3D Imaging of Biological Specimens by Electron Microscopy

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1 3D Imaging of Biological Specimens by Electron Microscopy Focus on Microscopy Hannover, May 2010 Elke Spiess, Wim Voorhout and Wim Busing FEI Company

2 The world of decreasing dimension From van Leeuwenhoek era to today From millimeter down to sub nanometer From organisms to macromolecules From 2D to 3D information From static to dynamic 2

3 3D information Confocal Light Microscopy (CLSM) For looking at thick sections, whole cells or tissue using fluorescent probes Serial sectioning (TEM) build up a 3D image from consecutive sections SDB Slice & View removing thin layers and block face imaging Tomography (TEM) acquisition of a series of images at different tilts Single Particle reconstruction acquire many images of isolated structures 3

4 How to get there? Source: SDI Lab Analytical and Life Science Instrument Market DNA/Genes Proteins Cells Organ/Systems Health 1. (Cryo) - Electron Tomography 2. Single Particle Analysis 3. Template Matching Segmentation High resolution Small volume Low resolution Large volume 4

5 Xplore3D Electron Tomography - Four Steps Acquisition 1 Reconstruction Alignment Visualization Courtesy: Dr. Kobayashi, National Institute of Advanced Industrial Science and Technology, Osaka, Japan 5

6 Rota Virus (negative stain) Tomography Volume rendering using ResolveRT 6 6

7 HIV Infected Cells (epon fixed) Tomography Aligned Data Set 3D Reconstruction nm epon sections from HIV infected cells. Data collected on Tecnai Spirit BioTwin Courtesy: Robert-Koch Institute, Berlin

8 How to get there? The Structural Biology Playground Source: SDI Lab Analytical and Life Science Instrument Market DNA/Genes Proteins Cells Organ/Systems Health 2D 3D 660x 6,600x 1. (Cryo) - Electron Tomography 2. Single Particle Analysis 60,000x 3. Template Matching Segmentation High resolution Small volume Low resolution Large volume 10

9 Cryo EM Imaging at cryo temperatures To observe structures as close as possible to their natural state! 2D 3D 660x 6,600x 38,000x 11

10 Cryo EM: Noisy and Ugly?? NOT AT ALL!!! 12 Herpes virus capsid Deng et al. J. Virol. 81: (2007)

11 Examples: Cryo-Electron Tomography Direct visualization of a bacteriophage-like portal in the Kaposi s sarcoma-associated herpesvirus capsid by cryo-electron tomography 3D reconstruction Hexons Portal Penton 50nm 13 Deng et al. J. Virol. 81: (2007)

12 3D Reconstruction of SARS Virus on Tecnai G 2 T20 at 200kV SARS CoV 3D structure determined by single particle analysis Beniac, et al. PLoS ONE 2(10): e1082 (2007) and Nat. Stru. Mol. Bio. 13(8), (2006) National Microbiology Laboratory, Public Health Agency of Canada

13 SPA Tomography (Hybrid method) 3D reconstruction of 80S ribosome fragment Recording cryo tomogram of in-situ ribosomerich area 254 particles of which tomograms have been taken are selected Particle averaging on tomograms increases image information Imaged at TU/e Titan Krios Jason Pierson et al 17

14 Single Particle Resolution Why? Secondary Structure Elements at different resolutions Segment extracted from the atomic model of HK97 capsid protein. An alpha-helix and a betahairpin joined together by a loop and filtered to different resolutions. At 4Å resolution, strands in the b-hairpin begin to separate, the pitch of the a-helix becomes visible and bulky side chains can start to be seen. At 2Å resolution, the hole in each aromatic ring is resolved (red arrow). 19 Towards atomic resolution structural determination by single-particle cryo-electron microscopy Current Opinion in Structural Biology, Volume 18, Issue 2, April 2008, Pages Z Hong Zhou

15 Titan Krios Next generation TEM Automation in a box Especially designed for high throughput cryo electron microscopy experiments Autoloader for instant loading of 12 autogrids Dual axis tilt holder (+/- 70 degrees) Parallel illumination minimizes magnification errors during tomography - additional condenser lens High resolution at low temperature High stability lenses (constant power, regardless of lens setting) Upgradable with Cs aberration corrector Thermal and acoustic shielding by box (room requirements less stringent) 20

16 Conclusions Single Particle Best resolution for macro molecular machines/viruses Only works for identical structures 3.3A Cryo-EM Structure of a Non-enveloped Virus Reveals a Priming Mechanism for Cell Entry Xing Zhang,1 Lei Jin,1 Qin Fang,2 Wong H. Hui,3 and Z. Hong Zhou1,3,* 1. Department of Microbiology, Immunology & Molecular Genetics, University of California, Los Angeles, Los Angeles, CA ,USA 2. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei , China, 3. California Nano-Systems Institute, University of California, Los Angeles, Los Angeles, CA , USA Cell 141, 1 11, April 30,