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...
