Bijvoet Center Utrecht University
Heidelberglaan 8, 3584 CH Utrecht
Harry Heijnen and Willie Geerts
+31 (0)88 7557654 / +31 (0)30 2532885
Traditionally transmission electron microscopy (TEM) uses ultra-thin sections of chemically fixed and plastic-embedded cells or tissues to observe the subcellular structure and membrane dynamics of organelles. Chemical fixation of samples does not perfectly preserve the ultrastructure of organelles. To circumvent this limitation, physical immobilization approaches (vitrification by high pressure freezing or plunging) have been developed. Cryo-electron microscopy tomography (cryo-ET) is becoming a mainstream technology for studying membrane architecture and high-order macromolecular complexes in their close to native cellular environment at nm resolution. The major advantage of the physical fixation method is that it immobilizes cellular structures within milliseconds, which is much faster than conventional chemical fixation procedures. In addition, over the past decade real-time imaging and 3D electron tomography have increasingly replaced classical static light microscopy imaging and 2D electron microscopy. In our hotel approaches are being developed to combine light microscopy with high-resolution (cryo) electron tomography (Correlative Light and Electron Microscopy, CLEM). CLEM utilizes complementary visual techniques that allow capturing dynamic cellular processes by (immuno) fluorescence and then zooms in on these events to identify their ultrastructure by EM tomography. 3D reconstruction techniques allow snap-frozen structures to be reassembled visually into 3D models thereby providing detailed z-axis information. The Cryo-Electron Microscopy Unit harbors the latest instrumentation in FIB-SEM, cryo-EM and tomography technology, 3D reconstruction software and data analysis, all serving the needs of strategic themes in life sciences, including the analyses of biomolecules at the atomic level.
- Biomedical & health
- Cellular dynamics and organization in Bacteria, Yeast, Cell cultures
- Visualization of viral and bacterial outer membrane protein complexes
- Platelet adhesion dynamics and secretion
- Autophagy and red cell development
- Characterization of Intercellular signaling devices - Microvesicles and exosomes
Expertise and Track Record
We have a broad expertise in applying immuno and cryo EM, 3D electron tomography and correlative methods in life sciences and numerous collaborations (see references) with national and international groups. Our expertise includes 2D and 3D immunogold applications (frozen sections, whole mounts), Cryo-EM of whole cells, Correlative IF and cryo-EM (iCorr).
- Galina V. Beznoussenko, Sergei S. Pilyugin, Willie J.C. Geerts, Michael M. Kozlov, Koert N.J. Burger, Alberto Luini, Jure Derganc, Alexander A. Mironov. Trans-Membrane Area symmetry Controls the Shape of Cellular Organelles. International journal of Molecular Sciences (2015) Vol. 16.
- Eckly A, Heijnen H, Pertuy F, Geerts W, Proamer F, Rinckel JY, Léon C, Lanza F, Gachet C.. Biogenesis of the demarcation membrane system (DMS) in megakaryocytes. Blood (2014) Vol. 123(6):921-30.PMID:24152908.
- Hoenscher, C., Mari, M., Auffarth, K., Bohner, M., Griffith, J., Geerts, W. van der Laan, M., Carbrera, M., Reggiori, F., Ungermann, C. Cellular metabolism regulates contact sites between vacuoles and mitochondria. Developmental Cell, (2014), Vol. 30 (1) 86-94
- Muriel Mari, Willie J.C. Geerts and Fulvio Reggiori. Immuno- and correlative light microscopy-Electron tomography methods for 3D protein localization in yeast. Traffic (2014) Vol. 15 (10), 1164-1178
- Kondylis V, van Nispen Tot Pannerden HE, van Dijk S, Ten Broeke T, Wubbolts R, Geerts WJ, Seinen C, Mutis T, Heijnen HF. Endosome-mediated autophagy: an unconventional MIIC-driven autophagic pathway operational in dendritic cells. Autophagy (2013). Vol. 6: 861-880. PMID: 23481895.
- Van Nispen tot Pannerden HE, Geerts WJ, Kleijmeer MJ, Heijnen HF. Spatial organization of the transforming MHC class II compartment. Biology of the cell (2010). Vol. 102(11):581-91.PMID: 20712599.
- Van Nispen tot Pannerden H, de Haas F, Geerts W, Posthuma G, van Dijk S, and Heijnen HF The platelet interior revisited: electron tomography reveals tubular alpha-granule subtypes. Blood (2010). Vol. 116(7):1147-56. PMID: 20439620.
- Delevoye C., Hurbain I., Tenza D., Sibarita J.B., Uzan-Gafsou S., Ohno H., Geerts W.J.C., Verkleij, A.J., Salamero J., Marks M.S., and Raposo G. AP-1 and KIF13A coordinate endosomal sorting and positioning during melanosome biogenesis. J. Cell Biology (2009),187: 247–264
- Zeuschner D., Geerts W.J.C., van Donselaar E.G., Slot J,, Koster A.J., Klumperman J. Immuno-electron tomography reveals the existence of free COPII-coated vesicular and tubular transport carriers. Nature Cell Biology (2006) Vol. 8 Nr 4 pp 377–383
- M. Grabenbauer, W.J.C.Geerts, J. Fernadez-Rodriguez, A. Honger, A.J. Koster and T. Nilsson. Correlative microscopy and electron tomography of GFP through photooxidation Nature Methods (2005) Vol. 2 No.11 pp 857–862
- 2 Scientific Staff Members
- 1 Post-doc
- 1 Technical Staff Member
- Tecnai 12 Twin TEM with integrated LED-based fluorescence microscope (iCorr) / Tecnai 20 LaB6 with 4K CCD camera for BF and DF tomography / Talos F200X with Ceta 16 M camera and Super-X G2 EDS detector / Talos Arctica with Falcon 3 direct detection camera / Cryo-TEM holders/ Vitrobot / High Pressure Freezer / Helios Cryo-FIB-SEM / Nova 600 NanoLab / XL30S FEG
Raw and Final data will be stored in the Electron Microscopy Pilot Image Archive (http://www.ebi.ac.uk/pdbe/emdb/empiar/deposit), and in the EM Databank : http://www.emdatabank.org/deposit.html