2. Electron Microscopy
ïThe source of illumination for an electron microscope is a beam of electron.
ïThe resolution of electron microscope is between 2-4 Angstrom.It means we
can differentiate two points that are 2-4 Angstrom apart.
ïIt makes the object 500X greater than the compound microscope and 250,000
greater than the naked eye.
ïThe electrons make the limit of resolution very small. The wavelength of an
electron decreases as its velocity increases.
3.
4. Commonly used Electron Microscopes
1. Transmission Electron Microscope (TEM)
2. Scanning Electron Microscope (SEM)
5. Transmission Electron Microscope
ï¶TEM is a microscopy technique whereby a beam of electrons is
transmitted through an ultra-thin specimen.
ï¶Beam of electrons at high speed is directed towards specimen and
after interaction it passes through it.
ï¶Image is magnified and focused onto an imaging fluorescent screen
such as photographic film or detected through CCD camera
7. Structure of TEM
1. Electron Gun And Condenser System:
The electron gun produces the electron beam. A tungsten filament is placed
in the electron gun as a source of electron.
The condenser system focuses the beam onto the object.
Condenser:
The intensity and angular aperture of beam are controlled by condenser
lens system. It is present between the gun and the specimen.
8. 2. Image Producing System:
It consists of objective lens, moveable specimen
stage and projector lenses. They focus electrons passing through the
specimen to form a real, highly magnified image
3. Image recording system:
It consists of a fluorescent screen for viewing
and focusing the image and a digital camera for permanent records.
Fluorescent screen is fitted at the base of the microscope column.
9. TEM Working
i. Vacuum System: To direct electron beam to pass
through the EM-lenses and strike specimen
ii. Electron Emission Source: At 100-300kV electrons
are generated from tungsten / lanthanum hexaboride
iii. Electromagnetic Lenses and plates: To direct and
manipulate the electron beam as required
iv. Sample holder & Insertion device: To hold and
insert the sample into the column while maintaining
the vacuum
v. Imaging Device: To generate & display image from
transmitted beam of electrons
10. Specimen Preparation for TEM
1. Fixation:
Fixative (Chemical Products) are used that denature or
precipitated the cellular macromolecules
2. Sample Staining:
The most widely used stains in electron microscopy are
the heavy metals. Osmium Tetroxide is a good fixative
and excellent stain for lipids in membranous structures
and vesicles.
11. 3. Dehydration:
Specimen is dehydrated by incubation in a series of ethanol or acetone
solutions. Solvent concentration is increased gradually so that water is
removed gently, without causing shrinkage.
4. Embedding in resin:
Following dehydration, the solvent is replaced with a gradually
increasing concentration of liquid resin (typically epoxy resin for
ultrastructure studies). The specimen is placed in a mold filled with
liquid resin and cured into a hard block using heat or UV light. After
this, a sample can be stored indefinitely.
12. 5. Ultra-thin sectioning:
A specimen embedded in hardened resin can be sectioned extremely thinly, at
less than 100 ”m. This is done using ultra microtome.This allows for the
electron beam to pass from the electron gun through the specimen to the
detector. The sections are mounted on specimen grids which fit into microscope
sample holder.
6. Contrasting (poststaining):
Biological specimens are naturally not very electron opaque as they are composed of
atoms with low atomic numbers and the beam passes through them easily.
13. To increased ample contrast, the sections can be post-stained with lead citrate. This
heavy metal salt, similarly to osmium tetroxide and uranyl acetate, binds to cell
components and scatters the incident beam electrons. The areas of specimen section
which scatter electrons more are recorded as darker pixels, which stand out against the
brighter background.
15. Limitations of TEM
i. Extensive Sample Preparation:
Range of consumables and chemicals required
ii. Ultra-Thin Sectioning:
Very difficult part of sample preparation, diamond coated ultra-
microtome blades required & skillful handling to perform
iii. Time Consuming:
Relatively longer time required upto weeks for sample preparation
completion
iv. Large Number of Samples to be Imaged:
A single field of view may not yield conclusive results
17. SCANNING ELECTRON MICROSCOPE
SEM is a microscopy technique whereby a beam of electrons is used
to scan a sample.
ï Electrons interact with the atoms that make up the sample,
producing signals that contain information about sample
topography, composition and related properties.
19. SEM
Working
ï Electron beam is generated from electron gun (tungsten
wire)
ï Beam acceleration by the Anode
ï Electron beam focusing by controlling the electromagnetic
fields and lenses towards the specimen
ï Scanning the specimen surface by a mechanism of
deflection coils (Raster pattern)
ï Production of Secondary electrons (SE) , Back Scattered
electrons (BSE) and X-Rays
ï Detectors in imaging region convert SE, BSE and X-rays
into signals and corresponding images are generated
24. SEM Analysis Reveals
Topography:
Surface features of an object and its texture
Morphology:
The shape and size of the particles making up the object
Composition:
Elements and compounds that the object is composed of and relative
amount of them
âą Numbers of BSE depends on the atomic number of element and hence
helps to distinguish between different phases, providing image with
sampleâs composition information.
Crystallographic Information:
The arrangements of the atoms in the object
Cu atoms scatter more electrons
back towards detector than the
lighter aluminum atoms
25. SEM Sample preparation
Cleaning surface of
Specimen
Stabilizing / Fixing
the Specimen
Rinsing the
Specimen
Dehydration
Coating the
Specimen
26. Limitation of SEM
ï Artifacts during sample preparation. No absolute way to eliminate
or identify all potential artifacts
ï Conductive Specimen needed
ï Expensive metals for sample preparations
