An automated workflow for parallel processing of large multiview SPIM recordings

Multiview light sheet fluorescence microscopy (LSFM) allows to image developing organisms in 3D at unprecedented temporal resolution over long periods of time. The resulting massive amounts of raw image data requires extensive processing interactively via dedicated graphical user interface (GUI) applications. The consecutive processing steps can be easily automated and the individual time points can be processed independently, which lends itself to trivial parallelization on a high performance cluster (HPC). Here we introduce an automated workflow for processing large multiview, multi-channel, multi-illumination time-lapse LSFM data on a single workstation or in parallel on a HPC. The pipeline relies on snakemake to resolve dependencies among consecutive processing steps and can be easily adapted to any cluster environment for processing LSFM data in a fraction of the time required to collect it.Comment: 13 pages with supplement, LATEX; 1 table, 1 figure, 2 supplementary figures, 2 supplementary lists, 2 supplementary tables; corrected error in results table, results unchange

Light-sheet microscopy for everyone? Experience of building an OpenSPIM to study flatworm development.

Background: Selective plane illumination microscopy (SPIM a type of light-sheet microscopy) involves focusing a thin sheet of laser light through a specimen at right angles to the objective lens. As only the thin section of the specimen at the focal plane of the lens is illuminated, out of focus light is naturally absent and toxicity due to light (phototoxicity) is greatly reduced enabling longer term live imaging. OpenSPIM is an open access platform (Pitrone et al. 2013 and OpenSPIM.org) created to give new users step-by-step instructions on building a basic configuration of a SPIM microscope, which can in principle be adapted and upgraded to each laboratory’s own requirements and budget. Here we describe our own experience with the process of designing, building, configuring and using an OpenSPIM for our research into the early development of the polyclad flatworm Maritigrella crozieri – a non-model animal. Results: Our OpenSPIM builds on the standard design with the addition of two colour laser illumination for simultaneous detection of two probes/molecules and dual sided illumination, which provides more even signal intensity across a specimen. Our OpenSPIM provides high resolution 3d images and time lapse recordings, and we demonstrate the use of two colour lasers and the benefits of two color dual-sided imaging. We used our microscope to study the development of the embryo of the polyclad flatworm M. crozieri. The capabilities of our microscope are demonstrated by our ability to record the stereotypical spiral cleavage pattern of M. crozieri with high-speed multi-view time lapse imaging. 3D and 4D (3D + time) reconstruction of early development from these data is possible using image registration and deconvolution tools provided as part of the open source Fiji platform. We discuss our findings on the pros and cons of a self built microscope. Conclusions: We conclude that home-built microscopes, such as an OpenSPIM, together with the available open source software, such as MicroManager and Fiji, make SPIM accessible to anyone interested in having continuous access to their own light-sheet microscope. However, building an OpenSPIM is not without challenges and an open access microscope is a worthwhile, if significant, investment of time and money. Multi-view 4D microscopy is more challenging than we had expected. We hope that our experience gained during this project will help future OpenSPIM users with similar ambitions

Molecular, Cellular and Mechanical basis of Epithelial Morphogenesis during Tribolium Embryogenesis

Embryonic development entails a series of morphogenetic events which require a precise coordination of molecular mechanisms coupled with cellular dynamics. Phyla such as arthropods show morphological and gene expression similarities during middle embryogenesis (at the phylotypic germband stage), yet early embryogenesis adopts diverse developmental strategies. In an effort towards understanding patterns of conservation and divergence during development, investigations are required beyond the traditional model systems. Therefore, in the past three decades, several insect species representing various insect orders have been established as experimental model systems for comparative developmental studies. Among these, the red flour beetle Tribolium castaneum has emerged as the best studied holometabolous insect model after the fruit fly Drosophila melanogaster. Unlike Drosophila, Tribolium is a short-germ insect that retains many ancestral characters common to most insects. The early embryogenesis of Tribolium shows dynamic epithelial rearrangements with an epibolic expansion of the extraembryonic tissue serosa over the embryo, the folding of the embryo in between the serosa and the second extra embryonic tissue amnion and the folding of the amnion underneath the embryo. These extensive tissues are evolutionarily conserved epithelia that undergo different tissue movements and are present in varying proportions in different insects, providing exceptional material to compare and contrast morphogenesis during early embryogenesis. However, most of the previous work on insects including Tribolium have largely focused on the conservation and divergence of gene expression patterns and on gene regulatory interactions. Consequently, very little studies on dynamic cell behaviour have been done and we lack detailed information about the cellular and tissue dynamics during these early morphogenetic events. During my PhD, I first established a live imaging and data analysis pipeline for studying Tribolium embryogenesis in 4-D. I combined live confocal and lightsheet imaging of transgenic or transiently labelled embryos with mechanical or genetic perturbations using laser ablations and gene knockdowns. Using this pipeline quantifications of cell dynamics and tissue behaviours can be done to compare different regions of the embryo as the development proceeds. In the second and third part of my thesis, I describe the actomyosin dynamics and associated cell behaviours during the stages of serosa epibolic expansion, amniotic fold formation and serosa window closure. I cloned and characterised the cellular dynamics of the Tribolium spaghetti squash gene (Tc-squash) - the non-muscle Myosin II regulatory light chain, which is the main molecular force generator in epithelial cells. Interestingly, the analysis of Tc-squash dynamics indicates a conserved role of Myosin II in controlling similar cell behaviours across short germ and long germ embryos. In the last part of the thesis, I report the dynamics of an actomyosin cable that emerges at the interface of the serosa and amnion. This cable increases in tension during development, concomitant with serosa tissue expansion and increased tensions in the serosa. It behaves as a modified purse string as it’s circumference shrinks due to a decrease in the number of cable forming cells over time. This shrinkage is an individual contractile property of the cells forming the cable. This indicates that a supracellular and contractile actomyosin cable might be functional during serosa window closure in insects with distinct serosa and amnion tissues. Further, the tension in the cable might depend on the relative proportion of the serosa, amnion and embryonic regions. Using these integrated approaches, I have correlated global cellular dynamics during early embryogenesis with actomyosin behaviours, and then performed a high-resolution analysis and perturbations of selected events. The established imaging, image processing and perturbation tools can serve as an important basis for future investigations into the tissue mechanics underlying Tribolium embryogenesis and can also be adapted for comparisons of morphogenesis in other insect embryos. More broadly, correlating the existing genetic, mechanical and biochemical understanding of developmental processes from Drosophila with species such as Tribolium, could help identify deeply conserved design principles that lead to different morphologies through differences in underlying regulation.:Page List of Tables v List of Figures vii 1 Introduction 1 1.1 Evo-Devo of insects 3 1.2 Tribolium castaneum 5 1.3 Fluorescence live imaging and lightsheet microscopy 10 1.4 Morphogenesis 15 1.5 Thesis objective 29 2 4D lightsheet imaging and analysis pipeline of Tribolium embryos 33 2.1 Standardisation of an injection protocol for sample mounting and imaging with the Zeiss LZ1 SPIM 35 2.2 Double labelling of Tribolium embryos 37 2.3 Image processing with Fiji 37 2.4 Long term timelapse imaging of Tribolium embryogenesis with SPIM 44 2.5 2D cartographic projections of 3D data as a method to visualise and analyse SPIM data 47 2.6 Summary 59 3 Cellular dynamics of the non muscle Myosin II regulatory light chain - Tc-Squash 61 3.1 Tc-Squash dynamics during Tribolium embryogenesis 64 3.2 Myosin drives basal cell closure during blastoderm cellularisation 66 3.3 Myosin shows planar polarity in the embryonic tissue 69 3.4 Myosin accumulation and apical constriction of putative germ cells at the posterior pole 71 3.5 Myosin pulses during apical constriction of mesoderm cells 74 3.6 Myosin accumulates at the extraembryonic-embryonic boundary to form a contractile supracellular cable 77 3.7 Summary 77 4 A supracellular actomyosin cable operates during serosa epiboly 79 4.1 Actin and Myosin accumulate at the extraembryonic-embryonic boundary 81 4.2 The actomyosin assembly migrates ventrally till it forms the rim of the serosa window 82 4.3 The actomyosin cable shows dynamic shape changes during serosa window closure 87 4.4 Serosa cells increase in area till circular serosa window stage 89 4.5 Tension in the serosa tissue increases during epibolic expansion 89 4.6 Serosa cells decrease their apical areas after laser ablation 92 4.7 Tension in the actomyosin cable increases during serosa epiboly 93 4.8 Myosin dynamics at the cable changes between early and serosa window stage 96 4.9 Individual cell membrane shrinkage and cell rearrangements decrease the cable circumference 98 4.10 Myosin dynamics at the cable during serosa window closure 101 4.11 Tension in the cable is not relieved after multiple laser cuts 103 4.12 Analysis of the actomyosin cable in Tc-zen 1 knockdown 105 4.13 Summary 109 5 Discussion 111 5.1 Reconstruction of insect embryogenesis using lightsheet microscopy and tissue cartography 111 5.2 Conserved Myosin II behaviours and its implications on morphogenesis across insects 114 5.3 A contractile supracellular actomyosin cable functions serosa window closure in Tribolium 119 6 Materials and Methods 123 6.1 Tribolium stock maintenance 123 6.2 RNA extraction and cDNA synthesis 124 6.3 Cloning of templates for mRNA synthesis and transgenesis 124 6.4 dsRNA synthesis for RNAi experiments 126 6.5 Capped, single stranded RNA synthesis 126 6.6 Fluorescence image acquisition 27 A Appendix 131 Bibliography 14

BigStitcher: reconstructing high-resolution image datasets of cleared and expanded samples.

Light-sheet imaging of cleared and expanded samples creates terabyte-sized datasets that consist of many unaligned three-dimensional image tiles, which must be reconstructed before analysis. We developed the BigStitcher software to address this challenge. BigStitcher enables interactive visualization, fast and precise alignment, spatially resolved quality estimation, real-time fusion and deconvolution of dual-illumination, multitile, multiview datasets. The software also compensates for optical effects, thereby improving accuracy and enabling subsequent biological analysis

Automated processing of zebrafish imaging data: a survey

Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines

Bioinformatická analýza komplexních multidimensionálních mikroskopických dat

Microscopy is embedded in the history of life sciences and vice versa. Recent advances in the field present new challenges as new revolutionary technologies arise. Sample prepa- ration, microscope operation and data analysis have become particularly demanding re- quiring specific interdisciplinary expertise. Bioimaging data analysis is computationally demanding, as microscopy technologies can easily acquire data of exceptional size, often in terabytes. Correct analysis requires computer vision knowledge, as well as knowledge of studied biological systems and last, but not least deep understanding of microscopy technology. Tools available for the analysis of the imaging data vary from open-source customizable software with a coverage of multiple tasks to a task specific proprietary software. To choose the best tools for the analysis, analysts should know their options and tasks at hand. In bioimage analysis the tasks needed to be employed depend on the desired outcome and the acquisition technology. Amongst the possible tasks to con- sider belong deconvolution, segmentation and registration. Amount of approaches and algorithms available is progressively growing, resulting in a complex field, difficult to be easily familiar with. My thesis covers different microscopy technologies with emphasis on...Mikroskopia je odbor, ktorý je súčasťou biologických vied a ich histórie. Rast tohto odboru, vznik nových revolučných technológií, so sebou prináša nové výzvy. Príprava vzoriek, obsluha mikroskopu a spracovanie biologických obrazových dát sa stávajú ne- ustále náročnejšími a vyžadujú medzidisciplinárnu expertízu špecifickú pre dané oblasti. Dnešné mikroskopické technológie generujú obrovské množstvo dát, často v terabajtoch, vďaka čomu sa analýza biologických obrazových dát stáva výpočtovo náročná. K analýze je potrebná znalosť počítačového videnia, mikroskopických technológií a skúmaných bio- logických systémov. K dispozícii sú nástroje v rôznorodej škále, od open-source riešení, ktoré pokrývajú veľké množstvo úkonov, k proprietárnym softwarom zameraným na je- den úkon. Analytici by sa mali orientovať v možnostiach spracovania a v úkonoch, ktoré plánujú vykonať, aby vybrali najlepšie nástroje. Úkony potrebné vykonať závisia od po- žadovaného výsledku a technológie, ktorá bola použitá na získanie obrazových dát. Medzi možné úkony patrí dekonvolúcia, segmentácia a registrácia dát. Neustále vznikajú nové prístupy a algoritmy na analýzu, vďaka čomu je analýza obrazových dát zložitá. Táto práca sa zaoberá rôznymi mikroskopickými technológiami so zameraním na light sheet fluorescence mikroskopiu,...Katedra buněčné biologieDepartment of Cell BiologyFaculty of SciencePřírodovědecká fakult

Comparative assessment of induced abnormal mitotic events by high-throughput light sheet imaging and image analysis

In recent years, three-dimensional (3D) in vitro cell culture models such as spheroids and organoids have revolutionized life science research by providing a much more reliable context resembling the in vivo microenvironment. These systems yield important cell-to-cell interactions and induce cell differentiation. However, no conventional microscopy setup can provide sufficient imaging throughput as well as spatial and temporal resolution to enable full 3D live imaging and analysis down to subcellular processes. In this project, we established state-of-the-art light sheet microscopy for live, long-term imaging of a short interfering ribonucleic acid (siRNA) treated 3D cell culture model. Due to the high temporal and special resolution of the light sheet microscope, we minimized imaging artifacts and achieved unprecedented visual representations of spheroids throughout development and upon gene knock-down by siRNAs. Furthermore, we deployed a high-throughput image analysis pipeline and machine learning classification to evaluate global, cellular and subcellular features for a precise, quantitative gene knock-down phenotype description. The RNA interference (RNAi) induced gene knock-down phenotypes were replicated and compared by a novel molecular, site-specificepigenome modifying method. Throughout this project, we carefully evaluated every step of the workflow to improved its throughput and increased its reproducibility and usability. We addressed the key challenges in light sheet microscopy, such as sample preparation, data handling, image processing and analysis, thereby establishing quantitative light sheet microscopy screening of 3D cell culture models for many research applications. In total, we believe that our workflow can provide the basis for high-content analysis of 3D cell culture models for future research, enabling much more detailed functional experiments and basic research studies

Pushing Light-Sheet Microscopy to Greater Depths

Light-sheet fluorescence microscopy (LSFM) has established itself as an irreplaceable imaging technique in developmental biology over the past two decades. With its emergence, the extended recording of in toto datasets of developing organisms across scales became possible. Remarkably, LSFM opened the door to new spatio-temporal domains in biology, offering cellular resolution on the one hand, and temporal resolution on the order of seconds on the other hand. As in any fluorescence microscopy technique, LSFM is also affected by image degradation at greater tissue depths. Thus far, this has been addressed by the suppression of scattered light, use of fluorophores emitting in the far red spectrum, multi-view detection and fusion, adaptive optics, as well as different illumination schemes. In this work, I investigate for the first time in vivo optical aberration reduction via refractive index matching in LSFM. Examples are shown on common model organisms as Arabidopsis thalina, Oryzias latipes, Mus musculus, as well as Drosophila. Additionally, I present a novel open-top light-sheet microscope, tailored for high-throughput imaging of mammalian samples, such as early stage mouse embryos. It is based on a three objective geometry, encompassing two opposing detection objective lenses with high light collection efficiency, and an invertedly mounted illumination lens. It bridges the spatial scale between samples by employing an extendible light-sheet illumination via a tunable acoustic gradient index lens. Both parts of this work improve the image quality across the 3D volume of specimens, paving the way for more quantitative recordings at greater tissue depths

Development of optical methods for real-time whole-brain functional imaging of zebrafish neuronal activity

Each one of us in his life has, at least once, smelled the scent of roses, read one canto of Dante’s Commedia or listened to the sound of the sea from a shell. All of this is possible thanks to the astonishing capabilities of an organ, such as the brain, that allows us to collect and organize perceptions coming from sensory organs and to produce behavioural responses accordingly. Studying an operating brain in a non-invasive way is extremely difficult in mammals, and particularly in humans. In the last decade, a small teleost fish, zebrafish (Danio rerio), has been making its way into the field of neurosciences. The brain of a larval zebrafish is made up of 'only' 100000 neurons and it’s completely transparent, making it possible to optically access it. Here, taking advantage of the best of currently available technology, we devised optical solutions to investigate the dynamics of neuronal activity throughout the entire brain of zebrafish larvae

Recent advances in optical tomography in low scattering media

Low scattering media is the best scenario for optical imaging in thick samples and deep tissue, as it allows to obtain high resolution images without suffering the limitations that the diffusion phenomenon imposes. The high contribution of ballistic light in this regime enabled the development of light sheet microscopy and optical projection tomography, two of the most common techniques nowadays in research laboratories. Their revolutionary approach and wide spectrum of applications and possibilities has lead to a frenetic rhythm of new works and techniques arising every year. The large amount of information available often overwhelms scientists and researchers trying to keep up to date with the last cutting edge advances of the field. This paper aims to give a brief review of the origins and fundamental aspects of these two techniques to focus on the most recent and yet non reviewed works. Apart from novel methods, this document also covers combined multimodal approaches and systems. To conclude, we put a spotlight on the important role that open-source microscopy systems play in the field, as they improve the accessibility to these techniques and promote collaborative networks across the optical imaging community