Microscopy Conference 2023

Session chairs

Throughout the last two decades different volume scanning electron microscopy (vEM) methods have gained more importance in the life sciences allowing new questions to be answered for samples from cells to organisms and tissues. New developments for hardware and software for transmission as well as scanning electron microscopy have paved the road to new exciting research topics to be addressed. This session will illustrate the diversity of methods and applications of the current volume SEM modalities, namely array tomography (AT), serial block face SEM (SBEM) and focused ion beam SEM (FIB-SEM), as well as plastic tomography focusing on higher resolution. Besides different research topics, the challenges linked to the sample preparation, imaging and data analysis will be covered.  

Neuropathology on tape – applications of ATUM-based volume EM

Abstract text (incl. figure legends and references) Volume Electron Microscopy (vEM) reveals biological structures at nanometer resolution in three dimensions and resolves ambiguities of two-dimensional representations. However, as the structures of interest – like disease hallmarks emerging from neuropathology – are often rare but the field of view is small, this can easily turn a vEM project into a needle in a haystack problem. One solution for this is correlated light and electron microscopy (CLEM), which requires precise coordinate transfer between the two imaging modalities. With array tomography methods, serial ultrathin sections are collected into a storable tissue library, thus restoring precious samples like human biopsies and enabling repetitive imaging at different resolution levels for an SEM-based search strategy. For this, automated tape collecting ultramicrotomy (ATUM) has been developed to reliably collect serial ultrathin sections via a conveyor belt onto a plastic tape that is later mounted onto silicon wafers for serial scanning EM (SEM) (Kasthuri, Hayworth et al., 2015). The ATUM-SEM procedure is highly modular and enables targeting and high resolution imaging of specific structures. The variation of section thickness determines axial resolution and screening speed. We have developed ATUM hybrid methods that enable both, fast screening and targeted imaging at isotropic voxels (Kislinger, Gnägi et al., 2020). We exemplify targeted ATUM-SEM in mouse models for the reconstruction of Alzheimer"s disease plaques and for the visualization of Blood Brain Barrier leakage (Khalin, Adarsh et al., 2022). In order to correlate transcript levels with ultrastructure, we designed an indirect correlative method called SPcEM which combines ATUM-SEM with Spatial Transcriptomics (Androvic, Schifferer et al., 2022). In a mouse demyelination model adjacent cryosections are inspected by either of the two methods for sophisticated microglia classification. Androvic P, Schifferer M, Anderson KP, Cantuti-Castelvetri L, Ji H, Liu L, Besson-Girard S, Knoferle J, Simons M, Gokce O (2022) Spatial Transcriptomics-correlated Electron Microscopy. bioRxiv: 2022.05.18.492475 Khalin I, Adarsh N, Schifferer M, Wehn A, Groschup B, Misgeld T, Klymchenko A, Plesnila N (2022) Size-Selective Transfer of Lipid Nanoparticle-Based Drug Carriers Across the Blood Brain Barrier Via Vascular Occlusions Following Traumatic Brain Injury. Small (Weinheim an der Bergstrasse, Germany) n/a: 2200302 Kislinger G, Gnägi H, Kerschensteiner M, Simons M, Misgeld T, Schifferer M (2020a) ATUM-FIB microscopy for targeting and multiscale imaging of rare events in mouse cortex. STAR Protocols 1: 100232Kislinger G, Gnägi H, Kerschensteiner M, Simons M, Misgeld T, Schifferer M (2020b) Multiscale ATUM-FIB Microscopy Enables Targeted Ultrastructural Analysis at Isotropic Resolution. iScience 23: 101290 Fig. 1 ATUM-SEM and ATUM-FIB of AD plaques Fig. 2 BBB-CLEM with fluorescent and gold nanoparticles Fig. 3 SPcEM in demyelinating disease

IM 3: SEM and FIB developments

LS 3: Imaging of large volumes and plastic section tomography

Invited talk

Prof.

Focus on the 3D architecture of mitotic spindles in human cells

Abstract text (incl. figure legends and references) Mitosis in eukaryotes follows organism- as well as cell-specific patterns. One common feature of mitosis is the assembly of a bipolar spindle apparatus that is involved in the segregation of the genetic material to the newly forming daughter cells. The spindle is a complex microtubule-based cellular machinery. Within the spindle, microtubules are interacting with molecular motors and passive crosslinkers to align chromosomes in metaphase and move chromosome halves in anaphase. It is crucial to study the 3D complexity of mitotic spindles in order to better understand chromosome missegregation. Almost any chromosome missegregation results in aneuploidy, which is a hallmark of most types of cancer. Studying the 3D ultrastructure of the mitotic spindle apparatus at the level of microtubule organization requires electron microscopy. For 3D analysis, we apply high-pressure freezing, freeze substitution and resin embedding. Subsequently, we pre-screen embedded cells using confocal light microscopy to identify cells in desired stages of mitotic cell division. Once a cell has been chosen, we apply serial sectioning followed by electron tomography to reconstruct spindles in silico with single-microtubule resolution. For 3D reconstruction and quantitative analysis of spindle organization, we are applying a routine pipeline, which involves various software tools such as IMOD and Amira. With this approach, we recently analyzed bipolar spindles in HeLa cells (Kiewisz et al., Elife, 2022). During the meeting we will also report on our newest results on the ultrastructure of kinetochore microtubules (KMTs) in monopolar spindles in retinal pigment epithelial 1 (RPE1) cells. In both bipolar and monopolar mammalian spindles, KMTs are organized in so-called kinetochore (k)-fibers. Comparing spindle organization in various cell types, it is our goal to refine our recently developed biophysical model of k-fiber self-organization in mammalian cells (Conway et al., Elife, 2022).

Structural analyses of the brown algae Ectocarpus

Abstract text (incl. figure legends and references) Brown algae belong to the stramenopiles and as such are phylogenetically distant from plants, animals and fungi. They have been evolving independently from plants and animals more than one billion years ago. Brown algae are one of only five eukaryotic lineages that have evolved independently to develop into complex multicellular organisms. To contribute to the understanding of developmental processes that lead to the observed complex multicellularity in brown algae we have analysed Ectocarpus siliculosus on the ultrastructural level both by scanning and transmission electron microscopy. Several sample preparation techniques were tested to find optimal conditions for structure preservation for this organism. In order to characterise the morphology and ultrastructure of Ectocarpus we have compared the wild-type and a mutant that shows defects in the first cell divisions of the developing organism. Potential differences in Golgi morphology were analysed in datasets obtained by focused-ion beam scanning electron microscopy of plastic embedded samples of both the wild-type and the mutant.

LS 4: Image analysis of large data sets

Abstract talk

Correlative light and serial block face electron microscopy for targeting and investigating oligodendrocyte-vasculature interactions

Abstract text (incl. figure legends and references) Cell-cell specific interactions occur between all major cell types in the brain. Although the investigation of cellular interactions with light microscopy is a standard approach, targeting specific cell-cell interactions with electron microscopy is highly challenging. In this project we investigated cellular interactions of oligodendrocyte cell bodies with the brain vasculature. This interaction has been has been previously identified, but organization and function of oligodendrocytes associated with the brain vasculature remains unknown. In preceding light microscopy experiments we estimated that in the neocortical grey matter about 17% of oligodendrocytes contact in majority capillaries. As the vasculature is highly organized and is formed by different cell types including endothelial cells, pericytes and astrocytes we wanted to further investigate vascular associated oligodendrocytes on the ultrastructural level to resolve the anatomical organization. To specifically target and analyze oligodendrocyte vascular ensembles and obtain 3D reconstructions with electron microscopy we applied a correlative light and serial sectioning electron microscopy approach. We obtained fluorescent oligodendrocytes by using a transgenic mouse line that expressed membrane targeted EGFP under an oligodendrocytic specific promoter. After transcardial fixation of mice with 4% paraformaldehyde and 2.5% glutaraldehyde, the vasculature was labelled with fluorescent tomato lectin, a specific marker for the vasculature. After fixation, EGFP fluorescence remained and we identified oligodendrocyte-vasculature ensembles in 50 µm thick vibratome sections of the motor cortex using confocal imaging. After identification of ensembles we applied 2-photon branding (910 nm) of ensemble locations and included orientation marks of larger size for improved sample recovery. Subsequently the brain slices were prepared for serial block face electron microscopy (SBF-SEM), embedded in epon and mounted with silver containing conductive resin on sample pins. Using confocal microscopy z-stacks that we acquired during sample preparation, we re-identified the marked positions and proceeded with SBF-EM (Zeiss) to acquire backscattered images of the target regions at nanoscale resolution (pixel size of 10 x 10 x 30 nm). Data analysis included image post-processing, reconstruction of the structures of interest with Microscopy image browser and 3D rendering with Imaris (Fig 1). By optimizing parameters of the sample preparation process we could detect the laser marked regions and identified oligodendrocyte-vasculature ensembles in electron microscopy. Analysis of the EM images revealed that the oligodendrocyte cell body and the vascular basement membrane are in direct contact (n = 3 cells, n = 2 mice,). Moreover, we detected Golgi apparatus, microtubules, endoplasmic reticulum, mitochondria and lipid inclusions in oligodendrocytes cell bodies (Fig 2). Astrocyte endfeet surrounded the oligodendrocyte cell body at non-contact sites suggesting a close interaction with astrocytes at the neuro-vascular-junction. Our presented approach shows that a combination of fluorescence labelling, light microscopy and SBF-EM allows the investigation of the nanoscale structure of an oligodendroglial vascular niche. Our data suggest that oligodendrocytes are a component of the neuro-glia-vascular unit raising the possibility of direct signaling pathways and metabolite exchange with vascular cells.

LS 5: Correlative and multimodal microscopy

It is not easy being green: the technical path for the ultrastructural study of the locomotory apparatus of the moss Physcomitrium patens

Abstract text (incl. figure legends and references) Electron microscopy (EM) as a whole technique enables the ultrastructural studies of many samples from individual cells to full organisms. For the visualization of a single image under the electron beam, extensive time investment is made in technical preparation and several EM techniques often need to be applied until an image or model can be presented. The path is not always easy, and optimizations are commonly needed to avoid unintentional artifacts that can impact the final ultrastructural observation. This problem is even more pronounced when it comes to processing plant samples, such as the moss Physcomitrium patens, where there is a need to consider the existence of the cell wall, that provides rigidity and strength to cells, but also functions as a barrier for the chemicals needed to observe the ultrastructure using room temperature EM. In this study, the challenge presented to our facility was the characterization of the locomotory apparatus of the moss P.patens, namely the de novo centriole assembly that is involved in the motility of the sperm cells during spermatogenesis. These sperm cells develop inside an antheridium located at the tip of the gametophore, a complex structure that contains leaves, rhizoids and both sexual organs (the antheridia and the archegonia). We addressed this question by combining the observations from Chemical Fixation, High-Pressure Freezing–Freeze Substitution, Correlative Light-Electron Microscopy and Electron Tomography. Each of the different EM techniques helped to address different parts of the biological question. Chemical Fixation allowed to carefully identify the main stages of development and to do the initial ultrastructural characterization. High-Pressure Freezing – Freeze Substitution was used to confirm that the identified structures were not artifacts induced by chemical fixation and allowed a more accurate and detailed ultrastructural characterization of each structure. Electron Tomography gave three-dimensional models of the structures. Correlative Light-Electron Microscopy (CLEM) helped to elucidate the protein composition of the sperm cells&quot; locomotory apparatus and associate the light microscopy results with the structures observed by electron microscopy. Overall, this work shows how complex the technical path in an EM study of a larger specimen can be and how crucial it is to keep optimizing and developing EM techniques that supports the answering of biological questions even when using bigger and more intricate specimens. Figure 1: 3D models of different stages in centriole assembly and maturation in Physcomitrium patens. a) Bicentriole stage; b) Sister centrioles associated with the multilayered structure (MLS); c) Asymmetrical sister centrioles docked to the cell membrane, serving as basal bodies for ciliogenesis. Cyan – astral microtubules; Light green – centriolar microtubules; Red – Centriolar cartwheel; Dark green – microtubular spline of the MLS; Yellow – lamellar strip of the MLS; Magenta – ciliary transition zone. Scale-bars = 200nm. Adapted from: The 3D architecture and molecular foundations of de novo centriole assembly via bicentrioles (2021).

LS 7: Advances in sample preparation

Structure of the endoplasmic reticulum during cell differentiation and stress

Abstract text (incl. figure legends and references) The endoplasmic reticulum (ER) consists of an elaborate network of flat cisternae (aka sheets) and tubules that extends throughout the cell and occupies a large fraction of the cytoplasmic volume. The ER contributes to the production and folding of approximately one third of cellular proteins, has a key role in coordinating lipid biosynthesis, and constitutes a reservoir of Ca2+ ions that act as signalling molecules to regulate essential cellular processes. The ER has a central role in organelle communication as it forms membrane contacts with most other organelles. Specific ER stress signaling pathways, collectively known as the unfolded protein response (UPR), are required for maintaining ER homeostasis. The UPR is triggered when ER protein folding capacity is overwhelmed by cellular demand. The UPR initially aims to restore ER homeostasis and normal cellular functions. However, prolonged UPR induces apoptosis to eliminate the defected cell. The continuity of the ER network, the extensive contacts with other organelles and its highly dynamic nature provides a mechanism to propagate various signals throughout the cell. The ER functions as a sensor of cell's well-being by integrating inter- and intracellular signals and inducing appropriate responses under various conditions during growth, division and differentiation. The dynamic and complex organization of ER poses a major challenge on understanding how its functioning – maintenance of the structure, distribution of its functions and communication with other organelles – is orchestrated. We are utilizing CLEM and various volumeEM techniques in imaging of organelles, cells and tissue to carry out a detailed quantitative morphological characterization of the ER under physiological or pathological conditions such as ER stress, and during differentiation processes where cells adopt specialized functions. This will increase our understanding of fundamental aspects on the regulation of ER functioning and organization of the organelle communication. Figure 1: hiPSC prior (left) and after (middle and right from stage 7) differentiation to pancreatic secretory cells. ER is depicted in yellow.

LS 3
Imaging of large volumes and plastic section tomography

Appointment

Session chairs

Description

Programme

LS 3Imaging of large volumes and plastic section tomography

Appointment

Session chairs

Description

Programme

LS 3
Imaging of large volumes and plastic section tomography