Electron Microscopy

EM microtome with Jemima.jpg

The electron microscopy (EM) laboratory is a collaborative unit, specialising in cell biological EM since the opening of the LMCB in 1993. With state-of-the-art specimen preparation and imaging technologies, and two experienced researchers, we support the needs of the LMCB groups in all their EM research.

We have experience using a wide range of electron microscopy techniques as well as experience working with a broad range of model organisms.

We provide a full range of support for LMCB researchers throughout the entire timeline of their projects, from planning experiments through to training, sample preparation, imaging and data analysis. The support given is tailored to each project, dependent on the technical difficulty of the experiments and the experience of the researcher. This can vary from advice and technical support for the more experienced, full training in required techniques and the equipment use for the less experiences researchers or the undertaking of the EM projects ourselves, a collaborative endeavor as part of the ongoing research.

Should you need any further information please contact us.

Dr. Jemima Burden (Head of Electron Microscopy)

Dr. Ian White

Electron Microscopy Publications                                                                                                                        

 

Equipment:

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  • FEI T12 Tecnai Spirit biotwin with tomography, LaB6, and SIS morada 11megapixel digital camera
  • Phillips CM10 coupled to a 1kx1k Gatan digital camera (tungsten).
  • Leica EMPACT high pressure freezer
  • Leica automated freeze substitution machine AFS
  • Leica automated freeze substitution machine AFS 2 with FSP (for programmable solution transfer)
  • Leica UC7 ultramicrotome with HD camera and live view monitor
  • Leica UCT ultramicrotome
  • Leica UCT cryo-ultramicrotome
  • BioRad 3100 critical point dryer
  • Baltec Med020 for rotary shadowing, carbon/platinum coating and freeze drying.
  • PC Workstations with image analysis software, tomography reconstruction and rendering software are also in place.

Techniques:

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  • Conventional aldehyde fixation and resin embedding
  • High pressure freezing with freeze substitution
  • Tokuyasu cryo preparation
  • Room temperature sectioning: semi-thins, ultra-thins, serial sectioning
  • Cryo-sectioning
  • Whole mounts
  • Rip-offs
  • Pre-embedding immunolabelling
  • Correlative Light and Electron microscopy (CLEM): organism, cellular and organelle levels. XY and XZ orientations.
  • 3D EM: Reconstruction of tilt tomograms, TEM images of serial sections, Serial Block Face SEM images or Focused ion beam SEM images.

Model Organisms:

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  • virus infected cells
  • primary and immortalized cell cultures
  • brain tissue
  • S. cerevisae 
  • S. pombe
  • C. elegans
  • D. melanogaster (embryos, retina, imaginal discs, thorax epithelium)
  • Danio rerio

Publications:


Ribeiro S, Napoli I, White IJ, Parrinello S, Flanagan AM, Siter U, Parada LF, Lloyd AC. Injury signals cooperate wtih Nf1 loss to relieve the tumor-suppressive environment of adult peripheral nerve. Cell Rep. 2013 Oct 17;5(1):126-36.

Fuchs O, Abitbol K, Burden JJ, Mercer A, Brown L, Iball J, Anne Stephenson F, Thomson AM, Jovanovic JN. GABA(A) receptors can initiate the formation of functional inhibitory GABAergic synapses. Eur J Neurosci. 2013 OCT;38(8):3146-58

Marra V, Burden JJ, Thorpe JR, Smith IT, Smith SL, Hausser M, Branco T, Staras K. A preferentially segregated recycling vesicle pool of limited size supports neurotransmission in native central synapses. Neuron. 2012 Nov8;76(3):579-89

Orenbuch A, Shalev L, Marra V, Sinai I, Lavy Y, Kahn J, Burden JJ, Staras K, Gitler D. Synapsin selectively controls the mobility of resting pool vesicles at hippocampal terminals. J Neurosci. 2012 Mar 21;32(12):3969-80.

Ratnayaka A, Marra V, Bush D, Burden JJ, Branco T, Staras K. Recruitment of resting vesicles into recycling pools supports NMDA receptor-dependent synaptic potentiation in cultured hippocampal neurons. J Physiol. 2012 Apr 1;590(Pt 7):1585-97.

Napoli I, Noon LA, Ribeiro S, Kerai AP, Parrinello S, Rosenberg LH, Collins MJ, Harrisingh MC, White IJ, Woodhoo A, Lloyd AC. A central role for the ERK-signaling pathway in controlling Schwann cell plasticity and peripheral nerve regeneration in vivo. Neuron. 2012 Feb 23;73(4):729-42.           

Vitureira N, Letellier M, White IJ, Goda Y. Differential control of presynaptic efficacy by postsynaptic N-cadherin and β-catenin Nat Neurosci. 2011 Dec 4;15(1):81-9.

Nightingale TD, White IJ, Doyle EL, Turmaine M, Harrison-Lavoie KJ, Webb KF, Cramer LP, Cutler DF.  Actomyosin II contractility expels von Willebrand factor from Weibel-Palade bodies during exocytosis.  J Cell Biol. 2011 Aug 22;194(4):613-29.

Willenborg C, Jing J, Wu C, Matern H, Schaack J, Burden J and Prekeris R. Interaction between FIP5 and S_X18 Regulates Epithelial Lumen Formation. Journal of CellBiology 2011

Llobet A, Gallop J, Burden J, Camdere G, Chandra P, Vallis Y, Hopkins C, Lagnado L, and McMahon H. Endophilin Drives the Fast Mode of Vesicle Retrieval in a Ribbon Synapse. Journal of Neuroscience 2011 Jun 8: 8512-9.

Henry AG, White IJ, Marsh M, von Zastrow M, Hislop JN. The role of ubiquitination in lysosomal trafficking of ∂-opioid receptors. Traffic. 2011 Feb;12(2):170-84.

Jing J, Junutula JR, Wu C, Burden J, Matern H, Peden AA, Prekeris R. FIP1/RCP  binding to Golgin-97 regulates retrograde transport from recycling endosomes to thetrans-Golgi network. Mol Biol Cell. 2010 Sep;21(17):3041-53.

Pears MR, Codlin S, Haines RL, White IJ, Mortishire-Smith RJ, Mole SE, Griffin JL. Deletion of btn1, an orthologue of CLN3, increases glycolysis and perturbs amino acid metabolism in the fission yeast model of Batten disease. Mol Biosyst.  2010 Jun 18;6(6):1093-102

Staras K, Branco T, Burden JJ, Pozo K, Darcy K, Marra V, Ratnayaka A, Goda Y.  A vesicle superpool spans multiple presynaptic terminals in hippocampal neurons.Neuron. 2010 Apr 15;66(1):37-44.

Eden ER, White IJ, Tsapara A, Futter CE. Membrane contacts between endosomes and ER provide sites for PTP1B-epidermal growth factor receptor interaction. Nat Cell Biol. 2010 Mar;12(3):267-72.

Georgiou M, Marinari E, Burden J, Baum B. Cdc42, Par6, and aPKC regulate Arp2/3-mediated endocytosis to control local adherens junction stability. Curr Biol. 2008 Nov 11;18(21):1631-8.

Schonteich E, Wilson GM, Burden J, Hopkins CR, Anderson K, Goldenring JR, Prekeris R. The Rip11/Rab11-FIP5 and kinesin II complex regulates endocytic protein recycling. J Cell Sci. 2008 Nov 15;121(Pt 22):3824-33.

Codlin S, Haines RL, Burden JJ, Mole SE. Btn1 affects cytokinesis and cell-wall deposition by independent mechanisms, one of which is linked to dysregulation of vacuole pH. J Cell Sci. 2008 Sep 1;121(Pt 17):2860-70.

Silva EA, Burden J, Franc NC. In vivo and in vitro methods for studying apoptotic cell engulfment in Drosophila. Methods Enzymol. 2008;446:39-59.

Silva E, Au-Yeung HW, Van Goethem E, Burden J, Franc NC. Requirement for a Drosophila E3-ubiquitin ligase in phagocytosis of apoptotic cells. Immunity. 2007 Oct;27(4):585-96.

Zenner HL, Collinson LM, Michaux G, Cutler DF. High-pressure freezing provides insights into Weibel-Palade body biogenesis. J Cell Sci. 2007 Jun 15;120(Pt 12):2117-25.

Darcy KJ, Staras K, Collinson LM, Goda Y. An ultrastructural readout of fluorescence recovery after photobleaching using correlative light and electron microscopy. Nat Protoc. 2006;1(2):988-94.

Harrison-Lavoie KJ, Michaux G, Hewlett L, Kaur J, Hannah MJ, Lui-Roberts WW, Norman KE, Cutler DF. P-selectin and CD63 use different mechanisms for delivery to Weibel-Palade bodies. Traffic. 2006 Jun;7(6):647-62.

Darcy KJ, Staras K, Collinson LM, Goda Y. Constitutive sharing of recycling synaptic vesicles between presynaptic boutons. Nat Neurosci. 2006 Mar;9(3):315-21.

Millán J, Hewlett L, Glyn M, Toomre D, Clark P, Ridley AJ. Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains. Nat Cell Biol. 2006 Feb;8(2):113-23.

Michaux G, Abbitt KB, Collinson LM, Haberichter SL, Norman KE, Cutler DF. The  physiological function of von Willebrand's factor depends on its tubular storage inendothelial Weibel-Palade bodies. Dev Cell. 2006 Feb;10(2):223-32.

Pinal N, Goberdhan DC, Collinson L, Fujita Y, Cox IM, Wilson C, Pichaud F. Regulated and polarized PtdIns(3,4,5)P3 accumulation is essential for apical membrane morphogenesis in photoreceptor epithelial cells. Curr Biol. 2006 Jan 24;16(2):140-9.

Lui-Roberts WW, Collinson LMHewlett LJ, Michaux G, Cutler DF. An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade  bodies of endothelial cells. J Cell Biol. 2005 Aug 15;170(4):627-36.

Garcia E, Pion M, Pelchen-Matthews A, Collinson L, Arrighi JF, Blot G, Leuba F, Escola JM, Demaurex N, Marsh M, Piguet V. HIV-1 trafficking to the dendritic cell-T-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse. Traffic. 2005 Jun;6(6):488-501.

Signoret N, Hewlett L, Wavre S, Pelchen-Matthews A, Oppermann M, Marsh M. Agonist-induced endocytosis of CC chemokine receptor 5 is clathrin dependent. Mol Biol Cell. 2005 Feb;16(2):902-17.

Burnett C, Makridou P, Hewlett L, Howard K. Lipid phosphate phosphatases dimerise, but this interaction is not required for in vivo activity. BMC Biochem. 2004 Jan 16;5:2.

Andrawiss M, Takeuchi Y, Hewlett L, Collins M. Murine leukemia virus particle  assembly quantitated by fluorescence microscopy: role of Gag-Gag interactions and membrane association. J Virol. 2003 Nov;77(21):11651-60.

Hannah MJ, Hume AN, Arribas M, Williams R, Hewlett LJ, Seabra MC, Cutler DF. Weibel-Palade bodies recruit Rab27 by a content-driven, maturation-dependent mechanism that is independent of cell type. J Cell Sci. 2003 Oct 1;116(Pt 19):3939-48.

Michaux G, Hewlett LJ, Messenger SL, Goodeve AC, Peake IR, Daly ME, Cutler DF. Analysis of intracellular storage and regulated secretion of 3 von Willebrand disease-causing variants of von Willebrand factor. Blood. 2003 Oct 1;102(7):2452-8.

Hannah MJ, Williams R, Kaur J, Hewlett LJ, Cutler DF. Biogenesis of Weibel-Palade bodies. Semin Cell Dev Biol. 2002 Aug;13(4):313-24.

Connolly JO, Simpson N, Hewlett L, Hall A. Rac regulates endothelial morphogenesis and capillary assembly. Mol Biol Cell. 2002 Jul;13(7):2474-85.

Futter CE, Pearse A, Hewlett LJ, Hopkins CR. Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly  withlysosomes. J Cell Biol. 1996 Mar;132(6):1011-23.