10th Anniversary Light Sheet Fluorescence Microscopy Conference

Name: 10th Anniversary Light Sheet Fluorescence Microscopy Conference
Start: 2018-08-12T16:00:00+02:00
End: 2018-08-15T15:00:00+02:00
Location: MPI-CBG

12 Aug 2018, 16:00 → 15 Aug 2018, 15:00 Europe/Berlin

MPI-CBG

Pfotenhauerstraße 108 01307 Dresden Germany

Pavel Tomancak, Jan Peychl

Description

Light sheet microscopy has emerged in the last 15 years as a powerful tool to interrogate living biological systems with unprecedented imaging speeds for as long as it takes. Large, fixed, cleared or expanded biological samples can be imaged with high resolution in a fraction of time compared to conventional microscopy approaches. When combined, the spatial coverage and temporal resolution leads to in toto recording of complex biological phenomena.

Light sheets opened entirely new windows into the inner workings of cells and organisms and re-invigorated several research fields including developmental and cell biology, physiology, neurobiology and biodiversity research. This conference marks the 10th Anniversary of Light Sheet Fluorescence Microscopy (LSFM) meetings that started in 2009 in Dresden. It will bring together the technology developers and users of light sheets, showcase the latest light sheet set-ups and how to best deploy them for the benefit of the biological research community.

A unique feature of the conference is that it immediately follows the 3rd EMBO practical course on light sheet microscopy held in Dresden between August 2nd and 11th 2018. This course teaches light sheet microscopy to the next generation of scientists who will present the results of their light sheet experiments at the conference. Additionally, a full range of commercial light sheet microscopy hardware used at the course will be demonstrated at the conference.

Sandy Schneider & Christina Kuß

lsconference@mpi-cbg.de

+49 351 210-2756/2805

Sunday, 12 August
- 16:00 → 18:00
  
  Special session: Registration
- 18:00 → 18:30
  
  Special session: Welcome and retrospect
- 18:30 → 21:30
  
  Dinner at CBG 3h
Monday, 13 August
- 09:00 → 10:30
  Light sheet hardware 1
  - 09:00
    
    Resolution and applications of LSFM in the life sciences 15m
    
    Speaker: Prof. Ernst Stelzer
  - 09:15
    
    Applications and extensions of SCAPE microscopy 15m
    
    Speaker: Elizabeth Hillman (Columbia University)
  - 09:30
    
    Adding Dimensions to Light Sheet Imaging 15m
    
    Speaker: Prof. Scott Fraser
  - 09:45
    
    Dual illumination inverted light sheet microscope for long term live imaging 15m
    
    Organoid cultures have been recently established to study organ formation and tissue morphogenesis. However, imaging of these samples has been hampered by their long and often inefficient development and their light sensitivity. Light sheet microscopy would be the imaging technique of choice due to its low photo-toxicity and high acquisition speed. However, many current light sheet microscopes suffer from complicated sample mounting that also limits sample survival (e.g. mounting in FEP tube or agarose embedding) and lack of multi-position imaging. To overcome these limitations we have built an inverted light sheet microscope system with two illumination objectives, one high NA imaging objective and a beam scanning and alignment module. The sample is easily accessible and completely isolated from immersion medium and multiple samples can be imaged in parallel. Using this microscope we were able to acquire a continuous five-day long time-lapse capturing the formation of fully grown intestinal organoid starting from a single stem cell embedded in a matrigel. By imaging the stem cell marker (LGR5-GFP) we could follow for the first time the dynamics of stem cells during a complete intestinal organoid development at single cell resolution. Moreover, we demonstrated the versatility of this microscope by imaging different organoid models and the development of several living samples across different scales (e.g. zebrafish, mouse embryo and C. elegans).
    
    Speaker: Dr Petr Strnad (Ecole polytechnique federale de Lausanne)
  - 10:00
    
    The mesoSPIM initiative – open-source light-sheet microscopes for imaging in cleared tissue 15m
    
    Tissue clearing methods have recently seen a renaissance with a wide variety of clearing approaches now available. In neuroscience, the combination of tissue clearing with light-sheet microscopy is ideal to bridge scales from the µm to cm-level, thus providing a link on the mesoscale for detailed 3D anatomical investigations. To optimally image cleared samples, we set out to design a modular light-sheet microscope that combines extremely simple sample mounting and exchange with large field-of-views (FOV) of 2-22 mm to provide users with overview datasets within minutes. Especially for such large FOVs, common light-sheet microscopes suffer from non-uniform axial resolution due to the varying thickness of the light-sheet. To circumvent this problem, we are using tuneable lenses to shift the excitation beam waist through the sample in synchrony with the rolling shutter of the camera. For whole mouse brains, typical datasets are isotropic (5 µm sampling), small (12-16 GB), and generated quickly (7-8 minutes). Together with standardized quick-exchange sample holders, these features allow fast screening of samples for clearing, imaging, and labelling quality and thus speed up data acquisition considerably.
    After creating overview datasets, users can zoom in and acquire high-resolution data.
    The microscope has been tested and validated in combination with common clearing methods ranging from hydrogel-based techniques such as CLARITY to organic solvent approaches such as iDISCO – by using a modular design of the imaging chambers, switching between different imaging media can be done in less than a minute. Recently, we have realized four such microscopes at various institutions across Switzerland as part of the mesoSPIM initiative (mesospim.org) – a project aimed at creating a community to accelerate the exchange of tissue clearing and mesoscale imaging expertise. Microscope hard- and software are open-source and we welcome suggestions for improvements.
    
    Speaker: Fabian Voigt (Brain Research Institute, University of Zurich)
  - 10:15
    
    Single- and multi-photon shaped illumination for light-sheet fluorescence microscopy 15m
    
    The use of exotic optical modes is becoming increasingly widespread in microscopy. Particularly, propagation-invariant beams, such as Airy and Bessel beams and optical lattices, have been particularly useful in light-sheet fluorescence microscopy (LSFM) as they enable high-resolution imaging over a large field-of-view (FOV), possess a resistance to the deleterious effects of specimen induced light scattering, and can potentially reduce photo-toxicity (e.g. [1]).
    
    Although these propagation-invariant beams can resist the effects of light scattering to some degree, and there has been some interest in adaptive-optical methods to correct for beam aberrations when they cannot, scattering and absorption of the illuminating light-sheet limit the penetration of LSFM into tissues and results in non-uniform intensity across the FOV.
    
    A new degree of control over the intensity evolution of propagation-invariant beams can overcome beam losses across the FOV, restoring uniform illumination intensity and therefore image quality. This concept is compatible with all types of propagation-invariant beams and is characterised in the context of light-sheet image quality [2].
    
    Another property to control is the wavelength of light used. Optical transmission through tissue is greatly improved at longer wavelengths into the near-infrared due to reduced Rayleigh scattering and two-photon excitation has proved beneficial for imaging at greater depth in LSFM. Three-photon excitation has already been demonstrated as a powerful tool to increase tissue penetration in deep brain confocal microscopy, and when combined with beam shaping can also be a powerful illumination strategy for LSFM [3].
    
    Recent progress in shaping optical fields for LSFM will be presented.
    
    [1] T. Vettenburg et al, Nat. Methods 11, 541-544 (2014), doi:10.1038/nmeth.2922
    [2] J. Nylk et al, Sci. Adv. 4, eaar4817 (2018), doi:10.1126/sciadv.aar4817
    [3] A. Escobet-Montalbán et al, bioRxiv 323790 (2018), doi: 10.1101/323790
    
    Speaker: Dr Jonathan Nylk (University of St Andrews)
- 10:30 → 11:15
  
  Coffee break 45m
- 11:15 → 12:45
  Light sheet hardware 2
  - 11:15
    
    Flamingos for you and me 15m
    
    Speaker: Dr Jan Huisken (Morgridge Institute for Research)
  - 11:30
    
    Lightwedge and Lightsheet-Raman Microscopy 15m
    
    Speaker: Prof. Rainer Heintzmann
  - 11:45
    
    Light sheet microscopy in Nature Methods 15m
    
    Speaker: Rita Strack (Nature Methods)
  - 12:00
    
    Fast nearly isotropic imaging of large samples with light sheets beyond the diffraction limit 15m
    
    We were able to generate extremely long thin sheets of light in the one micron range and a vastly increased Rayleigh length by breaking the diffraction limit of light sheets of low numerical aperture. We measured the thickness of the light sheets with different methods including standard point spread function measurement with fluorescent beads. By using these light sheets in our ultramicroscope fast 3D imaging of whole mouse brains with objectives with a large field of view was possible. Due to the extremely low divergence of the light sheets mouse brains could be reconstructed from a single stack of optical sections with nearly isotropic resolution. The light sheets used were essentially non-Gaussian generated by new optics we developed. Compared to a Gaussian light sheet of the same NA our new light sheet is much thinner. Thus the diffraction limit which holds also for low NA Gaussian light sheets was significantly surpassed. These optics will allow the application of ultramicroscopy to ever increasing samples beyond the whole mouse brain range including human cancers.
    Besides mouse brains we imaged also cleared whole adult drosophilae. We were able to get good transparency for all developmental stages of the insect from larvae to adult animals with fully preserved GFP signal. We showed that also dualview imaging of cleared adult drosophilae is possible and and allows easy isotropic resolution also with standard light sheet microscopy for such kind of specimens.
    .
    
    Speaker: Prof. Hans-Ulrich Dodt (TU Wien)
  - 12:15
    
    Focused-light-induced cytoplasmic streaming (FLUCS). A new paradigm to probe the physiology on intracellular transport. 15m
    
    Throughout the last decades, access to genetic perturbations, fluorescent labels and modern microscopy advanced our molecular understanding of cell-biological processes tremendously.
    
    The spatio-temporal organization of cells and developing embryos that we observe under these microscopes is widely believed to depend on physical processes such as diffusion and motor-driven intracellular flows. Thus far, however, it remains a challenge to unravel physiology of these physical transport processes, which is due to the lack of suitable perturbation methods.
    
    Here, we exploit thermoviscous expansion phenomena to optically induce hydrodynamic flow in single cells and developing embryos. By controlling such flows inside the cytoplasm of the C. elegans zygote, we reveal the causal implications of intracellular flows during PAR polarization. Specifically, we show i) that hydrodynamic flows inside the cytoplasm localize PAR-2 proteins at the posterior membrane. ii) Induced cortical flows transported membrane-bound PAR molecules and rotated the membrane polarization, leading to iii) the down-stream phenotype of an inverted body axis.
    
    Furthermore, we utilize flow perturbations for probe-free active micro-rheology of the cytoplasm and within subcellular compartments. We conclude by emphasizing the opportunities and challenges of combining FLUCS with light-sheet-microscopy.
    
    Mittasch et al., Nat Cell Biol 20 (2018)
    Kruse, Chiaruttini, and Roux, Nat Cell Biol 20 (2018)
    
    Speaker: Moritz Kreysing
  - 12:30
    
    12 years with Light Sheet Microscopy in core imaging facilities on Dresden campus 15m
    
    Light Sheet Microscopy made great progress during last 15 years. We can now see its transition from the hands of developers to the hands of ''ordinary'' scientists who work in the field of biomedical research. Core imaging facilities can, in our opinion, play great role in supporting such transition. In our talk we will share the lessons which we learned during last 12 years when we have been bringing light sheet microscopy technology to users of core imaging facilities on Dresden campus. We will reflect the perspective of core imaging facilities who provide open access to broad range of imaging technologies to hundreds of users per year.
    
    Our light sheet story started in 2006, when the first Zeiss light sheet ''concept'' system got installed in the the multi user environment of the core Light Microscopy Facility (LMF) of the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden (Germany). And then our journey continued with the first Z.1 system (Zeiss) which was installed in January 2013. This system was used around 9.500 hours during first two years of its operation. Facing the capacity limits of this system, we installed second Z.1.system in March 2015 (modified also for imaging of cleared samples). Total usage of both Z.1 systems from January 2015 till June 2018 reached circa 15 300 hours reflecting popularity of this technology among our users.
    
    Two Z.1. setups at the MPI-CBG got complemented by the Ultramicroscope setup (LaVision Biotech Germany) which was installed at the core imaging facility of our partner institute on Dresden campus – Center of Molecular and Cellular Bioengineering (CMCB) in 2014. In 2017 the MPI-CBG established new Advanced Imaging Facility which provides users with an access to home-built lattice light sheet setup. All four light sheet setups are now available (together with other imaging technologies) via Biopolis Dresden Imaging Platform (www.biodip.de) to more then 600 users on Dresden campus and beyond.
    
    Speaker: Dr Jan Peychl (Head, Light Microscopy Facility, MPI-CBG, Dresden, Germany)
- 12:45 → 14:00
  
  Lunch at CBG canteen 1h 15m
- 14:00 → 15:00
  Techno bites: Techno bites 1
  - 14:00
    
    Zeiss 5m
  - 14:05
    
    Acquifer: Large Image Data Solutions by Acquifer 5m
  - 14:10
    
    Applied Scientific Instrumentation: ASI makes DIY light sheet easy 5m
  - 14:15
    
    ANDOR/Bitplane: Quantitative 3D and 4D image analysis for Light Sheet Microscopy in Imaris 5m
  - 14:20
    
    Hamamatsu: Extended Focus Device 5m
  - 14:25
    
    LaVision BioTec: Cleared tissue imaging 5m
  - 14:30
    
    Leica: SP8 DLS – Leica`s Lightsheet on a Confocal at a glance 5m
  - 14:35
    
    Luxendo: Luxendo Light-Sheet Microscopes: Seeing Life from a Different Angle 5m
  - 14:40
    
    PCO: To cool or not to cool? How cooling influences the performance of sCMOS-cameras 5m
  - 14:45
    
    PhaseView: Alpha3, a new generation of light sheet microscope 5m
  - 14:50
    
    Intelligent Imaging Innovations GmbH: Imaging Life with 3i LightSheet Systems 5m
- 15:00 → 17:00
  
  Company demos
- 15:00 → 17:00
  
  Posters: Posters I
- 17:00 → 18:30
  Image analysis of light sheet data
  - 17:00
    
    Different Rigs for Different Gigs 15m
    
    Speaker: Prof. Eugene W. Myers
  - 17:15
    
    Bioimaging information: combining light sheet imaging with single cell transcriptomics 15m
    
    Speaker: Dr Lars Hufnagel
  - 17:30
    
    Content-Aware Image Restoration and Quantitative Downstream Analysis 15m
    
    Speaker: Florian Jug
  - 17:45
    
    Towards next generation imaging pipelines using the Adaptive Particle Representation. 15m
    
    The Adaptive Particle Representation (APR) is an alternative image representation to pixel images for realizing the next generation of imaging pipelines using light-sheet fluorescence microscopy. The APR addresses computational, memory, and storage bottlenecks by adapting the image resolution to the local image content. Unlike standard image compression, the adaptive computational and memory benefits of the APR can be used for all processing and visualization tasks, without returning to the original full pixel image. Here we will present recent developments and extensions for the APR including GPU pipeline and processing acceleration, adaptation through time, and block-wise APR transforms for large images. Also, we will discuss current software support including, Python and Java wrappers for the C++ LibAPR library, and integration with Fiji and BigDataViewer.
    
    Speaker: Bevan Cheeseman
  - 18:00
    
    Light propagation in retinal tissue and in silico 15m
    
    Light penetration and formation of image inside biological tissues is of physiological relevance in the eye and practical importance in the context of tissue and whole organism microscopy. We present here how the vertebrate retina, having a counter intuitive inverted structure deals with incoming light. We show by direct transmission measurements how the unique chromatin arrangement within photoreceptor nuclei impacts image quality at the level of the photoreceptors. The experimental findings are complemented by wave optical simulations of forward scattering. The simulations from the anatomically faithful tissue models successfully predicts the loss of image contrast due to the large angle scattering occurring in the tissue and also provides a physical and mechanistic understanding of the image formation process.
    
    We further show, how these simulations can also be used to mimic the imaging process in tissue microscopy. Our open source software, biobeam [1] has the flexibility to implement multiple modalities ranging from laser scanning to light sheet fluorescence imaging. It can reproduce aberrations, distortions, adaptive optical effects and most intricate wave optical phenomena relevant to microscopy. With the multiplexed, GPU accelerated implementation of the in silico light propagation our software pushes the frontiers of computer model guided microcopy enabling highest resolution deep tissue imaging.
    
    [1] Weigert, Martin, Kaushikaram Subramanian, Sebastian T. Bundschuh, Eugene W. Myers, and Moritz Kreysing. "Biobeam—Multiplexed wave-optical simulations of light-sheet microscopy." PLoS computational biology 14, no. 4 (2018): e1006079.
    
    Speaker: Kaushikaram Subramanian
  - 18:15
    
    BigStitcher: Reconstructing high-resolution image datasets of cleared and expanded samples 15m
    
    Continuous advancements in microscopy and sample preparation methods such as clearing or expansion allow for the investigation of ever larger samples at high resolution. This entails increasingly large datasets that may consist of hundreds of images of one sample that are not aligned, suffer from optical disturbances and often cannot even be opened as a whole, which can pose a serious bottleneck to scientific inquiries. With terabyte-sized datasets becoming more and more common, development of software tools that make handling and analysis of large and complex image data available to the broader scientific community is an urgent issue.
    
    To allow efficient handling and reconstruction of large multi-tile and multi-view image data, we developed the BigStitcher software. It enables import from most file formats, interactive handling, fast and precise alignment, as well as deconvolution and real-time fusion of large image datasets. We additionally support the alignment of multi-tile acquisitions taken from different orientations, effectively doubling the size of objects that can be imaged. We also enable the correction of a variety of optical distortions, e.g. via automatic illumination selection, flat-field correction and interest-point-based correction of chromatic aberrations.
    
    We implemented BigStitcher using the state-of-the-art frameworks ImgLib2 and BigDataViewer. By combining multi-resolution data representation and sub-pixel accurate registration algorithms, even very large datasets can be reconstructed on conventional, off-the-shelf hardware. In an effort to make large sample reconstruction available as a routine task, we provide a user-friendly graphical user interface (GUI) to manually guide the alignment and interactively display the intermediate results using BigDataViewer. BigStitcher is open-source and provided as a Fiji-plugin, making it a powerful, scalable tool for automated processing of very large image datasets.
    
    Speaker: David Hörl (Department of Biology II, Ludwig-Maximilians-Universität München)
- 18:30 → 20:00
  
  Dinner at CBG 1h 30m
- 20:00 → 21:30
  
  Posters: Posters II
Tuesday, 14 August
- 09:00 → 10:30
  Sample preparation, clearing and expansion
  - 09:00
    
    Mastodon -- a large-scale track-editing framework for light sheet data 15m
    
    Light sheet microscopy allows live 3D imaging of entire developing embryos with
    high spatial and temporal resolution. Computational analysis of these recordings
    promises new insights in developmental biology. However, a single dataset often
    comprises many terabytes, which makes storage, processing, and visualization of
    the data a challenging problem. The open-source Fiji platform provides tools to
    address this challenge. In this talk, I will present Mastodon, a track-editing
    framework for cell tracking and lineage tracing in Fiji.
    
    Large-scale automated tracking in biological datasets is a very active field of
    research. To support machine learning methods, editing tools are needed to
    facilitate curation, proof-reading, and the manual generation of ground truth
    data. To make such tools accessible to biologist researchers, they should be
    easy to obtain, learn, and use. Additionally they must be intuitively usable and
    remain responsive in the face of millions of tracked objects and terabytes of
    image data. To make them useful for researchers in automated tracking, they need
    to be open source, adaptable, and extensible. Mastodon is our effort to provide
    such a tool in Fiji.
    
    Speaker: Tobias Pietzsch (MPI-CBG)
  - 09:15
    
    Cellular basis of limb morphogenesis: lessons from the crustacean Parhyale hawaiensis 15m
    
    During development coordinated cell behaviors orchestrate tissue and organ morphogenesis to suit the lifestyle of the organism. We have used here the crustacean Parhyale hawaiensis to study the cellular basis of limb development. Transgenic Parhyale embryos with fluorescently labeled nuclei were imaged at high spatiotemporal resolution using multi-view light-sheet fluorescence microscopy over several days of embryogenesis spanning appendage morphogenesis from early specification up to late differentiation stages. To be able to analyze the terabyte-sized data sets we used a new tool called Massive Multi-view Tracker (MaMuT) for cell tracking that enabled us to reconstruct the complete cell lineage of an outgrowing thoracic limb with single-cell resolution. The quantitative analyses about cell behaviors show that the limb primordium in Parhyale becomes subdivided from an early stage into anterior-posterior and dorsal-ventral compartments whose boundaries intersect at the distal tip of the growing limb. Limb bud formation is associated with the spatial modulation of cell proliferation, while limb elongation is also driven by the preferential orientation of division of epidermal cells along the proximal-distal axis of growth. Our findings validate the boundary model originally proposed by Hans Meinhardt. This model postulates that a secondary developmental field, i.e. the proximodistal axis of a limb that is specified during embryogenesis de novo relative to the main anteroposterior and dorsoventral body axes, and is initiated around the intersection of the anteroposterior and dorsoventral compartment boundaries.
    
    Speaker: Carsten Wolff (Humboldt University Berlin, Germany)
  - 09:30
    
    Whole-body/organ Cell Profiling by a Hydrophilic Tissue-clearing method, CUBIC 15m
    
    Speaker: Hiroki Ueda (The University of Tokyo/RIKEN (BDR))
  - 09:45
    
    High resolution and quantitative super-resolution microscopy: from cells to tissue imaging 15m
    
    Speaker: Francesca Cella Zanacchi
  - 10:00
    
    Attenuation Artifacts in LSFM Corrected by OPTiSPIM 15m
    
    Speaker: Jim Swoger (EMBL Barcelona)
  - 10:15
    
    Multi-sample SPIM image acquisition, processing and analysis of embryonic zebrafish vasculature 15m
    
    To quantitatively understand biological processes that occur over long time periods, it is desirable to image multiple samples simultaneously, and automatically process and analyze the resulting datasets. Here, we present a comprehensive and dedicated multi-sample image acquisition and processing workflow using selective plane illumination microscopy (SPIM) to image several embryos up to 4 days and demonstrate its value for understanding the formation of embryonic zebrafish vasculature.
    
    To process and analyze the large amount of data generated, we designed customized, automated and parallelized image processing tools in Fiji and FunImageJ. With a novel approach of vascular segmentation, a precise quantification of the vascular network’s growth over the first days of development was obtained. Further analysis of the imagery data revealed that parts of the vasculature showed different degrees of symmetry and variation. Moreover, analysis of calcium signaling suggested that variation on a macroscopic level was already established on a signaling level.
    
    Our multi-sample imaging pipeline further paves the way for many other quantitative long-term imaging studies such as xenotransplantation experiments or small-scale screens. It advocates a holistic approach based on multi-sample imaging using SPIM with integrated data processing and analysis to reveal and understand biological processes that occur over long time periods.
    
    Speaker: Dr Jan Huisken (Morgridge Institute for Research)
- 10:30 → 11:15
  
  Coffee break 45m
- 11:15 → 12:45
  Neurobiology applications of light sheet microscopy
  - 11:15
    
    Light sheet theta microscopy for quantitative imaging of large cleared samples. 15m
    
    Speaker: Dr Raju Tomer (Department of Biological Sciences, Columbia University)
  - 11:30
    
    Photoreceptors catching the light(sheet): Fate, movements and lamination 15m
    
    Speaker: Dr Caren Norden (Max Planck Institute of Molecular Cell Biology and Genetics)
  - 11:45
    
    A non-cell autonomous actin redistribution enables isotropic retinal growth 15m
    
    For many developing tissues, their shape is established early in development. In order to maintain this shape during subsequent growth, these tissues need to scale isotropically. The way by which cells inside tissues enable coordinated, isotropic tissue scaling is not understood, however, as most studies focused on changing, rather than maintaining tissue shapes during development. In this study, using light sheet fluorescence microscopy of both fixed and live samples, we follow tissue shape with cellular resolution in the zebrafish retinal neuroepithelium. This vertebrate neural progenitor tissue forms a smooth cup early in development and keeps its architecture as it grows. By combining 3D analysis and theory, we identify global cell elongation as a cellular mechanism to maintain retinal shape during growth. Timely cell height increase occurs concurrently with a non-cell autonomous actin redistribution, during which actin gets depleted from the lateral cell-cell interfaces. Blocking actin redistribution and cell height increase perturbs isotropic tissue scaling and we observe, using long light sheet timelapses, the emergence of the resulting disturbed, folded tissue shape. Taken together, from our whole-tissue imaging and analysis, we propose a model in which timely tissue-wide actin redistribution permits global cell elongation, which enables isotropic growth of the developing retinal neuroepithelium, a concept that could be applied to other systems.
    
    Speaker: Marija Matejcic
  - 12:00
    
    Brainwide optical circuit interrogation guided by online analysis of neuronal function 15m
    
    Understanding the brain requires measuring and perturbing neuronal activity. Tools for this are typically applied locally, but behavior is generated by the coordinated activity of neurons widely distributed across the brain. Thus, ideally we want to measure activity patterns of all neurons in the brain during behavior, use this information to decide which neurons to perturb, and record the brainwide effects of the perturbation.
    
    We introduce an experimental and computational system that enables such experiments at the brainwide scale. In behaving larval zebrafish, we measure neuronal activity in the entire brain during behavior using light-sheet imaging. Concurrently, through fast distributed computational analysis, we generate whole-brain functional maps relating neuronal activity to stimuli/behavior. Any subset of neurons can be selected from the maps and then optically ablated with a two-photon laser. The resulting changes in whole-brain activity and behavior are subsequently analyzed, all in the same animal.
    
    We apply this method to brainwide neuronal responses during visually-evoked swimming and find that a widely distributed set of nuclei mediate the behavior. Deleting specific functional neuron types from any of these nuclei has profound effects on brainwide responses consistent with a distributed implementation of the sensorimotor transformation.
    
    We extend the method to cellular-resolution targeted optogenetic activation during whole-brain imaging. These methods allow for concurrent whole-brain activity and causality mapping in the same animal, which will enable delineating the contributions of neurons to brainwide circuit dynamics and behavior.
    
    Speaker: Dr Nikita Vladimirov (Max-Delbrück Centre; Janelia Research Campus)
  - 12:15
    
    High-Throughput LSFM Imaging Of 3D-3-Culture Models To Unveil Macrophage Plasticity In The Tumour Microenvironment 15m
    
    Drug screens on complex cell models and organisms are a key factor to understand and treat human diseases. However, fast and effective conclusions have been hindered by the lack of robust and predictable models amenable to high-throughput (HT) analysis. Recently, important advances have been made towards the development of 3D co-culture models using distinct cell types that better recapitulates its in vivo features. These models bridge the gap between adherent cell culture and animal models, providing a powerful in vitro model for preclinical research.
    
    A major hurdle, hampering the widespread utilization of complex in vitro models, is the lack of robust imaging tools. Light sheet fluorescence microscopy (LSFM) has been proposed to overcome those limitations [1, 2]. Few years ago, we created the first flow cytometry system based on LSFM, SPIM-Fluid [3, 4], allowing the massive interrogation of a large set of biological parameters in hundreds of 3D cell cultures, thus providing statistical relevance. Now we have developed a new LSFM platform, Flexi-SPIM, which combines automatic fluidic loading of the samples and traditional scanning, overcoming the limitations of previous systems while keeping its HT capabilities.
    
    Using Flexi-SPIM, we are able to image more than 150 sample in only two imaging sessions of complex 3D-3 culture models including a co-culture of tumour cell spheroids of a non-small cell lung carcinoma cell line (tdTomato); cancer-associated fibroblasts (GFP) and a monocytic cell line (THP-1) (Cell tracker) in alginate capsules [5]. We observed phenotypic changes over time as well as how myeloid cells infiltrate into the tumour spheroids and display an immunosuppressive phenotype typical of tumour-associated macrophages.
    
    Ref
    [1] Gualda EJ,et al. Nat Methods 10 (2013)
    [2] Gualda EJ,et al. Front Cell Neurosci 8 (2014)
    [3] Gualda EJ,et al. Biomed Opt Express 6 (2015)
    [4] Estrada MF,et al. Biomaterials 78 (2016)
    [5] Rebelo SP,et al. Biomaterials 163 (2018)
    
    Speaker: Dr Emilio J Gualda (ICFO- The Institute of Photonic Sciences, BIST- The Barcelona Institute of Science and Technology)
  - 12:30
    
    Hybrid Optically-Gated Light Sheet Microscopy Allows Long-term Timelapse Imaging in the Developing Zebrafish Heart 15m
    
    Although the low-impact nature of light sheet microscopy has opened up new avenues for developmental timelapse imaging, the heart remains a particularly challenging organ to image in 3D timelapse. To image processes on timescales of minutes to hours (such as heart development, cell migration, repair and regeneration) demands some form of synchronized image acquisition in order to separate the high-frequency heartbeat motion from the lower-speed morphological changes of interest. Although current postacquisition synchronization methods are attractive for imaging the beat process, or for acquiring small numbers of timepoints, the accumulated light dose precludes longer-term timelapse imaging. Indeed, we will show that this rapidly induces catastrophic photobleaching, phototoxicity and heart arrhythmia.
    
    We have previously developed prospective optically-gated light sheet microscopy, to allow synchronised 3D imaging of the in vivo beating zebrafish heart with a laser dose no higher than required for imaging static tissue. However, sustained timelapse imaging over 24h or more presents significant additional challenges, since the dramatic morphological changes undergone by the heart frustrate existing synchronization approaches. We will describe how we have been able to overcome this barrier by using hybrid prospective-retrospective optical gating technologies, and present 24h 3D-timelapse video imaging of cardiac development and immune response to cardiac injury.
    
    Just as light sheet microscopy minimizes the distribution of the light dose to the specimen in the spatial domain, our approach offers the same gain in the time domain. Our work opens up the unperturbed, beating heart to direct timelapse imaging studies that have until now been restricted to stationary organs. Our new approach also points the way towards integrated light-sheet microscopy studies of the developmental coupling between heart structure, fluid flow and electrical activity.
    
    Speaker: Jonathan Taylor
- 12:45 → 14:00
  
  Lunch at CBG canteen 1h 15m
- 14:00 → 16:00
  
  Company demos
- 14:00 → 16:00
  
  Posters: Posters III
- 16:00 → 16:45
  
  EMBO practical course presentations
- 17:00 → 18:30
  Cell biology applications of light sheet microcopy
  - 17:00
    
    In Vivo Cellular Dynamics imaged using Adaptive Optics Lattice Light Sheet Microscopy 15m
    
    Speaker: Srigokul Upadhyayula (Harvard Medical School)
  - 17:15
    
    Imaging organoids in diSPIM 15m
    
    Speaker: Christian Conrad (DKFZ)
  - 17:30
    
    Light-sheet microscopy with subcellular resolution: technology and applications 15m
    
    Speaker: Reto Fiolka (UT Southwestern Medical Center)
  - 17:45
    
    Integrated development of a methodology for studying the spatio-temporal dynamic of the DNA damage response within Multi Cellular Tumor Spheroids 15m
    
    Speaker: Corinne LORENZO
  - 18:00
    
    Interplay between cell size and cell polarity 15m
    
    The PAR network polarizes a broad range of cell types by localizing proteins to opposing membrane domains. Despite its abundance, we know almost nothing about how the PAR proteins adapt to this vast diversity of cell sizes and shapes. In many systems, maintenance of polarity has been described as a reaction-diffusion network of the proteins involved.
    
    Here, by first using theoretical modelling, we show that these reaction diffusion systems break below a certain cell size, resulting in a uniform, unpolarized membrane distribution. This predicts that cells below a size threshold should be unable to maintain polarity in vivo. The precise nature of this threshold depends on parameters such as membrane diffusion and turnover.
    
    Next, by combining light sheet microscopy-based 3D reconstruction of the plasma membrane with single molecule measurements of key biophysical parameters, we have revealed this size limit in vivo, in a developmental lineage of the C. elegans embryo. These findings are in remarkable quantitative agreement with our theoretical predictions.
    
    Thus, intrinsic properties of polarity proteins impose physical limits on the ability of cells to polarize, pointing to an unappreciated link between the size of a cell and its ability to polarize and establish cell fate.
    
    Speaker: Lars Hubatsch (The Francis Crick Insititute)
  - 18:15
    
    High content 3-D light sheet microscopy in 96 and 384-well plate formats applied to study cancer cell size, and invasiveness and morphology in 3D matrices 15m
    
    Conventional light sheet fluorescence microscopy (LSFM) requires two microscope objective lenses orientated at 90° to one another. However, their proximity to one another and the sample makes high content imaging of samples mounted on conventional 96 and 384-well plates difficult. Oblique plane microscopy (OPM)1 uses a single high numerical aperture microscope objective to provide both fluorescence excitation and detection whilst maintaining the advantages of LSFM.
    
    We present the development and application of a stage scanning OPM (ssOPM)2 approach for high content light sheet fluorescence imaging in commercially available glass and plastic-bottomed 96 and 384-well plates. 3D images of cells were acquired by scanning the sample through the tilted light sheet at a constant velocity. Methods for implementing autofocus during acquisition together with the data acquisition pipeline will be discussed.
    
    The ssOPM system was used to perform functional screens for regulators of cell size and 3D invasiveness. In the screen for regulators of size, melanoma cells were grown as 2D cultures in 384-well plates and genes were systematically knocked down with a library of 120 siRNAs. For 3D invasion assays, 9 siRNAs were used and plates were incubated for 24 hours allowing cells to invade vertically into the gel prior to fixation and staining. For both assays, 100s-1000s cells were quantified per condition to allow 3D cytometric data analysis.
    
    We developed a MATLAB 3D image analysis pipeline for automated segmentation and morphological quantification of the image data. This allowed determination of cell size in 2D and 3D, measurement of cell invasiveness into the collagen matrix, and quantification of cell morphology of invading cells. The ssOPM approach will enable a better understanding of which genes are responsible for cancer cell size determination and invasion in 3D cultures.
    
    1 Dunsby, C. Opt. Express 16.25 (2008): 20306-20316; 2 Maioli, V., et al. Sci Rep 6:37777 (2016).
    
    Speaker: Dr Chris Dunsby (Imperial College London)
- 18:30 → 22:30
  
  Gala dinner 4h
Wednesday, 15 August
- 09:00 → 10:30
  Developmental biology applications of light sheet microscopy
  - 09:00
    
    3D+time imaging of developing embryos for the reconstruction of their cell lineage 15m
    
    Speaker: Dr Nadine Peyrieras (BioEmergences, CNRS USR3695 Gif-sur-Yvette, France)
  - 09:15
    
    Exploring a new contrast mechanism for light-sheet microscopy: fast harmonic generation imaging 15m
    
    Speaker: Willy Supatto
  - 09:30
    
    Imaging and dynamic analysis of the post-implantation mouse embryo at the cellular level using light-sheet microscopy 15m
    
    Speaker: Dr Katie McDole
  - 09:45
    
    The Physics of Blastoderm Flow during Early Gastrulation of Tribolium castaneum 15m
    
    The early embryo of the red flour beetle, Tribolium castaneum, initially consists of a single-layered blastoderm covering the yolk uniformly that differentiates into an embryonic rudiment as well as extraembryonic amnion and serosa. The germband anlage forms inside the egg during gastrulation when the embryonic rudiment condenses and folds along the ventral midline; this process is accompanied by large‐scale flow and expansion of the extraembryonic serosa which ultimately covers the entire surface of the egg, thus engulfing the growing embryo. The mechanical properties of these tissues and the forces governing these processes in Tribolium, as well as in other species, are poorly understood. Here, we present our findings on the dynamics of myosin in the early blastoderm of Tribolium using multiview lightsheet live imaging of transiently labeled wild type embryos. We quantitatively measure the global distribution of myosin throughout the flow phase and present a physical description that couples the contractile forces generated by myosin to the mechanical properties of the blastoderm. In particular, we describe the overall tissue as a thin, actively contractile, viscous bulk medium that exhibits friction with the vitelline membrane. This description accurately captures the large‐scale deformation the tissue undergoes during the initial stages of gastrulation. Our findings lay a foundation for the physical description of gastrulation in Tribolium and will allow, in combination with the well-studied Drosophila paradigm, for the first time the comparative analysis of blastoderm tissue morphogenesis
    
    Speaker: Dr Stefan Münster (TU Dresden - MPI-PKS - MPI-CBG)
  - 10:00
    
    Smart adaptive multi-sample imaging for multi-view light sheet microscopy – when the microscope decides what to look at 15m
    
    Light sheet microscopy of early embryo developmental stages is challenging. A significant number of time-lapse acquisitions, started at the onset of development, need to be stopped due to suboptimal sample orientation, poor image quality, not-fertilised eggs or because the development process arrests due to the phototoxicity induced by the imaging process itself. To increase the likelihood of success, we devised a strategy based on simultaneous multi-sample imaging at a low frame rate coupled with smart, image-aware microscope control. The open source ClearControl framework allows flexible user-defined assembly of instructions, such as device-based instructions (e.g. image acquisition), GPU-accelerated image post-processing and analysis, adaptive instructions (e.g. auto focus) and smart instructions (e.g. sample selection).
    
    The advantages of our strategy are twofold: first, it reduces phototoxic effects by minimizing sample exposure, and second, it allows the screening of several samples in parallel, until an operator-independent algorithm decides for a sample to image in more temporal detail and with higher signal-to-noise-ratio.
    
    We implemented our multi-sample imaging strategy on a multi-view light sheet microscope – the XWingScope. FEP tubes with typically five embryos of Drosophila melanogaster in the early cleavage stage were mounted and serially imaged by using a motorised stage. The acquired volumetric images were analysed by searching for a rapid rise in an entropy based image quality metric, which is used to detect the formation of the syncytial blastoderm and to predict when the first cells will invaginate. The presented strategy allows to select a sample automatically, auto-focus it, and continue imaging to capture the beginning of gastrulation with increased temporal resolution. Further improvements, more sophisticated control and decision-making algorithms, and other applications are made possible by our highly modular and extensible framework.
    
    Speaker: Dr Robert Haase (MPI CBG)
  - 10:15
    
    Imaging axial emergence though self-organization in embryonic organoids. 15m
    
    Emergence of multicellular forms (tissues, organs and organisms) from cells through changes in their shape, size, number and organization is central to understanding the process of morphogenesis. While molecular players are known, we do not know how the activity of genes and proteins is translated into 3D structures in space and time. Preexisting spatial cues, species-specific geometry and extraembryonic signaling centers, confound studying these processes in vivo. We have recently shown that 3D cell aggregates from different species (mouse embryonic stem cells and zebrafish blastula cells, which we term gastruloids and pescoids respectively) generate spatial asymmetries in gene expression and cell behavior within otherwise equivalent groups of cells, to develop a global coordinate system (body axes) de novo. Combining light-sheet imaging with germ-layer specific labelling of cells we are now gaining some insights into the spatio-temporal precision and species-independent manner, with which such 3D embryonic cell aggregates generate the major body axes even in the absence of any embryonic information. Using these embryonic organoids, as a minimal alternate system, sufficient to generate embryonic axes, we aim to understand early development in embryos as an emergent phenomenon of the self-organization of pluripotent cells.
    
    Speaker: Vikas Trivedi (EMBL Barcelona)
- 10:30 → 11:15
  
  Coffee break 45m
- 11:15 → 12:45
  Miscellaneous applications of light sheet microscopy
  - 11:15
    
    Imaging photosensitive reef-building corals 15m
    
    Speaker: Dr Philippe Laissue
  - 11:30
    
    Root organogenesis is biased towards the availability of water 15m
    
    Speaker: Daniel von Wangenheim (University of Nottingham)
  - 11:45
    
    Macro LSFM with sTSLIM (scanning Thin-Sheet Laser Microscopy) 15m
    
    Speaker: Peter Santi (University of Minnesota)
  - 12:00
    
    Conditional control of fluorescent protein degradation by an auxin-dependent nanobody 15m
    
    Most biological processes involve spatial-temporal changes in the concentration of proteins that ensure that the right protein acts at the right place at the right time. Due to its high temporal resolution and minimal photo bleaching light sheet microscopy is ideally suited to visualize such protein dynamics given that the protein of interest is labelled with a fluorescent probe. Indeed, GFP-traps and increasingly CRISPR/Cas9-mediated fluorescent knock in’s exist in several experimental systems ranging from tissue cell culture to model organisms and thus are great resources for light sheet microscopy experiments. To understand complex biological systems, however, we do not only need to visualize the emergent behaviour of protein, cells or organisms, but also to have to ability to interrogate the system. Here, we present an auxin-dependent GFP-nanobody to regulate the levels of overexpressed and endogenous GFP-tagged proteins in a conditional and reversible manner. We demonstrate efficient and reversible inactivation of the anaphase promoting complex/cyclosome (APC/C) in human tissue cell culture and thus provide new means to study the functions of this essential ubiquitin E3 ligase. Further, utilizing light sheet imaging, we show that the auxin-dependent GFP-nanobody can be applied to zebrafish. Hence, in principle the auxin-dependent GFP-nanobody has the potential to make any existing GFP-line in this and other model organisms compatible with auxin-mediated protein degradation thus enabling advanced functional studies.
    
    Speaker: Dr Jörg Mansfeld (Cell Cycle, Biotechnology Center, Technische Universität Dresden)
  - 12:15
    
    Imaging Intestinal Invasion by Vibrio cholerae, and other Stories from the Gut 15m
    
    Our digestive tracts are home to trillions of microbes that immigrate, emigrate, reproduce, and compete with one another. Little is known about the physical structure and temporal dynamics of gut microbial communities, which must necessarily influence the function not only of normal, commensal communities but also community invasion by pathogens. To address this, my lab applies light sheet fluorescence microscopy to a model system that combines a realistic in vivo environment with a high degree of experimental control: larval zebrafish with defined subsets of commensal bacterial species. I will focus here on experiments in which a native bacterial species is challenged by the invasion of a second species, specifically Vibrio cholerae, the pathogen that causes cholera. Using live imaging and genetic manipulation of Vibrio’s Type VI Secretion System (T6SS), with which the bacterium stabs adjacent cells, we have found that Vibrio cholerae can displace resident bacteria through a surprising ability to induce strong mechanical contractions of the host gut [S. L. Logan, J. Thomas, J. Yan, R. P. Baker, D. S. Shields, J. B. Xavier, B. K. Hammer, and R. Parthasarathy. Proc. Natl. Acad. Sci. 115: E3779-E3787 (2018)]. This suggests not only previously unknown mechanisms for bacterial manipulation of animal physiology, but also potential paths for microbiome engineering. I will also describe other experiments in which the spatial and temporal dynamics of gut microbes are key determinants of responses to such challenges as antibiotic perturbation and inhibition of motility.
    
    Speaker: Raghuveer Parthasarathy (University of Oregon, USA)
  - 12:30
    
    Molecular mapping of developmental disease progression using HiTS-FAST 15m
    
    Three-dimensional molecular mapping of RNA expression within intact biological tissue is allowing for new insight into the relationship structure and function. Current RNA quantification approaches are depth-limited to less than 200 $\mu m$ by the requirement for single-molecule read out of sequential barcoded RNA fluorescence in-situ hybridization (RNA-FISH) or hydrogel embedded in-situ RNA sequencing. An alternative approach to single-molecule readout is to sacrifice intra-cellular localization by using amplified RNA-FISH to detect RNA expression on a per cell basis. This strategy has the potential to extend three-dimensional molecular mapping of RNA expression to cubic millimeters or centimeters of tissue. Building on our previous work on autofocusing, inertia-free light sheet fluorescence microscopy for cleared tissue, we designed a platform for rapid multiplexed molecular interrogation of cleared tissue samples. The High Throughput Scalable Fluorescence Assay for Spatial Transcriptomics (HiTS-FAST) platform combines closed-loop feedback to maintain co-planar alignment of the exciting light sheet and optical detection plane in thick samples, programmable fluidics for sample clearing and sequential multiplex labeling, and third generation single-molecule hairpin chain reaction (smHCR v3.0) to reliably label RNA expression millimeters deep in cleared tissue samples. I will present our hardware and software implementation to maintain co-planarity, automated sample handling, and present initial results from multiplexed labeling of both RNA and protein in healthy and developmentally disrupted whole rat lungs.
    
    Speaker: Prof. Douglas Shepherd (University of Colorado Anschutz Medical Campus)
- 12:45 → 13:00
  
  Special session: Closing of the conference
- 13:00 → 14:00
  
  Lunch at CBG canteen 1h

10th Anniversary Light Sheet Fluorescence Microscopy Conference

MPI-CBG

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