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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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...
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,...
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...
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...
Metazoans specify germ layers during early development in a process called gastrulation. Gastrulation involves massive cell movements during which the specified germ layers are divided into molecularly distinct domains, which later gives rise to diverse differentiated cell types. Gastrulation movements have been described at both the cellular and molecular levels in vertebrates and insects in...
Total internal reflection microscopy (TIRFM) has been the method of choice for many years to image insulin secretory granule (SG) dynamics and secretion in primary beta cells and insulinoma cell lines. However, it only allows for imaging of SGs located <200 nm from the surface of the cell attached to the glass, thereby restricting the view only to events happening on one side of the cell....
In Expansion Microscopy (ExM) a sample with fluorophores linked to a swellable gel is expanded homogeneously by a factor of approx. 4 [1]. This leads to a virtual optical resolution of up to 60 nm laterally and 250 nm axially. Applied to mouse brain samples this allows for resolution of neuronal network details on length scales of 100 nm, which are normally below the diffraction limit of...
Lightsheet microscopy is a fluorescence imaging technique that allows visualization of whole organs or small organisms while preserving their physical integrity i.e. without the need to slice them prior imaging. Although the principle of operation of this technology was developed more than 100 years ago, it is only in the last fifteen years that biologists have commonly started to use such...
The labyrinthine structure of the vertebrate inner ear is vital for the perception of gravity, and linear and angular acceleration, to help control balance. The formation of this complex organ involves dynamic changes in cell shape and movement in the otic epithelia during embryo development. Optical sectioning microscopy, in particular light-sheet fluorescence microscopy coupled with the...
ariadne.ai offers tailored solutions for the analysis of increasingly demanding high-throughput light and electron microscopy datasets by combining the state-of-the-art convolutional neural networks with the experience of our professional image annotator team. Fed with high quality ground truth, our novel machine-learning tool for the automated segmentation of somata analyzes whole mouse-brain...
The aim of our project is to depict the dynamics of mouse antigen presenting cells (APCs) in a mammary gland during the ontogenesis and breastfeeding period. Additionally, the theory postulating transport of bacteria from the small intestine to the mammary gland will be tested.
Recent studies show that a subset of bacteria in milk could be transported by dendritic cells (DCs) from the...
Medaka (Oryzias latipes) is amenable to in vivo-imaging by light-sheet microscopy due to its comparably slow development, its very large available toolbox and mostly transparent embryos. Its lifelong growth allows extensive studies of stem cells. However, following these stem cells and their descendants by in vivo-imaging is very challenging and imaging conditions needed optimization.
In...
The evolution of distinct head and trunk domains revolutionized animal morphology. Sophisticated anterior structures evolved to sense the environment while powerful posterior appendages propelled bilaterians to diversify and occupy every ecosystem on the planet. Pioneering work on developmental genetics revealed that the head and the trunk identities are specified by distinct regulatory...
To understand the structure and function of a living matter, scientists rely on microscopic imaging methods. The behavior and interactions between cells in the developing embryos happen in a three dimensional environment. Hence, we need a microscope to record data in three dimensions. Light sheet microscopy is a novel technique which has the capability to acquire a 3D images with high...
Several novel Light Sheet Fluorescence Microscopy techniques have emerged in recent years in pursue of good axial resolution over a long field of view. These include Bessel, Lattice and Airy beam light-sheet microscopy, among others. There has not been a direct comparison in the literature of their dimensions, and often different criteria are used among publications. Most of them present...
Lattice Light Sheet Microscope (LLSM) represents the novel generation of 3D fluorescence microscopes dedicated to live single-cell analysis. LLSM[1] uses ultrathin light sheets derived from 2D optical lattices. These are scanned plane-by-plane through the specimen to generate a 3D image. The thinness of the sheet leads to high axial resolution and negligible photobleaching and background...
Long-term in toto imaging of developing vertebrate embryos to achieve the full reconstruction of their lineage tree is still a major challenge. Based on Single Plane Illumination Microscopy (SPIM by PhaseView http://phaseview.com/alpha3/), we developed a methodology for long-term in toto imaging of zebrafish and rabbit embryos and demonstrate its performance in terms of cell detection and...
ANNEXIN 1 (ANN1) is the most abundant member of the evolutionary conserved multigene protein superfamily of annexins. Annexins participate in diverse cellular processes, such as cell growth, differentiation, vesicle trafficking and stress responses. Moreover, they can associate with cytoskeleton and membrane phospholipids in a calcium dependent manner. Expression of annexins is developmentally...
Light sheet microscopy imaging allows recording entire embryos in 3D and over time (3D+t) for many hours. Fluorescently labeled structures can be tracked automatically in these 3D+t images [1-4]. Analyzing the resulting cell migration trajectories can provide detailed insights in large-scale tissue reorganization and morphological changes in early developmental stages at the cellular level....
Lattice light-sheet microscopy [1] provides fast volumetric images with subcellular resolution from cells to embryos. To expand its capacity to a wider range of biological applications, we implemented an inverted version of a lattice light-sheet microscope [2]. The inverted lattice light-sheet microscope adopts a two chamber-design, which separates the biological samples from the immersion...
Understanding the architecture of neural circuits is an important but formidable task. Critical details of neuronal connectivity - the synapses - occur on length scales of about 100 nm. Thus, imaging techniques reaching optical super resolution are required. However, neurites extend over distances of millimeters and centimeters, thus optical sectioning, a large field of view and a high imaging...
Rabies virus-based retrograde tracing is a powerful approach for visualizing synaptically connected neurons. Combined with a refined tissue clearing technology [1], this approach enables e.g. the visualization of transplanted neurons and synaptically connected host cells in whole-mouse brain preparations [2]. In order to visualize and 3D reconstruct such a transplant connectome we optimized a...
Plant growth and development is a complex process evolving through continuous qualitative and quantitative changes in four (x-, y-, z- and t-) dimensions. Classical microscopy methods pose some serious limitations for long-term live cell and developmental plant imaging. Out-of-focus fluorescence, phototoxicity, photobleaching, restricted temporal resolution and limitations in imaging depth are...
Light sheet fluorescence microscopes have high spatial resolution but sequential scanning of the planes limits the maximum imaging speed that can be achieved. On the other hand, light field microscopes and other extended depth of field imaging methods have high temporal resolution but suffer from low spatial resolution. Here we show a method to dynamically increase the spatial resolution of a...
The microtubule network is an essential part of the cell, providing structure and shape. It is also important for intracellular transport of cargos, which is crucial for correct embryo morphogenesis. Microtubules are dynamic structures that undergo continual assembly and disassembly within the cell.
In particular, yolk microtubule organization undergoes several changes over the various...
Precise regulation of cargo trafficking and axonal transport is critical for neuronal development and function. Cargos are delivered to specific locations through the activity of diverse kinesins and their cargo-linking adaptor proteins. However, the roles of these kinesins and adaptor proteins as well as the mechanisms of cargo localization during neuronal morphogenesis remain poorly...
With the introduction of optical clearing in neuroscience, considerable advances in tissue clearing and large volume microscopy have been made1-4. However, volume imaging and cytoarchitectonic characterization of large human brain samples, scalable in terms of time and cost to cover a significant portion of a cortical area, has so far remained challenging. This is especially true for adult...
Symmetry-breaking events are fundamental biological processes for the formation of specialized tissues. In particular for intestinal organoids, symmetry-breaking is a paradoxical event: only a fraction of cells, part of a genetically identical population forming a cyst and immersed in unchanged medium, undergo differentiation. Although striking, the underlying mechanisms that drive...
A classical Selective Plane Illumination Microscope (single light-sheet, generated using a cylindrical lens) suffers from a number of issues, such as shadow artefacts, scattered out-of-focus background and limited FoV (Field of View). A variety of advanced techniques in light-sheet microscopy have been proposed to tackle these issues, and previous publications have shown how image quality can...
The Francis Crick Institute is a young, large and ambitious biomedical research centre that aims to understand the biology underpinning human health. The Crick Advanced Light Microscopy facility (CALM) supports basic and advanced light microscopy with the institute. We recently acquired a Luxendo-Bruker MuVi light-sheet microscope in order to support research in many different fields,...
The vertebrate inner ear contains three orthogonally arranged semicircular canals that function to detect angular accelerations (turning movements of the head). Each canal comprises a curved duct with a swelling (ampulla) at the base that houses sensory hair cells. We are using light-sheet fluorescence imaging of transgenic zebrafish to examine formation of the semicircular canals from...
The emergence of Selective Plane Illumination Microscopy (SPIM) about a decade ago enabled scientists to study the development of whole embryos (e.g. fruit fly, zebrafish, mouse) with unmatched spatiotemporal resolution and low phototoxicity. Yet, true in toto recordings of only few model organisms have been realized thus far. As any light microscopy technique, SPIM suffers from optical...
Despite the successes of entry-level open source projects, with new frontiers for light sheet imaging apparent, the demand for the technology outstrips supply. Furthermore, these projects are in need of rejuvenation, do not reflect the state of the art and remain dependent on an abundance of adventurous life-scientists capable of tackling the technical challenges. Similarly, commercial...
A symmetric light-sheet microscope is presented, featuring two high numerical aperture objectives arranged in 120°. Both objectives are capable of illuminating the sample with a tilted light-sheet and detecting the fluorescence signal. This configuration allows for multi-view, isotropic imaging of delicate samples where rotation is not possible, while collecting more than twice as much light...
Until recently, imaging deep into intact organs using fluorescence microscopy posed a significant challenge. Imaging depth for conventional one-photon excitation microscopy is limited to a few tens of microns due to appreciable light scattering and absorbance in dense, turbid tissue. This limited penetration depth is not significantly improved even with the use of genetically encoded...
Tackling current biomedical challenges calls for in-depth understanding of biological systems, particularly their structures, functions, and interactions on both the molecular and the cellular level. Biological imaging constitutes an important field of scientific investigation and one of its most valuable techniques is fluorescence microscopy. State-of-the-art imaging devices, such as light...
Intratumoral heterogeneity is a critical factor when diagnosing and treating patients with cancer. Marked differences in the genetic and epigenetic backgrounds of cancer cells have been revealed by advances in genome sequencing, yet little is known about the phenotypic landscape and the spatial distribution of intratumoral heterogeneity within solid tumours. Here, we developed a pipeline for...
