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

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

4

DNA/Genes Proteins Cells Organ/Systems Health

High resolutionSmall volume

Source: SDI Lab Analytical and Life Science Instrument Market ’06-’10

How to get there?

1. (Cryo) - Electron Tomography2. Single Particle Analysis

3. Template MatchingSegmentation

Low resolutionLarge volume

5

Acquisition

Alignment

Reconstruction

Visualization

Electron Tomography - Four Steps

Courtesy: Dr. Kobayashi, National Institute of Advanced Industrial Science and Technology, Osaka, Japan

1

2 4

3

Xplore3D

6 6

Volume rendering using ResolveRT

Rota Virus (negative stain) Tomography

7 7

3D ReconstructionAligned Data Set

HIV Infected Cells (epon fixed) Tomography

250 nm epon sections from HIV infected cells.Data collected on Tecnai Spirit BioTwinCourtesy: Robert-Koch Institute, Berlin

10

DNA/Genes Proteins Cells Organ/Systems Health

Source: SDI Lab Analytical and Life Science Instrument Market ’06-’10

How to get there?

1. (Cryo) - Electron Tomography2. Single Particle Analysis

3. Template MatchingSegmentation

The Structural Biology Playground

6,600x 60,000x660x

2D 3D

High resolutionSmall volume

Low resolutionLarge volume

11

Cryo EM – Imaging at cryo temperatures

6,600x

38,000x660x

2D 3D

To observe structures as close as possible to their natural state!

12

NOT AT ALL !!!

Cryo EM: Noisy and Ugly??

Herpes virus capsidDeng et al. J. Virol. 81:3640-3644 (2007)

13

3D reconstruction

Portal

50nmPenton

Hexons

Direct visualization of a bacteriophage-like portal in the Kaposi’s sarcoma-associated herpesvirus capsid by cryo-electron tomography

Deng et al. J. Virol. 81:3640-3644 (2007)

Examples: Cryo-Electron Tomography

14 14

3D Reconstruction of SARS Virus on Tecnai G2

T20 at 200kV

Beniac, et al. PLoS ONE 2(10): e1082 (2007) and Nat. Stru. Mol. Bio. 13(8), 751–752 (2006)National Microbiology Laboratory, Public Health Agency of Canada

SARS – CoV 3D structure determined by single particle analysis

17

SPA – Tomography (Hybrid method)3D reconstruction of 80S ribosome fragment

Recording cryo tomogram of in-situ ribosome-rich 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

19

Single Particle Resolution – Why?Secondary Structure Elements at 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.

Segment extracted from the atomic model of HK97 capsid protein. An alpha-helix and a beta-hairpin joined together by a loop and filtered to different resolutions.

At 2Å resolution, the hole in each aromatic ring is resolved (red arrow).

Towards atomic resolution structural determination by single-particle cryo-electron microscopyCurrent Opinion in Structural Biology, Volume 18, Issue 2, April 2008, Pages 218-228 Z Hong Zhou

20

Titan Krios – Next generation TEM

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)

Automation in a box

22

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 90095-7364,USA 2. State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071, China, 3. California Nano-Systems Institute, University of California, Los Angeles, Los Angeles, CA 90095-7364, USA

Cell 141, 1–11, April 30, 2010.