Andreas Schertel

Counting synapses using FIB/SEM microscopy: a true revolution for ultrastructural volume reconstruction

The advent of transmission electron microscopy (TEM) in the 1950s represented a fundamental step in the study of neuronal circuits. The application of this technique soon led to the realization that the number of synapses changes during the course of normal life, as well as under certain pathological or experimental circumstances. Since then, one of the main goals in neurosciences has been to define simple and accurate methods to estimate the magnitude of these changes. Contrary to analysing single sections, TEM reconstructions are extremely time-consuming and difficult. Therefore, most quantitative studies use stereological methods to define the three-dimensional characteristics of synaptic junctions that are studied in two dimensions. Here, to count the exact number of synapses per unit of volume we have applied a new three-dimensional reconstruction method that involves the combination of focused ion beam milling and scanning electron microscopy (FIB/SEM). We show that the images obtained with FIB/SEM are similar to those obtained with TEM, but with the advantage that FIB/SEM permits serial reconstructions of large volumes of tissue to be generated rapidly and automatically. Furthermore, we compared the estimates of the number of synapses obtained with stereological methods with the values obtained by FIB/SEM reconstructions. We concluded that FIB/SEM not only provides the actual number of synapses per volume but it is also much easier and faster to use than other currently available TEM methods. More importantly, it also avoids most of the errors introduced by stereological methods and overcomes the difficulties associated with these techniques.

Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4‐mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin‐induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.

Cryo FIB-SEM: Volume imaging of cellular ultrastructure in native frozen specimens

Volume microscopy at high resolution is increasingly required to better understand cellular functions in the context of three-dimensional assemblies. Focused ion beam (FIB) milling for serial block face imaging in the scanning electron microscope (SEM) is an efficient and fast method to generate such volume data for 3D analysis. Here, we apply this technique at cryo-conditions to image fully hydrated frozen specimen of mouse optic nerves and Bacillus subtilis spores obtained by high-pressure freezing (HPF). We established imaging conditions to directly visualize the ultrastructure in the block face at -150 °C by using an in-lens secondary electron (SE) detector. By serial sectioning with a focused ion beam and block face imaging of the optic nerve we obtained a volume as large as X=7.72 μm, Y=5.79 μm and Z=3.81 μm with a lateral pixel size of 7.5 nm and a slice thickness of 30 nm in Z. The intrinsic contrast of membranes was sufficient to distinguish structures like Golgi cisternae, vesicles, endoplasmic reticulum and cristae within mitochondria and allowed for a three-dimensional reconstruction of different types of mitochondria within an oligodendrocyte and an astrocytic process. Applying this technique to dormant B. subtilis spores we obtained volumes containing numerous spores and discovered a bright signal in the core, which cannot be related to any known structure so far. In summary, we describe the use of cryo FIB-SEM as a tool for direct and fast 3D cryo-imaging of large native frozen samples including tissues.

A vacuole-like compartment concentrates a disordered calcium phase in a key coccolithophorid alga

Coccoliths are calcitic particles produced inside the cells of unicellular marine algae known as coccolithophores. They are abundant components of sea-floor carbonates, and the stoichiometry of calcium to other elements in fossil coccoliths is widely used to infer past environmental conditions. Here we study cryo-preserved cells of the dominant coccolithophore Emiliania huxleyi using state-of-the-art nanoscale imaging and spectroscopy. We identify a compartment, distinct from the coccolith-producing compartment, filled with high concentrations of a disordered form of calcium. Co-localized with calcium are high concentrations of phosphorus and minor concentrations of other cations. The amounts of calcium stored in this reservoir seem to be dynamic and at a certain stage the compartment is in direct contact with the coccolith-producing vesicle, suggesting an active role in coccolith formation. Our findings provide insights into calcium accumulation in this important calcifying organism.

Microglia remodel synapses by presynaptic trogocytosis and spine head filopodia induction

Microglia are highly motile glial cells that are proposed to mediate synaptic pruning during neuronal circuit formation. Disruption of signaling between microglia and neurons leads to an excess of immature synaptic connections, thought to be the result of impaired phagocytosis of synapses by microglia. However, until now the direct phagocytosis of synapses by microglia has not been reported and fundamental questions remain about the precise synaptic structures and phagocytic mechanisms involved. Here we used light sheet fluorescence microscopy to follow microglia–synapse interactions in developing organotypic hippocampal cultures, complemented by a 3D ultrastructural characterization using correlative light and electron microscopy (CLEM). Our findings define a set of dynamic microglia–synapse interactions, including the selective partial phagocytosis, or trogocytosis (trogo-: nibble), of presynaptic structures and the induction of postsynaptic spine head filopodia by microglia. These findings allow us to propose a mechanism for the facilitatory role of microglia in synaptic circuit remodeling and maturation.Direct visualization of microglia-mediated synapse pruning has been lacking. This study shows direct evidence of microglia-synapse interaction where microglia do not necessarily ‘eat’ post-synaptic structure but ‘nibble’ on pre-synaptic terminals, much akin to trogocytosis by lymphocytes.

Mineral-bearing vesicle transport in sea urchin embryos.

Sea urchin embryos sequester calcium from the sea water. This calcium is deposited in a concentrated form in granule bearing vesicles both in the epithelium and in mesenchymal cells. Here we use in vivo calcein labeling and confocal Raman spectroscopy, as well as cryo-FIB-SEM 3D structural reconstructions, to investigate the processes occurring in the internal cavity of the embryo, the blastocoel. We demonstrate that calcein stained granules are also present in the filopodial network within the blastocoel. Simultaneous fluorescence imaging and Raman spectroscopy show that these granules do contain a calcium mineral. By tracking the movements of these granules, we show that the granules in the epithelium and primary mesenchymal cells barely move, but those in the filopodial network move long distances. We could however not detect any unidirectional movement of the filopodial granules. We also show the presence of mineral containing multivesicular vesicles that also move in the filopodial network. We conclude that the filopodial network is an integral part of the mineral transport process, and possibly also for sequestering calcium and other ions. Although much of the sequestered calcium is deposited in the mineralized skeleton, a significant amount is used for other purposes, and this may be temporarily stored in these membrane-delineated intracellular deposits.

Calcium transport into the cells of the sea urchin larva in relation to spicule formation

Significance A major challenge in biomineralization is to determine the pathways by which calcium is transferred from external sources to the mineralization site. Using the membrane-impermeable calcium-binding dye calcein and fluorescent dextran, in conjunction or not with a calcium channel blocker, we show that both molecules readily enter the body cavity of sea urchin larvae and the cells responsible for skeleton formation. The documented existence of vesicles in these cells that form openings to the body cavity supports the notion that a major calcium uptake pathway involves direct incorporation of sea water into the cells by endocytosis. This pathway, if proven to be widespread among organisms of other phyla, would radically change our understanding of calcium transport in biomineralization. We investigated the manner in which the sea urchin larva takes up calcium from its body cavity into the primary mesenchymal cells (PMCs) that are responsible for spicule formation. We used the membrane-impermeable fluorescent dye calcein and alexa-dextran, with or without a calcium channel inhibitor, and imaged the larvae in vivo with selective-plane illumination microscopy. Both fluorescent molecules are taken up from the body cavity into the PMCs and ectoderm cells, where the two labels are predominantly colocalized in particles, whereas the calcium-binding calcein label is mainly excluded from the endoderm and is concentrated in the spicules. The presence of vesicles and vacuoles inside the PMCs that have openings through the plasma membrane directly to the body cavity was documented using high-resolution cryo-focused ion beam-SEM serial imaging. Some of the vesicles and vacuoles are interconnected to form large networks. We suggest that these vacuolar networks are involved in direct sea water uptake. We conclude that the calcium pathway from the body cavity into cells involves nonspecific endocytosis of sea water with its calcium.

Cryo-FIB-SEM serial milling and block face imaging: large volume structural analysis of biological tissues preserved close to their native state.

Many important biological questions can be addressed by studying in 3D large volumes of intact, cryo fixed hydrated tissues (⩾10,000μm3) at high resolution (5-20nm). This can be achieved using serial FIB milling and block face surface imaging under cryo conditions. Here we demonstrate the unique potential of the cryo-FIB-SEM approach using two extensively studied model systems; sea urchin embryos and the tail fin of zebrafish larvae. We focus in particular on the environment of mineral deposition sites. The cellular organelles, including mitochondria, Golgi, ER, nuclei and nuclear pores are made visible by the image contrast created by differences in surface potential of different biochemical components. Auto segmentation and/or volume rendering of the image stacks and 3D reconstruction of the skeleton and the cellular environment, provides a detailed view of the relative distribution in space of the tissue/cellular components, and thus of their interactions. Simultaneous acquisition of secondary and back-scattered electron images adds additional information. For example, a serial view of the zebrafish tail reveals the presence of electron dense mineral particles inside mitochondrial networks extending more than 20μm in depth in the block. Large volume imaging using cryo FIB SEM, as demonstrated here, can contribute significantly to the understanding of the structures and functions of diverse biological tissues.

Biologically controlled synthesis and assembly of magnetite nanoparticles

Magnetite nanoparticles have size- and shape-dependent magnetic properties. In addition, assemblies of magnetite nanoparticles forming one-dimensional nanostructures have magnetic properties distinct from zero-dimensional or non-organized materials due to strong uniaxial shape anisotropy. However, assemblies of free-standing magnetic nanoparticles tend to collapse and form closed-ring structures rather than chains in order to minimize their energy. Magnetotactic bacteria, ubiquitous microorganisms, have the capability to mineralize magnetite nanoparticles, the so-called magnetosomes, and to direct their assembly in stable chains via biological macromolecules. In this contribution, the synthesis and assembly of biological magnetite to obtain functional magnetic dipoles in magnetotactic bacteria are presented, with a focus on the assembly. We present tomographic reconstructions based on cryo-FIB sectioning and SEM imaging of a magnetotactic bacterium to exemplify that the magnetosome chain is indeed a paradigm of a 1D magnetic nanostructure, based on the assembly of several individual particles. We show that the biological forces are a major player in the formation of the magnetosome chain. Finally, we demonstrate by super resolution fluorescence microscopy that MamK, a protein of the actin family necessary to form the chain backbone in the bacteria, forms a bundle of filaments that are not only found in the vicinity of the magnetosome chain but are widespread within the cytoplasm, illustrating the dynamic localization of the protein within the cells. These very simple microorganisms have thus much to teach us with regards to controlling the design of functional 1D magnetic nanoassembly.

Ultra-structural analysis of the brain in a Drosophila model of Alzheimer's disease using FIB/SEM microscopy.

Alzheimers disease (AD), one of the most prevalent neurodegenerative brain diseases, has been extensively researched for years. However, its synaptic structure, which is a basis for understanding neurodegenerative disorders, has not yet been understood clearly. Defining the structures of neurons and their synaptic connections is the significant goal of brain research. To study synaptic connectivity, three-dimensional (3D) reconstructions of the nervous system are very helpful. In this study, the 3D structure of brain synapses in the Drosophila melanogaster Swedish amyloid precursor protein (APP) mutant, which is characterized by early onset AD, was analyzed using focused ion beam/scanning electron microscopy (FIB/SEM). This technique is one of the most useful for 3D reconstruction, as the process of obtaining serial images is fully automated and thus avoids the problems inherent in hand-operated ultrathin serial sectioning. The 3D images of normal and AD brains reported in this study reveal characteristic features of AD such as appearance of autophagy, abnormal axon formation and increased mitochondrial size. This 3D analysis reveals structural change as a basis for understanding neurodegenerative disorder.