Conventional cell-biological TEM applications and correlative microscopy

Our group supports a broad range of “classical” cell-biological TEM methods. This includes ultra-thin sectioning of resin-embedded tissues, cells and isolated cellular components such as e.g. synaptosomes, immunogold-labelling techniques including Tokuyasu-cryo-sectioning as well as metal-shadowing of nucleic acids and nucleic acid-protein complexes. In TEM, we collaborate with all departments and research groups of the institute having a wet lab but also with external collaboration partners. TEM sample preparation is technically difficult and time-consuming. In most cases, sample preparation requires careful optimization for each sample to achieve optimal image contrast depending on the nature of the tissue and cells, respectively. TEM projects are therefore performed as full-service projects and are considered as scientific collaboration rather than routine service. This includes both, individual projects to answer a specific scientific question as well as long-lasting collaborations.

 

Figure 1: (A) Montage of 1080 TEM images showing the entire mushroom body calyx region from the Drosophila brain. (B) Zoom into the region marked in (a). Several bouton regions are visible, one of which showing a synaptic active zone is highlighted in (C).

One example for a long-term project is the analysis of the structure, assembly- and infection-mechanism of the bacterial phage SPP1 which infects the Gram-positive model bacterium Bacillus subtilis. Originally introduced by the funding director of the MPI-MG, Thomas Trautner, SPP1 phages until today serve as a model system for eukaryotic dsDNA viruses and have been intensively studied in the institute over many years. After the retirement of Rudi Lurz, we are continuing the close collaboration with Paulo Tavares (former member of the Trautner lab; CNRS, Gif-sur-Yvette) and have now applied Tokuyasu cryo-sectioning and immunogold electron microscopy to analyze the spatiotemporal compartmentalization of viral components during virus replication in the bacterial cell. We could show that newly replicated viral genomes assemble into a single DNA compartment. Later during infection, SPP1 procapsids localize at the periphery of the viral DNA compartment for genome packaging whereas DNA-filled capsids segregate into flanking warehouses that are temporally and spatially independent from the freshly replicated viral DNA (Labarde et al., manuscript under revision). We also use immunogold-labeling, negative-stain screening and single particle cryo-EM to study the assembly of the SPP1 phage at a structural and molecular level (for more information please visit the cryo-EM section of our homepage).

Conventional TEM on ultrathin-sectioned resin-embedded samples provides ultrastructural details at nanometer resolution. However, the field of view of a single TEM image is limited to some µm squared depending on the final magnification and given size of the camera used, which often makes evaluation of biological events and related disease processes difficult. We therefore implemented automated data acquisition based on the Leginon System (Suloway et al., 2005, J Struct Biol), which enables us to routinely image large regions of interest up to 500 x 500 µm2 at a pixel size of ~1 nm. The acquired micrographs (> 1000 images per region of interest) are then stitched using the TrakEM plugin in FIJI (Cardona et al., 2012, PLoS One) to one large single montage (Fig. 1) which can then be inspected for statistical analysis. In collaboration with Stephan Sigrist (FU-Berlin), we applied this approach to proof the hypothesis, that age induced memory impairment (AMI) which is also observed in flies and which can be suppressed by restoring juvenile polyamine levels (Gupta et al., 2013, Nature Neuroscience) is related to structural rearrangements at the active zone. Imaging the calyx-region of drosophila melanogaster brains derived from animals at different age and grown under different feeding conditions, we could show statistically that the number of active zones as well as the density of synaptic vesicles per synaptic bouton decreased with age. Spermidine treatment did not restore these changes, instead, spermidine suppressed an increase of both, the average size of T-bars and the average number of Bruchpilot molecules within this scaffold, indicating significant restructuring at the active zone with increasing age (Fig. 1; Gupta et al., 2017, PLoS Biol).

We also use automated TEM to study the differentiation of induced pluripotent stem cells (iPSCs) at ultrastructural level. In collaboration with J. Adjaye (now University Düsseldorf), we found that bile canaliculi, a unique ultrastructural feature of hepatocytes, start to form within the hepatic endoderm (HE) state (Matz et al., 2017, Sci Rep.). Similarly, we could show that iPSCs derived from patient fibroblasts which potentially can be used as a model system to study rare mtDNA-linked genetic diseases, develop a typical triangular shape upon differentiation towards neuron progenitor cells (collaboration with A. Prigione, MDC Berlin, Lorenz et al., 2017, Cell Stem Cell). We also apply automated TEM to study disease processes such as cancer or segmental progeroid disorders (collaboration with Björn Fischer-Zirnsak, research group Mundlos).

Many research groups within our institute exploit 3D cell-culture model systems such as spheroids, organoids and gastruloids to study cell differentiation, tissue development and embryogenesis as well as related disease processes including cancer. We therefore aim to extend automated imaging towards 3D ultrastructure visualization and currently testing and implementing new technologies and workflows including automated serial sectioning, array tomography, block-face imaging as well as FIB-SEM imaging. This involves correlative approaches combining light and electron microscopy. Automated widefield microscopy and live cell imaging is used to monitor the formation and growth of biological 3D structures. Once a given time point is reached or a structure of interest has formed, confocal laser scanning and light sheet microscopy, respectively, are applied to derive a coarse 3D model prior to embedding the samples for further TEM or SEM analysis of ultrastructural details beyond the Abbe diffraction limit of light microscopy. Once established using HEK cell derived spheroids, we will apply these methodologies to study biological processes such as sprouting angiogenesis (with Petra Knaus, FU Berlin), scaffold formation in synapsis (with Stephan Sigrist, FU Berlin), cancer (with Gilbert Schönfelder, BfR Berlin) and reorganization in the nucleus related to regulation of gene expression or the cell cycle (AGs Mundlos, Hnisz, Actas, Kinkley).

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