Microscopy Conference 2023

Session chairs

Prof. Dr.

Structural and metabolic dynamics of plant cells in context of heat acclimation

Abstract text (incl. figure legends and references) In order to survive sub-lethal heat stress, plants possess the ability to acclimate to moderately elevated temperatures. They are altering their physiology in many different ways to maintain cellular homeostasis despite the change in environmental conditions. The carbohydrate metabolism is important for energy storage and biomass production and is also involved in regulation of heat acclimation response. To accurately resolve subcellular metabolic fluxes and compartment-specific metabolite concentrations in the model organism Arabidopsis thaliana, it is necessary to combine metabolic and ultrastructural data. To achieve this, we combined a non-aqueous fractionation procedure to generate subcellular metabolite concentrations with 3D imaging of leaf tissue by serial block-face scanning electron microscopy, which resulted in two datasets of control and heat treated leaf tissue, spanning approximately 150 µm in x and y and 50 µm in z-direction. We were able to calculate effective metabolite concentrations in three compartments of the cell, namely chloroplast, cytosol and vacuole. Applying a kinetic model of carbohydrate metabolism, subcellular fluxes were estimated which revealed metabolic heat acclimation strategies of plant metabolism and provide evidence for a tightly regulated metabolic network. Denk, W., &amp; Horstmann, H. (2004). Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol, 2(11), e329. https://doi.org/10.1371/journal.pbio.0020329 Fürtauer, L., Weckwerth, W., &amp; Nägele, T. (2016). A benchtop fractionation procedure for subcellular analysis of the plant metabolome. Front Plant Sci, 7, 1912. https://doi.org/10.3389/fpls.2016.01912 Seydel, C., Kitashova, A., Fürtauer, L., &amp; Nägele, T. (2022). Temperature-induced dynamics of plant carbohydrate metabolism. Physiol. Plant, 174(1). e13602. https://doi.org/10.1111/ppl.13602

LS 3: Imaging of large volumes and plastic section tomography

LS 4: Image analysis of large data sets

Poster

3D tomographic analysis to reveal intermediate stages of primary cilia formation

Abstract text (incl. figure legends and references) Introduction The intracellular ciliogenesis pathway includes binding of vesicles to the distal appendage and their fusion to generate one ciliary vesicle. In the following, it elongates into the ciliary membrane and the axoneme grows out. Objectives We aim to describe these consecutive stages of the ciliogenesis pathway using electron tomographic investigations in three dimensions (3D) and also by CLEM (correlative light and electron microscopy). Materials &amp; methods We use a protocol for correlative light and electron microscopy (CLEM) to analyse the timing of Rab8 recruitment to the basal body and to visualize membrane remodelling processes which take place in early stages of cilia formation. From human retinal pigment epithelial 1 (hRPE1) cells, stably expressing CENTRIN1 and RAB8A with fluorescent tags, fluorescence light microscopy images (focal stacks) are recorded after formaldehyde fixation, specifically the hRPE1 centriolar region. For electron microscopy, cells were fixed using glutardialdehyde, en-bloc contrasted (OsO4, UAc), dehydrated, resin-embedded, and sectioned (600 – 900 nm). On cells containing fluorescent signals at the centrosom, STEM tomograms were recorded, reconstructed (IMOD), and segmented. Correlation of positions of fluorescent proteins with ultrastructural features was done using the software eC-CLEM. Results By using fluorescence microscopy, we identified a dot-like pattern (mCherry-Rab8a) to one of the two Centrin1-GFP-positive centrioles in. In STEM tomograms of the centrosomal regions and in 3D models (by manual segmentation), novel intermediate stages of cilia formation were revealed; we named them the donut-shaped stage, and the biconcave, erythrocyte-shaped stage. Our data let us conclude that distal appendage vesicles fuse side-by-side to first build a C-shaped structure, which later employs the shape of a donut. Centripetal fusion events result in a biconcave membrane structure, later forming a cap-shaped ciliary membrane. Overlays (CLEM) showed that the earliest stage of ciliogenesis in which mCherry-Rab8a localized to the basal body in our set-up was the C-shaped ciliary membrane structure. To get insight into the cellular compartments that serve as a source for the early ciliary membrane compartment, we incubated cells with the lectin wheat germ agglutinin (WGA) conjugated to horseradish peroxidase (HRP), a tag helping to visualise the fusion protein by EM within its cellular context. Interestingly, we detected electron dense membrane material generated by HRP at the developing ciliary membrane during early cilia formation. This observation suggests that endocytosed membrane material contributes to the formation of the early ciliary membrane compartment. Conclusion We extend the stages of primary cilia formation during the intracellular cilia assembly pathway by identifying the donut-shaped and the biconcave stage, and we present evidence that endocytic trafficking routes contribute to the early ciliary membrane compartment. Lit.: Rachel et al 2020 J Struct Biol 211: 107551 Buerger et al 2021 Meth Cell Biol 162: 171-203 Fig. 1: images show (left, middle) two xy planes (thickness 2.7 nm; 54 nm apart from each other) of a STEM tomogram (IMOD). The slice was cropped to the region of the basal body. Bar, 200 nm; (right) 3D model of the reconstructed volume after manual segmentation of the STEM tomogram; top view, to best visualize the donut shape of the centriolar vesicle

LS 5: Correlative and multimodal microscopy

SEM array tomography with TEM like resolution in X and Y

Abstract text (incl. figure legends and references) Several approaches for volume scanning electron microscopy have been established during the last years. This article deals with array tomography by collecting SEM images of serial sections of high pressure frozen, freeze substituted and plastic embedded cultivated cells. The objective of the study is to obtain TEM like resolution with an SEM on serial thin sections mounted on silicon chips. Human cytomegalovirus (HCMV) infected human foreskin fibroblasts were grown on sapphire disks, high pressure frozen, freeze substituted with 0.2 % osmium tetroxide, 0.1 % uranyl acetate and 5 % of water in acetone and embedded in Epon. Serial thin sections (100 nm) were collected on silicon chips, partially post stained with lead citrate trihydrate (3% liquid) and imaged in a Hitachi S-5200 SEM at an accelerating voltage of 2 kV for recording the so-called composite signal, which consists of secondary electrons type 3 (that are produced by backscattered electrons) for intermediate magnifications (Fig. A), or with 5 kV accelerating voltage and recording the regular secondary electron signal for high magnifications (Figs. B and C). When the sections were not poststained with lead, the secondary electron as well as the backscattered electron signal were very noisy. We first tried plasma cleaning to remove some of the Epon and to make the heavy metal better amenable, but in our hands, this did not significantly improve image quality. The breakthrough was post staining of the sections on the silicon chips with lead citrate. It was then possible to record 62 consecutive sections of cells infected with HCMV and make a three-dimensional reconstruction. Figure A shows a portion of one section at an intermediate magnification. Chromosomes, microtubules and the centriols are visible on this section. Figures B and C show higher magnification images of an area of the cytoplasm with a viral particle in a vesicle. The images were recorded at 2 and at 5 kV. Best resolution was obtained at an accelerating voltage of 5 kV. The leaflets of the membrane bilayer are well visible and better resolved at 5 kV. These results fit well with our earlier studies of freeze substituted samples in FIB-SEM (Villinger et al., 2012, Histochem Cell Biol 138, 549-556) With the presented approach it was possible to obtain very good resolution in X and Y of serial sections. All details visible in TEM on similar samples can also be seen in our SEM images. Best results were obtained after lead post staining at 5 kV accelerating voltage and using the secondary electron signal. The best accelerating voltage is a tradeoff of the smaller beam diameter and the higher penetration depth at 5 kV compared to 2 kV. The resolution in Z is obviously defined by the thickness of the sections and is 100 nm in the presented dataset.

LS 7: Advances in sample preparation

Why software is key for designing your volume EM experiment.

Abstract text (incl. figure legends and references) The biological questions that drive a volume EM study set the key parameters of an acquisition such as the desired resolution, the volume extent and the number of replicates. These determine the imaging method and corresponding limitations in throughput and dimensions. Software can play a crucial role in overcoming limitations during acquisition by means of automation. Turning the acquired raw data into the final volume representation requires dedicated software for aligning serial images or reconstructing tomographic tilt series. Transforming voxel intensities into quantitative measures can be a final important procedure towards biological interpretation of the data that also heavily relies on software. Often these post-acquisition procedures are a bottleneck and thus need to be considered thoroughly when planning a volume EM study. Various projects from EMBL's EM Core Facility illustrate how software development enable scaling up different volume EM pipelines enabling new scientific applications.

Prof.

Comparative morphology of mitochondria in optic nerve and cell body of retinal ganglion cells in a mouse model of spastic paraplegia type 2

Abstract text (incl. figure legends and references) Oligodendrocytes facilitate rapid action potential propagation by ensheathing central nervous system axons, thereby existing in a close metabolic relationship with the myelinated axon. This "axo-glial" unit critically depends on proper energy supply by axonal mitochondria to meet their high energy demands for saltatory action potential propagation. In neurodegenerative diseases such as PLP1-related disorders, axonal swellings together with alterations in morphology and function of axonal mitochondria serve as hallmarks for dys- and demyelinating disease states. So far it remained elusive how lack of proteolipid protein (PLP) in oligodendrocytes and myelin impairs axonal function, but a deficit in axonal energy supply due to mitochondrial dysfunction is one plausible explanation. Here we used retinal ganglion cells (RGCs) with their myelinated axons in the optic nerve as model tissue for studying axonal mitochondria in a Plp-deficient mouse model of spastic paraplegia type 2. To investigate the influence of myelin on the local mitochondrial heterogeneity in RGC neurons we compared mitochondrial morphology in myelinated and non-myelinated parts of the axon. For this purpose, we developed a method to reliably target RGC mitochondria in distinct regions: the optic nerve, the optic nerve head (ONH), and the retinal ganglion cell body for electron microscopy and 3D reconstruction. A hybrid method was established in combining chemical fixation with high-pressure freezing (HPF) and freeze substitution to obtain a well-preserved mitochondrial ultrastructure. HPF is a well-established method to study the ultrastructure of cells and tissue in a close-to-native state, but complex tissue requires dissection before freezing. To minimize handling artefacts and disturbance of tissue organization, prefixed tissue was sliced with a vibratome producing sections of required thickness, from which the most suitable sections were selected for HPF. Careful vibratome sectioning of the eye was indispensable for targeting the optic nerve head. For the structural investigation, we applied transmission electron microscopy (TEM) and electron tomography for 3D analysis of individual mitochondria. Mitochondria in the RG cell body and optic nerve head region revealed very little morphologic difference between wild-type mice and Plp−/y mice. However, we observed inclusions in RGC mitochondria of Plp−/y mice, a finding that requires further investigation. At the ONH, mitochondrial clusters containing mitochondria with a round shape and mostly tubular cristae were prominent in both genotypes. The optic nerve revealed clear genotype-dependent changes in mitochondrial morphology. This is consistent with a recent study by Steyer et al. 2020, J Struc Biol 210(2):107492), in which 3D reconstruction of data sets obtained by focused ion beam scanning electron microscopy (FIB-SEM) showed that axons in Plp−/y mice contain twice as many mitochondria per 10 µm axon, with the mitochondria additionally appearing shorter and rounder. Overall, we were able to develop a method to reliably target the optic nerve head with attached retina and optic nerve to study mitochondrial heterogeneity in RGC axons in a mouse model of spastic paraplegia type 2. This will help us to better understand the consequences for axonal mitochondria architecture and function in Plp-deficiency.

Ultrastructural characterization and reconstruction of cultured xenapses

Abstract text (incl. figure legends and references) In our lab, we have developed a new model to study the neurotransmission process in neurons. Using click-chemistry microstructured glass coverslips were functionalized to grow xenapses - TIRF-amenable presynaptic boutons on the glass surface. The new model system required not only functional but also ultrastructural characterization, and comparison to synapses in conventional neuronal cultures and slices. Given the size of structures of interest, and the resolution required, we chose to perform 3D reconstructions from serial sections of plastic-embedded xenapses. Mouse neurons, grown on functionalized, carbon-coated coverslips, were fixed by immersion in a mix of 1% glutaraldehyde with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer on ice, additionally post-fixed at room temperature with 1% osmium tetroxide in the same buffer, and en bloc stained with either uranyl or samarium acetate. Samples were dehydrated in an ethanol series and embedded in Epon. The cured Epon blocks had a medium hardness. The region of interest was identified by visual inspection in a light microscope after removal of the glass and then additionally embedded in Epon for stabilization and protection of the sample during sectioning. After trimming the block with a diamond knife to obtain highly polished edges, the serial sections were cut at 40-50 nm and ribbons of 40-50 sections were collected on Formvar-coated slot TEM grids. Imaging was performed with a TEM Philips CM-10 equipped with a bottom-mounted TemCam F-416 camera. Image series were aligned and rendered in Reconstruct. Morphological analysis (size and distribution of synaptic vesicles (SV) within the acquired volume) was performed with MetaMorph software. Data obtained from EM are in good accordance with our live cell imaging, electrophysiology, and immunofluorescence data and confirmed that xenapses showed all the hallmarks of presynaptic boutons in culture or in brain slices. Xenapses are filled with numerous SVs, analogous in size to SVs in standard cultures; they also harbor other structures typical to presynaptic boutons – like mitochondria, endosomes/cisternae, cytoskeleton elements, multi-vesicular bodies, large dense core and clathrin-coated vesicles. SVs distributed within xenapse's volume may represent the resting and recycling pools of vesicles, whereas the ones at the footprint are analogous to the readily releasable pool of SVs in normal synapses as revealed in experiments with TIRF microscopy. Taken together our data indicate that the new model shows all structural features of standard presynapses and enables live-cell imaging of single vesicle exo- and endocytosis in space and time.

PS 9
Poster session LS 3: Imaging of large volumes and plastic section tomography

Appointment

Session chairs

Programme

PS 9Poster session LS 3: Imaging of large volumes and plastic section tomography

Appointment

Session chairs

Programme

PS 9
Poster session LS 3: Imaging of large volumes and plastic section tomography