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, 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 and, in most cases, 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, with many projects resulting in long-term 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 long-term project for example a is the analysis of the structure, assembly- and infection-mechanism of the bacterial phage SPP1 that 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 in 2012, 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., 2021, PNAS). We also use immunogold-labeling, negative-staining 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, and finding certain biological events therefore often corresponds to the proverbial search for a needle in a haystack. 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 ~1-2 nm. The acquired micrographs (often more than 1000 images per region of interest) are then stitched using the TrakEM plugin in FIJI (Cardona et al., 2012, PLoS One) to one single montage (Figure 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, and that Spermidine treatment caused significant restructuring at the active zone with increasing age (Figure 1; Gupta et al., 2017, PLoS Biol).
This collaboration led to several follow-up projects on neuronal stem cells obtained from iPSCs that where derived from patient fibroblasts (Lorenz et al., 2017, Cell Stem Cell), hepatocyte-like cells generated from human Induced pluripotent stem cells (Matz et al., 2017, Sci Rep.) and cancer model systems (Brandt et al., 2019, Nat Commun; Bischoff et al., 2020, iScience). In ongoing projects, we now use automated TEM imaging to study early mouse embryo development at the blastocyst state (Bulut-Karslioglu lab), mouse brain development (Kraushar lab), as well as fibroblast cultures derived from patients showing severe progeroid features (FG Mundlos).
A new focus of our service group is the implementation of correlative light and electron microscopy techniques. Correlative approaches have been used to study transmission of equine herpes viruses between peripheral blood mononuclear cells from the lung to endothelial cells (Kamel et al., 2020, iScience) and SPP1 phage assembly in bacteria (Labarde et al., 2021, PNAS). As test system, we also established a simple HEK cell spheroid model system, which can be used for high-throughput and 3D light microscopy as well as TEM and SEM imaging. Starting with TEM montages of mouse blastocysts, we aim at establishing robust image analysis workflows to analyze ultrastructural details such as organelles, vesicles, fat droplets or cell-cell contacts in collaboration with the Bulut-Karslioglu lab (van der Weijden et al., 2022, bioRxix). In collaboration with the Kinkley lab, we also test and implement new embedding strategies (e.g. Crome-EM) which potentially can be used for studying chromatin compaction or other gene regulatory events inside the nucleus.