Welcome to the Amira-Avizo Software Use Case Gallery

Intravital microscopy provides dynamic understanding of multiple cell biological processes, but its limited resolution has so far precluded structural analysis. Because it is difficult to capture rare and transient events, only a few attempts have been made to observe specific developmental and pathological processes in animal models using electron microscopy. The multimodal correlative approach that we propose here combines intravital microscopy, microscopic X-ray computed tomography and thr... Read more

During cell division, spindle microtubules ensure an equal repartition of chromosomes between the two daughter cells. While the kinetochore-dependent mechanisms that drive mitotic chromosome segregation are well understood, in oocytes of most species atypical spindles assembled in absence of centrosomes entail poorly understood mechanisms of chromosome segregation. In particular, the structure(s) responsible for force generation during meiotic chromosome separation in oocytes is unclear. Usin... Read more

HIV-1 maturation occurs via multiple proteolytic cleavages of the Gag polyprotein, causing rearrangement of the virus particle required for infectivity. (…) How individual cleavages contribute to changes in protein structure and interactions, and how the mature, conical capsid forms, are poorly understood. Here, we employed cryoelectron tomography to determine morphology and high-resolution CA lattice structures for HIV1 derivatives in which Gag cleavage sites are mutated. These analyse... Read more

Mitochondria undergo spontaneous transient elevations in matrix pH associated with drops in mitochondrial membrane potential. These mitopHlashes require a functional respiratory chain and the profusion protein optic atrophy 1, but their mechanistic basis is unclear. To gain insight on the origin of these dynamic events, we resolved the ultrastructure of flashing mitochondria by correlative light and electron microscopy. HeLa cells expressing the matrix-targeted pH probe mitoSypHer were screen... Read more

The fruit fly, Drosophila melanogaster, is an important experimental model to address central questions in neuroscience at an organismic level. However, imaging of neural circuits in intact fruit flies is limited due to structural properties of the cuticle. Here we present a novel approach combining tissue clearing, ultramicroscopy, and data analysis that enables the visualisation of neuronal networks with single-cell resolution from the larval stage up to the adult Drosophila. (…) This... Read more

With the advent of neurogenetic methods, the neural basis of behavior is presently being analyzed in more and more detail. This is particularly true for visually driven behavior of Drosophila melanogaster where cell-specific driver lines exist that, depending on the combination with appropriate effector genes, allow for targeted recording, silencing and optogenetic stimulation of individual cell-types. Together with detailed connectomic data of large parts of the fly optic lobe, this has rece... Read more

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath beta’–COP. We find that all coat components disassemble... Read more

Protein aggregation and dysfunction of the ubiquitin-proteasome system are hallmarks of many neurodegenerative diseases. Here, we address the elusive link between these phenomena by employing cryo-electron tomography to dissect the molecular architecture of protein aggregates within intact neurons at high resolution. We focus on the poly-Gly-Ala (poly-GA) aggregates resulting from aberrant translation of an expanded GGGGCC repeat in C9orf72, the most common genetic cause of amyotrophic latera... Read more

Analysis of neuronal arborization and connections is a powerful tool in fundamental and clinical neuroscience. Changes in neuronal morphology are central to brain development and plasticity and are associated with numerous diseases. Golgi staining is a classical technique based on a deposition of metal precipitate in a random set of neurons. Despite their versatility, Golgi methods have limitations that largely precluded their use in advanced microscopy. We combined Golgi staining with fluore... Read more

The mitotic spindle ensures the faithful segregation of chromosomes. Here we combine the first large-scale serial electron tomography of whole mitotic spindles in early C. elegans embryos with live-cell imaging to reconstruct all microtubules in 3D and identify their plus- and minus-ends. We classify them as kinetochore (KMTs), spindle (SMTs) or astral microtubules (AMTs) according to their positions, and quantify distinct properties of each class. While our light microscopy and muta... Read more

We investigated the synaptic innervation of apical tufts of cortical pyramidal cells in a region between layers 1 and 2 using 3-D electron microscopy (3D-EM) applied to four cortical regions in mouse. Across all cortices, we found the relative inhibitory input at the apical dendrite’s main bifurcation to be more than 3-fold stronger for layer 2 pyramidal cells than for all other pyramidal cells. Towards the distal tuft dendrites in upper layer 1, however, the relative inhibitory input was a... Read more

The major mammalian bloodstream form of the African sleeping sickness parasite Trypanosoma bruceimultiplies rapidly, and it is important to understand how these cells divide. Organelle inheritance involves complex spatiotemporal re-arrangements to ensure correct distribution to daughter cells…

Podocytes present a unique 3D architecture specialized for glomerular filtration. However, several 3D morphological aspects on podocyte development remain partially understood because they are difficult to reveal using conventional scanning electron microscopy (SEM). Here, we adopted serial block-face SEM imaging…

Macroautophagy is morphologically characterized by autophagosome formation. Autophagosomes are double-membraned vesicles that sequester cytoplasmic components for further degradation in the lysosome. Basal autophagy is paramount for intracellular quality control in post-mitotic cells but, surprisingly, the number of autophagosomes in post-mitotic neurons is very low, suggesting that alternative degradative structures could exist in neurons…

In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of inform... Read more

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of…

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light–SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present four examples of uses for this workflow that are not practical by light microscopy and/or transmission electron microscopy. First, distinguishing closely associated microtubules within K-fibers; second, resolving brid... Read more

Serial block-face electron microscopy (SBEM) provides nanoscale 3D ultrastructure of embedded and stained cells and tissues in volumes of up to 10⁷ µm³. In SBEM, electrons with 1–3 keV energies are incident on a specimen block, from which backscattered electron (BSE) images are collected with x, y resolution of 5–10 nm in the block-face plane, and successive layers are removed by an in situ ultramicrotome. Sp... Read more

Centrioles are vital cellular structures that form centrosomes and cilia. The formation and function of cilia depends on a set of centriole’s distal appendages. In this study, we use correlative super resolution and electron microscopy to precisely determine where distal appendage proteins localize in relation to the centriole microtubules and appendage electron densities. Here we characterize a novel distal appendage protein ANKRD26 and detail, in high resolution, the initial steps of dist... Read more

Neurons project axons to local and distal sites and can display heterogeneous morphologies with limited physical dimensions that may influence the structure of large organelles such as mitochondria. Using cryo-electron tomography (cryo-ET), we characterized native environments within axons and presynaptic varicosities to examine whether spatial restrictions within these compartments influence the morphology of mitochondria. Segmented tomographic reconstructions revealed distinctive morphologi... Read more

The orchestration of intercellular communication is essential for multicellular organisms. One mechanism by which cells communicate is through long, actin-rich membranous protrusions called tunneling nanotubes (TNTs), which allow the intercellular transport of various cargoes, between the cytoplasm of distant cells in vitro and in vivo. Here, we use correlative FIB-SEM, light- and cryo-electron microscopy approaches to elucidate the structural organization of neuronal TNTs. Our data indicate ... Read more