tesis.Postprint (published version

Nectaries and reproductive biology of croton sarcopetalus (euphorbiaceae)

Flower morphology, nectary structure, nectar chemical composition, breeding system, floral visitors and pollination were analysed in Croton sarcopetalus, a diclinous-monoecious shrub from Argentina. Male flowers have five receptacular nectaries, with no special vascular bundles, that consist of a uniserial epidermis with stomata subtended by a secretory parenchyma. Female flowers bear two different types of nectaries: inner (IN) and outer (ON) floral nectaries. IN, five in all, are structurally similar to the nectaries of male flowers. The five ON are vascularized, stalked, and composed of secretory, column-shaped epidermal cells without stomata subtended by secretory and ground parenchyma. In addition, ON act as post-floral nectaries secreting nectar during fruit ripening. Extrafloral nectaries (EFN) are located on petioles, stipules and leaf margins. Petiolar EFN are patelliform, stalked and anatomically similar to the ON of the female flower. Nectar sampled from all nectary types is hexose dominant, except for the ON of the female flower at the post-floral stage that is sucrose dominant. The species is self-compatible, but geitonogamous fertilization is rarely possible because male and female flowers are not usually open at the same time in the same individual, i.e. there is temporal dioecism. Flowers are visited by 22 insect species, wasps being the most important group of pollinators. No significant differences were found in fruit and seed set between natural and hand pollinated flowers. This pattern indicates that fruit production in this species is not pollen/pollinator limited and is mediated by a wide array of pollinators. © 2001 The Linnean Society of London.Fil: Freitas, Leandro. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Bernardello, Gabriel Luis Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Galetto, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Paoli, Adelita. Universidade Estadual Paulista Julio de Mesquita Filho; Brasi

Tribu LYCIEAE Hunz.

Se describe la familia Solanaceae y se presenta una clave con los generos presentes en la provincia (en coautoria con G. Barboza y F. Chiarini). Asimismo, y de mi autoria, se tratan los dos géneros de la Tribu Lycieae y la descripcion de las especies autóctonas de la provincia de Mendoza.Fil: Bernardello, Gabriel Luis Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Instituto Multidisciplinario de Biología Vegetal (p); Argentina; Argentin

Chapter A Bridge Between East and West: Frank Lloyd Wright’s Drawing as Synthesis of Two Different Cultures

The 43rd UID conference, held in Genova, takes up the theme of ‘Dialogues’ as practice and debate on many fundamental topics in our social life, especially in these complex and not yet resolved times. The city of Genova offers the opportunity to ponder on the value of comparison and on the possibilities for the community, naturally focused on the aspects that concern us, as professors, researchers, disseminators of knowledge, or on all the possibile meanings of the discipline of representation and its dialogue with ‘others’, which we have broadly catalogued in three macro areas: History, Semiotics, Science / Technology. Therefore, “dialogue” as a profitable exchange based on a common language, without which it is impossible to comprehend and understand one another; and the graphic sign that connotes the conference is the precise transcription of this concept: the title ‘translated’ into signs, derived from the visual alphabet designed for the visual identity of the UID since 2017. There are many topics which refer to three macro sessions: - Witnessing (signs and history) - Communicating (signs and semiotics) - Experimenting (signs and sciences) Thanks to the different points of view, an exceptional resource of our disciplinary area, we want to try to outline the prevailing theoretical-operational synergies, the collaborative lines of an instrumental nature, the recent updates of the repertoires of images that attest and nourish the relations among representation, history, semiotics, sciences

Floral nectaries, nectar production dynamics, and chemical composition in six Ipomoea species (Convulvulaceae) in relation to pollinators

Background and Aims Floral nectaries and nectar features were compared between six Argentinian Ipomoea species with differences in their pollinator guilds: I. alba, I. rubriflora, I. cairica, I. hieronymi var. hieronymi, I. indica, and I. purpurea. • Methods Pollinators were recorded in natural populations. The morpho-anatomical study was carried out through scanning electron and light microscopy. Nectar sugars were identified via gas chromatography. Nectar production and the effect of its removal on total nectar sugar amount were determined by using sets of bagged flowers. • Key Results Hymenopterans were visitors of most species, while hummingbirds visited I. rubriflora and sphingids I. alba. All the species had a vascularized discoidal nectary surrounding the ovary base with numerous open stomata with a species-specific distribution. All nectar samples contained amino acids and sugars. Most species had sucrose-dominant nectars. Flowers lasted a few hours. Mean nectar sugar concentration throughout the lifetime of the flower ranged from 34·28 to 39·42 %, except for I. cairica (49·25 %) and I. rubriflora (25·18 %). Ipomoea alba had the highest nectar volume secreted per flower (50·12 µL), while in the other taxa it ranged from 2·42 to 12·00 µL. Nectar secretion began as soon as the flowers opened and lasted for a few hours (in I. purpurea, I. rubriflora) or it was continuous during the lifetime of the flower (in the remaining species). There was an increase of total sugar production after removals in I. cairica, I. indica and I. purpurea, whereas in I. alba and I. rubriflora removals had no effect, and in I. hieronymi there was a decrease in total sugar production. • Conclusions The chemical composition, production dynamics and removal effects of nectar could not be related to the pollinator guild of these species. Flower length was correlated with nectary size and total volume of nectar secreted, suggesting that structural constraints may play a major role in the determination of nectar traits of these species.Fil: Galetto, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Bernardello, Gabriel Luis Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentin

Floral nectaries, nectar production dynamics, and chemical composition in six Ipomoea species (Convulvulaceae) in relation to pollinators

Background and Aims Floral nectaries and nectar features were compared between six Argentinian Ipomoea species with differences in their pollinator guilds: I. alba, I. rubriflora, I. cairica, I. hieronymi var. hieronymi, I. indica, and I. purpurea. • Methods Pollinators were recorded in natural populations. The morpho-anatomical study was carried out through scanning electron and light microscopy. Nectar sugars were identified via gas chromatography. Nectar production and the effect of its removal on total nectar sugar amount were determined by using sets of bagged flowers. • Key Results Hymenopterans were visitors of most species, while hummingbirds visited I. rubriflora and sphingids I. alba. All the species had a vascularized discoidal nectary surrounding the ovary base with numerous open stomata with a species-specific distribution. All nectar samples contained amino acids and sugars. Most species had sucrose-dominant nectars. Flowers lasted a few hours. Mean nectar sugar concentration throughout the lifetime of the flower ranged from 34·28 to 39·42 %, except for I. cairica (49·25 %) and I. rubriflora (25·18 %). Ipomoea alba had the highest nectar volume secreted per flower (50·12 µL), while in the other taxa it ranged from 2·42 to 12·00 µL. Nectar secretion began as soon as the flowers opened and lasted for a few hours (in I. purpurea, I. rubriflora) or it was continuous during the lifetime of the flower (in the remaining species). There was an increase of total sugar production after removals in I. cairica, I. indica and I. purpurea, whereas in I. alba and I. rubriflora removals had no effect, and in I. hieronymi there was a decrease in total sugar production. • Conclusions The chemical composition, production dynamics and removal effects of nectar could not be related to the pollinator guild of these species. Flower length was correlated with nectary size and total volume of nectar secreted, suggesting that structural constraints may play a major role in the determination of nectar traits of these species.Fil: Galetto, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; ArgentinaFil: Bernardello, Gabriel Luis Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentin

Anatomía floral de dos especies de Larnax (Solanaceae)

Se estudia comparativamente la anatomía floral de dos especies peruanas del género Larnax Miers (tribu Solaneae): L. lutea S. Leiva y L. parviflora N. W. Sawyer & S. Leiva, con especial referencia a la vasculatura floral. Los datos obtenidos son novedosos a nivel genérico. Dentro de las características importantes a este nivel se destacan la presencia de cáliz acrescente, petalostemo y nectario ovárico. Se analiza el valor de los datos obtenidos en la delimitación de Larnax con géneros afines, concluyéndose que es más cercano a Athenaea y Aureliana que a Deprea, lo cual apoya el reciente nuevo sistema de Hunziker (2001).The floral anatomy of two Peruvian species of the genus Larnax Miers (tribe Solaneae) is studied: L. lutea S. Leiva and L. parviflora N. W. Sawyer & S. Leiva, with emphasis on their floral vasculature. The obtained results are new at the generic level. Among the most important features for the genus, are mentioned the presence of an accrescent calyx, the stapet, and an ovarian nectary. These data are analyzed in the delimitation of Larnax with allied genera, reaching the conclusion that it is closer to Athenaea and Aureliana than is to Deprea, supporting the recent system proposed by Hunziker (2001).Fil: Cabrera, Mario Fernando. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; ArgentinaFil: Bernardello, Gabriel Luis Mario. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto Multidisciplinario de Biología Vegetal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto Multidisciplinario de Biología Vegetal; Argentin

The ceab's marine observatory in the catalan sea : consolidating long time series observations?

Peer Reviewe

Oxygen minimum zones in the tropical Pacific across CMIP5 models: mean state differences and climate change trends

We analyse simulations of the Pacific Ocean oxygen minimum zones (OMZs) from 11 Earth system model contributions to the Coupled Model Intercomparison Project Phase 5, focusing on the mean state and climate change projections. The simulations tend to overestimate the volume of the OMZs, especially in the tropics and Southern Hemisphere. Compared to observations, five models introduce incorrect meridional asymmetries in the distribution of oxygen including larger southern OMZ and weaker northern OMZ, due to interhemispheric biases in intermediate water mass ventilation. Seven models show too deep an extent of the tropical hypoxia compared to observations, stemming from a deficient equatorial ventilation in the upper ocean, combined with too large a biologically driven downward flux of particulate organic carbon at depth, caused by particle export from the euphotic layer that is too high and remineralization in the upper ocean that is too weak. At interannual timescales, the dynamics of oxygen in the eastern tropical Pacific OMZ is dominated by biological consumption and linked to natural variability in the Walker circulation. However, under the climate change scenario RCP8.5, all simulations yield small and discrepant changes in oxygen concentration at mid depths in the tropical Pacific by the end of the 21st century due to an almost perfect compensation between warming-related decrease in oxygen saturation and decrease in biological oxygen utilization. Climate change projections are at odds with recent observations that show decreasing oxygen levels at mid depths in the tropical Pacific. Out of the OMZs, all the CMIP5 models predict a decrease of oxygen over most of the surface and deep ocean at low latitudes and over all depths at high latitudes due to an overall slow-down of ventilation and increased temperature

Seven years of marine environmental changes monitoring at coastal OOCS stations (Catalan Sea, NW Mediterranean)

Since March 2009 up to the present (more than 7 years now), the Operational Observatory of the Catalan Sea (OOCS; http://www2.ceab.csic.es/ oceans/) remains a witness of persistent marine environmental changes. The OOCS has two fixed observation stations at the head of the Blanes Canyon (200 m depth, 41.66°N; 2.91°E) and at the Blanes bay (20 m depth, 41.67°N; 2.80°E) in the Catalan Sea, NW Mediterranean. At the canyon station, a multi-parametric buoy presently installed delivers high frequency (by 30 min) and multi-parametric oceanographic (i.e. salinity, temperature, chlorophyll, turbidity, as well as light intensity in the PAR range for the upper 50 m depth) and atmospheric (air temperature, relative humidity, wind speed and direction and PAR) data. Subsurface photos and videos by an IP high resolution fisheye camera attached to the buoy are also delivered at 4-hour basis. Data and multimedia are transmitted in near real time for public access, via combined GSM/GPRS and 3G connections. At both stations, CTD profiles and water samples (collected for nutrients and picoplankton analyses) are carried out on board a research vessel at fortnightly basis. Numerical simulations along with the time series of in-situ observations show inter-annual seasonality anomalies possibly linked to global environmental changes. The lower-atmosphere and upper-sea environmental time series data collected prove the occurrence of shifting patterns of heat and matter fluxes impacting pelagic and benthic organisms.Peer Reviewe