ELECTRON MICROSCOPYELECTRON MICROSCOPY

Presented by

sem.mov

Siddhartha Swarup JenaRAD/10-30

Ph.D. Mol. Bio & Biotech

IntroductionIntroduction

Microscopes magnify & resolve images

‘Its not how much they magnify that is key - but how well they resolve…’

Invented in 1930s, but not used much until after WW-II.

1932, produced the world's first transmission electron microscope (TEM).

German physicist Ernst Ruska and German electrical engineer Max Knoll constructed the prototype electron microscope.

S S Jena

Invention of EM

In 1932, invented by E. Ruska et al. In 1986, Ruska received the Nobel Prize.

S S Jena

Contd…

The transmission electron microscope (TEM) was the first type of Electron Microscope to be developed

The first scanning electron microscope (SEM) debuted in 1938 ( Von Ardenne) with the first commercial instruments around 1965.

Its late development was due to the electronics involved in "scanning" the beam of electrons across the sample.

S S Jena

Imaging with a simple lens

semi-angular aperture

object plane

image plane

<<< conjugate planes of focus >>>

axis of lens

S S Jena

Focal length

f

f = focal length of lens

parallel

rays of light

axis

back focal plane of lens >>>

Distance between center of lens and focal point is the focal length

S S Jena

Optics – A Simple Lens

S S Jena

Resolution

The limit of resolution of a microscope is the smallest distance between 2 points that can be seen using a microscope

This is a measure of the clarity of the image

As Magnification increases, resolution decreases.

Resolving power is inversely proportional to the wavelength of the radiation it uses

S S Jena

Resolution

dmin =0.61 n sin

n = refractive index = wavelength

d

note: resolving power independent of lens properties

: for green (500nm) light dmin = c. 0.2 µm

S S Jena

The Light Microscope

Series of lenses through which ordinary white light can be focused.

Optical microscopes can not resolve 2 points closer together than about half (0.45) the wavelength of the light used (450-600nm).

The total magnification is the eyepiece magnification multiplied by the objective magnification.

The maximum magnification of a light microscope is x1500 & resolution up to 0.2 m.

S S Jena

The Electron Microscope Electrons (negatively charged very small

particles) can behave as waves. The wavelength of electrons is about 0.005nm

Electrons are ‘fired’ from an electron gun at the specimen and onto a fluorescent screen or photographic plate

Electrons scatter when they pass through thin sections of a specimen

There are 2 major types of electron microscopy - transmission and scanning

Both focus an electron beam onto the specimen using electromagnets

S S Jena

Comparison of Optical and Electron MicroscopesComparison of Optical and Electron Microscopes

Electron microscopes are operated in vacuum because the mean free path of electrons in air is short – this mean biological samples should not degas – they can either be dehydrated or frozen

Electron microscopes have higher resolution than optical microscopes – atomic resolution is possible.

Chemical imaging and spectroscopy – mapping π and σ bonds at 1nm resolution can be done.

S S Jena

Why high vacuum ?

Mean free path of electron is very short in air

Tungsten filaments burn out in air

Columns must be kept dust free

Achieved by two fold pumping:

Rotary (mechanical) pump + Diffusion pump or + turbo pump

S S Jena

S S Jena

In transmission EM the electrons pass through the specimen

Specimen needs to be extremely thin - 10nm to 100nm

TEM can magnify objects up to 500 000 times

TEM has made it possible to see the details of interior views and discover new organelles

Cells or tissues are killed and chemically ‘fixed’ in a complicated and harsh treatment

Transmission Electron Transmission Electron

Microscope (TEM)Microscope (TEM)

S S Jena

TEM contd…

Has a resolution 1000 times better than light microscope (0.2nm)

Transmitted electrons (those that do not scatter) are used to produce image

Denser regions in specimen, scatter more electrons and appear darker

S S Jena

The process

Electrons are emitted by an electron gun, commonly fitted with a tungsten filament cathode

Electric field accelerates Magnetic (and electric) field control path

of electrons Electron wavelength @ 200KeV 2x10-

12 m Resolution normally achievable @

200KeV 2 x 10-10 m 2Å

S S Jena

Components of a Transmission Components of a Transmission MicroscopeMicroscope

Thermionic Gun:

Electron source.

Triode or self-biasing gun

W, LaB6, CeB6

If misaligned, low intensity & other alignments may also be out

S S Jena

Electron gun

Brightness = electron current by a source with unit area and unit solid angle

Bias (Wehnelt)Cylinder

Filament (20-100 KV)

Anode

stream of electrons originating from outer shell of filament atoms

S S Jena

Lenses

Provide means to (de)focus the electron beam on the specimen, to focus the image, to change the magnification, and to switch between image and diffraction

Electromagnetic lenses are based on the fact the moving electrons are forced into a spiral trajectory, i.e. focused into one point

S S Jena

The electromagnetic lens

Works at fixed focal distance and variable focal length.

(like the human eye lens but unlike light optics)

windings

soft iron pole piece

windings

e-

electrons are charged, and are therefore deflected when they cross a magnetic field

S S Jena

Lens systema) Condenser lens: Uniformly illuminate the sample. Usually 2; C1 and C2 lens If misaligned, we will lose the beam when changing

magnification

b) Objective lens: Image sample – determines resolution. If misaligned, the image will be distorted, blurry.

c) Projector lens: magnifies image/ forms diffraction pattern – should not alter

resolution. If misaligned, the image will be distorted, diffraction pattern

may be blurry.S S Jena

S S Jena

Mechanistic View of TEM

S S Jena

S S Jena

Sample preparation

1. Chemical fixation: Proteins with formaldehyde and glutaraldehyde and

lipids with osmium tetroxide.

2. Cryofixation: Freezing a specimen so rapidly, to liquid nitrogen or even

liquid helium temperatures, that the water forms vitreous (non-crystalline) ice.

3. Dehydration: Freeze drying, or replacement of water with organic

solvents such as ethanol or acetone, followed by critical point drying or infiltration with embedding resins.

S S Jena

Contd…4. Embedding The tissue is passed through a 'transition solvent' such as

epoxy propane and then infiltrated with a resin such as Araldite epoxy resin

Tissues may also be embedded directly in water-miscible acrylic resin

After the resin has been polymerised (hardened) the sample is thin sectioned (ultrathin sections) and stained - it is then ready for viewing.

5. Sectioning These can be cut on an ultramicrotome with a diamond

knife to produce ultrathin slices about 60-90 nm thick. Disposable glass knives are also used because they can be

made in the lab and are much cheaper. S S Jena

6. Staining Uses heavy metals such as lead,

uranium or tungsten to scatter imaging electrons and thus give contrast between different structures, since many biological materials are nearly "transparent" to electrons (weak phase objects).

Contd…

S S Jena

Freeze-fracture or freeze-etch

A preparation method particularly useful for examining lipid membranes and their incorporated proteins in "face on" view.

The fresh tissue or cell suspension is frozen rapidly (cryofixed), then fractured by simply breaking or by using a microtome while maintained at liquid nitrogen temperature.

The cold fractured surface is then shadowed with evaporated platinum or gold at an average angle of 45° in a high vacuum evaporator.

A second coat of carbon, evaporated perpendicular to the average surface plane is often performed to improve stability of the replica coating.

S S Jena

The specimen is returned to room temperature and pressure, then the extremely fragile "pre-shadowed" metal replica of the fracture surface is released from the underlying biological material by careful chemical digestion with acids, hypochlorite solution or SDS detergent.

The still-floating replica is thoroughly washed from residual chemicals, carefully fished up on fine grids, dried then viewed in the TEM.

Contd…

S S Jena

TEM images

Transmission electron micrograph of epithelial cells

from a rat small intestine. Scale bar = 5 mm.

TEM view of a plant cell

S S Jena

TEM Limitations

Specimen dead. Specimen preparation uses

extreme chemicals so artifacts are always a concern.

S S Jena

Live specimens possible. No sectioning is required.

Magnify objects up to two million times.

Lower magnifications than the TEM.

Resolving power is about 20nm

In Scanning EM microscopes the electrons bounce off the surface of the specimen

Produce images with a three-dimensional appearance

Allow detailed study of surfaces.

Scanning Electron Microscope Scanning Electron Microscope

(SEM)(SEM)

S S Jena

S S Jena

Schematic view of SEM

S S Jena

S S Jena

Sample preparation

All water must be removed from the samples because the water would vaporize in the vacuum.

All metals are conductive and require no preparation before being used.

All non-metals need to be made conductive by covering the sample with a thin layer of conductive material.

This is done by using a device called a "sputter coater.“

E.g. gold coating

S S Jena

SEM images

Tumor spheroid

Insect head

S S Jena

TEM Vs SEM images

TEM- interior SEM- surfaceS S Jena

EM VariationsEM Variations

High Voltage TEM Scanning tunneling microscope Scanning transmission electron

microscope (STEM) Scanning probe microscope Atomic force microscope Environmental scanning electron

microscope Elemental Composition SEM

S S Jena

ApplicationsApplications

Morphology (imaging) Crystal structures (diffraction) Protein localization Electron & Cellular tomography Toxicology Biological production and viral load monitoring Particle analysis Materials qualification Structural biology Virology Forensics Mining (mineral liberation analysis)

S S Jena

Disadvantages of EMDisadvantages of EM

Expensive to build and maintain

Requires extremely stable high-voltage supplies, extremely stable currents to each electromagnetic coil/lens, continuously-pumped high- or ultra-high-vacuum systems, and a cooling water supply circulation through the lenses and pumps.

As they are very sensitive to vibration and external magnetic fields, must be housed in stable buildings (sometimes underground) with special services such as magnetic field cancelling systems.

The samples largely have to be viewed in vacuum

S S Jena

Light Vs Electron Microscopes

Feature Light Microscope Electron Microscope

Radiation used

Radiation source

Nature of lenses

Lenses used

Image seen

Radiation medium

Magnification

Limit of resolution

What it can show

S S Jena

….Thank You

S S Jena