3D Volume rendering of invertebrates using light-sheet fluorescence microscopy

Light-Sheet Fluorescence Microscopy has recently emerged as the technique of choice for obtaining high quality three-dimensional (3D) images of whole organisms, with low photo-damage and fast acquisition rates. Unlike conventional optical and confocal microscopy or scanning electron microscopy systems, it offers the possibility of obtaining multiple views of the sample by rotating it. We show that the use of light-sheet fluorescence microscopy, for the analysis of invertebrates, provides a fair compromise compared to scanning electron microscopy in terms of resolution, but avoids some of its drawbacks, such as sample preparation or limited three-dimensional perspectives. In this paper, we will show how LSFM techniques can provide a cheap, high quality, multicolor, 3D alternative to classic microscopes, for the study of the morphological structure of insects and invertebrates in morphogenesis studies of the whole animal.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

Development of microscopic techniques for the visualization of plant–root-knot nematode interaction

Plant-parasitic nematodes are a significant cause of yield losses and food security issues. Specifically, nematodes of the genus Meloidogyne can cause significant production losses in horticultural crops around the world. Understanding the mechanisms of the ever-changing physiology of plant roots by imaging the galls induced by nematodes could provide a great insight into their control. However, infected roots are unsuitable for light microscopy investigation due to the opacity of plant tissues. Thus, samples must be cleared to visualize the interior of whole plants in order to make them transparent using clearing agents. This work aims to identify which clearing protocol and microscopy system is the most appropriate to obtain 3D images of tomato cv. Durinta and eggplant cv. Cristal samples infected with Meloidogyne incognita to visualize and study the root–nematode interaction. To that extent, two clearing solutions (BABB and ECi), combined with three different dehydration solvents (ethanol, methanol and 1-propanol), are tested. In addition, the advantages and disadvantages of alternative imaging techniques to confocal microscopy are analyzed by employing an experimental custom-made setup that combines two microscopic techniques, light sheet fluorescence microscopy and optical projection tomography, on a single instrument.Postprint (published version

Autofluorescence of stingray skeletal cartilage: hyperspectral imaging as a tool for histological characterization

Tessellated cartilage is a distinctive composite tissue forming the bulk of the skeleton of cartilaginous fishes (e.g. sharks and rays), built from unmineralized cartilage covered at the surface by a thin layer of mineralized tiles called tesserae. The finescale structure and composition of elasmobranch tessellated cartilage has largely been investigated with electron microscopy, micro-computed tomography and histology, but many aspects of tissue structure and composition remain uncharacterized. In our study, we demonstrate that the tessellated cartilage of a stingray exhibits a strong and diverse autofluorescence, a native property of the tissue which can be harnessed as an effective label-free imaging technique. The autofluorescence signal was excited using a broad range of wavelengths in confocal and light sheet microscopy, comparing several sample preparations (fresh; demineralized and paraffin-embedded; non-demineralized and plastic-embedded) and imaging the tissue at different scales. Autofluorescence varied with sample preparation with the signal in both plastic- and paraffin-embedded samples strong enough to allow visualization of finescale (=¿1 µm) cellular and matrix structures, such as cell nuclei and current and former mineralization fronts, identifiable by globular mineralized tissue. A defined pericellular matrix (PCM) surrounding chondrocytes was also discernible, described here for the first time in elasmobranchs. The presence of a PCM suggests similarities with mammalian cartilage regarding how chondrocytes interact with their environment, the PCM in mammals acting as a transducer for biomechanical and biochemical signals. A posterior analysis of hyperspectral images by an MCR-ALS unmixing algorithm allowed identification of several distinct fluorescence signatures associated to specific regions in the tissue. Some fluorescence signatures identified could be correlated with collagen type II, the most abundant structural molecule of cartilage. Other fluorescence signatures, however, remained unidentified, spotlighting tissue regions that deserve deeper characterization and suggesting the presence of molecules still unidentified in elasmobranch skeletal cartilage. Our results show that autofluorescence can be a powerful exploratory imaging tool for characterizing less-studied skeletal tissues, such as tessellated cartilage. The images obtained are largely comparable with more commonly used techniques, but without the need for complicated sample preparations or external staining reagents standard in histology and electron microscopy (TEM, SEM).Postprint (published version

Modeling Biochemical Gradients In Vitro to Control Cell Compartmentalization in a Microengineered 3D Model of the Intestinal Epithelium

Gradients of signaling pathways within the intestinal stem cell (ISC) niche are instrumental for cellular compartmentalization and tissue function, yet how are they sensed by the epithelium is still not fully understood. Here a new in vitro model of the small intestine based on primary epithelial cells (i), apically accessible (ii), with native tissue mechanical properties and controlled mesh size (iii), 3D villus-like architecture (iv), and precisely controlled biomolecular gradients of the ISC niche (v) is presented. Biochemical gradients are formed through hydrogel-based scaffolds by free diffusion from a source to a sink chamber. To confirm the establishment of spatiotemporally controlled gradients, light-sheet fluorescence microscopy and in-silico modeling are employed. The ISC niche biochemical gradients coming from the stroma and applied along the villus axis lead to the in vivo-like compartmentalization of the proliferative and differentiated cells, while changing the composition and concentration of the biochemical factors affects the cellular organization along the villus axis. This novel 3D in vitro intestinal model derived from organoids recapitulates both the villus-like architecture and the gradients of ISC biochemical factors, thus opening the possibility to study in vitro the nature of such gradients and the resulting cellular response.© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH

BIOLOGIA GENERAL: BiE Plantas: Módulo VI

2022/20231r quadrimestr

3D volume rendering of invertebrates using light-sheet fluorescence microscopy

Light-Sheet Fluorescence Microscopy (LSFM) has recently emerged as the technique of choice for obtaining high quality 3D images of whole organisms with low photo-damage and fast acquisition rates. Unlike conventional optical microscopy or scanning electron microscopy systems it offers the possibility of obtaining multi-views of the sample by rotating it. Here we show that the use of light sheet microscopy for the analysis of invertebrates provides a fair compromise compared to scanning electron microscopy (SEM) in terms of resolution but avoiding some its drawbacks such as sample preparation or limited three-dimensional perspectivesFundação para a Ciência e Tecnologia, grant SFRH/BD/80717/2011Postprint (published version