PANOS Spring Meeting 2021 - Building an Imaging Network

01307 Dresden
Thomas Kurth, Thomas Müller-Reichert, Wiebke Moebius

Dear Attendees,

due to the Covid-19 pandemic we had to cancel the PANOS-Meeting 2020 and postponed it to spring 2021.

It will now be held march 25 and 26 as an online Meeting (via Zoom)

PANOS (Präparation und Abbildung Nativer Organischer Systeme) is a subgroup of the German Society of Electron Microscopy (DGE). This year's PANOS Spring Meeting 2020 will be hosted at the Center for Regenerative Therapies Dresden of the TUD in Dresden. Main topic will be the collaboration of imaging facilities across institution boundaries, epecially the formation of common centralized imaging platforms and networks offering access to state-of-the-art instruments, services and expertise. Contributions illustrating the benefits of such imaging networks are encouraged. In addition, also other EM-related topics as well as tips-and-tricks are welcome, and we encourage especially young scientists to present their work.

Registration is free!

Abstract submission deadline: 02 march 2021

Invited Speakers: Hella Hartmann (Dresden), Paul Verkade (Bristol, UK), Erin Tranfield (Oeiras/PT), Andreas Müller (PLID, Dresden)

Supported by: The German Society of Electron Microscopy (DGE)



  • Thursday, 25 March
    • 09:00 09:10
      Welcome Note 10m
    • 09:10 09:40
      BiodIP: The Biopolis Dresden Imaging Platform - history, benefits, and challenges 30m
      Speakers: Dr Hella Hartmann, Dr Jan Peychl
    • 09:40 09:55
      Microscopy goes to school 15m
      Speaker: Silke Tulok
    • 09:55 10:25
      The importance of imaging networks - from the COMULIS COST action to the TechEM virtual seminar series 30m
      Speaker: Dr Erin Tranfield
    • 10:25 11:00
      Virtual coffee break, breakout sessions for discussions in small groups 35m
    • 11:00 11:30
      Networking to advance Science 30m
      Speaker: Dr Paul Verkade
    • 11:30 12:00
      A collaborative effort to reconstruct microtubule-organelle interaction in pancreatic beta cells 30m
      Speaker: Dr Andreas Müller
    • 12:00 12:30
      NET - Network Electron Microscopy Tübingen 30m
      Speakers: Dr Katharina Hipp, Dr Stefan Fischer, Dr York-Dieter Stierhof
    • 12:30 12:45
      PANOS affairs, Announcements 15m
    • 12:45 14:00
      Lunch 1h 15m
    • 14:00 16:00
      Breakout sessions for poster presentations 2h

      Poster presentations in separate breakout rooms (one room per presentation)

      • Dissecting a specialist’s sensory jewel: 3D analysis of anterior sensilla of C. elegans dauer larvae by FIB-SEM acquisition 20m
        Speaker: Sebastian Britz
      • Functionalizing the microtubule lumen 20m
        Speaker: Foram Joshi
      • Atomic resolution in life sciences 20m
        Speaker: Emanuel Katzmann
      • Automated CLEM registration without fiducials using cross-modal deep learning 20m
        Speaker: Rick Seifert
      • Identification of microfossils of hominin tools from late Pleistocene using microscopy and energy dispersive X-ray analysis (EDS) 20m
        Speaker: Tatiana Miranda
      • Do More With Your Microscope - Adding an in situ toolkit 20m
        Speaker: Andrew Smith
    • 09:00 09:20
      Multimodal Imaging Approach for Visualizing Deoxy-Sphingolipid Accumulation on Ultra-Structural and Temporal Resolution in Cells 20m

      We developed and applied a novel lipid-labeling approach for 3D-electron microscopy aiming to understand the enigmatic sub-cellular action of non-canonical 1-deoxy-sphingolipids (deoxySLs) − A lipid class relevant for diseases such as type 2 diabetes mellitus (T2DM), hereditary sensory and autonomic neuropathy type 1 (HSN-1) and cancer. [1,2,3,4]
      Earlier studies indicated that intracellular deoxySL accumulation is related to mitochondrial dysfunction and defects in sub-cellular membrane structure and trafficking [2,6].
      In the present study, we investigated altered organelle dynamics by applying 1 µM DOXSA and organelle live dyes to MEF cells. Live-cell imaging showed impaired cell motion, mitochondrial dysfunction and tube formation of ER and auto-lysosomal compartments.
      Based on these findings, cultured mammalian cells (MEF cells) were treated with alkyne -1- deoxysphinganine (alkyne-DOXSA) − A traceable analogue of 1-deoxysphinganine (DOXSA).
      Alkyne-lipids are lipid tracer which act essentially native in biological systems [5].
      Subsequently we investigated deoxySL related lipid membrane defects on ultrastructural level using TEM tomography. For visualization of alkyne-lipids, we employed a novel labeling method. We call that approach the "Golden-Click-Method" (GCM). GCM is based on functionalized gold-nanoparticles containing azide groups as electron-dense reporters. These are covalently conjugated to alkyne-deoxySLs during a copper-catalyzed “click-reaction”. This labeling approach for EM was further complemented by STED microscopy of fluorescently tagged alkyne-deoxySLs enabling co-localization studies of alkyne-lipids and specific organelle markers at the sub-organelle level. In conclusion, our examinations revealed auto-lysosomal activity in MEF cells after exogeneous DOXSA-application. Emerging intra-cellular membrane distortion and toxic acting lipid aggregates may be a plausible explanation for deoxySL-mediated organelle dysfunctions leading ultimately to neurodegeneration. − Here, we emphasize on sub-cellular processes that connect deoxySL mediated membrane damage with cell biological mechanisms, such as ER-Stress and mitochondrial dysfunction. Moreover, our findings in adherent MEF cells corroborate that deoxySL mediated autophagy and intra-lysosomal enrichment of deoxySL derivates may display relevant factors for neurodegenerative diseases.

      [1] Penno et al., Hereditary sensory neuropathy type 1 is caused by the accumulation of two neurotoxic sphingolipids (2010)
      [2] Alecu et al., Localization of 1-deoxysphingolipids to mitochondria induces mitochondrial dysfunction (2017)
      [3] Othman et al., Plasma 1-deoxysphingolipids are predictive biomarkers for type 2 diabetes mellitus. (2015)
      [4] Massard et al. Phase I dose-escalating study of ES-285 given as a three-hour intravenous infusion every three weeks in patients with advanced malignant solid tumors. (2012)
      [5] Thiele et al., Tracing Fatty Acid Metabolism by Click-Chemistry (2012)
      [6] Jimenez-Rojo et al., Biophysical Properties of Novel 1-Deoxy-(Dihydro)ceramides Occurring in Mammalian Cells.” (2014) Cells.” (2014).

      Speaker: Marina Hesse (German Center for Neurodegenerative Diseases (DZNE), Bonn)
    • 09:20 09:40
      Correlative light and electron microscopy of RPE1 cells to study ciliogenesis 20m
      Speakers: Ralph Witzgall (University of Regensburg, Biology and Preclinical Medicine, Centre for EM/Anatomy) , Reinhard Rachel (University of Regensburg, Biology and Preclinical Medicine, Centre for EM/Anatomy)
    • 09:40 10:00
      Volume imaging: from HeLa cells to the human nervous system 20m

      New developments in volume imaging by different scanning electron microscopy techniques have been paving the way to address new scientific questions in the life sciences. The opportunity to explore large areas in 2D as well as growing volumes in 3D at high resolution has started giving us a better understanding of complex biological systems from organismal level down to sub-organelle complexes. I would like to present some of our current projects we are running in the facility taking advantage of the expertise of different research groups in different imaging techniques. Combing super resolution light microscopy, TEM tomography, as well as focused ion beam scanning electron microscope (FIB-SEM) from nm up to mm scale. We are using the FIB-SEM to get a better understanding of a variety of samples from different model organisms and different sample preparation modalities such as microwave-assisted processing, high-pressure freezing or critical point drying. Topics such as the organization of the cristae in mitochondria (HeLa cells) up to the organization of nerves in mice have been studied. Furthermore the large area tile scan capability of the scanning electron microscope allows the investigation of mm sized samples at nm resolution, such as optic nerves from human patients. These specific samples pose special challenges such as availability of material and sample preparation of autopsy material. Therefore every sample preparation as well as imaging regiment has to be tailored to its biological specimen as well as its individual research question.

      Speaker: Dr Anna Steyer (Max-Planck-Institut für Experimentelle Medizin)
    • 10:00 10:20
      The Role of Tubulin Post-Translational Modifications in Ciliary Mechanics 20m

      Tubulin post-translational modification signify a hugely unexplored platform for spatiotemporal encoding of information within cells that guide the assembly and function of stable cytoskeletal manifestations. In the presented work, the roles of tubulin polyglycylation and polyglutamylation in ciliary mechanics are addressed by combining different experimental approaches, including immunolabelling, cryo-ET and sub-tomogram averaging. We conclude that polyglycylation is involved in axonemal dynein activity regulation while polyglutamylation acts as a passive regulator of axonemal microtubule sliding resistance. This work could have only been carried out thanks to a well-stablished collaboration network that involves different institutions and facilities, posing a great example of how interdisciplinary, collaborative research brings about radically new discoveries that permeate different aspects of science.

      Speaker: Gonzalo Alvarez Viar (MPI-CBG)
    • 10:20 10:50
      Virtual coffee break, breakout sessions for discussions in small groups 30m
    • 10:50 11:10
      Cryo-FIB-SEM for the characterisation of biomaterials: potentiality and challenges 20m
      Speaker: Dr Luca Bertinetti
    • 11:10 11:30
      High-Throughput Electron Tomography - Challenges, Strategies, and Applications 20m
      Speaker: Martin Schorb
    • 11:30 11:50
      High-throughput screening of mitotic mammalian cells for electron microscopy using classic histological dyes 20m

      The identification and investigation of rare or fast events by electron microscopy can be a challenging task. The identification of cells in metaphase or early anaphase might be comparable to finding a needle in a haystack as only about 10 % of cells in culture are usually in mitosis. In addition, only a fraction of those cells is in the desired phase of cell division, and this specific stage usually lasts only a few minutes. We introduced a new workflow that allows screening and selection of mammalian cells in mitosis prior to subsequent electron microscopy. We mainly describe four improved steps of specimen preparation. Firstly, we describe a method to efficiently enrich mammalian cells and attach them to sapphire discs; secondly, we report on the use of 3D-printed containers to seed cells on coated sapphire discs for high-pressure freezing (HPF); thirdly, we take advantage of specimen carriers that allow for an upside-down placing of the sapphire discs without a second carrier or spacer ring to close the “sandwich” for HPF; and fourthly, we use histological dyes to stain DNA/chromatin during freeze-substitution. Out of 14 tested histological dyes, four of them where especially well-suited for visual inspection of mitotic cells by light microscopy. Applying this streamlined workflow, HeLa or RPE-1 cells at different stages of mitosis were selected for further ultrastructural analysis. This method can be applied routinely to already existing sample preparation protocols.

      Speaker: Gunar Fabig (TU Dresden)
    • 11:50 12:10
      Infos about PANOS 2022 and Farewell 20m