3D Cryo-EM Workshop An Introduction to Electron Microscopy

A Brief History of Microscopes: Achieving the Best Resolution Resolution: The ability to see two closely spaced objects as separate. 1) Light Microscopes a) Antony van Leeuwenhoek (1632-1723) - 1695: Arcana Naturae Detecta (Secrets of Nature Revealed)

b)  Robert  Hooke’s  Micrographia (1665)

Compound Microscope

Cork Cells

Early microscope lenses suffered from spherical and chromatic aberrations.

Carl Zeiss and Ernst Abbe (1860’s  - 70’s)  in  Jena,  Germany   Spherical Aberration

Ernst Abbe

Achieving the Best Resolution Abbe’s  Formula  for  Resolution  (diffraction  limit,  d) • Want resolution (d) to be as small as possible: 0.61( ) d = n (sin ) , use blue light at 450 nm n, index of refraction, as large as possible , half angle of light cone (70o) n (sin ) = Numerical Aperture (NA) – usually stamped on the side of the objective lens (e.g. 1.30)

Best resolution for a good light microscope : 0.187 μm (0.2 μm)

Pursuit  of  Resolution  →  Berlin,  Germany

Inception of Electron Microscopy Early  1930’s:  Electromagnets  as  lenses  to  focus  electron  beams  

Ernst Ruska

Max Knoll

Bodo von Borries

The First Electron Microscope: Knoll and Ruska (1932)

1931: Ruska and Knoll build the first electron microscope with two lenses; resolution was no better than a light microscope.

1932: Knoll and Ruska report their work. 1934: Ruska employs a condenser lens.

1935: Driest and Muller modify Ruska’s  design  to  achieve  resolutions   better than a light microscope. 1938: von Borries and Ruska achieve 100 Ångstrøm resolution. 1938 First scanning transmission electron microscope developed (M. von Ardenne)

1939: Commercialization of the von Borries-Ruska TEM by Siemens and Halske. Resolution down to ~10 nm. ~1940: Basic theoretical work on electron optics and electro-magnetic lenses (W. Glaser, O. Scherzer) 1943: Electron energy-loss spectroscopy EELS (J. Hillier) 1948: First TEM at Swiss Federal Institute of Technology (ETH) Zurich Resolution down to ~2 nm.

1951: X-ray spectroscopy (R. Castaing) 1956: First lattice image (J. Menter) 1964: First commercial SEM by Cambridge Instruments

~1970: First High Resolution (HR)-TEM microscopes with a resolution better than 4 Å. 1986: Nobel prize for E. Ruska (together with G. Binning and H. Rohrer, who developed the Scanning Tunneling Microscope)

~2000: Development of aberration-corrected TEM (H. Rose, M. Haider, K. Urban) ~2003: Aberration-corrected HR-TEM microscopes; Resolution = sub-Ångstrøm

Transmission Electron Microscopy de Broglie

• Electrons have wavelength (λ) • Wavelength of an electron beam depends upon the potential, V, through which it has been accelerated.

• The greater the accelerating voltage, the smaller the λ. • According  to  Abbe’s  equation, the smaller the λ, the better the resolution. • TEM accelerating voltages range from 40 - 300 kV

=

=

h mv 12.3 −− √V

0.61( ) d= n (sin )

Å

Wide-Field Light Microscopy vs. Transmission Electron Microscopy

Nearly 85 Years of TEM Development

Ruska’s  Microscope  (1931): JEOL’s  New  JEM-ARM300F resolution no better than High Resolution 300 kV TEM (d = 63 pm) d = 0.2 μm = 200 nm (1 Angstrom = 100 pm)

LSU’s  New  JEOL  1400  TEM  Operating  at  120  kV;;  d ≈  0.20  nm  (2  Å)

LSU’s  new  Transmission  Electron  Microscope  (TEM):  CMS  Building Looking behind the column during installation, before the  panels  were  added… …a  bit  more  complicated than  Ruska’s  instrument!!

Rule of Thumb: 80 – 120 kV for biological applications (samples)

120 – 300 kV for materials applications

The Electron Beam Travels Though a High Vacuum Cathode

10-7 - 10-10 mbar

IGP

Ion getter pump

10-5 - 10-7 mbar

Specimen holder IGP

Turbo molecular pump Oil diffusion pump

TMP

10-0 - 10-2 mbar Rotary pump

Viewing screen RP

Atmosphere: 1000 mbar

TEM for Biological Applications 1) Classic thin sections of cells and tissues - 1950’s  &  60’s,  a  time  of  great exploration/discovery George Palade Keith Porter (Porter-Blum ultra-microtome) Hans Ris, Morris Karnovsky, etc. - Structure/Function 2) Immunogold labeling 3) Nucleic acid (DNA and RNA) spreading 4) Negative staining 5) Cryo-EM

1) Thin sections for TEM

TEM is an art!!

1) Thin sections for TEM

Copper Grids: 3 mm diam.

Four Grid Holder for JEM 1400

Lead  and  uranium  salts  as  “stains”  to  deflect electrons and thus provide a contrasting image.

TEM of a root tip cell.

1) Thin sections for TEM Mitochondrion with its ATP Synthases almost discernible

rough Endoplasmic Reticulum studded with ribosomes

Golgi Stacks Centriole

2) Immuno-Electron Microscopy

In the peroxisomes

Protein A from Staphylococcus aureus Fig. 9-29a

2) Immuno-Electron Microscopy

The gold particles are electron dense, so they deflect electrons.

3) Nucleic Acid Spreads Miller and Beatty, 1969 First to visualize actively transcribing genes by TEM. “Miller’s  Christmas  Trees”

with  “ornaments” “It  is  arguably  one  of  the  best   known biological images produced in the last 40 years, amply  demonstrating  that  ‘a picture is worth a thousand words.’”              - Joe Gall, 2001

Basic Chromatin Structure in Eukaryotes Classic  “beads-on-astring”  11  nm  fiber Each bead is a Nucleosome 146 base pairs of DNA wrap around the core protein particle nearly twice. 40-60 base pairs link one bead to the next

R-Looping  with  the  TEM,  late  1970’s:   - First Indications of Pre-mRNA Splicing Nuclear Pre-mRNA for β-globin Exon 1

Intron

Exon 2

Cytoplasmic (spliced) β-globin mRNA Exon 1

Exon 2

From Beyer, Bouton and Miller, 1981

Pre-mRNA splicing occurs cotranscriptionally

Ann Beyer, U. of Virginia

4) Negative Staining - with uranyl acetate, phosphotungstic acid, osmium tetroxide, etc. Bacteriophage T4

Rabbit Hemorrhagic Disease Virus

Nuclear Pore Complexes - Note their 8-fold rotational symmetry

Ebola

Virus Particles

5) Cryo-TEM

The Ribosome

The Kinetochore

TEM for Materials Applications

Catalytic nano-crystals From Younan Xia at Georgia Tech

LSU’s  nano-materials viewed with the 1400 at 120 kV

Generating and Focusing the Beam

Electron – specimen interactions Primary electrons (E0) Backscattered electrons (E=E0)

Elastic (higher angle, E=E0)

Inelastic (low angle, E=E0-ΔE) Unscattered (E=E0)

Electron – specimen interactions Primary electrons

Backscattered electrons X-rays

Secondary electrons Cathode luminescense

SEM analysis

Heat

Auger electrons Specimen

Interaction volume

TEM analysis Elastically scattered electrons Inelastically scattered electrons

Unscattered electrons

Rough Endoplasmic Reticulum (rER) - Ribosomes attached to membranes made of phospholipids. - Post-fixed with osmium tetroxide (phospholipids). - Stained with uranyl acetate and lead citrate.

No heavy metal staining: Primary electron beam

Plastic embedded specimen

phospholipids

ribosomes

Objective aperture

Signal Intensity Specimen profile

With  heavy  metal  “staining”:  Scattering  of  electrons Primary electron beam

…Heavy  metal  ions

Plastic embedded specimen

phospholipids

ribosomes

Objective aperture

Signal Intensity Specimen profile

Membranes and Ribosomes appear dark.

Image Capture with Gatan’s CCD cameras UltraScan (bottom-mount) and ORIUS (side-mount) cameras

Fiber Optical Coupling

keV electrons

scintillator

fiber-optical coupling CCD detector

The ORIUS Camera

Scanning Electron Microscopy

Scanning EM:

Cilia projecting from hair cell of inner ear

• Surface features • 0.5 – 30 kV • 1 nm resolution.

• Tissue: fixed, criticalpoint dried (freeze-dried), coated with a thin layer of gold or platinum. • Electron beam scans coated specimen. • Secondary electrons are collected by detector. • Forms 3D images.

Primary electrons Backscattered electrons X-rays

Secondary electrons Cathode luminescense Heat

Auger electrons Specimen

SEM analysis

Interaction volume

TEM analysis Elastically scattered electrons Inelastically scattered electrons Unscattered electrons

Imaging in the scanning electron microscope Scanning and signal detection …Primary  electron beam

secondary electrons

Imaging in the scanning electron microscope

Secondary electron detector Primary electrons

+200-500V – Collector voltage +7-12kV HV

SE

Electrons

Photons

Photomultiplier

Electrons

Imaging in the scanning electron microscope Contrast formation in SEM using secondary electrons (SE) Different number of electrons from different spots of the specimen

Dependent on composition of the specimen topography of the specimen acceleration voltage of primary electrons location of the detector

Imaging in the scanning electron microscope Contrast formation of biological objects Uncoated

Coated with 4 nm platinum

Primary electron beam

Primary electron beam

Platinum

Only few electrons escape from specimen Signal from a large volume (“unsharp, noisy” image)

Localization of the signal to the surface

Imaging in the scanning electron microscope Contrast based on SE - topography PE SE R

PE

SE R

SE

R

Specimen

Primary electrons Secondary electrons Excited volume

Imaging in the scanning electron microscope Contrast based on SE (Detector position)

Transmission Electron Microscopy • Electrons have wavelength (λ) • Wavelength of an electron beam depends upon the potential, V, through which it has been accelerated.

• The greater the accelerating voltage, the smaller the λ. • According  to  Abbe’s  equation, the smaller the λ, the better the resolution. • TEM acceleration voltages range from 40 - 300 kV