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

Modular multimodal platform for classical and high throughput light sheet microscopy

Light-sheet fluorescence microscopy (LSFM) has become an important tool for biological and biomedical research. Although several illumination and detection strategies have been developed, the sample mounting still represents a cumbersome procedure as this is highly dependent on the type of sample and often this might be time consuming. This prevents the use of LSFM in other promising applications in which a fast and straightforward sample-mounting procedure and imaging are essential. These include the high-throughput research fields, e.g. in drug screenings and toxicology studies. Here we present a new imaging paradigm for LSFM, which exploits modularity to offer multimodal imaging and straightforward sample mounting strategy, enhancing the flexibility and throughput of the system. We describe its implementation in which the sample can be imaged either as in any classical configuration, as it flows through the light-sheet using a fluidic approach, or a combination of both. We also evaluate its ability to image a variety of samples, from zebrafish embryos and larvae to 3D complex cell cultures.The authors acknowledge financial support from the Spanish Ministerio de Economía y Competitividad (MINECO) through the “Severo Ochoa” program for Centres of Excellence in R&D (CEX2019-000910-S [MCIN/ AEI/10.13039/501100011033]), Fundació Privada Cellex, Fundació Mir-Puig, and Generalitat de Catalunya through CERCA program; MINECO/FEDER Ramón y Cajal program (RYC-2015-17935); Laserlab- Europe EU-H2020 GA no. 871124; European Union’s Horizon 2020 Framework Programme (H2020 Marie Skłodowska-Curie Innovative Training Networks ImageInLife N. 721537). We thank Verena Ruprecht (CRG- Center of Genomic Regulation, Barcelona), Paz Herráez (Universidad de León), Ester Antón-Galindo and Noelia Fernández-Castillo (Universitat de Barcelona), Marymar Becerra (Universidad Nacional Autónoma de México), Georges Lutfalla, Mai Nguyen Chi and Tamara Sipka (Université de Montpellier), Catarina Brito (ITQB/IBEQ, Lisbon), Antonia Weberling and Magdalena Zernicka-Goetz (University of Cambridge), and Corinne Lorenzo (ITAV – CNRS, Toulouse) for the samples provided. We also thank Maria Marsal and Jordi Andilla for many fruitful discussions.Postprint (published version

Multiple asters organize the yolk microtubule network during dclk2-GFP zebrafish epiboly

It is known that the organization of microtubule (MT) networks in cells is orchestrated by subcellular structures named MT organizing centers (MTOCs). In this work, we use Light Sheet Fluorescence and Confocal Microscopy to investigate how the MT network surrounding the spherical yolk is arranged in the dclk2-GFP zebrafish transgenic line. We found that during epiboly the MT network is organized by multiple aster-like MTOCS. These structures form rings around the yolk sphere. Importantly, in wt embryos, aster-like MTOCs are only found upon pharmacological or genetic induction. Using our microscopy approach, we underscore the variability in the number of such asters in the transgenic line and report on the variety of global configurations of the yolk MT network. The asters’ morphology, dynamics, and their distribution in the yolk sphere are also analyzed. We propose that these features are tightly linked to epiboly timing and geometry. Key molecules are identified which support this asters role as MTOCs, where MT nucleation and growth take place. We conclude that the yolk MT network of dclk2-GFP transgenic embryos can be used as a model to organize microtubules in a spherical geometry by means of multiple MTOCs.This project and MB have received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 721537 “ImageInLife”. EG received funding from MINECO/FEDER Ramon y Cajal program (RYC-2015-17935). Authors also acknowledge financial sup- port from Fundació Privada Cellex, Fundación Mig-Puig; the Generalitat de Catalunya through the CERCA program; the Spanish Ministry of Economy and Competitiveness through the “Severo Ochoa” program for Centres of Excellence in R&D (CEX2019-000910-S) and from Laserlab-Europe EU-H2020 (871124). We thank Virginie Lecaudey, IZNF Germany, and Darren Gilmour, IMLS Switzerland, for the EB3-mCherry and γ-tubulin- TdTomato DNA constructs, Marina Mione, CIBIO, University of Trento, for the dclk2-GFP DNA construct and Esteban Hoijman, CRG Spain, for the DCX-GFP DNA construct. We thank Verena Ruprecht, CRG Spain, Jordi Andilla, ICFO, Spain, and Pilar Pujol, ICFO, Spain, for helpful discussions. We thank Senda Jiménez, CRG, Spain, all SLN-ICFO members, and ICFO Biology lab members for their supporPostprint (published version

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

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.Horizon 2020 Framework Programme (Marie Sklodowska-Curie 721537); Laserlab-Europe (871124); Ministerio de Economía y Competitividad (RYC-2015-17935); Generalitat de Catalunya (CERCA Program); Fundación Cellex (Fundación Mir-Puig); Ministerio de Economía y Competitividad (CEX2019-000910-S).Peer ReviewedPostprint (published version

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

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.Ministerio de Economía y Competitividad (RYC-2015-17935, CEX2019-000910-S); Fundación Cellex (Mir-Puig); Laserlab-Europe (871124); Generalitat de Catalunya (CERCA); Fundación Cellex; Horizon 2020 Framework Programme (721537).Peer ReviewedPostprint (published version

Comunicazioni ottiche in spazio libero

Scopo di questa tesina è caratterizzare i sistemi ottici non guidati, in cui la portante risiede nella regione dell'infrarosso, visibile o ultravioletto e viene propagata nello spazio circostante fino al ricevitore. La prima parte dell'elaborato presenta la struttura di un sistema ottico generico, descrivendone i componenti quali le sorgenti, il canale e il ricevitore. La seconda parte si focalizzerà nei sistemi ottici ad infrarosso in utilizzi indoor, confrontandoli con i sistemi a radiofrequenza e discutendo problematiche quali riflessioni e rumore generato dall'ambiente circostant

Sviluppo sperimentale di un tomografo a coerenza ottica

Scopo dellat tesi è descrivere la tecnica Tomografia a Coerenza Ottica nonché descrivere i risultati ottenuti da un apparato sperimentale implementato in laboratorio e testato su alcuni campioni. La tecnica OCT permette di ottenere immagini a due o tre dimensioni di tessuti biologici e non solo, attraverso l'analisi della luce retrodiffusa dal campion

High-throughput live imaging using Light Sheet Microscopy

© 2020 IEEE. Personal use of this material is permitted. Permission from IEEE must be obtained for all other uses, in any current or future media, including reprinting/republishing this material for advertising or promotional purposes,creating new collective works, for resale or redistribution to servers or lists, or reuse of any copyrighted component of this work in other works.We have developed a compact, multi-modal lightsheet microscope platform where different kind of experiments may be addressed with a single equipment. We will evaluate the performance of the Flexi-SPIM platform and its ability to carry out high-throughput quantitative analysis with high resolution, high speed and minimal photo-damage on complex 3D cell cultures and the microtubule network during zebrafish development.EJG is supported by MINECO/FEDER projects (SEV-2015-0522 and RYC-2015-17935). MB is supported by Marie Skłodowska-Curie ITN ImageInLife programmePeer ReviewedPostprint (author's final draft